Several bioactive dehydroabietylamine Schiff-bases (L1L4), amides (L5L11) and complex CuL3(NO3)2, Cu(L5)3, Co(L6)2Cl2 had been synthesized successfully for developing more efficient but lower toxic antiproliferative compounds. Their antiproliferative activities to Hela (cervix), HepG2 (liver), MCF-7 (breast), A549 (lung) and HUVEC (umbilical vein, normal cell) were investigated in vitro. The toxicity of all compounds was less than dehydroabietylamine (L0). For HepG2 cells, L1, L2 and L3 had higher anti-HepG2 activity, especially L1 (0.52 µM) had highest anti-HepG2 activity but low toxicity. For MCF-7 cells, L1, L2, L3 and L4 had higher anti-MCF-7 activity, especially L3(0.49 µM) had highest anti-MCF-7 activity but low toxicity. For A549 cells, L2 and L3 had higher anti-A549 activity. Furthermore, L1 and L3 may be the great promise antiproliferative drugs with nontoxic side effects, due to the high anti-HepG2 and anti-MCF-7 inhibition rate in vivo, 65% and 61%, respectively. L1, L2 and L3 could induce apoptosis through intercalating into DNA.

Introduction

Recently, to develop efficient chemotherapy from natural products and their modified products has attracted remarkable attention. Among them, a number of well-defined bioactive natural products like rosin have drawn more attention on account of their promising antibacterial, antiproliferative and antifungal activities [1–4]. As an important modified product from rosin, dehydroabietylamine (L0) and its derivatives have been widely researched with their antitumor, cytotoxic, antifungal, antibacterial and antiparasitic activities. For example, Moreira and co-workers found antileishmanial activities of several dehydroabietic acid derivatives and dehydroabietylamine amides (IC50 = 0.4–9.0 µM) [5]. Similarly, Muhammad's group reported one dehydroabietylamine derivative with notable growth attenuation of MCF-7 cell lines (IC50 = 4.8 µM) [6]. So far, our group has successfully modified some Schiff-base and amides compounds with high inhibitory activities to HepG2, A549 and MCF-7 cells, which showed the smallest IC50 values of 0.14, 0.24, 0.75 and 1.85 µM. Moreover, these compounds could induce apoptosis through intercalating into DNA [7].

The success of platinum (Pt) complexes for anticancer therapy in clinics encourages the development of new metallodrugs. As essential metal element, copper and cobalt distribute widely in biological systems like body and cells and play vital roles in biology and biomedicine [8–10]. Copper complexes are reported as novel oxidative cleaving agents of DNA with their high nucleobase affinity and biologically accessible redox potentials [11–13]. Similarly, cobalt complexes have attracted much attentions of the interaction with DNA.

Schiff base ligands have been reported with various bioactivities like antifungal, antibacterial, herbicidal, analytical and clinical fields [14–16]. Many researchers are interested in transition metal complexes of unusual configuration with nitrogen and oxygen donor Schiff bases [17–19]. Schiff base copper(II) or cobalt complexes ow antiproliferative, antifungal, antimicrobial, antiviral and antioxidant activities with diverse structurally characters [20–25].

However, compared with dehydroabietylamine's organic derivatives, the valuable investigation of their transition metal complexes’ cytotoxicity is fewer [26–29]. Our group has reported a new copper(II) complex Cu(L1)2 with its antitumor activity in our previous work [25].

In continuation of our previous work with exploring dehydroabietylamine derivatives for their potential inhibitory abilities to cancer cells, herein, we report a series of L1L4 and amides L5L11 (8), which were used commercially available dehydroabietylamine (L0) and pyridine aldehydes and carboxylic acids with different functional group as starting materials on purpose of studying the effect of structure-activity relationship. Pyridine is the original ring system of various natural products and important pharmaceutical, industrial and agricultural chemicals. It's expected that the combination of dehydroabietylamine and pyridine derivatives will produce a better biological activity of novel compounds, we have designed and synthesized Schiff-bases, amides and some metal complexes. Then screened their antiproliferative activity and toxicity by MTT assay in vitro, which could inhibit the growth of Hela, HepG2, MCF-7, A549 and HUVEC cell lines. The result indicated that most compounds possessed higher antiproliferative activities compared with L0. It was further identified L1 high anti-HepG2 activity and L3 had high anti-MCF-7 activity in vivo with the cancer inhibition rate up to 65% and 61%, respectively. The result demonstrated that compound L1 and L3 may be the great promise antiproliferative drugs with nontoxic side effects. The inducing-apoptosis of L1, L2 and L3 to HepG2, A549 and MCF-7 cells respectively were verified by Annexin V-FITC/PI assay. The result showed L1, L2 and L3 inhibited the growth of cancer cells by inducing apoptosis. The way of interaction with Herring Sperm DNA (fDNA) were also investigated by UV-Vis, fluorescence and viscosity methods, respectively. The result indicated that the binding modes between L1, L2, L3 and fDNA was interaction. The structure of DNA would be changed or destroyed when compounds bound to the DNA with intercalation mode, which would cause cell apoptosis and inhibit the growth of cancer cells.

Syntheses and physicochemical data of L1–L11, CuL3(NO3)2 and Co(L6)2Cl2 (the stereo center of L1–L11 same as L0).

Scheme 1.
Syntheses and physicochemical data of L1–L11, CuL3(NO3)2 and Co(L6)2Cl2 (the stereo center of L1–L11 same as L0).
Scheme 1.
Syntheses and physicochemical data of L1–L11, CuL3(NO3)2 and Co(L6)2Cl2 (the stereo center of L1–L11 same as L0).

Materials and methods

Biological assays

Cell culture, antiproliferative activities and cytotoxicity assay

Hela, HepG2, MCF-7, A549 and HUVEC cells were used in the antiproliferative activity assay. Cancer cells were seeded in 96-well plates with a density of 104 cells per well, after 12 h of incubation at 5% CO2 and 37°C, the culture media was removed and cells were incubated with L1L11, complex CuL3(NO3)2, Cu(L5)3 and Co(L6)2Cl2 dissolved in DMEM at different concentrations (each concentration repeated three times) for 36 h at 5% CO2 and 37°C. Subsequently, removed the culture media and the new culture medium containing MTT (1 mg/ml) was added, followed by incubating for 4 h to allow the formation of formazan dye. After removing the medium, 200 µl DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 595 nm in a microplate photometer. Cell viability values were determined (at least three times) according to the following formulae: cell viability (%) = the absorbance of experimental group/the absorbance of blank control group × 100%.

Induction of apoptosis by flow cytometric analysis

We further investigated whether L1 and L2 could induce apoptosis, DMSO was used as negative control. HepG2, A549 and MCF-7cells (1 × 106) were cultured in 35 mm dishes and incubated at 37°C for 24 h. After incubation with DMSO at 5 µg/ml, L1 at 0.1, 0.5, 5 µg/ml, L2 and L3 at 0.1, 1, 5 µg/ml to HepG2 cells, L2 at 0.1, 1, 5 µg/ml to A549 cells and L3 at 0.1, 0.5, 5 µg/ml for 24 h (each concentration repeated thre times, the incubation time is optimum), the treated cells were washed, trypsinized (non-EDTA), and centrifuged (2000 rpm/min). Then the cells were collected and resuspended in 500 µl of buffer solutions loaded with Annexin V-FITC apoptosis detection reagent (with 5 µl Annexin V-FITC and 5 µl PI). The Annexin V-FITC-stained cells were incubated for 5–15 min in the dark, and approximate 10 000 cells were collected for flow cytometry analysis with a single 488 nm argon laser.

Confocal fluorescence images

In this work, HeLa cells were seeded on 35 mm glass dishes for 24 h, then incubated with L0-FITC (100 nM) in the dark for 2 h or 24 h at 5% CO2 and 37°C before fluorescence imaging. As such, cells were treated with L0-FITC (100 nM) at 4°C in the dark for 2 h. After incubation, the culture media was removed and cells were washed by PBS (pH = 7.4), then incubated with fresh medium. Confocal fluorescence images were detected with the excitation of 488 nm and emission of 519 nm.

