European Journal of Medicinal Chemistry 97 (2015) 235e244
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and antitumor activity of triterpenoid pyrazine derivatives from 23-hydroxybetulinic acid Hengyuan Zhang a, b, Yiwei Wang a, b, Peiqing Zhu a, b, Jie Liu a, c, Shengtao Xu a, b, Hequan Yao a, b, *, Jieyun Jiang d, Wencai Ye e, Xiaoming Wu a, b, Jinyi Xu a, b, * a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China d Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA e College of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2014 Received in revised form 26 April 2015 Accepted 27 April 2015 Available online 29 April 2015
Pyrazine-fused 23-hydroxybetulinic acid was synthesized by introducing a pyrazine ring between C-2 and C-3 position and further modifications were carried out by substitution of C-28 carboxyl group by ester and amide linkage to enhance the antitumor activity. The biological screening results showed that all of the derivatives exhibited more significant antiproliferative activity than the parent compound. In particular compound 12a exhibited the most potent activity with IC50 values of 3.53 mM, 4.42 mM and 5.13 mM against cell lines SF-763, B16 and Hela, respectively. In the preliminary mechanism study, 12a caused cell arrest in G1 phase and significantly induced apoptosis of B16 cells in a dose-dependent manner. Furthermore, the in vivo antitumor activity of 12a was validated (tumor inhibitory ratio of 55.6% and 62.7%, respectively) in mice with H22 liver cancer and B16 melanoma. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Triterpenoid 23-Hydroxybetulinic acid Pyrazine Apoptosis Antitumor activity
1. Introduction Natural products have been the single most productive source of leads for the development of drugs for the treatment of cancer. In fact, the majority of antitumor agents are of natural origin [1]. Betulinic acid (3-Hydroxy-lup-20(29)-en-28-oic acid, Fig. 1) is a pentacyclic lupane-type triterpene that is widely distributed throughout the plant kingdom. A variety of biological activities have been ascribed to betulinic acid including anti-inflammatory, antibacterial, antimalarial and antioxidant properties. However, betulinic acid is most highly regarded for its anti-HIV-1 activity and specific cytotoxicity against a variety of tumor cell lines. Previous experimental and epidemiological studies have indicated that betulinic acid as well as its analog- 23-hydroxybetulinic acid (HBA, 1, Fig. 1) may be developed as potent anti-HIV and antitumor drugs [2e4].
* Corresponding authors. State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China. E-mail addresses: [email protected] (H. Yao), [email protected] (J. Xu). http://dx.doi.org/10.1016/j.ejmech.2015.04.057 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
The 23-hydroxybetulinic acid, isolated from the root of Pulsatilla chinensis, displayed similar anti-HIV and antitumor activities as its analog betulinic acid [5]. To date numerous derivatization studies have been performed on betulinic acid leading to the production of an array of betulinic acid derivatives [3]. Nevertheless, few studies have been devoted to the structural modifications of 23hydroxybetulinic acid. In general, due to various reasons original natural products are not particularly good candidates, and a need therefore exists for improving their drug-like properties by chemical modification [1]. It has been found that heterocyclic ring-fused triterpenoids at C-2 and C-3 position impart the desired characteristics [6]. Nitrogen heterocycles are important pharmacophores in drug design, especially pyrazine derivatives, which are among the most frequently cited heterocyclic compounds [7]. Previously, Urban et al. described the partial synthesis of triterpenic heterocycles, in which some lupane pyrazine derivatives were significantly cytotoxic in vitro to warrant the extension of the in vitro studies to in vivo testing in mice [8]. The cytotoxic activity of some triterpenic compounds was also successfully increased by using heterocyclic modifications [9e11].
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H. Zhang et al. / European Journal of Medicinal Chemistry 97 (2015) 235e244
Fig. 1. Chemical structures of betulinic acid and 23-hydroxybetulinic acid.
Previously, a set of C-17-carboxylic acid-modified 23hydroxybetulinic acid ester and amide derivatives have been reported by our group [12,13]. The promising anti-proliferative of triterpenic pyrazines encouraged us to continue to design and synthesize novel betulinic pyrazine derivatives from 23hydroxybetulinic acid. Base on the structureeactivity relationships (SARs) studies of betulinic acid and 23-hydroxybetulinic acid, further modifications were carried out by substitution of C-28 carboxyl group by ester and amide linkage to enhance the antitumor activity. The obtained derivatives were evaluated for their in vitro antitumor activity against five cancer cell lines. Furthermore, the in vivo antitumor activity of the representative compound was validated in mice with H22 liver cancer and B16 melanoma. The primary antitumor mechanism was also investigated. 