Synthesis and Antitumor Activity of 1-Substituted 1,2,3-Triazole-Mollugin Derivatives

A new series of mollugin-1,2,3-triazole derivatives were synthesized using a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of corresponding O-propargylated mollugin with aryl azides. All the compounds were evaluated for their cytotoxicity on five human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7) using MTS assays. Among the synthesized series, most of them showed cytotoxicity and most of all, compounds 14 and 17 exhibited significant cytotoxicity of all five cancer cell lines.

Although mollugin has promising anticancer activity, it has little effect on the viability of cancer cells directly. Therefore, we tried to introduce new groups based on mollugin to enhance direct cytotoxicity of mollugin on cancer cells in the further investigation. Through literature research, we found that mollugin derivatives have been synthesized through modification of the ester group (C-2) and substitution reactions (C-4, C-6, C-7, C-1' and C-2') [12,13]. To our surprise, the hydroxyl group (C-1) of mollugin has not been modified and we synthesized mollugin derivatives by modifying this group.
1,2,3-Triazoles are attractive connecting units, as they are stable with metabolic degradation and capable of hydrogen bonding, which can be favorable in binding of biomolecular targets and solubility [14,15]. Therefore, 1,2,3-Triazole is often used as a functional group that needs to be considered in the process of drug design [16,17]. In addition, the click the click reaction of copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition has been widely used to covalently link two molecular fragments between a terminal alkyne and an azide to generate substituted 1,2,3-triazoles [18,19]. It is worth mentioning that the reaction was generally regiospecific in forming only the 1,4-substituted 1,2,3-triazole, which facilitates the further purification of the target product [20,21].
In this manuscript, the key intermediate was obtained by proparylation of the hydroxyl group (C-1) of mollugin (Figure 1). Then a series of mollugin derivatives were synthesized through the click chemistry approach by introducing different substituted aromatic azides [22][23][24]. Further the synthesized derivatives were screened for cytotoxicity against five different human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7).
As we can see from Scheme 2, 40 mollugin derivatives were obtained via the key click reaction. All the compounds present different substituents at the triazole moiety to evaluate their influence on the antitumor activity. Thus, mollugin derivatives with an aromatic ring with electron-donating groups or electron-withdrawing groups were prepared. All the synthesized triazolyl derivatives (5-44) were characterized by 1 H NMR, 13

Evaluation of Biological Activity
Compound 1 and its synthesized derivatives were screened against a group of five different human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7) to evaluate their cytotoxic potential using MTS assay [29,30]. Cisplatin (DDP) and Taxol (TAX)
In this manuscript, the key intermediate was obtained by proparylation of the hydroxyl group (C-1) of mollugin (Figure 1). Then a series of mollugin derivatives were synthesized through the click chemistry approach by introducing different substituted aromatic azides [22][23][24]. Further the synthesized derivatives were screened for cytotoxicity against five different human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7).
As we can see from Scheme 2, 40 mollugin derivatives were obtained via the key click reaction. All the compounds present different substituents at the triazole moiety to evaluate their influence on the antitumor activity. Thus, mollugin derivatives with an aromatic ring with electron-donating groups or electron-withdrawing groups were prepared. All the synthesized triazolyl derivatives (5-44) were characterized by 1 H NMR, 13 C NMR, and HRMS spectroscopic study (see Supplementary Materials).
As we can see from Scheme 2, 40 mollugin derivatives were obtained via the key click reaction. All the compounds present different substituents at the triazole moiety to evaluate their influence on the antitumor activity. Thus, mollugin derivatives with an aromatic ring with electron-donating groups or electron-withdrawing groups were prepared. All the synthesized triazolyl derivatives (5-44) were characterized by 1 H NMR, 13 C NMR, and HRMS spectroscopic study (see Supplementary Materials).

Evaluation of Biological Activity
Compound 1 and its synthesized derivatives were screened against a group of five different human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7) to evaluate their cytotoxic potential using MTS assay [29,30]. Cisplatin (DDP) and Taxol (TAX) were taken as reference drugs and their IC 50 data were present in Table 1. More than half of the derivatives exhibited better cytotoxic activity than mollugin. Some of derivatives displayed good cytotoxicity (IC 50 < 20 µM) and even more potent than the control drug DDP, compounds These data have allowed us to carry out a structure and activity relationship (SAR) study on the influence of the modifications of different group in the cytotoxicity. The main results can be summarized as follows: derivatives containing electron-donating groups such as hydroxyl, methoxy, and alcohol hydroxyl groups tend to have good cytotoxicity. By comparing IC 50 value of compounds 5, 11, 14, and 15, it could be concluded that cytotoxicity increased with the growth of methoxy group number in those derivatives. According to the experimental results, derivatives that contain electron-withdrawing groups do not have cytotoxicity except for compound 36. Compound 36 possesses notable cytotoxicity against A549 cancer cells with IC 50 value of 4.82 ± 0.84 µM, which is triple and quadruple improvement in cytotoxicity compared to the control drug DDP.

General Experimental Procedures
All the reagents and solvents used for purification and synthesis were purchased from Meryer. All synthesized derivatives were purified by column chromatography (silica gel, petroleum ether/ethyl acetate, 20:1 to 1:1 and petroleum ether/acetone, 20:1 to 1:1) and their structures were elucidated by 1 H NMR, 13 C NMR, high-resolution mass spectrometry (HR-ESIMS). Mass spectra were performed on UPLC-IT-TOF (Shimadzu, Kyoto, Japan) spectrometer. NMR spectra were recorded on AVANCE III 400 MHz (Bruker, Bremerhaven, Germany) and Avance III 600 MHz (Bruker, Bremerhaven, Germany) instruments using CDCl 3 , CD 3 OD or acetone-d 6 as the solvent with TMS as the internal standard. Chemical shifts (δ) were reported in parts per million (ppm) and the coupling constants (J) were given in Hertz. Column chromatography was performed on silica gel (200-300 and 300-400 mesh, Qingdao Makall Group CO., Qingdao, China). All chemical reactions were monitored by TLC on silica gel 60 F254 plates and the spots were visualized by UV light and sprayed with 10% H 3 PO 4 ·12MoO 3 in EtOH, followed by heating. All compounds were named using the ACD40 Name-Pro program, which is based on IUPAC rules. Azides (4) were synthesized according to procedures previously described in the literature [27,28].

Conclusions
In conclusion, 40 1-substituted 1,2,3-triazole-mollugin derivatives were synthesized through Huisgen 1,3-dipolar cycloaddition reaction and evaluated for cytotoxicity against a series of five different human cancer cell lines (HL-60, A549, SMMC-7721, SW480, and MCF-7) along with the parent molecule. Most of the derivatives showed better cytotoxicity than parent molecule. It is worth mentioning that our experiment results showed that compound 14 and 17 exhibited cytotoxicity of all five cancer cell lines significantly and compound 36 could enhance the cytotoxicity of lung cancer cells (A549) specifically. Structure and activity relationship (SAR) analysis reveals that electron-donating groups including hydroxyl, methoxy, and alcohol hydroxyl groups are essential for retaining the cytotoxicity to derivatives. In addition, for derivatives containing methoxy groups that the cytotoxicity may increase with the number of methoxy groups. Based on the SAR studies, we believe that the enhancement of cytotoxicity of the derivatives may be caused by the aromatic ring becoming electron-rich or the electron-donating atoms with lone pairs provided by electron-donating groups available to serve as hydrogen bond acceptors with the active site, which is worthy of further study.