In vivo experiment

In vivo experiment was conducted by Nanjing Keygen Biotech. Co. LTD. In detail, the animal work took place in the Animal Room II at Laboratory Animal Center SPF Area. Experimental animal production license: SCXK (Shanghai) 2017-0005; Qualification certificate No.: 2015000565460; License for use of experimental animals: SYXK (Jiangsu) 2017-0015. To develop the tumor model, 1 × 106 HepG2 or MCF-7 cells were subcutaneously injected into the right armpit of every Balb/C nude mouse. And then two groups of HepG2-tumor-bearing mice with five mice per group were randomly chosen in our experiment: (1) PBS (as a control), (2) L1 (L3 for MCF-7-tumor-bearing mice). After the size of tumors reached 50–100 mm3, all agents including PBS, L1 (0.5 mg/kg) or L3 (0.6 mg/kg) solutions were administrated via an intravenous injection, respectively. During the next 25 days, the tumor size of every mouse in our experiments was measured by a vernier caliper every 3 days. Moreover, to accurately evaluate the growth inhibition of tumors, the mice were killed by CO2 euthanasia after 25 days, and then their tumors were collected, photographed, and weighed. Besides, the sections of tumor, heart, kidney, liver, lung, and spleen tissues of different groups harvested on the 25th day were observed using H&E staining, and then examined by a pathologist. The tumor size was calculated as the volume = 0.5 × (tumor length) × (tumor width)2. The inhibition efficiency of tumor growth was calculated according to the following equation: 
inhibitionefficiency(%)=(1theweightofexperimentalgroup/theweightofcontrolgroup))×100%
CD31 immumohistochemical staining with mice was taken on the 25th day after an intravenous injection of compound L1 (0.5 mg/kg), L3 (0.6 mg/kg) and PBS control. No anesthetics were used during this experiment.

General experimental procedures

All reagents and solvents were analytical reagent (AR) grade and were used as received unless otherwise indicated. The IR spectra were recorded on a Bruker Vectex 80 FT-IR spectrometer with KBr discs in the 4000–500 cm−1 range. The 1H, 13C {1H} NMR spectra were measured on a Bruker Avance III 600 or 500 MHz NMR spectrometer (Billerica, MA, U.S.A.). 1H and 13C {1H} NMR spectra were recorded in CDCl3 as solvent unless otherwise stated. Chemical shifts (δ) are given as parts per million (ppm) relative to the NMR solvent signals (CDCl3 7.26 and 77.00 ppm for 1H and 13C{1H} NMR, respectively). J values are given in Hz. HRMS were measured to determine purity of all tested compounds by LTQ Orbitrap XL mass spectrometer (Thermo Electron, U.S.A.). Reactions were monitored by TLC using silica gel 60 F-254 in 0.25 mm-thick plates. Compounds on TLC plates were detected under UV light at 254 nm. Purifications were performed by flash chromatography on silica gel (300–400 mesh). The DNA binding modes were investigated by Lambda 950 (PerkinElmer) and LS55 Fluorescence Spectrophotometer (PerkinElmer). The pKb value was conducted with pH by METTLER FE20K. Cell imaging was carried out on a confocal fluorescence microscope (Nikon, Eclipes TE2000-E). Reagents and compounds Dulbecco's Modified Eagle's Medium (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS) and penicillin/streptomycin were all purchased from Nanjing Keygen Biotech. Co. LTD. Induction apoptosis assay was operated by Becton Dickinson Ultra-high speed separation flow cytometry instrument and Annexin V-FITC/PI was purchased from Nanjing Keygen Biotech. Co. LTD.

Synthesis of N-dehydroabietyl-pyridine-2-Schiff-base (L1)

L0 (1.43 g, 5.0 mmol), 2-pyridylaldehyde (0.54 g 5.0 mmol) with acetic acid as the catalyst was dissolved in ethanol (50 ml), stirred and refluxed overnight. When reaction mixture was cooled to the room temperature, lots of green needle-like crystal precipitation appeared. Then the green crystals were purified by recrystallization from ethanol solution. (1.63 g, 87%), mp: 104.2–105.5°C; IR (neat) νmax 3428, 2922, 2860, 1638, 1561, 1439, 1371, 1044, 976, 771, 606 cm−1; 1H NMR (CDCl3, 600 MHz): δ 1.05 (3H, s), 1.21–1.24 (9H, s), 1.38–1.44 (2H, m), 1.56–1.57 (1H, m), 1.60–1.67 (2H, m), 1.71–1.81 (2H, m), 1.89–1.92 (1H, m), 2.25–2.30 (1H, m), 2.76–2.91 (3H, m), 3.42–3.61 (2H, dd, J = 12 Hz), 6.87 (1H, s), 6.98 (1H, dd, J = 7.2 Hz), 7.14 (1H, d, J = 7.2 Hz), 7.18 (1H, d, J = 8.4 Hz), 7.29–7.30 (1H, m), 7.69–7.72 (1H, m), 8.01–8.05 (1H, m), 8.34 (1H, s), 8.61(1H, d, J = 3.6 Hz); 13C{1H} NMR (CDCl3, 151 MHz): δ 18.76, 18.90, 19.64, 23.96, 25.64, 30.48, 33.40, 36.60, 37.66, 38.20, 38.42, 45.63, 58.45, 72.91, 121.16, 123.80, 124.41, 124.61, 126.87, 135.00, 136.55, 145.39, 147.47, 149.18, 154.68, 161.83; MS [M + H]+m/z 375.2800 (calcd for C26H34N2 374.2722). Anal. calcd for C26H34N2: C, 83.37; H, 9.15; N, 7.48. Found: C, 83.43; H, 9.28; N, 7.29.

Synthesis of N-dehydroabietyl-6-methylpyridine-2-Schiff-base (L2)

A mixture of L0 (1.43 g, 5.0 mmol), 6-methyl-2-pyridylaldehyde (0.61 g 5.0 mmol) with acetic acid as the catalyst was dissolved in ethanol (50 ml) and refluxed for 24 h. Then the mixture was cooled to the room temperature, removed the solvent by reduced pressure distillation, and received the brown oil compound. Finally, the brown powders were obtained by recrystallization from methanol solution and dried in vacuum. (1.36 g, 70%), mp: 35.7–37.9°C; IR (neat) νmax 3434, 2925, 2867, 1650, 1587, 1450, 1371, 1039, 981, 795; 1H NMR (CDCl3, 600 MHz): δ 1.00 (3H, s), 1.20–1.24 (9H, s), 1.38–1.44 (2H, m), 1.56–1.57 (1H, m), 1.60–1.67 (2H, m), 1.71–1.81 (1H, m), 1.89–1.92 (1H, m), 2.25–2.30 (1H, m), 2.58 (3H, s), 2.79–2.82 (2H, m), 2.85–2.86 (1H, m), 3.41–3.57 (2H, dd, J = 12 Hz), 6.86 (1H, s), 6.98 (1H, dd, J = 7.2 Hz), 7.14 (1H, d, J = 7.2 Hz), 7.18 (1H, d, J = 8.4 Hz), 7.58 (1H, m), 7.81 (1H, d, J = 7.8 Hz), 8.31 (1H, s); 13C{1H} (CDCl3, 151 MHz): δ 18.74, 18.86, 19.56, 23.91, 24.22, 25.56, 30.43, 33.35, 36.61, 37.61, 38.13, 38.41, 45.68, 72.95, 118.09, 123.73, 124.10, 124.33, 126.80, 134.95, 136.64, 145.31, 147.44, 154.15, 157.78, 162.27; MS [M + H]+m/z 389.29 (calcd for C27H36N2 388.2878). Anal. calcd for C27H36N2: C, 83.45; H, 9.34; N, 7.21. Found: C, 83.69; H, 9.29; N, 7.02.

