Design and synthesis of novel artemisinin derivatives with potent activities against colorectal cancer in vitro and in vivo

European Journal of Medicinal Chemistry 182 (2019) 111665

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design and synthesis of novel artemisinin derivatives with potent activities against colorectal cancer in vitro and in vivo Liang-Liang Wang a, b, c, 1, Lingmei Kong a, b, c, 1, Hui Liu d, Yunqin Zhang a, b, c, Li Zhang d, Xingyong Liu d, Feng Yuan a, b, c, Yan Li a, b, c, **, Zhili Zuo a, b, c, d, * a

State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming, 650201, PR China University of Chinese Academy of Sciences, Beijing, 100049, PR China d School of Chemical Engineering, Sichuan University of Science & Engineering, Zigong, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2019 Received in revised form 29 August 2019 Accepted 29 August 2019 Available online 30 August 2019

A series of novel derivatives of artemisinin-4-(arylamino)quinazoline have been designed and synthesized, and most of them showing potent in vitro cytotoxic activity against HCT116 and WM-266-4 cell lines. Compound 32 was the most active derivative against HCT116 cell line with an IC50 of 110 nM, and significantly improved the antitumor activity of the parent compounds dihydroartemisinin (DHA) (IC50 ¼ 2.85 mM) and Gefitinib (IC50 ¼ 19.82 mM). In vivo HCT116 xenografts assay showed that compound 32 exhibited potent antitumor activity with obvious tumor growth delay and tumor shrunken after 18 days treatment on xenografted mice, and especially without loss of body weight. Our results indicate that compounds 32 may represent a safe, novel structural lead for developing new chemotherapy of colorectal cancer. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Artemisinin Quinazoline Hybridization Colorectal cancer Chemotherapy

1. Introduction Colorectal cancer (CRC) has been ranked as the third most common cancer causing nearly 0.5 million deaths each year and over 1.3 million new cases annually, while in China, morbidity rate was continuously increasing in the last decade [1e3]. Though surgical resection combined with chemotherapy for colorectal cancer has effectively inhibited colorectal cancers, the 5-year survival rate has been hardly improved due to the drug resistance and undesirable side effects because of lacking in selectivity of currently available drugs, which makes it highly urgent to develop new and more effective anti-colorectal cancer medicine [4e6]. One feasible approach of seeking new chemotherapy drug candidate is to modify the current clinically available anti-CRC medicines. Artemisinin (ART) was isolated from the traditional Chinese

medicinal herb Artemisia annua L. in 1972 by Youyou Tu [7,8]. ART and its derivatives such as Dihydroartemisinin (DHA), and Artesunate (AS) etc. are frontline drugs against malarial infections (Fig. 1) [9e11]. While in the last decades, the broad anticancer activities of ART derivates including anti Leukemia, breast cancer, lung cancer, pancreatic cancer, liver cancer etc. have also been extensively documented [12e20]. Yet, the anticancer mechanism is not fully understood, it is believed that the 1,2,4-trioxane moiety in the molecule underlies the potentially similar mechanism for both antitumor and antimalarial properties via reactive oxygen species (ROS) and carbon-centered free radicals generated from peroxide bridge cleavage by intracellular Fe(II) [21e24]. Recently, Krishna's team reported an oral artesunate neoadjuvant therapy with good tolerance in a randomized double-blind pilot clinical phase II trial to treat colorectal cancer (CRC), indicating that ART derivates have

* Corresponding authors: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China. ** Corresponding author. State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China. E-mail addresses: [email protected] (Y. Li), [email protected] (Z. Zuo). 1 Contribute equally to this work. https://doi.org/10.1016/j.ejmech.2019.111665 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

Fig. 1. The design of Artemisinin-4-(arylamino)quinazoline hybrids.

potential to be developed as an anti-CRC drug [25e28]. To seek more potent ART derivatives possessing potent pharmacological properties, we envisioned that one straightforward approach is to utilize the concept of molecular hybridization [29,30] to hybridize ART with one pharmacophore existing in approved drug, which would generate tremendous combinations and produce new analogues with improved bioactivities compared to the parent compounds. Protein kinases inhibitors for instance Gefitinib, Vandetanib, Afatinib, etc. have become one type of significant anticancer agents in various cancer chemotherapy by deregulation of protein kinases involved in cancer progression in the past fifteen years (Fig. 1) [31]. Many of those kinase inhibitor drugs harbor the same crucial motif 4-(arylamino)quinazoline as the core pharmacophore. In consideration of the great potential of ART derivatives as anticancer agent as well as their unique antitumor mechanism and the prevalence of 4-(arylamino)quinazoline fragment in the anticancer drug, we have tried to combine those two entity together in expectation of discovering novel ART-4-(arylamino)quinazoline hybrids with more potent anti-CRC effect. Herein, we report the syntheses of a set of ART-4-(arylamino)quinazoline hybrids with various types of linkers featured with ether, ester, amide and chiral elements (Fig. 1). To the best of our knowledge, No ART-4-(arylamino) quinazoline hybrids have been reported to date. Their antitumor activities on human colorectal cancer HCT116 cells and human melanoma cancer WM-266-4 were examined in vitro. The in vivo study on xenografted mice showed this type of artemisinin hybrids possessed potent antitumor activities.

2. Result and discussion 2.1. Chemistry 2.1.1. Synthesis of artemisinin hybrids with ether linker We initiated the synthesis of the first series of artemisinin hybrids 9a-9h featured with an ether linker from starting materials 2amino-5-methylbenzoic acid 1 which was converted to 4-(3chloro-4-fluorophenylamino)quinazoline 2 by known procedure [32], followed by acetylation and radical bromination of bezylic methyl to afford bromide 3. Subsequent nucleophilic substitution of 3 by water or diol under basic condition delivered 4e7 in good yields. Subjection of commercially available dihydroartemisinin (DHA) 8 to alcohol 4e7 in the presence of BF3$Et2O resulted in the hybrid mixtures of 10b-isomer 9a-9d and 10a-isomer 9e-9h, of which both are readily separated by flash column chromatography (Scheme 1). The stereochemistry of both diastereomers was confirmed by analyzing 1He1H NOESY spectrum or by calculating the vicinal coupling constant JH10-H9. Generally J10a-isomer (7e10 Hz) with trans configuration is bigger than J10b-isomer (3e5 Hz) with cis configuration [33,34]. 2.1.2. Synthesis of artemisinin hybrids with ester and amide linker 10a-Dihydroartemisinin (DHA) 8 coupled with dicarboxylic acid 10e12 to predominately deliver 13e15 respectively. The following esterification reaction with alcohol 4 or 16 could afford the 10ahybrids 17a-17c or 17d-17f (Scheme 2). 10a-aminoartemisinin 18 could be obtained from

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

3

Scheme 1. Synthesis of hybrids with ether linker.

Scheme 2. Synthesis of hybrids with ester linker.

Dihydroartemisinin (DHA) 8 by azidation and subsequent Staudinger reduction [35]. The following amidation reaction between 18 and dicarboxylic acid 10e12 afforded intermediates 19e21 respectively, which then reacted with alcohol 4 or 16 to produce hybrids 10a-hybrids 22a-22c or 22d-22f (Scheme 3).

reaction between DHA 8 and D-Phenylglycine derived alkamine 29 to deliver a-isomer 30. The following coupling reaction between 30 and acid 31 afforded the target compound 32. The structure of 10aisomer 32 was unambiguously confirmed by full NMR spectrum analysis (Scheme 4) (See supporting information).

2.1.3. Synthesis of artemisinin hybrids with chiral linkers The preparation of Hybrids (S)-27 began with esterification of TBS protected (S)-Mandelic acid 23 with alcohol 4, followed by desilylation to afford alcohol (S)-25. The following acetalation reaction between 25 and DHA in the presence of BF·3Et2O delivered the inseparable a and b mixture of hybrid (S)-27. Hybrid (R)-28 was synthesized by the same way as hybrid (S)-27 from TBS protected (R)-Mandelic acid 24 (Scheme 4). The synthesis of hybrids 32 commenced with acetylation

2.2. Biological evaluation 2.2.1. In vitro cytotoxicity activity To evaluate the cytotoxic effect of the novel hybrids, we investigated cell viability in human colorectal cancer HCT116 cells and human melanoma cancer WM-266-4 cells by MTS assay using Dihydroartemisinin (DHA), Artesunate (AS), Cisplatin (cis-DDP) and Taxol as control. The results of IC50 were shown in Table 1. We first evaluated the hybrids 9a-9d with various length of

4

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

Scheme 3. Synthesis of hybrids with amide linker.

