Chem Biol Drug Des 2014 Research Article
Synthesis of Novel Heterocyclic Ring-Fused 18b-Glycyrrhetinic Acid Derivatives with Antitumor and Antimetastatic Activity Cheng Gao1, Fu-Jun Dai2, Hai-Wei Cui1, Shi-Hong Peng2, Yuan He2, Xue Wang2, Zheng-Fang Yi2,* and Wen-Wei Qiu1,3,* 1
Department of Chemistry, East China Normal University, Shanghai 200062, China 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China 3 Shanghai Engineering Research Center for Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, China *Corresponding authors: Zheng-Fang Yi, [email protected], Wen-Wei Qiu, [email protected] Glycyrrhetinic acid (GA) is one of the most important triterpenoic acids shows many pharmacological effects, especially antitumor activity. GA triggers apoptosis in various tumor cell lines. However, the antitumor activity of GA is weak, thus the synthesis of new synthetic analogs with enhanced potency is needed. By introducing various five-member fused heterocyclic rings at C-2 and C-3 positions, 18 novel GA derivatives were obtained. These compounds were evaluated for their inhibitory activity against the growth of eight different tumor cell lines using a SRB assay. The most active compound 37 showed IC50 between 5.19 and 11.72 lM, which was about 11-fold more potent than the lead compound GA. An apoptotic effect of GA and 37 was determined using flow cytometry and trypan blue exclusion assays. We also demonstrated here for the first time that GA and the synthetic derivatives exhibited inhibitory effect on migration of the tested tumor cells, especially 37 which was about 20-fold more potent than GA on antimetastatic activity. Key words: antitumor, apoptosis, glycyrrhetinic acid, migration, triterpenoic acids Received 14 January 2014 and accepted for publication 12 February 2014
Natural products have been used to treat human disease for thousands of years and play an increasingly significant role in drug discovery and development process in recent years (1). Pentacyclic triterpenoids are one of the most ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12308
important classes of natural products occurring and have been studied intensively for their diverse biological and pharmacological activities (2–5). Glycyrrhetinic acid (GA, Scheme 1), a natural pentacyclic triterpene acid, is known to show many biological effects including anti-inflammatory, antiviral, and hepatoprotective (6). Recently, GA attracted the focus of research interests not only for it can be accessed abundantly from the roots of licorice [up to 24% (7,8)], but also for its triggering apoptosis on tumor cells (9–12). However, GA has only weak inhibitory effect against various tumor cell lines (IC50 is approximately 80 lM), and some structural modifications have been previously performed (13–20). We also become interested in producing more active GA analogs. Recently, we and other groups have found that introduction of the nitrogencontaining heterocyclic rings, especially five-membered heterocyclic rings to the pentacyclic triterpenoids, such as maslinic acid and betulinic acid, can improve the biological activities (21–24). Thus, to obtain more potent anticancer analogs, we designed and synthesized a series of novel five-membered heterocyclic ring-fused GA derivatives at C-2 and C-3 positions, and their inhibitory activity against the growth of 8 different tumor cell lines was evaluated using a SRB assay. GA and its derivative were screened by flow cytometry and trypan blue exclusion assays to determine the apoptotic behavior, and their inhibitory effect on tumor cell migration was tested for the first time.
Methods and Materials Chemistry All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. When needed, the reactions were carried out in flame or oven-dried glassware under a positive pressure of dry N2. Column chromatography was performed on silica gel (QinDao, 200–300 mesh) using the indicated eluents. Thin-layer chromatography was carried out on silica gel plates (QinDao) with a layer thickness of 0.25 mm. Melting points were determined using the MELTEMP 3.0 apparatus and are uncorrected. 1H (300, 400, and 500 MHz) and 13C (100 and 125 MHz) NMR spectra were recorded on Varian Mercury-300, Bruker AM-400, and Bruker AV-500 spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal 1
Gao et al.
COOR'
COOH
COOH
O
O
O H
H
a
H
d O
O
HO GA
6 R'= -Me 7 R'= -Bn
1 b
b
COOH
COOH
O R
H
COOR'
O
R c O
HN N
O
R
H
H
O O
O 2 R= -H 3 R= -CF3
4 R= -H 5 R= -CF3
c COOR'
8 9 10 11
R= -H R= -CF3 R= -H R= -COOEt
R'= -Me R'= -Me R'= -Bn R'= -Bn
O R
H
HN N 12 13 14 15
R= -H R= -CF3 R= -H R= -COOEt
R'= -Me R'= -Me R'= -Bn R'= -Bn
Scheme 1: Reagents and conditions: (a) Jone’s reagent, THF, 0 °C, 1 h, (92%); (b) RCOOEt (R = -H, -CF3, -COOEt), NaH (60%), THF, rt, 8 h (91% for 2, 82% for 3, 86% for 8, and 74% for 9); (c) NH2NH2H2O, AcOH, rt, 12 h (88% for 4, 78% for 5, 92% for 12, 68% for 13; 88% and 69% for 14 and 15, respectively, over two steps). (d) MeI or BnBr, K2CO3, DMF, rt, 2 h (87% for 6 and 92% for 7).
standard. All chemical shift values were reported in units of d (ppm). The following abbreviations were used to indicate the peak multiplicity: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad. High-resolution mass data were obtained on a Bruker micrOTOF-Q II spectrometer.
Preparation of compound 26 To a solution of 12 (203 mg, 0.4 mmol) and DMAP (49 mg, 0.4 mmol) in dry DCM (20 mL) was added mesyl chloride (0.15 mL, 2.0 mmol) under N2. The reaction mixture was stirred for 12 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 4/1 v/v) to give 26 as a white solid (168 mg, 73%); mp. 245.0–247.0 °C. 1H NMR (400 MHz, CDCl3) d 7.63 (s, 1H), 5.74 (s, 1H), 3.83 (d, J = 15.6 Hz, 1H), 3.70 (s, 3H), 3.22 (s, 3H), 2.52 (s, 1H), 2
1.39 (s, 3H), 1.35 (s, 3H), 1.29 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.60, 176.98, 169.99, 163.80, 128.67, 127.92, 118.21, 59.93, 53.29, 51.89, 48.47, 45.31, 44.13, 43.46, 41.32, 41.09, 37.89, 37.86, 36.67, 34.91, 31.95, 31.53, 31.22, 28.69, 28.39, 26.65, 26.51, 24.73, 23.35, 18.65, 18.28, 15.48. ESI-HRMS (m/z) [M + Na]+ calcd for C33H48N2NaO5S, 607.3182, found 607.3160.
Preparation of compound 27 To a solution of 12 (150 mg, 0.3 mmol) and DMAP (36 mg, 0.3 mmol) in dry DCM (20 mL) was added Ac2O (151 mg, 1.5 mmol) under N2. The reaction mixture was stirred for 12 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 27 as a white solid, (148 mg, Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
91%); mp. 225.0–227.0 °C. 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 2.63 (s, 3H), 2.53 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.61, 176.95, 169.79, 169.48, 162.24, 128.69, 124.78, 119.32, 59.92, 53.29, 51.85, 48.43, 45.30, 44.10, 43.42, 41.29, 37.94, 37.84, 36.91, 34.71, 31.95, 31.93, 31.56, 31.20, 28.66, 28.36, 26.63, 26.50, 24.75, 23.33, 21.66, 18.67, 18.25, 15.46. ESI-HRMS (m/z) [M + Na]+ calcd for C34H48N2NaO4, 571.3512, found 571.3541.
Preparation of compounds 29 and 33–37 Compound 28 To a solution of N-Boc-piperidine-3-carboxylic acid (275 mg, 1.2 mmol) in dry DCM (20 mL) was added N,N’carbonyldiimidazole (194 mg, 1.2 mmol). The reaction mixture was stirred for 0.5 h at 23 °C, and then 12 (203 mg, 0.4 mmol) and DMAP (146 mg, 1.2 mmol) was added. The reaction mixture was stirred for 3 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 28 as a white solid (213 mg, 76%). 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19 (d, J = 7.1 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.70 (s, 4H), 3.15 (d, J = 10.4 Hz, 1H), 2.86 (t, J = 11.1 Hz, 1H), 2.52 (s, 1H), 1.43(s,9H), 1.39 (s, 3H), 1.33, 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.09 (s, 3H), 0.83 (s, 3H).