2. Results and discussions 2.1. Chemistry The 23-hydroxybetulinic acid was isolated from the roots of P. chinensis (Bge) Regel [5,14], and characterized by 1H NMR, 13C NMR and high-resolution mass spectra. As described in Scheme 1, it was treated with BnBr and K2CO3 in DMF to yield 28-benzyl-23hydroxybetulinic ester 2, the hydroxyl group of 2 was protected using tert-butyldimethylsilyl chloride in the presence of DMAP to give siloxane 3. Oxidation of 3 with pyridinium chlorochromate afforded the ketone 4, which was then converted to pyrazine derivative 5 in the presence of ethylenediamine and sulfer in morpholine [15]. Deprotection of 5 with hydrochloric acid in acetone gave alcohol 6. The NMR spectrum displayed readily recognizable signals for the fused pyrazine ring [proton signals at d 8.35 (H-20 ), 8.38 (H-10 ) and d 2.45 (H-1), 3.08 (H-10 ) and olefinic carbon signals at d 152.3 (C-2), 157.8 (C-3), 142.2 (C-10 ), 141.5 (C-20 )]. Previous study indicated that substitution of C-23 hydroxyl group may benefit the potency [16]. Therefore, a simple acetyl group was introduced to this position and selected for further investigation. The carboxylic pyrazine 8 was obtained by debenzylation of ester 7 in THF with Pd/C as catalyst under atmospheric pressure of hydrogen. Following deacetylation of 8 produced pyrazine derivative 9. Derivatives 10a-10e were synthesized by the reaction of compound 9 with corresponding bromides under basic condition. Hydrolysis of derivative 10d and 10e afforded compound 10f and 10g. To synthesize derivatives 12a-12d, C-28 carboxylic group was converted to acyl chloride intermediate 11, which was further reacted with the corresponding alcohols and amides (Scheme 2). 2.2. Biological evaluation 2.2.1. In vitro antiproliferative activity The in vitro antiproliferative activity of these novel betulinic
pyrazine derivatives was evaluated on five cancer cell lines (HL-60 human promyelocytic leukemia cell, BEL-7402 human hepatocellular carcinoma, HeLa human cervical adenocarcinoma, SF-763 human brain adenocarcinoma and B16 mice melanoma cells) by MTT assay with doxorubicin as the positive control. The results summarized in Table 1 are presented as the concentration of drug inhibiting 50% cell growth (IC50). The data indicated that all of the derivatives exhibited stronger antiproliferative activities than parent compound 23hydroxybetulinic acid in the five cancer cell lines, especially against human brain adenocarcinoma SF-763 and mice melanoma B16 cells. Pyrazine-fused compound 9 was about 2- to 3-fold more potent against five cancer cell lines than HBA, indicating that introduction of the pyrazine ring imparted potent antiproliferative activity. The acetylated derivative 8 showed lower IC50 values than compound 9, which is consistent with the case of benzoylation [16]. Compounds 10f, 12a and 12d were the most promising derivatives with IC50 values lower than 10 mM on all tested cell lines, in particular compound 12a was about 7- to 10-fold more potent against five cancer cell lines than HBA and 2- to 4-fold than 9. These findings revealed that electron-donating and/or polar substituents especially groups bearing the amide linkage at C-28 side chain would benefit the potency. The results also demonstrated that the existence of a structural constraint such as benzyl, cyclohexyl, biphenyl and phenyl at C-28 is favorable to the inhibitory activity, which is exemplified by compounds 6, 10g, 10f and 12d. The obtained structureeactivity relationships of C-28 position have also confirmed the results from previous comparative molecular field analysis (CoMFA), in which the contour maps illustrated that bulky and/or electron-donating groups at C-28 would be favorable to the antitumor activity [17]. Furthermore, selectivity of compound 12a was assessed on normal human hepatocyte (HL-7702 cell line). The selectivity index was calculated by IC50 value in HL-7702 cell line (75.43 ± 2.05 mM) divided by IC50 value in cancer cell lines (see supplementary material). It was observed that 12a was 11e20 times more selective towards cancer cells than to non-tumor cell. The doseeresponse curves for the test compound towards different tumor cell lines and non-tumor (HL-7702) cell line are given in Fig. 2. 2.2.2. Compound 12a induces cell cycle arrest To determine whether the suppression of cell growth by triterpenic pyrazines is caused by a cell-cycle effect, we detected the DNA content of cell nuclei by flow cytometry (Fig. 3). B16 cells were treated with compound 12a at concentrations of 1.25, 2.5, and 5 mM, which resulted in accumulation of 29.67, 34.67, and 33.46% of cells at the G1 phase, respectively. These results indicated that compound 12a inhibited the growth of the cancer cells by inhibiting the cell cycle via G1-phase arrest. 2.2.3. Compound 12a induces apoptosis To clarify whether the loss of cancer cell viability promoted by triterpenic pyrazines is associated with apoptosis, an annexin VeFITC/propidium iodide (PI) binding assay was performed. As shown in Fig. 4, compound 12a exhibited potent dose-dependent activity in the induction of apoptosis. Treatment of B16 cells with 12a at 1.25, 2.5, and 5 mM for 72 h resulted in 16.51, 31.62, and 57.44% apoptotic cells (early and late), as compared with 5.66% in an untreated vehicle control, indicating that compound 12a was able to induce apoptotic cell death in B16 cells. 2.2.4. In vivo antitumor activity of compound 12a Based on the in vitro results and intensive mechanistic studies, we further tested the antitumor activity of compound 12a in vivo by performing an assay in mice with H22 liver cancer and B16
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Scheme 1. Synthesis of pyrazine-fused 23-hydroxybetulinic acid. Reagents and conditions: (a) BrBn, K2CO3, DMF, rt, 10 h; (b) TBSCl, DMAP, CH2Cl2; rt, 4 h (c) PCC, CH2Cl2, rt, 4 h; (d) sulfur, ethylenediamine, morpholine, reflux, 4 h; (e) 10% HCl, acetone, rt, 10 h; (f) Ac2O, pyridine, rt, 3 h; (g) H2, 10% Pd/C, THF, rt, 2 h; (h) 4 N NaOH, THF/CH3OH, rt, 1 h.
Scheme 2. Synthesis of 23-hydroxybetulinic pyrazine analogs 10a-10h and 12a-12d. Reagents and conditions: (a) R1Br, K2CO3, DMF; (b) oxalyl chloride, cat. DMF, CH2Cl2, rt, 3 h; (c) R2X (X ¼ O, NH), DMAP, CH2Cl2. (d) triethylamine, H2O, CH3OH/THF, rt, 6 h; (e) NaOH, H2O, CH3OH/THF, reflux, 1 h.