Scheme 4. Synthesis of hybrids with chiral linkers.

ether linkers against both HCT116 cells and WM-266-4 cells. Promising antiproliferative activity was observed for 9d with a lowest IC50 (0.24 mM) (Entry 4, Table 1), which is 10-fold more potent than the parent drugs dihydroartemisinin (DHA) with an IC50 of 2.85 mM (Entry 27, Table 1). However, its corresponding b-

anomers 9e-9h showed less potent antiproliferative activity against both cells in comparison with 9a-9d (Entries 5e8 vs 1e4, Table 1), indicating that the stereochemistry on the anomeric carbon of hybrid exerts impact on its bioactivities. Generally, hybrids 9a-9d selectively inhibited HCT116 cells compared with WM-266-4 cells.

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665 Table 1 The IC50 values of artemisinin derivatives to the HCT116 and WM-266-4 cells. Entry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Compounds No.

9a 9b 9c 9d 9e 9f 9g 9h 17a 17b 17c 17d 17e 17f 22a 22b 22c 22d 22e 22f 32 27 28 Gefitinib 30 31 DHA AS DDP Taxol

IC50(mM)a HCT116

WM-266-4

0.29 ± 0.11 2.11 ± 0.34 0.36 ± 0.02 0.24 ± 0.06 0.42 ± 0.20 6.54 ± 1.20 2.42 ± 0.33 0.35 ± 0.06 0.18 ± 0.03 0.29 ± 0.05 0.31 ± 0.03 0.28 ± 0.11 0.78 ± 0.14 0.66 ± 0.14 2.78 ± 0.22 5.56 ± 0.36 5.68 ± 0.77 12.88 ± 0.76 5.35 ± 0.28 11.78 ± 0.46 0.11 ± 0.03 0.89 ± 0.12 0.68 ± 0.11 19.82 ± 2.54 16.24 ± 1.26 >40 2.85 ± 0.65 0.89 ± 0.02 10.98 ± 1.01 0.11 ± 0.01 pM

10.75 ± 0.20 5.18 ± 0.57 3.81 ± 0.36 2.91 ± 0.12 4.56 ± 0.91 15.75 ± 1.47 12.60 ± 2.86 5.70 ± 1.11 10.24 ± 1.73 11.36 ± 0.58 3.51 ± 0.68 9.29 ± 0.54 14.99 ± 2.39 18.76 ± 3.36 9.88 ± 0.12 15.29 ± 0.25 25.27 ± 0.32 15.43 ± 1.29 5.08 ± 0.10 27.79 ± 1.27 6.67 ± 0.41 ND ND 17.42 ± 1.56 15.12 ± 0.25 >40 >40 8.46 ± 0.24 3.92 ± 1.13 0.32 ± 0.08 nM

a The IC50 values were calculated from three independent experiments and represented the mean ± SD (n ¼ 3). ND: not determined.

The selectivity could be attributed to the Artemisinin pharmacophore. Artemisinin derivatives were reported to selectively attenuate colon cancers [28] due to the inhibition of the constitutively activated STAT3 signaling [36], which is particularly high and required for the growth of colon cancer cells [37e39]. Then hybrids 17a-17c with diester linker was tested on HCT116 cell and the sub-micromole level IC50 were observed with 0.18, 0.29 and 0.31 mM, respectively (Entries 9e11, Table 1). In this case the length of linkers exerted tiny influence on the bioactivities. The similar tendency was observed that hybrids 17a-17c as well as N-acetylated 17d-17f showed more potency against HCT116 cells than WM-266-4 cells, while hybrids 17d-17f were less potent than the corresponding N-Hydrogen analogues 17a-17c on both types of cells (Entries 9e11 vs 12e14, Table 1). When the diester linker was slightly modified to be amide-ester linker as shown in derivatives 22a-22c as well as 22d-22f, the antiproliferative potency were dramatically reduced 10e20 fold on HCT116 cells compared with corresponding counterpart 17a-17c and 17d-17f (Entries 15e17 vs 9e11, 18e20 vs 12e14), while the similar tendency of decreased activities on WM-266-4 cells were also observed in spite of less degree of variation. Besides the flexible ether, diester and amide linker mentioned above, we introduced more rigidly chiral linker to adjust the spatial conformation of hybrids 27e28 and 32 in expectation of restricting fragment rotation between ART motif and 4-(arylamino)quinazoline subunit around the linker (Entries 21e23, Table 1). To our delight, compound 32 with (S)-alkamine linker showed the best antiproliferative potency against HCT116 cells with an IC50 of 110 nM. The other two hybrids 27e28 with a chiral liker derived from (S) or (R)-Mandelic acid also exhibited good antiproliferative activities against HCT116 cells with an IC50 of 0.89 and 0.68 mM,

5

respectively. Hence, both the (S)-alkamine linker-contained ART fragment and 4-(arylamino)quinazoline fragment on hybrid 32 are crucial to its significantly enhanced potency against HCT116 cell since the corresponding analogues 30 and 31 of those two fragments showed almost neglectable activities on HCT116 cells with IC50 > 15 mM, respectively (Entries 25e26, Table 1). With this most active compound 32 in hand, we are able to further investigate its anti-CRC activity in vivo. 2.2.2. In vivo anti- CRC activity Compound 32 was used to evaluate the in vivo anti-CRC efficacy in HCT116 mouse xenograft models. The following treatments were administered to female nude BALB/c mice inoculated with visible tumors: (1) control (Solvent), (2) and (3) compound 32, (4) Gefitinib, (5) DHA. Compound 32 showed obvious tumor growth delay in comparison with both control and drug Gefitinib or DHA treatment groups, even though the dosage of compound 32 used in the experiment is less than the control group. 5 mg/kg and 2.5 mg/kg were used in the test, while 10 mg/kg of Gefitinib and DHA were used as control. Meanwhile, no difference in body weight was observed between the Control and 32 treated mice, indicating the safety of compound 32. These results demonstrated that 32 has potential anti-tumor activity in vivo (Fig. 2). 3. Conclusion A series of novel artemisinin derivatives were designed and synthesized by junction of ART and 4-(arylamino)quinazoline pharmacophore via various types of linkers. All those artemisinin analogues were tested against the human colorectal cancer HCT116 cells and human melanoma cancer WM-266-4 and showed potent antiproliferative activities. In particular, the compound 32 was the most active derivative and effectively suppressed the tumor growth in vitro with an IC50 of 110 nM and significantly improved the antitumor activity of the parent compounds. Moreover, in vivo study on xenografted mice disclosed that compound 32 effectively delayed tumor growth after 18 days treatment, while the weight of the mice remained unchanged compared with the control group. This promising results indicated that compound 32 represent a new structural lead for development of new anti-CRC cancer drug. 4. Experimental section All commercially available reagents were used without further purification unless otherwise stated. All reactions were performed under an argon atmosphere unless otherwise stated. Flash column chromatography on silica gel (200e300 mesh) was used for purification of reaction products. NMR spectra were recorded with tetramethylsilane as the internal standard. 1H NMR spectra were recorded at 400 MHz、500 MHz、600 MHz、800 MHz, respectively, and 13C NMR spectra were recorded at 100 MHz、125 MHz、 150 MHz (Bruker Avance), respectively. Chemical shift values of NMR data are reported as values in ppm relative to (residual undeuterated) solvent signal as internal standard. Multiplicities for 1 H NMR signals are described using the following abbreviations: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet; High resolution mass spectra were obtained with the Q-TOF-Premier mass spectrometer. 4.1. Chemical synthesis 4.1.1. General procedure for preparation of hybrids (9a-9h) To a solution of dihydroartemisinin 8 (1.2 eq.) and corresponding alcohol 4e7 (1 eq.) in dry dichloromethane under argon was

6

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

Fig. 2. In vivo efficacy of compound 32 in human colorectal cancer xenograft models. (A) Two days after subcutaneous implantation of HCT116 cells, female nude mice received the following treatments: (1) Ctrl (Solvent), (2) (3) compound 32, (4) Gefitinib, (5) DHA via intraperitoneal injection every day, and tumor volumes were estimated every day. Each data point represents mean tumor volume of 8 animals in each group ± SE. p value, *<0.05; **<0.01; (B) The individual body weight was measured and expressed as mean ± SD (n ¼ 8); (C) (D) Tumors were isolated and tumor weights were measured at the end of treatment. Representative tumor images are shown and tumor weights are presented as mean ± SD (n ¼ 8).