Synthesis of N-dehydroabietyl-5-brominepyridine-2-Schiff-base (L3)

The mixture was cooled to the room temperature, lots of orange needle-like crystal precipitation appeared. Then removed the solvent, the pale orange block-shaped single crystals were purified by recrystallization from ethanol solution. (2.06 g, 91%), mp: 114.8–116.3°C; IR (neat) νmax 3428, 2923, 2854, 1644, 1562, 1439, 1371, 1036, 1002, 831, 702, 620 cm−1; 1H NMR (CDCl3, 600 MHz): δ 1.05 (3H, s), 1.22–1.25 (9H, s), 1.39–1.44 (2H, m), 1.56 (1H, m), 1.60–1.67 (2H, m), 1.71–1.81 (2H, m), 1.88–1.90 (1H, m), 2.25–2.30 (1H, m), 2.76–2.91 (3H, m), 3.57–3.59 (2H, dd, J = 12 Hz), 6.87 (1H, s), 6.99 (1H, dd, J = 7.2 Hz), 7.18–7.19 (1H, d, J = 8.4 Hz), 7.82–7.83 (1H, m), 7.89–7.90 (1H, m), 8.29 (1H, s), 8.67(1H, d, J = 1.8 Hz); 13C{1H} NMR (CDCl3, 151 MHz): δ 18.76, 18.89, 19.65, 23.97, 23.98, 25.63, 30.47, 33.41, 36.62, 37.66, 38.22, 38.42, 45.61, 72.88, 122.26, 123.85, 124.40, 126.88, 134.92, 139.18, 147.42, 150.30, 153.17, 160.80; MS [M + H]+m/z 453.1921 (calcd for C26H33BrN2 452.1827). Anal. calcd for C26H33BrN2: C, 68.87; H, 7.33; N, 6.18. Found: C, 68.79; H, 7.48; N, 6.21.

Synthesis of N-dehydroabietyl-6-fluorinepyridine-2-Schiff-base (L4)

When reaction mixture was cooled to the room temperature, evaporation of ethanol yielded brown oil compound, which was purified by recrystallization from methanol to give the pure product. (1.49 g, 76%), mp: 58.5–60.2°C; IR (neat) νmax 3423, 2929, 2861, 1649, 1598, 1450, 1375, 1036, 977, 747, 637 cm−1; 1H NMR (CDCl3, 600 MHz): δ 1.08 (3H, s), 1.20–1.24 (9H, s), 1.38–1.40 (2H, m), 1.53–1.59 (1H, m), 1.61–1.75 (2H, m), 1.71–1.82 (2H, m), 1.85–1.93 (1H, m), 2.25–2.27 (1H, m), 2.79–2.88 (3H, m), 3.54–3.61 (2H, dd, J = 12 Hz), 6.86 (1H, s), 6.97 (1H, dd, J = 8.4 Hz), 7.16 (1H, d, J = 7.2 Hz), 7.28–7.30 (1H, m), 7.38–7.47 (1H, m), 8.48–8.51(2H, d, J = 23.4 Hz); 13C{1H} NMR (CDCl3, 151 MHz): δ18.89, 19.49, 24.03, 24.04, 25.65, 30.39, 33.46, 36.73, 37.71, 38.31, 38.42, 45.99, 74.75, 123.84, 124.31, 124.42, 125.65, 125.68, 126.86, 134.92, 142.12, 142.17, 145.39, 145.68, 145.72, 147.47, 156.76, 156.79, 158.25, 160.02; MS [M + H]+m/z 393.2709 (calcd for C26H33FN2 392.2628). Anal. calcd for C26H34N2: C, 79.55; H, 8.47; N, 7.14. Found: C, 79.43; H, 8.38; N, 7.21.

Synthesis of N-dehydroabietyl-pyridine-2-carboxamide (L5)

2-picolinic acid (1.23 g, 10.0 mmol) and HOBT (1.35 g, 10.0 mmol) were dissolved in ethyl acetate (40 ml), then stirred at 0°C for 0.5 h. To the stirred solution at 0°C was slowly added DCC (2.06 g, 10.0 mmol), then stirred at 0°C for 2.5 h. The ethyl acetate (10 ml) of Dehydroabietylamine (2.85 g, 10.0 mmol) was added to the reaction system slowly, the reaction mixture was stirred at room temperature for 8 h. Filter to remove DCU, filtrate was diluted to 200 ml, it was washed with 5% NaHCO3 (3 × 20 ml), 10% citric acid (2 × 20 ml) and saturated salt water. Ethyl acetate layer was dried with anhydrous Na2SO4 for 2 h, and solvents were evaporated to give amide L5 as yellow powder. (3.58 g, 92%), mp: 69.3–70.2°C; IR(KBr) 3392 (N−H), 2926 (−CH3, −CH2), 1678 (O = C−N), 1526 (N−H); 1H NMR (CDCl3, 600 MHz) δ 1.022 (3H, s), 1.21–1.24 (9H, m), 1.41–1.81 (7H, m), 2.03–2.05 (1H, m), 2.28 (1H, brd, J = 12 Hz), 2.81–2.91 (3H, m), 3.28–3.50 (2H, m), 6.89 (1H, t, J = 6 Hz, −CONH), 6.99 (1H, s), 7.17 (1H, d, J = 8.4 Hz), 7.40 (1H, d, J = 12 Hz), 7.81–8.52 (4H, m); 13C NMR (CDCl3, 151 MHz) δ 18.71, 18.90, 19.14, 23.95, 23.97, 25.52, 30.49, 33.42, 36.32, 37.61, 37.85, 38.31, 45.50, 49.96, 122.27, 123.85, 124.29, 126.05, 126.94, 134.94, 137.71, 145.56, 147.16, 148.06, 149.95, 164.33; MS(ESI) m/z 391.33 [M + H]+, 413.33 [M + Na]+ (calcd for C26H34N2O 390.2671). Anal. calcd for C26H34N2O: C, 79.96; H, 8.77; N, 7.17. Found: C, 79.85; H, 8.69; N, 7.21.

Synthesis of N-dehydroabietyl-pyridine-3-carboxamide (L6)

The condensation reaction used HOBT and DCC. The solution was dried with anhydrous Na2SO4 and evaporated to get faint yellow powders. (3.05 g, 78%), mp: 167.0–167.9°C; IR(KBr) 3426 (N−H), 2927 (−CH3, −CH2), 1647 (O = C−N), 1542 (N−H); 1H NMR(CDCl3, 600 MHz) δ 1.03 (3H, s), 1.22–1.24 (9H, m), 1.36–1.81 (7H, m), 1.99–2.01 (1H, m), 2.30 (1H, brd, J = 13.2 Hz), 2.79–2.93 (3H, m), 3.34–3.49 (2H, m), 6.50 (1H, t, J = 6 Hz, −CONH), 6.88 (1H, s), 6.98 (1H, d, J = 12 Hz), 7.16 (1H, d, J = 7.8 Hz), 7.46–9.12 (4H, m); 13C NMR(CDCl3, 151 MHz) δ 18.61, 18.80, 19.13, 23.94, 23.96, 25.38, 30.36, 33.42, 36.48, 37.56, 37.74, 38.35, 45.83, 50.42, 123.58, 123.95, 124.19, 126.96, 130.59, 134.61, 135.19, 145.72, 146.94, 147.59, 152.10, 165.69; MS(ESI) m/z:391.58 [M + H]+, 413.42 [M + Na]+ (calcd for C26H34N2O 390.2671). Anal. calcd for C26H34N2O: C, 79.96; H, 8.77; N, 7.17. Found: C, 79.81; H, 8.64; N, 7.28.

Synthesis of N-dehydroabietyl-6-methylpyridine-3-carboxamide (L7)

HOBT and DCC were used in the condensation reaction. Ethyl acetate was dried with and evaporated to get yellow powders. (0.71 g, 71%), mp: 151.8–153.3°C; IR(KBr) 3419 (N−H), 2927 (−CH3, −CH2), 1645 (O = C−N), 1540 (N−H); 1H NMR (CDCl3, 600 MHz) δ 0.987 (3H, s), 1.20–1.25 (9H, m), 1.33–1.78 (7H, m), 1.93–1.97 (1H, m), 2.28 (1H, brd, J = 12.6 Hz), 2.56–2.62 (3H, m), 2.77–2.93 (3H, m), 3.29–3.44 (2H, m), 6.46 (1H, t, J = 6 Hz, −CONH), 6.86 (1H, s), 6.98 (1H, d, J = 9.6 Hz), 7.14–7.18 (2H, m), 7.96 (1H, d, J = 10.2 Hz), 8.83 (1H, s); 13C NMR (CDCl3, 151 MHz) δ 18.63, 18.81, 19.13, 24.01, 24.40, 25.45, 30.41, 33.44, 36.43, 37.54, 37.82, 38.33, 45.74, 50.34, 123.23, 123.93, 124.23, 126.97, 127.81, 134.68, 135.62, 145.66, 146.98, 147.14, 161.48, 165.92; MS(ESI) m/z 405.42 [M + H]+, 427.33 [M + Na]+ (calcd for C27H36N2O 404.2828). Anal. calcd for C27H36N2O: C, 80.15; H, 8.97; N, 6.92. Found: C, 80.06; H, 8.89; N, 7.03.

