AKT and MAPK signaling pathway

Journal Pre-proof Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway Jin Yang, Shengcao Hu, Chunlin Wang, Junrong Song, Chao Chen, Yanhua Fan, Yaacov Ben-David, Weidong Pan PII:

S0223-5234(19)31050-5

DOI:

https://doi.org/10.1016/j.ejmech.2019.111898

Reference:

EJMECH 111898

To appear in:

European Journal of Medicinal Chemistry

Received Date: 19 September 2019 Revised Date:

16 November 2019

Accepted Date: 16 November 2019

Please cite this article as: J. Yang, S. Hu, C. Wang, J. Song, C. Chen, Y. Fan, Y. Ben-David, W. Pan, Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111898. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Graphical abstract

Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway J Jin Yang 1, Shengcao Hu1, Chunlin Wang, Junrong Song, Chao Chen, Yanhua Fan**, Yaacov Ben-David **, and Weidong Pan*

Fangchinoline derivatives induce cell cycle arrest and apoptosis in human leukemia cell lines via suppression of the PI3K/AKT and MAPK signaling pathway Jin Yang a,c, 1, Shengcao Hu a,c, 1, Chunlin Wang b,c, Junrong Song b,c, Chao Chen b,c, Yanhua Fan **,b,c, Yaacov Ben-David **,b,c, and Weidong Pan*,b,c a

College of Pharmacy, Zunyi Medical University, Zunyi 563000, PR China

b

State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou

Medical University, Guiyang 550014, China c

The Key Laboratory of Chemistry for Natural Products of Guizhou Province and

Chinese Academy of Sciences/Guizhou Provincial Engineering Research Center for Natural Drugs, Guiyang 550014, China E-mail address: [email protected] (W. Pan), [email protected] (Y. Fan), [email protected] (Y. B. David). 1 These authors contributed equally to this work.

Keywords: Fangchinoline; Hit compound; Leukemia; PI3K/AKT pathway; c-MYC; Apoptosis; Cell cycle arrest.

ABSTRACT Fangchinoline, a bisbenzylisoquinoline alkaloid extracted from Stephania tetrandra S. Moore, is known to exert anti-cancer activity. A series of new fangchinoline derivatives have been synthesized and evaluated for their anti-cancer activity. In cell viability assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on leukemia and breast cancer cell lines than the parental compound. Among them, 3e exhibited strongest cell growth inhibitory activity in a dose- and time-dependent manner on leukemia cell line HEL through induction of G0/G1 cell cycle arrest and apoptosis. Treatment of HEL cells with 3e also resulted in significant suppression of the MAPK and PI3K/AKT pathway as well as c-MYC downregulation, which may responsible for induction of apoptosis and cell cycle arrest. In docking analysis, high affinity interaction between 3e and Akt1 was responsible for drastic kinase inhibition by the compound. This derivative compound may be useful as a potent anti-cancer agent for treatment of leukemia. 1. Introduction Leukemia, a malignant blood disease, is the leading cause of cancer death in children, which compromises about 30% of all childhood cancers [1]. In developing countries, the cure rate for childhood leukemia is under 35% [2]. In addition, drug resistance is still a major limitation hindering successful chemotherapy in leukemia treatment. Thus, there is an urgent need for development of drug with better anti-leukemia activity.

The PI3K/AKT pathway plays an important role in the survival, proliferation and maturation of early erythroid progenitor cells [3]. This signaling pathway is hyperactivated in 50% of acute myeloid leukemia (AML) patients [4]. Pharmacological inhibitors of PI3K suppress proliferation and cell cycle progression as well as specific inhibition of this signaling pathway can induce apoptosis [4-8]. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is associated with cell proliferation, differentiation, migration, aging and apoptosis [9]. In numerous studies, inhibition of ERK and JNK efficiently promoted apoptosis of leukemia cells, indicating their therapeutic benefit for this malignancy [10]. The above observations also suggest that dual inhibition of PI3K and MAPK pathway could produce an additive anti-cancer effect in AML cells. Fangchinoline, a bisbenzylisoquinoline alkaloid, was first isolated from traditional Chinese medicinal herb Stephania tetrandra S. Moore [11]. In many reports, fangchinoline displayed extensive biologic activities, including anti-tumor [12], anti-inflammation [13] and anti-hypertension [14]. In 1976, Kuroda et al. first discovered the anti-tumor effect of fangchinoline [15]. Hyun Sun Cho et al. found that fangchinoline

caused

apoptosis

of

lung

cancer

cells,

mediated

through

down-regulation of Akt and ERK phosphorylation. Treatment of lung cancer with fangchinoline was also enhanced the efficacy of chemotherapy and radiation therapy [16]. In another independent study, fangchinoline activated caspase-9, caspase-8, caspase-3 and PARP to promote cell death of the human bladder cancer cell lines 5637 and T24, representing high-risk superficial bladder cancer and high-grade

bladder cancer, respectively [17]. These observations raised up the likelihood of fangchinoline derivatives to show better anti-cancer activity. Previous studies of our research group shown that fangchinoline derivatives protected by benzoyl at 7-phenol position and nitrified at 14-position exhibiting significant inhibitory effects on five cancer cell lines, especially WM9 melanoma cell lines. This anti-cancer inhibitory activity was associated with down-regulation of anti-apoptotic factors bcl-xl, Survivin and bcl-2 [18-19]. However, the molecular target remained to be further explored. We performed a target screen using molecular docking and identified Akt1 as the potential target of these derivatives. Thus, we carried out a rational design base on the binding model of fangchinoline and Akt1 to further improve the kinase inhibitory activity. Hence, we generated additional derivatives of this ester side chain in the C7 position of fangchinoline to improve its biological activity. This effort resulted in the synthesis of a series of new fangchinoline derivatives. In this study, we evaluated their anti-proliferation activity against human leukemia cells (HEL and K562) and breast cancer cells (MDA-MB-231, MCF7), as well as determined their underlying mechanism of anti-cancer activity. 2. Results and discussion 2.1. Chemistry The synthetic routes and reagents for twenty-five new C7 substituted fangchinoline derivatives 1a-4h are shown in Scheme 1.

Scheme 1. Synthesis of fangchinoline derivatives. Reagents and conditions: (a) DMAP (0.2 eq.), DCM, RCOCl (1.1 eq.), Ar, 0 °C to rt, 2-4 h (74-90%).

2.2. Biological evaluation 2.2.1. Effects of fangchinoline derivatives on proliferation MTT assay was carried out to test the anti-proliferation activity of synthesized compounds against human leukemia cell lines HEL, K562 and breast cancer cell lines MDA-MB-231, MCF-7. As shown in Table 1, most of fangchinoline derivatives displayed better anti-proliferation activity against HEL, K562 MDA-MB-231 and MCF-7 cells than the positive controls, including vincristine, tetrandrine and the parent compound fangchinoline. The preliminary structure-activity relationship analysis (SAR) parallel the results of

cell viability assay. SAR indeed showed mono-substituted heterocycle derivatives exhibited better cell inhibitory activities than that of halogenated aryl and alkyl substituted derivatives. Introduction of large steric hindrance groups significantly reduced the inhibitory activity. For alkyl chain substituents, longer carbon chains and fewer branches increased the inhibitory effect of the derivatives. In addition, mono-halogenated aryl substituted derivatives showed better cell inhibitory activity than that of dihalogenated aryl and trihalogenated aryl substituted derivatives. Compared with the ortho-substitution, introduction of para-halogenated aryl and meta-haloaryl groups significantly improved their activity. Among the 25 fangchinoline derivatives, 3e showed the strongest inhibitory effect on the four tested cancer cell lines. Compared with fangchinoline, the anti-cancer activity of 3e increased nearly 15-fold in HEL cells, 36-fold in MDA-MB-231 cells and 9-fold in MCF-7. In addition, 3e showed 10-fold more cytotoxicity on these cells than vincristine. Because 3e had the best inhibitory effect on HEL cells, this compound was selected for further mechanism studies.

Table 1 The IC50 values of fangchinoline derivatives against HEL, K562, MDA-MB-231 and MCF-7 cell lines. IC50 (µM) compounds HEL

K562

MDA-MB-231

MCF-7

1a

10.81±0.89

12.29±1.10

10.01±0.94

30.08±1.36

1b

4.84±0.93

6.47±0.73

9.47±0.98

18.76±1.27

1c

7.86±0.90

6.55±0.82

8.68±0.94

12.36±1.09

1d

25.93±1.41

14.26±1.04

17.82±1.17

22.12±1.35

1e

3.54±0.48

3.30±0.52

4.05±0.61

7.14±0.70

2a

3.73±0.47

4.03±0.55

8.56±0.93

7.69±0.89

2b

5.16±0.71

5.39±0.63

10.31±1.30

﹥100

2c

4.27±0.47

6.77±0.83

5.03±0.94

7.28±0.22

2d

10.20±1.01

9.43±1.10

7.90±0.90

16.05±1.21

2e

15.72±1.20

10.41±1.02

13.04±1.12

8.57±0.93

2f

21.01±1.32

13.26±1.24

11.31±1.05

11.95±0.97

2g

21.34±1.33

17.51±1.24

13.49±1.13

﹥100

3a

3.46±0.43

5.89±0.77

9.89±1.00

20.64±1.32

3b

2.98±0.42

3.74±0.50

9.04±0.96

23.70±1.38

3c

17.63±1.07

12.41±1.09

10.12±1.01

20.67±1.32

3d

40.22±1.60

15.86±1.20

15.09±1.18

25.25±1.40

3e

1.52±0.03

1.53±0.19

1.60±0.21

4.01±0.60

4a

3.22±0.46

3.58±0.32

5.41±0.74

6.26±0.77

4b

4.26±0.57

3.79±0.58

8.03±0.91

4.42±0.65

4c

2.81±0.40

2.80±0.45

7.55±0.88

8.63±0.94

4d

10.19±1.01

6.11±0.79

27.97±1.45

﹥100

4e

4.56±0.60

8.52±0.93

18.27±1.26

10.85±1.04

4f

7.65±0.70

7.60±0.80

23.00±1.36

18.24±1.26

4g

2.31±0.14

3.06±0.49

5.58±0.75

6.84±0.84

4h

3.46±0.43

4.59±0.59

16.54±1.13

8.19±0.91

vinblastine

15.98±1.02

9.49±0.75

17.74±0.65

37.49±1.57

tetrandrine

19.74±1.30

6.43±0.81

18.45±1.27

19.58±1.29

fangchinoline

22.71±1.36

5.94±0.77

58.61±1.77

17.41±1.24

Result of MTT assay after 48 h drug treatment; the values are average for at least 3 independent experiments; variation ± 10%.