added BF3$Et2O (2 eq.) dropwise at 0  C. The mixture was then stirred overnight at this temperature and quenched with saturated NaHCO3 aqueous solution. The reaction mixture was then extracted with ethyl acetate. After combining with organic phase and dried over Na2SO4, the solvent was removed under reduced pressure to give the crude product. The a, b-isomers could be readily separated in each case by flash chromatography to afford hybrids 9a-9h. Hybrid 9a (25%): 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 8.01e7.93 (m, 2H), 7.91 (d, J ¼ 8.6 Hz, 1H), 7.77 (d, J ¼ 8.3 Hz, 1H), 7.64e7.54 (m, 1H), 7.17 (t, J ¼ 8.7 Hz, 1H), 5.46 (s, 1H), 4.94 (d, J ¼ 12.5 Hz, 1H), 4.89 (d, J ¼ 3.3 Hz, 1H), 4.74 (d, J ¼ 12.5 Hz, 1H), 2.73e2.61 (m, 1H), 2.38 (td, J ¼ 14.1, 3.8 Hz, 2H), 2.08e1.99 (m, 1H), 1.94e1.84 (m, 1H), 1.83e1.73 (m, 2H), 1.61 (dd, J ¼ 13.2, 2.6 Hz, 1H), 1.55e1.46 (m, 2H), 1.43 (s, 3H), 1.33e1.22 (m, 3H), 0.94 (s, 3H), 0.92 (s, 3H); 13C NMR (101 MHz, CDCl3) d 156.56, 153.99, 153.45, 148.01, 135.94, 133.85, 132.19, 127.70, 123.27, 120.83, 120.10, 118.95, 115.6, 113.49, 103.40, 100.23, 87.17, 80.06, 68.52, 51.40, 43.22, 36.45, 35.32, 33.46, 29.82, 25.10, 23.60, 23.50, 19.27, 12.02; HRMS (ESI) Calcd. for C30H34ClFN3O5 [MþH]þ: 570.2171; Found: 570.2166. Hybrid 9b (31%): 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 8.16 (s, 1H), 8.11e7.97 (m, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.46 (s, 1H), 4.79 (d, J ¼ 3.1 Hz, 1H), 4.67 (s, 2H), 4.09e3.99 (m, 1H), 3.66 (t, J ¼ 6.0 Hz, 2H), 3.57e3.48 (m, 1H), 2.69e2.59 (m, 1H), 2.27 (dd, J ¼ 18.6, 8.8 Hz, 1H), 1.92 (dd, J ¼ 12.1, 5.9 Hz, 3H), 1.79 (s, 1H), 1.78e1.72 (m, 2H), 1.61e1.54 (m, 1H), 1.49e1.40 (m, 1H), 1.28 (d, J ¼ 5.7 Hz, 1H), 1.24 (s, 3H), 1.21e1.15 (m, 2H), 1.12 (d, J ¼ 7.2 Hz, 1H), 0.89 (d, J ¼ 7.2 Hz, 6H); 13C NMR (151 MHz, CDCl3) d 157.43, 154.77, 154.53, 149.34, 137.55, 135.16,

132.10, 128.62, 123.88, 121.39, 121.35, 121.07, 118.90, 116.59, 115.03, 102.18, 88.16, 72.12, 67.81, 65.20, 52.48, 44.34, 37.54, 36.33, 34.57, 30.98, 29.79, 25.81, 24.50, 24.49, 20.30, 13.01; HRMS (ESI) Calcd. for C33H40ClFN3O6 [MþH]þ: 628.2590; Found: 628.2592. Hybrid 9c (28%): 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 8.17 (d, J ¼ 7.6 Hz, 2H), 8.06 (dd, J ¼ 6.5, 2.5 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.74e7.66 (m, 2H), 7.16 (t, J ¼ 8.8 Hz, 1H), 5.55 (s, 1H), 4.73e4.72 (m, 2H), 4.56 (d, J ¼ 12.7 Hz, 1H), 3.93 (dd, J ¼ 13.5, 8.1 Hz, 1H), 3.59 (dd, J ¼ 9.7, 4.7 Hz, 1H), 3.55e3.42 (m, 1H), 3.40e3.27 (m, 1H), 2.70e2.57 (m, 1H), 2.34 (td, J ¼ 14.1, 3.7 Hz, 1H), 2.03e1.90 (m, 2H), 1.90e1.79 (m, 2H), 1.71 (ddd, J ¼ 16.7, 9.4, 5.0 Hz, 4H), 1.65e1.58 (m, 2H), 1.51e1.40 (m, 2H), 1.30 (s, 3H), 1.28e1.20 (m, 2H), 0.94 (d, J ¼ 6.1 Hz, 3H), 0.88 (d, J ¼ 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 156.62, 153.84, 153.63, 148.51, 136.50, 134.19, 131.52, 127.60, 123.24, 120.76, 119.99, 118.39, 115.52, 113.99, 103.15, 101.08, 87.19, 80.13, 71.27, 69.93, 66.89, 51.55, 43.42, 36.50, 35.40, 33.62, 29.97, 26.27, 25.04, 24.95, 23.66, 23.49, 19.36, 12.02; HRMS (ESI) Calcd. for C34H42ClFN3O6 [MþH ]þ: 642.2746; Found: 642.2743. Hybrid 9d (21%): 1H NMR (400 MHz, CDCl3) d 8.76 (s, 1H), 8.04e7.93 (m, 2H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 8.7 Hz, 1H), 7.65e7.58 (m, 1H), 7.18 (t, J ¼ 8.7 Hz, 1H), 5.44 (s, 1H), 4.73 (d, J ¼ 3.1 Hz, 1H), 4.68 (d, J ¼ 7.3 Hz, 2H), 3.80 (dd, J ¼ 15.7, 6.6 Hz, 1H), 3.55 (t, J ¼ 6.4 Hz, 2H), 3.45e3.29 (m, 1H), 2.67e2.53 (m, 1H), 2.36 (td, J ¼ 14.1, 3.8 Hz, 1H), 2.05e1.96 (m, 1H), 1.92e1.83 (m, 1H), 1.83e1.77 (m, 1H), 1.77e1.72 (m, 1H), 1.72e1.65 (m, 3H), 1.63e1.57 (m, 3H), 1.46 (dd, J ¼ 11.2, 5.2 Hz, 3H), 1.38 (s, 3H), 1.34e1.27 (m, 1H), 1.26e1.18 (m, 2H), 0.93 (d, J ¼ 6.1 Hz, 3H), 0.87 (d, J ¼ 7.3 Hz, 3H); 13 C NMR (101 MHz, CDCl3) d 158.99, 156.66, 153.08, 137.01, 131.97,