Compound 29 By a similar procedure described for 28, 29 was obtained as a white solid (206 mg, 73%). 1H NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 5.73 (s, 1H), 4.15 (d, J = 12.6 Hz, 2H), 3.83 (d, J = 15.7 Hz, 1H), 3.78–3.65 (m, 4H), 2.90 (t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.46(s, 9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 30 By a similar procedure described for 28, 30 was obtained as a white solid (228 mg, 78%). 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 4.07 (d, J = 13.5 Hz, 2H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 2.95-3.07 (m, 2H), 2.75 (t, J = 12.0 Hz, 2H), 2.52 (s, 1H), 1.45(s,9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 31 By a similar procedure described for 28, 31 was obtained as a white solid (235 mg, 79%). 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 4.08 (d, J = 13.3 Hz, 2H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 3.10 (td, Chem Biol Drug Des 2014
J = 7.4, 4.2 Hz, 2H), 2.67 (t, J = 11.8 Hz, 2H), 2.53 (s, 1H), 1.45 (s,9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 32 By a similar procedure described for 28, 32 was obtained as a white solid (237 mg, 78%). 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19 (d, J = 7.1 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.70 (s, 4H), 3.15 (d, J = 10.4 Hz, 1H), 2.86 (t, J = 11.1 Hz, 1H), 2.52 (s, 1H),1.45 (s, 9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 33 To a solution of 28 (159 mg, 0.21 mmol) in Et2O (5 mL) was added boron trifluoride etherate (2 mL). The reaction mixture was stirred for 0.5 h at 23 °C and then poured into ice water (20 mL). The precipitate was filtered and washed with Et2O (5 mL), then dried to give 33 as a white solid (104 mg, 80%); mp. 218.0–221.0 °C. 1H NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 5.73 (s, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.75–3.63 (m, 4H), 3.43 (d, J = 13.1 Hz, 1H), 3.23 (d, J = 11.4 Hz, 1H), 3.09 (d, J = 10.3 Hz, 1H), 2.87 (s, 1H), 2.52 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.80, 177.07, 171.90, 170.09, 163.19, 128.68, 125.18, 119.90, 59.92, 53.21, 51.91, 48.47, 45.36, 44.15, 43.48, 41.30, 37.97, 37.88, 36.88, 36.69, 34.78, 31.96, 31.54, 31.24, 28.70, 28.40, 26.66, 26.53, 25.15, 24.95, 24.80, 23.37, 18.66, 18.29, 15.56, 14.43. ESI-HRMS (m/z) [M + Na]+ calcd for C38H55N3NaO4, 640.4090, found 640.4119.
Compound 34 By a similar procedure described for 33, 34 was obtained as a white solid (99 mg, 77%); mp. 227.0–231.0 °C. 1H NMR (500 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.76 (s, 1H), 3.70 (s, 3H), 3.25 (s, 2H), 2.86 (t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.85, 177.10, 171.75, 170.16, 163.25, 128.75, 125.28, 120.00, 59.97, 53.28, 51.97, 48.53, 45.41, 44.19, 44.15, 43.53, 41.36, 38.02, 37.93, 36.94, 36.59, 34.84, 32.01, 31.60, 31.29, 28.74, 28.45, 26.71, 26.59, 25.18, 25.01, 24.85, 23.41, 18.72, 18.34, 15.61, 14.47. ESI-HRMS (m/z) [M + Na]+ calcd for C38H55N3NaO4, 640.4090, found 640.4085.
Compound 35 By a similar procedure described for 33, 35 was obtained as a white solid (90 mg, 68%); mp. 219.0–220.0 °C. 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.74 (s, 1H), 4.17 3
Gao et al.
(s, 1H), 3.85 (d, J = 16.0 Hz, 1H), 3.70 (d, J = 5.7 Hz, 3H), 3.60 (d, J = 15.4 Hz, 2H), 3.42 (s, 2H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.16 (s, 3H), 1.07 (s, 3H), 0.84 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.76, 177.08, 171.67, 169.95, 162.52, 128.74, 124.95, 119.50, 59.98, 53.28, 51.93, 48.50, 45.37, 45.33, 44.18, 43.49, 41.34, 38.00, 37.91, 36.95, 34.79, 33.16, 31.99, 31.60, 31.26, 30.70, 30.51, 28.72, 28.67, 28.44, 26.69, 26.57, 24.85, 23.39, 18.71, 18.32, 15.57. ESI-HRMS (m/z) [M + H]+ calcd for C39H58N3O4, 632.4427, found 632.4408.
Compound 36 By a similar procedure described for 33, 36 was obtained as a white solid (95 mg, 71%); mp. 210.0–211.0 °C. 1H NMR (400 MHz, CDCl3) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.71 (s, 3H), 3.56 (d, J = 12.4 Hz, 2H), 3.13–2.93 (m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.70, 177.01, 171.85, 169.89, 162.24, 128.67, 124.82, 119.20, 59.93, 53.24, 51.86, 48.44, 45.31, 45.09, 44.11, 43.43, 41.28, 37.94, 37.84, 36.90, 35.19, 34.72, 33.64, 33.55, 31.93, 31.57, 31.20, 28.70, 28.67, 28.37, 26.62, 26.50, 24.79, 23.34, 21.48, 18.65, 18.26, 15.50. ESI-HRMS (m/ z) [M + H]+ calcd for C40H60N3O4, 646.4584, found 646.4573.
Compound 37 By a similar procedure described for 33, 37 was obtained as a white solid (92 mg, 67%); mp. 201.0–204.0 °C. 1H NMR (400 MHz, CDCl3) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.70 (s, 3H), 3.52 (s, 2H), 3.11–2.92 (m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13 C NMR (100 MHz, CDCl3) d 199.70, 177.08, 171.62, 170.03, 163.91, 128.71, 125.21, 120.55, 59.91, 53.23, 53.13, 51.93, 48.49, 45.67, 45.36, 45.15, 44.17, 43.50, 41.34, 37.98, 36.89, 36.25, 34.83, 31.99, 31.53, 31.45, 31.26, 28.72, 28.43, 26.69, 26.56, 25.66, 24.83, 24.77, 23.42, 23.39, 20.54, 18.69, 18.30, 15.62, 15.57. ESIHRMS (m/z) [M + H]+ calcd for C41H62N3O4, 660.4740, found 660.4731.
Biology Cell lines and culture conditions Cell lines used in this study were obtained from the American Type Culture Collection (ATCC). HCT-116, HCT-8, HT-29, CT-26, PC-3, and DU-145 cell lines were cultured in RPMI1640 medium. Mouse 4T1 breast cancer cells were cultured in DMEM medium, and human MDA-MB231 breast cancer cells were cultured in MEM medium supplemented with 1% sodium pyruvate and 1% nonessential amino acids. Medium was supplemented with 4
10% FBS, penicillin (100 units/mL), and streptomycin (100 units/mL). All cells were incubated at 37 °C and 5% CO2 incubator.
Viability assay The cell viability of eight tumor cell lines in the presence of our synthetic compounds was determined by SRB (Sigma Aldrich, St.Louis, MO, USA) assay (14) In brief, cells were seeded into 96-well plates at the appropriate cell densities to prevent confluence during the experiment. After incubation for 24 h, the cells were treated with various concentrations of the compound (0–50 lM) for 96 h. Control cells were exposed to DMSO at a concentration equivalent to that of the compound-treated cells, and the final dilution of DMSO solvent used in this assay never exceeded 0.4%. After treatment for 96 h, the supernatant medium was removed from the 96-well plates, and the cells were fixed by 50% TCA at 4 °C. After fixation for 1 h, the plates were washed by water for five times. The plates were allowed to dry using hair dryer followed by being dyed with 100 lL of 0.4% SRB for 10 min. After dying, the plates were washed by 1% acetic acid to remove the dye and allowed to dry using hair dryer. One hundred microliter of 10 mM Tris base solution was added to each well, and absorbance was examined using a 96-well plate reader. The IC50 was calculated using GraphPad Software (San Diego, CA, USA).
Trypan blue exclusion assay Apoptosis of mouse CT-26 colorectal cancer cells was assessed by trypan blue exclusion assay (25). In short, cells were plated onto 6-well plates and were exposed to GA and 37 for 24 h. After treatment with various doses of the compounds for 24 h, all cells including the supernatant were collected and centrifuged to collect the viable, apoptotic, and dead cells. The cells were suspended in serum-free medium. Equal volume of trypan blue and cell suspension were mixed and analyzed by an inverted microscope (Olympus, Tokyo, Japan). The colorless cells were considered as viable cells, and the colored cells were considered as apoptotic and dead cells.
Flow cytometry assay Apoptosis of mouse CT-26 colorectal cancer cells induced by GA and 37 was also quantified by an annexin V-FITC/PI (BD Bioscience, San Diego, CA, USA) dual-staining assay (26). Briefly, cells were seeded into 6-well plates and were allowed to incubate for 24 h, then compound GA or 37 was added. After treatment for 24 h, cells were collected, centrifuged, and resuspended with PBS. After centrifugation, cells were resuspended with binding buffer and incubated with annexin V and PI for 30 min in dark condition. Analysis was performed by flow cytometry. Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
Transwell migration assay The inhibitory effect on migration of mouse CT-26 colorectal cancer cells was assessed by the modified Boyden’s chamber (Millipore, Billerica, MA, USA) assay in 24-well cell culture plate with 8.0-lm pore (27). After overnight starvation, cells were collected, centrifuged, and resuspended with serum-free medium. The top chambers were seeded with 10 9 104 mouse CT-26 colorectal cancer cells in 100 lL serum-free RMPI 1640 medium containing different concentrations of GA and 37. The bottom chambers were filled with 700 lL medium supplemented with different concentrations of GA and 37. Cells were allowed to migrate for 12 h. 4% paraformaldehyde was added to the wells with the chamber in order to fix the cells. After fixation for 0.5 h, the non-migrated cells on the top surface of the membrane were scraped with cotton swab. The cells were washed with PBS for three times, and cells were stained with 0.2% crystal violet. Then the chambers were washed using water, and the membrane was left to dry. Images
were taken using an inverted microscope (Olympus), and cells from six random areas per filter were counted.