2.2.2. The effect of 3e compound on cell proliferation We assessed the effects of 3e on HEL cell proliferation at 12, 24, 36, 48 and 72 h, using MTT assay. Compared to the control group, 3e inhibited the proliferation of HEL cells in a time- and dose- dependent manner (Fig. 1).

Fig. 1. The inhibitory effect of 3e on human leukemia HEL cells. (A) Morphological changes of HEL cells at different concentrations of 3e after 24 h of drug treatment. (B1) Inhibitory effects of 3e on HEL cell proliferation at the indicated times. (B2) Percentage of cell viability at the time of protein extraction used for western blotting. 2.2.3. Induction of cell cycle arrest by 3e in HEL cells Unrestricted proliferation, inhibition of differentiation is the ultimate characteristic of malignant cells [20-21]. Indeed, variety of cell cycle regulators are currently used for treatment of various cancers [22]. Since inhibition of PI3K/AKT and MAPK/ERK pathways also causes cell cycle arrest [23], we further assessed the effects of 3e on cell cycle progression, using HEL cells. As shown in Fig. 2, 3e treatment led to an accumulation of cells in the G0/G1 phase. c-MYC proto-oncogene induces cell cycle through a variety of mechanisms, participates in the activity of DNA polymerase, and regulate cell proliferation and differentiation [24]. Suppression of c-MYC enhances p21WAF1/CIP1-mediated G1 cell cycle arrest through the modulation of ERK phosphorylation by ascochlorin [25]. Western blotting indeed demonstrated significant reduction in the expression of c-MYC and subsequently upregulated p21 expression in HEL cells treated with 3e (Fig. 2). These results suggested that 3e may induce G0/G1 arrest via c-MYC down-regulation and p21 up-regulation.

Fig. 2. The effect of 3e on cell cycle parameters of HEL cells. (A) 3e treatment results in cell cycle arrest of human leukemia cells. (B) Changes in p21 and c-MYC protein levels in human leukemia cells treated with 3e.

2.2.4. Induction of apoptosis by 3e in HEL cells Since 3e treatment caused morphological changes and cell death, we also tested the effects of this compound on apoptosis. Annexin V/PI staining and western blot was then conducted to test the effect of 3e on apoptosis. 3e treatment significantly increased the percentage of early apoptotic cells from 2.25% to 19.97% and late apoptotic cell from 0.78% to 27.92% (Fig. 3A). While apoptosis is the main form of

cell death, caspase activation is the final executive stage of apoptosis [26]. Treatment of HEL cells with 3e resulted in cleavage of caspase 3, caspase 9 and PARP along with decreased in expression of these proteins (Fig. 3B). Interestingly, 3e treatment caused no obvious change in caspase 8, suggesting this compound induced cell apoptosis independent of this caspase. Caspase activation causes a cascade of cell responses that lead to apoptosis. Activity of the intrinsic or extrinsic pathway normally leads to caspase activation. Cell death receptor pathway relies on the recruitment of ligands through death receptor to activate receptor proteins and downstream caspase-8 [27]. Our results indicated that 3e activated caspases and increased cleavage of caspase-9, caspase-3 and PARP, required for execution of apoptosis [28-31].

Fig. 3. Induction of apoptosis by 3e in HEL cells. (A) Apoptosis results was detected by flow cytometry. (B) Western blotting using the indicated proteins after treatment with 3e. Ratio of protein to β-actin shown in Figure 4B, right panel. p < 0.05**. Values represent the means ± SD. β-actin was used as loading control.

2.2.5. Inhibitory effect of 3e on the PI3K/AKT and MAPK Signaling Pathways Previous studies indicated that fangchinoline displayed anti-cancer activity by inhibiting the PI3K/AKT and MAPK signaling pathways in human malignant gliomas and other cancer cell lines [32]. We hypothesized that fangchinoline derivatives synthesized in this study might also exhibited anticancer activity via these signaling pathways, but stronger than parental compound. We then further evaluated the effects of 3e on the PI3K/AKT and MAPK signaling pathways by western blotting. As shown in Fig. 4, treatment with 3e decreased the levels of p-AKT and p-ERK, which are known mediators of cell proliferation and inhibition of apoptosis [33-36]. Furthermore, 3e significantly increased the expression of p-JNK. As higher expression of JNK is involved in apoptosis [37], our results suggested that 3e may block cell proliferation and induce apoptosis through suppression of the PI3K and MAPK activity and activation of JNK.

Fig. 4. The 3e compound suppressed the PI3K/AKT and MAPK signaling pathway in HEL cells. HEL cells were treated with 2.5 µM of 3e for 8 h. Total protein was collected from HEL cells and subjected to western blotting, as described [38]. (A) Western blotting of the indicated proteins in response to 3e and control DMSO treatment. (B) Ratio of protein to β-actin. p < 0.05**. Values represent the means ± SD. β-actin was used as loading control.

2.2.6. Akt kinase inhibition by 3e To further explore the mechanism of Akt inhibition by the compound, we performed the Akt kinase assay using 3e treated HEL cells. As shown in Table 2, 3e dose-dependently inhibited the Akt kinase activity with an IC50 values of 0.55 µM compared to the lead compound fangchinoline (IC50 > 20 µM). A molecular docking study was then performed to predict binding model of 3e and Akt. The data presented in Fig. 5 showed the difference in ligand-bound conformations within the Akt active

site between the cocrystal ligand G4K (Fig. 5D) and 3e (Fig. 5B). While both compounds formed similar van der waals interactions with the residuals including Glu17, Thr82, Ile84, Val271, Asp274 and Thr291, 3e formed other interactions such as amide-Pi stacking with Phe293 and Pi-Alkyl Tyr272 and Arg273. In comparison to fangchinoline, 3e formed three additional hydrogen bonds with Akt (Arg273 and Lys276) (Fig. 5B-C). These additional interactions likely make the interactions with Akt more stable, resulting in higher enzyme inhibition (Fig. 5B-5C). These data indicated that the structure modification of fangchinoline enhanced the Akt kinase inhibitory activity of fangchinoline.

Fig. 5. The predicted binding mode of 3e and Akt1. (A) The binding conformation of 3e (Gray) and fangchinoline (Yellow) in the active site of Akt1. (B) the 2D binding interaction of 3e and Akt1. (C) The 2D binding interaction of fangchinoline and Akt1. (D) The 2D binding interaction of the cocrystal ligand G4K and Akt1.

Table 2 Akt kinase inhibition by 3e and fangchinoline. Compounds

Akt kinase inhibition (IC50)

3e

0.55 (µM)

fangchinoline

>20 (µM)

3. Conclusion This study investigated the mechanism of anti-leukemic activity of 3e compound on human leukemia cells. Our results suggested that cell growth inhibition of HEL cells was associated with G0/G1 cell cycle arrest and induction of apoptosis via inhibition of the PI3K and MAPK pathway. Docking analysis indeed identified high affinity interactions between 3e and Akt resulting in kinase inactivation. However, future investigation may be needed to uncover the mechanism of MAPK/ERK inhibition by the compound. Downregulation of c-MYC by MAPK inhibition [39] may underlined cell cycle arrest by the compound, which may be mediated through upregulation of P21Cip1. Interestingly, induction of apoptosis by 3e appeared to be dependent of caspase 3/9, but independent of caspase 8, suggesting a death receptor independent mechanism of apoptosis, a notion that may need be investigated in future studies. Overall, these findings support the continued investigation on the use of fangchinoline derivatives as new anti-cancer agents.

4. Experimental section 4.1. Chemistry 4.1.1. General Fangchinoline was obtained with purity ≥ 98%. Reagents and solvents were purchased from Adamas, JK chemical and local commercial sources. The reagents and the solvents were purified according to the guidelines in Purification of Laboratory Chemicals. Column chromatography was performed on silica gel (Qingdao, 200-300 mesh) using the indicated eluents. Thin-layer (0.25 mm, GF254) chromatography was carried out on silica gel plates (Qingdao, China). 1H NMR spectra were recorded on 600 MHz (Bruker, USA) and 500 MHz (Wipm, China) and 400 MHz (Varian, USA) spectrometers in appropriate solvents using TMS as internal standard or the solvent signals as secondary standards and the chemical shifts are shown in δ scales. Multiplicities of NMR signals are designated as s (singlet), d (doublet), t (triplet), br (broad), and m (multiplet, for unresolved lines).

13

C NMR spectra were recorded on

150 MHz or 125 MHz or 100 MHz spectrometers. High-resolution mass spectra were obtained by using Bruker ESI-QTOF mass spectrometry. IR spectra were recorded by using FTIR Spectrometer (IR 200) and the KBr disk method was adopted. Melting point (uncorrected) were determined on WRX-4 Micro Melting Point Apparatus. 4.1.2. General produce for the synthesis of 1a to 4h Acyl chloride (0.18 mmol, 1.1 eq) was added at 0 °C to a solution of fangchinoline (100 mg, 0.16 mmol) and DMAP (0.032 mmol, 0.2 eq) in 2 mL dry CH2Cl2 under argon and stirred for 2-4 h. The reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate and extracted three times with CH2Cl2. The

combined organic phase was dried over anhydrous magnesium sulfate before vacuum suction filtration. The removal of the solvent in vacuo afforded the crude product, which was chromatographied on silica gel (CH2Cl2/MeOH, 50/1 v/v, 0.1% TEA) to provide the pure product 1a-1e, 2a-2g, 3a-3e and 4a-4h.

