Novel Thioethers of Dihydroartemisinin Exhibiting Their Biological Activities

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Introduction
Artemisinin, a sesquiterpene lactone, has been isolated on a large-scale from Artemisia annua L. in Vietnam [1]. Artemisinin has excellent antimalarial activity due to an endoperoxide bridge that is an active center for biological activity [2]. Discovery of artemisinin was considered a revolution in the treatment of severe malaria in the 90s of the last century [3]. In a short time, many artemisinin derivatives were introduced and screened for antimalarial activity to fnd new drugs for the treatment of this disease with the aim of improving efcacy and resistance [4]. Among these derivatives, dihydroartemisinin 1 (DHA), artemether 1a, and artesunate 1b (Figure 1) are three candidates that have the earliest been approved for use in the treatment of malarial disease [5,6]. Although the antimalarial activity is improved compared with artemisinin, over time resistance to these drugs has also been discovered, and their efectiveness is also signifcantly reduced [7,8]. Tus, various artemisinin derivatives were prepared and probed for antimalarial activity, for example, DHA α-alkylbenzylic ethers [9], derivatives containing a sugar moiety [10], watersoluble derivatives [11,12]. Apparently, artemisinin scafold is still the best selection for new antimalarial drug development.
In addition to antimalarial activity, artemisinin and its derivatives have also been discovered as potential candidates for anti-infammatory and antitumor activities [13][14][15][16][17][18][19]. Reports show that artesunate is a potent inhibitor of many cancer cell lines and efectively prevents metastasis of tumor cells [20][21][22][23]. Interestingly, artesunate 1b is less toxic to healthy cells, and suitable for clinical trial studies. Based on the artesunate 1b structure, a dimethylene bridge in the ether C-O-C linkage at C10 was used as a template for the synthesis of dihydroartemisinin derivatives. Several structures like 1c and 1d were also reported to have potential in vitro anticancer activity [24,25]. Terefore, artemisinin 1 scafold is an ideal target for searching and developing a new drug that can be used for both antimalarial and antitumor purposes.
In pharmaceutical chemistry research, thiols are considered as substances with a broad spectrum of activities, including anti-infammatory and anticancer activities [26][27][28][29]. Tey are widely used in drug design, and several thiols are important components of drugs such as omeprazole and lansomeprazole [30,31]. In our previous report [32], the conjugates of thiol with zerumbone, another naturally occurring sesquiterpene, exhibited activity against HepG2, A549, and HeLa cell lines with the IC 50 ranging from 0.93 to 22.40 μM, that were approximately 4-fold to 20fold stronger cytotoxic activity than that of zerumbone. Terefore, the strategy of using thiols in combination with the artemisinin skeleton can be efective. Currently, there are few studies on the conjugates of thiols with DHA [33][34][35]. Notably, three thioethers between DHA and mercaptothiadiazoles, mercaptotetrazole via a dimethylene bridge as well as their antibacterial activity were reported [35]. In line with our continuing study into the chemical modifcation at C-10 of DHA, in the present work, new derivatives of DHA with a series of heterocyclic thiols via thioether and ether linkages were designed, synthesized, and evaluated for their cytotoxic, anti-infammatory, and α-glucosidase inhibitory activities.

2.1.
Chemistry. Dihydroartemisinin is commercially available in Vietnam. Other chemicals were purchased from Sigma-Aldrich and used without further purifcation. 1 H-NMR and 13 C-NMR spectra were recorded at ambient temperature on a Bruker Avance 600 MHz spectrometer in DMSO-d 6 . Chemical shifts δ are quoted in parts per million (ppm) referenced to the residual solvent peak, (DMSO-d 6 at 2.49 ppm and 39.5 ppm) relative to TMS. High-resolution mass spectra were recorded by using a X500R QTOF LC-MS system, SCIEX, US. Tin-layer chromatography (TLC) was performed on a precoated aluminum sheet of Silica Gel 60 F254 (Merck), and products were visualized by using a UV lamp at 254 nm. Column chromatography was carried out on silica gel (40-230 mesh).
Te alpha-glucosidase inhibitory activity of test compounds was determined based on the conversion of substrate p-nitrophenyl-α-D-glucopyranoside (pNPG) to yellowcolored product p-nitrophenol (pNP) following the described method of Ting and coworkers [40] with modifcations.
Te in vitro cytotoxic evaluation against Hep-G2 and LU-1 cancer cell lines was carried out according to the described protocols [41,42].

