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Abstract

Sixteen conjugates of dihydroartemisinin and zerumbone with 2-mercapto-1,3,4-oxadiazoles were synthesized and structurally elucidated by 1D NMR, 2D NMR, and HRMS spectra. The cytotoxic screening results showed that all the conjugates of dihydroartemisinin with 2-mercapto-1,3,4-oxadiazoles (19a-h) exhibited cytotoxic activity against two human cancer cell lines, HepG2 and LU-1, with the IC₅₀ values ranging from 2.22 to 40.69 µM. Among dihydroartemisinin conjugates, conjugate 19b displayed the strongest activity against both HepG2 and LU-1 cell lines, with the IC₅₀ values of 3.49 and 2.22 μM, respectively. The zerumbone conjugates (20a-h) expressed their cytotoxic activity stronger than that of 19a-h series, with IC₅₀ values ranging from 1.54 to 2.00 µM. In addition, all 16 compounds exhibited an impressively inhibitory effect against EGFR tyrosine kinase with binding affinities ranging from −8.61 to −10.2 kcal/mol, higher than that of the erlotinib drug (−7.50 kcal/mol), a co-crystallized inhibitor of EGFR receptor. Both conjugates (19a and 20a) containing the 2-hydroxyphenyl-2mercapto-1,3,4-oxadiazole moiety had the best binding energies (−10.2 and −9.494 kcal/mol, respectively) on the EGFR tyrosine kinase. Furthermore, the potential interactions and binding patterns between the compounds and the relevant amino acid residues revealed the most significant contribution to amplifying their efficacy against this protein, according to docking studies, which identified both hydrogen bonds and hydrophobic contacts at the active site of the EGFR protein.

Keywords

anticancer artemisinin conjugation oxadiazole sesquiterpene zerumbone

Introduction

Oxadiazole rings constitute an important group of heterocycles. In general, oxadiazoles are synthetic and rarely found in nature, except for three oxadiazoles: phidiniadine A (1), phidiniadine B (2), and quisqualic acid, which were isolated from the aeolid opisthobranch Phidiana militaris and the seeds of Quisqualis indica and Q. fructus.^1,2 Structurally, two nitrogen atoms occupy different positions in the five-membered ring to form four oxadiazole isomers, including 1,2,3-, 1,2,4-, 1,3,4-, and 1,2,5-oxadiazole.³ Among these isomers, 1,2,4- and 1,3,4-oxadiazoles are two oxadiazoles that are found to have more applications in pharmaceuticals.⁴ Some 1,2,4- and 1,3,4-oxadiazole derivatives are important drugs for the treatment of diseases, such as ataluren (4) for Duchenne muscular dystrophy (DMD) disease, pleconaril (5), and raltegravir (6) for the treatment of viral infection⁵ (Figure 1). Studies on the role of the oxadiazole ring in anti-inflammatory, anticancer, antidiabetic, and antimicrobial activities by molecular modeling indicate that the heteroatoms and π electrons of the oxadiazole ring are preferred centers for the formation of hydrogen and π-π bonds with important amino acids in the active site of the protein and prevent their overexpression.^6–10 Therefore, oxadiazoles can be used as substrates to incorporate with other bioactive compounds.

Figure 1.

The chemical structure of oxadiazole isomers, natural oxadiazoles, and some oxadiazole drugs.

Artemisinin (7) and zerumbone (9) (Figure 2) are two sesquiterpenes that were isolated on a large scale from two Vietnamese traditional medicinal plants, Artemisia anua L.¹¹ and Zingiber zerumbet (L.) Sm.,¹² respectively. Both artemisinin and zerumbone exhibit excellent biological activities, including antimalarial and cytotoxic activities.^13–16 Artemisinin possesses an endoperoxide bridge that is key for biological activities such as antimalarial, anticancer, and antiviral activities.^17–20 However, its antimalarial activity is the most prominent. For this reason, artemisinin and several of its derivatives are approved for use to treat malarial diseases throughout the world.^21,22 In addition to antimalarial activity, artemisinin and its derivatives have been reported as potential inhibitors of cytotoxic activities.^23,24 In this field, the excellent role of the endoperoxide bridge is again exhibited by interacting with iron ions in the cancer cells to produce the alkylating carbon-centered radicals and radical oxygen species (ROS). These radicals prevent the growth of cancer cells, promote their apoptosis and DNA damage, and inhibit the formation of vessels in tumor metastasis.^23,25

Figure 2.

The chemical structures of artemisinin 7, zerumbone 9, and some of their conjugates.

Another bioactive center motif is the natural pentadienone moiety found in zerumbone (9).²⁶ The α,β-unsaturated ketone group of zerumbone has an important role as a Michael receptor, which has preferential activity toward the thiol group (SH) of glutathiones (GSHs).^27,28 The inhibition of nF-кB factor activity by zerumbone is interpreted by the Michael addition of a thiol group to the α,β-unsaturated ketone group of zerumbone. Moreover, zerumbone is reported to inhibit the Wnt/β-catenin signaling pathway, which is related to cancer cell proliferation and metastasis.²⁹ In molecular biology, some diseases are related to the overexpression of target proteins, which are controlled by important amino acids in their active sites. Zerumbone interacts with these amino acids to prevent protein overexpression, such as COX-2 protein in in-inflammatory activity,³⁰ EGFR,³¹ nF-kB, Wnt protein, TNF-α, IKK-α, and IKK-β enzymes in anticancer activity.^32–36

Nowadays, discovering particular and efficient chemotherapy drugs is a task given to medicinal chemists. These medications work by interacting with certain molecular targets to limit the growth of cancer cells, severely harming the malignant cells in the process. However, various issues, including poor delivery, selectivity, and/or resistance, may slow down the medication development process. Understanding the cellular and molecular pathways that contribute to the process of cancer development and/or spread is, therefore, of significant interest.³⁷

Protein kinases (PKs) constitute crucial protein families in the process of diverse disease propagation, such as cancer, diabetes, and/or inflammation.³⁸ Therefore, they constitute a very promising target for new drug discovery due to their prominent roles in several cellular functions such as apoptosis, cell cycle, DNA damage/repair, and metabolism.³⁹ The overexpression of EGFR (epidermal growth factor receptor) causes a variety of cancer types, including breast, colon, and ovarian subtypes. EGFR is a transmembrane PK receptor that is responsible for cell proliferation and/or apoptosis through different signal transduction pathways.⁴⁰ It was recorded to be involved in the process of angiogenesis which increases the proliferation of tumor cells, invasiveness, and metastasis. EGFR inhibitors (e.g. erlotinib)⁴¹ are one of the most important FDA-approved drugs for cancer treatment. Our initial in silico study shows that 2-mercapto-1,3,4-oxadiazole conjugates with zerumbone and DHA have better binding affinities to EGFR receptors than erlotinib due to the direct interactions of crucial amino acids in the active site of EGFR with the oxazole ring and other moieties of these conjugates.

As mentioned earlier, the strategy of building structural blocks based on 1,3,4-oxadiazole and these sesquiterpenes is reasonable. As a continuous result of our recent studies^42–44 on the synthesis and biological activities of dihydroartemisinin (DHA) and zerumbone conjugates with the thiols, in the present work, sixteen conjugates of 2-mercapto-1,3,4-oxadiazoles with DHA and zerumbone via thioether linkage have been designed, synthesized, and evaluated for cytotoxic activity against HepG2, LU-1 cell lines, as well as in silico inhibitory activity against EGFR tyrosine kinase.