4.1.2.1. 7-O-(2-Chloroacetate)-fangchinoline (1a). White amorphous solid (Yield 89%), mp: 162-164 °C. IR (KBr, cm–1): 2933, 2841, 2794, 1786, 1611, 1508, 1445, 1360, 1269, 1119. 1H NMR (400 MHz, CDCl3) δ 7.40 - 7.34 (m, 1H), 7.08 (dd, J = 2.0, 2.0 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.83 - 6.77 (m, 1H), 6.55 (s, 1H), 6.40 (s, 2H), 6.27 (d, J = 8.0 Hz, 1H), 5.96 (s, 1H), 4.30 (t, J = 7.2 Hz, 2H), 4.23 (s, 1H), 3.88 (s, 3H), 3.68 (s, 3H), 3.53 (t, J = 6.4 Hz, 2H), 3.43 (s, 3H), 3.05 (d, J = 9.2 Hz, 5H), 2.84 (s, 3H), 2.71 (d, J = 14.8 Hz, 1H), 2.54 (s, 3H), 2.45 (t, J = 8.2 Hz, 2H), 2.26 2.18 (m, 2H), 2.03 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 177.7, 154.0, 150.7, 149.5, 149.3, 147.8, 146.2, 142.8, 132.3, 130.5, 126.8, 123.7, 122.2, 115.1, 112.2, 111.7, 105.7, 68.4, 63.6, 56.0, 55.9, 55.6, 43.8, 43.1, 41.1, 30.0, 29.5, 27.7, 27.3, 27.2, 23.6, 22.0. HRMS (ESI-QTOF) m/z Calcd. for C39H41ClN2O7+ [M+H]+: 685.2675 , found: 685.2684. 4.1.2.2. 7-O-(3-Chloropropanoate)-fangchinoline (1b). Light yellow amorphous solid (Yield 83%), mp: 165-167°C. IR (KBr, cm–1): 3006, 2934, 2837, 2798, 1747, 1616, 1509, 1445, 1363, 1329, 1127. 1H NMR (500 MHz, CDCl3) δ 7.41 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.14 - 7.11 (m, 1H), 6.92 - 6.83 (m, 2H), 6.58 (d, J = 11.5 Hz, 1H), 6.45 (t, J = 7.0 Hz, 2H), 6.33 - 6.25 (m, 2H), 6.20 - 6.12 (m, 1H), 5.95 (s, 1H), 5.76 - 5.70 (m, 2H), 5.31 (s, 1H), 4.20 - 4.16 (m, 2H), 3.93 (s, 3H), 3.85 (d, J = 13.5 Hz, 1H),

3.73 (s, 3H), 3.49 (s, 3H), 3.30 - 2.99 (m, 9H), 2.91 (s, 1H), 2.85 (d, J = 12.0 Hz, 1H), 2.81 (s, 2H), 2.75 (d, J = 15.0 Hz, 1H), 2.58 (s, 3H), 1.40 (t, J = 7.5 Hz, 2H).

13

C

NMR (125 MHz, CDCl3) δ 170.5, 168.5, 154.1, 150.9, 149.3, 147.9, 146.4, 143.0, 132.4, 132.1, 131.6, 130.6, 128.2, 126.6, 126.3, 122.3, 122.2, 122.1, 117.1, 105.8, 63.8, 56.0, 55.9, 55.6, 45.7, 44.4, 42.9, 41.0, 29.6, 23.9, 22.7, 8.5. HRMS (ESI-QTOF) m/z Calcd. for C40H43ClN2O7+ [M+H]+: 699.2832, found: 699.2837. 4.1.2.3. 7-O-(4-Chlorobutanoate)-fangchinoline (1c). Light yellow amorphous solid (Yield 76%), mp: 170-173°C. IR (KBr, cm–1): 3032, 2932, 2840, 2800, 1763, 1606, 1465, 1417, 1357, 1268, 1122. 1H NMR (400 MHz, CDCl3) δ 7.46 - 7.38 (m, 1H), 7.12 (dd, J = 2.0, 2.0 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 - 6.81 (m, 1H), 6.60 (s, 1H), 6.44 (d, J = 3.2 Hz, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.00 (s, 1H), 4.35 (t, J = 7.0 Hz, 2H), 4.27 (s, 1H), 3.92 (s, 3H), 3.72 (s, 3H), 3.57 (t, J = 6.2 Hz, 2H), 3.47 (s, 3H), 3.31 - 2.98 (m, 7H), 2.88 (s, 3H), 2.76 (d, J = 15.2 Hz, 1H), 2.58 (s, 3H), 2.49 (t, J = 8.2 Hz, 2H), 2.27 (dd, J = 15.2, 7.6 Hz, 2H), 2.07 (s, 2H), 1.96 - 1.84 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 177.7, 169.0, 154.0, 150.7, 149.5, 149.3, 146.2, 142.8, 132.3, 130.5, 126.8, 123.6, 122.2, 120.1, 115.1, 112.2, 111.7, 105.7, 68.4, 63.6, 56.0, 55.9, 55.6, 43.8, 43.1, 41.4, 29.9, 29.5, 27.7, 27.3, 27.2, 23.6, 22.0. HRMS (ESI-QTOF) m/z Calcd. for C41H46ClN2O7+ [M+H]+: 713.2988, found: 713.2971. 4.1.2.4. 7-O-(2-Bromoacetate)-fangchinoline (1d). Yellow amorphous solid (Yield 90%), mp: 160-163 °C. IR (KBr, cm–1): 3028, 2934, 2839, 2798, 1786, 1611, 1584, 1466, 1445, 1360, 1270, 1121. 1H NMR (500 MHz, CDCl3) δ 7.34 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 8.0, 2.5 Hz, 1H), 6.90 - 6.85 (m, 2H), 6.80 (dd, J = 8.0, 2.5 Hz, 1H),

6.53 (s, 1H), 6.48 (s, 1H), 6.35 (s, 1H), 6.25 (d, J = 8.0 Hz, 1H), 5.97 (s, 1H), 3.93 (s, 3H), 3.85 (dd, J = 10.5, 5.5 Hz, 1H), 3.75 (d, J = 10.0 Hz, 1H), 3.72 (s, 3H), 3.55 (dd, J = 13.0, 8.5 Hz, 2H), 3.41 (s, 3H), 3.28 (dd, J = 12.0, 5.5 Hz, 2H), 3.00 - 2.85 (m, 5H), 2.82 - 2.67 (m, 4H), 2.64 (s, 3H), 2.49 (d, J = 13.5 Hz, 2H), 2.34 (s, 3H).

13

C

NMR (125 MHz, CDCl3) δ 153.7, 149.6, 149.3, 148.5, 147.0, 146.7, 142.3, 134.8, 134.7, 132.5, 132.5, 131.4, 130.2, 129.3, 122.7, 122.1, 122.0, 116.1, 112.4, 111.4, 105.7, 66.0, 63.8, 61.1, 56.1, 56.1, 55.9, 55.4, 53.4, 49.0, 45.5, 44.7, 43.8, 42.6, 42.5, 42.2, 41.5, 40.1, 39.7, 34.6, 29.7, 27.5, 25.6, 25.0, 22.1. HRMS (ESI-QTOF) m/z Calcd. for C39H42BrN2O7+ [M+H]+: 753.1876, found: 753.1884. 4.1.2.5. 7-O-(2,2,2-Trichloroacetate)-fangchinoline (1e). White amorphous solid (Yield 80%), mp: 173-176 °C. IR (KBr, cm–1): 3032, 2932, 2868, 2839, 2796, 1765, 1611, 1466, 1445, 1360, 1270, 1125. 1H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.31 (dd, J = 8.0, 2.0 Hz, 1H), 7.21 (dd, J = 8.0, 2.4 Hz, 1H), 6.65 (s, 1H), 6.59 (dd, J = 8.4, 2.4 Hz, 1H), 6.48 (s, 1H), 6.29 (s, 1H), 6.14 (dd, J = 8.4, 2.0 Hz, 1H), 5.94 (s, 1H), 3.96 (s, 3H), 3.77 (s, 3H), 3.48 (tdd, J = 16.0, 11.2, 6.4 Hz, 2H), 3.34 (s, 3H), 3.14 2.71 (m, 9H), 2.68 (s, 3H), 2.49 (t, J = 12.0 Hz, 3H), 2.41 (s, 3H).

13

C NMR (100

MHz, CDCl3) δ 160.8, 155.8, 149.3, 148.2, 146.2, 146.1, 143.9, 142.4, 134.9, 132.8, 130.7, 129.8, 126.7, 122.0, 121.9, 121.0, 120.5, 112.3, 107.4, 104.7, 94.0, 63.9, 61.7, 56.3, 55.8, 45.5, 45.0, 42.2, 41.8, 40.4, 37.5, 24.0, 21.5. HRMS (ESI-QTOF) m/z Calcd. for C39H39Cl3N2O7+ [M+H]+: 753.1831, found: 753.1823. 4.1.2.6. 7-O-(Trimethylacetyl)-fangchinoline (2a). Light yellow amorphous solid (Yield 79%), mp: 166-169 °C. IR (KBr, cm–1): 2929, 2851, 2800, 1766, 1610, 1507,

1445, 1360, 1270, 1124. 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H), 6.84 (s, 3H), 6.35 (s, 2H), 3.92 (s, 3H), 3.69 (s, 3H), 3.56 (s, 2H), 3.39 (s, 3H), 3.00 - 2.87 (m, 4H), 2.81 - 2.70 (m, 3H), 2.59 (s, 5H), 2.31 (s, 3H), 1.22 (s, 6H), 0.93 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 163.7, 153.7, 149.5, 149.3, 146.9, 146.7, 142.2, 134.8, 134.6, 132.5, 131.5, 130.2, 129.5, 122.7, 122.0, 121.9, 116.1, 111.4, 105.6, 63.9, 61.1, 56.1, 55.9, 45.6, 42.6, 42.2, 40.1, 25.0, 22.0. HRMS (ESI-QTOF) m/z Calcd. for C42H49N2O7+ [M+H]+: 693.3534, found: 693.3514. 4.1.2.7. 7-O-(3-Methylcrotonoyl)-fangchinoline (2b). Light yellow amorphous solid (Yield 83%), mp: 161-164°C. IR (KBr, cm–1): 3028, 2999, 2932, 2835, 2798, 1745, 1610, 1591, 1461, 1446, 1360, 1271, 1123, 970. 1H NMR (600 MHz, CDCl3) δ 7.30 (dd, J = 8.2, 2.1 Hz, 1H), 7.11 (dd, J = 8.4, 2.4 Hz, 1H), 6.89 – 6.84 (m, 2H), 6.77 (dd, J = 8.2, 2.4 Hz, 1H), 6.49 (d, J = 13.8 Hz, 2H), 6.34 (s, 1H), 6.23 (d, J = 7.8 Hz, 1H), 5.89 (s, 1H), 5.17 (s, 1H), 3.92 (s, 3H), 3.75 (d, J = 9.6 Hz, 1H), 3.70 (s, 3H), 3.58 – 3.42 (m, 3H), 3.40 (s, 3H), 3.21 (dd, J = 12.6, 6.0 Hz, 1H), 2.98 – 2.66 (m, 8H), 2.55 (s, 1H), 2.54 (s, 3H), 2.44 (dd, J = 15.6, 4.8 Hz, 1H), 2.34 (s, 3H), 2.02 (s, 3H), 1.80 – 1.76 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 153.7, 150.0, 149.3, 148.6, 147.5, 146.9, 142.8, 135.0, 132.5, 130.6, 130.1, 128.7, 122.7, 121.9, 121.8, 120.8, 116.2, 114.4, 112.3, 111.4, 105.4, 64.1, 61.1, 56.1, 55.9, 45.5, 42.5, 42.2, 27.4, 24.7, 22.0, 20.2. HRMS (ESI-QTOF) m/z Calcd. for C42H46N2O7+ [M+H]+: 691.3383, found: 691.3370. 4.1.2.8. 7-O-Acryloyl-fangchinoline (2c). White amorphous solid (Yield 73%), mp: 154-159°C. IR (KBr, cm–1): 3028, 2999, 2932, 2835, 2798, 1745, 1610, 1591, 1461,

