A Concise Synthesis of Three Branches Derived from Polysaccharide RN1 and Anti-Pancreatic Cancer Activity Study

Abstract

RN1, a polysaccharide from flowers of Panax pseudo-ginsieng Wall. Var. notoginseng (Burkill) Hoo & Tseng, is a potential multi-targeting drug candidate for pancreatic cancer treatment. However, the active targeting domain of RN1 is still unknown. Herein, three RN1 derived branches were synthesized via [3+2] or [2+2] strategies, efficiently. Two pentasaccharides, 18 and 27, showed similar inhibition effect on pancreatic cancer BxPC-3 cells to that of RN1 at same concentration. Interestingly, tetrasaccharide 21 potently inhibited gemcitabineresistant cell line Panc-1 at high concentration. These suggest that the branches of RN1 might be the active targeting domain and tetrasaccharide 21 might be a potential leading compound for pancreatic cancer with gemcitabine resistance.

1. Introduction

Pancreatic cancer is a cancer with high mortality, which is close to incidence [1]. Gemcitabine is currently one of the major treatments for pancreatic cancers, apart from operation in clinical. However, the response rate of gemcitabine had been decreased year by year [2]. Despite new adjuvant gemcitabine, gemcitabine combination therapy, and new active compounds were reported [3,4,5,6,7], the low survival rate of patients with pancreatic cancer never changed essentially. Carbohydrate-based drug possess the specific advantage in cytotoxicity because of the consisting of saccharide residues. Sugar drugs usually function in a specific mechanism and offer new opportunities for drug discovery [8].

Recently, an arabinogalactan polysaccharide RN1, a multi-targeting polysaccharide from flowers of Panax notoginseng that has anti-pancreatic cancer activity, was reported in this lab [9,10]. The complexity of the structure of polysaccharides prevent their investigation of polysaccharide, including drug discovery. The possible unknown impurities in natural extract polysaccharides may cause adverse effects. In addition, quality control of polysaccharide-based drugs is always a challenge. So, discovering the active domain of polysaccharide RN1 and synthesis the corresponding structure and well-defined sugar chains are necessary. As shown in Figure 1a, the putative repeating unit of RN1 is a Gal-β-1,6-Gal tetrasaccharide backbone carrying one of the two possible arabinogalactan branches [9]. In this paper, we chemically synthesized two branches of RN1 to explore the role of branches in anti-pancreatic cancer. Besides, a common tetrasaccharide 21 of these two branches was synthesized to compare with the other two branch compounds (18, 27 in Figure 1b). Certainly, it’s better to protect the reducing end of these three oligosaccharides with benzyl than free hydroxyl, because of the possible π–π interacting with targeting proteins. There are also other choices in the reducing end, but the structure-activity relationship would be investigated in the future.

2. Materials and Method

2.1. General Experimental Procedures

All chemical reagents (AR) and solvents (AR) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China) and used without further purification. Reactions were monitored by TLC (Thin layer chromatography) on glass Silica Gel HSGF254 (Sinopharm Chemical Reagent Co., Shanghai, China) plates with UV 254 nm detection. TLC staining for carbohydrate samples was performed by dipping the plates into 10% H₂SO₄ in ethanol and drying with a heat gun. Anhydrous dichloromethane (DCM) was freshly distilled from calcium hydride under nitrogen prior to use. Molecular sieves 4Å powder was purchase from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA), and was activated by heating at 200 °C in vacuum for 2 h. Nuclear magnetic resonance (NMR) spectra were measured on a Varian-MERCURY Plus (400 MHz) (Varian, Inc., Palo Alto, CA, USA) and Bruker AVANCE III (500 MHz) (Bruker Corporation, Billerica, MA, USA). The chemical shifts were assigned in ppm and the coupling constants in Hz. ESI-HRMS spectra were measured on an Agilent 6230 LC-TOF MS spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA).

Detail experimental operations and the corresponding data are available in the supplementary materials. The structures of each new compound were shown in Figures S2–S15 in Supplementary Materials. The pictorial form of NMR data of each new compound were shown in Figures S16–S74. These figures were arranged according to the numeric name of new compounds from small to large.

