Synthesis and Antileukemia Activity Evaluation of Benzophenanthridine Alkaloid Derivatives

Thirty-three benzophenanthridine alkaloid derivatives (1a–1u and 2a–2l) were synthesized, and their cytotoxic activities against two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were evaluated in vitro using a Cell Counting Kit-8 (CCK-8) assay. Nine of these derivatives (1i–l, 2a, and 2i–l) with IC50 values in the range of 0.18–7.94 μM showed significant inhibitory effects on the proliferation of both cancer cell lines. Analysis of the primary structure–activity relationships revealed that different substituent groups at the C-6 position might have an effect on the antileukemia activity of the corresponding compounds. In addition, the groups at the C-7 and C-8 positions could influence the antileukemia activity. Among these compounds, 2j showed the strongest in vitro antiproliferative activity against Jurkat Clone E6-1 and THP-1 cells with good IC50 values (0.52 ± 0.03 μM and 0.48 ± 0.03 μM, respectively), slightly induced apoptosis, and arrested the cell-cycle, all of which suggests that compound 2j may represent a potentially useful start point to undergo further optimization toward a lead compound.


Introduction
Leukemia is a broad term for a group of blood cell cancers that begin in stem cells found in the bone marrow. Leukemia occurs most often in adults older than 55, but it is also the most common cancer in children younger than 15; in particular, the incidence rates of leukemia are the highest in early childhood and later adulthood [1,2]. Patients with leukemia usually have serious complications, such as autoimmune cytopenia [3], bleeding [4], electrolyte imbalance, and hyperuricemia [5]; therefore, leukemia seriously threatens human health and quality of life.
Currently, chemotherapy and hematopoietic stem-cell bone marrow transplantation are still the main treatments for leukemia. However, bone marrow transplantation involves a complicated process that requires antigen compatibility between the donor and recipient. Although these methods can lead to remission in most patients, the recurrence rate is very high, and the long-term survival rate is low. Notably, high-dose combination chemotherapy can cause patients to develop drug resistance and serious side-effects, such as bone marrow suppression, gastrointestinal reactions, and cardiotoxicity [6][7][8][9].
Among the many available drugs to treat leukemia, imatinib and all-trans-retinoic acid (ATRA) plus arsenic trioxide (ATO) are widely used worldwide. Although there has been great success with reducing the symptoms of patients with leukemia after treatment with these drugs, the side-effects and early mortality they can cause remain significant, which are major barriers to treating leukemia patients [10,11]. In recent years, a number of new treatments for leukemia emerged. For example, CEP-701, which is an FLT3 inhibitor, was of new treatments for leukemia emerged. For example, CEP-701, which is an FLT3 inhibitor, was assessed in leukemia, with the hope that it represents the development of an important new molecularly targeted therapy for this disease [12,13]. Therefore, whether to improve the treatment and long-term survival rates of patients with leukemia, or to find new treatments for leukemia, it is extremely important to research and develop effective new drugs.
Natural products, as an important source of drugs and drug lead compounds, have the advantages of unique mechanisms, remarkable results, low toxicity, and few side-effects. Many well-known natural products with various applications, such as artemisinin, paclitaxel, and vinblastine, come from a wide variety of Chinese herbal medicines found abundantly in China. Therefore, drug candidates for treating leukemia could be obtained through the structural optimization of natural lead compounds. Z. nitidum is an important Chinese herbal medicine that possesses various antitumor active ingredients. Benzophenanthridine alkaloids are some of the most important active ingredients abundantly found in this plant. At present, this type of alkaloid has been found to have a variety of biological activities with antibacterial [14][15][16], analgesic, anti-inflammatory [17], antiviral [18], anti-phytopathogenic [19] and antitumor [20][21][22][23][24][25] effects. However, there are few reports on the antileukemia activity of benzophenanthridine alkaloids.
Our previous studies indicated that certain benzophenanthridine alkaloids showed strong inhibitory effects on leukemia cell lines [26,27]. To continue our research, two active benzophenanthridine alkaloids, chelerythrine (1) and sanguinarine (2) (Figure 1), which were found in high abundance, were selected as the starting compounds for structural modification to obtain antileukemia drug candidates with better activity. Therefore, thirty-three benzophenanthridine alkaloid derivatives (1a-1u and 2a-2l) were synthesized, and their antileukemia activities against two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were evaluated in vitro (According to the preliminary screening results in Table S2 in the Supplementary Materials, we selected these two leukemia cells for activity test). Among them, nine derivatives (1i-l, 2a and 2i-l) showed significant inhibitory effects on the proliferation of Jurkat Clone E6-1 and THP-1 cells. In particular, compound 2j displayed the strongest inhibition against Jurkat Clone E6-1 and THP-1 cells, with IC50 values of 0.52 ± 0.03 μM and 0.48 ± 0.03 μM, respectively. Furthermore, the influence of compound 2j on the cell-cycle and apoptosis in both leukemia cell lines was tested. Herein, we report the synthesis and antileukemia activity evaluation of a series of novel benzophenanthridine alkaloid derivatives of chelerythrine (1) and sanguinarine (2) (1a-1u and 2a-2l). Their cytotoxic activities and initial structure-activity relationships (SARs) are also reported.

