Design, synthesis of amide derivatives of scutellarin and their antileukemia and neuroprotective activities

A unique series of amide-scutellarin derivatives were designed and synthesized in order to develop the function of scutellarin further. The antiproliferative activity of all target compounds against two human leukemia cell line was evaluated. Among them, compounds 6g and 7c displayed the most antitumor activities against HL-60 and THP-1 cells. Moreover, all compounds were also assayed for their neuroprotective activity against hydrogen peroxide (H2O2)-induced PC12 cell injury, and the majority of the compounds had moderate to good neuroprotective properties. These findings confirmed that these target compounds could be used as antileukemia or neuroprotective candidates in the future. Graphical abstract Graphical abstract


Introduction
Cancer is the second cause of mortality worldwide and continuing to be a serious public health problem in globally [1]. The incidence and mortality of cancer have been increasing precipitously, especially in developing countries, such as China [2]. Leukemia is a type of haemopoietic malignancy that is more common in teenagers, even though it shows relatively low morbidity and mortality compared to other tumors [3,4]. According to the latest statistics, leukemia is the leading cause of death among all cancers in the adolescents and young adults of China [5]. Traditional chemotherapeutic agents are found to be successful in destroying leukemic cells. However, they are restricted by the chemo-resistant and side-effect of anticancer drugs [6,7]. Therefore, the development of new antileukemia drugs is urgent.
Natural products have been the major sources of new anticancer drugs, with nearly 64% of currently marketed antitumor drugs owing their origins to natural compounds over the nearly four decades [8]. Flavonoids are natural products with important medicinal values, which are the main active ingredients in the plant kingdom [9]. They have a variety of pharmacological activities against cancers, cardiovascular diseases, and neurodegenerative disorders [10][11][12]. Scutellarin is one of flavones that extracted from traditional Chinese herb Erigeron breviscapus (vant.) Hand-Mazz. And the antitumor activity of scutellarin has attracted the attention of researchers for it could suppress proliferation, migration and invasion of cancer cells [13][14][15][16]. There were also accumulating evidences suggesting that scutellarin could significantly induce the apoptosis of tumor cells and sensitize cancer cells to chemotherapy [17][18][19].
Moreover, the anticancer activity of scutellarin on human leukemia cells has also been investigated. For example, scutellarin could suppress viability, migration, and invasion of human leukemia cell line K562 via downregulated Raf/ MEK/ERK signaling pathway [20]. Meanwhile it has been founded scutellarin induced apoptosis in HL-60 cells by targeting AKT/MAPK signaling pathway and displayed antileukemic activity in HL-60, NB4, and U937 cell lines by affecting proteins related to GADD45, PTEN, and ILK signaling pathways [21]. In addition, scutellarin could also inhibit the proliferation of B-lymphoma Namalwa cells though inducing cell cycle arrest and down-regulating cyclin D1 and CDK4 expressions [22]. Furthermore, scutellarin is the main effective component of breviscapine, which has been used to treating cardiovascular and cerebrovascular diseases for many years [23]. The recent studies showed that scutellarin exhibited strong neuroprotective properties and produced a beneficial effect to neurodegenerative diseases, such as Alzheimer's disease [24] and Parkinson's disease [25]. Mechanism studies have shown that the neuroprotective potential of scutellarin attributed to its antioxidant activity [26]. Since oxidative stress is currently considered to be one of the key factors in the pathogenesis of these diseases [27]. The above evidence showed that scutellarin was worthy of further study as a lead compound.
However, the weak to moderate antitumor activity and neuroprotection, as well as poor pharmacokinetics properties limited its further application [28,29]. Esterification and amidation were effective approaches to improve the pharmacokinetics properties and biological activities of natural products, and it was anticipated that similar modifications could improve scutellarin activity [30].
On the basis of these reasons, a series of amide derivatives of scutellarin were designed and synthesized. Eleven anilines with different substitutions were introduced to carboxyl groups on sugars of scutellarin in order to enhance the bioavailability and bioactivity. Meanwhile, methylation or benzylation at 6-OH and 4′-OH were conducted to investigate the impact on the antitumor activity with different linking groups. The antiproliferative activities of all target compounds against two human leukemia cell lines, HL-60 and THP-1, were evaluated, and the structure-activity relationship were discussed. Furthermore, the neuroprotective activity of these derivatives was also investigated.