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Table 1 Antiproliferative activities of triterpenic pyrazine analogs against different tumor cell lines. Comp.
Cell lines (IC50a, mM ± SD) HL-60
b
HBA 6 8 9 10a 10b 10c 10e 10f 10g 10h 12a 12b 12c 12d ADMc a b c
45.15 18.89 20.35 24.14 22.51 23.56 19.11 23.62 11.69 21.06 19.52 7.11 14.71 22.15 10.19 0.17
BEL-7402 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.11 2.24 1.41 3.42 1.33 3.04 2.05 1.41 1.54 1.19 1.88 0.36 1.80 2.02 0.76 0.01
39.67 10.05 16.79 19.10 14.76 17.41 11.88 16.99 9.37 17.65 13.10 7.03 11.06 15.73 8.09 0.22
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.22 2.01 0.92 2.07 1.09 1.70 0.89 1.36 22.01 1.49 1.53 0.22 1.04 1.72 0.33 0.02
SF-763 43.40 9.54 11.09 15.43 10.02 13.53 8.62 13.14 5.63 10.97 8.86 3.53 9.26 13.03 5.77 0.16
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
HeLa 7.20 1.32 1.67 1.46 1.21 4.12 0.61 1.79 0.44 0.89 0.92 0.19 0.02 1.26 0.25 0.03
52.39 12.80 14.61 19.57 15.09 20.74 10.96 17.01 9.32 15.90 10.08 5.13 10.89 17.53 7.91 0.21
B16 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.02 3.51 2.02 5.32 2.38 1.30 1.20 1.49 0.87 2.67 0.34 0.17 0.34 1.52 0.81 0.03
29.87 8.56 9.90 14.89 9.97 13.06 8.98 11.26 7.15 12.75 9.41 4.42 8.08 12.10 6.02 0.18
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.64 0.87 1.05 0.89 0.43 1.05 0.67 0.95 0.59 2.53 0.91 0.15 0.87 1.16 0.24 0.01
Each data represents mean ± S.D. from three different experiments performed in triplicate. HBA: 23-Hydroxybetulinic acid. ADM: Doxorubicin.
melanoma, respectively. As illustrated in Tables 2 and 3, 12a showed more potent antitumor activity (tumor inhibitory ratio of 55.6%, 62.7%) than parent 23-hydroxybetulinic acid (tumor inhibitory ratio of 27.8%, 32.4%). In the H22 liver cancer group, 12a exhibited stronger activity than positive control cyclophosphamide (tumor inhibitory ratio of 53.7%). In the B16 melanoma group, 12a also significantly suppressed tumor growth by inhibitory ratio of 62.7%, which was only slightly less potent than that of 5fluorouracil (tumor inhibitory ratio of 70.3%). Thus, compound 12a is worthy of further investigation as a potential anticancer drug candidate. 3. Conclusion In summary, a series of novel triterpenic pyrazine derivatives has been synthesized and evaluated for their in vitro antitumor activity against five human cancer cell lines. The screening results showed that all of the target pyrazine derivatives exhibited improved significant antitumor activity than parent compound against tested cancer cell lines, in particular compound 12a exhibited the most potent activity with IC50 values of 3.53 mM,
4.42 mM and 5.13 mM against cell lines SF-763, B16 and Hela, respectively. Remarkably, 12a was highly selective towards tumor cell lines and the selectivity index value was as high as 20. The action mechanism of 12a was investigated by flow cytometric analysis, which indicated that 12a significantly induced apoptosis of B16 cells in a dose-dependent manner and caused cell arrest in G1 phase. Furthermore, the in vivo evaluation showed that 12a exhibited remarkable antitumor efficacies against mice bearing H22 liver cancer and B16 melanoma with tumor inhibition rate of 55.6% and 62.7%, respectively. Preliminary structureeactivity analysis indicated that (1) the introduction of pyrazine ring between C-2 and C-3 position facilitated antitumor potencies; (2) bulky and electron-donating groups at the C-28 position would be favorable to the activity. It is expected that the mechanistic and biological studies described, together with our previous reports about 23-hydroxybetulinic acid derivatives, could expedite the development of new natural product-based therapeutic agents for clinical cancer intervention. 4. Experimental section 4.1. Chemistry 4.1.1. General Most chemicals and solvents were purchased from commercial sources. Further purification and drying by standard methods were employed when necessary. Melting points were determined on an XT-4 micro melting point apparatus and uncorrected. IR spectra were recorded in CDCl3 or KBr pellets on a Nicolet Impact 410 spectrophotometer. 1H NMR and 13C NMR spectra were recorded with a Bruker AV-300 or ACF 500 spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts are expressed in d values (ppm) and the coupling constants (J) in Hz. Purity of all tested compounds was 95%, as estimated by HPLC analysis. The major peak of the compounds analyzed by HPLC accounted for 95% of the combined total peak area when monitored by a UV detector at 210 nm. EI-MS spectra were recorded on an Agilent1100- LC-MSD-Trap/SL spectrometer and High-resolution mass spectra were recorded using an Agilent QTOF 6520.
Fig. 2. Survival of tumor and non-tumor (HL-7702) cells after 72 h of treatment with compound 12a.
4.1.2. Benzyl 3,23-dihydroxy-lup-20(29)-en-28-oate (2) To a solution of 1 (1.00 g, 2.12 mmol) in DMF (20 mL) was successively added K2CO3 (1.00 g, 7.24 mmol) and benzyl bromide
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Fig. 3. Effect of compound 12a on cell cycle progression of B16 cells. B16 cells were incubated with the indicated concentrations of 12a for 72 h before staining with PI. Cellular DNA content for cell-cycle distribution analysis was measured by flow cytometry.