Design and Synthesis of the Benzophenanthridine Alkaloid Derivatives
In our previous studies, bocconoline (Figure 1), a benzophenanthridine alkaloid isolated from Z. nitidum, showed good antiproliferative effects on leukemia cells and low toxicity [27]. It differs structurally from other benzophenanthridine alkaloids due to the hydroxymethyl group at the C-6 position. Therefore, it was speculated that this substitution might play a crucial role in reducing the toxicity of this compound. In addition, Herein, we report the synthesis and antileukemia activity evaluation of a series of novel benzophenanthridine alkaloid derivatives of chelerythrine (1) and sanguinarine (2) (1a-1u and 2a-2l). Their cytotoxic activities and initial structure-activity relationships (SARs) are also reported.
through literature investigation, we found that the introduction of appropriate groups (malonic esters, dialkyl phosphites, nitro alkanes, or indoles) at the C-6 position could enhance its activities [21]. Hence, in order to discover more benzophenanthridine alkaloid derivatives with good antileukemia activity and low toxicity, two natural benzophenanthridine alkaloids with good antileukemia activity, chelerythrine (1) and sanguinarine (2), were chosen as starting points, and a series of their derivatives, 1a-1u and 2a-2l, were synthesized by changing the substituent at the C-6 position (The 1 H-and 13 C-NMR, HR-ESI-MS and HPLC spectra of all compounds S5-S103, S106-S138 are shown in the Supplementary Materials). The synthetic routes for the target compounds are shown in Figure 2. Briefly, structural modification of chelerythrine (1) and sanguinarine (2) mainly involved changing the substituent at the C-6 position by nucleophilic addition, including the introduction of cyano [28], indole, malonic ester [21], ester [29], allyl [30], and acetonyl units. To obtain compounds 1b and 2b, reduction of the C=N double bond at the C-6 position was achieved by treatment with NaBH4 [14]. The ethyl acetate units in compounds 1e and 2e were converted to hydroxyethyls in compounds 1f and 2f with LiAlH4 [31]. Additionally, compounds 1n-q were synthesized via the Claisen-Schmidt reaction [32].  The cytotoxic activities of the 33 synthesized derivatives tested at 20 µM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were ated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Countin 8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The r of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compou and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μ THP-1, respectively. From the results of the activity data, it could be seen that comp 2a showed good activity, but 2a could not show a good dose dependence in the f study of the cell cycle and apoptosis. However, compound 2j showed a better do pendence than 2a. Therefore, compound 2j might be a potential antileukemia comp and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation. Table 1. IC50 values of 33 derivatives against leukemia cell lines in vitro ( x ± SD, n = 3).

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation.