Results and Discussion Synthesis of GA derivatives To determine which five-membered heterocyclic ring contributed the most antitumor activity, a series of GA derivatives with heterocyclic rings (pyrazole, 5-(trifluoromethyl)-1H-pyrazole, ethyl 1H-pyrazole-5-carboxylate, 1,2,3thiadiazole, and 2-methyloxazole) fused at C-2 and C-3 positions were synthesized (Schemes 1 and 2) based on our previous research (21,22). The synthesis of pyrazole derivatives is outlined in Scheme 1. Oxidation of GA with Jone’s reagent gave intermediate 1, which was then converted to 6 or 7 by methyl or benzyl esterification with MeI or BnBr, respectively. Intermediates 2, 3, 8, 9, 10, and 11 were obtained by Claisen condensation of 1 with
COOR'
COOR'
COOR'
O
O
O H
a
H
HON
O
b
H N
O
H
O
O 6 R'= -Me 7 R'= -Bn
18 19
16 R= -Me 17 R= -Bn
e
R'= -Me R'= -Bn
c
COOMe O H2N HN
COOR'
COOMe O
O H
O
H
H f
S
N
N
O
N N 23
20 R'= -Me 21 R'= -Bn
24 d
22 R'= -H
g COOH O H S N N 25 Scheme 2: Reagents and conditions: (a) isoamyl nitrite, t-BuOK, t-BuOH, rt, 5 h (65% for 16 and 56% for 17); (b) Zn, Ac2O, AcOH, rt, 12 h (68% for 18 and 81% for 19); (c) POCl3, pyridine, rt, 12 h (93% for 20 and 87% for 21); (d) H2 (1 atm), Pd/C, MeOH, 12 h (94% for 22); (e) NH2NHCONH2HCl, AcONa, EtOH, reflux, 2 h; (f) SOCl2, CH2Cl2, rt, 12 h (84% for 24 over two steps); (g) LiI, DMF, reflux, 48 h (43% for 25).
Chem Biol Drug Des 2014
5
Gao et al.
because of the strong electron-withdrawing group CF3, made the acylation of NH group in prazole unsuccessfully.
corresponding esters in the presence of NaH. Then the pyrazole derivatives 4, 5, 12, 13, 14, and 15 were synthesized by condensation of corresponding intermediates with hydrazine hydrate in AcOH.
Antiproliferative activity To evaluate the anticancer potency of these synthesized heterocyclic ring-fused GA derivatives, the antiproliferative activity of 4–25 (Scheme 1 and 2) was first screened in an SRB assay against eight cancer cell lines of different origin: HCT-116, HCT-8, HT-29, CT-26, PC-3, DU-145, MDA-MB-231, 4T1 (Table 1). The results showed that derivatives having a free carboxylic group in position C-30 (4 and 5) resulted in a lower inhibition of cell growth compared with the corresponding methyl or benzyl esters (12, 14, and 13), and this was in accordance with previously reports (13,14), thus they were excluded from further investigations. The 2-methyloxazole was not a favorable substituted group to improve antitumor activity, because of both derivatives having a free carboxylic group in position C-30 (22), and the corresponding methyl or benzyl esters (20 or 21) gave IC50 values higher than 50 lM. Compound 24 was found to be insoluble in dimethylsulfoxide (DMSO), therefore it was untested, and its corresponding C-30 free carboxylic compound 25 displaying expected poor activity. Most of the pyrazole derivatives showed significantly improved IC50 values, such as 12 and 13, especially 14 (average IC50 = 13.94 lM) and 15 (average IC50 = 14.25 lM) exhibited approximately 6.5 times more potent than GA (average IC50 = 90.86 lM). The praz-
The synthesis of 1,2,3-thiadiazole and 2-methyloxazole derivatives is outlined in Scheme 2. The 2-methyloxazole derivatives 18 and 19 were obtained by treatment of 6 and 7 with isoamyl nitrite in the presence of t-BuOK in t-BuOH, followed by reaction with zinc powder in HOAc/ Ac2O, respectively. Compound 20 and 21 were synthesized by cyclization of 18 and 19 in the presence of POCl3 in pyridine. Hydrogenation of 21 with Pd/C under H2 afforded 22. Intermediate 23 was obtained by reaction of 6 with semicarbazide hydrochloride. Reaction of 23 with SOCl2 produced 24. The 1,2,3-thiadiazole derivative 25 was obtained by demethylation of C-30 methyl ester with LiI in DMF. The synthesis of piperidine derivatives 26–37 is outlined in Scheme 3. Compounds 26 and 27 were produced by amidation of 12 with MeSO2Cl and Ac2O, respectively. Intermediates 28, 29, 30, 31, and 32 were obtained by amidation of 12 with corresponding piperidine caboxylic acids in the presence of CDI and DMAP in CH2Cl2. Compound 33 was produced by deprotection of Boc in the presence of boron trifluoride etherate. Compounds 34, 35, 36, and 37 were prepared in a manner similar to that for 33. We also tried to amidation of 13 in a similar way, and
COOMe
COOMe
COOMe
O
O
O H
H
N
R N
HN
N
N
N
H
O
a or b
12
33
NH 26 R= -SO2Me 27 R= -Ac 28 R=
c n O
N Boc
29 R=
n= 0 COOMe
n NBoc n= 0
O
O
30 R=
HN
n NBoc n= 1
O 31 R=
c
H
O n N
N
n NBoc n= 2
O 32 R= n O
34 35 36 37
n= 0 n= 1 n= 2 n= 3
NBoc n= 3 OH
n Scheme 3: Reagents and conditions: (a) MeSO2Cl or Ac2O, DMAP, DCM, rt, 12 h (73% for 26 and 91% for 27); (b)BocN (n = 0) O OH n (n = 0, 1, 2, and 3), CDI (N,N’-carbonyldiimidazole), DMAP, CH2Cl2, rt, 3 h (76% for 28, 73% for 29, 78% for 30, 79% for 31, orBocN O and 82% for 32); (c) Et2OBF3, Et2O, rt, 0.5 h (80% for 33, 77% for 34, 68% for 35, 71% for 36, and 67% for 37).
6
Chem Biol Drug Des 2014
Chem Biol Drug Des 2014
75.08 >50 15.36 13.25 11.90 7.49 8.20 >50 >50 >50 33.17
SD)
0.79 0.15 0.48 0.25 0.41
1.64
3.96
HCT-116
82.22 >50 36.75 31.48 19.96 13.26 9.96 >50 >50 >50 47.51
HCT-8
0.60 0.89 0.79 0.34 0.84
2.10
4.96 77.23 >50 29.90 31.97 27.60 15.22 12.49 >50 >50 >50 >50
HT-29
0.66 0.73 0.79 0.73 0.60
1.59 70.48 >50 38.56 26.10 22.04 19.78 17.65 >50 >50 >50 45.67
CT-26
1.99 1.92 1.36 1.08 0.88
1.82
0.65 88.04 >50 31.27 26.51 19.80 12.27 19.56 >50 >50 >50 >50
PC-3
5.0 0.39 1.61 1.70 0.35
5.31 136.40 >50 >50 30.31 33.60 19.03 23.83 >50 >50 >50 >50
DU-145
0.78 3.59 2.66 0.55
3.80
8.70 14.12 >50 21.98 23.19 12.13 6.36 6.99
SD)
0.69 0.38 0.26 0.25 0.15
2.20 1.00
HCT-116
24.47 29.23 >50 23.82 22.31 8.98 13.55 11.72
HCT-8
1.62 0.69 0.37 0.60 0.87
1.25 0.97 18.48 30.99 >50 21.78 25.21 12.12 12.46 8.76
HT-29
0.95 1.02 0.52 0.64 0.31
1.73 1.44 15.25 33.02 >50 23.74 21.38 9.87 12.95 8.08
CT-26
0.65 1.41 0.44 0.24 0.50
0.85 1.63
18.90 25.41 >50 23.12 22.36 11.71 12.00 5.19
PC-3
2.11 1.87 0.31 1.69 0.10
2.28 1.09
>50 36.70 >50 21.94 20.76 13.26 13.91 7.54
DU-145
3.60 1.07 1.07 1.32 0.38
1.29
From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments; data not calculated.
26 27 29 33 34 35 36 37
Compound
IC50 (lM
Table 2: IC50 values of 26–37 against the growth of eight cancer cell lines
From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments; data not calculated.