Synthesis of N-dehydroabietyl-6-chloropyridine-3-carboxamide (L8)

After the condensation reaction of 6-chloro-3-picolinic acid (0.393 g, 2.5 mmol) and L0 (1.43 g, 5.0 mmol) used HOBT and DCC, solvents were evaporated to get white powders. (0.77 g, 73%), mp: 178.6–179.6°C; IR(KBr) 3440 (N−H), 2927 (−CH3, −CH2), 1658 (O = C−N), 1586 (N−H); 1H NMR (CDCl3, 600 MHz) δ 1.01 (3H, s), 1.21–1.23 (9H, m), 1.31–1.81 (7H, m), 1.93–2.04 (1H, m), 2.30 (1H, brd, J = 12.6 Hz), 2.77–2.95 (3H, m), 3.30–3.46 (2H, m), 6.33 (1H, t, J = 6 Hz, −CONH), 6.88 (1H, s), 6.99 (1H, d, J = 9.6 Hz), 7.16 (1H, d, J = 7.8 Hz), 7.37 (1H, s), 8.02 (1H, d, J = 10.2 Hz), 8.71 (1H, s); 13C NMR (CDCl3, 151 MHz) δ 18.59, 18.80, 19.13, 23.99, 25.42, 25.55, 30.36, 32.64, 33.44, 33.83, 36.47, 37.54, 37.61, 37.80, 38.30, 45.77, 50.52, 123.99, 124.21, 124.41, 126.98, 129.44, 134.59, 137.91, 145.75, 146.91, 147.86, 154.13, 164.83; MS(ESI) m/z 425.25 [M + H]+, 447.33 [M + Na]+ (calcd for C26H33ClN2O 424.2281). Anal. calcd for C26H33ClN2O: C, 73.48; H, 7.83; N, 6.59. Found: C, 73.52; H, 7.79; N, 6.61.

Synthesis of N-dehydroabietyl-pyridine-4-carboxamide (L9)

HOBT and DCC were used in the condensation reaction. Ethyl acetate was dried with and evaporated to get faint yellow powders. (2.70 g, 69%), mp: 143.9–144.8°C; IR(KBr) 3437 (N−H), 2922 (−CH3, −CH2), 1650 (O = C−N), 1543 (N−H); 1H NMR (CDCl3, 600 MHz) δ 1.02 (3H, s), 1.23–1.29 (9H, m), 1.37–1.84 (7H, m), 1.96–2.06 (1H, m), 2.33 (1H, brd, J = 12.6 Hz), 2.83–2.95 (3H, m), 3.35–3.49 (2H, m), 6.25 (1H, t, J = 6 Hz, −CONH), 6.91 (1H, s), 7.01 (1H, d, J = 8.4 Hz), 7.18 (1H, d, J = 8.4 Hz), 7.61–8.75 (4H, m); 13C NMR (CDCl3, 151 MHz) δ 18.59, 18.80, 19.13, 23.93, 23.95, 25.40, 30.36, 33.41, 36.46, 37.57, 37.73, 38.32, 45.86, 50.46, 120.77, 120.81, 123.98, 124.21, 126.97, 134.60, 141.86, 145.77, 146.90, 150.65, 165.70; MS(ESI) m/z 391.50 [M + H]+, 413.50 [M + Na]+ (calcd for C26H34N2O 390.2671). Anal. calcd for C26H34N2O: C, 79.96; H, 8.77; N, 7.17. Found: C, 79.87; H, 8.71; N, 7.23.

Synthesis of N-dehydroabietyl-2-methylpyridine-4-carboxamide (L10)

The condensation reaction used HOBT and DCC. The solution was dried with anhydrous Na2SO4 and evaporated to get white powders. (1.61 g, 80%), mp: 82.1–82.9°C; IR(KBr) 3331 (N−H), 2926 (−CH3, −CH2), 1650 (O = C−N), 1546 (N−H); 1H NMR (CDCl3, 600 MHz) δ 1.01 (3H, s), 1.20–1.26 (9H, m), 1.31–1.80 (7H, m), 1.94–1.97 (1H, m), 2.29 (1H, brd, J = 13.2 Hz), 2.58–2.59 (3H, m), 2.79–2.94 (3H, m), 3.28–3.45 (2H, m), 6.43 (1H, t, J = 6 Hz, −CONH), 6.88 (1H, s), 6.99 (1H, d, J = 6 Hz), 7.16 (1H, d, J = 8.4 Hz), 7.34–8.55 (3H, m); 13C NMR (CDCl3, 151 MHz) δ 18.52, 18.70, 19.06, 23.86, 23.88, 24.33, 24.35, 24.85, 25.31, 25.54, 30.25, 33.33, 33.86, 36.37, 37.47, 37.70, 38.23, 45.72, 50.42, 117.80, 120.58, 123.85, 124.09, 126.87, 134.56, 142.29, 145.63, 146.87, 149.67, 159.44, 166.01; MS(ESI) m/z 405.42 [M + H]+, 427.33 [M + Na]+ (calcd for C27H36N2O 404.2828). Anal. calcd for C27H36N2O: C, 80.15; H, 8.97; N, 6.92. Found: C, 80.19; H, 8.86; N, 6.87.

Synthesis of N-dehydroabietyl-2, 6-dichloro-pyridine-4-carboxamide (L11)

HOBT and DCC were used in the condensation reaction. Ethyl acetate was dried with and evaporated to get white powders. (1.64 g, 72%), mp: 86.9–87.6°C; IR(KBr) 3345 (N−H), 2926 (−CH3, −CH2), 1650 (O = C−N), 1541 (N−H); 1H NMR (CDCl3, 600 MHz) δ 1.01 (3H, s), 1.21–1.24 (9H, m), 1.31–1.81 (7H, m), 1.92–1.95 (1H, m), 2.31 (1H, brd, J = 12.6 Hz), 2.79–2.96 (3H, m), 3.28–3.46 (2H, m), 6.37 (1H, t, J = 6 Hz, −CONH), 6.89 (1H, s), 6.99 (1H, d, J = 9.6 Hz), 7.16 (1H, d, J = 7.8 Hz), 7.53–7.54 (2H, m);13C NMR (CDCl3, 151 MHz) δ 18.55, 18.74, 19.17, 23.97, 24.00, 25.40, 30.24, 33.44, 36.46, 37.53, 37.83, 38.22, 45.74, 50.84, 120.69, 124.01, 124.16, 126.99, 134.56, 145.82, 146.85, 147.21, 151.46, 163.38; MS(ESI) m/z 459.25 [M + H]+, 481.25 [M + Na]+ (calcd for C26H32Cl2N2O 458.1892). Anal. calcd for C26H32Cl2N2O: C, 67.97; H, 7.02; N, 6.10. Found: C, 67.83; H, 7.11; N, 6.05.

Synthesis of CuL3(NO3)2

Cu(NO3)2·3H2O (0.024 g, 0.1 mmol) in ACN (4 ml) was dropwise added into a solution of 5-bromine-2-pyridine-dehydroabietylamine-Schiff-base (L3) (0.045 g, 0.1 mmol) in DCM (4 ml) with stirring at 60°C. It was kept at room temperature to obtain the green block-shaped single crystals in 52% yield.

Synthesis of Cu(L5)3

N-dehydroabietyl-pyridine-2-carboxamide (0.117 g, 0.3 mmol) was dissolved in ethanol (6 ml). Then a solution of Cu(NO3)2·3H2O (0.024 g, 0.1 mmol) in ethanol (4 ml) was added dropwise to the above mixture, continue to reflux with stirring for 5 h until light blue precipitate was formed. The precipitate was collected by filtration and washed with ethanol and distilled water. Then the precipitate was recrystallized with ethanol to obtain pure powder in 68% yield.

