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.

1. 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) isozymes that have been identified, CA IX is a tumor-associated isoform which over-expressed in a broad range of solid tumor types, while in normal tissues, the expression was much more limited, mainly in the glandular mucosa region in stomach [8,9]. In anoxia environments, the expression of CA IX is important in tumor growth and metastasis by increasing the tumor cell survival and invasion, consequently, CA IX is considered to be a promising anti-cancer drug target [10,11,12] and developing high selective inhibitors of CA IX may reduce side effects compared to classical anticancer agents [13]. In the last years, several approaches have been reported that the coumarins are truly CA IX-selective inhibitors [14,15,16,17] which exhibit a very different binding mode [17].

Using the principle of hybridization, we designed and synthesized four new series of artemisinin derivatives by means of click chemistry (Scheme 1), in which the coumarin moiety was attached to C-10 position of dihydroartemisinin to afford new hybrids. The details of the synthesis of target compounds and evaluation of their activities against cancer cell lines are presented herein.

Scheme 1

The design of target compounds.

2. Results and Discussion

2.1. Chemistry

The synthetic approaches toward the target compounds 10a–e, 11a–e, 12a–e, and 13a–e were illustrated in Scheme 2, proceeded in five steps, commencing from 1,3-dihydroxybenzene or 2,4-dihydroxybenzaldehyde, and DHA.

Scheme 2

Synthetic routes to target compounds 10a–e, 11a–e, 12a–e and 13a–e. Reagents and conditions: (1) H2SO4/r.t. 12 h; (2) piperidine/EtOH/reflux 8–10 h; (3) 1,2-dibromoethane or 1,3-dibromoproane/Me2CO/K2CO3/56 °C 10 h; (4) 3-bromo-1-propyne/DMF/K2CO3/60 °C 4–5 h; (5) NaN3/ DMF/70 °C 4–5 h; (6) 2-bromoethanol or 3-bromo-1-propanol/BF3·Et2O/CH2Cl2/0 °C 6–8 h; (7) NaN3/DMF/70 °C 4–5 h; (8) CuSO4·5H2O/sodium ascorbate/DMF/r.t. 8–10 h. (9) 2-propyn-1-ol/BF3·Et2O/CH2Cl2/0 °C 6–8 h; (10) CuSO4·5H2O/sodium ascorbate/DMF/r.t. 8–10 h.

The coumarin derivatives 1a–e were synthesized according to the literature [18,19]. Reaction of 1,3-dihydroxybenzene with corresponding substituted ethyl acetoacetate in H2SO4 afforded substituted coumarins (1a–d) [18], while 1e was synthesized by 2,4-dihydroxybenzaldehyde cyclized with diethyl malonate [19]. The substituted coumarins (1a–e) were then reacted with 1,2-dibromoethane, 1,3-dibromopropane or 3-bromo-1-propyne, respectively, to give 2a–e, 3a–e, 4a–e, 7a, 7b, and 9a were obtained by dihydroartemisinin(DHA) reacted with 2-bromoethanol, 3-bromo-1-propanol, or 3-bromo-1-propyne, respectively, as β configuration (indicated by the small coupling constants between 9-H and 10-H in 1H NMR, J9,10 = 3–4 Hz [20]) by using boron trifluoride ethyl ether as a catalyst [21]. Further azidation of 2a–e, 3a–e, 7a, and 7b gave compounds 5a–e, 6a–e, 8a, and 8b [22].

Finally, the intermediate bearing azido group (5a–e, 6a–e, 8a, 8b) reacted with the intermediate possessing terminal alkyne group (4a–e, 9a), respectively, (5a–e, 6a–e with 9a; 8a, 8b, with 4a–e) via click chemistry [23] to get the target compounds.

2.2. 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.

Table 1

IC50 values of tested compounds against MRC-5, HCT-116, MDA-MB-231, and HT-29.

Compd.	IC50 (μM)
	MRC-5	HCT-116			MDA-MB-231			HT-29
	Normoxia	Anoxia	Normoxia	Value 1	Anoxia	Normoxia	Value1	Anoxia	Normoxia	Value 1
AAZ	>100	>100	>100	-	>100	>100	-	>100	>100	-
DOX	0.34	0.15	0.06	0.39	0.37	0.27	0.73	0.05	0.09	1.74
10a	27.98	11.94	10.31	0.86	36.74	52.02	1.42	1.47	12.29	8.35
10b	33.56	8.31	6.46	0.78	24.68	28.01	1.13	2.18	10.88	4.99
10c	22.31	6.89	6.51	0.95	33.19	32.34	0.97	1.82	20.97	11.50
10d	50.72	5.30	4.18	0.79	29.19	61.41	2.10	0.05	17.70	333.96
10e	52.47	28.80	51.51	1.79	91.21	>100	1.10	8.69	31.83	3.66
11a	15.49	6.02	8.21	1.36	17.82	51.18	2.87	1.22	8.78	7.17
11b	43.79	9.33	12.86	1.38	36.90	72.62	1.97	5.06	8.23	1.63
11c	60.86	16.56	20.97	1.27	39.75	56.00	1.41	23.20	19.28	0.83
11d	>100	6.00	7.68	1.28	22.51	51.60	2.29	1.34	3.90	2.90
11e	38.74	28.14	44.14	1.57	50.67	120.72	2.38	12.03	51.09	4.25
12a	37.40	18.27	27.44	1.50	47.74	125.40	2.63	10.64	33.05	3.11
12b	18.51	24.35	8.47	0.35	14.67	40.05	2.73	2.46	11.34	4.62
12c	33.84	12.07	16.01	1.33	33.16	>100	3.02	2.87	17.32	6.04
12d	62.33	5.86	15.89	2.71	43.51	>100	2.30	3.63	22.37	6.16
12e	69.55	35.67	57.87	1.62	65.06	>100	1.54	20.75	38.96	1.88
13a	26.17	9.65	28.49	2.95	36.06	>100	2.77	6.40	27.51	4.30
13b	21.40	2.75	20.44	7.43	26.57	>100	3.76	5.31	19.83	3.74
13c	27.78	2.13	19.07	8.95	4.17	59.60	14.29	2.80	11.86	4.23
13d	31.47	0.43	17.96	41.38	3.62	72.50	20.03	4.68	8.76	1.87
13e	27.24	17.60	64.28	3.65	25.94	53.68	2.07	7.09	15.74	2.22