2.2. Cells and Materials

All cells were obtained from the Cell Bank in the Type Culture Collection Center of the Chinese Academy of Sciences, Shanghai, China. The human pancreatic ductal adenocarcinoma cell lines Panc-1, BxPC-3 and the normal hepatic cell line LO2 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All reagents used for the cell culture studies were purchased from Gibco (Pittsburgh, PA, USA). All cells were maintained at 37 °C, in a humidified atmosphere, with 5% CO₂ in air, and subcultured every 3–5 days. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, used to determine cell viability, was obtained from Sigma-Aldrich (Louis, MO, USA).

2.3. Cell Viability Assessment

Cell viability was determined using MTT assays. Briefly, cancer cells and normal liver cells were treated with certain concentrations of compounds (0–500 μM) for 72 h. MTT (20 μL, 0.5 mg/mL) was added and incubated for another 4 h, and then the supernatant was removed and the dye crystals were dissolved in 200 μL DMSO. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

3. Result and Discussion

3.1. Retrosynthetic Analysis

As shown in Figure 2, these three oligosaccharides were synthesized via [3+2] or [2+2] strategies. Trisaccharides or disaccharide donors were used for the corresponding target oligosaccharides. Gal-Gal disaccharide 14 was shared as common acceptor. With the protection of 4,6-O-benzylidene, 3-OH will be more active, facilitate further glycosylation. Donor 7 and donor 9 were reacted with acceptor 14 through directly activating p-tolyl under NIS/TMSOTf (N-Iodosuccinimide/Trimethylsilyl Triflate). But, donor 25 was a trichloroacetimidate, for the failure of the synthesis of its p-tolyl structure.

3.2. Synthesis of Donors 7, 9, and Acceptor 14

To prepare the l-arabinose trisaccharide donor for petasaccharide 18, full Bz protected l-Ara donor 1 (Scheme 1) and the known acceptor 6 were used at the beginning according to the published d-arabinose trisaccharide by employing a full Bz protected d-Ara donor [11]. Interestingly, donor 1 cannot work in the glycosylation, but results in a thiolphenol byproduct. A similar result had also been reported in the synthesis of glucans [12,13]. According to the previous work [14], Bz is a disarmed protect group, which reduces the reactivity of the donor. Additional two Bz lead to the disparity of the reactivity of donor 1 and acceptor 6, so then p-tolylthiol was transferred. In the next step, a more reactive l-Ara, donor 5, was designed. The 5-O-Bz of donor 1 was replaced by TBS (t-Butyldimethylsilyl). There were three reasons to choose TBS: (1) TBS can selectively protect primary hydroxyl in high yield; (2) TBS is a good armed protect group, even better than benzyl [15]; (3) TBS can be removed under acidic condition together with benzylidene of Gal to simplify deprotection. The synthesis of donor 5 start from the known compound 2 [16], over four steps. Controlling the ratio of donor 5 and acceptor 6 in glycosylation may lead to different yields of trisaccharide 7 and disaccharide 8. The free hydroxyl of disaccharide 8 was then Levprotected to afford disaccharide donor 9 under EDCI/DMAP/LevOH (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride/4-Dimethylaminopyridine/Levulinic acid).

To prepare disaccharide acceptor 14, donor 10 and the known acceptor 11 [17] were reacted under TMSOTf at −78 °C to afford disaccharide 12. Low temperature is necessary in this reaction. Activating of 12 to couple with benzyl alcohol, and the resulting mixture continued to deprotect Lev without further purification, to afford important acceptor 14.

3.3. Exploration of the Synthesis of the Gal-Containing Trisaccharide Donor

It is difficult to synthesize the remaining Gal-containing trisaccharide donor. Similar to trisaccharide 7, disaccharide 8 was used as acceptor, along with two easily prepared and one already available Gal donors—15, 16 and 10—but none of them can afford the target trisaccharide in a major product, instead a thiolphenol byproduct (Scheme 2). Perhaps more active Gal donors would work, such as p-Tol 2-O-Benzoyl-3,4,6-O-tribenzylthiogalactoside, but access to those donors need additional several steps and extra deprotection of benzyl will be needed, which will make the synthetic route lower efficient.