Results and discussion

Chemistry

The conjugates 19a-h and 20a-h were created by the S-alkylation reaction of eight 2-mercapto-1,3,4-oxadiazoles 16a-h with the halides 17 and 18 (Scheme 1) that were prepared from DHA⁴⁵ and zerumbone,⁴⁶ respectively. First, the 2-(bromoethyl) ether of DHA 17 was obtained in a 46% yield using the etherification of DHA with 2-bromoethanol catalyzed by BF₃. Et₂O in CH₂Cl₂ at 0°C to rt.⁴⁵ For the preparation of 18, NBS in MeOH-H2O (1:1 v/v) was employed as the brominating agent and afforded the product in quantitative yield. Although not definitively established a free radical mechanism has been invoked for this reaction.⁴⁶ This intermediate was used for the S-alkylation reaction without purification.

Scheme 1.

Synthesis of DHA and zerumbone conjugates: 19a-h, and 20a-h. Reagents and condition: i) EtOH/H₂SO₄, reflux, ii) H₂N-NH₂.H₂O/EtOH, reflux, iii) CS₂/KOH, CH₃COOH, reflux, 14 h, iv) 2-bromoethanol/BF₃. Et₂O, CH₂Cl₂, 0°C to rt, 4 h, v) NBS, CH₃CN:H₂O (1:1, v: v), 1 min., vi) 17, K₂CO₃, DMF, overnight, vii) 18, K₂CO₃, DMF, overnight.

In the next step, 5-(hetero)aryl-2-mercapto-1,3,4-oxadiazoles 16a-h were synthesized from the corresponding acids: 2-hydroxybenzoic acid 13a, 4-methoxybenzoic acid 13b, 3,4,5-trimethoxybenzoic acid 13c, 4-methoxyphenyl acetic acid 13d, 2-pyridine carboxylic acid 13e, 3-pyridine carboxylic acid 13f, 4-pyridine carboxylic acid 13g, and furan-2-carboxylic acid 13h by a three-step procedure as described by Du et al.⁴⁷ and Hanif et al.⁴⁸ The acids were first esterified with ethanol and then reacted with hydrazine hydrate to give the corresponding hydrazides 15a-h that were refluxed with CS₂ in glacial acetic acid to yield 2-mercapto-1,3,4-oxadiazoles 16a-h.

In the final step, 2-mercapto-1,3,4-oxadiazoles 16a-h were S-alkylated with 17 and 18 catalyzed by K₂CO₃ in DMF at room temperature to yield the corresponding derivatives 19a-h, and 20a-h as white solids that were purified by column chromatography/silica gel eluted with n-hexane: acetone in 63%–79%, and 51%–68% yields, respectively.

The structures of 19a-h and 20a-h conjugates were determined by ¹H NMR, ¹³C NMR, and HRMS spectra and compared with those reported by Chinh and colleagues for the related conjugates.^43,44 In a series of 19a-h, the signal of protons and carbons was divided into two regions. In the downfield region, the signals of protons were found to be similar to those in the 1,3,4-oxadiazoles 16a-h. The oxadiazole ring was characterized by two carbon signals of C-2' and C-5' carbons within the regions of 164.0-166.8, and 162.8-164.5 ppm, respectively. In the up-field region, the signals of protons and carbons in the DHA moieties were similar to those of the compound 17. The resonance of protons in the O-CH₂-CH₂-S bridges gave rise to three multiplet signals found within the region of 3.45–4.05 ppm. The first multiplet at 3.95–4.05 ppm was assigned to a proton in O-CH₂-, and the remaining proton in this group appeared by another multiplet with δ_H in the region of 3.66–3.73 ppm. Two protons of the S-CH₂- group were determined in the remaining multiplet signal appearing at 3.45–3.64 ppm. In their ¹³C NMR spectra, two signals of carbon atoms in O-CH₂-CH₂-S bridges were found at 65-66 ppm (O-CH₂-) and 32.3-33.6 ppm (S-CH₂-). The spectral data were in excellent agreement with their structure as designed.

Biology

As shown in Table 1, the conjugates 20a-h of zerumbone with 2-mercapto-1,3,4-oxadiazoles expressed cytotoxic activity against HepG2 and LU-1 cell lines stronger than that of the conjugates 19a-h of DHA with the same oxadiazoles. The cytotoxic activities of both series 19a-h and 20a-h were stronger than those of their parent compounds, DHA and zerumbone. Specifically, in a series 19a-h, the structures of R in 2-mercapto-1,3,4-oxadiazole moieties had various influences on the cytotoxic activity of their conjugates. The pyridine nuclei of 2-mercapto-1,3,4-oxadiazoles were not useful for the cytotoxicity of conjugates 19e-g. The remaining conjugates in this series exhibited strong activity against HepG2 and LU-1, with IC₅₀ values within the region of 2.22–11.11 µM. Among these conjugates, 19b, containing the 4-methoxyphenyl-2-mercapto-1,3,4-oxadiazole moiety, showed the strongest activity against both HepG2 and LU-1, with IC₅₀ values of 3.49 and 2.22 µM, respectively.

Table 1.

In vitro cytotoxic activity of the synthesized conjugates.

No	Compounds	IC₅₀ values^a (µM)
No	Compounds	Hep-G2	LU-1
1	Ellipticine	1.18 ± 0.03	1.30 ± 0.06
2	19a	3.83 ± 0.06	3.95 ± 0.04
3	19b	3.49 ± 0.04	2.22 ± 0.02
4	19c	3.56 ± 0.09	3.42 ± 0.08
5	19d	3.72 ± 0.02	3.71 ± 0.04
6	19e	39.53 ± 0.06	26.72 ± 0.10
7	19f	39.52 ± 0.08	20.91 ± 0.04
8	19g	–	40.69 ± 0.10
9	19h	5.98 ± 0.02	11.11 ± 0.06
10	DHA	35.88 ± 5.06	–
11	20a	1.73 ± 0.06	1.54 ± 0.09
12	20b	1.82 ± 0.14	1.60 ± 0.04
13	20c	1.61 ± 0.10	1.57 ± 0.04
14	20d	1.76 ± 0.04	1.76 ± 0.08
15	20e	1.92 ± 0.06	1.75 ± 0.12
16	20f	1.95 ± 0.08	1.75 ± 0.02
17	20g	1.95 ± 0.14	1.75 ± 0.04
18	20h	2.00 ± 0.04	1.77 ± 0.10
19	Zerumbone	76.40 ± 5.19	28.47 ± 3.02

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

In a series 20a-h, all the conjugates expressed impressive cytotoxicity toward HepG2 and LU-1 with IC₅₀ values ranging from 1.54 to 2.00 µM, much stronger than those of the series 19a-h. It was surprising to know that the difference in the cytotoxic activity of conjugates 20a-h with individual cell lines HepG2 and LU-1 was not significant. Moreover, a small difference in IC₅₀ values between the two cell lines was also observed. Finally, the influence of R structures on the cytotoxicity of conjugate 20a-h was not clear. Therefore, the structural moiety of the 1,3,4-oxadiazole ring connected to the zerumbone scaffold through the thioether bridge may be the required structure for the expression of their cytotoxic activity. This finding is useful for the design of novel candidates bearing this required structural moiety to improve their anticancer activity.