1 The values stand for the activity promotion for each cancer cell line. Value = IC50(normoxia)/IC50(anoxia).

The line chart that depicted the IC50 values was shown in Figure 1, Figure 2 and Figure 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).

Figure 1

The line chart of the IC50 values of tested compounds against MRC-5 and HT-29.

Figure 2

The line chart of the IC50 values of tested compounds against MRC-5 and MDA-MB-231.

Figure 3

The line chart of the IC50 values of tested compounds against MRC-5 and HCT-116.

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 (Figure 1, Figure 2 and Figure 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 (Figure 1 and Figure 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 one- to 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.

Figure 4

The cytotoxic activities of anoxia condition compared with normoxia codition.

Above all, these results indicated that our design of CA IX inhibitors does correspond with its action mode, and deserved further investigation.

3. Materials and Methods

3.1. Chemistry

Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration. The 1H-NMR and 13C-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.

3.1.1. 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 H2SO4 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].

7-Hydroxy-4-methyl-2H-chromen-2-one (1a): White solid; yield 73%. ESI-HRMS [M + H]+: (m/z) Calcd. for C10H9O3: 177.0546. Found: 177.0556.

7-Hydroxy-4-(trifluoromethyl)-2H-chromen-2-one (1b): White solid; yield 52%. ESI-HRMS [M − H]−: (m/z) Calcd. for C10H6F3O3: 231.0275. Found: 231.0162.

7-Hydroxy-3,4-dimethyl-2H-chromen-2-one (1c) White solid; yield 76%. ESI-HRMS [M + H]+: (m/z) Calcd. for C11H11O3: 191.0703. Found: 191.0712.

3-Chloro-7-hydroxy-4-methyl-2H-chromen-2-one (1d): White solid; yield 74%. ESI-HRMS [M + H]+: (m/z) Calcd. for C10H8ClO3: 211.0156. Found: 211.0166.

3.1.2. The Synthesis of Compound 1e

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].

Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (1e): Light yellow solid; yield 81%. ESI-HRMS [M + Na]+: (m/z) Calcd. for C12H10O5Na: 257.0420. Found: 257.0434.

3.1.3. General procedure for the synthesis of compounds 2a–e, 3a–e

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 mL × 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure.

7-(2-Bromoethoxy)-4-methyl-2H-chromen-2-one (2a): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C12H12BrO3: 282.9964. Found: 282.9981.

7-(2-Bromoethoxy)-4-(trifluoromethyl)-2H-chromen-2-one (2b): White solid; yield 53%. ESI-HRMS [M + H]+: (m/z) Calcd. for C12H9BrF3O3: 336.9682. Found: 336.9712.

7-(2-Bromoethoxy)-3,4-dimethyl-2H-chromen-2-one (2c): White solid; yield 67%. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H14BrO3: 297.0121. Found: 297.0129.

7-(2-Bromoethoxy)-3-chloro-4-methyl-2H-chromen-2-one (2d): White solid; yield 70%. ESI-HRMS [M + H]+: (m/z) Calcd. for C12H11BrClO3: 316.9575. Found: 316.9589.

Ethyl 7-(2-bromoethoxy)-2-oxo-2H-chromene-3-carboxylate (2e): Yellow solid; yield 65%. ESI-HRMS [M + H]+: (m/z) Calcd. for C14H14BrO5: 341.0019. Found: 341.0036.

7-(3-Bromopropoxy)-4-methyl-2H-chromen-2-one (3a): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H14BrO3: 297.0121. Found: 297.0132.

7-(3-Bromopropoxy)-4-(trifluoromethyl)-2H-chromen-2-one (3b): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H11BrF3O3: 350.9838. Found: 350.9851.

7-(3-Bromopropoxy)-3,4-dimethyl-2H-chromen-2-one (3c): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C14H16BrO3: 311.0277. Found: 311.0284.

7-(3-Bromopropoxy)-3-chloro-4-methyl-2H-chromen-2-one (3d): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H13BrClO3: 330.9731. Found: 330.9748.

Ethyl 7-(3-bromopropoxy)-2-oxo-2H-chromene-3-carboxylate (3e): Yellow solid; yield 62%. ESI-HRMS [M + H]+: (m/z) Calcd. for C15H16BrO5: 355.0176. Found: 355.0183.