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

127.02, 123.56, 121.12, 120.00, 118.90, 118.28, 115.61, 113.62, 103.16, 101.88, 101.10, 87.04, 80.13, 71.17, 69.80, 67.37, 51.52, 43.41, 36.49, 35.40, 33.61, 29.90, 28.32, 23.24, 25.12, 23.65, 23.45, 21.76, 19.34, 11.99; HRMS (ESI) Calcd. for C35H44ClFN3O6 [MþH]þ: 656.2903; Found: 656.2901. Hybrid 9e (23%): 1H NMR (400 MHz, CDCl3) d 8.73 (s, 1H), 8.19 (s, 1H), 8.06 (dd, J ¼ 6.5, 2.4 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.71e7.60 (m, 2H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.40 (s, 1H), 5.11 (d, J ¼ 13.0 Hz, 1H), 4.82 (d, J ¼ 13.0 Hz, 1H), 4.64 (d, J ¼ 9.2 Hz, 1H), 2.60e2.52 (m, 1H), 2.44e2.34 (m, 2H), 2.07e1.98 (m, 1H), 1.91 (d, J ¼ 11.1 Hz, 1H), 1.78 (dd, J ¼ 13.4, 3.4 Hz, 1H), 1.73e1.66 (m, 1H), 1.64e1.56 (m, 1H), 1.54e1.43 (m, 1H), 1.37 (s, 3H), 1.29 (dd, J ¼ 9.9, 4.6 Hz, 2H), 1.25 (s, 1H), 0.99e0.93 (m, 6H); 13C NMR (101 MHz, CDCl3) d 156.56, 153.83, 153.24, 148.01, 135.94, 133.85, 132.19, 127.70, 123.27, 120.83, 120.10, 118.95, 115.64, 113.49, 103.40, 100.23, 87.17, 80.06, 68.52, 51.40, 43.22, 36.45, 35.32, 33.46, 29.82, 25.10, 23.65, 23.52, 19.27, 12.02; HRMS (ESI) Calcd. for C30H34ClFN3O5 [MþH]þ: 570.2171; Found: 570.2169. Hybrid 9f (27%): 1H NMR (400 MHz, CDCl3) d 8.72 (s, 1H), 8.14 (s, 1H), 7.97 (dd, J ¼ 6.4, 2.3 Hz, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.74 (d, J ¼ 8.5 Hz, 1H), 7.69e7.62 (m, 1H), 7.15 (t, J ¼ 8.8 Hz, 1H), 5.35 (s, 1H), 4.66 (d, J ¼ 4.2 Hz, 2H), 4.44 (d, J ¼ 9.2 Hz, 1H), 4.07 (dt, J ¼ 9.1, 6.8 Hz, 1H), 3.59 (d, J ¼ 6.4 Hz, 2H), 2.41e2.30 (m, 2H), 2.04e1.95 (m, 1H), 1.86 (dd, J ¼ 11.8, 5.7 Hz, 3H), 1.76e1.63 (m, 3H), 1.50 (dd, J ¼ 11.2, 6.8 Hz, 1H), 1.42 (dd, J ¼ 11.0, 4.6 Hz, 1H), 1.34 (s, 3H), 1.26 (s, 1H), 1.25 (s, 2H), 1.19 (d, J ¼ 13.2 Hz, 1H), 0.94 (d, J ¼ 5.4 Hz, 3H), 0.80 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 168.64, 156.94, 154.18, 148.50, 137.00, 133.92, 132.01, 126.78, 123.76, 121.50, 119.95, 118.80, 115.5, 113.68, 103.47, 99.16, 90.25, 79.53, 71.19, 66.19, 64.62, 50.56, 44.24, 36.40, 35.29, 33.13, 31.65, 28.98, 24.96, 23.63, 21.15, 19.22, 11.55; HRMS (ESI) Calcd. for C33H40ClFN3O6 [MþH ]þ: 628.2590; Found: 628.2583. Hybrid 9g (33%): 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 7.98 (dd, J ¼ 6.4, 2.4 Hz, 1H), 7.88 (d, J ¼ 8.5 Hz, 1H), 7.73 (d, J ¼ 8.2 Hz, 1H), 7.64e7.58 (m, 1H), 7.16 (t, J ¼ 8.8 Hz, 1H), 5.33 (s, 1H), 4.64 (s, 2H), 4.41 (d, J ¼ 9.2 Hz, 1H), 4.00e3.92 (m, 1H), 3.53 (t, J ¼ 5.7 Hz, 2H), 3.44 (dd, J ¼ 6.2, 3.0 Hz, 1H), 2.42e2.31 (m, 2H), 2.04e1.95 (m, 2H), 1.92e1.83 (m, 1H), 1.68 (dd, J ¼ 14.0, 6.6 Hz, 5H), 1.55e1.43 (m, 2H), 1.37 (s, 3H), 1.31e1.20 (m, 4H), 0.95 (d, J ¼ 5.7 Hz, 3H), 0.83 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 156.70, 153.98, 153.52, 148.36, 136.70, 134.06, 131.69, 127.62, 123.67, 121.26, 119.96, 118.20, 115.5, 113.84, 103.35, 99.26, 90.23, 79.44, 71.10, 69.63, 67.83, 50.59, 44.27, 36.38, 35.30, 33.17, 31.64, 25.36, 25.29, 24.96, 23.66, 21.15, 19.24, 11.58; HRMS (ESI) Calcd. for C34H42ClFN3O6 [MþH]þ: 642.2746; Found: 642.2744. Hybrid 9h (19%): 1H NMR (400 MHz, CDCl3) d 8.75 (s, 1H), 8.01 (s, 1H), 7.97 (dd, J ¼ 6.5, 2.5 Hz, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.74 (d, J ¼ 8.4 Hz, 1H), 7.62e7.55 (m, 1H), 7.17 (t, J ¼ 8.7 Hz, 1H), 5.33 (s, 1H), 4.66 (d, J ¼ 3.2 Hz, 2H), 4.41 (d, J ¼ 9.2 Hz, 1H), 3.95 (dd, J ¼ 15.3, 6.4 Hz, 1H), 3.53 (t, J ¼ 5.5 Hz, 2H), 3.41 (dd, J ¼ 15.7, 6.6 Hz, 1H), 2.36 (ddd, J ¼ 22.6, 12.6, 5.4 Hz, 2H), 2.00 (dd, J ¼ 14.1, 3.5 Hz, 1H), 1.93e1.83 (m, 1H), 1.78e1.68 (m, 4H), 1.63 (dd, J ¼ 15.8, 7.4 Hz, 4H), 1.50e1.47 (m, 1H), 1.46 (s, 1H), 1.38 (s, 3H), 1.26 (dd, J ¼ 9.4, 4.8 Hz, 4H), 0.95 (d, J ¼ 5.9 Hz, 3H), 0.84 (d, J ¼ 7.1 Hz, 3H); 13C NMR (151 MHz, CDCl3) d 157.55, 155.87, 154.38, 137.92, 134.85, 132.69, 130.88, 128.79, 124.59, 122.17, 121.01, 118.72, 116.62, 114.68 (s), 104.30, 100.23, 91.22, 80.41, 72.08, 70.88, 68.91, 51.61, 45.28, 37.37, 36.30, 34.19, 32.62, 29.30, 29.22, 25.99, 24.66, 22.73, 22.17, 20.26, 12.59; HRMS (ESI) Calcd. for C35H44ClFN3O6 [MþH]þ: 656.2903; Found: 656.2902. 4.1.2. General procedure for preparation of hybrids 17a-17f Alcohol 4 (1 eq.) and corresponding carboxylic acid intermediates 13e15 (1.2 eq.) were dissolved in dry dichloromethane under argon respectively. Then N-(3-dimethylaminopropyl)-N0 -

7

ethylcarbodiimide hydrochloride (EDCI, 2 eq.) and DMAP (0.5 eq.) were added to above mixture at ambient temperature. The reaction was monitored by TLC. After completion, the reaction was quenched by saturated NaHCO3 aqueous solution and extracted by ethyl acetate. The combined organic phase was then removed under reduced pressure and purified by chromatography to give the products 17a-17c. Products 17d-17f were prepared by the same procedure as mentioned above with Alcohol 16 and carboxylic acid intermediates 13e15 respectively. Hybrid 17a (77%): 1H NMR (400 MHz, CDCl3) d 8.76 (s, 1H), 8.64 (s, 1H), 8.41 (dd, J ¼ 6.4, 2.2 Hz, 1H), 7.87 (d, J ¼ 9.9 Hz, 2H), 7.67 (dd, J ¼ 5.2, 3.3 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz, 1H), 7.19 (t, J ¼ 8.7 Hz, 1H), 5.57 (d, J ¼ 14.2 Hz, 1H), 5.46 (d, J ¼ 9.8 Hz, 1H), 5.27 (d, J ¼ 14.2 Hz, 1H), 4.72 (s, 1H), 2.97e2.78 (m, 4H), 2.43 (s, 1H), 2.23 (td, J ¼ 14.1, 3.5 Hz, 1H), 2.12 (s, 1H), 1.90 (d, J ¼ 14.2 Hz, 1H), 1.85e1.73 (m, 1H), 1.50 (d, J ¼ 12.1 Hz, 1H), 1.41 (dd, J ¼ 17.2, 8.1 Hz, 2H), 1.33e1.18 (m, 2H), 1.14 (s, 3H), 0.91 (s, 3H), 0.82 (d, J ¼ 10.0 Hz, 1H), 0.63 (d, J ¼ 7.0 Hz, 3H), 0.53 (dt, J ¼ 24.5, 8.3 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 172.53, 170.24, 156.50, 153.52, 153.42, 148.04, 134.88, 133.97, 129.57, 127.47, 122.64, 120.12, 120.06, 116.60, 115.5, 114.19, 103.27, 92.08, 90.04, 78.72, 63.90, 50.17, 43.70, 36.09, 34.96, 32.76, 30.35, 28.56, 28.11, 24.47, 23.44, 20.46, 19.12, 10.82; HRMS (ESI) Calcd. for C34H38ClFN3O8 [MþH ]þ: 670.2332; Found: 670.2336. Hybrid 17b (69%): 1H NMR (400 MHz, CDCl3) d 8.73 (s, 1H), 8.17 (s, 1H), 8.05e7.96 (m, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.71 (d, J ¼ 8.5 Hz, 1H), 7.65e7.55 (m, 1H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.62 (d, J ¼ 9.8 Hz, 1H), 5.29 (d, J ¼ 6.4 Hz, 2H), 5.22 (s, 1H), 2.53e2.38 (m, 5H), 2.36e2.25 (m, 2H), 2.04e1.94 (m, 3H), 1.85 (dd, J ¼ 8.9, 4.6 Hz, 1H), 1.64 (d, J ¼ 11.9 Hz, 2H), 1.54 (dt, J ¼ 13.3, 3.9 Hz, 1H), 1.39 (ddd, J ¼ 19.7, 12.3, 5.6 Hz, 1H), 1.31 (s, 3H), 1.22 (dd, J ¼ 12.3, 6.0 Hz, 2H), 1.12e0.99 (m, 1H), 0.94 (d, J ¼ 5.3 Hz, 3H), 0.77 (d, J ¼ 7.1 Hz, 3H); 13 C NMR (151 MHz, CDCl3) d 172.42, 172.26, 157.96, 154.88, 154.77, 149.34, 135.07, 134.67, 132.61, 128.46, 124.91, 122.51, 120.71, 120.49, 116.38, 115.02, 104.44, 92.30, 91.35, 79.99, 65.37, 51.29, 44.94, 37.11, 36.02, 33.85, 33.04, 32.73, 31.51, 25.66, 24.41, 21.72, 20.07, 19.57, 11.96; HRMS (ESI) Calcd. for C35H40ClFN3O8 [MþH ]þ: 684.2488; Found: 684.2484. Hybrid 17c (63%): 1H NMR (400 MHz, CDCl3) d 8.74 (s, 1H), 8.42 (s, 1H), 8.15 (s, 1H), 8.04e7.97 (m, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 8.5 Hz, 1H), 7.63e7.53 (m, 1H), 7.15 (t, J ¼ 8.7 Hz, 1H), 5.65 (d, J ¼ 9.8 Hz, 1H), 5.25 (s, 3H), 2.56e2.45 (m, 1H), 2.38e2.27 (m, 5H), 2.01e1.93 (m, 1H), 1.89e1.79 (m, 1H), 1.64 (d, J ¼ 10.4 Hz, 6H), 1.58e1.50 (m, 1H), 1.45e1.35 (m, 1H), 1.32 (s, 3H), 1.22 (dd, J ¼ 11.1, 5.3 Hz, 2H), 1.11 (dd, J ¼ 27.0, 13.8 Hz, 1H), 0.97 (d, J ¼ 14.3 Hz, 1H), 0.94 (d, J ¼ 5.2 Hz, 3H), 0.75 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 171.97, 171.63, 156.85, 154.0, 153.81, 148.40, 133.97, 133.81, 132.29, 127.73, 123.77, 121.32, 119.93, 115.5, 113.94, 103.47, 91.15, 90.43, 79.08, 64.61, 50.42, 44.06, 36.18, 35.14, 32.97, 32.65, 30.65, 24.78, 23.50, 23.08, 22.83, 20.81, 19.15, 11.04; HRMS (ESI) Calcd. for C36H42ClFN3O8 [MþH ]þ: 698.2645; Found: 698.2642. Hybrid 17d (68%): 1H NMR (400 MHz, CDCl3) d 9.24 (s, 1H), 8.11 (d, J ¼ 8.7 Hz, 1H), 7.95 (s, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.53 (dd, J ¼ 6.2, 2.2 Hz, 1H), 7.38e7.30 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 5.76 (d, J ¼ 9.8 Hz, 1H), 5.41 (s, 1H), 5.30 (d, J ¼ 6.2 Hz, 2H), 2.81e2.64 (m, 4H), 2.60e2.48 (m, 1H), 2.36 (td, J ¼ 14.1, 3.7 Hz, 1H), 2.17 (s, 3H), 2.02 (d, J ¼ 14.5 Hz, 1H), 1.92e1.82 (m, 1H), 1.70 (d, J ¼ 9.9 Hz, 3H), 1.63e1.54 (m, 1H), 1.52e1.42 (m, 1H), 1.40 (s, 3H), 1.36e1.23 (m, 3H), 0.95 (d, J ¼ 5.8 Hz, 3H), 0.79 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.72, 170.09, 169.89, 160.68, 156.42, 153.99, 151.59, 136.19, 136.03, 133.28, 129.17, 128.62, 126.94, 122.22, 120.84, 120.41, 116.26, 103.45, 91.32, 90.47, 79.10, 64.50, 50.52, 44.17, 36.22, 35.19, 33.05, 30.75, 28.10, 27.88, 24.91, 23.54, 22.64, 20.90, 19.18, 10.99; HRMS (ESI) Calcd. for C36H40ClFN3O9 [MþH]þ: 712.2437; Found: 712.2440. Hybrid 17e (74%): 1H NMR (400 MHz, CDCl3) d 9.23 (s, 1H), 8.11