Synthesis of Co(L6)2Cl2

N-dehydroabietyl-pyridine-3-carboxamide (0.078 g, 0.2 mmol) was dissolved in ethanol (6 ml). Then a solution of CoCl2·6H2O (0.0237 g, 0.1 mmol) in ethanol (4 ml) was added dropwise to the above mixture, continue to reflux with stirring for 6.5 h. Down to room temperature and filter. Several days later, sapphire block-shaped single crystals suitable for X-ray analysis were obtained by slowly evaporating the solvent in 64% yield.

X-ray crystal structure determination

Diffraction data were collected on a Bruker D8 VENTURE PHOTON 100 diffractometer using a graphite-monochromated MoKα radiation (0.71073 Å) at 293 K in the ω-2θ scan mode. In all cases, an empirical absorption correction by SADABS was applied to the intensity data. The structures were solved by direct methods and refined on F2 by full-matrix least-squares methods using the SHELXTL crystallographic software package. All non-hydrogen atoms were refined anisotropically with hydrogen atoms included in calculated positions (riding model). Crystallographic data for compound L3, the copper(II) complex CuL3(NO3)2 and the cobalt(II) complex Co(L6)2Cl2 is given in Supplementary Table S1. CCDC 1816225 contain the supplementary crystallographic data for compound L3. CCDC 1816226 contain the supplementary crystallographic data for the copper(II) complex CuL3(NO3)2. CCDC 1476583 contain the supplementary crystallographic data for the cobalt(II) complex Co(L6)2Cl2. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/data_request/cif.

Results and discussion

Chemistry

Dehydroabietylamine derivatives, Schiff-bases L1L4 and amides L5L11 (8), which were used commercially available (+)-dehydroabietylamine (L0, [α]20D = 55.1°(c 2.4 pyridine) and pyridine aldehydes and carboxylic acids with different functional group as starting materials on purpose of studying the effect of structure-activity relationship.

The pale orange block-shaped single crystal of L3 was acquired with monoclinic crystal system. Its molecular structure is made up of one pyridine ring, one aromatic ring and two aliphatic rings. The pyridine ring was coplanar with C7, N2, C6 and H6 (Figure 1). According to Lu's report, the length of imine double bond C10−N2 was 1.2913(3) Å [30], which was consistent with our result of C6−N2 (1.249(6) Å) and our previous work C7−N1 (1.2690(4) Å). Selected bond angles and lengths are displayed in Table 1. Hydrogen bonds are shown in Table 2. The crystallographic data is shown in Supplementary Table S1.

Molecular structure.

Figure 1.
Molecular structure.

(a) L3; (b) CuL3(NO3)2 (all hydrogen atoms omitted for clarity).

Figure 1.
Molecular structure.

(a) L3; (b) CuL3(NO3)2 (all hydrogen atoms omitted for clarity).

Table 1
Selected bond lengths (Å) and angles (deg) for L3, CuL3(NO3)2 and Co(L6)2Cl2
L3CuL3(NO3)2Co(L6)2Cl2
Br1–C1 1.890(5) Cu1–N1 1.989(6) O1–Cu1–N2 98.0(2) Co1–N1 2.049(6) 
N2–C6 1.249(6) Cu1–N2 1.990(7) O4–Cu1–N3 93.3(2) Co1–Cl1 2.225(3) 
N2–C7 1.454(6) Cu1–O2 1.959(7) O1–Cu1–N2 155.9(2) C6–O1 1.219(11) 
C1–N1–C5 116.6(4) Cu1–O4 1.963(5) N1–Cu1–N2 82.9(2) C6–N2 1.361(13) 
N1–C1–C2 123.8(5) Cu1–O1 2.395(8) O4–Cu1–O5 57.8(3) C7–N2 1.475(9) 
C1–C2–Br1 120.0(4) Cu1–O5 2.524(6) O1–Cu1–O4 109.3(2) N1–Co1–Cl1 110.28(18) 
C3–C2–Br1 120.3(3) O4–Cu1–O1 109.3(3) N2–Cu1–O5 106.8(2) O1–C6–N2 125.0(10) 
C6–N2–C7 119.4(4) O4–Cu1–N2 97.7(3) N3–Cu1–O5 93.3(2) C1–N1–C5 117.3(9) 
L3CuL3(NO3)2Co(L6)2Cl2
Br1–C1 1.890(5) Cu1–N1 1.989(6) O1–Cu1–N2 98.0(2) Co1–N1 2.049(6) 
N2–C6 1.249(6) Cu1–N2 1.990(7) O4–Cu1–N3 93.3(2) Co1–Cl1 2.225(3) 
N2–C7 1.454(6) Cu1–O2 1.959(7) O1–Cu1–N2 155.9(2) C6–O1 1.219(11) 
C1–N1–C5 116.6(4) Cu1–O4 1.963(5) N1–Cu1–N2 82.9(2) C6–N2 1.361(13) 
N1–C1–C2 123.8(5) Cu1–O1 2.395(8) O4–Cu1–O5 57.8(3) C7–N2 1.475(9) 
C1–C2–Br1 120.0(4) Cu1–O5 2.524(6) O1–Cu1–O4 109.3(2) N1–Co1–Cl1 110.28(18) 
C3–C2–Br1 120.3(3) O4–Cu1–O1 109.3(3) N2–Cu1–O5 106.8(2) O1–C6–N2 125.0(10) 
C6–N2–C7 119.4(4) O4–Cu1–N2 97.7(3) N3–Cu1–O5 93.3(2) C1–N1–C5 117.3(9) 
Table 2
Hydrogen bonds of the title complex Co(L6)2Cl2
D–H···Ad(D–H)(Å)d(H···A)(Å)d(D···A)(Å)∠DHA(°)
C1–H1A···Cli 0.9290 (86) 2.7044(28) 3.4864(90) 142.332(500) 
D–H···Ad(D–H)(Å)d(H···A)(Å)d(D···A)(Å)∠DHA(°)
C1–H1A···Cli 0.9290 (86) 2.7044(28) 3.4864(90) 142.332(500) 

Symmetry code: ix, −1+y, z.

Some copper(II) and cobalt(II) complexes with higher antitumor activities have been reported because of their biocompatible ion properties [26–28]. In this work, a copper(II) complex CuL3(NO3)2 in a shape of green block single crystal is co-ordinated through L3 and copper(II) nitrate. Its structure is a distorted octahedral geometry that formed by a six-co-ordinate copper center, two nitrogen atoms of L3 and four oxygen of NO3. To be specific, this distorted octahedral geometry is composed of copper as the center, N2 as the vertex, O2 as the bottom and a distorted quadrangle consisted of O4, O5, O1 and N1. The bond length between the copper and the oxygen atom or nitrogen atom, Cu1−O1 is 2.3958(72) Å, Cu1−O2 is 1.9597(66) Å, Cu1−O4 is 1.9626(57) Å, Cu1−O5 is 2.5236(61) Å, Cu1−N1 is 1.9887(64) Å and Cu1−N2 is 1.9904(70) Å. The bond angle of O2−Cu1−O1 is 58.818(301)°, O5−Cu1−O4 is 55.630(229)°, which is smaller than that of N2−Cu1−N1 (82.997(260)°). The crystal crystallographic data of CuL3(NO3)2is shown in Tables 1, 2, Supplementary Table S1.

X-ray crystallographic analysis of the cobalt(II) complex reveals that its crystallization is in a chiral space group C2 with the Flack parameter of 0.05(4) due to chiral carbons in the ligand [31]. Its asymmetric unit includes half of a cobalt(II) ion, one ligand (L6), half CH3OH and one Cl ion (Figure 2a). In this complex, cobalt is four-co-ordinate to shape a similarly distorted tetrahedral CoN2Cl2 geometry consisted of two chloride ions and two nitrogen atoms from two ligands (Figure 2b). The infinite 1-D chain structure is formed by molecular hydrogen bond with C1-H1A···Cl (Figure 3). The Co-Cl distance is 2.225(3) Å and the Co-N distance is 2.049(6) Å, which are shorter than that reported for cobalt complexes (2.055(3) Å) [32].

Molecular structure.