1446, 1360, 1271, 1123, 970. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.30 - 7.20 (m, 2H), 6.62 (s, 1H), 6.56 (dd, J = 8.4, 2.4 Hz, 1H), 6.51 - 6.40 (m, 2H), 6.30 (s, 1H), 6.19 (dd, J = 16.8, 10.4 Hz, 1H), 6.12 (dd, J = 8.4, 2.0 Hz, 1H), 5.93 (s, 1H), 5.72 (dd, J = 10.2, 1.4 Hz, 1H), 4.02 (d, J = 9.0 Hz, 1H), 3.95 (s, 3H), 3.86 (dd, J = 11.0, 5.4 Hz, 1H), 3.76 (d, J = 7.6 Hz, 3H), 3.66 - 3.57 (m, 1H), 3.39 (s, 1H), 3.34 (s, 2H), 3.26 (dd, J = 12.2, 5.6 Hz, 1H), 3.07 - 2.85 (m, 5H), 2.80 - 2.67 (m, 2H), 2.63 (s, 3H), 2.51 (s, 3H), 2.46 (d, J = 14.8 Hz, 2H), 2.01 (s, 1H), 1.84 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 163.7, 156.4, 149.1, 148.4, 146.4, 144.8, 143.7, 142.6, 135.0, 133.3, 132.8, 132.7, 132.0, 128.3, 127.5, 126.4, 125.2, 122.2, 121.8, 121.6, 120.8, 120.5, 112.2, 106.2, 104.8, 61.1, 56.1, 56.0, 55.6, 44.9, 43.5, 42.2, 40.6, 25.1, 25.0, 20.3, 17.8. HRMS (ESI-QTOF) m/z Calcd. for C40H43N2O7+ [M+H]+: 663.3065, found: 663.3042. 4.1.2.9. 7-O-Cinnamoyl-fangchinoline (2d). Light yellow amorphous solid (Yield 87%), mp: 169-174°C. IR (KBr, cm–1): 3031, 2933, 2837, 2796, 1609, 1572, 1464, 1444, 1362, 1269, 1242, 1115. 1H NMR (500 MHz, CDCl3) δ 7.46 (s, 1H), 7.37 (d, J = 4.5 Hz, 4H), 7.28 (d, J = 4.5 Hz, 3H), 7.20 (dd, J = 8.0, 2.0 Hz, 1H), 6.60 (dd, J = 8.5, 2.0 Hz, 2H), 6.46 (s, 1H), 6.27 (s, 1H), 6.17 (dd, J = 8.5, 1.5 Hz, 1H), 5.94 (s, 1H), 4.62 (dd, J = 15.0, 6.0 Hz, 1H), 4.45 (dd, J = 15.0, 6.0 Hz, 1H), 3.95 (d, J = 9.5 Hz, 1H), 3.89 (s, 3H), 3.87 (dd, J = 12.0, 6.0 Hz, 2H), 3.76 (s, 3H), 3.68 – 3.59 (m, 1H), 3.40 - 3.35 (m, 1H), 3.33 (s, 3H), 3.25 (dd, J = 12.5, 5.0 Hz, 1H), 3.04 – 2.83 (m, 6H), 2.74 - 2.69 (m, 2H), 2.61 (s, 3H), 2.44 (s, 3H).

13

C NMR (125 MHz, CDCl3) δ

156.1, 148.8, 148.7, 146.4, 145.8, 143.4, 141.2, 132.8, 132.1, 131.8, 128.7, 126.8, 126.5, 121.4, 120.8, 120.7, 112.2, 17.3, 104.7, 63.7, 61.3, 56.2, 56.0, 55.6, 44.9, 42.4,

42.4, 40.0, 39.2, 37.4, 25.3, 20.2. HRMS (ESI-QTOF) m/z Calcd. for C46H47N2O7+ [M+H]+: 739.3378, found: 739.3356. 4.1.2.10. 7-O-(Undec-10-enoly)-fangchinoline (2e). Light yellow amorphous solid (Yield 70%), mp: 182-187°C. IR (KBr, cm–1): 2929, 2851, 2800, 1766, 1610, 1507, 1445, 1360, 1270, 1124. 1H NMR (400 MHz, CDCl3) δ 7.33 (dd, J = 8.2, 2.0 Hz, 1H), 7.13 (dd, J = 8.0, 2.4 Hz, 1H), 6.86 (t, J = 7.0 Hz, 2H), 6.79 (dd, J = 8.2, 2.4 Hz, 1H), 6.50 (d, J = 10.4 Hz, 2H), 6.34 (s, 1H), 6.26 (d, J = 7.2 Hz, 1H), 5.94 (s, 1H), 5.85 5.75 (m, 1H), 3.78 - 3.73 (m, 2H), 3.92 (s, 3H), 3.80 – 3.73 (m, 2H), 3.70 (s, 3H), 3.59 - 3.44 (m, 2H), 3.40 (s, 3H), 3.25 (dd, J = 12.4, 5.6 Hz, 1H), 2.95 - 2.69 (m, 7H), 2.58 (s, 3H), 2.53 -2.44 (m, 2H), 2.34 (s, 3H), 2.04 (q, J = 7.2 Hz, 2H), 1.78 (s, 1H), 1.42 - 1.33 (m, 4H), 1.29 - 1.19 (m, 9H). 13C NMR (100 MHz, CDCl3) δ 153.7, 151.5, 149.5, 148.6, 148.5, 147.0, 143.8, 136.4, 135.1, 134.7, 134.0, 132.7, 130.2, 128.3, 128.1, 127.5, 122.9, 122.8, 122.0, 121.9, 120.4, 116.9, 116.0, 112.7, 111.4, 105.7, 73.5, 64.3, 61.3, 56.1, 55.8, 55.7, 45.5, 44.1, 42.6, 42.3, 41.9, 40.8, 24.2, 22.1. HRMS (ESI-QTOF) m/z Calcd. for C48H59N2O7+ [M+H]+: 775.2370, found: 775.2371. 4.1.2.11. 7-O-Hexanoyl-fangchinoline (2f). White amorphous solid (Yield 81%), mp: 176-179°C. IR (KBr, cm–1): 3032, 2932, 2868, 2839, 2796, 1765, 1611, 1466, 1445, 1360, 1270, 1125. 1H NMR (400 MHz, CDCl3) δ 7.37 (dd, J = 8.2, 2.0 Hz, 1H), 7.17 (dd, J = 8.0, 2.4 Hz, 1H), 6.90 (dd, J = 7.8, 4.8 Hz, 2H), 6.83 (dd, J = 8.2, 2.4 Hz, 1H), 6.54 (d, J = 10.0 Hz, 2H), 6.38 (s, 1H), 6.30 (dd, J = 8.4, 2.0 Hz, 1H), 5.98 (s, 1H), 5.33 (s, 1H), 3.96 (s, 3H), 3.80 (dd, J = 16.8, 7.6 Hz, 2H), 3.74 (s, 3H), 3.63 - 3.47 (m, 2H), 3.44 (s, 3H), 3.28 (dd, J = 12.4, 5.6 Hz, 1H), 3.00 - 2.72 (m, 7H), 2.63 (s, 3H),

2.55 - 2.50 (m, 2H), 2.38 (s, 3H), 1.44 (dd, J = 14.4, 7.1 Hz, 2H), 1.39 - 1.15 (m, 5H), 0.92 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, CDCl3) δ 153.7, 149,8, 149.3, 148.6,

147.2, 146.9, 142.7, 135.0, 134.8, 132.5, 130.7, 130.2, 128.9, 128.0, 122.7, 122.0, 116.2, 112.5, 111.4, 105.5, 64.1, 61.2, 56.1, 55.9, 55.6, 53.4, 45.6, 43.8, 42.7, 42.2, 41.6, 39.8, 32.9, 31.1, 25.0, 24.2, 22.4, 14.0. HRMS (ESI-QTOF) m/z Calcd. for C43H51N2O7+ [M+H]+: 707.3687, found: 707.3618. 4.1.2.12. 7-O-Cyclopropyl-fangchinoline (2g). White amorphous solid (Yield 85%), mp: 168-171°C. IR (KBr, cm–1): 3028, 2997, 2932, 2835, 2796, 1738, 1611, 1585, 1462, 1445, 1360, 1228, 1114. 1H NMR (500 MHz, CDCl3) δ 7.34 (dd, J = 8.0, 2.0 Hz, 1H), 7.13 (dd, J = 8.0, 2.5 Hz, 1H), 6.91 - 6.79 (m, 3H), 6.57 (d, J = 2.0 Hz, 1H), 6.52 (s, 1H), 6.34 - 6.28 (m, 2H), 6.05 (s, 1H), 3.92 (s, 3H), 3.91 - 3.87 (m, 1H), 3.80 (d, J = 10.0 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 1H), 3.55 - 3.49 (m, 1H), 3.45 (s, 2H), 3.40 (s, 2H), 3.35 (s, 2H), 3.27 (dd, J = 12.5, 5.5 Hz, 1H), 3.00 - 2.70 (m, 9H), 2.63 (s, 3H), 2.57 (d, J = 13.5 Hz, 1H), 2.46 (d, J = 16.5 Hz, 1H), 2.35 (s, 3H).

