Design, Synthesis and Cytotoxicity of Novel Dihydroartemisinin-Coumarin Hybrids via Click Chemistry

In order to develop novel chemotherapeutic agents with potent anticancer activities, we designed four series of novel compounds employing hybridization strategy. Twenty novel dihydroartemisinin-coumarin hybrids, 10a–e, 11a–e, 12a–e, 13a–e, were synthesized via click chemistry in this study and their structures were characterized by HRMS and NMR. The cytotoxic activities were measured by MTT assay against three cancer cell lines (HCT-116, MDA-MB-231, and HT-29) under normoxic or anoxic conditions, respectively. The target compounds exhibited moderate activity with IC50 values in the 0.05–125.40 μM range, and these compounds exhibited better activity against HT-29 cell line under anoxic condition. The cytotoxic activities of most compounds under anoxic condition displayed one- to 10-fold greater activity than under normoxic condition. Compounds 10a–e showed better selectivity against the HT-29 cell line than the other two cell lines. These results indicated that our design of CA IX inhibitors does correspond with its action mode to some degree and deserves further investigation.


Introduction
Currently, the mortality rate associated with cancer shows an increasing tendency worldwide. Chemotherapy is the most important therapeutic strategy against all types of cancer; however, the available anticancer drugs usually cause adverse effects and lead to the development of resistance. Therefore, there is a pressing need for novel chemotherapeutic agents with higher selectivity and more potent anticancer activities [1,2].
Many natural products have been found to possess antineoplastic activity with minimal side effects [3]. Artemisinin, a well-known anti-malarial agent, has been reported to possess a potent and broad antitumor spectrum against human cancer cell lines [4]. However, the drawback, such as drug resistance or unsatisfactory metabolism, constrains artemisinin to be employed as an antitumor drug clinically. Numbers of artemisinin derivatives have been designed and synthesized to overcome the imperfection mentioned above and some of them have shown excellent anticancer activities and pharmacokinetic properties [5].
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc metalloenzymes [6] that catalyze a very simple, but important, reaction: the reversible hydration of carbon dioxide to bicarbonate ion and proton, following a two-step catalytic mechanism [7]. Out of the sixteen human CA (hCA) Scheme 1. The design of target compounds.

Cytotoxicity
We focused our attention on the cytotoxicity of the four series of newly-synthesized compounds (10a-e, 11a-e, 12a-e, and 13a-e) under normoxic or anoxic conditions. The cytotoxic activities of target compounds were measured by MTT assay, working with MRC-5 (human fetal lung fibroblast cells, normal lung cell), HCT-116 (human colorectal cancer cell line, do not express CA IX in response to anoxia), MDA-MB-231 (human breast carcinoma cell line, CA IX negative), and HT-29 (human colon carcinoma cell line, overexpresses high amounts of CA IX) [24,25], using doxorubicin as a positive control, and acetazolamide (AAZ, standard carbonic anhydrase inhibitor) as reference drug.
The data was calculated by the Logit method and presented as IC50 values in Table 1. In 96 h MTT assay results, cytotoxic activities for the twenty target compounds against four cell lines were found to shown moderate activity compared to AAZ, which shows poor cytotoxicity (IC50 > 100 μM) with IC50 values in the 0.05-125.40 μM range, but the cytotoxic activities were less than the positive control DOX.
The line chart that depicted the IC50 values was shown in Figures 1-3. In order to gain a clearer understanding of the activity data, we set the data ">100" in Table 1

Cytotoxicity
We focused our attention on the cytotoxicity of the four series of newly-synthesized compounds (10a-e, 11a-e, 12a-e, and 13a-e) under normoxic or anoxic conditions. The cytotoxic activities of target compounds were measured by MTT assay, working with MRC-5 (human fetal lung fibroblast cells, normal lung cell), HCT-116 (human colorectal cancer cell line, do not express CA IX in response to anoxia), MDA-MB-231 (human breast carcinoma cell line, CA IX negative), and HT-29 (human colon carcinoma cell line, overexpresses high amounts of CA IX) [24,25], using doxorubicin as a positive control, and acetazolamide (AAZ, standard carbonic anhydrase inhibitor) as reference drug.
The data was calculated by the Logit method and presented as IC 50 values in Table 1. In 96 h MTT assay results, cytotoxic activities for the twenty target compounds against four cell lines were found to shown moderate activity compared to AAZ, which shows poor cytotoxicity (IC 50 > 100 µM) with IC 50 values in the 0.05-125.40 µM range, but the cytotoxic activities were less than the positive control DOX. Table 1. IC 50 values of tested compounds against MRC-5, HCT-116, MDA-MB-231, and HT-29.

