Synthesis and protective effect of new ligustrazine-vanillic acid derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells

Ligustrazine-vanillic acid derivatives had been reported to exhibit promising neuroprotective activities. In our continuous effort to develop new ligustrazine derivatives with neuroprotective effects, we attempted the synthesis of several ligustrazine-vanillic acid amide derivatives and screened their protective effect on the injured PC12 cells damaged by CoCl2. The results showed that most of the newly synthesized derivatives exhibited higher activity than ligustrazine, of which, compound VA-06 displayed the highest potency with EC50 values of 17.39 ± 1.34 μM. Structure-activity relationships were briefly discussed.Graphical abstract New series of ligustrazine-vanillic acid amide derivatives were synthesized and evaluated for their protective effect on the injured PC12 cells damaged by CoCl2. VA-06 was found to be the most active one


Background
Ischemic stroke is one of the leading causes of death and disability in the world [1][2][3]. It is clear that even a brief ischemic stroke may trigger complex cellular events that ultimately lead to the neuronal cell death and loss of neuronal function [1,4,5]. Although remarkable progress has been made in treating stroke, effective approaches to recover damaged nerve are not yet to be found [6][7][8][9]. Therefore, it is necessary to develop new generation of neuroprotective agents with neural repair-promoting effect.
Ligustrazine (tetramethylpyrazine, TMP) ( Fig. 1) is a major effective component of the traditional Chinese medicine Chuanxiong (Ligusticum chuanxiong hort), which is currently widely used in clinic for the treatment of stroke in China. It has been reported to show beneficial effect on ischemic brain injury in animal experiments and in clinical practice [10][11][12][13][14].
In continuation of our research, we decided to undertake a study of the ligustrazinyl amides, because amides relatively have metabolic stability when compared to ligustrazinyl esters [24]. In this study, we reported the design, synthesis of the novel T-VA amide analogues containing different types of amide fragments, as well Open Access *Correspondence: wpl581@126.com; hm_lei@126.com 1 School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China Full list of author information is available at the end of the article as in vitro neuroprotective activities screening on the injured PC12 cells. And the structure-activity relationships (SARs) of these novel compounds were also briefly discussed.

Protective effect on injured PC12 Cells
Setting ligustrazine and T-VA as the positive control drug, the neuroprotective activity of target compounds was evaluated on the neuronal-like PC12 cells damaged by CoCl 2 . The results, expressed as proliferation rate (%) at different concentration and EC 50 , were summarized in Table 2. As shown in Table 2, most of the ligustrazine-vanillic acid amide derivatives showed better protective effects than the positive control drug TMP (EC 50 = 64.35 ± 1.47 µM) on injured differentiated PC12 cells. Among the candidates, the compound VA-06  From the obtained results, it was observed that esterification at the carboxylic group of vanillic acid may contribute to enhance the neuroprotective activity, such as VA-01 > VA-02. This was in agreement with our previous research [20]. It should be noticed that introduction of a large lipophilic aromatic amine residue leaded to complete loss of neuroprotective activity (with exception of VA-06), such as VA-13-VA-16. But the compounds that introduced an aromatic amine residue at the carboxylic group of vanillic acid performed better neuroprotective activities than VA-02 without any group substituted, such as VA-03, VA-04, VA-05, VA-08 > VA-02. Furthermore, the structure-activity relationship analysis among the T-VA aromatic amide derivatives revealed that the neuroprotective activities were mainly influenced by the type, but not the alkyl chain length of amine substituents, as exemplify by VA-04 > VA-03, VA-05. Although none of the newly synthesized T-VA derivatives showed more effect than the positive control drug T-VA, the structure-activity relationship (SAR) analysis above provided important information for further design of new neuroprotective ligustrazine derivatives.

Protective effect of VA-06 on injured PC12 cells
To further characterize the protective effect of VA-06 on injured PC12 cells, the cell morphology changes were observed under an optical microscopy. As shown in Fig. 2, the morphology of undifferentiated PC12 cells was normal, the cells were small and proliferated to form clone-like cell clusters without neural characteristics ( Fig. 2A); By exposure to NGF, normal differentiated PC12 cells showed round cell bodies with fine dendritic networks similar to those nerve cells (Fig. 2B). Moreover, the mean value expressed as percent of neurite-bearing cells in NGF treated cells was 65.4% (Fig. 3). When the differentiated PC12 cells treated with 250 mM CoCl 2 for 12 h, almost all cells showed typical morphological changes such as cell body shrinkage and the disruption of the dendritic networks (Fig. 2C); the mean value of neurite-bearing cells (9.4%, Fig. 3) showed a significant decrease. While pretreatment with 60 μM VA-06 before delivery of CoCl 2 dramatically alleviated the damage caused by CoCl 2 to cell morphology ( Fig. 2D) and showed significant difference in the number of neuritebearing cells (47.5%, Fig. 3) from that of CoCl 2 treatment alone.

Conclusions
In this study, we successfully synthesized 20 novel T-VA amide derivatives by combining T-VA with different amines. Their protective effects against CoCl 2 -induced neurotoxicity in differentiated PC12 cells were determined by the MTT assay. The result indicated that most of T-VA amide derivatives showed protective effects on injured differentiated PC12 cells. Among them, a large portion of the derivatives were more active (with lower EC 50 values) than the positive control drug TMP, of which compound VA-06 displayed the highest neuroprotective effect with EC 50 values of 17.39 ± 1.34 µM.
Although none of the newly synthesized T-VA derivatives showed more effect than the positive control drug T-VA, the results enriched the study of ligustrazine derivatives with neuroprotective activity. Further bioassay of compound VA-06 on neuroprotective activity on animal models is underway.