13

C NMR (125

MHz, CDCl3) δ 153.7, 149.8, 149.3, 148.4, 147.3, 146.9, 142.8, 135.0, 134.9, 132.5, 130.1, 128.8, 122.6, 121.9, 120.5, 116.2, 111.4, 105.6, 63.9, 61.2, 56.1, 55.9, 45.7, 43.8, 42.7, 42.2, 38.7, 25.7, 22.0, 12.0, 8.8, 8.7. HRMS (ESI-QTOF) m/z Calcd. for C41H45N2O7+ [M+H]+: 677.3221, found: 677.3201. 4.1.2.13. 7-O-(Ethyl(methyl)carbamyl)-fangchinoline (3a). White amorphous solid (Yield 74%), mp: 154 -157°C. IR (KBr, cm–1): 3031, 2997, 2933, 2837, 2798, 1731, 1612, 1581, 1469, 1446, 1361, 1231, 1125. 1H NMR (400 MHz, CDCl3) δ 7.33 (dd, J = 8.0, 2.0 Hz, 1H), 7.17 (dd, J = 8.0, 2.4 Hz, 1H), 6.84 (s, 1H), 6.68 (dd, J = 8.4, 2.4

Hz, 1H), 6.63 (s, 1H), 6.50 (s, 1H), 6.30 (s, 1H), 6.21 (dd, J = 8.4, 2.0 Hz, 1H), 5.99 (s, 1H), 3.88 (s, 3H), 3.77 (s, 3H), 3.34 (s, 3H), 3.28 (dd, J = 12.6, 5.4 Hz, 3H), 3.09 2.84 (m, 7H), 2.82 - 2.71 (m, 3H), 2.63 (s, 3H), 2.47 (s, 3H), 2.42 (s, 1H), 1.36 (t, J = 7.2 Hz, 1H), 1.29 (d, J = 6.4 Hz, 3H), 1.19 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 178.7, 154.5, 149.0, 148.0, 147.0, 146.1, 143.7, 142.1, 134.6, 134.5, 133.0, 130.4, 129.9, 129.3, 128.1, 127.6, 121.3, 121.1, 121.0, 120.3, 112.5, 108.0, 104.9, 63.7, 56.2, 56.0, 55.8, 46.8, 44.9, 42.2, 40.9, 25.1, 22.6, 22.5, 20.4. HRMS (ESI-QTOF) m/z Calcd. for C41H48N3O7+ [M+H]+: 694.3487, found: 694.3491. 4.1.2.14. 7-O-Diethylcarbamyl-fangchinoline (3b). Light yellow amorphous solid (Yield 77%), mp: 161-163°C. IR (KBr, cm–1): 3031, 2941, 2931, 2837, 2796, 1614, 1583, 1462, 1447, 1321, 1270, 1125. 1H NMR (400 MHz, CDCl3) δ 7.32 (dd, J = 8.2, 2.0 Hz, 1H), 7.12 (dd, J = 8.2, 2.4 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.79 (dd, J = 8.2, 2.4 Hz, 1H), 6.50 (s, 2H), 6.36 (d, J = 5.0 Hz, 1H), 6.24 (dd, J = 8.2, 2.0 Hz, 1H), 5.90 (s, 1H), 5.18 (s, 1H), 3.92 (s, 3H), 3.84 (d, J = 9.6 Hz, 1H), 3.75 (dd, J = 10.8, 5.6 Hz, 1H), 3.70 (d, J = 3.6 Hz, 3H), 3.60 - 3.51 (m, 2H), 3.45 (d, J = 6.4 Hz, 3H), 3.42 (s, 2H), 3.32 (dd, J = 12.4, 5.6 Hz, 1H), 3.11 - 2.66 (m, 9H), 2.61 (d, J = 6.8 Hz, 1H), 2.57 (s, 3H), 2.38 (s, 3H), 2.03 (s, 3H), 1.79 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 153.7, 150.1, 149.2, 148.7, 147.3, 147.0, 142.8, 134.4, 134.2, 130.1, 128.1, 122.9, 121.9, 121.8, 120.8, 116.0, 114.7, 112.2, 111.4, 105.4, 64.0, 61.4, 56.0, 55.9, 50.6, 45.1, 42.2, 42.0, 41.4, 40.0, 27.4, 24.1, 22.3, 22,0, 20.2. HRMS (ESI-QTOF) m/z Calcd. for C42H50N3O7+ [M+H]+: 708.3649, found: 708.3615.

4.1.2.15.

7-O-(Phenothiazine-10-carboxyl)-fangchinoline

(3c).

Light

yellow

amorphous solid (Yield 72%), mp: 172-175°C. IR (KBr, cm–1): 3028, 2997, 2932, 2835, 2796, 1738, 1611, 1585, 1462, 1445, 1360, 1228, 1114. 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 1.6 Hz, 1H), 7.88 – 7.80 (m, 3H), 7.76 – 7.70 (m, 1H), 7.55 (tt, J = 7.1, 5.3 Hz, 2H), 7.28 (s, 1H), 7.23 (dd, J = 8.3, 2.1 Hz, 1H), 7.14 (dd, J = 8.1, 2.5 Hz, 1H), 6.52 (dd, J = 8.4, 2.6 Hz, 1H), 6.44 (s, 1H), 6.40 (s, 1H), 6.25 (s, 1H), 6.10 (dd, J = 8.4, 2.2 Hz, 1H), 5.86 (s, 1H), 3.91 (s, 3H), 3.87 (d, J = 9.4 Hz, 1H), 3.79 (dd, J = 11.1, 5.4 Hz, 1H), 3.73 (s, 3H), 3.47 – 3.33 (m, 2H), 3.31 (s, 3H), 3.18 (dd, J = 12.6, 5.2 Hz, 2H), 3.03 – 2.80 (m, 4H), 2.74 – 2.66 (m, 2H), 2.58 (s, 3H), 2.53 (s, 3H), 2.45 (ddd, J = 16.9, 14.6, 7.1 Hz, 3H), 2.17 – 2.12 (m, 1H).

13

C NMR (100 MHz,

CDCl3) δ 156.1, 148.9, 148.8, 146.4, 145.7, 143.4, 142.3, 138.4, 134.8, 134.5, 133.8, 132.8, 132.0, 132.0, 129.4, 129.1, 129.0, 128.5, 128.3, 127.8, 127.5, 127.2, 126.7, 122.3, 122.2, 121.4, 120.8, 120.8, 120.7, 112.2, 106.9, 104.7, 77.3, 77.0, 76.7, 63.8, 61.3, 56.2, 56.0, 55.7, 44.9, 42.5, 42.4, 40.0, 39.3, 37.4, 25.3, 20.3. HRMS (ESI-QTOF) m/z Calcd. for C50H48N3O7S+ [M+H]+: 834.3189, found: 834.3197. 4.1.2.16. 7-O-(1,3-Oxazinane-3-carboxyl)-fangchinoline (3d). White amorphous solid (Yield 82%), mp: 167-171°C. IR (KBr, cm–1): 3028, 2931, 2848, 2796, 2773, 1729, 1612, 1586, 1466, 1444, 1362, 1225, 1116, 1070, 1024; 1H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.29 - 7.25 (m, 1H), 7.19 (dd, J = 8.0, 2.4 Hz, 1H), 6.57 (dd, J = 8.0, 2.8 Hz, 2H), 6.46 (s, 1H), 6.29 (s, 1H), 6.14 (dd, J = 8.4, 2.0 Hz, 1H), 5.92 (s, 1H), 4.30 (d, J = 6.8 Hz, 1H), 4.21 - 4.10 (m, 1H), 3.92 (s, 3H), 3.82 (dd, J = 11.0, 5.2 Hz, 1H), 3.76 (s, 3H), 3.66 (dd, J = 15.2, 5.2 Hz, 1H), 3.33 (s, 3H), 3.19 (dd, J = 12.4, 5.2

Hz, 1H), 3.02 - 2.80 (m, 6H), 2.73 - 2.67 (m, 2H), 2.59 (s, 3H), 2.48 (s, 3H), 2.42 (d, J = 14.4 Hz, 2H), 2.04 (s, 3H), 1.74 - 1.56 (m, 5H), 1.47 - 1.35 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 156.3, 156.2, 149.0, 148.6, 146.4, 144.3, 143.6, 142.6, 135.0, 133.8, 133.7, 132.8, 129.5, 128.6, 128.3, 125.4, 122.3, 122.0, 121.7, 121.4, 120.8, 120.6, 112.4, 105.9, 104.9, 63.9, 61.3, 56.2, 56.0, 55.7, 51.9, 45.0, 43.5, 42.4, 40.9, 40.0, 37.4, 33.8, 33.7, 25.4, 23.6, 20.4. HRMS (ESI-QTOF) m/z Calcd. for C42H48N3O8+ [M+H]+: 722.3436, found: 722.3417. 4.1.2.17. 7-O-(Tetrahydrothiophene-2-carboxyl)-fangchinoline (3e). Light yellow amorphous solid (Yield 83%), mp: 158-164°C. IR (KBr, cm–1): 3032, 2932, 2837, 2794, 1737, 1611, 1445, 1415, 1360, 1272, 1122. 1H NMR (400 MHz, CDCl3) δ 7.34 (dd, J = 8.2, 2.0 Hz, 1H), 7.13 (dd, J = 8.2, 2.4 Hz, 1H), 6.90 - 6.85 (m, 2H), 6.80 (dd, J = 8.0, 2.4 Hz, 1H), 6.51 (d, J = 20.0 Hz, 2H), 6.35 (s, 1H), 6.29 - 6.23 (m, 1H), 5.96 (s, 1H), 3.93 (s, 3H), 3.78 (dd, J = 11.6, 6.0 Hz, 1H), 3.71 (s, 3H), 3.60 - 3.51 (m, 1H), 3.42 (s, 3H), 3.25 (dd, J = 12.4, 5.6 Hz, 1H), 2.96 - 2.69 (m, 7H), 2.58 (s, 3H), 2.53 2.43 (m, 2H), 2.34 (s, 3H), 2.03 (s, 2H), 1.60 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 153.7, 149.7, 149.3, 148.6, 142.5, 134.9, 134.8, 132.5, 130.0, 130.2, 129.0, 128.0, 122.7, 122.0, 120.5, 116.1, 112.4, 111.4, 105,5, 64.0, 61.1, 56.1, 55.9, 45.5, 43.8, 42.7, 42.2, 39.8, 24.8, 22.0, 19.7. HRMS (ESI-QTOF) m/z Calcd. for C42H47N2O7S+ [M+H]+: 723.3098, found: 723.3086. 4.1.2.18. 7-O-(2-Chlorobenzoyl)-fangchinoline (4a). White amorphous solid (Yield 88%), mp: 172-175°C. IR (KBr, cm–1): 3028, 2931, 2839, 2798, 1737, 1626, 1464, 1446, 1360, 1270, 1232, 1125. 1H NMR (600 MHz, CDCl3) δ 7.48 - 7.44 (m, 3H),

7.41 – 7.36 (m, 3H), 7.30 - 7.28 (m, 1H), 7.13 (dd, J = 8.4, 2.4 Hz, 1H), 6.92 (dd, J = 8.4, 1.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.79 (dd, J = 8.4, 2.4 Hz, 1H), 6.53 (s, 2H), 6.40 (s, 1H), 6.24 (d, J = 7.2 Hz, 1H), 5.99 (d, J = 15.6 Hz, 1H), 3.95 (s, 3H), 3.80 (d, J = 9.6 Hz, 1H), 3.75 (s, 3H), 3.61 – 3.55 (m, 2H), 3.47 (s, 3H), 3.30 (ddd, J = 12.6, 9.6, 5.4 Hz, 1H), 3.14 (dd, J = 12.6, 5.4 Hz, 1H), 3.02 - 2.95 (m, 2H), 2.82 - 2.45 (m, 9H), 2.39 (s, 3H), 2.07 (s, 3H).