Figure 2.
Molecular structure.

(a) The asymmetric unit of Co(L6)2Cl2; (b) molecular structure of Co(L6)2Cl2.

Figure 2.
Molecular structure.

(a) The asymmetric unit of Co(L6)2Cl2; (b) molecular structure of Co(L6)2Cl2.

1D chain structure.

Figure 3.
1D chain structure.

(a,b) The 1D chain structure of Co(L6)2Cl2 formed by hydrogen bonds.

Figure 3.
1D chain structure.

(a,b) The 1D chain structure of Co(L6)2Cl2 formed by hydrogen bonds.

Biological evaluation

Antiproliferative activities

L0, DOX (Doxorubicin, positive control [33]) and all obtained compounds were assessed by MTT assay in vitro. All results are in summary with IC50 values in Table 3.

Table 3
Cytotoxicity of L0, L1–L11, CuL3(NO3)2, Cu(L5)3 and Co(L6)2Cl2 to selected axenic human cancer cells and normal cells
IC50  ± SE1 (µM)
complexHelaHepG2MCF-7A549HUVEC
L0 2.02 ± 0.02 2.56 ± 0.04 19.45 ± 0.39 5.02 ± 0.19 1.27 ± 0.03 
L1 2.78 ± 0.07ns5 0.52 ± 0.04*4 5.14 ± 0.29***2 5.16 ± 0.51ns5 22.53 ± 1.10***2 
L2 12.32 ± 0.42***2 1.31 ± 0.03ns5 2.85 ± 0.09***2 1.37 ± 0.04*4 31.49 ± 5.57***2 
L3 12.57 ± 0.21***2 1.63 ± 0.29ns5 0.49 ± 0.03***2 3.10 ± 0.42ns5 29.69 ± 1.78***2 
CuL3(NO3)2 11.70 ± 0.10***2 8.75 ± 0.51***2 11.11 ± 0.34***2 14.28 ± 0.38***2 20.95 ± 3.81***2 
L4 4.31 ± 1.57ns5 3.08 ± 0.04ns5 1.49 ± 0.19***2 8.54 ± 1.65*4 34.59 ± 0.27***2 
L5 38.07 ± 1.71***2 24.56 ± 1.09***2 11.98 ± 0.36***2 43.74 ± 0.94***2 88.43 ± 2.47***2 
Cu(L5)3 23.57 ± 1.47***2 9.48 ± 0.12***2 30.42 ± 0.48***2 18.10 ± 0.60***2 7.45 ± 0.17ns5 
L6 15.90 ± 0.14***2 14.13 ± 0.41***2 15.04 ± 1.16**3 27.95 ± 2.06***2 41.13 ± 0.55***2 
Co(L6)2Cl2 6.35 ± 0.33**3 5.68 ± 0.32**3 30.65 ± 2.32***2 17.37 ± 0.53***2 5.75 ± 0.03ns5 
L7 8.24 ± 0.42***2 15.54 ± 0.74***2 48.37 ± 0.24***2 41.70 ± 0.71***2 15.93 ± 0.37***2 
L8 9.87 ± 0.45***2 18.88 ± 1.64***2 35.15 ± 0.04***2 27.95 ± 1.23***2 15.83 ± 0.47***2 
L9 17.57 ± 0.37***2 18.79 ± 0.37***2 29.34 ± 1.03***2 20.98 ± 0.18***2 102.05 ± 6.62***2 
L10 16.48 ± 0.18***2 12.84 ± 0.20***2 41.28 ± 2.00***2 47.29 ± 2.17***2 13.48 ± 0.44**3 
L11 59.38 ± 1.79***2 61.44 ± 1.05***2 56.21 ± 1.03***2 72.28 ± 1.55***2 26.68 ± 1.02***2 
DOX 3.55 ± 0.17ns5 1.20 ± 0.04ns5 14 ± 1.03***2 3.35 ± 0.69ns5 4.40 ± 0.55***2 
IC50  ± SE1 (µM)
complexHelaHepG2MCF-7A549HUVEC
L0 2.02 ± 0.02 2.56 ± 0.04 19.45 ± 0.39 5.02 ± 0.19 1.27 ± 0.03 
L1 2.78 ± 0.07ns5 0.52 ± 0.04*4 5.14 ± 0.29***2 5.16 ± 0.51ns5 22.53 ± 1.10***2 
L2 12.32 ± 0.42***2 1.31 ± 0.03ns5 2.85 ± 0.09***2 1.37 ± 0.04*4 31.49 ± 5.57***2 
L3 12.57 ± 0.21***2 1.63 ± 0.29ns5 0.49 ± 0.03***2 3.10 ± 0.42ns5 29.69 ± 1.78***2 
CuL3(NO3)2 11.70 ± 0.10***2 8.75 ± 0.51***2 11.11 ± 0.34***2 14.28 ± 0.38***2 20.95 ± 3.81***2 
L4 4.31 ± 1.57ns5 3.08 ± 0.04ns5 1.49 ± 0.19***2 8.54 ± 1.65*4 34.59 ± 0.27***2 
L5 38.07 ± 1.71***2 24.56 ± 1.09***2 11.98 ± 0.36***2 43.74 ± 0.94***2 88.43 ± 2.47***2 
Cu(L5)3 23.57 ± 1.47***2 9.48 ± 0.12***2 30.42 ± 0.48***2 18.10 ± 0.60***2 7.45 ± 0.17ns5 
L6 15.90 ± 0.14***2 14.13 ± 0.41***2 15.04 ± 1.16**3 27.95 ± 2.06***2 41.13 ± 0.55***2 
Co(L6)2Cl2 6.35 ± 0.33**3 5.68 ± 0.32**3 30.65 ± 2.32***2 17.37 ± 0.53***2 5.75 ± 0.03ns5 
L7 8.24 ± 0.42***2 15.54 ± 0.74***2 48.37 ± 0.24***2 41.70 ± 0.71***2 15.93 ± 0.37***2 
L8 9.87 ± 0.45***2 18.88 ± 1.64***2 35.15 ± 0.04***2 27.95 ± 1.23***2 15.83 ± 0.47***2 
L9 17.57 ± 0.37***2 18.79 ± 0.37***2 29.34 ± 1.03***2 20.98 ± 0.18***2 102.05 ± 6.62***2 
L10 16.48 ± 0.18***2 12.84 ± 0.20***2 41.28 ± 2.00***2 47.29 ± 2.17***2 13.48 ± 0.44**3 
L11 59.38 ± 1.79***2 61.44 ± 1.05***2 56.21 ± 1.03***2 72.28 ± 1.55***2 26.68 ± 1.02***2 
DOX 3.55 ± 0.17ns5 1.20 ± 0.04ns5 14 ± 1.03***2 3.35 ± 0.69ns5 4.40 ± 0.55***2 
1.

Average IC50 values ≥3 independent replicates.

2.

***P < 0.001.

3.

** P < 0.01.

4.

* P < 0.05.

5.

ns = not significant, compounds compared with L0.

For HUVEC cells, here synthesized compounds were less toxic (IC50 = 5.75−102.05 µM) than DOX (4.40 µM) and dehydroabietylamine (L0) (1.27 µM). L0 had slightly lower antiproliferative activity (except Hela cells) compared with DOX. For HepG2 cells, the IC50 value of L1 (0.52 µM), L2(1.31 µM) and L3 (1.63 µM) was lower than that of L0 (2.56 µM), which suggested they all had higher antiproliferative activities to HepG2 cells than L0, especially L1 owned high anti-HepG2 activity (0.52 µM) but low toxicity (22.53 µM). For MCF-7 cells, compound L1 (5.14 µM), L2 (2.85 µM), L3 (0.49 µM) and L4 (1.49 µM) have higher antiproliferative activities than DOX (14 µM) and L0 (19.45 µM) with their apparently lower IC50 values. For A549 cells, the IC50 value of L2 (1.37 µM) and L3 (3.10 µM) was relatively lower than L0 (5.02 µM) and DOX (3.35 µM), which revealed L2 and L3 had higher antiproliferative activities to A549 cells. Complex CuL3(NO3)2, Cu(L5)3 and Co(L6)2Cl2 had high antiproliferative activity to Hela and HepG2 cells. In addition, we explored the security index (SI) value. SI value for L1 is 43.3 and L3 was 60.6, which meant they are safer than L0(0.5) and DOX (3.7). In short, L1 had obviously higher anti-HepG2 activity while L3 had higher anti-MCF-7 activity. It was worth noting that L3 had great high anti-MCF-7 activity (0.49 µM) but low toxicity (29.69 µM), which indicated it may be a potential antiproliferative drug.