Compd.
The line chart that depicted the IC 50 values was shown in Figures 1-3. In order to gain a clearer understanding of the activity data, we set the data ">100" in Table 1 as 100, for it would not affect the result. Then we set out to evaluate the structure-activity relationship (SAR) against three cancer cell lines (HCT-116, MDA-MB-231, and HT-29).
First, we found that for the four series of compounds: (i) in the coumarin ring, the 3-chloro, 4-methyl substituent exhibited better activity; (ii) on the contrary, the 3-ethoxycarbonyl group in the 3-position of coumarin showed poorer activity than others; and (iii) the 4-methyl or 4-trifluoromethyl substituent showed no significant differences in the activity.
Then we analyzed the activity data against three cancer cell lines under normoxic or anoxic condition separately compared with MRC-5 (normal cell line) under normoxic conditions (Figures 1-3). In general, the target compounds exhibited better activity against HT-29 cell line under anoxic conditions. The cytotoxicity of these compounds against HT-29 and MDA-MB-231 (CA IX negative [25]) had a general promotion (Figures 1 and 2) under anoxic condition, but the cytotoxicity to MDA-MB-231 exhibited a comparable activity to MRC-5 which mean poor selectivity to normal cells; while the activity for HT-29 under anoxic conditions exhibited better selectivity and appeared less toxic to normal cell MRC-5. For the HCT-116 cell line, which does not express CA IX in response to anoxia [24], there was no significant change in cytotoxicity ( Figure 3).
Moreover, the cytotoxic activities of most compounds under anoxic condition displayed oneto 10-fold greater activity than under normoxic condition ( Figure 4). For HT-29 cell line which over expresses high amounts of CA IX, the first series 10a-e exhibited better selectivity compared to other two cell lines.
Above all, these results indicated that our design of CA IX inhibitors does correspond with its action mode, and deserved further investigation.  1 The values stand for the activity promotion for each cancer cell line. Value = IC50(normoxia)/IC50(anoxia).     1 The values stand for the activity promotion for each cancer cell line. Value = IC50(normoxia)/IC50(anoxia).    First, we found that for the four series of compounds: (i) in the coumarin ring, the 3-chloro, 4-methyl substituent exhibited better activity; (ii) on the contrary, the 3-ethoxycarbonyl group in the 3-position of coumarin showed poorer activity than others; and (iii) the 4-methyl or 4-trifluoromethyl substituent showed no significant differences in the activity.
Then we analyzed the activity data against three cancer cell lines under normoxic or anoxic condition separately compared with MRC-5 (normal cell line) under normoxic conditions (Figures 1-3). In general, the target compounds exhibited better activity against HT-29 cell line under anoxic conditions. The cytotoxicity of these compounds against HT-29 and MDA-MB-231 (CA IX negative [25]) had a general promotion (Figures 1 and 2) under anoxic condition, but the cytotoxicity to MDA-MB-231 MRC-5. For the HCT-116 cell line, which does not express CA IX in response to anoxia [24], there was no significant change in cytotoxicity ( Figure 3).
Moreover, the cytotoxic activities of most compounds under anoxic condition displayed oneto 10-fold greater activity than under normoxic condition ( Figure 4). For HT-29 cell line which over expresses high amounts of CA IX, the first series 10a-e exhibited better selectivity compared to other two cell lines. Above all, these results indicated that our design of CA IX inhibitors does correspond with its action mode, and deserved further investigation.

Chemistry
Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration. The 1 H-NMR and 13 C-NMR spectra were measured by a Bruker AV-400 spectrometer (Bruker Bioscience, Billerica, MA, USA), with tetramethylsilane as an internal standard. High-resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) in ESI mode. Reaction progress was monitored by TLC on silica gel precoated GF254 plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Preparative flash column chromatography was performed on the 200-300 mesh silica gel (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Unless otherwise noted, all solvents and reagents were commercially available and used without further purification.