GA 4 5 12 13 14 15 20 21 22 25
Compound
IC50 (lM
Table 1: IC50 values of 4–25 against the growth of eight cancer cell lines
0.46 0.67 0.48 0.32 0.51
3.00
28.63 32.28 >50 24.00 19.16 10.05 9.56 9.76
1.03 2.53 1.29 0.97 0.62
2.21 1.00
MDA-MB-231
101.20 >50 35.30 33.18 27.36 18.14 16.58 >50 >50 >50 >50
MDA-MB-231
12.47 20.10 >50 9.68 5.34 5.80 5.77 6.09
4T1
96.22 >50 33.71 14.73 15.66 6.32 5.75 >50 >50 >50 >50
4T1
1.26 0.66 0.97 0.24 0.68
1.48 1.97 0.78 0.88 0.25
1.65 0.36
2.22
27.73 >50 21.26 19.96 10.49 10.82 8.02
_
1.52 1.37 0.63 0.82 0.40
1.10
0.77 1.26 0.91 0.60
3.19
Average
90.86 >50 _ 25.94 22.24 13.94 14.25 >50 >50 >50 _
Average
Novel GA Derivatives with Antitumor Activity
7
Gao et al.
ole and ethyl 1H-pyrazole-5-carboxylate groups almost contributed equally activity to corresponding derivatives (14 and 15). Prazole-substituted derivative with benzyl esterification of the C-30 carboxylic group (14) exhibited about twofold higher cytotoxic activity against tumor cells than its corresponding methyl ester (12). Considering GA itself has poor water solubility and exhibits low bioavailability and if the hydrophobic benzyl ester group is formed at the C-30, free carboxylic acid position will make the derivative more insoluble in water than corresponding methyl ester, and therefore, the methyl ester 12 was selected as a new lead for further modification. We also considered 13 as a further modifying lead and found the NH group of its prazole ring was hard to substitute because of the strong electron-withdrawing group CF3 in presence. We considered 12 as a new starting modifying structure for more potent activity derivatives. Novel piperidine compounds 26–37 were synthesized from 12 with amidation and N-Boc deprotection reactions. Screening of these derivatives (compounds 26–37) with the SRB assay revealed that all of the piperidine compounds showed more potent activity than their parent compound 12 (Table 2). The most potent compound 37 showed the average IC50 8.02 lM, which was about 11-fold more potent than the lead compound GA. Compound 34 (substituted with 4-piperidinecarboxylic acid), possessed a
slightly more cytotoxic activity than compound 33 (substituted with 3-piperidinecarboxylic acid). Perhaps more distance between the prazole to the NH group of piperidine will afford more potency, and this speculation was further confirmed by the results of 34 (n = 0), 35 (n = 1), 36 (n = 2), and 37 (n = 3). The free NH group in piperidine was important for keeping the cytotoxic activity against tumor cells, and if the group was protected with the Boc (29), the activity would be decreased dramatically.
Apoptosis study using flow cytometry assay and trypan blue staining As previously reported (14), GA induces apoptosis on various tumor cell lines. To examine whether this effect remains or is lost on derivatization, we tested GA and 37 for the induction of apoptosis using flow cytometry and trypan blue exclusion assays. As shown in Figures 1 and 2, we found that the ability of GA and 37 to trigger apoptosis is displayed obviously on mouse CT-26 colorectal cancer cells.
Migration assay Migration is a key step during the metastasis of cancer (28), and to our best knowledge, there are no reports describing inhibitory effect of GA and its analogs on
2.31%
4.85%
12.29%
0.65%
0.59%
1.77%
Control
GA (80 μM)
GA212 (8 μM)
Figure 1: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in flow cytometry assay compared with control group. After treatment of GA or 37 for 24 h, CT-26 colorectal cancer cells with the supernatant were collected, centrifuged, and resuspended with PBS. After centrifugation, cells were resuspended with binding buffer and stained with annexin V and PI for 0.5 h. Analysis was performed by flow cytometry, and the percentage displayed the apoptosis cells of all cells.
Control
GA (80 μM)
37 (8 μM)
Figure 2: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in trypan blue exclusion assay compared with control group. Mouse CT-26 colorectal cancer cells were seeded onto 6-well plates. After treatment of GA or 37 for 24 h, cells with the supernatant were collected, centrifuged, and resuspended with serum-free medium. The cells were stained with trypan blue, and images were taken using an inverted microscope. Arrows indicated the apoptotic or dead cells.
8
Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
GA (80 μM)
Control
37 (4 μM) Migration of cancer cells (% of control)
Figure 3: GA and 37 suppressed migration of mouse CT-26 colorectal cancer cells in transwell migration assay compared with control group. After starvation overnight, the cells containing the tested compounds were seeded onto top side of the chambers. Cells were allowed to migrate for 12 h and fixed with 4% paraformaldehyde. After fixation for 0.5 h, the non-migrated cells on the top side were scraped using cotton swab. Migrated cells were stained with 0.2% crystal violet, and images were taken using an inverted microscope. Cells from six random areas per filter were counted.
37 (16 μM)
120 100 80
*** ***
60
***
40
***
20 0
migration of tumor cell lines. To determine this effect, we performed transwell migration assay. As shown in Figure 3, we found that GA inhibited the migration of mouse CT-26 colorectal cancer cells obviously, compared with the control group. We also tested the antimetastatic activity of 37 and other derivatives (such as 34 and the data were not shown) and found that these derivatives, especially 37, significantly inhibited the migration (IC50 was about 4 lM) of mouse CT-26 colorectal cancer cells in a dose-dependent manner. Surprisingly, 37 suppressed migration completely at medium concentration (16 lM). In addition, the results showed that the inhibitory effect on migration of 37 at 4 lM was similar to that of GA at 80 lM, it means that 37 was about 20-fold more potent than GA on antimetastatic activity of mouse CT-26 colorectal cancer cells.
Conclusion In this study, we synthesized a class of novel heterocyclic ring-fused of GA analogs and screened them for antiproliferative activity on cancer cell lines. Most of these compounds exhibited an obvious increase in inhibitory potency compared with GA. An unsubstituted carboxylic group in C-30 resulted in a lower inhibition of cell growth Chem Biol Drug Des 2014
37 (8 μM)
C
GA
4
8
16
37 (μM)
compared with the corresponding esters. Introduction of prazole substituent was quite promising. Most of the pyrazole derivatives showed significantly improved IC50 values, especially 14 and 15. The further modification of prazole compound 12 with various piperidinecarboxylic acids was efficiently afforded the most active compound 37. As demonstrated by flow cytometry and trypan blue exclusion assays, tumor cell death trigged by the compounds resulted from apoptotic processes. In addition, we also described here for the first time that GA and the synthetic compounds exhibited antimetastatic activity on the tested cancer cell line. In conclusion, we report GA heterocyclic derivatives as a series of new chemical entities for the first time. Especially 37, which exhibited good antitumor and antimetastatic activity, could be used as a promising lead for the development of a new class of antitumor and antimetastatic agents.
Acknowledgments Shanghai Science and Technology Council (Grant 12ZR1408500), the Fundamental Research Funds for the Central Universities, Innovation Program of Shanghai Munici9
Gao et al.
pal Education Commission (13zz034), and National Natural Science Foundation of China (81272463) are appreciated for their financial support.
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€hl D. (2010) 14. Csuk R., Schwarz S., Kluge R., Stro Synthesis and biological activity of some antitumor active derivatives from glycyrrhetinic acid. Eur J Med Chem;45:5718–5823. 15. Gao Y., Guo X., Li X., Liu D., Song D., Xu Y., Sun M., Jing Y., Zhao L. (2010) The synthesis of glycyrrhetinic acid derivatives containing a nitrogen heterocycle and their antiproliferative effects in human leukemia cells. Molecules;15:4439–4449. re C., Ghostin 16. Lallemand B., Chaix F., Bury M., Bruye J., Becker J.P., Delporte C., Gelbcke M., Mathieu V., vost M., Jabin I., Kiss R. (2011) N-(2-{3Dubois J., Pre [3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)- glycyrrhetinamide (6b): a novel anticancer glycyrrhetinic acid derivative that targets the proteasome and displays anti-kinase activity. J Med Chem;54:6501–6513. €hl D. 17. Csuk R., Schwarz S., Siewert B., Kluge R., Stro (2011) Synthesis and antitumor activity of ring A modified glycyrrhetinic acid derivatives. Eur J Med Chem;46:5356–5369. 18. Logashenko E.B., Salomatina O.V., Markov A.V., Korchagina D.V., Salakhutdinov N.F., Tolstikov G.A., Vlassov V.V., Zenkova M.A. (2011) Synthesis and pro-apoptotic activity of novel glycyrrhetinic acid derivatives. ChemBioChem;12:784–794. €hl D. (2012) Does 19. Csuk R., Schwarz S., Kluge R., Stro one keto group matter? Structure-activity relationships of glycyrrhetinic acid derivatives modified at position C-11. Arch Pharm Chem Life Sci;345:28–32. €hl D. 20. Csuk R., Schwarz S., Siewert B., Kluge R., Stro (2012) Conversions at C-30 of glycyrrhetinic acid and their impact on antitumor activity. Arch Pharm Chem Life Sci;345:223–230. 21. Qiu W.W., Shen Q., Yang F., Wang B., Zou H., Li J.Y., Li J., Tang J. (2009) Synthesis and biological evaluation of heterocyclic ring-substituted maslinic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B. Bioorg Med Chem Lett;19:6618–6622. 22. Xu J., Li Z., Luo J., Yang F., Liu T., Liu M., Qiu W.W., Tang J. (2012) Synthesis and biological evaluation of heterocyclic ring-fused betulinic acid derivatives as novel inhibitors of osteoclast differentiation and bone resorption. J Med Chem;55:3122–3134. 23. Santos R.C., Salvador J.A., Marın S., Cascante M. (2009) Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity. Bioorg Med Chem;17:6241–6250. 24. Kumar V., Rani N., Aggarwal P., Sanna V.K., Singh A.T., Jaggi M., Joshi N., Sharma P.K., Irchhaiya R., Burman A.C. (2008) Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives. Bioorg Med Chem Lett;18:5058–5062. 25. Dong Y., Lu B., Zhang X., Zhang J., Lai L., Li D., Wu Y., Song Y., Luo J., Pang X., Yi Z., Liu M. (2010) Cucurbitacin E, a tetracyclic triterpenes compound from Chinese medicine, inhibits tumor angiogenesis through VEGFR2-mediated Jak2-STAT3 signaling pathway. Carcinogenesis;31:2097–2104. Chem Biol Drug Des 2014
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26. Dai F., Chen Y., Song Y., Huang L., Zhai D., Dong Y., Lai L., Zhang T., Li D., Pang X., Liu M., Yi Z. (2012) A natural small molecule harmine inhibits angiogenesis and suppresses tumour growth through activation of p53 in endothelial cells. PLoS ONE;7:e52162. 27. Song Y., Dai F., Zhai D., Dong Y., Zhang J., Lu B., Luo J., Liu M., Yi Z. (2012) Usnic acid inhibits breast tumor angiogenesis and growth by suppressing VEGFR2-mediated AKT and ERK1/2 signaling pathways. Angiogenesis;15:421–432. 28. Zhang T., Li J., Dong Y., Lai L., Dai F., Deng H., Chen Y., Liu M., Yi Z. (2012) Cucurbitacin E inhibits breast tumor metastasis by suppressing cell migration and invasion. Breast Cancer Res Treat;135:445–458.