To analyze the relationship between structure and cytotoxicity of Schiff-base compounds L1L4, amide compounds L5L11, we could find that Schiff-base compounds (0.49–14.28 µM) had higher antiproliferative activities than amide compounds (5.68–72.28 µM), particularly Schiff-base compounds L1, L2 and L3. The reason might be the special bond −CH = N− within Schiff-base compounds could conjugate with the pyridine ring, which made contributions to the structural stability, additionally, the solubility of Schiff-base was slightly better. Among these amide compounds, compounds with methyl (L7, L10) and chlorine (L8, L11) could enhance antiproliferative activity with L7L8 to Hela cells and L10L11 to Hela and HepG2 cells, but toxicity had been also increased. According to the previous report, anticancer activities could be certainly improved by introducing a polar group containing a hydrogen bond donor [33]. Simultaneously, there is a great number of amino and carboxyl groups in cancer cells, which can easily form hydrogen bonds with the ‘N' with lone pair electrons in pyridine ring.

Induction of apoptosis

DOX is a well-known anticancer drug that can induce apoptosis to prevent cancer proliferation. Apoptosis is a physiologically programmed cell death mode, which plays a crucial role in cancer, the evolution of organisms and the stability of internal environment [34]. There are different phases in the apoptosis process including initiation, execution and degradation. It's reported that the process is activated by two major pathways and one major route called the ‘intrinsic’ cell death pathway, which can result in apoptosis and easily be activated by proapoptotic factors from mitochondria containing Apaf-1 and cytochrome c [35,36]. To investigate the potential mechanism of antiproliferative activities, here we have taken the apoptosis assay of L1, L2 and L3 to HepG2, A549 and MCF-7 cells respectively by Annexin V-FITC/PI and flow cytometry with DMSO as negative control, the result was revealed in Figure 4.

The flow cytometric analysis of inducing-apoptosis treated with L1, L2 and L3.
Figure 4.
The flow cytometric analysis of inducing-apoptosis treated with L1, L2 and L3.

(a) H-C: HepG2 cells incubated with DMSO (negative control), then L1 at 0.1, 0.5, 5 µg/ml, L2 and L3 at 0.1, 1, 5 µg/ml, respectively; (b) A–C: A549 cells incubated with DMSO, then L2 at 0.1, 1, 5 µg/ml, respectively; (c) M-C: MCF-7 cells incubated with DMSO, then L3 at 0.1, 0.5, 5 µg/ml, respectively.

Figure 4.
The flow cytometric analysis of inducing-apoptosis treated with L1, L2 and L3.

(a) H-C: HepG2 cells incubated with DMSO (negative control), then L1 at 0.1, 0.5, 5 µg/ml, L2 and L3 at 0.1, 1, 5 µg/ml, respectively; (b) A–C: A549 cells incubated with DMSO, then L2 at 0.1, 1, 5 µg/ml, respectively; (c) M-C: MCF-7 cells incubated with DMSO, then L3 at 0.1, 0.5, 5 µg/ml, respectively.

Apoptotic cells increased and live cells decreased after incubation with HepG2, A549 or MCF-7 cells, which is distinctly exhibited the dosage-dependent manner for L1, L2 and L3with the concentration enhanced from 0.1 to 5 µg/ml. For HepG2 cells, the apoptotic ratio was 16.73%, 81.89% and 97.56%, respectively when the concentration of L1 was 0.1, 0.5, 5 µg/ml. With the concentration at 0.1, 1 and 5 µg/ml respectively, the apoptotic ratio of L2 was 12.41%, 25.21% and 95.62% while L3 was 9.24%,23.26% and 46.23%. After incubation with L2, the apoptotic ratio of A549 cells was 13.96%, 20.30% and 80.64%, respectively with the concentration at 0.1, 1 and 5 µg/ml. Besides, the apoptotic ratio of MCF-7 cells was 14.63%, 23.55% and 83.05%, respectively with the concentration of L3 at 0.1, 1 and 5 µg/ml. In general, L1 owned better ability of inducing HepG2 cells apoptosis, while L2 to A549 cells and L3 to MCF-7 cells, they all could inhibit the proliferation of cancer cells through inducing apoptosis.

For further exploration, log P and pKb of all compounds were calculated (Table 4), which suggest compounds L1L11 are optimal for potential antiproliferation drugs.

Table 4
Physicochemical data (logP and pKb) of L0–L11, DOX
L0L1L2L3L4L5L6L7L8L9L10L11DOX
logP 0.40 1.18 0.81 1.23 0.99 3.37 3.37 4.33 4.26 3.37 3.53 3.05 1.50 
pKb 7.00 7.76 7.58 8.32 7.52 14.80 14.70 13.76 13.84 14.62 13.62 13.90 5.80 
L0L1L2L3L4L5L6L7L8L9L10L11DOX
logP 0.40 1.18 0.81 1.23 0.99 3.37 3.37 4.33 4.26 3.37 3.53 3.05 1.50 
pKb 7.00 7.76 7.58 8.32 7.52 14.80 14.70 13.76 13.84 14.62 13.62 13.90 5.80 

Confocal fluorescence images

Moreover, we tried to make the inducing apoptosis process visible by combining L0 (starting material) and FITC (a green fluorescent dye) as an example with confocal fluorescence images.

After the co-incubation of L0-FITC (100 nM) and Hela cells at 4°C for 2 h in the dark, there was hardly any green fluorescence signal inside the cells at 4°C (Figure 5a), and the obvious green fluorescence signal (Figure 5b) appeared at 37°C, which meant the inhibited internalization of L0-FITC at a lower temperature. This result displayed the uptake of L0-FITC followed the temperature-dependent endocytic pathway [37]. When the incubation time was up to 24 h, Hela cells morphology changed into quite integrated (Figure 5d). The result indicated L0 could penetrate into the cytomembrane, translocate into cytoplasm through endocytosis, damage cells and trigger cells to die.

Confocal fluorescence images of Hela cells incubated with L0-FITC.
Figure 5.
Confocal fluorescence images of Hela cells incubated with L0-FITC.

(a) Co-incubation of L0-FITC (100 nM) and Hela cells at 4°C for 2 h; (b) 37°C for 2 h; (c) Co-incubation of DMEM medium and Hela cells at 4°C for 2 h (control); (d) Co-incubation of L0-FITC (100 nM) and Hela cells at 37°C for 24 h. λex = 488 nm. Scale bars are 10 µm.

Figure 5.
Confocal fluorescence images of Hela cells incubated with L0-FITC.

(a) Co-incubation of L0-FITC (100 nM) and Hela cells at 4°C for 2 h; (b) 37°C for 2 h; (c) Co-incubation of DMEM medium and Hela cells at 4°C for 2 h (control); (d) Co-incubation of L0-FITC (100 nM) and Hela cells at 37°C for 24 h. λex = 488 nm. Scale bars are 10 µm.

DNA binding modes

As reported, DOX can efficiently penetrate into tissues and stay inside nucleated cells since its lipophilic characteristics and the ability to intercalate or bind to DNA [11]. For further investigation, we explored the properties of L1L3 to bind to DNA. Here, we carried out EB (ethidium bromide) fluorescence displacement experiments to evaluate DNA (Salmon Sperm DNA) binding modes. EB had strong ability to intercalate toDNA base pairs, so the fluorescence intensity was obviously increased upon adding DNA, although there wasn't any appreciable emission of EB in the buffer solution [11,38,39]. The emission of DNA−EB is reduced or quenched by the addition of a compound, it can be at least confirmed the intercalation of this compound to DNA base pairs [40]. Subsequently, adding L1L3 into DNA-EB led its emission intensity to decrease, which was further proved thatL1L3 had the ability of binding to DNA to replace the sites of EB by intercalation (shown in Figure 6). Next, we applied absorption spectra to investigate DNA binding modes of L1L3 (shown in Supplementary Fig. S1), which usually changed with hypochromism or bathochromism. During the experiment, enhancing DNA concentration could generate a hypochromic shift, suggesting the intercalative binding mode. The results of fluorescence and absorption spectra all revealed L1L3 could bind to DNA by intercalation.