Molecular docking study

The EGFR protein is found on the surface of some normal cells (including cancer cells) and is involved in the processes of cell development and division. Blocking the EGFR pathway can control the growth of cancer cells, and so EGFR inhibitors are used in cancer treatment. Recent studies have shown that tyrosine kinase inhibitors (TKIs) associated with mutations in EGFR are used to treat initial lung cancer.⁴⁹ The three-dimensional crystal structure of the inactive EGFR tyrosine kinase domain (PDB ID: 4HJO, resolution: 2.75 Å) was obtained from the RCSB protein data bank and was used for molecular docking and in silico analysis. Before virtual screening was conducted, the initial crystallographic ligand was re-docked into the active site of the EGFR protein with an RMSD value <2 Å (Figure 3) to show that the docking protocol was suitable for this study. The crystallographic ligand, Erlotinib, is a tyrosine kinase inhibitor known to exhibit activity by binding to the active conformation of the EGFR-TKD (tyrosine kinase domain) used to block EGFR (epidermal growth factor receptor) signaling in cancer. Therefore, this compound was chosen as the reference compound for the current study. Next, two series of compounds were subjected to molecular docking to evaluate their effects on the EGFR target. The binding energies, the number of hydrogen bonds, and the residues involved in the hydrogen bonding of sixteen compounds (19a-h and 20a-h) against EGFR are presented in Table 2. As shown in Table 2, the binding affinities of the two series of compounds are in the range of −8.61 to −10.2 kcal/mol, with the 19a-h series exhibiting higher binding affinity than the 20a-h series, with mean values of −9.594 and −8.9795 kcal/mol, respectively. In addition, the appearance of hydrogen bonds formed by the compounds was mainly with amino acid residues Thr830, Asp831, Phe699, Gly700, Lys721, and Arg817.

Figure 3.

Plot illustrating a 3D representation of the superimposition of the co-crystallized (green) and the re-docked (yellow) Erlotinib in the active site of EGFR protein (PDB ID: 4HJO) with an RMSD value of 1.45 Å.

Table 2.

Binding energies, number of hydrogen bonds, and residues involved in hydrogen bonding of compounds (19a-h and 20a-h) against EGFR (PDB ID: 4HJO).

No.	Code docking	Binding energy (kcal.mol⁻¹)	Number of hydrogen bonds	Residues involved in hydrogen bonding	Distance of hydrogen bond(Å)
1	19a	−10.2	2	Thr 830, Asp 831	1.96, 2.08 (2.26)
2	19b	−9.081	3	Thr 830, Lys 721, Arg 817	2.34, 2.36 (2.39), 2.16
3	19c	−8.893	3	Cys 773, Arg 817, Lys 721	2.67, 2.39, 2.78
4	19d	−9.91	3	Thr 766, Thr 830, Leu 753	2.53, 2.73, 2.97
5	19e	−10.02	5	Lys 721, Arg 817, Thr 766, Asp 831, Thr 830	2.33 (2.30), 2.14, 3.00, 2.36, 2.51 (2.17)
6	19f	−9.577	2	Thr 830, Asp 831	1.97, 2.33
7	19g	−9.661	2	Thr 830, Asp 831	1.96, 2.33
8	19h	−9.41	2	Thr 830, Asp 831	2.37, 2.92, 2.06
9	20a	−9.494	2	Phe 699, Gly 700	2.89, 2.47
10	20b	−9.142	2	Phe 699, Gly 700	2.69, 2.43
11	20c	−8.8	2	Lys 721, Met 769	2.42 (2.50), 2.43
12	20d	−9.078	0	-	-
13	20e	−9.106	3	Thr 830, Asp 831, Thr 766	2.81, 2.18, 3.01
14	20f	−8.853	4	Lys 721, Thr 766, Phe 699, Gly 700	2.86, 2.56, 2.77, 2.41
15	20g	−8.753	3	Phe 699, Gly 700, Lys 721	2.75, 2.47, 2.98
16	20h	−8.61	3	Thr 830, Asp 831, Thr 766	2.65, 2.08, 3.02
17	Erlotinib	−7.50	1	Met 769	2.92

The details of the interactions presented in Table 2 and Figure 4 indicate that both tested compound series exhibit better interactions with EGFR than Erlotinib. Figure 4 shows the best fit of 19a within the DHA-2-mercapto-1,3,4-oxadiazole hybrid series, forming a complex with the EGFR receptor. Docking results reveal that the amino acids Thr830, Asp831 (forming hydrogen bonds), Leu753, Leu764, Met742 (forming π-alkyl interactions), Val702, Cys773, Leu694, Leu820 (forming alkyl interactions), and Lys721 (forming π-cation interactions) are the most active and responsible positions for the interaction with 19a. Within this region, two strong hydrogen bonds were observed with Thr830 and Asp831, at distances of 1.96 Å and 2.08 (2.26) Å, respectively. Compared to other compounds, the hydrogen bond distances of these two positions with compound 19a are the shortest, which may explain the best binding affinity value of this compound. Furthermore, from previous studies, the conclusion that 1, 3, 4-oxadiazole ring-containing derivatives, when docked into EGFR usually form hydrogen bonds at the Thr830 and Asp831 positions suggests that these are two important positions that contribute to the inhibitory potential and significant cytotoxicity activity of this derivative.^50–53 In addition, several hydrophobic interactions have been observed with the amino acids Thr830, Asp831, Leu753, Leu764, Met742, Val702, Cys773, Leu694, Leu820, and Lys721. In the zerumbone 2-mercapto-1,3,4-oxadiazole hybrid series, particularly in compound 20a, there are two strong hydrogen bonds with the binding pocket residues at positions Phe699 and Gly700, along with other hydrophobic interactions as shown in Figure 4. This compound has a carbonyl group on the zerumbone ring with a high electron-withdrawing capability. This oxygen position forms a hydrogen bond with the N-H of Phe699 and Gly700, enhancing the stability of the complex. The compound also forms alkyl and π-alkyl interactions with Ala719, Leu820, Lys721, Val702, and Leu834, making the complex thermodynamically more stable and potentially capable of inhibiting the overexpression of EGFR.

Figure 4.

2D (the left) & 3D (the right) views of the top-leads 19a and 20a in the active site of EGFR (PDB ID: 4HJO) using Discovery Studio Visualizer software.

Conclusion

Sixteen novel conjugates 19a-h and 20a-h of DHA and zerumbone with 2-mercapto-1,3,4-oxadiazoles were synthesized and their cytotoxic activity against HepG2 and LU-1 cell lines were evaluated. The cytotoxic effect of dihydroartemisinin and zerumbone that were linked to the 1,3,4-oxadiazole ring through a thioether linker was significantly improved against HepG2 and LU-1 cell lines. The conjugates of zerumbone with 2-mercapto-1,3,4-oxadiazoles via a thioether linkage exhibited in vitro cytotoxic activity stronger than those of the DHA.

In addition, the subsequent docking studies revealed that conjugates 19a and 20a have higher binding energies and a binding manner similar to that of erlotinib on the EGFR enzyme. Docking analysis showed important interactions between conjugates (19a, 20a) and specific amino acid residues in the 4HJO active site, as well as the favorable binding modes of these compounds. The knowledge of the structural prerequisites for anticancer activity against human cancer cells is being improved by this investigation. The findings are basic to propose that the two series of the 1,3,4-oxadiazoles with DHA and zerumbone serve as the appropriate motifs for designing potent EGFR inhibitors, exhibiting a mechanism of action like erlotinib. These inhibitors are promising candidates for the development of new drugs in targeted cancer therapy.