(0.3 mL, 2.52 mmol). The mixture was stirred for 12 h at room temperature and then poured into water (15 mL) and extracted with ethyl acetate (30 mL 3). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether-ethyl acetate 2:1) to afford compound 2 as a white solid (1.07 g, 89.9%). 1H NMR (CDCl3, 300 MHz) d: 0.75 (3H, s), 0.84 (6H, s), 0.93 (3H, s), 1.67 (3H, s), 2.11e2.22 (1H, m), 2.23e2.29 (1H, m), 3.01 (1H, m, H-19), 3.37 (1H, d, J ¼ 9.9 Hz, H-23a), 3.59 (1H, m, H-3), 3.67 (1H, d, J ¼ 9.8 Hz, H23b), 4.59, 4.72 (each 1H, s, H-29), 5.05e5.17 (2H, m, eCH2Ar), 7.23e7.42 (5H, m, HeAr); 13C NMR (CDCl3, 75 MHz) d: 11.3, 14.7, 15.8, 16.5, 18.4, 19.4, 20.9, 25.5, 26.8, 29.6, 30.6, 32.1, 34.0, 36.9, 37.0, 38.1, 38.4, 40.6, 41.8, 42.4, 46.9, 49.4, 49.9, 50.5, 56.5 (C-17), 65.7(eCH2Ar), 71.8 (C-23), 76.6 (C-3), 109.6 (C-29), 128.1 (CeAr), 128.2 (CeAr), 128.5 (CeAr), 136.4 (CeAr), 150.5 (C-20), 175.8 (C-28); ESI-MS m/z: 563.3 [M þ H]þ, 585.3 [M þ Na]þ, 601.4 [M þ K]þ. 4.1.3. Benzyl 3-hydroxy-23-t-butyldimethylsilyloxy-lup-20(29)-en28-oate (3) To a solution of 2 (1.00 g, 1.78 mmol) in dichloromethane (30 mL) was added successively DMAP (300 mg, 2.46 mmol) and tert-Butyldimethylchlorosilane (360 mg, 2.39 mmol). The mixture was stirred for 4 h at room temperature, and then, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and washed with 10% HCl, water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether-ethyl acetate 20:1) to give compound 3 as a white solid (1.11 g, 92.2%). The crude product can be also used for the next step without further purification. 1H NMR (CDCl3, 300 MHz): d 0.06 (6H, s, Si-(CH3)2), 0.75 (3H, s), 0.84 (6H, s), 0.90 (9H, s, t-Bu), 0.93 (3H, s), 1.67 (3H, s), 2.13e2.20 (1H, m), 2.25e2.28 (1H, m), 3.01 (1H, m, H-19), 3.33 (1H, d, J ¼ 9.3 Hz, H-23a), 3.56 (1H, m, H-3), 3.65 (1H, d, J ¼ 9.3 Hz, H-23b), 4.59, 4.72 (each 1H, s, H-29), 5.04e5.18 (2H, m, eCH2Ar), 7.32e7.35 (5H, m, HeAr). 4.1.4. Benzyl 3-oxo-23-t-butyldimethylsilyloxylup-20(29)-en-28oate (4) To a solution of 3 (1.03 g, 1.52 mmol) in dry dichloromethane (30 mL) was added pyridinium chlorochromate (500 mg, 2.32 mmol). The mixture was stirred at room temperature for 3 h, then filtrated over Celite and concentrated in vacuo. The residue was chromatographed on silica gel (petroleum ether-ethyl acetate 30:1) to give the product as a white solid (910 mg, 88.8%). Mp. 151e154 C; 1H NMR (CDCl3, 300 MHz): d 0.07 (6H, s, Si-(CH3)2), 0.80 (3H, s), 0.83 (3H, s), 0.86 (3H, s), 0.87 (9H, s, t-Bu), 0.96 (3H, s), 1.68 (3H, s), 2.18e2.32 (2H, m), 2.37e2.42 (2H, m, H-2), 3.04 (1H, m, H-19), 3.28, 3.56 (each 1H, d, J ¼ 9.1 Hz, H-23), 4.61, 4.73 (each 1H, d, J ¼ 1.2 Hz, H-29), 5.06e5.20 (2H, m, eCH2Ar), 7.35e7.38 (5H, m, HeAr); 13C NMR (CDCl3, 75 MHz) d: e5.7 (Si-(CH3)2), e5.5 (Si(CH3)2), 14.4, 15.6, 15.7, 17.1, 18.1, 19.4, 19.6, 21.5, 25.7, 25.8, 29.5, 30.6, 32.1, 33.2, 36.2, 36.3, 36.9, 37.7, 38.3, 40.5, 42.4, 45.9, 46.9, 49.3, 49.4, 51.9, 56.5 (C-17), 65.7 (eCH2Ar), 68.4 (C-23), 109.6 (C29), 128.1 (CeAr), 128.3 (CeAr), 128.5 (CeAr), 136.5 (CeAr), 150.5
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Fig. 4. Induction of apoptosis on B16 cells by compound 12a. B16 Cells were treated with vehicle, 12a at 1.25, 2.5, and 5 mM, respectively, for 72 h. Apoptosis was measured by Annexin V/PI staining by flow cytometry. The values are the mean ± SE of at least three independent experiments.