Chem Biol Drug Des 2014
Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Synthetic procedures for the preparation of compounds 4, 5, 12, 13, 14, 15, 20, 21, 22, 22, 24, 25. Appendix S2. Compound 34 suppressed migration of mouse CT-26. Figure S1. Compounds 34 suppressed migration of mouse CT-26 colorectal cancer cells in transwell migration assay compared with control group.
11
Synthesis of Novel Heterocyclic Ring-Fused 18b-Glycyrrhetinic Acid Derivatives with Antitumor and Antimetastatic Activity Cheng Gao1, Fu-Jun Dai2, Hai-Wei Cui1, Shi-Hong Peng2, Yuan He2, Xue Wang2, Zheng-Fang Yi2,* and Wen-Wei Qiu1,3,* 1
Department of Chemistry, East China Normal University, Shanghai 200062, China 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China 3 Shanghai Engineering Research Center for Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, China *Corresponding authors: Zheng-Fang Yi, [email protected], Wen-Wei Qiu, [email protected] Glycyrrhetinic acid (GA) is one of the most important triterpenoic acids shows many pharmacological effects, especially antitumor activity. GA triggers apoptosis in various tumor cell lines. However, the antitumor activity of GA is weak, thus the synthesis of new synthetic analogs with enhanced potency is needed. By introducing various five-member fused heterocyclic rings at C-2 and C-3 positions, 18 novel GA derivatives were obtained. These compounds were evaluated for their inhibitory activity against the growth of eight different tumor cell lines using a SRB assay. The most active compound 37 showed IC50 between 5.19 and 11.72 lM, which was about 11-fold more potent than the lead compound GA. An apoptotic effect of GA and 37 was determined using flow cytometry and trypan blue exclusion assays. We also demonstrated here for the first time that GA and the synthetic derivatives exhibited inhibitory effect on migration of the tested tumor cells, especially 37 which was about 20-fold more potent than GA on antimetastatic activity. Key words: antitumor, apoptosis, glycyrrhetinic acid, migration, triterpenoic acids Received 14 January 2014 and accepted for publication 12 February 2014
Natural products have been used to treat human disease for thousands of years and play an increasingly significant role in drug discovery and development process in recent years (1). Pentacyclic triterpenoids are one of the most ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12308
important classes of natural products occurring and have been studied intensively for their diverse biological and pharmacological activities (2–5). Glycyrrhetinic acid (GA, Scheme 1), a natural pentacyclic triterpene acid, is known to show many biological effects including anti-inflammatory, antiviral, and hepatoprotective (6). Recently, GA attracted the focus of research interests not only for it can be accessed abundantly from the roots of licorice [up to 24% (7,8)], but also for its triggering apoptosis on tumor cells (9–12). However, GA has only weak inhibitory effect against various tumor cell lines (IC50 is approximately 80 lM), and some structural modifications have been previously performed (13–20). We also become interested in producing more active GA analogs. Recently, we and other groups have found that introduction of the nitrogencontaining heterocyclic rings, especially five-membered heterocyclic rings to the pentacyclic triterpenoids, such as maslinic acid and betulinic acid, can improve the biological activities (21–24). Thus, to obtain more potent anticancer analogs, we designed and synthesized a series of novel five-membered heterocyclic ring-fused GA derivatives at C-2 and C-3 positions, and their inhibitory activity against the growth of 8 different tumor cell lines was evaluated using a SRB assay. GA and its derivative were screened by flow cytometry and trypan blue exclusion assays to determine the apoptotic behavior, and their inhibitory effect on tumor cell migration was tested for the first time.
Methods and Materials Chemistry All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. When needed, the reactions were carried out in flame or oven-dried glassware under a positive pressure of dry N2. Column chromatography was performed on silica gel (QinDao, 200–300 mesh) using the indicated eluents. Thin-layer chromatography was carried out on silica gel plates (QinDao) with a layer thickness of 0.25 mm. Melting points were determined using the MELTEMP 3.0 apparatus and are uncorrected. 1H (300, 400, and 500 MHz) and 13C (100 and 125 MHz) NMR spectra were recorded on Varian Mercury-300, Bruker AM-400, and Bruker AV-500 spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal 1
Gao et al.
COOR'
COOH
COOH
O
O
O H
H
a
H
d O
O
HO GA
6 R'= -Me 7 R'= -Bn
1 b
b
COOH
COOH
O R
H
COOR'
O
R c O
HN N
O
R
H
H
O O
O 2 R= -H 3 R= -CF3
4 R= -H 5 R= -CF3
c COOR'
8 9 10 11
R= -H R= -CF3 R= -H R= -COOEt
R'= -Me R'= -Me R'= -Bn R'= -Bn
O R
H
HN N 12 13 14 15
R= -H R= -CF3 R= -H R= -COOEt
R'= -Me R'= -Me R'= -Bn R'= -Bn
Scheme 1: Reagents and conditions: (a) Jone’s reagent, THF, 0 °C, 1 h, (92%); (b) RCOOEt (R = -H, -CF3, -COOEt), NaH (60%), THF, rt, 8 h (91% for 2, 82% for 3, 86% for 8, and 74% for 9); (c) NH2NH2H2O, AcOH, rt, 12 h (88% for 4, 78% for 5, 92% for 12, 68% for 13; 88% and 69% for 14 and 15, respectively, over two steps). (d) MeI or BnBr, K2CO3, DMF, rt, 2 h (87% for 6 and 92% for 7).
standard. All chemical shift values were reported in units of d (ppm). The following abbreviations were used to indicate the peak multiplicity: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad. High-resolution mass data were obtained on a Bruker micrOTOF-Q II spectrometer.
Preparation of compound 26 To a solution of 12 (203 mg, 0.4 mmol) and DMAP (49 mg, 0.4 mmol) in dry DCM (20 mL) was added mesyl chloride (0.15 mL, 2.0 mmol) under N2. The reaction mixture was stirred for 12 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 4/1 v/v) to give 26 as a white solid (168 mg, 73%); mp. 245.0–247.0 °C. 1H NMR (400 MHz, CDCl3) d 7.63 (s, 1H), 5.74 (s, 1H), 3.83 (d, J = 15.6 Hz, 1H), 3.70 (s, 3H), 3.22 (s, 3H), 2.52 (s, 1H), 2
1.39 (s, 3H), 1.35 (s, 3H), 1.29 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.60, 176.98, 169.99, 163.80, 128.67, 127.92, 118.21, 59.93, 53.29, 51.89, 48.47, 45.31, 44.13, 43.46, 41.32, 41.09, 37.89, 37.86, 36.67, 34.91, 31.95, 31.53, 31.22, 28.69, 28.39, 26.65, 26.51, 24.73, 23.35, 18.65, 18.28, 15.48. ESI-HRMS (m/z) [M + Na]+ calcd for C33H48N2NaO5S, 607.3182, found 607.3160.
Preparation of compound 27 To a solution of 12 (150 mg, 0.3 mmol) and DMAP (36 mg, 0.3 mmol) in dry DCM (20 mL) was added Ac2O (151 mg, 1.5 mmol) under N2. The reaction mixture was stirred for 12 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 27 as a white solid, (148 mg, Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
91%); mp. 225.0–227.0 °C. 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 2.63 (s, 3H), 2.53 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.61, 176.95, 169.79, 169.48, 162.24, 128.69, 124.78, 119.32, 59.92, 53.29, 51.85, 48.43, 45.30, 44.10, 43.42, 41.29, 37.94, 37.84, 36.91, 34.71, 31.95, 31.93, 31.56, 31.20, 28.66, 28.36, 26.63, 26.50, 24.75, 23.33, 21.66, 18.67, 18.25, 15.46. ESI-HRMS (m/z) [M + Na]+ calcd for C34H48N2NaO4, 571.3512, found 571.3541.