Experimental

Chemistry

While dihydroartemisinin is commercially available in Vietnam, zerumbone is isolated from the rhizome of Zingiber zerumbet according to a protocol described by Akhtar et al.⁵⁴ Other chemicals were purchased from Sigma Aldrich (Singapore) and used without further purification. ¹H NMR and ¹³C NMR spectra were recorded at the 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 ppm, 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).

2-mercapto-1,3,4-oxadiazoles 16a-h were prepared from the corresponding acids: 2-hydroxybenzoic acid 13a, 4-methoxybenzoic acid 13b, 3,4,5-trimethoxybenzoic acid 13c, 4-methoxyphenyl acetic acid 13d, 2-pyridine carboxylic acid 13e, 3-pyridine carboxylic acid 13f, 4-pyridine carboxylic acid 13g, and furan-2-carboxylic acid 13h by a procedure as reported by Du et al.⁴⁷ and Hanif et al.⁴⁸

The 2-(bromo)ethyl ether of DHA 17 was prepared from DHA according to a recipe as described by Liu et al.⁴⁵ while the intermediate 18 was obtained by a method reported by Kitayama et al.⁴⁶

General procedure for the synthesis of conjugates 19a-h, and 20a-h

To a stirred solution of each 2-mercapto-1,3,4-oxadiazole (16a-h) (1.0 mmol) and potassium carbonate (152.0 mg, 1.1 mmol) in 5 mL dry dimethylformamide at room temperature, 17 or 18 (1.0 mmol) was added. The mixture was stirred at room temperature overnight until TLC (n-hexane:acetone) indicated the absence of 2-mercapto-1,3,4-oxadiazole. The mixture was poured into cool water and acidified by the diluted solution to a pH = 3. The precipitate was filtered on a Buchner funnel, washed thoroughly with water, and dried in a vacuum. Crude 19a-h and 20a-h were purified by column chromatography on silica gel eluted by n-hexane:acetone 3:1 and 4:1, respectively.

2-(5-((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)thio)-1,3,4-oxadiazol-2-yl)phenol (19a)

Yield 73%, white powder, m.p. 120-121 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.76 (dd, J₁ = 1.8 Hz, J₂ = 8.1 Hz, 1H, H-6), 7.45 (dt, J₁ = 1.2 Hz, J₂ = 8.1 Hz, 1H, H-4), 7.07 (d, J = 8.1 Hz, 1H, H-3), 7.00 (dt, J₁ = 1.2 Hz, J₂ = 8.1 Hz, 1H, H-5), 5.32 (s, 1H, H-12"), 4.75 (d, J = 3.6 Hz, 1H, H-10"), 4.03 (m, 1H, O-CH_2a-), 3.71 (m, 1H, O-CH_2b-), 3.58 (m, 2H, S-CH₂-), 2.38 (m, 1H, H-9"), 2.15 (dt, J₁ = 3.9 Hz, J₂ = 14.1 Hz, 1H, H-4₁"), 1.96 (m, 1H, H-4₂"), 1.76 (m, 1H, H-5₁"), 1.56 (ddd, J₁ = J₂ = 3.6 Hz, J₃ = 13.5 Hz, 1H, H-8₁"), 1.48 (m, 1H, H-8₂"), 1.34 (m, 3H, H-7₁", H-5₂", H-8"a), 1.26 (s, 3H, H-15"), 1.23 (m, 1H, H-6"), 1.10 (m, 1H, H-5"a), 0,82(d, J₁ = 6.0 Hz, 3H, H-13"), 0.78 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 164.0 (C-5'), 163.1 (C-2'), 156.1 (C-2), 133.3 (C-4), 128.6 (C-6), 119.6 (C-5), 117.0 (C-3), 109.4 (C-1), 103.3 (C-3"), 100.8 (C-10"), 87.0 (C-12"), 80.4 (C-12"a), 66.0 (10"-O-CH₂-), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 36.0 (C-4"), 34.0 (C-7"), 32.5 (S-CH₂), 30.4 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₅H₃₃N₂O₇S: [M+H]⁺ (m/z): 505.2008, found: 505.1994.

2-(4-methoxyphenyl)-5-((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)thio)-1,3,4-oxadiazole (19b)

Yield 79%, white powder, m.p. 135-136 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.91 (dd, J₁ = 2.1 Hz, J₂ = 8.7 Hz, 2H, H-2, H-6), 7.13 (dd, J₁ = 2.1 Hz, J₂ = 8.7 Hz, 2H, H-3, H-5), 5.33 (s,1H, H-12"), 4.75 (d, J = 3.6 Hz, 1H, H-10"), 4.03 (m, 1H, O-CH_2a-), 3.85 (s, 3H, 4-OCH₃), 3.71 (m, 1H, O-CH_2b-), 3.57 (m, 2H, S-CH₂-), 2.38 (m, 1H, H-9"), 2.16 (dt, J₁ = 3.3 Hz, J₂ = 14.1 Hz, 1H, H-4"a), 1.96 (m, 1H, H-4₁"), 1.76 (m, 1H, H-5₁"), 1.55 (ddd, J₁ = J₂ = 3.3 Hz, J₃ = 13.8 Hz, 1H, H-8₁"), 1.47 (m, 1H, H-8₂"), 1.33 (m, 3H, H-7₁", H-5₂", H-8"a), 1.26 (s, 3H, H-15"), 1.22 (m, 1H, H-6"), 1.09 (m, 1H, H-5"a), 0.81(d, J = 6.6 Hz, 3H, H-14"), 0.80 (d, J = 7.2 Hz, 3H, H-13"), 0.78 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 164.9 (C-5'), 162.8 (C-2'), 162.0 (C-4), 128.1 (C-2, C-6), 115.4 (C-1), 114.8 (C-3, C-5), 103.3 (C-3"), 100.8 (C-10"), 87.0 (C-12"), 80.4 (C-12"a), 66.0 (10"-O-CH₂-), 55.5 (4-OCH₃), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 33.9 (C-7"), 32.5 (S-CH₂-), 30.3 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₆H₃₅N₂O₇S: [M+H]⁺ (m/z): 519.2165, found: 519.2141.

2-((2-(((3R,5aR,8aS,9R,10S,12R,12aR)-3,9-dimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl)thio)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (19c)

Yield 78%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.23 (s, 2H, H-2, H-6), 5.33 (s, 1H, H-12"), 4.75 (d, J = 3.6 Hz, 1H, H-10"), 4.05 (m, 1H, O-CH_2a-), 3.88 (s, 6H, 3-OCH₃, 5-OCH₃), 3.75 (s, 3H, 4-OCH₃), 3.71 (m, 1H, O-CH_2b-), 3.63 (m, 1H, S-CH_2a-), 3.57 (m, 1H, S-CH_2b-), 2.38 (m, 1H, H-9"), 2.15 (dt, J₁ = 3.9 Hz, J₂ = 14.1 Hz, 1H, H-4₁"), 1.95 (m, 1H, H-4₂"), 1.76 (m, 1H, H-5₁"), 1.54 (ddd, J₁ = J₂ = 3.9 Hz, J₃ = 13.5 Hz, 1H, H-8₁"), 1.46(m, 1H, H-8₂"), 1.32 (m, 3H, H-7₁", H-5₂", H-8"a), 1.25 (s, 3H, H-15"), 1.22 (m, 1H, H-6"), 1.09 (m, 1H, H-5"a), 0.81 (d, J = 6.6 Hz, 3H, H-14"), 0.80 (d, J = 7.8 Hz, 3H, H-13"), 0.77 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 164.9(C-5'), 163.5 (C-2'), 153.4 (C-3, C-5), 140.5 (C-4), 118.3 (C-1), 103.8 (C-2, C-6), 103.3 (C-3"), 100.8 (C-10"), 87.0 (C-12"), 80.4 (C-12"a), 66.0 (10"-O-CH₂-), 60.2 (4-OCH₃), 56.2 (3-OCH₃, 5-OCH₃), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 34.0 (C-7"), 32.5 (S-CH₂-), 30.3 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₈H₃₉N₂O₉S: [M+K]⁺ (m/z): 579.2376, found: 579.2345.