3.1.4. 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 mL × 2). The organic layer was dried over anhydrous Na2SO4 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.

4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4a): White solid; yield 91%; m.p. 129–132 °C. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H10O3: 215.0703. Found: 215.0715.

7-(Prop-2-yn-1-yloxy)-4-(trifluoromethyl)-2H-chromen-2-one (4b): White solid; yield 78%; m.p. 82–84 °C. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H8F3O3: 269.0420. Found: 269.0437.

3,4-Dimethyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4c): White solid; yield 85%; m.p. 118–122 °C. ESI-HRMS [M + H]+: (m/z) Calcd. for C14H13O3: 229.0859. Found: 229.0875.

3-Chloro-4-methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4d):White solid; yield 82%; m.p. 131–134 °C. ESI-HRMS [M + H]+: (m/z) Calcd. for C13H10ClO3: 249.0313. Found: 249.0323.

Ethyl 2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromene-3-carboxylate (4e): White solid; yield 79%; m.p. 122–124 °C. ESI-HRMS [M + H]+: (m/z) Calcd. for C15H13O5: 273.0757. Found: 273.0764.

3.1.5. General Procedure for the Synthesis of Compounds 7a, 7b, 9a

To a solution of DHA (10.0 mmol) and 2-bromoethanol (10.0 mmol) in CH2Cl2 (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 NaHCO3 solution (20 mL × 3). The organic layer was dried over anhydrous Na2SO4 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.

The compounds 7b and 9a were synthesized by the same operation procedure of compound 7a.

1-Bromo-2-(10β-dihydroartemisinoxy)ethane (7a): White solid; yield 87%; m.p. 72–74 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.94 (d, J = 7.2 Hz, 3H), 0.96 (d, J = 6.4 Hz), 1.21–1.40 (m, 3H), 1.44 (s, 3H), 1.45–1.56 (m, 2H), 1.63–1.68 (m, 1H), 1.73–1.79 (m, 1H), 1.84–1.96 (m, 2H), 2.01–2.07 (m, 1H), 2.33–2.41 (m, 1H), 2.61–2.69 (m, 1H), 3.52 (t, J = 5.2 Hz, 2H), 3.77–3.82 (m, 1H), 4.09–4.15 (m, 1H), 4.85 (d, J = 3.2 Hz, 1H), 5.49 (s, 1H). ESI-HRMS [M + Na]+: (m/z) Calcd. for C17H27BrO5Na: 413.0934. Found: 413.0942.

1-Bromo-3-(10β-dihydroartemisinoxy)propane (): White solid; yield 89%; m.p. 68–71 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.92 (d, J = 7.6 Hz, 3H), 0.96 (d, J = 6.0 Hz, 3H), 1.19–1.28 (m, 1H), 1.29–1.39 (m, 1H), 1.44 (s, 3H), 1.45–1.56 (m, 2H), 1.60–1.69 (m, 1H), 1.72–1.78 (m, 2H), 1.85–1.92 (m, 1H), 2.01–2.14 (m, 3H), 2.37 (dt, J = 4.0 Hz, J = 13.6 Hz, 1H), 2.60–2.68 (m, 1H), 3.47–3.52 (m, 3H), 3.98–4.03 (m, 1H), 4.81 (d, J = 3.6 Hz, 1H), 5.43 (s, 1H). ESI-HRMS [M + Na]+: (m/z) Calcd. for C18H29BrO5Na: 427.1091. Found: 427.1099.

3-(10β-Dihydroartemisinoxy)prop-1-yne (: White solid; yield 87%; m.p. 105–108 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 1.20–1.40 (m, 3H), 1.44 (s, 3H), 1.47–1.55 (m, 2H), 1.62–1.66 (m, 1H), 1.74–1.79 (m, 2H), 1.85–1.92 (m, 1H), 2.01–2.07 (m, 1H), 2.33–2.41 (m, 2H), 2.64–2.71 (m, 1H), 4.31 (d, J = 1.2 Hz, 2H), 4.98 (d, J = 3.2 Hz), 5.42 (s, 1H). ESI-HRMS [M + Na]+: (m/z) Calcd. for C18H26O5Na: 345.1672. Found: 345.1683.

3.1.6. General Procedure for the Synthesis of Compounds 5a–e, 6a–e, 8a, 8b

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 mL × 3). The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and the crude products were used in next step immediately without further purification.

3.1.7. General Procedure for the Synthesis of Compounds 10a–e, 11a–e, 12a–e, 13a–e

To a solution of compound 8a (10 mmol) and 4a (10 mmol) in CH2Cl2 (20 mL), a mixture of CuSO4·5H2O (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 Na2SO4 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.

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).