13

C NMR (150 MHz, CDCl3) δ 153.6, 149.9, 149.3,

147.4, 146.9, 142.7, 135.0, 134.9, 132.2, 132.5, 130.4, 130.1, 129.1, 128.7, 128.2, 122.7, 121.9, 116.2, 111.4, 105.5, 63.8, 61.2, 56.1, 55.9, 45.4, 43.8, 42.2, 42.1, 40.0, 24.9, 22.0. HRMS (ESI-QTOF) m/z Calcd. for C44H44ClN2O7+ [M+H]+: 747.2832, found: 747.2814. 4.1.2.19. 7-O-(3-Chlorobenzoyl)-fangchinoline (4b). White amorphous solid (Yield 85%), mp: 175-177°C. IR (KBr, cm–1): 3014, 2931, 2871, 2802, 2773, 1746, 1612, 1584, 1460, 1444, 1361, 1227, 1121. 1H NMR (400 MHz, CDCl3) δ 7.53 (t, J = 2.0 Hz, 1H), 7.44 (s, 1H), 7.29 (d, J = 8.0 Hz, 2H), 7.22 - 7.17 (m, 2H), 7.01 (d, J = 8.0 Hz, 1H), 6.63 – 6.59 (m, 2H), 6.47 (s, 1H), 6.29 (s, 1H), 6.17 (dd, J = 8.4, 2.0 Hz, 1H), 5.95 (s, 1H), 3.98 (d, J = 9.2 Hz, 1H), 3.94 (s, 3H), 3.87 (t, J = 5.6 Hz, 1H), 3.76 (s, 3H), 3.71 - 3.62 (m, 1H), 3.34 (s, 3H), 3.24 (dd, J = 12.4, 5.6 Hz, 1H), 3.06 - 2.85 (m, 6H), 2.79 – 2.68 (m, 3H), 2.62 (s, 3H), 2.51 (s, 3H), 2.46 (s, 1H), 2.04 (d, J = 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 155.9, 153.4, 149.0, 148.6, 146.4, 143.5, 142.4, 140.2, 135.0, 134.6, 133.9, 132.8, 132.3, 130.0, 129.8, 129.6, 128.5, 128.0, 122.7, 122.6, 122.0, 121.5, 120.9, 120.7, 119.1, 118.8, 117.1, 116.8, 112.3, 106.7, 104.8,

63.7, 61.5, 56.2, 56.0, 55.7, 44.9, 43.5, 42.3, 40.8, 39.6, 25.3, 20.3. HRMS (ESI-QTOF) m/z Calcd. for C44H44ClN2O7+ [M+H]+: 747.2814, found: 747.2832. 4.1.2.20. 7-O-(4-Chlorobenzoyl)-fangchinoline (4c). White amorphous solid (Yield 90%), mp: 171-174°C. IR (KBr, cm–1): 3033, 2997, 2932, 2839, 2798, 1758, 1611, 1444, 1436, 1360, 1271,1124. 1H NMR (400 MHz, CDCl3) δ7.31 (dd, J = 8.4, 2.0 Hz, 1H), 7.12 (dd, J = 8.2, 2.2 Hz, 1H), 6.88 (dd, J = 8.4, 1.6 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.78 (dd, J = 8.4, 2.4 Hz, 1H), 6.49 (s, 2H), 6.38 (d, J = 10.8 Hz, 1H), 6.24 (dd, J = 8.4, 2.0 Hz, 1H), 6.12 – 6.05 (m, 1H), 5.88 (s, 1H), 5.68 – 5.65 (m, 1H), 5.29 (s, 1H), 3.92 (s, 3H), 3.77 – 3.74 (m, 1H), 3.71 (s, 3H), 3.63 – 3.43 (m, 3H), 3.41 (s, 3H), 3.23 (dd, J = 12.6, 5.8 Hz, 1H), 3.00 – 2.60 (m, 8H), 2.55 (s, 3H), 2,52 – 2.42 (m, 1H), 2.35 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 153.7, 149.7, 149.2, 148.4, 147.2, 146.9, 142.7, 134.9, 134.8, 132.5, 130.2, 128.9, 126.8, 122.7, 122.0, 121.9, 116.1, 112.4, 111.4, 105.5, 63.9, 61.2, 56.1, 56.0, 55.9, 55.7, 45.5, 43.8, 42.6, 42.2, 41.6, 40.6, 24.4, 22.0. HRMS (ESI-QTOF) m/z Calcd. for C44H44ClN2O7+ [M+H]+: 747.2814, found: 747.2837. 4.1.2.21. 7-O-(2,4-Dichlorobenzoyl)-fangchinoline (4d). Light yelow amorphous solid (Yield 90%) mp: 176-179°C. IR (KBr, cm–1): 3032, 2931, 2835, 2800, 1757, 1612, 1464, 1412, 1358, 1267, 1126. 1H NMR (400 MHz, CDCl3) δ 7.67 (dd, J = 8.8, 5.4 Hz, 2H), 7.21 - 7.18 (m, 1H), 7.15 (s, 1H), 7.10 (dd, J = 8.0, 2.4 Hz, 1H), 6.98 (t, J = 8.6 Hz, 2H), 6.47 (dd, J = 8.4, 2.4 Hz, 1H), 6.38 (d, J = 12.8 Hz, 2H), 6.21 (s, 1H), 6.05 (dd, J = 8.4, 2.0 Hz, 1H), 5.83 (s, 1H), 3.85 (s, 3H), 3.82 – 3.76 (m, 2H), 3.68 (s, 3H), 3.40 - 3.30 (m, 1H), 3.26 (s, 3H), 3.21 (d, J = 5.2 Hz, 1H), 3.05 (dd, J = 13.8, 5.6

Hz, 1H), 2.90 - 2.63 (m, 6H), 2.56 (s, 3H), 2.43 (s, 3H), 2.40 - 2.28 (m, 2H), 2.15 2.09 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 165.7, 163.6, 156.2, 149.1, 148.9, 146.4, 145.9, 143.6, 142.2, 137.4, 134.8, 133.5, 132.9, 131.8, 129.6, 129.1, 129.0, 128.1, 126.8, 122.2, 121.4, 121.3, 120.9, 120.8, 120.6, 116.0, 115.8, 115.8, 112.2, 107.3, 104.8, 63.9, 61.3, 56.3, 56.0, 55.7, 44.9, 42.5, 42.2, 40.0, 39.2, 24.9, 20.3. HRMS (ESI-QTOF) m/z Calcd. for C44H43Cl2N2O7+ [M+H]+: 781.2447, found: 781.2425. 4.1.2.22. 7-O-(3,4-Dichlorobenzoyl)-fangchinoline (4e). Light yelow amorphous solid (Yield 86%), mp: 179-183°C. IR (KBr, cm–1): 3033, 2999, 2933, 2835, 2798, 1788, 1612, 1463, 1445, 1360, 1270, 1120. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.27 (s, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 8.0, 2.4 Hz, 1H), 6.53 (dd, J = 8.4, 2.2 Hz, 1H), 6.44 (d, J = 10.4 Hz, 2H), 6.28 (s, 1H), 6.13 (dd, J = 8.4, 1.6 Hz, 1H), 5.90 (s, 1H), 5.30 (s, 1H), 3.94 (s, 3H), 3.80 (dd, J = 11.2, 5.6 Hz, 1H), 3.76 (s, 3H), 3.34 (s, 1H), 3.33 (s, 3H), 3.21 - 3.15 (m, 2H), 3.01 - 2.82 (m, 5H), 2.74 - 2.68 (m, 2H), 2.59 (s, 3H), 2.55 (s, 3H), 2.39 (dd, J = 14.4, 9.6 Hz, 1H), 2.24 (d, J = 14.8 Hz, 1H).

13

C NMR (100 MHz, CDCl3) δ 156.0, 149.4,

149.0, 148.8, 147.6, 145.9, 143.2, 142.2, 135.0, 134.2, 132.9, 132.1, 129.5, 128.9, 128.3, 127.5, 123.2, 123.9, 121.6, 121.4, 121.3, 120.9, 120.8, 119.6, 112.1, 106.7, 104.6, 63.8, 56.3, 56.0, 55.6, 45.8, 44.9, 42.5, 39.6, 37.2, 25.4, 20.2. HRMS (ESI-QTOF) m/z Calcd. for C44H43Cl2N2O7+ [M+H]+: 781.2447, found: 781.2425. 4.1.2.23. 7-O-(2,4,6-Trichlorobenzoyl)-fangchinoline (4f). Light yelow amorphous solid (Yield 71%), mp: 173-175°C. IR (KBr, cm–1): 3031, 2995, 2933, 2837, 2798, 1764, 1611, 1464, 1445, 1360, 1271, 1107. 1H NMR (600 MHz, CDCl3) δ 7.32 (dd, J

= 7.8, 2.4 Hz, 1H), 7.26 (s, 2H), 7.13 (dd, J = 8.4, 2.4 Hz, 1H), 6.86 (s, 2H), 6.80 (dd, J = 8.4, 2.4 Hz, 1H), 6.48 (s, 1H), 6.42 (s, 1H), 6.37 (s, 1H), 6.28 (dd, J = 8.4, 2.4 Hz, 1H), 5.86 (s, 1H), 3.93 (s, 3H), 3.77 (s, 3H), 3.73 (d, J = 10.2 Hz, 1H), 3.61 (dd, J = 12.8, 5.4 Hz, 1H), 3.54 (ddd, J = 13.6, 11.2, 5.4 Hz, 1H), 3.40 (s, 3H), 3.24 (ddd, J = 12.6, 9.0, 6.6 Hz, 1H), 3.18 (dd, J = 12.6, 5.4 Hz, 1H), 3.01 – 2.88 (m, 2H), 2.72 (dd, J = 14.4, 11.2 Hz, 1H), 2.68 – 2.63 (m, 2H), 2.59 – 2.53 (m, 3H), 2.52 – 2.48 (m, 1H), 2.48 (s, 3H), 2.31 (s, 3H).