2-(4-methoxybenzyl)-5-((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)thio)-1,3,4-oxadiazole (19d)

Yield 74%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.23 (d, J = 8.7 Hz, 2H, H-2, H-6), 6.91 (d, J = 8.7 Hz, 2H, H-3, H-5), 5.32 (s, 1H, H-12"), 4.73 (d, J = 3.6 Hz, 1H, H-10"), 4.16 (s, 2H, 1-CH₂-), 3.95 (m, 1H, O-CH_2a-), 3.73 (s, 3H, 4-OCH₃), 3.66 (m, 1H, O-CH_2b-), 3.45 (m, 2H, S-CH₂-), 2.39 (m, 1H, H-9"), 2.17 (dt, J₁ = 3.9 Hz, J₂ = 14.1 Hz, 1H, H-4₁"), 1.99 (m, 1H, H-4₂"), 1.79 (m, 1H, H-5₁"), 1.64 (ddd, J₁ = J₂ = 3.0 Hz, J₃ = 13.8 Hz, 1H, H-8₁"), 1.52 (m, 2H, H-8₂", H-5₂"), 1.33 (m, 2H, H-7₁", H-8"a), 1.29 (m, 1H, H-6"), 1.27 (s, 3H, H-15"), 1.12 (m, 1H, H-5"a), 0.88 (d, J = 6.0 Hz, 3H, H-14"), 0.83 (m, 1H, H-7₂"), 0.80 (d, J = 7.2 Hz, 3H, H-13"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 166.8 (C-5'), 163.4 (C-2'), 158.4 (C-4), 129.9 (C-2, C-6), 126.0 (C-1), 114.1 (C-3, C-5), 103.3 (C-3"), 100.7 (C-10"), 87.0 (C-12"), 80.4 (C-12"a), 65.5 (10"-O-CH₂-), 55.0 (4-OCH₃), 52.0 (C-5"a), 43.7 (C-8"a), 36.6 (C-6"), 36.0 (C-4"), 34.1 (C-7"), 32.3 (S-CH₂-), 30.4 (C-9"), 29.9 (1-CH₂-), 25.6 (C-13"), 24.2 (C-5"), 23.8 (C-8"), 20.1 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₇H₃₇N₂O₇S: [M+H]⁺ (m/z): 533.2321, found: 533.2300.

2-(pyridin-2-yl)-5-((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)thio)-1,3,4-oxadiazole (19e)

Yield 70%, white powder, m.p. 121-122 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.77 (m, H-6), 8.15 (ddd, J₁ = 0.6 Hz, J₂ = 1.2 Hz, J₃ = 7.8 Hz, 1H, H-3), 8.05 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-5), 7.63 (m, 1H, H-4), 5.32 (s, 1H, H-12"), 4.76 (d, J = 3.0 Hz, 1H, H-10"), 4.05 (m, 1H, O-CH_2a-), 3.73 (m, 1H, O-CH_2b-), 3.61 (m, 2H, S-CH₂-), 2.38 (m, 1H, H-9"), 2.15 (dt, J₁ = 3.9 Hz, J₂ = 7.8 Hz, 1H, H-4₁"), 1.95 (m, 1H, H-4₂"), 1.76 (m, 1H, H-5₁"), 1.55 (ddd, J₁ = J₂ = 3.3 Hz, J₃ = 13.5 Hz, 1H, H-8₁"), 1.47 (m, 1H, H-8₂"), 1.33 (m, 3H, H-7₁", H-5₂", H-8"a), 1.25 (s, 3H, H-15''), 1.21 (m, 1H, H-6''), 1.09 (m, 1H, H-5''A), 0.80 (d, J = 6.0 Hz, 3H, H-14"), 0.79 (d, J = 7.2 Hz, 3H, H-13"), 0.75 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 164.8 (C-5'), 164.5 (C-2'), 150.2 (C-1), 142.5 (C-3), 137.8 (C-5), 126.3 (C-6), 122.6 (C-4), 103.3 (C-3"), 100.8 (C-10"), 87.0 (C-12"), 80.3 (C-12"a), 65.9 (10"-O-CH₂-), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 33.9 (C-7"), 32.5 (S-CH₂-), 30.3 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₄H₃₂N₃O₆S: [M+H]⁺ (m/z): 490.2012, found: 490.1983.

2-(pyridin-3-yl)-5-((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)thio)-1,3,4-oxadiazole (19f)

Yield 71%, white powder, m.p. 130-131 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 9.16 (dd, J₁ = 0.9 Hz, J₂ = 8.1 Hz, 1H, H-2), 8.80 (dd, J₁ = 1.8 Hz, J₂ = 4.8 Hz, 1H, H-4), 8.36 (m, 1H, H-6), 7.64 (m, 1H, H-5), 5.33 (s, 1H, H-12"), 4.76 (d, J = 3.6 Hz, 1H, H-10"), 4.05 (m, 1H, O-CH_2a-), 3.72 (m, 1H, O-CH_2b-), 3.64 (m, 1H, S-CH_2a-), 3.59 (m, 1H, S-CH_2b-), 2.38 (m, 1H, H-9"), 2.15 (dt, J₁ = 3.9 Hz, J₂ = 14.1 Hz, 1H, H-4₁"), 1.95 (m, 1H, H-4₂"), 1.77 (m, 1H, H-5₁"), 1.56 (ddd, J₁ = J₂ = 4.2 Hz, J₃ = 13.2 Hz, 1H, H-8₁"), 1.47 (m, 1H, H-8₂"), 1.32 (m, 3H, H-7₁", H-5₂", H-8"a), 1.25 (s, 3H, H-15''), 1.21 (m, 1H, H-6"), 1.11 (m, 1H, H-5"A), 0.81 (d, J = 7.2 Hz, 3H, H-14"), 0.80 (d, J = 7.2 Hz, 3H, H-13"), 0.78 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 164.4 (C-5'), 163.2 (C-2'), 152.4 (C-2), 147.0 (C-4), 133.9 (C-6), 124.3 (C-5), 119.7 (C-1), 103.3 (C-3"), 100.8 (C-10"), 87.0 (C-12"), 80.3 (C-12"a), 65.9 (10"-O-CH₂-), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 34.0 (C-7"), 32.5 (S-CH₂-), 30.3 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₄H₃₂N₃O₆S: [M+H]⁺ (m/z): 490.2012, found: 490.1983.