Results and Discussion
3.1. Chemistry. Te objective of this research was to synthesize the thioethers of dihydroartemisinin that were obtained by the connection of thiols 4a-4k with the DHA via ether and thioether linkages as outlined in Scheme 1. Te ether bridge of intermediate 3 was created by an etherifcation between DHA with 2-bromoethanol according to a known procedure [36], followed by the S-alkylation of thiols 4a-4k with 3 at room temperature catalyzed by a weak base K 2 CO 3 in DMF to produce thioethers 5a-k in 31-70% yields.
Most of the conjugates between DHA 1 and thiols were successfully obtained as designed. However, some thiols are tautomerized leading to the formation of products that are not as originally designed. Besides, dimer products of both Sand N-alkylation were also observed and isolated. Te frst is the case of 2-mercaptoimidazole 4g. TLC showed that two products were formed including 5g2 and 5g1 that were easily separated by column chromatography/silica gel eluting with n-hexane : acetone � 2 : 1. Teir 1 H and 13 C-NMR spectra indicated that 5g1 is a product of S-alkylation, and 5g2 is another product of both the S-alkylation and N-alkylation reactions that contains two DHA units (Scheme 1). Te assignment of signals was accomplished based on the current NMR data of 5g1 and references [43]. Accordingly, the assigned data of the DHA moiety are also in good agreement with the 1D-and 2D-NMR spectra of 5f.
In the case of 6-mercaptopurine 4h, this compound can exist in several structural forms due to the tautomerization [42]. Teoretically, the alkylation reaction can create products with diferent structures. However, the observation of the TLC of the reaction under a UV lamp at 254 nm revealed that only one product 5h was formed and easily purifed by column chromatography/silica gel eluted with nhexane: acetone � 2 : 1. Te NMR spectra of the product show that alkylation occurred at both -SH and -NH groups, evidenced by the observation that all signals of the protons and carbons in the two DHA moieties appear in pairs at the same positions, respectively. For example, 2 doublets of H-10 and H-10″ (J � 3.3 Hz) can be found in the 1 H-NMR spectrum at 4.75 and 4.64 ppm, respectively. Te signals of C-10 and C-10″ atoms were also found to be 100.7 and 100.4 ppm, respectively.
Lastly, in the cases of 5k, the tautomerization of the benzimidazole ring also resulted in the formation of a mixture of two isomers. Te TLC of the reaction (nhexane : acetone � 2 : 1) gives an unique spot visualized at 254 nm under a UV lamp. However, the NMR signal in the aromatic region of the obtained product suggests that this is a mixture of 5-OMe and 6-OMe forms [44] (Figure 2), these isomers could not separate from each other by column chromatography.
Spectral analysis of the mixture of 5k1 and 5k2 isomers also revealed that the signals of H-4′, H-6′ of the 5-OMe isomer and H-5′, H-7′ of 6-OMe one were split as pairs, corresponding to a 5 : 4 ratio, which was determined by its 1 H-NMR. While the signal region of aromatic carbon atoms of this moiety in the 13 C-NMR spectrum was very weak and difcult to attribute. Figure 3 depicts the cytotoxic half-maximal inhibitory concentrations (IC 50 ) of test compounds on RAW 264.7 cells, as well as concentrations eliciting no signifcant cytotoxicity for further assessment.

Anti-Infammatory Activity. (1) Cell Viability.
Te efects of test compounds on RAW 264.7 viability were determined in terms of viable cell percentages measured by the MTT test. Results revealed cell viabilities ranging from 0 to 23.77 ± 2.18% when incubating with test compounds at a concentration of 20 µg/mL (data not shown), suggesting lower amounts of test compounds for application in nitrite assay.

Reduction of NO Production.
Te in vitro antiinfammatory activity of test compounds was evaluated by measuring the reduced NO production in cell culture supernatants of LPS-stimulated RAW 264.7 cells. Te results showed that nine out of ten compounds inhibited NO production with percentages of inhibition ranging from 59.74 ± 3.04 to 77.93 ± 0.88% (Table 1). Among the tested compounds, 5i was the sole causing no NO reduction. While the inhibition of NO production to the culture medium in wells treated with LPS was taken as 0%, the percentage by incubation with positive control (dexamethasone) was 62.46 ± 1.27% (Table 1). Table 2 presented the result of the α-glucosidase inhibitory assay for the obtained conjugates. Accordingly, three products 5a, 5e, and 5i expressed a good efect in suppressing the α-glucosidase, especially compound 5i with the IC 50 of 0.21 mM was found to be stronger than the positive control. Tis is expected to be a promising fnding in research of antidiabetes activity of DHA derivatives that has not drawn a worthy attention.

Cytotoxic
Activity. Te result of in vitro cytotoxicity evaluation of the prepared products was summarized in Table 3. Te synthesized thioethers were screened for cytotoxic activity against HepG2 and LU-1 cell lines together with DHA. Te results showed that eight compounds had stronger cytotoxic activity than DHA against both tested  cancer cell lines except for 5c and 5g1 that contained heterocycles 2-mercaptopyrimidine and 2-mercaptoimidazole. Among synthesized thioethers, the conjugate of DHA with 6-mercaptopurine 5h exhibited the most potent activity against HepG2 and LU-1 cell lines with the IC 50 of 2.73 and 13.06 µM, that is about 6-fold to 8-fold stronger than that of DHA. Notably, compounds 5a and 5e displayed an efect in all three tested assays.

Conclusions
A convenient procedure was applied to prepare novel derivatives of DHA containing both C-O-C and C-S-C linkages. Anomalies of the signal in NMR spectra were discussed and explained by the tautomerization of thiols bearing imidazole and benzimidazole rings. Screening for the in vitro cytotoxicity has indicated that almost all the synthesized compounds had a stronger activity than DHA against HepG2 and LU-1 cancer cell lines. For evaluation of in vitro anti-infammatory activity, apart from 5c, nine out of ten tested products showed NO production inhibition with the IC 50 values ranging from 1.48 to 8.39 µM. Additionally, three compounds 5a, 5e, and 5i showed inhibitory activity on α-glucosidase with the IC 50 values of 0.47, 0.41, and 0.21 mM, respectively. In view of the above obtained fndings, it is appropriate to conclude that the presence of 2mercaptopyridine or 2-mercaptobenzothiazole can be benefcial structures for the biological activities of the conjugates of DHA in this study.

Data Availability
Te data used to support the study can be made available upon request to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.