8

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

(d, J ¼ 8.6 Hz, 1H), 7.96e7.86 (m, 2H), 7.50 (dd, J ¼ 6.4, 2.5 Hz, 1H), 7.35e7.27 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 5.77 (d, J ¼ 9.8 Hz, 1H), 5.42 (s, 1H), 5.28 (d, J ¼ 6.1 Hz, 2H), 2.59e2.50 (m, 1H), 2.47 (t, J ¼ 7.3 Hz, 4H), 2.36 (td, J ¼ 14.1, 3.9 Hz, 1H), 2.17 (s, 3H), 2.06e1.94 (m, 3H), 1.92e1.83 (m, 1H), 1.78e1.68 (m, 3H), 1.61 (dt, J ¼ 13.8, 4.2 Hz, 1H), 1.53e1.43 (m, 1H), 1.40 (s, 3H), 1.37e1.22 (m, 3H), 0.95 (d, J ¼ 5.9 Hz, 3H), 0.82 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 171.42, 170.61, 169.88, 160.60, 156.41, 153.99, 151.59, 136.17, 136.10, 133.37, 129.07, 128.68, 126.83, 122.36, 120.88, 116.28, 103.45, 90.96, 90.48, 79.10, 64.32, 50.54, 44.20, 36.24, 35.19, 33.05, 32.05, 30.73, 24.91, 23.55, 22.68, 20.96, 19.19, 18.73, 11.10; HRMS (ESI) Calcd. for C37H42ClFN3O9 [MþH]þ: 726.2594; Found: 726.2599. Hybrid 17f (71%): 1H NMR (400 MHz, CDCl3) d 9.24 (s, 1H), 8.12 (d, J ¼ 8.5 Hz, 1H), 7.91 (d, J ¼ 8.5 Hz, 2H), 7.50 (dd, J ¼ 6.4, 2.4 Hz, 1H), 7.34e7.27 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 5.77 (d, J ¼ 9.8 Hz, 1H), 5.42 (s, 1H), 5.28 (d, J ¼ 9.0 Hz, 2H), 2.59e2.48 (m, 1H), 2.45e2.31 (m, 5H), 2.17 (s, 3H), 2.02 (dd, J ¼ 13.9, 3.6 Hz, 1H), 1.93e1.84 (m, 1H), 1.74e1.66 (m, 7H), 1.63e1.56 (m, 1H), 1.53e1.44 (m, 1H), 1.41 (s, 3H), 1.37e1.21 (m, 3H), 0.96 (d, J ¼ 5.9 Hz, 3H), 0.82 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 171.79, 170.96, 169.85, 160.59, 156.39, 154.00, 151.60, 136.16, 133.42, 129.06, 128.69, 126.71, 122.37, 120.89, 120.32, 116.26, 103.44, 90.84, 90.48, 79.12, 64.27, 50.55, 44.22, 36.25, 35.20, 33.07, 32.75, 30.76, 24.93, 23.56, 23.20, 22.99, 22.67, 20.97, 19.19, 11.10; HRMS (ESI) Calcd. for C38H44ClFN3O9 [MþH]þ: 740.2750; Found: 740.2745. 4.1.3. General procedure for preparation of hybrid 22a-22f Alcohol 4 (1 eq.) and corresponding carboxylic acid intermediates 19e21 (1.2 eq.) were dissolved in dry dichloromethane under argon respectively. Then N-(3-dimethylaminopropyl)-N0 ethylcarbodiimide hydrochloride (EDCI, 2 eq.) and DMAP (0.5 eq.) were added to above mixture at ambient temperature. The reaction was monitored by TLC. After completion, the reaction was quenched by saturated NaHCO3 aqueous solution and extracted by ethyl acetate. The combined organic phase was then removed under reduced pressure and purified by chromatography to give the products 22a-22c. Products 22d-22f were prepared by the same procedure as mentioned above with Alcohol 16 and carboxylic acid intermediates 19e21 respectively. Hybrid 22a (64%): 1H NMR (400 MHz, CDCl3) d 9.43 (s, 1H), 8.77 (s, 1H), 8.60 (d, J ¼ 4.5 Hz, 1H), 7.85 (d, J ¼ 8.2 Hz, 1H), 7.76 (s, 1H), 7.55 (d, J ¼ 8.1 Hz, 1H), 7.17 (t, J ¼ 8.4 Hz, 1H), 6.66 (s, 1H), 5.59 (d, J ¼ 13.4 Hz, 1H), 5.28 (d, J ¼ 13.9 Hz, 1H), 4.94 (t, J ¼ 10.0 Hz, 1H), 4.56 (s, 1H), 3.03 (d, J ¼ 14.6 Hz, 1H), 2.85e2.61 (m, 3H), 2.31e2.12 (m, 2H), 1.97 (t, J ¼ 24.0 Hz, 3H), 1.79 (d, J ¼ 7.8 Hz, 1H), 1.48 (d, J ¼ 12.3 Hz, 1H), 1.33 (s, 1H), 1.28 (s, 3H), 1.18 (d, J ¼ 12.1 Hz, 1H), 1.07 (d, J ¼ 5.2 Hz, 1H), 0.91 (s, 4H), 0.80 (s, 1H), 0.47 (d, J ¼ 6.5 Hz, 3H), 0.38 (d, J ¼ 12.7 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 171.72, 171.04, 156.55, 153.46, 153.3, 148.22, 135.44, 134.16, 128.91, 127.41, 122.47, 120.05, 119.77, 116.25, 115.30, 114.36, 103.25, 90.21, 79.08, 75.29, 63.43, 50.32, 43.92, 36.07, 35.09, 32.69, 31.03, 29.80, 27.99, 24.86, 23.48, 19.93, 19.17, 11.68; HRMS (ESI) Calcd. for C34H39ClFN4O7 [MþH]þ: 669.2491; Found: 669.2484. Hybrid 22b (67%): 1H NMR (400 MHz, CDCl3) d 9.33 (s, 1H), 8.71 (s, 1H), 8.32 (s, 1H), 8.07e8.00 (m, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.68e7.56 (m, 2H), 7.15 (t, J ¼ 8.8 Hz, 1H), 6.66 (d, J ¼ 9.5 Hz, 1H), 5.31 (q, J ¼ 13.7 Hz, 2H), 5.08 (t, J ¼ 10.0 Hz, 1H), 4.99 (s, 1H), 2.58e2.47 (m, 1H), 2.42 (dd, J ¼ 14.8, 7.9 Hz, 1H), 2.37e2.20 (m, 4H), 2.00 (dt, J ¼ 20.6, 13.3 Hz, 4H), 1.82 (dd, J ¼ 7.6, 3.6 Hz, 1H), 1.56 (d, J ¼ 10.5 Hz, 1H), 1.49e1.41 (m, 2H), 1.31 (s, 3H), 1.19e1.04 (m, 2H), 0.92 (d, J ¼ 5.5 Hz, 3H), 0.89e0.74 (m, 2H), 0.68 (d, J ¼ 7.0 Hz, 3H); 13 C NMR (101 MHz, CDCl3) d 172.01, 171.60, 157.19, 153.94, 153.80, 148.35, 134.51, 133.86, 130.69, 127.43, 124.27, 121.89, 119.69, 118.76, 115.35, 114.27, 103.37, 90.45, 79.29, 75.20, 64.33, 50.47, 44.26, 36.16, 35.16, 34.60, 32.82, 32.18, 31.13, 24.88, 23.49, 20.25, 19.31, 19.14,