Preparation of compounds 29 and 33–37 Compound 28 To a solution of N-Boc-piperidine-3-carboxylic acid (275 mg, 1.2 mmol) in dry DCM (20 mL) was added N,N’carbonyldiimidazole (194 mg, 1.2 mmol). The reaction mixture was stirred for 0.5 h at 23 °C, and then 12 (203 mg, 0.4 mmol) and DMAP (146 mg, 1.2 mmol) was added. The reaction mixture was stirred for 3 h at 23 °C and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 28 as a white solid (213 mg, 76%). 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19 (d, J = 7.1 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.70 (s, 4H), 3.15 (d, J = 10.4 Hz, 1H), 2.86 (t, J = 11.1 Hz, 1H), 2.52 (s, 1H), 1.43(s,9H), 1.39 (s, 3H), 1.33, 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.09 (s, 3H), 0.83 (s, 3H).
Compound 29 By a similar procedure described for 28, 29 was obtained as a white solid (206 mg, 73%). 1H NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 5.73 (s, 1H), 4.15 (d, J = 12.6 Hz, 2H), 3.83 (d, J = 15.7 Hz, 1H), 3.78–3.65 (m, 4H), 2.90 (t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.46(s, 9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 30 By a similar procedure described for 28, 30 was obtained as a white solid (228 mg, 78%). 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 4.07 (d, J = 13.5 Hz, 2H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 2.95-3.07 (m, 2H), 2.75 (t, J = 12.0 Hz, 2H), 2.52 (s, 1H), 1.45(s,9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 31 By a similar procedure described for 28, 31 was obtained as a white solid (235 mg, 79%). 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 5.73 (s, 1H), 4.08 (d, J = 13.3 Hz, 2H), 3.82 (d, J = 15.7 Hz, 1H), 3.70 (s, 3H), 3.10 (td, Chem Biol Drug Des 2014
J = 7.4, 4.2 Hz, 2H), 2.67 (t, J = 11.8 Hz, 2H), 2.53 (s, 1H), 1.45 (s,9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 32 By a similar procedure described for 28, 32 was obtained as a white solid (237 mg, 78%). 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 4.19 (d, J = 7.1 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.70 (s, 4H), 3.15 (d, J = 10.4 Hz, 1H), 2.86 (t, J = 11.1 Hz, 1H), 2.52 (s, 1H),1.45 (s, 9H), 1.39 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H).
Compound 33 To a solution of 28 (159 mg, 0.21 mmol) in Et2O (5 mL) was added boron trifluoride etherate (2 mL). The reaction mixture was stirred for 0.5 h at 23 °C and then poured into ice water (20 mL). The precipitate was filtered and washed with Et2O (5 mL), then dried to give 33 as a white solid (104 mg, 80%); mp. 218.0–221.0 °C. 1H NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 5.73 (s, 1H), 3.83 (d, J = 15.8 Hz, 1H), 3.75–3.63 (m, 4H), 3.43 (d, J = 13.1 Hz, 1H), 3.23 (d, J = 11.4 Hz, 1H), 3.09 (d, J = 10.3 Hz, 1H), 2.87 (s, 1H), 2.52 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.80, 177.07, 171.90, 170.09, 163.19, 128.68, 125.18, 119.90, 59.92, 53.21, 51.91, 48.47, 45.36, 44.15, 43.48, 41.30, 37.97, 37.88, 36.88, 36.69, 34.78, 31.96, 31.54, 31.24, 28.70, 28.40, 26.66, 26.53, 25.15, 24.95, 24.80, 23.37, 18.66, 18.29, 15.56, 14.43. ESI-HRMS (m/z) [M + Na]+ calcd for C38H55N3NaO4, 640.4090, found 640.4119.
Compound 34 By a similar procedure described for 33, 34 was obtained as a white solid (99 mg, 77%); mp. 227.0–231.0 °C. 1H NMR (500 MHz, CDCl3) d 7.82 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.76 (s, 1H), 3.70 (s, 3H), 3.25 (s, 2H), 2.86 (t, J = 11.8 Hz, 2H), 2.52 (s, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.85, 177.10, 171.75, 170.16, 163.25, 128.75, 125.28, 120.00, 59.97, 53.28, 51.97, 48.53, 45.41, 44.19, 44.15, 43.53, 41.36, 38.02, 37.93, 36.94, 36.59, 34.84, 32.01, 31.60, 31.29, 28.74, 28.45, 26.71, 26.59, 25.18, 25.01, 24.85, 23.41, 18.72, 18.34, 15.61, 14.47. ESI-HRMS (m/z) [M + Na]+ calcd for C38H55N3NaO4, 640.4090, found 640.4085.
Compound 35 By a similar procedure described for 33, 35 was obtained as a white solid (90 mg, 68%); mp. 219.0–220.0 °C. 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 5.74 (s, 1H), 4.17 3
Gao et al.
(s, 1H), 3.85 (d, J = 16.0 Hz, 1H), 3.70 (d, J = 5.7 Hz, 3H), 3.60 (d, J = 15.4 Hz, 2H), 3.42 (s, 2H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.16 (s, 3H), 1.07 (s, 3H), 0.84 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.76, 177.08, 171.67, 169.95, 162.52, 128.74, 124.95, 119.50, 59.98, 53.28, 51.93, 48.50, 45.37, 45.33, 44.18, 43.49, 41.34, 38.00, 37.91, 36.95, 34.79, 33.16, 31.99, 31.60, 31.26, 30.70, 30.51, 28.72, 28.67, 28.44, 26.69, 26.57, 24.85, 23.39, 18.71, 18.32, 15.57. ESI-HRMS (m/z) [M + H]+ calcd for C39H58N3O4, 632.4427, found 632.4408.
Compound 36 By a similar procedure described for 33, 36 was obtained as a white solid (95 mg, 71%); mp. 210.0–211.0 °C. 1H NMR (400 MHz, CDCl3) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.71 (s, 3H), 3.56 (d, J = 12.4 Hz, 2H), 3.13–2.93 (m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.70, 177.01, 171.85, 169.89, 162.24, 128.67, 124.82, 119.20, 59.93, 53.24, 51.86, 48.44, 45.31, 45.09, 44.11, 43.43, 41.28, 37.94, 37.84, 36.90, 35.19, 34.72, 33.64, 33.55, 31.93, 31.57, 31.20, 28.70, 28.67, 28.37, 26.62, 26.50, 24.79, 23.34, 21.48, 18.65, 18.26, 15.50. ESI-HRMS (m/ z) [M + H]+ calcd for C40H60N3O4, 646.4584, found 646.4573.
Compound 37 By a similar procedure described for 33, 37 was obtained as a white solid (92 mg, 67%); mp. 201.0–204.0 °C. 1H NMR (400 MHz, CDCl3) d 7.84 (s, 1H), 5.73 (s, 1H), 3.82 (d, J = 15.8 Hz, 1H), 3.70 (s, 3H), 3.52 (s, 2H), 3.11–2.92 (m, 4H), 2.53 (s, 1H), 1.40 (s, 3H), 1.33 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 0.83 (s, 3H). 13 C NMR (100 MHz, CDCl3) d 199.70, 177.08, 171.62, 170.03, 163.91, 128.71, 125.21, 120.55, 59.91, 53.23, 53.13, 51.93, 48.49, 45.67, 45.36, 45.15, 44.17, 43.50, 41.34, 37.98, 36.89, 36.25, 34.83, 31.99, 31.53, 31.45, 31.26, 28.72, 28.43, 26.69, 26.56, 25.66, 24.83, 24.77, 23.42, 23.39, 20.54, 18.69, 18.30, 15.62, 15.57. ESIHRMS (m/z) [M + H]+ calcd for C41H62N3O4, 660.4740, found 660.4731.
Biology Cell lines and culture conditions Cell lines used in this study were obtained from the American Type Culture Collection (ATCC). HCT-116, HCT-8, HT-29, CT-26, PC-3, and DU-145 cell lines were cultured in RPMI1640 medium. Mouse 4T1 breast cancer cells were cultured in DMEM medium, and human MDA-MB231 breast cancer cells were cultured in MEM medium supplemented with 1% sodium pyruvate and 1% nonessential amino acids. Medium was supplemented with 4
10% FBS, penicillin (100 units/mL), and streptomycin (100 units/mL). All cells were incubated at 37 °C and 5% CO2 incubator.
Viability assay The cell viability of eight tumor cell lines in the presence of our synthetic compounds was determined by SRB (Sigma Aldrich, St.Louis, MO, USA) assay (14) In brief, cells were seeded into 96-well plates at the appropriate cell densities to prevent confluence during the experiment. After incubation for 24 h, the cells were treated with various concentrations of the compound (0–50 lM) for 96 h. Control cells were exposed to DMSO at a concentration equivalent to that of the compound-treated cells, and the final dilution of DMSO solvent used in this assay never exceeded 0.4%. After treatment for 96 h, the supernatant medium was removed from the 96-well plates, and the cells were fixed by 50% TCA at 4 °C. After fixation for 1 h, the plates were washed by water for five times. The plates were allowed to dry using hair dryer followed by being dyed with 100 lL of 0.4% SRB for 10 min. After dying, the plates were washed by 1% acetic acid to remove the dye and allowed to dry using hair dryer. One hundred microliter of 10 mM Tris base solution was added to each well, and absorbance was examined using a 96-well plate reader. The IC50 was calculated using GraphPad Software (San Diego, CA, USA).