13

C NMR (150 MHz, CDCl3) δ159.97, 153.58, 149.78,

149.44, 148.33, 146.91, 146.71, 142.71, 136.29, 135.34, 134.67, 133.79, 132.52, 131.72, 130.05, 129.67, 128.60, 128.49, 128.31, 127.95, 122.83, 122.73, 122.08, 120.12, 115.94, 112.27, 111.46, 106.05, 63.97, 61.21, 56.09, 56.01, 55.88, 46.07, 45.33, 43.85, 42.63, 42.36, 41.46, 37.73, 25.65, 22.30, 11.08. HRMS (ESI-QTOF) m/z Calcd. for C44H42Cl3N2O7+ [M+H]+: 815.2050, found: 815.2058. 4.1.2.24. 7-O-(4-Bromobenzoyl)-fangchinoline (4g). White amorphous solid (Yield 83%), mp: 167-169°C. IR (KBr, cm–1): 3035, 2998, 2932, 2835, 2798, 1743, 1611, 1446, 1460, 1360, 1271,1114. 1H NMR (500 MHz, CDCl3) δ 7.31 (dd, J = 8.0, 2.0 Hz, 1H), 7.12 (dd, J = 8.0, 2.5 Hz, 1H), 6.87 (dd, J = 20.0, 8.0 Hz, 2H), 6.78 (dd, J = 8.0, 2.5 Hz, 1H), 6.49 (s, 2H), 6.37 (s, 1H), 6.24 (d, J = 7.5 Hz, 1H), 6.12 - 6.03 (m, 1H), 5.88 (s, 1H), 5.73 - 5.58 (m, 2H), 5.29 (s, 1H), 3.92 (s, 3H), 3.77 (d, J = 10.0 Hz, 1H), 3.71 (s, 3H), 3.51 (ddd, J = 10.5, 8.5, 3.0 Hz, 2H), 3.41 (s, 3H), 3.25 (dd, J = 12.5, 5.5 Hz, 1H), 2.86 -2.78 (m, 8H), 2.56 (s, 3H), 2.53 - 2.41 (m, 2H), 2.35 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 155.8, 153.3, 149.0, 148.7, 146.5, 143.5, 142.4, 142.2, 135.0, 133.9, 132.8, 132.5, 132.1, 129.7, 128.5, 128.0, 126.0, 126.2, 121.9, 121.5, 120.9,

120.7, 118.7, 118.4, 118.2, 112.3, 104.8, 63.7, 56.3, 56.0, 55.7, 44.9, 43.5, 42.3, 40.8, 39.1, 25.4, 20.3. HRMS (ESI-QTOF) m/z Calcd. for C44H44BrN2O7+ [M+H]+: 791.2325, found: 791.2332. 4.1.2.25. 7-O-(1-Naphthoyl)-fangchinoline (4h). White amorphous solid (Yield 88%), mp: 159-163°C. IR (KBr, cm–1): 3032, 2997, 2931, 2835, 2796, 1738, 1611, 1584, 1462, 1445, 1360, 1227, 1114. 1H NMR (400 MHz, CDCl3) δ 8.46 - 8.38 (m, 1H), 7.98 - 7.89 (m, 2H), 7.72 (d, J = 6.4 Hz, 1H), 7.54 (dd, J = 6.9, 2.8 Hz, 3H), 7.31 7.19 (m, 2H), 6.65 - 6.59 (m, 2H), 6.43 (s, 1H), 6.17 - 6.10 (m, 2H), 5.92 (s, 1H), 4.04 (s, 3H), 3.90 (d, J = 9.2 Hz, 1H), 3.82 (dd, J = 11.0, 5.4 Hz, 1H), 3.69 (s, 3H), 3.38 3.30 (m, 1H), 3.27 (s, 3H), 3.20 (dd, J = 12.2, 5.4 Hz, 1H), 2.98 - 2.64 (m, 7H), 2.59 (s, 3H), 2.55 - 2.37 (m, 2H), 2.02 - 1.99 (m, 1H), 1.97 (s, 3H), 1.33 (dd, J = 14.0, 5.8 Hz, 1H).

13

C NMR (100 MHz, CDCl3) δ 168.1, 146.2, 134.8, 133.0, 132.8, 129.5,

126.9, 126.3, 125.4, 124.7, 124.6, 122.3, 121.7, 121.7, 112.3, 106.3, 104.7, 63.8, 56.3, 55.9, 55.6, 44.9, 42.4, 41.9, 40.1, 37.3, 25.4, 20.0. HRMS (ESI-QTOF) m/z Calcd. for C48H47N2O7+ [M+H]+: 763.3378, found: 763.3356.

4.2 Biological tests 4.2.1. Cell lines and cell culture Human leukemic HEL and K562, as well as breast cell lines MDA-MB-231, MCF-7 were obtained from University of Toronto. Cells cultured in RPMI (HEL and K562) or DMEM (MDA-MB-231 and MCF-7) medium (high glucose) supplemented with 5% fetal bovine serum FBS (HyClone, GE Healthcare, Australia) and maintained in a humidified incubator of 5% CO2 at 37 °C. When the growing cells reached approximately 70-90% confluence, they were treated with 3e.

4.2.2. Cell viability assay A stock solution of 3e (20 µM) was prepared in DMSO and stored in the refrigerator at -20 degrees. The cytotoxicity of 3e on HEL, K562 and MDA-MB-231 cells was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide MTT (Solarbio, China) method [40]. The cells were plated at a density of 1×104 / well in a 96-well plate and incubated in a 37 ℃ incubator for 24 h to allow the cells to adapt the environment prior to dosing. Cells then treated with 3e for 12, 24, 48, 36, 48, 72 h. Each well was added with 20 µL diphenyl tetrazolium bromide (MTT) and incubate for 4 hours, medium removed and 100 µL of Dimethyl sulfoxide (DMSO) was added. The cell viability was detected by measuring the absorbance at 490 nm on a plate reader (Bio Tek, Winooski, USA). All experiments were in triplicates and repeated at least three times. 4.2.3. Apoptosis analysis by annexin V and propidium iodide Staining HEL cells (2×105/well) were seeded in 6 well-plates and treated with DMSO or 3e for 24 h. Drug treated cells were collected and washed with cold PBS three times, then re-suspended in the binding buffer and stained with annexin V and PI, according to manufacturer instruction (BD Biosciences, Franklin lakes, NJ). Apoptotic cells were analyzed by flow cytometer (ACEA Biosciences Inc, San Diego, USA). 4.2.4. Cell cycle analysis by flow cytometry HEL cells (2x105/well) were plated in 6-well plates and treatment with DMSO or 3e. The drug treated cells were harvested, washed with cold PBS, fixed with iced 70% ethanol at 4 °C overnight and then centrifuged and washed with PBS three times. The washed cells were re-suspended and incubated with 0.5 mL of PBS containing 100 µg/mL RNase and 50 µg/mL PI for 1 h in the dark at 37 °C. The cellular DNA content was analyzed by flow cytometer.

4.2.4. Akt Kinase Activity Assay The inhibitory activity against Akt kinase was determined by using an Akt kinase activity assay kit (ab139436, Abcam, USA) according to the manufacturer’s protocol. In brief, cell Lysates were purified by Mono Q anionexchange column and measured the kinase assay of 3e. 4.2.5. Molecular Modeling Molecular docking was performed according to the previous study [41]. The crystal structure of Akt1 (PDB entry code: 6ffh) in complex with G4K was used for molecular modeling. The AutoDock 4.2 was employed for docking calculations. Accelrys Discovery Studio Visualizer 4.5 was used for graphic display. 4.2.6. Western blot analysis Total protein was collected from HEL cells using RIPA (Radio Immunoprecipitation Assay) lysis buffer and separated on a 10% SDS Page. The protein was then transferred to a polyvinylidene fluoride (PVDF, 0.2 m) membrane and blocked with 5% BSA solution for 1 h at room temperature. The membrane was then incubated with the specific primary antibody overnight at 4 °C. Primary antibodies Akt (ab8805), p-Akt (ab81283), JNK (AB208035), p-JNK (ab124956), Caspase-3 (ab179517), Caspase-9 (ab202068), DNA repair enzyme PARP (ab32138), ERK (ab17942), p-ERK (ab50011) were purchased from Abcam (Abcam, Cambridge, United Kingdom). Primary antibodies C-myc (#9402), p21 (#8831) were obtained from CST (CST, Trask Lane, Danvers, USA). Beta-actin is from Proto-Technology (Protein-Tech, Bucuresti, Romania). Blots were incubated with the corresponding HRP-conjugated secondary antibody for 2 hours at room temperature. Membranes washed with PVDF three times with cold

TBS for five minutes each time. The expression of a particular protein was measured by applying an ECL selection substrate (Li-Cor, Lincoln, USA) 4.2.7. Statistical analysis Data from above experiments were repeated in triplicates at least in three independent times. The t-test was used to determine statistical differences between treated groups and controls, and P<0.05** was considered statistically significant. The values were presented as mean ± SD. The significance level was calculated using one-way analysis of variance to assess the differences between experimental groups. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 81360479, 21867009, 81472609 and U1812403), and the Science and Technology

Department

of

Guizhou

Province

(QKHRC[2016]4037

and

QKHPTRC[2017]5737), and Guizhou Provincial Engineering Research Center for Natural Drugs. Appendix A. Supplementary data The 1H,

13

C-NMR and HRMS spectral data were provided in the Supplementary

material. Author contributions Shengcao Hu synthesized compounds. Jin Yang conducted biology experiments. Chu nlin Wang assisted in apoptosis experiments. Jin Yang, Shengcao Hu, Yanhua Fan, Yaacov Ben David and Weidong Pan wrote the manuscript. Yanhua Fan, Yaacov Ben-David and Weidong Pan contributed to experimental design and supervision.

References

1.

L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global

cancer

statistics,

2012,

CA.

Cancer

J.

Clin.

65

(2015)

87-108,

https://doi.org/10.3322/caac.21262. 2.

S.C. Howard, M. Pedrosa, M. Lins, A. Pedrosa, C.H. Pui, R.C. Ribeiro, F. Pedrosa, Establishment of a pediatric oncology program and outcomes of childhood acute lymphoblastic leukemia in a resource-poor area, JAMA, 291 (2004) 2471-2475, https:// doi.org /10.1001/jama.291.20.2471.

3.

J.H. Myklebust, H.K. Blomhoff, L.S. Rusten, T. Stokke, E.B. Smeland, Activation

of

phosphatidylinositol

3-kinase

is

important

for

erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells,

Exp.

Hematol.

30

(2002)

990-1000,

https://doi.org/10.1016/S0301-472X(02)00868-8. 4.

S. Park, N. Chapuis, J. Tamburini, V. Bardet, P. Cornillet-Lefebvre, L. Willems, A. Green, P. Mayeux, C. Lacombe, D. Bouscary, Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia, Haematol. 95 (2010) 819-828, https://doi.org/10.3324/haematol.2009.013797.

5.