Table 2 In vivo antitumor activity of compound 12a against mice bearing H22 liver cancer. Drugs
Dose
Injection
Number of mice Start
End
Start
Vehiclea HBAb 12a CPc
0.5 mL/mouse 30 mg/kg 30 mg/kg 30 mg/kg
iv ip ip iv
10 10 10 10
10 10 10 10
18.3 18.4 18.6 18.1
a b c
Weight of tumor X ± SD (g)
Weight of mice (g)
Ratio of inhibition (%)
P value
27.8 55.6 53.7
>0.05
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and antitumor activity of triterpenoid pyrazine derivatives from 23-hydroxybetulinic acid Hengyuan Zhang a, b, Yiwei Wang a, b, Peiqing Zhu a, b, Jie Liu a, c, Shengtao Xu a, b, Hequan Yao a, b, *, Jieyun Jiang d, Wencai Ye e, Xiaoming Wu a, b, Jinyi Xu a, b, * a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Department of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China d Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA e College of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 November 2014 Received in revised form 26 April 2015 Accepted 27 April 2015 Available online 29 April 2015
Pyrazine-fused 23-hydroxybetulinic acid was synthesized by introducing a pyrazine ring between C-2 and C-3 position and further modifications were carried out by substitution of C-28 carboxyl group by ester and amide linkage to enhance the antitumor activity. The biological screening results showed that all of the derivatives exhibited more significant antiproliferative activity than the parent compound. In particular compound 12a exhibited the most potent activity with IC50 values of 3.53 mM, 4.42 mM and 5.13 mM against cell lines SF-763, B16 and Hela, respectively. In the preliminary mechanism study, 12a caused cell arrest in G1 phase and significantly induced apoptosis of B16 cells in a dose-dependent manner. Furthermore, the in vivo antitumor activity of 12a was validated (tumor inhibitory ratio of 55.6% and 62.7%, respectively) in mice with H22 liver cancer and B16 melanoma. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Triterpenoid 23-Hydroxybetulinic acid Pyrazine Apoptosis Antitumor activity
1. Introduction Natural products have been the single most productive source of leads for the development of drugs for the treatment of cancer. In fact, the majority of antitumor agents are of natural origin [1]. Betulinic acid (3-Hydroxy-lup-20(29)-en-28-oic acid, Fig. 1) is a pentacyclic lupane-type triterpene that is widely distributed throughout the plant kingdom. A variety of biological activities have been ascribed to betulinic acid including anti-inflammatory, antibacterial, antimalarial and antioxidant properties. However, betulinic acid is most highly regarded for its anti-HIV-1 activity and specific cytotoxicity against a variety of tumor cell lines. Previous experimental and epidemiological studies have indicated that betulinic acid as well as its analog- 23-hydroxybetulinic acid (HBA, 1, Fig. 1) may be developed as potent anti-HIV and antitumor drugs [2e4].
* Corresponding authors. State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China. E-mail addresses: [email protected] (H. Yao), [email protected] (J. Xu). http://dx.doi.org/10.1016/j.ejmech.2015.04.057 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
The 23-hydroxybetulinic acid, isolated from the root of Pulsatilla chinensis, displayed similar anti-HIV and antitumor activities as its analog betulinic acid [5]. To date numerous derivatization studies have been performed on betulinic acid leading to the production of an array of betulinic acid derivatives [3]. Nevertheless, few studies have been devoted to the structural modifications of 23hydroxybetulinic acid. In general, due to various reasons original natural products are not particularly good candidates, and a need therefore exists for improving their drug-like properties by chemical modification [1]. It has been found that heterocyclic ring-fused triterpenoids at C-2 and C-3 position impart the desired characteristics [6]. Nitrogen heterocycles are important pharmacophores in drug design, especially pyrazine derivatives, which are among the most frequently cited heterocyclic compounds [7]. Previously, Urban et al. described the partial synthesis of triterpenic heterocycles, in which some lupane pyrazine derivatives were significantly cytotoxic in vitro to warrant the extension of the in vitro studies to in vivo testing in mice [8]. The cytotoxic activity of some triterpenic compounds was also successfully increased by using heterocyclic modifications [9e11].
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Fig. 1. Chemical structures of betulinic acid and 23-hydroxybetulinic acid.
Previously, a set of C-17-carboxylic acid-modified 23hydroxybetulinic acid ester and amide derivatives have been reported by our group [12,13]. The promising anti-proliferative of triterpenic pyrazines encouraged us to continue to design and synthesize novel betulinic pyrazine derivatives from 23hydroxybetulinic acid. Base on the structureeactivity relationships (SARs) studies of betulinic acid and 23-hydroxybetulinic acid, further modifications were carried out by substitution of C-28 carboxyl group by ester and amide linkage to enhance the antitumor activity. The obtained derivatives were evaluated for their in vitro antitumor activity against five cancer cell lines. Furthermore, the in vivo antitumor activity of the representative compound was validated in mice with H22 liver cancer and B16 melanoma. The primary antitumor mechanism was also investigated. 