Trypan blue exclusion assay Apoptosis of mouse CT-26 colorectal cancer cells was assessed by trypan blue exclusion assay (25). In short, cells were plated onto 6-well plates and were exposed to GA and 37 for 24 h. After treatment with various doses of the compounds for 24 h, all cells including the supernatant were collected and centrifuged to collect the viable, apoptotic, and dead cells. The cells were suspended in serum-free medium. Equal volume of trypan blue and cell suspension were mixed and analyzed by an inverted microscope (Olympus, Tokyo, Japan). The colorless cells were considered as viable cells, and the colored cells were considered as apoptotic and dead cells.
Flow cytometry assay Apoptosis of mouse CT-26 colorectal cancer cells induced by GA and 37 was also quantified by an annexin V-FITC/PI (BD Bioscience, San Diego, CA, USA) dual-staining assay (26). Briefly, cells were seeded into 6-well plates and were allowed to incubate for 24 h, then compound GA or 37 was added. After treatment for 24 h, cells were collected, centrifuged, and resuspended with PBS. After centrifugation, cells were resuspended with binding buffer and incubated with annexin V and PI for 30 min in dark condition. Analysis was performed by flow cytometry. Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
Transwell migration assay The inhibitory effect on migration of mouse CT-26 colorectal cancer cells was assessed by the modified Boyden’s chamber (Millipore, Billerica, MA, USA) assay in 24-well cell culture plate with 8.0-lm pore (27). After overnight starvation, cells were collected, centrifuged, and resuspended with serum-free medium. The top chambers were seeded with 10 9 104 mouse CT-26 colorectal cancer cells in 100 lL serum-free RMPI 1640 medium containing different concentrations of GA and 37. The bottom chambers were filled with 700 lL medium supplemented with different concentrations of GA and 37. Cells were allowed to migrate for 12 h. 4% paraformaldehyde was added to the wells with the chamber in order to fix the cells. After fixation for 0.5 h, the non-migrated cells on the top surface of the membrane were scraped with cotton swab. The cells were washed with PBS for three times, and cells were stained with 0.2% crystal violet. Then the chambers were washed using water, and the membrane was left to dry. Images
were taken using an inverted microscope (Olympus), and cells from six random areas per filter were counted.
Results and Discussion Synthesis of GA derivatives To determine which five-membered heterocyclic ring contributed the most antitumor activity, a series of GA derivatives with heterocyclic rings (pyrazole, 5-(trifluoromethyl)-1H-pyrazole, ethyl 1H-pyrazole-5-carboxylate, 1,2,3thiadiazole, and 2-methyloxazole) fused at C-2 and C-3 positions were synthesized (Schemes 1 and 2) based on our previous research (21,22). The synthesis of pyrazole derivatives is outlined in Scheme 1. Oxidation of GA with Jone’s reagent gave intermediate 1, which was then converted to 6 or 7 by methyl or benzyl esterification with MeI or BnBr, respectively. Intermediates 2, 3, 8, 9, 10, and 11 were obtained by Claisen condensation of 1 with
COOR'
COOR'
COOR'
O
O
O H
a
H
HON
O
b
H N
O
H
O
O 6 R'= -Me 7 R'= -Bn
18 19
16 R= -Me 17 R= -Bn
e
R'= -Me R'= -Bn
c
COOMe O H2N HN
COOR'
COOMe O
O H
O
H
H f
S
N
N
O
N N 23
20 R'= -Me 21 R'= -Bn
24 d
22 R'= -H
g COOH O H S N N 25 Scheme 2: Reagents and conditions: (a) isoamyl nitrite, t-BuOK, t-BuOH, rt, 5 h (65% for 16 and 56% for 17); (b) Zn, Ac2O, AcOH, rt, 12 h (68% for 18 and 81% for 19); (c) POCl3, pyridine, rt, 12 h (93% for 20 and 87% for 21); (d) H2 (1 atm), Pd/C, MeOH, 12 h (94% for 22); (e) NH2NHCONH2HCl, AcONa, EtOH, reflux, 2 h; (f) SOCl2, CH2Cl2, rt, 12 h (84% for 24 over two steps); (g) LiI, DMF, reflux, 48 h (43% for 25).
Chem Biol Drug Des 2014
5
Gao et al.
because of the strong electron-withdrawing group CF3, made the acylation of NH group in prazole unsuccessfully.
corresponding esters in the presence of NaH. Then the pyrazole derivatives 4, 5, 12, 13, 14, and 15 were synthesized by condensation of corresponding intermediates with hydrazine hydrate in AcOH.
Antiproliferative activity To evaluate the anticancer potency of these synthesized heterocyclic ring-fused GA derivatives, the antiproliferative activity of 4–25 (Scheme 1 and 2) was first screened in an SRB assay against eight cancer cell lines of different origin: HCT-116, HCT-8, HT-29, CT-26, PC-3, DU-145, MDA-MB-231, 4T1 (Table 1). The results showed that derivatives having a free carboxylic group in position C-30 (4 and 5) resulted in a lower inhibition of cell growth compared with the corresponding methyl or benzyl esters (12, 14, and 13), and this was in accordance with previously reports (13,14), thus they were excluded from further investigations. The 2-methyloxazole was not a favorable substituted group to improve antitumor activity, because of both derivatives having a free carboxylic group in position C-30 (22), and the corresponding methyl or benzyl esters (20 or 21) gave IC50 values higher than 50 lM. Compound 24 was found to be insoluble in dimethylsulfoxide (DMSO), therefore it was untested, and its corresponding C-30 free carboxylic compound 25 displaying expected poor activity. Most of the pyrazole derivatives showed significantly improved IC50 values, such as 12 and 13, especially 14 (average IC50 = 13.94 lM) and 15 (average IC50 = 14.25 lM) exhibited approximately 6.5 times more potent than GA (average IC50 = 90.86 lM). The praz-
The synthesis of 1,2,3-thiadiazole and 2-methyloxazole derivatives is outlined in Scheme 2. The 2-methyloxazole derivatives 18 and 19 were obtained by treatment of 6 and 7 with isoamyl nitrite in the presence of t-BuOK in t-BuOH, followed by reaction with zinc powder in HOAc/ Ac2O, respectively. Compound 20 and 21 were synthesized by cyclization of 18 and 19 in the presence of POCl3 in pyridine. Hydrogenation of 21 with Pd/C under H2 afforded 22. Intermediate 23 was obtained by reaction of 6 with semicarbazide hydrochloride. Reaction of 23 with SOCl2 produced 24. The 1,2,3-thiadiazole derivative 25 was obtained by demethylation of C-30 methyl ester with LiI in DMF. The synthesis of piperidine derivatives 26–37 is outlined in Scheme 3. Compounds 26 and 27 were produced by amidation of 12 with MeSO2Cl and Ac2O, respectively. Intermediates 28, 29, 30, 31, and 32 were obtained by amidation of 12 with corresponding piperidine caboxylic acids in the presence of CDI and DMAP in CH2Cl2. Compound 33 was produced by deprotection of Boc in the presence of boron trifluoride etherate. Compounds 34, 35, 36, and 37 were prepared in a manner similar to that for 33. We also tried to amidation of 13 in a similar way, and
COOMe
COOMe
COOMe
O
O
O H
H
N
R N
HN
N
N
N
H
O
a or b
12
33
NH 26 R= -SO2Me 27 R= -Ac 28 R=
c n O
N Boc
29 R=
n= 0 COOMe
n NBoc n= 0
O
O
30 R=
HN
n NBoc n= 1
O 31 R=
c
H
O n N
N
n NBoc n= 2
O 32 R= n O
34 35 36 37
n= 0 n= 1 n= 2 n= 3
NBoc n= 3 OH
n Scheme 3: Reagents and conditions: (a) MeSO2Cl or Ac2O, DMAP, DCM, rt, 12 h (73% for 26 and 91% for 27); (b)BocN (n = 0) O OH n (n = 0, 1, 2, and 3), CDI (N,N’-carbonyldiimidazole), DMAP, CH2Cl2, rt, 3 h (76% for 28, 73% for 29, 78% for 30, 79% for 31, orBocN O and 82% for 32); (c) Et2OBF3, Et2O, rt, 0.5 h (80% for 33, 77% for 34, 68% for 35, 71% for 36, and 67% for 37).