7-(2-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-methyl-2H-chromen-2-one (10a): White solid; yield 31%; m.p. 83–85 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.87 (d, J = 7.6 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 1.19–1.28 (m, 3H), 1.44 (s, 3H), 1.45–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.70–1.75 (m, 1H), 1.80–1.90 (m, 1H), 1.99–2.04 (m, 1H), 2.33–2.37 (m, 1H), 2.40 (s, 3H), 2.60–2.68 (m, 1H), 4.43–4.46 (m, 2H), 4.76–4.71 (d, J = 12.4 Hz, 2H), 4.80–4.83 (m, 2H), 4.91 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.16 (s, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.80–6.83 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 160.90, 160.65, 155.12, 152.29, 145.40, 125.85, 123.49, 114.39, 112.56, 112.07, 104.13, 101.77, 101.58, 87.98, 81.06, 66.80, 61.61, 52.51, 49.41, 44.35, 37.36, 36.40, 34.56, 30.78, 26.14, 24.64, 24.42, 20.31, 18.65, 12.97. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H37N3O8Na: 590.2473. Found: 590.2462.

7-(2-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-trifluoromethyl-2H-chromen-2-one (10b): White solid; yield 29%; m.p. 69–71 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.88 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.30 (m, 3H), 1.44 (s, 3H), 1.55–1.59 (m, 1H), 1.62–1.80 (m, 3H), 1.84–2.05 (m, 3H), 2.33–2.41 (m, 1H), 2.61–2.68 (m, 1H), 4.48 (t, J = 4.8 Hz, 2H), 4.69 (d, J = 12.4 Hz, 1H), 4.84 (m, 2H), 4.91 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.66 (s, 1H), 6.86 (d, J = 2.8 Hz, 1H), 6.89 (dd, J = 2.4 Hz, J = 9.2 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.69 (s, H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.42, 158.89, 156.07, 145.40, 130.83, 128.74, 126.57, 123.37, 113.10, 112.97, 112.93, 112.90, 112.86, 107.73, 104.06, 102.12, 101.52, 87.90, 80.97, 66.88, 65.47, 61.53, 52.40, 49.16, 44.24, 36.29, 34.46, 30.68, 26.05, 24.55, 24.33, 20.21, 12.88. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H34F3N3O8Na: 644.2190. Found: 644.2183.

7-(2-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-3,4-dimethyl-2H-chromen-2-one (10c): White solid; yield 37%; m.p. 82–84 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.87 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.20–1.31 (m, 3H), 1.44 (s, 3H), 1.47–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.71–1.78 (m, 2H), 1.83–1.89 (m, 1H), 2.00–2.13 (m, 2H), 2.19 (s, 3H), 2.32–2.40 (m, 1H), 2.37 (s, 3H), 2.6–2.68 (m, 1H), 4.43 (t, J = 4.8 Hz, 2H), 4.68 (d, J = 12.8 Hz, 1H), 4.80–4.83 (m, 2H), 4.91 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 12.8 Hz, 1H), 5.42 (s, 1H), 6.78 (s, 1H), 6.80 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.13, 159.52, 153.41, 145.96, 145.41, 125.56, 123.49, 119.67, 115.01, 111.87, 104.14, 101.60, 101.43, 87.99, 81.09, 66.71, 61.65, 52.51, 49.45, 44.34, 37.37, 36.40, 34.56, 30.79, 26.18, 24.64, 24.43, 20.34, 15.18, 13.23, 13.00. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2636.

3-Chloro-7-(2-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-methyl-2H-chromen-2-one (10d): White solid; yield 39%; m.p. 75–77 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.87 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 1.21–1.28 (m, 3H), 1.44 (s, 3H), 1.47–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.71–1.75 (m, 2H), 1.84–1.89 (m, 1H), 2.00–2.05 (m, 2H), 2.32–2.40 (m, 1H), 2.54 (s, 3H), 2.61–2.65 (m, 1H), 4.46 (m, 2H), 4.68–4.70 (d, J = 12.4 Hz, 2H), 4.81–4.84 (m, 2H), 4.90–4.91 (d, J = 3.6 Hz, 1H), 4.93–4.96 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.87 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 160.55, 157.08, 152.92, 147.70, 145.39, 126.18, 123.51, 118.42, 114.07, 112.76, 104.14, 101.67, 101.55, 87.99, 81.06, 66.88, 61.57, 52.50, 49.36, 44.34, 37.37, 36.39, 34.56, 30.77, 26.16, 24.65, 24.43, 20.33, 16.19, 12.98. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H36ClN3O8Na: 624.2083. Found: 624.2088.

Ethyl 2-oxo-7-(2-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-2H-chromene-3-carboxylate (10e): White solid; yield 23%; m.p. 153–155 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.87 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 1.19–1.30 (m, 3H), 1.40 (t, J = 6.8 Hz, 3H), 1.44 (s, 3H), 1.46–1.60 (m, 3H), 1.70–1.78 (m, 2H), 1.83–1.90 (m, 1H), 2.00–2.06 (m, 1H), 2.33–2.41 (m, 1H), 2.61–2.68 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.48 (t, J = 4.8 Hz, 2H), 4.69 (d, J = 12.8 Hz, 1H), 4.84 (m, 2H), 4.91 (d, J = 3.2 Hz, 1H), 4.95 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 163.21, 162.98, 157.26, 156.79, 148.62, 145.44, 130.98, 123.49, 114.93, 113.35, 112.33, 104.15, 101.58, 101.21, 87.99, 81.07, 67.01, 61.82, 61.58, 52.49, 49.25, 44.32, 37.38, 36.38, 34.55, 30.77, 26.17, 24.65, 24.43, 20.34, 14.27, 12.99. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H39N3O10Na: 648.2528. Found: 648.2525.