2. Results and discussions 2.1. Chemistry The 23-hydroxybetulinic acid was isolated from the roots of P. chinensis (Bge) Regel [5,14], and characterized by 1H NMR, 13C NMR and high-resolution mass spectra. As described in Scheme 1, it was treated with BnBr and K2CO3 in DMF to yield 28-benzyl-23hydroxybetulinic ester 2, the hydroxyl group of 2 was protected using tert-butyldimethylsilyl chloride in the presence of DMAP to give siloxane 3. Oxidation of 3 with pyridinium chlorochromate afforded the ketone 4, which was then converted to pyrazine derivative 5 in the presence of ethylenediamine and sulfer in morpholine [15]. Deprotection of 5 with hydrochloric acid in acetone gave alcohol 6. The NMR spectrum displayed readily recognizable signals for the fused pyrazine ring [proton signals at d 8.35 (H-20 ), 8.38 (H-10 ) and d 2.45 (H-1), 3.08 (H-10 ) and olefinic carbon signals at d 152.3 (C-2), 157.8 (C-3), 142.2 (C-10 ), 141.5 (C-20 )]. Previous study indicated that substitution of C-23 hydroxyl group may benefit the potency [16]. Therefore, a simple acetyl group was introduced to this position and selected for further investigation. The carboxylic pyrazine 8 was obtained by debenzylation of ester 7 in THF with Pd/C as catalyst under atmospheric pressure of hydrogen. Following deacetylation of 8 produced pyrazine derivative 9. Derivatives 10a-10e were synthesized by the reaction of compound 9 with corresponding bromides under basic condition. Hydrolysis of derivative 10d and 10e afforded compound 10f and 10g. To synthesize derivatives 12a-12d, C-28 carboxylic group was converted to acyl chloride intermediate 11, which was further reacted with the corresponding alcohols and amides (Scheme 2). 2.2. Biological evaluation 2.2.1. In vitro antiproliferative activity The in vitro antiproliferative activity of these novel betulinic
pyrazine derivatives was evaluated on five cancer cell lines (HL-60 human promyelocytic leukemia cell, BEL-7402 human hepatocellular carcinoma, HeLa human cervical adenocarcinoma, SF-763 human brain adenocarcinoma and B16 mice melanoma cells) by MTT assay with doxorubicin as the positive control. The results summarized in Table 1 are presented as the concentration of drug inhibiting 50% cell growth (IC50). The data indicated that all of the derivatives exhibited stronger antiproliferative activities than parent compound 23hydroxybetulinic acid in the five cancer cell lines, especially against human brain adenocarcinoma SF-763 and mice melanoma B16 cells. Pyrazine-fused compound 9 was about 2- to 3-fold more potent against five cancer cell lines than HBA, indicating that introduction of the pyrazine ring imparted potent antiproliferative activity. The acetylated derivative 8 showed lower IC50 values than compound 9, which is consistent with the case of benzoylation [16]. Compounds 10f, 12a and 12d were the most promising derivatives with IC50 values lower than 10 mM on all tested cell lines, in particular compound 12a was about 7- to 10-fold more potent against five cancer cell lines than HBA and 2- to 4-fold than 9. These findings revealed that electron-donating and/or polar substituents especially groups bearing the amide linkage at C-28 side chain would benefit the potency. The results also demonstrated that the existence of a structural constraint such as benzyl, cyclohexyl, biphenyl and phenyl at C-28 is favorable to the inhibitory activity, which is exemplified by compounds 6, 10g, 10f and 12d. The obtained structureeactivity relationships of C-28 position have also confirmed the results from previous comparative molecular field analysis (CoMFA), in which the contour maps illustrated that bulky and/or electron-donating groups at C-28 would be favorable to the antitumor activity [17]. Furthermore, selectivity of compound 12a was assessed on normal human hepatocyte (HL-7702 cell line). The selectivity index was calculated by IC50 value in HL-7702 cell line (75.43 ± 2.05 mM) divided by IC50 value in cancer cell lines (see supplementary material). It was observed that 12a was 11e20 times more selective towards cancer cells than to non-tumor cell. The doseeresponse curves for the test compound towards different tumor cell lines and non-tumor (HL-7702) cell line are given in Fig. 2. 2.2.2. Compound 12a induces cell cycle arrest To determine whether the suppression of cell growth by triterpenic pyrazines is caused by a cell-cycle effect, we detected the DNA content of cell nuclei by flow cytometry (Fig. 3). B16 cells were treated with compound 12a at concentrations of 1.25, 2.5, and 5 mM, which resulted in accumulation of 29.67, 34.67, and 33.46% of cells at the G1 phase, respectively. These results indicated that compound 12a inhibited the growth of the cancer cells by inhibiting the cell cycle via G1-phase arrest. 2.2.3. Compound 12a induces apoptosis To clarify whether the loss of cancer cell viability promoted by triterpenic pyrazines is associated with apoptosis, an annexin VeFITC/propidium iodide (PI) binding assay was performed. As shown in Fig. 4, compound 12a exhibited potent dose-dependent activity in the induction of apoptosis. Treatment of B16 cells with 12a at 1.25, 2.5, and 5 mM for 72 h resulted in 16.51, 31.62, and 57.44% apoptotic cells (early and late), as compared with 5.66% in an untreated vehicle control, indicating that compound 12a was able to induce apoptotic cell death in B16 cells. 2.2.4. In vivo antitumor activity of compound 12a Based on the in vitro results and intensive mechanistic studies, we further tested the antitumor activity of compound 12a in vivo by performing an assay in mice with H22 liver cancer and B16
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Scheme 1. Synthesis of pyrazine-fused 23-hydroxybetulinic acid. Reagents and conditions: (a) BrBn, K2CO3, DMF, rt, 10 h; (b) TBSCl, DMAP, CH2Cl2; rt, 4 h (c) PCC, CH2Cl2, rt, 4 h; (d) sulfur, ethylenediamine, morpholine, reflux, 4 h; (e) 10% HCl, acetone, rt, 10 h; (f) Ac2O, pyridine, rt, 3 h; (g) H2, 10% Pd/C, THF, rt, 2 h; (h) 4 N NaOH, THF/CH3OH, rt, 1 h.
Scheme 2. Synthesis of 23-hydroxybetulinic pyrazine analogs 10a-10h and 12a-12d. Reagents and conditions: (a) R1Br, K2CO3, DMF; (b) oxalyl chloride, cat. DMF, CH2Cl2, rt, 3 h; (c) R2X (X ¼ O, NH), DMAP, CH2Cl2. (d) triethylamine, H2O, CH3OH/THF, rt, 6 h; (e) NaOH, H2O, CH3OH/THF, reflux, 1 h.
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Table 1 Antiproliferative activities of triterpenic pyrazine analogs against different tumor cell lines. Comp.