2-(pyridin-4-yl)-5-((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)thio)-1,3,4-oxadiazole (19g)

Yield 68%, white powder, m.p. 123-125 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.82 (dd, J₁ = 1.8 Hz, J₂ = 4.2 Hz, 2H, H-3, H-5), 7.92 (dd, J₁ = 1.8 Hz, J₂ = 4.2 Hz, 2H, H-2, H-6), 5.32 (s, 1H, H-12"), 4.75 (d, J = 3.0 Hz, 1H, H-10"), 4.04 (m, 1H, O-CH_2a-), 3.71 (m, 1H, O-CH_2b-), 3.61 (m, 2H, S-CH₂-), 2.38 (m, 1H, H-9"), 2.15 (dt, J₁ = 3.6 Hz, J₂ = 14.4 Hz, 1H, H-4₁"), 1.96 (m, 1H, H-4₂"), 1.77 (m, 1H, H-5₁"), 1.53 (ddd, J₁ = J₂ = 3.3 Hz, J₃ = 13.5 Hz, 1H, H-8₁"), 1.48 (m, 1H, H-8₂"), 1.32 (m, 3H, H-7₁", H-5₂", H-8"a), 1.26 (s, 3H, H-15"), 1.21 (m, 1H, H-6"), 1.10 (m, 1H, H-5"a), 0.81 (d, J = 6.6 Hz, 3H, H-14"), 0.79 (d, J = 7.2 Hz, 3H, H-13"), 0.77 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 165.2 (C-5'), 163.5 (C-2'), 150.9 (C-3, C-5), 130.1 (C-1), 119.9 (C-2, C-6), 103.3 (C-3"), 100.8 (C-10"), 86.9 (C-12"), 80.3 (C-12"a), 65.9 (10"-O-CH₂-), 51.9 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 33.9 (C-7"), 32.5 (S-CH₂-), 30.3 (C-9"), 25.5 (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-15"); ESI-HRMS calculated for C₂₄H₃₂N₃O₆S: [M+H]⁺ (m/z): 490.2012, found: 490.1983.

2-(furan-2-yl)-5-((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)thio)-1,3,4-oxadiazole (19h)

Yield 63%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.04 (dd, J₁ = 0.9 Hz, J₂ = 1.5 Hz, 1H, H-3), 7.34 (dd, J₁ = 0.9 Hz, J₂ = 3.6 Hz, 1H, H-5), 3.79 (dd, J₁ = 1.5 Hz, J₂ = 3.6 Hz, 1H, H-4), 5.32 (s, 1H, H-12"), 3.75 (d, J = 3.0 Hz, 1H, H-10"), 4.02 (m, 1H, O-CH_2a-), 3.70 (m, 1H, O-CH_2b-), 3.56 (m, 2H, S-CH₂-), 2.38 (m, 1H, H-9"), 2.16 (dt, J₁ = 3.9 Hz, J₂ = 14.1 Hz, 1H, H-4₁"), 1.97 (m, 1H, H-4₂"), 1.78 (m, 1H, H-5₁"), 1.56 (ddd, J₁ = J₂ = 3.3 Hz, J₃ = 13.5 Hz, 1H, H-8₁"), 1.47 (m, 1H, H-8₂"), 1.38 (m, 1H, H-5₂"), 1.33 (m, 2H, H-7₁", H-8"a), 1.26 (s, 3H, H-15"), 1.23 (m, 1H, H-6"), 1.11 (m, 1H, H-5"A), 0.83 (d, J = 6.6 Hz, 3H, H-14"), 0.80 (d, J = 7.2 Hz, 3H, H-13"), 0.78 (m, 1H, H-7₂"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 163.1 (C-5'), 157.7 (C-2'), 146.8 (C-1), 138.2 (C-3), 114.4 (C-5), 112.5 (C-4), 103,3 (C-3"), 100.7 (C-10"), 87.0 (C-12"), 80.3 (C-12"a), 65.8 (10"-O-CH₂-), 52.0 (C-5"a), 43.6 (C-8"a), 36.6 (C-6"), 35.9 (C-4"), 33.9 (C-7"), 32.6 (S-CH₂-), 30.3 (C-9"), 25. (C-13"), 24.2 (C-5"), 23.7 (C-8"), 20.0 (C-14"), 12.5 (C-13"); ESI-HRMS calculated for C₂₃H₃₁N₂O₇S: [M+H]⁺ (m/z): 479.1852, found: 479.1828.

(2E,6Z,10E)-6-(((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-2,9,9-trimethylcycloundeca-2,6,10-trien-1-one (20a)

Yield 61%, white powder, m.p. 129-130 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.75 (dd, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-6), 7.45 (m, 1H, H-4), 7.08 (dd, J₁ = 0.6 Hz, J₂ = 7.8 Hz, 1H, H-3), 6.99 (dt, J₁ = 0.6 Hz, J₂ = 7.8 Hz, 1H, H-5), 5.95 (m, 2H, H-3", H-11"), 5,82 (d, J = 16.2 Hz, 1H, H-10"), 5.47 (t, J = 16.2 Hz, 1H, H-7"), 4.36 (s, brd, 1H, H-15"a), 3.57 (s, brd, 1H, H-15"b), 2.57 (m, 3H, H-4"a, H-5"a, H-8"a), 2.24 (s, 2H, H-4"b, H-5"b), 1.81 (s, 1H, H-8"b), 1.71 (m, 3H, H-12"), 1.15 (s, 3H, H-13" or H-14"), 1.05 (3, 3H, H-14" or H-13"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1"), 164.7 (C-5'), 162.7 (C-2'), 159.2 (C-10”), 156.1 (C-2), 148.6 (C-364.7 (C-5’), 162.7 (C-2’), 1), 137.8 (C-2"), 134.2 (C-6"), 133.4 (C-4), 130.9 (C-6), 128.7 (C-7"), 127.0 (C-11"), 119.6 (C-5), 117.0 (C-3), 109.4 (C-1), 41.7 (C-8"), 37.1 (C-9"), 34.9 (C-5"), 31.0 (C-15”), 28.8 (C-13" or C-14"), 24.0 (C-4"), 23.6 (C-14" or C-13"), 11.8 (C-12"); ESI-HRMS calculated for C₂₃H₂₇N₂O₃S: [M+H]⁺ (m/z): 411.1742, found: 411.1716.

(2E,6Z,10E)-6-(((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-2,9,9-trimethylcycloundeca-2,6,10-trien-1-one (20b)

Yield 66%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.91 (d, J = 8.7 Hz, 2H, H-2, H-6), 7.14 (d, J = 8.7 Hz, 2H, H-3, H-5), 5.96 (m, 2H, H-3", H-11"), 5,82 (d, J = 16.8 Hz, 1H, H-10"), 5.46 (t, J = 8.1 Hz, 1H, H-7"), 4.37 (s, brd, 1H, H-15"a), 3.85 (s, 3H, 4-OCH₃), 3.54 (s, brd, 1H, H-15"b), 2.57 (m, 3H, H-4"a, H-5"a, H-8"a), 2.22 (s, 2H, H-4"b, H-5"b), 1.81 (s, 1H, H-8"b), 1.71 (m, 3H, H-12"), 1.45 (s, 3H, H-13" or H-14"), 1.05 (3, 3H, H-14" or H-13"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1"), 165.3 (C-5'), 162.5 (C-2'), 162.1 (C-4), 159.2 (C-10"), 148.7 (C-3"), 137.8 (C-2"), 134.2 (C-6"), 130.7 (C-7"), 128.3 (C-2, C-6), 127.0 (C-11"), 115.3 (C-1), 114.9 (C-3, C-5), 55.5 (4-OCH₃), 41.7 (C-8"), 37.2 (C-9"), 34.9 (C-5"), 31.0 (C-15"), 28.8 (C-13" or C-14"), 24.0 (C-4"), 23.6 (C-14" or C-13"), 11.8 (C-12"); ESI-HRMS calculated for C₂₄H₂₉N₂O₃S: [M+H]⁺ (m/z): 425.1899, found: 425.1879.