Inhibitory Effects on Leukemia Cell Proliferation
The cytotoxic activities of the 33 synthesized derivatives tested at 20 μM were evaluated in two leukemia cell lines (Jurkat Clone E6-1 and THP-1) using a Cell Counting Kit-8 (CCK-8) assay with doxorubicin hydrochloride (DOX) as a positive control. The results of the preliminary bioassay are listed in Table 1.
As shown in Table 1, the in vitro activity data revealed that nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on these leukemia cell lines with IC50 values ranging from 0.5 to 8.0 μM and from 0.1 to 6.0 μM, respectively. Notably, compounds 2a and 2j exhibited excellent activities in both cell lines with good IC50 values of 0.53 ± 0.05 μM and 0.52 ± 0.03 μM for Jurkat Clone E6-1 and 0.18 ± 0.03 μM and 0.48 ± 0.03 μM for THP-1, respectively. From the results of the activity data, it could be seen that compound 2a showed good activity, but 2a could not show a good dose dependence in the further study of the cell cycle and apoptosis. However, compound 2j showed a better dose dependence than 2a. Therefore, compound 2j might be a potential antileukemia compound and was chosen for further evaluation. 1n >20 >20 1o >20 >20 1p >20 >20 1q >20 .>20 1r >20 >20 1s >20 >20 1t >20 >20 1u >20 >20 >20 >20 Two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were treated with compound 2j at concentrations of 0.25, 0.5, and 1.0 μM for 48 h at 37 °C. The apoptosis rates induced by treatment with compound 2j are shown in Figure 3.  Two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were treated with compound 2j at concentrations of 0.25, 0.5, and 1.0 μM for 48 h at 37 °C. The apoptosis rates induced by treatment with compound 2j are shown in Figure 3.  Two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were treated with compound 2j at concentrations of 0.25, 0.5, and 1.0 μM for 48 h at 37 °C. The apoptosis rates induced by treatment with compound 2j are shown in Figure 3. Compared with the control group (0.92% ± 0.09% for Jurkat Clone E6-1 cells and 0.69% ± 0.08% for THP-1 cells), after treating the cells with compound 2j for 48 h, the rates of apoptosis increased in a dose-dependent manner. When Jurkat Clone E6-1 and THP-1 cells were treated with 0.5 µM and 1.0 µM compound 2j, their apoptosis rates increased significantly from 7.07% ± 0.43% to 17.84% ± 0.65% and from 6.01% ± 0.52% to 16.23% ± 1.15%, respectively (p < 0.001 vs. control group). These results suggested that compound 2j could slightly induce apoptosis in these two leukemia cell lines.

Effects of Compound 2j on the Cell-Cycle
Cell-cycle assays in the Jurkat Clone E6-1 and THP-1 cell lines were performed using flow cytometry, and the results are shown in Figure 4. When these two types of cells were treated with compound 2j for 48 h at concentrations ranging from 0 µM to 1.0 µM, the numbers of cells in the G 0 /G 1 phase increased significantly in a dose-dependent manner, which was accompanied by decreases in the G 2 /M populations. However, the percentages of cells in the S phase were not significantly different. These results showed that compound 2j could induce cell-cycle arrest in the G 0 /G 1 phase.
2j induced apoptosis in Jurkat Clone E6-1 cell line. Jurkat Clone E6-1 cells were treated with 0.25, 0.5, and 1.0 µ M of compound 2j for 48 h, and cells were subsequently stained with Annexin V-FITC/PI and subsequently analyzed by flow cytometry. (B) Compound 2j induced apoptosis in THP-1 cell line. THP-1 cells were treated with 0.25, 0.5, and 1.0 µ M of compound 2j for 48 h, and cells were subsequently stained with Annexin V-FITC/PI and subsequently analyzed by flow cytometry. All data are presented as means ± SD (n = 3); *** p < 0.001 vs. the control group.

Effects of Compound 2j on the Cell-Cycle
Cell-cycle assays in the Jurkat Clone E6-1 and THP-1 cell lines were performed using flow cytometry, and the results are shown in Figure 4. When these two types of cells were treated with compound 2j for 48 h at concentrations ranging from 0 μM to 1.0 μM, the numbers of cells in the G0/G1 phase increased significantly in a dose-dependent manner, which was accompanied by decreases in the G2/M populations. However, the percentages of cells in the S phase were not significantly different. These results showed that compound 2j could induce cell-cycle arrest in the G0/G1 phase.

SAR Analysis
As shown in Table 1, preliminary SAR studies were undertaken on the basis of the above cytotoxicity evaluation. Among the compounds synthesized, nine derivatives (1il, 2a, and 2i-l) showed significant inhibitory effects on the proliferation of Jurkat Clone E6-1 and THP-1 cells with IC50 values ranging from 0.1 to 8.0 μM. However, the other derivatives displayed weak or no inhibitory activity against the two leukemia cell lines. These results indicated that compounds containing cyano and malonic esters groups at the C-6 position of the benzophenanthridine alkaloid scaffold showed higher cytotoxic activity than the other types of compounds, and compounds with different substituents at the C-6 position exhibited different inhibitory activities. Compounds 2a and 2i-l showed much stronger cytotoxicity, with IC50 values of 0.53 μM, 1.30 μM, 0.52 μM, 1.23 μM, and 0.91 μM (in Jurkat Clone E6-1 cells) and 0.18 μM, 1.46 μM, 0.48 μM, 1.38 μM, and 1.17 μM (in THP-1 cells), respectively, than compounds 1i-l. These results implied that the antileukemia activities of the derivatives substituted with a methylenedioxy moiety at the C-7 and C-8 positions were greater than those of the derivatives substituted with methoxyl groups at the C-7 and C-8 positions. In other words, the antileukemia activities of the sanguinarine derivatives were significantly better than those of the chelerythrine derivatives. Therefore, it could be speculated that suitable nucleophilic groups, such as malonic esters and cyano, might enhance the antileukemia activity. Moreover, the substituents at the C-7 and C-8 positions were key units that affected the inhibitory activity of the compounds against the tested leukemia cell lines.