3.4. Synthesis of Target Compound 18, 21

With donors 7, 9 and acceptor 14 in hand, pentasaccharide 17 and tetrasaccharide 19 were prepared via thiolphenol glycosidic method under NIS/TMSOTf. The resulting reaction mixture of 19 then removed Lev to afford 20 without further purification. The deprotection of 17 and 20 was similar. 17 or 20 was treated with 80% acetic aqueous solution at 80 °C to remove TBS and benzylidene, and the acyl groups were removed in methanol at pH about 10, the resulting mixture was purified by “P2” column to afford pentasaccharide 18 and tetrasaccharide 21, respectively. Tetrasaccharide 20 was also tried as acceptor for pentasaccharide 20a. Probably, for the steric hindrance, neither donor 10 nor donor 22 work, not to mention lower active donors 15 and 16.So, the challenging glycosidic bond should be formed with a disaccharide acceptor (Scheme 3).

3.5. Synthesis of the Target and Challenging 27

As shown in Scheme 4, in order to avoid the thiophenol byproduct, disaccharide 9 was coupled with p-methoxybenzyl alcohol and subsequently to remove Lev without further purification to afford a new disaccharide, acceptor 23. Compared to donor 15, donor 16 was more active and obtained much higher yields to afford 24. The PMB (4-Methoxybenzylchloride) of 24 was removed by DDQ (1,2-Dichloro-4,5-dicyanobenzoquinone) and then converted into trichloroacetimidate to afford trisaccharide donor 25. Then, 25 was successfully coupling with common disaccharide acceptor 14 to afford 26 in good yield, and through a similar deprotection to 17 and 20 to afford the remaining target pentasaccharide 27.

3.6. Anti-Pancreatic Cancer Activity Test

Similar to RN1, these three saccharides were then tested in pancreatic cancer cell line BxPC-3. As shown in Figure 3a, pentasaccharides 18 and 27 exhibited about 55% inhibition effect on BxPC-3 at around concentrations of 500 μM (414 μg/mL for 18 and 429 μg/mL for 27), which is similar to the published RN1 polysaccharide at same mass concentration. But, activity of tetrasaccharide 21 is not well than that of the pentasaccharides in BxPC-3. Meanwhile, these three oligosaccharides were also tested in the gemcitabineresistant pancreatic cancer cell line Panc-1. Surprisingly, tetrasaccharide 21 exhibit nearly 85% inhibition at 500 μM, which is similar to gemcitabine (Figure 3b). In Figure 3b, tetrasaccharide 21 exhibited different linear relationship to the other three compounds. This result suggested that 21 probably targeted a specific molecule which was expressed in Panc-1 cells, but not in BxPC-3 and LO2 cells, while such interaction might require high concentrations. These three oligosaccharides were also tested in AsPC-1 cell line, and tetrasacchride 21 exhibit similar effect on AsPC-1 cell line to that of gemcitabine (see Figure S1 in the Supplementary Materials). In addition, these three saccharides showed scarce cytotoxicity effect on the LO2 cell line (Figure 3c), which show great advantage to that of gemcitabine. Although there’re already some compounds for gemcitabineresistant pancreatic cancer, such as nocodazole and combretastatin [5,7], comparatively, sugar compounds often show low or even no cytotoxicity. So, 21 can be a potential novel leading compound for gemcitabine-resistant pancreatic cancer.

4. Conclusions

We had synthesized three branches derived from RN1 polysaccharide via [3+2] and [2+2] strategies. Disaccharide 14 was shared as a common acceptor, and different donors derived from 8 were employed. Many intermediates were shared to make the synthetic route more economical. The deprotection was also perform in an efficient way, requiring only two steps, for TBS can be removed with benzylidene together. The thiolphenol byproducts were solved by employing a more active donor (Scheme 1) or a more stable acceptor (Scheme 2).

The target three oligosaccharides were tested in two different pancreatic cancer cell lines. Pentasaccharides 18 and 27 exhibited similar inhibition at the same mass concentration (about 500 μg/mL) to the published RN1 in BxPC-3. Additionally, tetrasaccharide 21 exhibited high inhibition at 500 μM in Panc-1. All these three RN1derived branches show almost no cytotoxicity in LO2. In conclusion, we’ve found the possible active targeting domain of RN1 (18 and 27) and a potential leading compound (21) for pancreatic cancer with gemcitabine resistance. The biological mechanism will be investigated in the future.