6
Chem Biol Drug Des 2014
Chem Biol Drug Des 2014
75.08 >50 15.36 13.25 11.90 7.49 8.20 >50 >50 >50 33.17
SD)
0.79 0.15 0.48 0.25 0.41
1.64
3.96
HCT-116
82.22 >50 36.75 31.48 19.96 13.26 9.96 >50 >50 >50 47.51
HCT-8
0.60 0.89 0.79 0.34 0.84
2.10
4.96 77.23 >50 29.90 31.97 27.60 15.22 12.49 >50 >50 >50 >50
HT-29
0.66 0.73 0.79 0.73 0.60
1.59 70.48 >50 38.56 26.10 22.04 19.78 17.65 >50 >50 >50 45.67
CT-26
1.99 1.92 1.36 1.08 0.88
1.82
0.65 88.04 >50 31.27 26.51 19.80 12.27 19.56 >50 >50 >50 >50
PC-3
5.0 0.39 1.61 1.70 0.35
5.31 136.40 >50 >50 30.31 33.60 19.03 23.83 >50 >50 >50 >50
DU-145
0.78 3.59 2.66 0.55
3.80
8.70 14.12 >50 21.98 23.19 12.13 6.36 6.99
SD)
0.69 0.38 0.26 0.25 0.15
2.20 1.00
HCT-116
24.47 29.23 >50 23.82 22.31 8.98 13.55 11.72
HCT-8
1.62 0.69 0.37 0.60 0.87
1.25 0.97 18.48 30.99 >50 21.78 25.21 12.12 12.46 8.76
HT-29
0.95 1.02 0.52 0.64 0.31
1.73 1.44 15.25 33.02 >50 23.74 21.38 9.87 12.95 8.08
CT-26
0.65 1.41 0.44 0.24 0.50
0.85 1.63
18.90 25.41 >50 23.12 22.36 11.71 12.00 5.19
PC-3
2.11 1.87 0.31 1.69 0.10
2.28 1.09
>50 36.70 >50 21.94 20.76 13.26 13.91 7.54
DU-145
3.60 1.07 1.07 1.32 0.38
1.29
From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments; data not calculated.
26 27 29 33 34 35 36 37
Compound
IC50 (lM
Table 2: IC50 values of 26–37 against the growth of eight cancer cell lines
From SRB assay after 96 h of treatment; the values are averaged from at least 3 independent experiments; data not calculated.
GA 4 5 12 13 14 15 20 21 22 25
Compound
IC50 (lM
Table 1: IC50 values of 4–25 against the growth of eight cancer cell lines
0.46 0.67 0.48 0.32 0.51
3.00
28.63 32.28 >50 24.00 19.16 10.05 9.56 9.76
1.03 2.53 1.29 0.97 0.62
2.21 1.00
MDA-MB-231
101.20 >50 35.30 33.18 27.36 18.14 16.58 >50 >50 >50 >50
MDA-MB-231
12.47 20.10 >50 9.68 5.34 5.80 5.77 6.09
4T1
96.22 >50 33.71 14.73 15.66 6.32 5.75 >50 >50 >50 >50
4T1
1.26 0.66 0.97 0.24 0.68
1.48 1.97 0.78 0.88 0.25
1.65 0.36
2.22
27.73 >50 21.26 19.96 10.49 10.82 8.02
_
1.52 1.37 0.63 0.82 0.40
1.10
0.77 1.26 0.91 0.60
3.19
Average
90.86 >50 _ 25.94 22.24 13.94 14.25 >50 >50 >50 _
Average
Novel GA Derivatives with Antitumor Activity
7
Gao et al.
ole and ethyl 1H-pyrazole-5-carboxylate groups almost contributed equally activity to corresponding derivatives (14 and 15). Prazole-substituted derivative with benzyl esterification of the C-30 carboxylic group (14) exhibited about twofold higher cytotoxic activity against tumor cells than its corresponding methyl ester (12). Considering GA itself has poor water solubility and exhibits low bioavailability and if the hydrophobic benzyl ester group is formed at the C-30, free carboxylic acid position will make the derivative more insoluble in water than corresponding methyl ester, and therefore, the methyl ester 12 was selected as a new lead for further modification. We also considered 13 as a further modifying lead and found the NH group of its prazole ring was hard to substitute because of the strong electron-withdrawing group CF3 in presence. We considered 12 as a new starting modifying structure for more potent activity derivatives. Novel piperidine compounds 26–37 were synthesized from 12 with amidation and N-Boc deprotection reactions. Screening of these derivatives (compounds 26–37) with the SRB assay revealed that all of the piperidine compounds showed more potent activity than their parent compound 12 (Table 2). The most potent compound 37 showed the average IC50 8.02 lM, which was about 11-fold more potent than the lead compound GA. Compound 34 (substituted with 4-piperidinecarboxylic acid), possessed a
slightly more cytotoxic activity than compound 33 (substituted with 3-piperidinecarboxylic acid). Perhaps more distance between the prazole to the NH group of piperidine will afford more potency, and this speculation was further confirmed by the results of 34 (n = 0), 35 (n = 1), 36 (n = 2), and 37 (n = 3). The free NH group in piperidine was important for keeping the cytotoxic activity against tumor cells, and if the group was protected with the Boc (29), the activity would be decreased dramatically.
Apoptosis study using flow cytometry assay and trypan blue staining As previously reported (14), GA induces apoptosis on various tumor cell lines. To examine whether this effect remains or is lost on derivatization, we tested GA and 37 for the induction of apoptosis using flow cytometry and trypan blue exclusion assays. As shown in Figures 1 and 2, we found that the ability of GA and 37 to trigger apoptosis is displayed obviously on mouse CT-26 colorectal cancer cells.
Migration assay Migration is a key step during the metastasis of cancer (28), and to our best knowledge, there are no reports describing inhibitory effect of GA and its analogs on
2.31%
4.85%
12.29%
0.65%
0.59%
1.77%
Control
GA (80 μM)
GA212 (8 μM)
Figure 1: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in flow cytometry assay compared with control group. After treatment of GA or 37 for 24 h, CT-26 colorectal cancer cells with the supernatant were collected, centrifuged, and resuspended with PBS. After centrifugation, cells were resuspended with binding buffer and stained with annexin V and PI for 0.5 h. Analysis was performed by flow cytometry, and the percentage displayed the apoptosis cells of all cells.
Control
GA (80 μM)
37 (8 μM)
Figure 2: GA and 37 induced apoptosis of mouse CT-26 colorectal cancer cells in trypan blue exclusion assay compared with control group. Mouse CT-26 colorectal cancer cells were seeded onto 6-well plates. After treatment of GA or 37 for 24 h, cells with the supernatant were collected, centrifuged, and resuspended with serum-free medium. The cells were stained with trypan blue, and images were taken using an inverted microscope. Arrows indicated the apoptotic or dead cells.
8
Chem Biol Drug Des 2014
Novel GA Derivatives with Antitumor Activity
GA (80 μM)
Control
37 (4 μM) Migration of cancer cells (% of control)
Figure 3: GA and 37 suppressed migration of mouse CT-26 colorectal cancer cells in transwell migration assay compared with control group. After starvation overnight, the cells containing the tested compounds were seeded onto top side of the chambers. Cells were allowed to migrate for 12 h and fixed with 4% paraformaldehyde. After fixation for 0.5 h, the non-migrated cells on the top side were scraped using cotton swab. Migrated cells were stained with 0.2% crystal violet, and images were taken using an inverted microscope. Cells from six random areas per filter were counted.
37 (16 μM)
120 100 80
*** ***
60
***
40
***
20 0
migration of tumor cell lines. To determine this effect, we performed transwell migration assay. As shown in Figure 3, we found that GA inhibited the migration of mouse CT-26 colorectal cancer cells obviously, compared with the control group. We also tested the antimetastatic activity of 37 and other derivatives (such as 34 and the data were not shown) and found that these derivatives, especially 37, significantly inhibited the migration (IC50 was about 4 lM) of mouse CT-26 colorectal cancer cells in a dose-dependent manner. Surprisingly, 37 suppressed migration completely at medium concentration (16 lM). In addition, the results showed that the inhibitory effect on migration of 37 at 4 lM was similar to that of GA at 80 lM, it means that 37 was about 20-fold more potent than GA on antimetastatic activity of mouse CT-26 colorectal cancer cells.
Conclusion In this study, we synthesized a class of novel heterocyclic ring-fused of GA analogs and screened them for antiproliferative activity on cancer cell lines. Most of these compounds exhibited an obvious increase in inhibitory potency compared with GA. An unsubstituted carboxylic group in C-30 resulted in a lower inhibition of cell growth Chem Biol Drug Des 2014
37 (8 μM)
C
GA
4
8
16
37 (μM)
compared with the corresponding esters. Introduction of prazole substituent was quite promising. Most of the pyrazole derivatives showed significantly improved IC50 values, especially 14 and 15. The further modification of prazole compound 12 with various piperidinecarboxylic acids was efficiently afforded the most active compound 37. As demonstrated by flow cytometry and trypan blue exclusion assays, tumor cell death trigged by the compounds resulted from apoptotic processes. In addition, we also described here for the first time that GA and the synthetic compounds exhibited antimetastatic activity on the tested cancer cell line. In conclusion, we report GA heterocyclic derivatives as a series of new chemical entities for the first time. Especially 37, which exhibited good antitumor and antimetastatic activity, could be used as a promising lead for the development of a new class of antitumor and antimetastatic agents.
Acknowledgments Shanghai Science and Technology Council (Grant 12ZR1408500), the Fundamental Research Funds for the Central Universities, Innovation Program of Shanghai Munici9
Gao et al.
pal Education Commission (13zz034), and National Natural Science Foundation of China (81272463) are appreciated for their financial support.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Synthetic procedures for the preparation of compounds 4, 5, 12, 13, 14, 15, 20, 21, 22, 22, 24, 25. Appendix S2. Compound 34 suppressed migration of mouse CT-26. Figure S1. Compounds 34 suppressed migration of mouse CT-26 colorectal cancer cells in transwell migration assay compared with control group.
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