Chemistry
The synthetic route to target compounds (6a-k, 7a-k) is illustrated in Scheme 1. Scutellarin was used as the raw material, and compounds 2 and 3 were prepared by reacted with benzyl bromide and methyl iodide in the presence of K 2 CO 3 in dry DMF at room temperature for 24 h. Then they were hydrolyzed by KOH to generate the intermediates 4 and 5. Finally, various types of substituted anilines (including chloro, methyl, hydroxyl, methoxy) were treated with the derivatives (4 and 5) of scutellarin to give the target compounds 6a-k and 7a-k. The structures of all the derivatives were confirmed by 1 H NMR, 13 C NMR, and high resolution mass spectrum.

Biological evaluation
In vitro antitumor activity Twenty-two new amide derivatives of scutellarin (6a-k and 7a-k) were evaluated for their inhibitory effects against two different human leukemia cancer cell lines (human promyelocytic leukemia cell line HL-60 and human acute monocyte leukemia cell line THP-1), in comparison with scutellrein (1) and positive control 5-FU (5-fluorouracil) in each panel. The antiproliferative activities of benzylation and methylation at 6-hydroxyl and 4′-hydroxyl of scutellarin are summarized in Tables 1, 2, respectively. On the whole, most target compounds displayed more potent inhibitory activities than scutellarin, and some of them showed superior cytotoxic activities to 5-FU. Compounds 6a-k with benzyl groups at the 6-hydroxyl and 4′hydroxyl of scutellarin generally displayed more potential activities against HL-60 and THP-1 cells than corresponding ones with methyl groups. As shown in Table 1, the benzyl substituted compounds 6a-k showed significant antiproliferative activities against HL-60 and THP-1 cell lines with IC 50 values ranging from 1.7 to 58.86 μM and 5.75 to 35.37 μM, respectively. Compound 6g, 6i, and 6j performed a great activity in HL-60 cells than scutellarin and 5-FU, with an IC 50 value of 2.91, 1.70, and 4.28 μM, respectively. Whereas, compound 6f and 6g exhibited good activity in THP-1 cells than scutellarin and 5-FU, with an IC 50 value of 5.75 and 6.11 μM, respectively. Notably, the hydroxyl aniline compound 6g displayed the most potent antiproliferative activity among compounds 6a-k, with IC 50  values of 2.91 μM against HL-60 and 6.11 μM against THP-1, respectively. Especially, the IC 50 values of 6g is 7.8-fold more potent than scutellarin against HL-60 and 1.8-fold more potent than 5-FU. Compound 6f showed the most potent antiproliferative activity against THP-1, which is 8.4fold more potent than scutellarin and 1.3-fold more potent than 5-FU. As shown in Table 2, some methyl substituted compounds 7a-k showed prominent cytotoxic activities on HL-60 and THP-1 cell lines. Compound 7b, 7c, 7e, and 7j performed a great activity in HL-60 cells than scutellarin and 5-FU, with an IC 50 value of 3.37, 4.86, 5.49, and 2.95 μM, respectively. Whereas, compound 7b, 7c, and 7e exhibited good activity in THP-1 cells than scutellarin and 5-FU, with an IC 50 value of 7.02, 4.47, and 4.62 μM, respectively. Chloroaniline compounds 7b and 7c displayed more potent activities against HL-60 and THP-1 than scutellarin and 5-FU with IC 50 values of 3.37, 4.86 μM and 7.02, 4.47 μM, respectively. Especially 7c is nearly 11-fold more potent than scutellarin against THP-1 and 1.6-fold more potent than 5-FU. Moreover, methylaniline 7e showed comparable antitumor potency to 5-FU against HL-60 and more potent activities against THP-1. Compound 7j was the most potent compound against HL-60, which is 7.7-fold more potent than scutellarin and 1.8fold more potent than 5-FU.
According to the IC 50 values in 1 and 2, preliminary structure-activity relationship of amide derivatives of scutellarin could be summarized: (1) bulky benzyl group was preferred for antiproliferative activity, and the results were consistent with previous literatures [31,32]. (2) Compared to unsubstituted anilines, introduce various substituents can have a positive impact on the antiproliferative activity. (3) The substitutions of the Cl group (electron-withdrawing group) resulted in a significant improvement in the antitumor activity of 7a-k and an indistinct distinction for 6a-k. This seems to be due to the highly electronegative Cl can enhance a number of pharmacokinetic and physicochemical properties such as improved metabolic stability and enhanced membrane permeation. (4) The antiproliferative activities of compounds with orthoor metasubstituted anilines generally showed better antiproliferative activity than para-substituted ones. This seems to the electron-donating effect of the methyl/hydroxyl/methoxy groups group at the orthoand metapositions provided stronger affinity with the target than para-substituted ones. (5) There is no discernible difference among different types of electron-donating substituents according to the data of antiproliferative activity.