Cell lines (IC50a, mM ± SD) HL-60
b
HBA 6 8 9 10a 10b 10c 10e 10f 10g 10h 12a 12b 12c 12d ADMc a b c
45.15 18.89 20.35 24.14 22.51 23.56 19.11 23.62 11.69 21.06 19.52 7.11 14.71 22.15 10.19 0.17
BEL-7402 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.11 2.24 1.41 3.42 1.33 3.04 2.05 1.41 1.54 1.19 1.88 0.36 1.80 2.02 0.76 0.01
39.67 10.05 16.79 19.10 14.76 17.41 11.88 16.99 9.37 17.65 13.10 7.03 11.06 15.73 8.09 0.22
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.22 2.01 0.92 2.07 1.09 1.70 0.89 1.36 22.01 1.49 1.53 0.22 1.04 1.72 0.33 0.02
SF-763 43.40 9.54 11.09 15.43 10.02 13.53 8.62 13.14 5.63 10.97 8.86 3.53 9.26 13.03 5.77 0.16
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
HeLa 7.20 1.32 1.67 1.46 1.21 4.12 0.61 1.79 0.44 0.89 0.92 0.19 0.02 1.26 0.25 0.03
52.39 12.80 14.61 19.57 15.09 20.74 10.96 17.01 9.32 15.90 10.08 5.13 10.89 17.53 7.91 0.21
B16 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.02 3.51 2.02 5.32 2.38 1.30 1.20 1.49 0.87 2.67 0.34 0.17 0.34 1.52 0.81 0.03
29.87 8.56 9.90 14.89 9.97 13.06 8.98 11.26 7.15 12.75 9.41 4.42 8.08 12.10 6.02 0.18
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.64 0.87 1.05 0.89 0.43 1.05 0.67 0.95 0.59 2.53 0.91 0.15 0.87 1.16 0.24 0.01
Each data represents mean ± S.D. from three different experiments performed in triplicate. HBA: 23-Hydroxybetulinic acid. ADM: Doxorubicin.
melanoma, respectively. As illustrated in Tables 2 and 3, 12a showed more potent antitumor activity (tumor inhibitory ratio of 55.6%, 62.7%) than parent 23-hydroxybetulinic acid (tumor inhibitory ratio of 27.8%, 32.4%). In the H22 liver cancer group, 12a exhibited stronger activity than positive control cyclophosphamide (tumor inhibitory ratio of 53.7%). In the B16 melanoma group, 12a also significantly suppressed tumor growth by inhibitory ratio of 62.7%, which was only slightly less potent than that of 5fluorouracil (tumor inhibitory ratio of 70.3%). Thus, compound 12a is worthy of further investigation as a potential anticancer drug candidate. 3. Conclusion In summary, a series of novel triterpenic pyrazine derivatives has been synthesized and evaluated for their in vitro antitumor activity against five human cancer cell lines. The screening results showed that all of the target pyrazine derivatives exhibited improved significant antitumor activity than parent compound against tested cancer cell lines, in particular compound 12a exhibited the most potent activity with IC50 values of 3.53 mM,
4.42 mM and 5.13 mM against cell lines SF-763, B16 and Hela, respectively. Remarkably, 12a was highly selective towards tumor cell lines and the selectivity index value was as high as 20. The action mechanism of 12a was investigated by flow cytometric analysis, which indicated that 12a significantly induced apoptosis of B16 cells in a dose-dependent manner and caused cell arrest in G1 phase. Furthermore, the in vivo evaluation showed that 12a exhibited remarkable antitumor efficacies against mice bearing H22 liver cancer and B16 melanoma with tumor inhibition rate of 55.6% and 62.7%, respectively. Preliminary structureeactivity analysis indicated that (1) the introduction of pyrazine ring between C-2 and C-3 position facilitated antitumor potencies; (2) bulky and electron-donating groups at the C-28 position would be favorable to the activity. It is expected that the mechanistic and biological studies described, together with our previous reports about 23-hydroxybetulinic acid derivatives, could expedite the development of new natural product-based therapeutic agents for clinical cancer intervention. 4. Experimental section 4.1. Chemistry 4.1.1. General Most chemicals and solvents were purchased from commercial sources. Further purification and drying by standard methods were employed when necessary. Melting points were determined on an XT-4 micro melting point apparatus and uncorrected. IR spectra were recorded in CDCl3 or KBr pellets on a Nicolet Impact 410 spectrophotometer. 1H NMR and 13C NMR spectra were recorded with a Bruker AV-300 or ACF 500 spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts are expressed in d values (ppm) and the coupling constants (J) in Hz. Purity of all tested compounds was 95%, as estimated by HPLC analysis. The major peak of the compounds analyzed by HPLC accounted for 95% of the combined total peak area when monitored by a UV detector at 210 nm. EI-MS spectra were recorded on an Agilent1100- LC-MSD-Trap/SL spectrometer and High-resolution mass spectra were recorded using an Agilent QTOF 6520.
Fig. 2. Survival of tumor and non-tumor (HL-7702) cells after 72 h of treatment with compound 12a.
4.1.2. Benzyl 3,23-dihydroxy-lup-20(29)-en-28-oate (2) To a solution of 1 (1.00 g, 2.12 mmol) in DMF (20 mL) was successively added K2CO3 (1.00 g, 7.24 mmol) and benzyl bromide
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Fig. 3. Effect of compound 12a on cell cycle progression of B16 cells. B16 cells were incubated with the indicated concentrations of 12a for 72 h before staining with PI. Cellular DNA content for cell-cycle distribution analysis was measured by flow cytometry.