7-(3-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-methyl-2H-chromen-2-one (11a): White solid; yield 32%; m.p. 69–70 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.84 (d, J = 7.6 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.21–1.28 (m, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.65–1.76 (m, 3H), 1.84–1.89 (m, 1H), 1.99–2.05 (m, 2H), 2.41 (s, 3H), 2.46 (m, 2H), 2.61–2.64 (m, 1H), 4.04 (t, J = 5.6 Hz, 2H), 4.60–4.63 (m, 2H), 4.67 (d, J = 12.8 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H), 4.93 (d, J = 12.4 Hz, 1H), 5.39 (s, 1H), 6.16 (s, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 3.2 Hz, J = 8.8 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H), 7.51 (d, J = 5.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.35, 161.11, 155.21, 152.43, 145.23, 125.75, 122.83, 114.02, 112.29, 112.11, 104.13, 101.71, 101.66, 87.96, 81.08, 64.69, 61.72, 52.49, 46.87, 44.34, 37.38, 36.40, 34.55, 30.79, 29.70, 26.16, 24.67, 24.42, 20.32, 18.69, 12.96. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2618.

7-(3-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-trifluoromethyl-2H-chromen-2-one (11b): White solid; yield 19%; m.p. 67–69 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.85 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.29 (m, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 2H), 1.55–1.59 (m, 1H), 1.66–1.76 (m, 2H), 1.84–1.89 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.48 (m, 2H), 2.59–2.67 (m, 1H), 4.07 (t, J = 6.0 Hz, 2H), 4.60–4.64 (m, 2H), 4.68 (d, J = 12.4 Hz, 1H), 4.90 (d, J = 3.2 Hz, 1H), 4.93 (d, J = 12.4 Hz, 1H), 5.39 (s, 1H), 6.64 (s, 1H), 6.85 (d, J = 2.8 Hz, 1H), 6.91 (dd, J = 2.4 Hz, J = 9.2 Hz, 1H), 7.52 (s, 1H), 7.64 (dd, J = 1.6 Hz, J = 8.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.23, 159.19, 156.25, 145.29, 126.57, 122.807, 120.19, 113.27, 112.66, 112.60, 107.44, 104.14, 102.10, 101.71, 87.96, 81.07, 64.95, 61.70, 52.48, 46.76, 44.33, 37.38, 36.39, 34.54, 30.78, 29.60, 26.15, 24.67, 24.41, 20.31, 12.95. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H36F3N3O8Na: 658.2347. Found: 658.2350.

7-(3-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-3,4-dimethyl-2H-chromen-2-one (11c): White solid; yield 35%; m.p. 83–85 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.85 (d, J = 5.2 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H), 0.90–0.99 (m, 1H), 1.15–1.27 (m, 2H), 1.30–1.31 (m, 1H), 1.55 (s, 3H), 1.58–1.72 (m, 3H), 1.74–1.79 (m, 1H), 1.82–1.89 (m, 1H), 2.19 (s, 3H), 2.38 (s, 3H), 2.40–2.48 (m, 3H), 3.57 (m, 1H), 4.00–4.03 (m, 2H), 4.59–4.65 (m, 3H), 4.87 (d, J = 4.4 Hz, 1H), 4.90 (d, J = 8.8 Hz, 1H), 5.26 (s, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 2.8 Hz, J = 8.8 Hz, 1H), 7.49 (s, 1H), 7.51 (d, J = 9.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.30, 160.24, 153.48, 146.11, 145.32, 125.44, 122.75, 119.31, 114.61, 111.96, 107.99, 101.32, 99.46, 93.80, 84.08, 77.26, 69.56, 64.53, 61.81, 46.90, 42.41, 40.63, 34.82, 34.70, 30.32, 29.72, 24.98, 20.99, 18.82, 15.10, 13.19, 12.26. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H41N3O8Na: 618.2786. Found: 618.2784.

3-Chloro-7-(3-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-methyl-2H-chromen-2-one (11d): White solid; yield 34%; m.p. 150–152 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.85 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.28 (m, 3H), 1.44 (s, 3H), 1.45–1.49 (m, 1H), 1.55–1.59 (m, 1H), 1.65–1.81 (m, 3H), 1.83–1.90 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.47 (m, 2H), 2.56 (s, 3H), 2.59–2.66 (m, 1H), 4.05 (m, 2H), 4.61 (m, 2H), 4.67 (d, J = 12.8 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H), 4.92 (d, J = 12.8 Hz, 1H), 5.39 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.26, 157.26, 153.01, 147.84, 145.22, 126.09, 122.84, 118.10, 113.68, 112.81, 104.13, 101.67, 101.56, 87.96, 81.07, 64.84, 61.67, 52.48, 46.86, 44.33, 37.38, 36.39, 34.55, 30.78, 29.68, 26.16, 24.67, 24.42, 20.33, 16.21, 12.97. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H38ClN3O8Na: 638.2240. Found: 638.2243.