In vitro neuroprotective activity
Scutellarin, as main active ingredient of breviscapine injection, has been used in China to treat acute cerebral infarction and cerebral hemorrhages for many years in clinic [33]. Modern pharmacological studies have demonstrated that scutellarin exerts neuroprotective effect though antioxidant and anti-inflammatory actions. Thus, their neuroprotective effects against H 2 O 2 -induced oxidative injury in PC12 cells were evaluated using MTT test. The neuroprotective effects of 6a-k and 7a-k are summarized in Figs. 1, 2 respectively.
It can be inferred from Fig. 2 that compared to 6a-k, methyl substituted derivatives 7a-k showed improved neuroprotective activity, especially compound 7a-h induced an obvious increase in cell viability compared to model group, which is similar to scutellarin.

Conclusion
In this study, 22 derivatives (6a-k and 7a-k) of scutellarein were designed and synthesized. All the target compounds  were first evaluated for their antiproliferative activities against HL-60 and THP-1 cell lines. Among them, compounds 6g and 7c exhibited the strongest antiproliferative potency against HL-60 and THP-1 cell lines with an IC 50 values of 2.91, 6.11 μM, and 4.86, 4.47 μM, respectively. Furthermore, the neuroprotective effects of all compounds against H 2 O 2 -induced oxidative injury in PC12 cells were also investigated. Most of compounds distinctly improved the cell viability rates of PC12 compared to the model group. In conclusion, these findings were important to explore new chemical entities based on scutellarin for the treatment of leukemia or neurological diseases.

Materials
Chemicals and solvents were purchased from commercial sources. Further purification by standard methods were employed when necessary. 1 H NMR and 13 C NMR spectra were recorded on a Bruker ARX-400 NMR spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts were expressed in δ values (ppm) and the coupling constants (J) in Hz. HRESIMS data were obtained using Bruker micro-TOFQ-Q mass spectrometer.

General procedures to synthesize 6a-k and 7a-k
The intermediates 4 and 5 were synthesized according to our previous report [28]. A mixture of 4 (0.16 mmol) and HOBt (0.19 mmol), or 5 (0.16 mmol) and HOBt (0.19 mmol) in anhydrous DMF (5 mL) was stirred at room temperature for 0.5 h. After the addition of various types of substituted anilines (0.19 mmol) and EDCI (0.32 mmol) to the solution, the mixture was further stirred at room temperature for another 24 h. Then, the mixture was poured into 30 mL of H 2 O, and extracted with EtOAc (3 × 20 mL). The organic layer was combined, washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude product was purified using silica gel column chromatography eluting with dichloromethane/methanol system (20:1, v/v).