11.93; HRMS (ESI) Calcd. for C35H41ClFN4O7 [MþH]þ: 683.2648; Found: 683.2643. Hybrid 22c (58%): 1H NMR (400 MHz, CDCl3) d 9.28 (s, 1H), 8.71 (s, 1H), 8.41 (s, 1H), 8.02 (d, J ¼ 2.3 Hz, 1H), 7.88 (d, J ¼ 8.1 Hz, 1H), 7.71 (d, J ¼ 8.4 Hz, 1H), 7.63e7.52 (m, 1H), 7.15 (t, J ¼ 8.7 Hz, 1H), 6.87 (d, J ¼ 9.3 Hz, 1H), 5.35 (d, J ¼ 12.9 Hz, 1H), 5.18e5.15 (m, 2H), 5.08 (s, 1H), 2.42e2.23 (m, 4H), 2.19e2.01 (m, 2H), 1.95 (d, J ¼ 14.5 Hz, 1H), 1.82 (d, J ¼ 13.5 Hz, 1H), 1.60 (d, J ¼ 10.9 Hz, 4H), 1.49 (dd, J ¼ 9.6, 4.1 Hz, 2H), 1.43e1.21 (m, 6H), 1.21e1.11 (m, 2H), 1.09e0.97 (m, 1H), 0.92 (d, J ¼ 4.5 Hz, 3H), 0.69 (d, J ¼ 7.0 Hz, 3H); 13C NMR (151 MHz, CDCl3) d 173.26, 173.25, 158.04, 155.06, 154.85, 149.86, 135.48, 134.45, 132.75, 128.79, 124.92, 122.51, 121.38, 120.68, 116.42, 115.25, 104.34, 91.42, 80.29, 76.09, 65.99, 51.48, 45.28, 37.14, 36.15, 36.09, 33.85, 33.72, 32.20, 25.81, 24.46, 24.35, 24.17, 21.30, 20.16, 12.97; HRMS (ESI) Calcd. for C36H43ClFN4O7 [MþH]þ: 697.2804; Found: 697.2801. Hybrid 22d (53%): 1H NMR (400 MHz, CDCl3) d 9.23 (s, 1H), 8.10 (d, J ¼ 8.6 Hz, 1H), 7.94 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.53 (d, J ¼ 4.1 Hz, 1H), 7.39e7.30 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 6.36 (d, J ¼ 9.3 Hz, 1H), 5.38 (s, 1H), 5.31e5.27 (m, 3H), 2.86e2.74 (m, 1H), 2.72e2.61 (m, 1H), 2.57 (d, J ¼ 6.6 Hz, 2H), 2.37 (s, 2H), 2.17 (s, 3H), 2.01 (d, J ¼ 17.5 Hz, 2H), 1.87 (dd, J ¼ 7.2, 3.4 Hz, 2H), 1.69 (d, J ¼ 12.2 Hz, 2H), 1.56 (d, J ¼ 13.3 Hz, 1H), 1.39 (s, 3H), 1.34e1.18 (m, 3H), 0.94 (d, J ¼ 5.8 Hz, 3H), 0.77 (d, J ¼ 7.0 Hz, 3H); 13C NMR (151 MHz, CDCl3) d 172.31, 170.95, 170.93, 161.61, 157.40, 154.95, 152.54, 137.15, 134.25, 130.16, 129.55, 127.89, 123.10, 121.82, 121.36, 117.24, 104.31, 91.59, 80.38, 76.04, 65.37, 51.64, 45.51, 37.26, 36.22, 33.99, 32.64, 30.80, 29.10, 26.00, 24.56, 23.65, 21.56, 20.21, 12.92; HRMS (ESI) Calcd. for C36H41ClFN4O8 [MþH]þ: 711.2597; Found: 711.2591. Hybrid 22e (63%): 1H NMR (400 MHz, CDCl3) d 9.23 (s, 1H), 8.11 (d, J ¼ 8.7 Hz, 1H), 7.95 (s, 1H), 7.90 (dd, J ¼ 8.7, 1.5 Hz, 1H), 7.51 (dd, J ¼ 6.3, 2.5 Hz, 1H), 7.37e7.29 (m, 1H), 7.19 (t, J ¼ 8.6 Hz, 1H), 6.33 (d, J ¼ 9.5 Hz, 1H), 5.38 (s, 1H), 5.35e5.23 (m, 3H), 2.46 (td, J ¼ 7.1, 1.9 Hz, 2H), 2.38e2.29 (m, 2H), 2.26 (td, J ¼ 7.3, 3.9 Hz, 2H), 2.21e2.15 (m, 3H), 2.02 (s, 1H), 1.97 (dd, J ¼ 14.4, 7.1 Hz, 2H), 1.87 (ddd, J ¼ 13.2, 6.3, 3.1 Hz, 1H), 1.80 (s, 1H), 1.76e1.66 (m, 2H), 1.60e1.53 (m, 1H), 1.50e1.39 (m, 2H), 1.37 (s, 3H), 1.29e1.18 (m, 2H), 0.95 (d, J ¼ 6.1 Hz, 3H), 0.78 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 171.69, 170.75, 170.15, 160.64, 156.49, 154.02, 151.51, 136.10, 133.52, 129.22, 128.64, 127.00, 122.61, 120.91, 120.33, 116.31, 103.28, 90.61, 79.39, 74.90, 64.28, 50.68, 44.56, 36.28, 35.24, 34.15, 33.02, 32.24, 31.62, 24.99, 23.59, 22.63, 20.61, 19.33, 19.22, 12.03; HRMS (ESI) Calcd. for C37H42ClFN4O8Na [MþNa]þ: 747.2573; Found: 747.2570. Hybrid 22f (58%): 1H NMR (400 MHz, CDCl3) d 9.23 (s, 1H), 8.11 (d, J ¼ 8.6 Hz, 1H), 7.95e7.84 (m, 2H), 7.51 (dd, J ¼ 6.4, 2.5 Hz, 1H), 7.36e7.27 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 6.20 (d, J ¼ 9.2 Hz, 1H), 5.39 (s, 1H), 5.32 (t, J ¼ 10.1 Hz, 1H), 5.27 (s, 2H), 2.40 (s, 2H), 2.36e2.28 (m, 2H), 2.25e2.19 (m, 2H), 2.17 (s, 3H), 2.05e1.97 (m, 1H), 1.92e1.83 (m, 2H), 1.74e1.65 (m, 6H), 1.60e1.53 (m, 1H), 1.46 (dd, J ¼ 13.0, 3.1 Hz, 1H), 1.38 (s, 3H), 1.29e1.20 (m, 2H), 0.95 (d, J ¼ 6.1 Hz, 3H), 0.78 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 171.90, 171.20, 169.95, 160.58, 156.42, 153.99, 151.53, 136.22, 136.14, 133.44, 129.12, 128.64, 126.85, 122.34, 120.88, 120.31, 116.28, 103.29, 90.64, 79.41, 74.89, 64.23, 50.67, 44.56, 36.28, 35.24, 35.15, 33.01, 32.80, 31.67, 25.01, 23.61, 23.58, 23.38, 22.68, 20.62, 19.23, 12.05; HRMS (ESI) Calcd. for C38H45ClFN4O8 [MþH]þ: 739.2910; Found: 739.2903. 4.1.4. Preparation of hybrid 32 To a solution of dihydroartemisinin 8 (310.9 mg, 1.09 mmol) and (S)-alkamine 29 (100 mg, 0.729 mmol) in dry dichloromethane under argon was added BF3$Et2O (155.2 mmol, 1.09 mmol) dropwise at 0  C. The mixture was then stirred overnight at this