(2E,6Z,10E)-2,9,9-trimethyl-6-(((5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)cycloundeca-2,6,10-trien-1-one (20c)

Yield 68%, white powder, m.p. 72-73 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.24 (, 2H, H-2, H-6), 5.94 (m, 2H, H-3", H-11"), 5,82 (d, J = 16.5 Hz, 1H, H-10"), 5.46 (t, J = 8.1 Hz, 1H, H-7"), 4.44 (d, J = 10.8 Hz, 1H, H-15"a), 3.88 (s, 6H, 3-OCH₃, 5-OCH₃), 3.75 (s, 3H, 4-OCH₃), 3.53 (d, J = 10.8 Hz, 1H, H-15"b), 2.61 (m, 3H, H-4"a, H-5"a, H-8"a), 2.33 (m, 2H, H-4"b, H-5"b), 1.80 (m, 1H, H-8"b), 1.71 (m, 3H, H-12"), 1.15 (s, 3H, H-13" or H-14"), 1.05 (3, 3H, H-14" or H-13"). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 165.3 (C-5'), 163.1 (C-2'), 159.2 (C-10"), 153.5 (C-3, C-5), 148.7 (C-3"), 140.7 (C-4), 137.8 (C-2"), 134.2 (C-6"), 130.7 (C-7"), 127.0 (C-11"), 118.2 (C-1), 103.9 (C-2, C-6), 103.9 (C-2, C-6), 56.2 (3-OCH₃, 5-OCH₃), 55.1 (4-OCH₃), 41.6 (C-8"), 37.2 (C-9"), 34.8 (C-5"), 31.0 (C-15"), 28.8 (C-13" or C-14"), 23.9 (C-4"), 23.7 (C-14" or C-13"), 11.8 (C-12"); ESI-HRMS calculated for C₂₆H₃₃N₂O₅S: [M+H]⁺ (m/z): 485.2110, found: 485.2083.

(2E,6Z,10E)-6-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-2,9,9-trimethylcycloundeca-2,6,10-trien-1-one (20d)

Yield 60%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 7.21 (d, J = 7.5 Hz, 2H, H-2, H-6), 6.91 (dd, J₁ = 2.4 Hz, J₂ = 7.5 Hz, 2H, H-3, H-5), 5.91 (m, 2H, H-3'', H-11''), 5.75 (d, J = 16.8 Hz, 1H, H-10''), 5.36 (t, J = 8.1 Hz, 1H, H-7''), 4.17 (s, 2H, H-15''), 3.73 (s, 2H, 1-CH₂-), 2.50 (m, 2H, H-4''a, H-5''a), 2.33 (m, 1H, H-8''a), 2.17 (m, 2H, H-4''b, H-5''b), 1.66 (s, 3H, H-12''), 1.57 (m, 1H, H-8''b), 1.16 (s, 3H, H-13'' or H-14''), 0.99 (s, 3H, H-14'' or H-13''); ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 167.2 (C-5'), 163.0 (C-2'), 159.4 (C-10''), 158.5 (C-4), 148.6 (C-3''), 137.8 (C-2''), 133.9 (C-6''), 130.7 (C-7''), 129.9 (C-2, C-6), 126.9 (C-11''), 126.0 (C-1), 114.2 (C-3, C-5), 55.1 (4-OCH₃), 41.3 (1-CH₂-), 37.1 (C-9''), 34.7 (C-5''), 30.8 (C-15''), 29.9 (1-CH₂-), 28.7 (C-13'' or C-14''), 23.9 (C-4''), 23.5 (C-14'' or C-13''), 11.8 (C-12''); ESI-HRMS calculated for C₂₅H₃₁N₂O₃S: [M+H]⁺ (m/z): 439.2055, found: 439.2030.

(2E,6Z,10E)-2,9,9-trimethyl-6-(((5-(pyridin-2-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)cycloundeca-2,6,10-trien-1-one (20e)

Yield 54%, white powder, m.p. 75-76 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.77 (m, 1H, H-3), 8.15 (d, J = 7.8 Hz, 1H, H-6), 8.06 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-5), 7.64 (m, 1H, H-4), 5.97 (m, 2H, H-3'', H-11''), 5.84 (d, J = 16.8 Hz, 1H, H-10''), 5.50 (t, J = 8.4 Hz, 1H, H-7''), 4.39 (s, brd, 1H, H-15''a), 3.62 (s, brd, 1H, H-15''b), 2.62 (m, 3H, H-4''a, H-5''a, H-8''a), 2.24 (m, 2H, H-4''b, H-5''b), 1.86 (m, 1H, H-8''b), 1.72 (s, 3H, H-12''), 1.17 (s, 3H, H-13'' or H-14''), 1.07 (s, 3H, H-14'' or H-13''). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 164.7 (C-5'), 164.5 (C-2'), 159.2 (C-10''), 150.2 (C-6), 148.6 (C-3''), 142.4 (C-2), 137.9 (C-2''), 137.8 (C-4), 133.8 (C-6''), 131.1 (C-7''), 127.0 (C-11''), 126.4 (C-5), 122.7 (C-3), 41.8 (C-8''), 37.1 (C-9''), 34.9 (C-5''), 31.0 (C-15''), 29.0 (C-13'' or C-14''), 24.0 (C-4''), 23.8 (C-14'' or C-13''), 11.8 (C-12''); ESI-HRMS calculated for C₂₂H₂₆N₃O₂S: [M+H]⁺ (m/z): 396.1745, found: 396.1722.

(2E,6Z,10E)-2,9,9-trimethyl-6-(((5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)cycloundeca-2,6,10-trien-1-one (20f)

Yield 54%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 9.16 (dd, J₁ = 0.6 Hz, J₂ = 1.8 Hz, 1H, H-2), 8.80 (dd, J₁ = 1.5 Hz, J₂ = 5.1 Hz, 1H, H-4), 8.35 (m, 1H, H-6), 7.64 (m, 1H, H-5), 5.97 (m, 2H, H-3'', H-11''), 5.83 (d, J = 16.5 Hz, 1H, H-10''), 5.48 (t, J = 8.1 Hz, 1H, H-7''),4.41 (s, brd, 1H, H-15''a), 3.59 (d, J = 7.2 Hz, 1H, H-15''b), 2.60 (m, 3H, H-4''a, H-5''a, H-8''a), 2.24 (m, 2H, H-4''b, H-5''b), 1.80 (m, 1H, H-8''b), 1.71 (s, 3H, H-12''), 1.16 (s, 3H, H-13'' or H-14''), 1.06 (s, 3H, H-14'' or H-13''). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 164.0 (C-5'), 163.5 (C-2'), 159.2 (C-10''), 152.5 (C-2), 148.7 (C-3''), 147.0 (C-4), 137.8 (C-3''), 134.0 (C-6'', C-6), 130.9 (C-7''), 127.0 (C-11), 124.3 (C-1), 119.6 (C-5), 41.7 (C-8''), 37.2 (C-9''), 34.9 (C-5''), 31.0 (C-15''), 28.8 (C-13'' or C-14''), 24.0 (C-4''), 23.6 (C-14'' or C-13''), 11.8 (C-12''); ESI-HRMS calculated for C₂₂H₂₆N₃O₂S: [M+H]⁺ (m/z): 396.1745, found: 396.1726.