SAR Analysis
As shown in Table 1, preliminary SAR studies were undertaken on the basis of the above cytotoxicity evaluation. Among the compounds synthesized, nine derivatives (1i-l, 2a, and 2i-l) showed significant inhibitory effects on the proliferation of Jurkat Clone E6-1 and THP-1 cells with IC 50 values ranging from 0.1 to 8.0 µM. However, the other derivatives displayed weak or no inhibitory activity against the two leukemia cell lines. These results indicated that compounds containing cyano and malonic esters groups at the C-6 position of the benzophenanthridine alkaloid scaffold showed higher cytotoxic activity than the other types of compounds, and compounds with different substituents at the C-6 position exhibited different inhibitory activities. Compounds 2a and 2i-l showed much stronger cytotoxicity, with IC 50 values of 0.53 µM, 1.30 µM, 0.52 µM, 1.23 µM, and 0.91 µM (in Jurkat Clone E6-1 cells) and 0.18 µM, 1.46 µM, 0.48 µM, 1.38 µM, and 1.17 µM (in THP-1 cells), respectively, than compounds 1i-l. These results implied that the antileukemia activities of the derivatives substituted with a methylenedioxy moiety at the C-7 and C-8 positions were greater than those of the derivatives substituted with methoxyl groups at the C-7 and C-8 positions. In other words, the antileukemia activities of the sanguinarine derivatives were significantly better than those of the chelerythrine derivatives. Therefore, it could be speculated that suitable nucleophilic groups, such as malonic esters and cyano, might enhance the antileukemia activity. Moreover, the substituents at the C-7 and C-8 positions were key units that affected the inhibitory activity of the compounds against the tested leukemia cell lines.

General Chemistry
Unless otherwise noted, all solvents and reagents were purchased from commercial sources, and some reactions were carried out under inert atmosphere and drying solvents with relevant specifications (extra dry, with molecular sieves, water ≤ 50 ppm (by K.F.) EnergySeal) purchased from commercial sources. All reactions were monitored by thinlayer chromatography (TLC) on silica gel GF 254 plates (Qingdao Haiyang Chem. Ind. Ltd. P.R. Qingdao, China); spots were visualized with ultraviolet light (UV, Shanghai Jingke Ind. Co., Ltd., Shanghai, China) and 5% H 2 SO 4 in ethanol. The following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet, and br.s = broad singlet. All first-order splitting patterns were assigned on the basis of appearance. All derivatives were purified by silica gel column chromatography. 1 H-and 13 C-NMR data were recorded with an INOVA-600 MHz spectrometer in CDCl 3 , CD 3 OD, acetone-d 6 , or DMSO-d 6 (Anhui Zesheng Tech. Co., Ltd., Hefei, Anhui, China) at room temperature, and the chemical shifts are shown relative to tetramethylsilane (TMS). High-resolution mass spectra were obtained using a Bruker microTOF-Q mass spectrometer.

Preparation of Raw Materials
Due to the low content of chelerythrine (1) and sanguinarine (2) in Z. nitidum, and it being difficult to enrich, we purchased the Macleaya cordata total alkaloids with higher content for enrichment and separation to obtain raw materials. TLC was used to identify the components of 1 and 2. The gradient elution of petroleum ether/ethyl acetate system (v/v: 10:1→1:1) was carried out by silica gel column chromatography, and five fractions (Fr.1-Fr.5) were obtained. Fr.2-Fr.4 were separated and purified by repeated silica gel column chromatography (petroleum ether/ethyl acetate: 4/1, 2/1, 1/1; CH 3 Cl/CH 3 OH: 49/1, 20/1, etc.) to obtain these two raw materials. Their structure was further determined by 1 H-and 13 C-NMR (The 1 H-and 13 C-NMR and HPLC spectra of the two compounds S1-S4, S104-S105 are shown in the Supplementary Materials).