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

temperature and quenched with saturated NaHCO3 aqueous solution. The reaction mixture was then extracted with ethyl acetate. After combining with organic phase and dried over Na2SO4, the solvent was removed under reduced pressure to give the crude product. The pure b-isomers could be predominately isolated by flash chromatography to afford the amine 30 (118 mg, 40%). To a solution of amine 30 (118 mg, 0.292 mmol) and carboxylic acid 31 (112 mg, 0.35 mmol) in dry DMF under argon were added DMAP (36 mg, 0.292 mmol), EDCI (84 mg, 0.438 mmol) and DIPEA (95 mg, 0.735 mmol) at room temperature. The reaction mixture was stirred at 40  C overnight. Then the reaction was quenched by saturated NaHCO3 aqueous solution and extracted by ethyl acetate. The combined organic phase was then removed under reduced pressure and purified by chromatography to give the hybrids 32 (86.9 mg, 42%). Hybrid 32: 1H NMR (600 MHz, Acetone-d6) d 9.50 (s, 1H, NH), 8.85 (s, 1H, H50 ), 8.70 (s, 1H, H80 ), 8.53 (d, J ¼ 7.08 Hz, 1H, CONH), 8.36 (dd, J ¼ 1.62, 8.64 Hz, 1H, H110 ), 8.31 (dd, J ¼ 2.55, 6.75 Hz, 1H, H130 ), 7.94e7.92 (m, 1H, H170 ), 7.86 (d, J ¼ 8.64 Hz, 1H, H100 ), 7.50 (d, J ¼ 7.50 Hz, 2H, H220 , H180 ), 7.36e7.32 (m, 3H, H160 , H190 , H210 ), 7.25 (t, J ¼ 7.35 Hz, 1H, H200 ), 5.61 (s, 1H, H12), 5.32 (dd, J ¼ 5.70, 12.54 Hz, 1H, H20 ), 4.73 (d, J ¼ 9.36 Hz, 1H, H10), 4.10 (d, J ¼ 5.52 Hz, 2H, H10 ), 2.36e2.33 (m, 1H, H9), 2.24 (dt, J ¼ 3.72, 13.97 Hz, 1H, H4a), 1.95e1.93 (m, 1H, H4b), 1.82e1.79 (m, 1H, H5b), 1.72e1.70 (m, 1H, H8a), 1.66e1.63 (m, 1H, H7b), 1.51 (td, J ¼ 4.20, 13.63 Hz, 1H, H8a0 ), 1.44e1.40 (m, 1H, H8b), 1.34e1.26 (m, 2H, H6, H5a), 1.19 (t, J ¼ 7.11 Hz, 1H, H5a0 ), 1.12 (s, 3H, H14), 1.01e0.95 (m, 1H, H7a), 0.88 (d, J ¼ 6.36 Hz, 3H, H15), 0.81 (d, J ¼ 7.08 Hz, 3H, H16); 13C NMR (151 MHz, Acetone-d6) d 166.09 (C30 ), 159.19 (C70 ), 156.42 (C80 ), 155.08 (d, J CeF ¼ 244.0 Hz, C150 ), 152.61 (C90 ), 141.53 (C230 ), 137.39 (d, J CeF ¼ 3.2 Hz, 1C120 ), 133.58 (C40 ), 131.94 (C11’), 129.28 (100 ), 128.99 (2C, C190 , C210 ), 128.18 (2C, C180 , C220 ), 127.91 (C200 ), 124.66 (C130 ), 123.39 (C50 ), 123.08 (d, J CeF ¼ 6.9 Hz, C170 ), 120.59 (d, J 0 0 0 CeF ¼ 18.5 Hz, C14 ), 117.29 (d, J CeF ¼ 23.4 Hz, C16 ), 115.67 (C6 ), 0 104.78 (C3), 101.23 (C10), 91.97 (C12), 80.95 (C12a ), 71.79 (C10 ), 55.35 (C20 ), 52.30 (C5a0 ), 46.15 (C8a’), 37.88(C6), 36.84 (C4), 34.93 (C7), 32.89(C9), 25.92 (C14), 25.33(C5), 22.48 (C8), 20.41 (C15), 12.84 (C16); HRMS (ESI) Calcd. for C38H41ClFN4O6 [MþH]þ: 703.2699; Found: 703.2690. 4.2. Biological assay 4.2.1. Cell culture The human colon carcinoma cell lines (HCT116) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The melanoma cell lines (WM-266-4) were purchased form ATCC(American type culture collection). Cells were cultured in medium (DMEM for WM266-4, RPMI-1640 for HCT116) supplemented with 10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin (HyClone, Logan, UT, USA). All the cells were incubated at 37  C, 5% CO2 in a humidified atmosphere. 4.2.2. Cell viability assay Cell viability was determined by MTS assay. Briefly, 5  103 cells were seeded in 96-well plates and cultured overnight. Cells were next treated with compounds in duplicate for 48 h. The medium in the 96-well plate was removed and MTS solution was added in each well. The cells were incubated at 37  C for 1e2 h. The optical density (OD) was measured at 492 nm using a microplate reader (BioRad Laboratories). The IC50 values were calculated by the relative survival curve. 4.2.3. In vivo tumor growth assay All animal experiments were conformed to the guidelines of the

9

Animal Ethics Committee of Kunming Institute of Botany (Kunming, China). Three-week-old female BALB/C nude mice, purchased from Vital River Laboratory Animal Technology (Beijing, China) and kept in a pathogen-free environment, were used to establish the HCT116 xenograft model. Briefly, 3  106 HCT116 cells were subcutaneously injected into the right flank of nude mice for tumor formation. Each group consisted of 8 mice. Animals were daily treated by intraperitoneal injection for 17 days. Tumor size was measured in two dimensions with a digital caliper and calculated using the formula (length  width  width  0.5). At the end of the experiment, all mice were sacrificed. Tumors were resected, weighed and photographed. Conflict of interest The authors declare that we have no conflict of interest. Acknowledgements We thanks for the financial support from the National Natural Science Foundation of China [Grant No. 21772207]; CAS‘Light of West China’Program and Youth Innovation Promotion Association CAS; “The Thousand Talents Program”of Yunnan Province (L.-L. Wang); and the Strategic Priority Research Program of the Chinese Academy of Sciences [Grant No. XDA12030206]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111665. References [1] M.M. Center, A. Jemal, R.A. Smith, E. Ward, Ca-Cancer, J. Clin. 59 (2009) 366e378. [2] J. Ferlay, I. Soerjomataram, M. Ervik, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11, Lyon, France, International Agency for Research on Cancer, 2013. http://globocan.iarc.fr. accessed February12, 2014. [3] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, CA: a cancer, J. Clin. 66 (2016) 115e132. [4] 5-Fluorouracil (5-Fu) as the most commonly used chemotherapy drug for colorectal cancer got only 10-30% success therapy rate in clinical, see: D.B. Longley, D.P. Harkin, P.G. Johnston, 5-fluorouracil: mechanisms of action and clinical strategies Nat. Rev. Cancer 3 (2003) 330e338. [5] A.H. Braun, W. Achterrath, H. Wilke, U. Vanhoefer, A. Harstrick, P. Preusser, New systemic frontline treatment for metastatic colorectal carcinoma, Cancer 100 (2004) 1558e1577. [6] J.J.G. Marin, M.R. Romero, A.G. Blazquez, E. Herraez, E. Keck, O. Briz, Importance and limitations of chemotherapy among the available treatments for gastrointestinal tumours, anti-cancer agents, Med. Chem. 9 (2009) 162e184. [7] Y.Y. Tu, The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine, Nat. Med. 17 (2011) 1217e1220. [8] L.H. Miller, X. Su, Artemisinin: discovery from the Chinese herbal garden, Cell 146 (2011) 855e858. [9] C.J. Woodrow, N.J. White, The clinical impact of artemisinin resistance in Southeast asia and the potential for future spread, FEMS Microbiol. Rev. 41 (2017) 34e48. [10] R.T. Eastman, D.A. Fidock, Artemisinin-Based combination therapies: a vital tool in efforts to eliminate malaria, Nat. Rev. Microbiol. 7 (2009) 864e874. [11] Y. Li, Y.-L. Wu, An over four millennium story behind qinghaosu (Artemisinin)-A fantastic antimalarial drug from a traditional Chinese herb, Curr. Med. Chem. 10 (2003) 2197e2230. [12] For selected review of artemisinin on anticancer, see: Y. Zhang, G. Xu, S. Zhang, D. Wang, P.S. Prabha, Z. Zuo, Antitumor Research on artemisinin and its bioactive derivatives Nat. Prod. Bioprospecting. 8 (2018) 303e319. [13] S. Slezakova, J. Ruda-Kucerova, Anticancer activity of artemisinin and its derivatives, Anticancer Res. 37 (2017) 5995e6003. [14] D. Chaturvedi, A. Goswami, P.P. Saikia, N.C. Barua, P.G. Rao, Artemisinin and its derivatives: a novel class of anti-malarial and anti-cancer agents, Chem. Soc. Rev. 39 (2010) 435e454. €hlich, [15] (For selected examples of artmisinin on anticancer, see:) C. Reiter, T. Fro M. Zeino, M. Marschall, H. Bahsi, M. Leidenberger, O. Friedrich, B. Kappes, F. Hampel, T. Efferth, S.B. Tsogoeva, New efficient artemisinin derived agents