General Procedure for the Synthesis of Compounds 1a-d
A solution of 1,3-dihydroxybenzene (11.0 g, 100 mmol) in substituted ethyl acetoacetate (100 mmol) was added dropwise to stirring H 2 SO 4 kept at 0˝C. After completion of the addition, the reaction mixture was kept stirring for 12 h at room temperature and then poured onto ice-water. The crude products were filtrated and recrystallized from ethanol [18]. 2, 4-dihydroxybenzaldehyde (6.9 g, 0.05 mol), diethyl malonate (9.6 g, 0.06 mol) and piperidine (0.2 g, 2.5 mmol) were refluxing in 150 mL ethanol for 10 h. After cooled to room temperature, the crude products were filtrated and recrystallized from methanol [19]. The compounds 2a-e and 3a-e were synthesized according to the method from literature [26][27][28]. To a solution of compound 1a-e (10.0 mmol) in acetone (30 mL), 1,2-dibromoethane or 1,3-dibromopropane (30.0 mmol) and potassium carbonate (12.3 mmol) were added. The reaction mixture was stirred at 56˝C for 10 h, then poured into water, and extracted with ethyl acetate (30 mLˆ3). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.

General Procedure for the Synthesis of Compounds 4a-e
To a solution of compound 1a (10.0 mmol) in DMF (20 mL), propargyl bromide (1.30 g, 10.9 mmol) and potassium carbonate (1.70 g, 12.3 mmol) were added. The reaction mixture was stirred at 60˝C for 5 h, then was diluted with ethyl acetate (80 mL) and washed with water (100 mLˆ2). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude products 4a were used without purification. The compounds 4b-e were synthesized by the same operation procedure of compound 4a. To a solution of DHA (10.0 mmol) and 2-bromoethanol (10.0 mmol) in CH 2 Cl 2 (30 mL), boron fluoride ethyl ether (0.5 mL) was added dropwise at 0˝C. The reaction mixture was stirred at 0˝C for 8 h, then washed with saturated NaHCO 3 solution (20 mLˆ3). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude products were purified by silica gel column chromatography (PET/EtOAc = 10:1, v/v) to get the target compound 7a.

4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one
The compounds 7b and 9a were synthesized by the same operation procedure of compound 7a. These compounds were synthesized by the same operation in literature [20]. To a solution of compound 2a (10 mmol) in DMF (20 mL), sodium azide (30 mmol) was added, the reaction mixture was stirred at 70˝C for 4 h. the reaction mixture was poured into water and extracted with ethyl acetate (40 mLˆ3). The organic layer was dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure, and the crude products were used in next step immediately without further purification. 10a-e, 11a-e, 12a-e, 13a-e To a solution of compound 8a (10 mmol) and 4a (10 mmol) in CH 2 Cl 2 (20 mL), a mixture of CuSO 4¨5 H 2 O (12.5 mg) and sodium ascorbate (30 mg) in water (10 mL) was added, and the reaction mixture was stirred at room temperature for 8 h. The reaction mixture was filtered and the organic layer was washed with water, then dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PET/EtOAc = 2:1, v/v) to get the target compound 10a.

General Procedure for the Synthesis of Compounds
The compounds 10b-e, 11a-e, 12a-e, 13a-e were synthesized by the same operation procedure of compound 10a (5a-e, 6a-e with 9a; 8a, 8b with 4a-e).        13         in the 0.05-125.40 µM range, and these compounds exhibited better activity against HT-29 cell line under anoxic condition. Under anoxic conditions, cytotoxic activities of most compounds under anoxic conditions displayed one-to 10-fold greater activity than under normoxic condition. We were delighted to find that the cytotoxic activities of compounds 10a-e exhibited better activity against the HT-29 cell line under anoxic conditions compared with the other two cancer cell lines, which indicated that our design of CA IX inhibitors do correspond with its action mode in some degree. On the basis of the preliminary SARs we summarized, further investigations are currently in process.