Chemistry
Reagents were bought from commercial suppliers without any further purification. Melting points were measured at a rate of 5 °C/min using an X-5 micro melting point apparatus (Beijing, China) and were not corrected. Reactions were monitored by TLC using silica gel coated aluminum sheets (Qingdao Haiyang Chemical Co., Qingdao, China). NMR spectra were recorded on a BRUKER AVANCE 500 NMR spectrometer (Fällanden, Switzerland) with tetramethylsilane (TMS) as an internal standard; chemical shifts δ were given in ppm and coupling constants J in Hz. HR-MS were acquired using a Thermo Sientific TM LTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY, USA). Cellular morphologies were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan).

Synthesis of (3,5,6-trimethylpyrazin-2-yl)methyl 4-methylbenzenesulfonate (2)
To a solution of compound 1 (7.0 g, 46.3 mmol) and KOH (2.6 g, 46.3 mmol) in dry THF (100 ml), Tscl (8.82 g, 46.3 mmol) was added, then the mixture was stirred at 25 °C for 15 h. After completion of the reaction (as monitored by TLC), the reaction mixture was poured into water and the crude product was extracted with dichloromethane (3 × 100 ml), the combined organic layers were washed with brine (100 ml), anhydrous Na 2 SO 4 , filtered and the solvents were evaporated under vacuum. The crude products were purified by flash chromatography (Petroleum ether:Ethyl acetate = 4:1) to produce a white solid. The crude product, with 90% purity, was not purified further.

Synthesis of methyl 4-hydroxy-3-methoxybenzoate (3)
To a solution of vanillic acid (5.502 g, 32.7 mmol) in dry MeOH (100 ml), 3 ml SOCl 2 was added gradually with stirring and cooling. Upon completion of the addition, the mixture was stirred at 25 °C for 15 h. After completion of the reaction (as monitored by TLC), the reaction mixture was evaporated under vacuum to produce a white solid. The crude product, with 95% purity, was not purified further.
After drying the organic layer over anhydrous Na 2 SO 4 and evaporating the solvent under vacuum, the crude products were purified by flash chromatography (Dichloromethane: methyl alcohol = 40:1) to produce a white solid.

Cell culture
PC12 cells were obtained from the Chinese Academy of Medical Sciences & Peking Union Medical College. The cultures of the PC12 cells were maintained as monolayer in RPMI 1640 supplemented with 10% (v/v) heat inactivated (Gibco) horse serum, 5% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin (Thermo Technologies, New York, NY,USA) and incubated at 37 °C in a humidified atmosphere with 5% CO 2 . T-VA amide derivatives were dissolved in dimethyl sulfoxide (DMSO).

Protective effect on damaged differentiated pc12 cells
The neuroprotective effect of newly synthesized T-VA amide derivatives was evaluated in vitro via the MTT method on the differentiated PC12 cells damaged by CoCl 2 with ligustrazine as the positive control. PC12 cells growing in the logarithmic phase were incubated in the culture dishe and allowed to grow to the desired confluence. Then the cells were switched to fresh serum-free medium and incubated for 14 h. At the end of this incubation, the PC12 cells were collected and resuspended in 1640 medium supplemented with 10% (v/v) fetal bovine serum, then the cells were seeded in poly-l-lysine-coated 96-well culture plates at a density of 7 × 10 3 cells/well and incubated for another 48 h in the presence of 50 ng/ml NGF.
The differentiated PC12 cells were pretreated with serial dilutions of T-VA amide derivatives (60, 30, 15, 7.5, 3.75 µM) for 36 h, and then exposed to CoCl 2 (final concentration, 250 mM) for another 12 h. Control differentiated cells were not treated with T-VA amide derivatives and CoCl 2 . At the end of this incubation, 20 μl of 5 mg/ml methylthiazol tetrazolium (MTT) was added to each well and incubation proceeded at 37 °C for another 4 h. After the supernatant medium was removed carefully, 200 μl dimethylsulphoxide (DMSO) were added to each well and absorbance was measured at 490 nm using a plate reader (BIORAD 550 spectrophotometer, Bio-rad Life Science Development Ltd., Beijing, China). The proliferation rates of damaged PC12 cells were calculated by the formula [OD 490 (Compd) − OD 490 (CoCl 2 )]/[OD 490 (NGF) − OD 490 (CoCl 2 )] × 100%; The concentration of the compounds which produces a 50% proliferation of surviving cells corresponds to the EC 50 . And it was calculated using the following equation: −pEC 50 = log C max − log 2 × (∑P − 0.75 + 0.25P max + 0.25P min ), where C max = maximum concentration, ∑P = sum of proliferation rates, P max = maximum value of proliferation rate and P min = minimum value of proliferation rate [20][21][22].

Observation of morphologic changes
The changes in cell morphology after treatment with VA-06 were determined using light microscopy in this assay, it was performed as previously described [22]. Differentiation was scored as the cells with one or more growth cone tipped neurites greater than 2 cell bodies in length. The cell differentiation rate was calculated by the formula [the number of differentiated cells]/[the number of total cells] × 100%. Three fields were randomly chosen from different wells of three independent experiments. All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using SAS version 9.0 (SAS Institute Inc., Cary, NC, USA). Between-groups differences were assessed using Student t tests and p < 0.05 was considered significant.