10

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665

against human leukemia cells, human cytomegalovirus and plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids, Eur. J. Med. Chem. 97 (2015) 164e172. € z, T. Fro €hlich, V. Klein, M. Zeino, K. Viertel, J. Held, [16] C. Reiter, C.A. Karago € B. Mordmüller, S.E. Oztürk, H. Anıl, T. Efferth, S.B. Tsogoeva, Synthesis and

[17] [18]

[19] [20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

study of cytotoxic activity of 1,2,4-trioxane- and egonol-derived hybrid molecules against plasmodium falciparum and multidrug-resistant human leukemia cells, Eur. J. Med. Chem. 75 (2014) 403e412. W.E. Ho, H.Y. Peh, T.K. Chan, W.S.F. Wong, Artemisinins: pharmacological actions beyond anti-malarial, Pharmacol. Ther. 142 (2014) 126e139. H.-H. Chen, H.-J. Zhou, X. Fang, Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro, Pharmacol. Res. 48 (2003) 231e236. H.-H. Chen, H.-J. Zhou, W.Q. Wang, G.D. Wu, Antimalarial dihydroartemisinin also inhibits angiogenesis, Cancer Chemother. Pharmacol. 53 (2004) 423e432. N. Berdelle, T. Nikolova, S. Quiros, T. Efferth, B. Kaina, Artesunate induces oxidative DNA damage, sustained DNA doublestrand breaks, and the ATM/ ATR damage response in cancer cells, Mol. Cancer Ther. 10 (2011) 2224e2233. B. Meunier, A. Robert, Heme as trigger and target for TrioxaneContaining antimalarial drugs, Acc. Chem. Res. 43 (2010) 1444e1451. G.H. Posner, P.M. O'Neill, Knowledge of the proposed chemical mechanism of action and cytochrome P450 metabolism of antimalarial trioxanes like artemisinin allows rational design of new antimalarial peroxides, Acc. Chem. Res. 37 (2004) 397e404. Y.Y. Lu, T.S. Chen, X.P. Wang, L. Li, Single-cell analysis of dihydroartemisinininduced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques, J. Biomed. Opt. 15 (2010), 046028. L.H. Stockwin, B. Han, S.X. Yu, M.G. Hollingshead, M.A. ElSohly, W. Gul, D. Slade, A.M. Galal, D.L. Newton, Artemisinin dimer anticancer activity correlates with heme-catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction, Int. J. Cancer 125 (2009) 1266e1275. S. Krishna, S. Ganapathi, I.C. Ster, M.E.M. Saeed, M. Cowan, C. Finlayson, H. Kovacsevics, H. Jansen, P.G. Kremsner, T. Efferth, D. Kumar, EBioMedicine 2 (2015) 82e90. For other recent examples of artemisinin against colon cancer, see: Z. Yao, A. Bhandari, Y. Wang, Y. Pan, F. Yang, R. Chen, E. Xia, O. Wang, Dihydroartemisinin potentiates antitumor activity of 5-fluorouracil against a resistant colorectal cancer cell line Biochem. Biophys. Res. Commun. 501 (2018) 636e642. J. Wang, J. Zhang, Y. Shi, C. Xu, C. Zhang, Y.K. Wong, Y.M. Lee, S. Krishna, Y. He, T.K. Lim, W. Sim, Z.C. Hua, H.-M. Shen, Q. Lin, Mechanistic investigation of the specific anticancer property of artemisinin and its combination with aminolevulinic acid for enhanced anticolorectal cancer activity, ACS Cent. Sci. 3 (2017) 743e750. T. Frçhlich, B. Ndreshkjana, J.K. Muenzner, C. Reiter, E. Hofmeister, S. Mederer, M. Fatfat, C. El-Baba, H. GaliMuhtasib, R. Schneider-Stock, S.B. Tsogoeva,

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

Synthesis of novel hybrids of thymoquinone and artemisinin with high activity and selectivity against colon cancer, ChemMedChem 12 (2017) 226e234. (For selected review on the concept on Hybridization:) B. Meunier, Hybrid molecules with a dual mode of action: dream or reality?, Acc. Chem. Res. 41 (2008) 69e77. M. Congreve, G. Chessari, D. Tisi, A.J. Woodhead, Recent developments in fragment-based drug discovery, J. Med. Chem. 51 (2008) 3661e3680. S. Klaeger, S. Heinzlmeir, M. Wilhelm, H. Polzer, B. Vick, P.A. Koenig, M. Reinecke, B. Ruprecht, S. Petzoldt, C. Meng, J. Zecha, K. Reiter, H. Qiao, D. Helm, H. Koch, M. Schoof, G. Canevari, E. Casale, S.R. Depaolini, A. Feuchtinger, Z. Wu, T. Schmidt, L. Rueckert, W. Becker, J. Huenges, A.K. Garz, B.O. Gohlke, D.P. Zolg, G. Kayser, T. Vooder, R. Preissner, H. Hahne, N. Tonisson, K. Kramer, K. Gotze, F. Bassermann, J. Schlegl, H.C. Ehrlich, S. Aiche, A. Walch, P.A. Greif, S. Schneider, E.R. Felder, J. Ruland, G. Medard, I. Jeremias, K. Spiekermann, B. Kuster, The target landscape of clinical kinase drugs, Science 358 (2017) eaan4368. X. Zhang, R. Li, K. Qiao, Z. Ge, L. Zhang, T. Cheng, R. Li, Novel dithiocarbamic acid esters derived from 6-aminomethyl-4-anilinoquinazolines and 6aminomethyl-4-anilino-3-cyanoquinolines as potent EGFR inhibitors, Arch. Pharm. Chem. Life Sci. 346 (2013) 44e52. X. Yang, W. Wang, J. Tan, D. Song, M. Li, D. Liu, Y. Jing, L. Zhao, Synthesis of a series of novel dihydroartemisinin derivatives containing a substituted chalcone with greater cytotoxic effects in leukemia cells, Bioorg. Med. Chem. Lett. 19 (2009) 4385e4388. S. Oh, I.H. Jeong, C.M. Ahn, W.S. Shin, S. Lee, Synthesis and antiangiogenic activity of thioacetal artemisinin derivatives, Bioorg. Med. Chem. 12 (2004) 3783e3790. S. Cho, S. Oh, Y. Um, J.-H. Jung, J. Ham, W. Seob S, S. Lee, Synthesis of 10substituted triazolyl artemisinins possessing anticancer activity via Huisgen 1,3-dipolar cylcoaddition, Bioorg. Med. Chem. Lett 19 (2009) 382e385. L. Jia, Q. Song, C. Zhou, X. Li, L. Pi, X. Ma, H. Li, X. Lu, Y. Shen, Dihydroartemisinin as a putative STAT3 inhibitor, suppresses the growth of head and neck squamous cell carcinoma by targeting Jak2/STAT3 signaling, PLoS One 11 (2016), e0147157. S. Grivennikov, E. Karin, J. Terzic, D. Mucida, G.-Y. Yu, S. Vallabhapurapu, J. Scheller, S. Rose-John, H. Cheroutre, L. Eckmann, M. Karin, IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitisassociated cancer, Cancer Cell 15 (2009) 103e113. J. Bollrath, T.J. Phesse, V.A. von Burstin, T. Putoczki, M. Bennecke, T. Bateman, € Canli, S. Schwitalla, V. Matthews, T. Nebelsiek, T. Lundgren-May, O. R.M. Schmid, T. Kirchner, M.C. Arkan, M. Ernst, F.R. Greten, gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis, Cancer Cell 15 (2009) 91e102. Q. Li, D. Zhang, X. Chen, L. He, T. Li, X. Xu, M. Li, Nuclear PKM2 contributes to gefitinib resistance via upregulation of STAT3 activation in colorectal cancer, Sci. Rep. 5 (2015) 16082.