(2E,6Z,10E)-2,9,9-trimethyl-6-(((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)cycloundeca-2,6,10-trien-1-one (20g)

Yield 58%, white powder, m.p. 90-91 °C; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.83 (dd, J₁ = 1.5 Hz, J₂ = 4.5 Hz, 2H, H-3, H-5), 7.91 (dd, J₁ = 1.5 Hz, J₂ = 4.5 Hz, 2H, H-2, H-6), 5.96 (m, 2H, H-3'', H-11''), 5.83 (d, J = 16.5 Hz, 1H, H-10''), 5.49 (t, J = 8.4 Hz, 1H, H-7''), 4.43 (d, J = 8.4 Hz, 1H, H-15''a), 3.61 (d, J = 8.4 Hz, 1H, H-15''b), 2.60 (m, 3H, H-4''a, H-5''a, H-8''a), 2.24 (m, 2H, H-4''b, H-5''b), 1.84 (m, 1H, H-8''b), 1.71 (s, 3H, H-12''), 1.16 (s, 3H, H-13'' or H-14''), 1.07 (s, 3H, H-14'' or H-13''). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 164.8 (C-5'), 163.7 (C-2'), 159.2 (C-10''), 150.9 (C-3, C-5), 148.6 (C-3''), 137.8 (C-2''), 133.9 (C-6''), 131.0 (C-1), 130.0 (C-7''), 127.0 (C-11''), 119.9 (C-2, C-6), 41.7 (C-8''), 37.2 (C-9''), 34.9 (C-5''), 31.0 (C-15''), 28.8 (C-13'' or C-14''), 24.0 (C-4''), 23.6 (C-14' or C-13''), 11.8 (C-12''); ESI-HRMS calculated for C₂₂H₂₆N₃O₂S: [M+H]⁺ (m/z): 396.1745, found: 396.1721.

(2E,6Z,10E)-6-(((5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)thio)methyl)-2,9,9-trimethylcycloundeca-2,6,10-trien-1-one (20h)

Yield 51%, colorless oil; ¹ H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.05 (d, J =1.5 Hz, 1H, H-3), 7.33 (d, J = 1.5 Hz, 1H, H-5), 6.80 (dd, J₁ = 1.5 Hz, J₂ = 3.6 Hz, 1H, H-4), 5.95 (m, 2H, H-3'', H-11''), 5.81 (d, J = 16.5 Hz, 1H, H-10''), 5.47 (t, J = 8.4 Hz, 1H, H-7''), 4.35 (s, brd, 1H, H-15''a), 3.56 (s, brd, 1H, H-15''b), 2.60 (m, 3H, H-4''a, H-5''a, H-8''a), 2.30 (m, 2H, H-4''b, H-5''b), 1.82 (m, 1H, H-8''b), 1.70 (s, 3H, H-12''), 1.16 (s, 3H, H-13'' or H-14''), 1.06 (s, 3H, H-14'' or H-13''). ¹³ C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.6 (C-1''), 162.7 (C-5'), 159.2 (C-10''), 158.1 (C-2'), 148.6 (C-3''), 147.0 (C-1), 138.2 (C-3), 137.8 (C-2''), 133.9 (C-6''), 131.0 (C-7''), 127.0 (C-11''), 114.6 (C-5), 112.6 (C-4), 41.7 (C-8''), 37.1 (C-9''), 34.9 (C-5''), 31.2 (C-15''), 28.8 (C-13'' or C-14''), 24.0 (C-4''), 23.6 (C-14'' or C-13''), 11.8 (C-12''); ESI-HRMS calculated for C₂₁H₂₅N₂O₃S: [M+H]⁺ (m/z): 385.1586, found: 385.1563.

Biology

The in vitro cytotoxic evaluation against HepG2 and LU-1 cancer cell lines was carried out according to the described protocols.^55,56 Accordingly, stock solutions of the conjugates 19a-h, 20a-h were prepared in dimethyl sulfoxide (DMSO) at a concentration of 4 mM, then were diluted 200-fold to obtain solutions with the highest concentration (20 μM) tested on 96-well plates. These solutions were serially diluted for bioassay. Determination of IC₅₀ values was carried out using two human cancer cell lines: HepG2, and LU-1. Ellipticine was used as a positive control. The IC₅₀ values were determined from dose-dependent curves plotted from five different concentration regimens (0–20 μM). At each regimen, the meaning of triplicate experiments was used for a point in the curve.

Molecular docking study

Molecular docking is one of the most effective computational approaches for drug development to determine the efficacy of investigated drugs. The interactions of novel 2-mercapto-1,3,4-oxadiazole-dihydroartemisinin (19a) and 2-mercapto-1,3,4-oxadiazole-zerumbone (20a) hybrids, having the highest EGFR inhibitory activity, were examined with the active site of the target enzyme (PDB ID: 4HJO)⁵⁷ to study their binding modes and orientations (for more details, see in Supplemental Material File 2).

Supplemental Material

sj-pdf-1-chl-10.1177_17475198231199428 – Supplemental material for Synthesis and cytotoxic activity evaluation of novel dihydroartemisinin and zerumbone conjugates with 2-mercapto-1,3,4-oxadiazoles as potential EGFR inhibitors

Supplemental material, sj-pdf-1-chl-10.1177_17475198231199428 for Synthesis and cytotoxic activity evaluation of novel dihydroartemisinin and zerumbone conjugates with 2-mercapto-1,3,4-oxadiazoles as potential EGFR inhibitors by Duc Quan Tran, Ngoc Hung Truong, Thi Hoang Anh Nguyen, Thi Thuy Trinh, Thi Cham Ba, Thi Thuy Linh Nguyen, Xuan Ha Nguyen, Manh Cuong Nguyen and Van Chinh Luu in Journal of Chemical Research

Supplemental Material

sj-pdf-2-chl-10.1177_17475198231199428 – Supplemental material for Synthesis and cytotoxic activity evaluation of novel dihydroartemisinin and zerumbone conjugates with 2-mercapto-1,3,4-oxadiazoles as potential EGFR inhibitors

Supplemental material, sj-pdf-2-chl-10.1177_17475198231199428 for Synthesis and cytotoxic activity evaluation of novel dihydroartemisinin and zerumbone conjugates with 2-mercapto-1,3,4-oxadiazoles as potential EGFR inhibitors by Duc Quan Tran, Ngoc Hung Truong, Thi Hoang Anh Nguyen, Thi Thuy Trinh, Thi Cham Ba, Thi Thuy Linh Nguyen, Xuan Ha Nguyen, Manh Cuong Nguyen and Van Chinh Luu in Journal of Chemical Research

Footnotes

Acknowledgements

We thank Bachelor student, Van Tu Ngo for his help in the preparation of the references and the supplemental materials.

Declaration of conflicting interests

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

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: 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

Xuan Ha Nguyen

Van Chinh Luu

Supplemental material

Supplemental material for this article is available online.

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