(0.3 mL, 2.52 mmol). The mixture was stirred for 12 h at room temperature and then poured into water (15 mL) and extracted with ethyl acetate (30 mL 3). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether-ethyl acetate 2:1) to afford compound 2 as a white solid (1.07 g, 89.9%). 1H NMR (CDCl3, 300 MHz) d: 0.75 (3H, s), 0.84 (6H, s), 0.93 (3H, s), 1.67 (3H, s), 2.11e2.22 (1H, m), 2.23e2.29 (1H, m), 3.01 (1H, m, H-19), 3.37 (1H, d, J ¼ 9.9 Hz, H-23a), 3.59 (1H, m, H-3), 3.67 (1H, d, J ¼ 9.8 Hz, H23b), 4.59, 4.72 (each 1H, s, H-29), 5.05e5.17 (2H, m, eCH2Ar), 7.23e7.42 (5H, m, HeAr); 13C NMR (CDCl3, 75 MHz) d: 11.3, 14.7, 15.8, 16.5, 18.4, 19.4, 20.9, 25.5, 26.8, 29.6, 30.6, 32.1, 34.0, 36.9, 37.0, 38.1, 38.4, 40.6, 41.8, 42.4, 46.9, 49.4, 49.9, 50.5, 56.5 (C-17), 65.7(eCH2Ar), 71.8 (C-23), 76.6 (C-3), 109.6 (C-29), 128.1 (CeAr), 128.2 (CeAr), 128.5 (CeAr), 136.4 (CeAr), 150.5 (C-20), 175.8 (C-28); ESI-MS m/z: 563.3 [M þ H]þ, 585.3 [M þ Na]þ, 601.4 [M þ K]þ. 4.1.3. Benzyl 3-hydroxy-23-t-butyldimethylsilyloxy-lup-20(29)-en28-oate (3) To a solution of 2 (1.00 g, 1.78 mmol) in dichloromethane (30 mL) was added successively DMAP (300 mg, 2.46 mmol) and tert-Butyldimethylchlorosilane (360 mg, 2.39 mmol). The mixture was stirred for 4 h at room temperature, and then, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and washed with 10% HCl, water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether-ethyl acetate 20:1) to give compound 3 as a white solid (1.11 g, 92.2%). The crude product can be also used for the next step without further purification. 1H NMR (CDCl3, 300 MHz): d 0.06 (6H, s, Si-(CH3)2), 0.75 (3H, s), 0.84 (6H, s), 0.90 (9H, s, t-Bu), 0.93 (3H, s), 1.67 (3H, s), 2.13e2.20 (1H, m), 2.25e2.28 (1H, m), 3.01 (1H, m, H-19), 3.33 (1H, d, J ¼ 9.3 Hz, H-23a), 3.56 (1H, m, H-3), 3.65 (1H, d, J ¼ 9.3 Hz, H-23b), 4.59, 4.72 (each 1H, s, H-29), 5.04e5.18 (2H, m, eCH2Ar), 7.32e7.35 (5H, m, HeAr). 4.1.4. Benzyl 3-oxo-23-t-butyldimethylsilyloxylup-20(29)-en-28oate (4) To a solution of 3 (1.03 g, 1.52 mmol) in dry dichloromethane (30 mL) was added pyridinium chlorochromate (500 mg, 2.32 mmol). The mixture was stirred at room temperature for 3 h, then filtrated over Celite and concentrated in vacuo. The residue was chromatographed on silica gel (petroleum ether-ethyl acetate 30:1) to give the product as a white solid (910 mg, 88.8%). Mp. 151e154 C; 1H NMR (CDCl3, 300 MHz): d 0.07 (6H, s, Si-(CH3)2), 0.80 (3H, s), 0.83 (3H, s), 0.86 (3H, s), 0.87 (9H, s, t-Bu), 0.96 (3H, s), 1.68 (3H, s), 2.18e2.32 (2H, m), 2.37e2.42 (2H, m, H-2), 3.04 (1H, m, H-19), 3.28, 3.56 (each 1H, d, J ¼ 9.1 Hz, H-23), 4.61, 4.73 (each 1H, d, J ¼ 1.2 Hz, H-29), 5.06e5.20 (2H, m, eCH2Ar), 7.35e7.38 (5H, m, HeAr); 13C NMR (CDCl3, 75 MHz) d: e5.7 (Si-(CH3)2), e5.5 (Si(CH3)2), 14.4, 15.6, 15.7, 17.1, 18.1, 19.4, 19.6, 21.5, 25.7, 25.8, 29.5, 30.6, 32.1, 33.2, 36.2, 36.3, 36.9, 37.7, 38.3, 40.5, 42.4, 45.9, 46.9, 49.3, 49.4, 51.9, 56.5 (C-17), 65.7 (eCH2Ar), 68.4 (C-23), 109.6 (C29), 128.1 (CeAr), 128.3 (CeAr), 128.5 (CeAr), 136.5 (CeAr), 150.5
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Fig. 4. Induction of apoptosis on B16 cells by compound 12a. B16 Cells were treated with vehicle, 12a at 1.25, 2.5, and 5 mM, respectively, for 72 h. Apoptosis was measured by Annexin V/PI staining by flow cytometry. The values are the mean ± SE of at least three independent experiments.
Table 2 In vivo antitumor activity of compound 12a against mice bearing H22 liver cancer. Drugs
Dose
Injection
Number of mice Start
End
Start
Vehiclea HBAb 12a CPc
0.5 mL/mouse 30 mg/kg 30 mg/kg 30 mg/kg
iv ip ip iv
10 10 10 10
10 10 10 10
18.3 18.4 18.6 18.1
a b c
Weight of tumor X ± SD (g)
Weight of mice (g)
Ratio of inhibition (%)
P value
27.8 55.6 53.7
>0.05