Ethyl 2-oxo-7-(3-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-2H-chromene-3-carboxylate (11e): White solid; yield 29%; m.p. 124–126 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.85 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.20–1.32 (m, 3H), 1.41 (t, J = 7.2 Hz, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.64–1.81 (m, 3H), 1.83–1.90 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.48 (m, 2H), 2.59–2.67 (m, 1H), 4.08 (t, J = 6.0 Hz, 2H), 4.41 (q, J = 7.2 Hz, 2H), 4.61 (td, J = 2.0 Hz, J = 6.8 Hz, 2H), 4.68 (d, J = 12.4 Hz, 1H), 4.90 (d, J = 3.2 Hz, 1H), 4.93 (d, J = 12.8 Hz, 1H), 5.39 (s, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.87 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 6.4 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 163.78, 163.31, 157.37, 156.96, 148.80, 145.26, 130.91, 122.83, 114.48, 113.45, 111.97, 104.13, 101.69, 101.12, 87.95, 81.07, 65.11, 61.76, 61.68, 52.47, 46.82, 44.32, 37.37, 36.381, 34.54, 30.78, 29.62, 26.16, 24.66, 24.41, 20.32, 14.29, 12.97. ESI-HRMS [M + Na]+: (m/z) Calcd. for C33H41N3O10Na: 662.2684. Found: 662.2679.

7-(1-(2-(10β-Dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (12a): White solid; yield 32%; m.p. 79–81 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 5.6 Hz, 3H), 1.16–1.30 (m, 3H), 1.36–1.39 (m, 1H), 1.42 (s, 3H), 1.45–1.52 (m, 2H), 1.56–1.60 (m, 1H), 1.64–1.74 (m, 1H), 1.84–1.88 (m, 1H), 1.99–2.05 (m, 1H), 2.31–2.39 (m, 1H), 2.41 (s, 3H), 2.57–2.64 (m, 1H), 3.79–3.84 (m, 1H), 4.27–4.32 (m, 1H), 4.51–4.57 (m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz, 1H), 5.15 (s, 1H), 5.27 (s, 2H), 6.16 (s, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.95 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.72 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.13, 155.15, 152.42, 143.10, 125.73, 123.49, 114.09, 112.33, 112.31, 104.21, 102.18, 102.13, 87.87, 80.80, 77.24, 66.40, 62.33, 52.38, 50.50, 44.04, 37.34, 36.31, 34.45, 30.59, 26.10, 24.61, 24.34, 20.36, 18.69, 12.81. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H37N3O8Na: 590.2473. Found: 590.2471.

7-(1-(2-(10β-Dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-trifluoromethyl-2H-chromen-2-one (12b): White solid; yield 24%; m.p. 77–79 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 5.6 Hz, 3H), 1.16–1.31 (m, 3H), 1.42 (s, 3H), 1.48–1.76 (m, 4H), 1.84–1.88 (m, 1H), 2.00–2.05 (m, 1H), 2.31–2.34 (m, 1H), 2.59–2.62 (m, 1H), 3.80–3.85 (m, 1H), 4.25–4.32 (m, 1H), 4.51–4.70 (m, 2H), 4.76 (d, J = 2.7 Hz, 1H), 5.16 (s, 1H), 5.29 (s, 1H), 6.64 (s, 1H), 7.01–7.03 (m, 2H), 7.64 (d, J = 9.2 Hz, 1H), 7.73 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.928, 159.095, 156.088, 142.555, 126.430, 124.988, 123.535, 113.398, 112.573, 107.385, 104.107, 102.503, 102.073, 87.770, 80.677, 77.191, 76.979, 76.767, 66.282, 62.356, 52.277, 50.429, 43.935, 37.235, 36.202, 34.358, 30.480, 25.974, 24.517, 24.229, 20.241, 19.083, 12.691. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H34F3N3O8Na: 644.2190. Found: 644.2187.

7-(1-(2-(10β-Dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-3,4-dimethyl-2H-chromen-2-one (12c): White solid; yield 36%; m.p. 84–86 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H), 0.91 (d, J = 4.4 Hz, 3H), 0.97–1.29 (m, 3H), 1.42 (s, 3H), 1.45–1.55 (m, 2H), 1.55–2.05 (m, 5H), 2.18 (s, 3H), 2.30–2.34 (m, 1H), 2.37 (s, 3H), 2.57–2.61 (m, 1H), 3.79–3.84 (m, 1H), 4.27–4.32 (m, 1H), 4.51–4.57 (m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz, 1H), 5.15 (m, 1H), 5.25 (m, 2H), 6.90–6.95 (m, 2H), 7.52 (dd, J = 3.6 Hz, J = 8.8 Hz, 1H), 7.72 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.26, 160.01, 153.39, 146.12, 143.21, 125.44, 123.52, 119.29, 114.66, 112.10, 104.16, 102.13, 101.76, 87.84, 80.78, 66.38, 62.25, 52.37, 50.47, 44.03, 37.29, 36.30, 34.43, 30.58, 26.07, 24.58, 24.31, 20.34, 15.08, 13.17, 12.80. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2645.