Synthesis of Compounds 1a and 2a
Trimethylsilyl cyanlde (TMSCN) (200 µL, 0.194 mmol) and 4-dimethylaminopyridine (DMAP) (60 mg, 0.492 mmol) were added to a stirred solution of 1 or 2 (0.144 mmol) in dry dichloromethane (DCM) (10 mL) at room temperature, and the reaction mixture was stirred under reflux for 14 h. After the reaction was complete, the mixture was washed with saturated NaHCO 3 solution three times and filtered. The filtrate was washed with an aqueous hydrochloric acid solution (0.1 mol/L, 5 × 10 mL), and then the organic layer was collected, dried over anhydrous Na 2 SO 4 , and concentrated under vacuum. The crude products were washed with methanol and filtered, and then dried under vacuum to obtain target compounds 1a and 2a.

Synthesis of Compounds 1b and 2b
NaBH 4 (10 mg, 0.264 mmol) was added to a solution of 1 or 2 (0.052 mmol) in MeOH (5 mL) at room temperature. The reaction mixture was stirred for 0.5 h at the same temperature. After the reaction was complete, acetic acid was added to remove the excess NaBH 4 and concentrated under vacuum. The residue was dissolved in dry DCM and extracted with saturated aqueous NaCl (3 × 10 mL). The organic layer was collected, dried over anhydrous Na 2 SO 4 , and concentrated under vacuum. Finally, the crude products were purified by silica gel column chromatography (petroleum ether (PE)/ethyl acetate (EA), 10:1) to obtain the target compounds.
Compound  1c, 1d, 1r-u, and 2c, 2d The indole compounds (two equivalents) were added to a solution of 1 or 2 (0.052 mmol) in CH 3 CN (10 mL) at room temperature. Each reaction mixture was stirred at the same temperature until the reaction was complete and then concentrated under vacuum. After that, the crude products were purified by silica gel column chromatography to obtain the target compounds.

Cell-Cycle Assay
After the cells were treated with compound 2j, they were trypsinized, prepared as a single-cell suspension (1 × 10 6 /mL), and transferred to microcentrifuge tubes for centrifugation at 1500 rpm for 5 min at room temperature; to prevent cell clumping, the cells were fixed by adding ice-cold 70% ethanol (1 mL) and blocking for 15 min at 4 • C. Then, the cells were centrifuged at 1500 rpm for 5 min, and 500 µL of PI solution was added (50 µg/mL PI, 100 µg/mL RNase A, 0.05% Triton X-100) for 40 min of incubation at 37 • C. The cells were centrifuged at 1500 rpm for 5 min, and 1 mL of PBS (HyClone, Logan, UT, USA) was added. After another centrifugation at 1500 rpm for 5 min, the cells were resuspended in 500 µL PBS and analyzed by flow cytometry.

Conclusions
In summary, 33 derivatives of chelerythrine and sanguinarine were designed and synthesized by using suitable nucleophilic substances for addition reactions, and their antileukemia activities against the Jurkat Clone E6-1 and THP-1 cell lines were evaluated for the first time. By analyzing these derivatives, some initial SARs were revealed. For example, the presence of cyano and malonic esters groups at the C-6 position of the benzophenanthridine skeleton resulted in stronger antileukemia activity, whereas the introduction of hydroxyethyl, acetonyl, or other groups at this position led to decreased activity. Moreover, compounds containing methylenedioxy moieties at the C-7 and C-8 positions had better antileukemia activity. Thus, when methylenedioxy groups were at the C-7 and C-8-positions, the introduction of cyano or malonic esters groups at the C-6 position could result in the best antileukemia activity.
Further studies indicated that compound 2j induced apoptosis in both Jurkat Clone E6-1 and THP-1 cells in a dose-dependent manner, and these results were consistent with those from the CCK-8 assay. The inhibitory effects of compound 2j might be related to cell-cycle changes, and these data were consistent with the apoptosis detection results. These findings became clearer after treatment with 1.0 µM 2j for 48 h. In conclusion, compound 2j induced apoptosis in Jurkat Clone E6-1 and THP-1 cells and arrested these cells in the G 0 /G 1 phase, possibly by disrupting the cell-cycle, reducing DNA synthesis, and inducing apoptosis. These mechanisms led to inhibition of the proliferation and growth of leukemia cells.
Among all of the prepared compounds, compound 2j showed satisfactory activity against Jurkat Clone E6-1 and THP-1 cells, and it could be considered for further investigation and optimization. However, since we selected transformed leukemia cells, this might have some drawbacks. In order to explore whether the cytotoxicity observed is specific to the leukemia lines, we will continue to test the compounds on a non-transformed cell type in future experiments. Lastly, our research suggested that compound 2j might be a potentially useful starting point for further optimization to become a new lead compound, providing a rich and diverse material basis for the development of innovative antileukemia drugs.