J.T. Barata, A. Silva, J.G. Brandao, L.M. Nadler, A.A. Cardoso, V.A. Boussiotis, Activation of PI3K is indispensable for interleukin 7-mediated viability, proliferation, glucose use, and growth of T cell acute lymphoblastic leukemia cells, J. Exp. Med. 200 (2004) 659-669, https://doi.org/ 10.1084/jem.20040789.

6.

M.G. Kharas, R. Okabe, J.J. Ganis, M. Gozo, T. Khandan, M. Paktinat, D.G. Gilliland, K. Gritsman, Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice, Blood. 115 (2010) 1406-1415, https://doi.org/10.1182/blood-2009-06-229443.

7.

A. Silva, A. Girio, I. Cebola, C.I. Santos, F. Antunes, J.T. Barata, Intracellular reactive

oxygen

species

are

essential

for

PI3K/Akt/mTOR-dependent

IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells, Leukemia. 25 (2011) 960-967. https://doi.org/10.1038/leu.2011.56. 8.

T. Palomero, M. Dominguez, A.A. Ferrando, The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia, Cell cycle. 7 (2008) 965-970, https://doi.org/10.4161/cc.7.8.5753.

9.

C.D. Kang, S.D. Yoo, B.W. Hwang, K.W. Kim, D.W. Kim, C.M. Kim, S.H. Kim, B.S. Chung, The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells,

Leuk.

Res.

24

(2000)

527-534,

https://doi.org/10.1016/S0145-2126(00)00010-2. 10. R.Yu, A.A. Shtil, T.H. Tan, I.B. Roninson, A.N. Kong, Adriamycin activates c-jun N-terminal kinase in human leukemia cells: a relevance to apoptosis, Cancer lett. 107 (1996) 73-81, https://doi.org/10.1016/0304-3835(96)04345-5. 11. T. Liu, X. Liu, W. Li, Tetrandrine, a Chinese plant-derived alkaloid, is a potential candidate for cancer chemotherapy, Oncotarget. 7 (2016) 40800-40815, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130046/. 12. B.S. Teh, P. Chen, M.F. Lavin, W.K. Seow, Y.H. Thong, Demonstration of the induction of apoptosis (programmed cell death) by tetrandrine, a novel anti-inflammatory agent, Int. J. Immunopharmacol. 13 (1991) 1117-1126, https://doi.org/10.1016/0192-0561(91)90163-2. 13. H.S. Choi, H.S. Kim, K.R. Min, Y. Kim, H.K. Lim, Y.K. Chang, M.W. Chung, Anti-inflammatory effects of fangchinoline and tetrandrine, J. Ethnopharmacol. 69 (2000) 173-179, https://doi.org/10.1016/S0378-8741(99)00141-5. 14. H.S. Kim, Y.H. Zhang, K.W. Oh, H.Y. Ahn, Vasodilating and hypotensive effects of fangchinoline and tetrandrine on the rat aorta and the stroke-prone

spontaneously hypertensive rat, J. Ethnopharmacol. 58 (1997) 117-123, https://doi.org/10.1016/S0378-8741(97)00092-5. 15. H. Kuroda, S. Nakazawa, K. Katagiri, O. Shiratori, M. Kozuka, Antitumor effect of bisbenzylisoquinoline alkaloids, Chem. Pharm. Bull. 24 (1976) 2413-2420, https:// doi.org/ 10.1248/cpb.24.2413. 16. H.S. Cho, S.H. Chang, Y.S. Chung, J.Y. Shin, S.J. Park, E.S. Lee, S.K. Hwang, J.T. Kwon, A.M. Tehrani, M. Woo, M.S. Noh, H. Hanifah, H. Jin, C.X. Xu, M.H. Cho, Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells, J. Vet. Sci. 10 (2009) 23-28, https://doi.org/10.4142/jvs.2009.10.1.23. 17. X. Li, B. Su, R. Liu, D. Wu, D. He, Tetrandrine induces apoptosis and triggers caspase cascade in human bladder cancer cells, J. Surg. Res. 166 (2011) e45-51, https://doi.org/10.1016/j.jss.2010.10.034. 18. J. Lan, N. Wang, L. Huang, Y. Liu, X. Ma, H. Lou, C. Chen, Y. Feng, W. Pan, Design and synthesis of novel tetrandrine derivatives as potential anti-tumor agents against human hepatocellular carcinoma, Eur. J. Med. Chem.. 127 (2017) 554-566, https://doi.org/10.1016/j.ejmech.2017.01.008. 19. Y. Liu, B. Xia, J. Lan, S. Hu, L. Huang, C. Chen, X. Zeng, H. Lou, C. Lin, W. Pan, Design, Synthesis and Anticancer Evaluation of Fangchinoline Derivatives, Molecules. 22 (2017) 1923-1935, https://doi.org/10.3390/molecules22111923. 20. H. Zhou, X.M. Li, J. Meinkoth, R.N. Pittman, Akt regulates cell survival and apoptosis at a postmitochondrial level, J. Cell Boil. 151 (2000) 483-494, https://doi.org/1010.1083/jcb.151.3.483. 21.

C.J.

Sherr,

Cancer

cell

cycles,

Science.

https://doi.org/10.1126/science.274.5293.1672.

274

(1996)

1672-1677,

22. L.H. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science. 266 (1994) 1821-1828, https://doi.org/10.1126/science.7997877. 23. S. Shukla, S. Gupta, Apigenin-induced cell cycle arrest is mediated by modulation of MAPK, PI3K-Akt, and loss of cyclin D1 associated retinoblastoma dephosphorylation in human prostate cancer cells, Cell cycle. 6 (2007) 1102-1114, https://doi.org/10.4161/cc.6.9.4146. 24. S. van den Heuvel, Cell-cycle regulation, Worm Book. 21 (2005) 1-16, https://doi.org/10.1895/wormbook.1.28.1. 25. C.A. Spencer, M. Groudine, Control of c-myc regulation in normal and neoplastic cells,

Adv.

Cancer

Res.

56

(1991)

1-48,

https://doi.org/10.1016/S0065-230X(08)60476-5. 26. J.H. Jeong, Y.C. Chang, Ascochlorin, an isoprenoid antibiotic, induces G1 arrest via downregulation of c-Myc in a p53-independent manner, Biochem. Biophys. Res. Commun. 398 (2010) 68-73, https://doi.org/10.1016/j.bbrc.2010.06.037. 27. I. Budihardjo, H. Oliver, M. Lutter, X. Luo, X. Wang, Biochemical pathways of caspase activation during apoptosis, Annu. Rev. Cell Dev. Biol. 15 (1999) 269-290, https://doi.org/10.1146/annurev.cellbio.15.1.269. 28. H. Zhou, X.M. Li, J. Meinkoth, R.N. Pittman, Akt regulates cell survival and apoptosis at a postmitochondrial level, J. Cell Biol. 151 (2000) 483-494, https://doi.org/10.1083/jcb.151.3.483. 29. P.A. Jeggo, DNA repair: PARP - another guardian angel, Curr. Biol. 8 (1998) R49-51, https://doi.org/10.1016/s0960-9822(98)70032-6. 30. F.M. Ruemmele, S. Dionne, I. Qureshi, D.S. Sarma, E. Levy, E.G. Seidman, Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase

(PARP),

Cell

Death

Differ.6

(1999)

729-735,

https://doi.org/10.1038/sj.cdd.4400545. 31. P. Li, D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, X. Wang, Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates

an

apoptotic

protease

cascade,

Cell.

91

(1997)

479-489,

https://doi.org/10.1016/s0092-8674(00)80434-1. 32. Y. Chen, S.H. Tseng, The Potential of Tetrandrine against Gliomas, Anticancer agents

Med.

Chem.

10

(2010)

534-542,

https://doi.org/10.2174/187152010793498609. 33. R. Seger, E.G. Krebs, The MAPK signaling cascade, Encyclopedia of Cell Biology.

3

(2016)

122-127,

https://doi.org/10.1016/B978-0-12-394447-4.30014-1. 34. J. Desbarats, R.B. Birge, M. Mimouni-Rongy, D.E. Weinstein, J.S. Palerme, M.K. Newell, Fas engagement induces neurite growth through ERK activation and p35 upregulation, Nat. cell Boil. 5 (2003) 118-125, https://doi.org/10.1038/ncb916. 35. G.H. Yang, B.B. Jarvis, Y.J. Chung, J.J. Pestka, Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation, Toxicol. Appl. Pharmacol. 164 (2000) 149-160, https://doi.org/10.1006/taap.1999.8888. 36. Y.T. Ip, R.J. Davis, Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development, Curr. Opin. Cell Boil. 10 (1998) 205-219, https://doi.org/10.1016/S0955-0674(98)80143-9. 37. K. Lei, R.J. Davis, JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 2432-2437, https://doi.org/10.1073/pnas.0438011100.

38. Y.J. Li, X. Zhao, L.M. Vecchiarelli-Federico, Y. Li, A. Datti, Y. Cheng, Y. Ben-David, Drug-mediated inhibition of Fli-1 for the treatment of leukemia, Blood Cancer J. 2 (2012) e54, https://doi.org/10.1038/bcj.2011.52. 39. Q. Zhao, A.N. Assimopoulou, S.M. Klauck, H. Damianakos, I. Chinou, N. Kretschmer, J.L. Rios, V.P. Papageorgiou, R. Bauer, T. Efferth, Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells, Oncotarget. 6 (2015) 38934-38951, https://doi.org/10.18632/oncotarget.5380. 40. D. Gerlier, N. Thomasset, Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods. 94 (1986) 57-63, D. Gerlier, N. Thomasset, Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods. 94 (1986) 57-63, https://doi.org/10.1016/0022-1759(86)90215-2. 41. Y.H. Fan, H.W. Ding, D. Kim, D.H. Bach, J.Y. Hong, Y.N. Xu, S.K. Lee, Antitumor Activity of DFX117 by Dual Inhibition of c-Met and PI3Kα in Non-Small

Cell

Lung

Cancer.

https://doi.org/10.3390/cancers11050627.

Cancers.

11

(2019),

627,

Highlights Twenty-five fangchinoline 7-O-acyl fangchinoline derivatives displayed higher proliferation inhibitory activity on leukemia and breast cancer cell lines than the parental compound, especially 3e. The compound 3e induced G0/G1 cell cycle arrest and apoptosis of HEL (leukemia cell line), it was mannered by dose- and time-dependent. The induction of apoptosis by 3e was associated with the inhibition of anti-apoptosis proteins p-AKT and p-ERK, and activation of apoptotic protein p-JNK as well as JNK. The molecular docking study showed affinity interaction between 3e and Akt1, and it was confirmed by Akt kinase inhibition experiment.

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