3-Chloro-7-(1-(2-(10β-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (12d): White solid; yield 33%; m.p. 76–78 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 5.6 Hz, 3H), 1.16–1.32 (m, 3H), 1.32–1.42 (m, 2H), 1.42 (s, 3H), 1.44–1.69 (m, 5H), 1.84–1.88 (m, 1H), 1.99–2.15 (m, 2H), 2.31–2.39 (m, 1H), 2.58–2.62 (m, 1H), 3.79–3.85 (m, 1H), 4.27–4.32 (m, 1H), 4.51–4.58 (m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz, 1H), 5.15 (s, 1H), 5.27 (s, 2H), 6.94 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.73 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.04, 157.29, 152.97, 147.83, 142.90, 126.07, 123.56, 118.17, 113.78, 113.02, 104.21, 102.18, 102.06, 87.87, 80.79, 66.40, 62.38, 52.38, 50.51, 44.03, 37.33, 36.31, 34.46, 30.59, 26.09, 24.61, 24.34, 20.36, 16.20, 12.82. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H36ClN3O8Na: 624.2083. Found: 624.2111.

Ethyl 2-oxo-7-(1-(2-(10β-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-2H-chromene-3-carboxylate (12e): White solid; yield 26%; m.p. 71–73 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.6 Hz, 3H), 0.92 (d, J = 5.6 Hz, 3H), 1.16–1.28 (m, 3H), 1.39–1.42 (m, 8H), 1.47–1.52 (m, 1H), 1.55–1.60 (m, 1H), 1.65–1.69 (m, 1H), 1.84–1.89 (m, 1H), 1.99–2.05 (m, 1H), 2.31–2.39 (m, 1H), 2.57–2.64 (m, 1H), 3.80–3.85 (m, 1H), 4.27–4.32 (m, 1H), 4.40 (q, J = 3.2 Hz, 2H), 4.52–4.58 (m, 1H), 4.64–4.70 (m, 1H), 4.76 (d, J = 3.6 Hz, 1H), 5.15 (s, 1H), 5.29 (s, 2H), 6.94 (d, J = 2.0 Hz, 1H), 6.98 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.74 (s, 1H), 8.51 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 163.55, 163.36, 157.34, 156.98, 148.79, 142.60, 130.88, 123.64, 114.63, 113.63, 112.08, 104.23, 102.19, 101.62, 87.88, 80.78, 66.40, 62.52, 61.79, 52.37, 50.54, 44.02, 37.35, 36.31, 34.46, 30.58, 26.10, 24.62, 24.34, 20.36, 14.29, 12.82. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H39N3O10Na: 648.2528. Found: 648.2530.

7-(1-(3-(10β-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (13a): White solid; yield 39%; m.p. 75–77 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.2 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H), 1.22–1.33 (m, 3H), 1.43 (s, 3H), 1.46–1.55 (m, 2H), 1.63–1.69 (m, 1H), 1.76–1.78 (m, 1H), 1.86–1.93 (m, 1H), 2.02–2.13 (m, 2H), 2.17–2.25 (m, 2H), 2.33–2.38 (m, 1H), 2.41 (s, 3H), 2.62–2.69 (m, 1H), 3.36–3.42 (m, 1H), 3.87–3.93 (m, 1H), 4.44–4.52 (m, 2H),4.77 (d, J = 3.6 Hz, 1H), 5.27 (s, 2H), 5.40 (s, 1H), 6.15 (s, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.18, 161.13, 155.12, 152.50, 143.05, 125.74, 123.05, 114.07, 112.39, 112.29, 104.19, 102.19, 102.13, 87.94, 80.97, 64.65, 62.311, 52.50, 47.65, 44.27, 37.46, 36.37, 34.56, 30.81, 30.44, 26.15, 24.66, 24.55, 20.36, 18.70, 13.09. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2674.

7-(1-(3-(10β-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-4-trifluoromethyl-2H-chromen-2-one (13b): White solid; yield 25%; m.p. 69–71 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.94 (d, J = 7.2 Hz, 3H), 0.97 (d, J = 6.0 Hz, 3H), 1.22–1.39 (m, 3H), 1.43 (s, 3H), 1.46–1.55 (m, 2H), 1.64–1.78 (m, 3H), 1.87–1.92 (m, 1H), 2.02–2.06 (m, 1H), 2.16–2.28 (m, 2H), 2.34–2.41 (m, 1H), 2.63–2.70 (m, 1H), 3.37–3.43 (m, 1H), 3.87–3.93 (m, 1H), 4.45–4.54 (m, 2H), 4.78 (d, J = 3.2 Hz, 1H), 5.29 (s, 2H), 5.40 (s, 1H), 6.64 (s, 1H), 7.02–7.03 (m, 2H), 7.64–7.67 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.03, 159.26, 156.18, 142.64, 141.33, 126.52, 123.13, 113.55, 112.67, 112.62, 107.50, 104.21, 102.61, 102.21, 87.95, 80.96, 64.66, 62.45, 52.50, 47.68, 44.27, 37.47, 36.37, 34.56, 30.81, 30.44, 26.14, 24.66, 24.55, 20.36, 13.09. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H36F3N3O8Na: 658.2347. Found: 658.2345.

7-(1-(3-(10β-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-3,4-dimethyl-2H-chromen-2-one (13c): White solid; yield 37%; m.p. 68–70 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H), 1.22–1.40 (m, 3H), 1.43 (s, 3H), 1.46–1.52 (m, 2H), 1.54–1.78 (m, 3H), 1.86–1.95 (m, 1H), 2.01–2.17 (m, 2H), 2.19 (s, 3H), 2.22–2.35 (m, 2H), 2.38 (s, 3H), 2.60–2.69 (m, 1H), 3.36–3.41 (m, 1H), 3.87–3.93 (m, 1H), 4.42–4.53 (m, 2H), 4.77 (d, J = 3.6 Hz, 1H), 5.26 (s, 2H), 5.40 (s, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 162.33, 160.02, 153.40, 146.16, 143.22, 125.44, 122.99, 119.31, 114.68, 112.18, 104.18, 102.18, 101.82, 87.93, 80.97, 64.65, 62.28, 52.51, 47.62, 44.27, 37.45, 36.37, 34.56, 30.82, 30.44, 26.15, 24.66, 24.55, 20.36, 15.10, 13.19, 13.09. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H41N3O8Na: 618.2786. Found: 618.2785.

3-Chloro-7-(1-(3-(10β-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (13d): White solid; yield 38%; m.p. 71–73 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H), 1.22–1.29 (m, 2H), 1.32–1.38 (m, 1H), 1.43 (s, 3H), 1.46–1.51 (m, 2H), 1.58–1.82 (m, 3H), 1.87–1.92 (m, 1H), 2.02–2.07 (m, 1H), 2.17–2.25 (m, 2H), 2.33–2.41 (m, 1H), 2.56 (s, 3H), 2.62–2.70 (m, 1H), 3.36–3.42 (m, 1H), 3.87–3.92 (m, 1H), 4.41–4.56 (m, 2H), 4.77 (d, J = 3.2 Hz, 1H), 5.27 (s, 2H), 5.40 (s, 1H), 6.95 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 161.04, 157.34, 152.98, 147.86, 142.90, 126.07, 123.06, 118.17, 113.80, 113.08, 104.21, 102.21, 102.09, 87.95, 80.97, 64.65, 62.40, 52.51, 47.65, 44.28, 37.47, 36.38, 34.57, 30.82, 30.45, 26.16, 24.67, 24.56, 20.37, 16.21, 13.10. ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H38ClN3O8Na: 638.2240. Found: 638.2240.

Ethyl 2-oxo-7-(1-(3-(10β-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-2H-chromene-3-carboxylate (13e): White solid; yield 30%; m.p. 79–81 °C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H), 0.96 (d, J = 5.6 Hz, 3H), 1.22–1.32 (m, 2H), 1.39–1.43 (m, 6H), 1.47–1.56 (m, 2H), 1.61–1.73 (m, 2H), 1.76–1.78 (m, 1H), 1.86–1.93 (m, 1H), 1.97–2.07 (m, 2H), 2.18–2.26 (m, 2H), 2.33–2.41 (m, 1H), 2.62–2.68 (m, 1H), 3.37–3.43 (m, 1H), 3.87–3.92 (m, 1H), 4.40 (q, 2H), 4.45–4.55 (m, 2H), 4.78 (d, J = 3.2 Hz, 1H), 5.29 (s, 2H), 5.40 (s, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.99 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.68 (s, 1H), 8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 163.59, 163.40, 157.32, 157.03, 148.84, 146.18, 130.84, 114.59, 113.84, 112.13, 104.19, 102.13, 101.78, 87.97, 81.01, 80.63, 64.64, 62.26, 61.77, 52.54, 48.49, 44.34, 37.47, 36.39, 34.63, 30.85, 30.14, 29.71, 26.17, 24.68, 20.38, 14.29, 13.16. ESI-HRMS [M + Na]+: (m/z) Calcd. for C33H41N3O10Na: 662.2684. Found: 662.2680.

3.2. In Vitro Cytotoxicity Study (MTT Assay)

All of the newly-synthesized compounds were screened for in vitro cytotoxicity by MTT assay. The exponentially-growing MRC-5, HCT-116, MDA-MB-231, and HT-29 cells were allowed to grow until 80% confluency, the culture medium was discarded, digested with trypsin, separated centrifugally, then fresh culture medium was added and blown gently into single-cell suspension. Then, the cell suspension was seeded into each well of 96-well microculture plates at a concentration of 1.5 × 103–3 × 103 cells/well.

After incubation for 24 h at 37 °C, the four lines of cells were treated with varying concentrations (100, 50, 25, 12.5, and 6.25 μM) of DOX, AAZ, or the derivatives for 96 h. The cells were incubated with 50 μL of 2 mg/mL MTT solution for 4 h in a humidified incubator. The supernatant was discarded, and the media were then replaced with 200 μL of dimethyl sulfoxide to dissolve the purple colored formazan crystals formed in the wells, and their absorbances were measured at 492 nm with a microplate reader (Synergy-HT, BioTek Instruments, Winooski, VT, USA); 200 μL DMSO was set as the blank control.

The anoxia condition was achieved by placing cells in a sealed hypoxia incubator chamber (Catalog Number 27310, Stemcell Technologies, Inc., Vancouver, BC, Canada) filled with 5% CO2 and 95% N2. For the hypoxia group, the cells were treated with varying concentrations (100, 50, 25, 12.5, and 6.25 μM) of DOX, AAZ, or the derivatives under anoxic condition for 24 h [29], and then moved into nomoxic condition and cultured for additional 72 h.

4. Conclusions

In this study, four series of dihydroartemisinin–coumarin hybrids were designed and synthesized, the target compounds were characterized by HRMS, 1H-, and 13C-NMR. The cytotoxic activities were evaluated by MTT assay against HCT-116, MDA-MB-231, and HT-29 cell lines 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. 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.