Emission spectra of DNA–EB in the absence and the presence of increasing amounts of L1–L3 at room temperature ([EB] = 2 × 10−5 M, [DNA] = 1 × 10−4 M, [L1–L3] = 0–1.5 × 10−5 M).
Figure 6.
Emission spectra of DNA–EB in the absence and the presence of increasing amounts of L1–L3 at room temperature ([EB] = 2 × 10−5 M, [DNA] = 1 × 10−4 M, [L1–L3] = 0–1.5 × 10−5 M).

(a) L1; (b) L2; (c) L3.

Figure 6.
Emission spectra of DNA–EB in the absence and the presence of increasing amounts of L1–L3 at room temperature ([EB] = 2 × 10−5 M, [DNA] = 1 × 10−4 M, [L1–L3] = 0–1.5 × 10−5 M).

(a) L1; (b) L2; (c) L3.

To sum up all of aforementioned findings, highly potent antiproliferation activity and low cytotoxicity of L1, L2 and L3 were convinced. They could also induce apoptosis by intercalating into DNA to inhibit the proliferation of cancer cells. It is really meaningful to understand the detailed cytotoxicity for exploring new antiproliferative drugs.

In vivo experiment

For further investigation, we have taken compound L1 and L3 into the mice in vivo experiment. In this experiment, tumor mice injected intravenously with the dosage 0.5 mg/kg of L1 and the dosage 0.6 mg/kg of L3 from 1 day to 25 days with every 3 days. L1 was injected into mice with HepG2 cells when L3 with MCF-7 cells.

After injection with L1 and L3, the weight and volume of tumor mice were obviously reduced compared with PBS control from Figure 7(a)–(j). For L1, the average weight/volume of HepG2 tumor was 0.68 g/0.648 cm3 while that was 1.94 g/1.787 cm3 for PBS control. The relative proliferation rate of HepG2 cells (T/C) was 36% and the inhibitory rate was up to 65%. For L3, the average weight/volume of MCF-7 tumor was 0.92 g/0.806 cm3 while that was 2.32 g/2.063 cm3 for PBS control. The relative proliferation rate of MCF-7 cells (T/C) was 41% and the inhibitory rate was up to 61%. As shown in Figure 7(k), no obvious changes in the morphology of heart, liver, spleen, lung, kidney and brain tissues of the tumor mice after injected with L1 and L3, suggesting they had no significant toxicity. The tumor (HepG2/MCF-7 cells) mice with CD31 immumohistochemical staining were conducted on the 25th day after injecting intravenously with L1 and L3, the result observed clearly with decreased tumor angiogenesis rate in comparison with PBS control shown in Figure 7(l), suggesting the great inhibitory property of L1 and L3in vivo.

Experiment in vivo.
Figure 7.
Experiment in vivo.

(a) different appearance and (b) the tumor volume of mice injected with PBS control and compound L1 after 25 days; (c) change in body weight (d) volume (e) tumor weight of mice injected with PBS control and L1 (0.5 mg/kg); (f) different appearance and (g) the tumor volume of mice injected with PBS control and L3 after 25 days; (h) change in body weight (i) volume (j) tumor weight of mice injected with PBS control and L3 (0.6 mg/kg); (k) H&E staining of the brain, heart, liver, spleen, lung and kidney tissues of mice on the 25th day after an intravenous injection of L1 and L3 in comparison with PBS control; (l) tumor mice with CD31 immumohistochemical staining on the 25th day after intravenously injected with L1, L3 and PBS control. Scale bar = 20 µm. Error bars are based on standard errors of the mean (n = 5).

Figure 7.
Experiment in vivo.

(a) different appearance and (b) the tumor volume of mice injected with PBS control and compound L1 after 25 days; (c) change in body weight (d) volume (e) tumor weight of mice injected with PBS control and L1 (0.5 mg/kg); (f) different appearance and (g) the tumor volume of mice injected with PBS control and L3 after 25 days; (h) change in body weight (i) volume (j) tumor weight of mice injected with PBS control and L3 (0.6 mg/kg); (k) H&E staining of the brain, heart, liver, spleen, lung and kidney tissues of mice on the 25th day after an intravenous injection of L1 and L3 in comparison with PBS control; (l) tumor mice with CD31 immumohistochemical staining on the 25th day after intravenously injected with L1, L3 and PBS control. Scale bar = 20 µm. Error bars are based on standard errors of the mean (n = 5).

Conclusion

In summary, we reported the synthesis, antiproliferative activities of a series of dehydroabietylamine derivatives and several complexes including Schiff-base compounds L1–L4, amides compounds L5–L11, two copper(II) complex CuL3(NO3)2, Cu(L5)3 and a cobalt(II) complex Co(L6)2Cl2. All compounds and complexes had lower toxicity than DOX and L0. The compound L1 (0.52 µM) had higher antiproliferative activity to HepG2 cells, while L2 (1.37 µM) to A549 cells and L3 (0.49 µM) to MCF-7 cells in vitro. Additionally, they could also induce apoptosis of mentioned cells respectively (HepG2/A549/MCF-7) with intercalative binding modes to DNA. Complex CuL3(NO3)2, Cu(L5)3 and Co(L6)2Cl2 had relatively high antiproliferative activity to Hela and HepG2 cells. Moreover, L1 had high antiproliferative activity to HepG2 cells but nontoxicity in vivo while L3 to MCF-7, suggesting L1 and L3 may be the great promise antiproliferative drugs with nontoxic side effects.

All of these results here reported may offer a new idea to design novel compounds with cellular targeting property and be helpful for exploring future antiproliferative drugs. The possible structural and cellular mechanisms are worth a deeper research that we are working on.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

F.Z. performed the synthesis of Schiff-bases, characterization, analysis and biological assay of all compounds and wrote the manuscript. W.L. performed the X-ray analysis and writing guidance. Y.X. performed the synthesis of amides. L.X.* performed the task analysis and writing guidance. J.Z. performed the purification of some compounds. X.S. performed the fDNA testing. S.Y. performed the NMR testing. M.Z. performed the cell incubation instruction. F.S. performed the X-ray testing. F.L. performed the flow cytometry testing. F.C. performed the writing guidance.

Acknowledgements

Thanks for National Key Research and Development Project (2017YFD0600706), the Sate Key Laboratory for Medicinal Resources Chemistry and Pharmaceutical Molecular Engineering of Guangxi Normal University (CMEMR2017-B06).

Abbreviations

     
  • CCDC

    Cambridge Crystallographic Data Centre

  •  
  • SI

    security index

References

References
1
Auxiliadora
,
M.D.A.
,
Pablo
,
B.R.
,
Francisco
,
B.F.
and
Miguel
,
A.G.
(
2016
)
Synthesis and antileishmanial activity of C7- and C12-functionalized dehydroabietylamine derivatives
.
Eur. J. Med. Chem.
121
,
445
450
2
Wang
,
Y.Y.
,
He
,
Y.
,
Yang
,
L.F.
,
Peng
,
S.H.
,
He
,
X.L.
,
Yi
,
Z.F.
et al (
2016
)
Synthesis of novel diterpenoid analogs with in-vivo antitumor activity
.
Eur. J. Med. Chem.
120
,
13
25
3
Huang
,
X.C.
,
Huang
,
R.Z.
,
Li
,
L.X.
,
Gou
,
S.H.
and
Wang
,
H.S.
(
2017
)
Synthesis and biological evaluation of novel chalcone derivatives as a new class of microtubule destabilizing agents
.
Eur. J. Med. Chem.
132
,
11
25
4
Lin
,
T.T.
,
Tran
,
M.
,
González
,
M.A.
,
Gautam
,
L.N.
,
Connelly
,
M.
,
Wood
,
R.K.
et al (
2015
)
(+)-Dehydroabietylamine derivatives target triple-negative breast cancer
.
Eur. J. Med. Chem.
102
,
9
13
5
Pirttimaa
,
M.
,
Nasereddin
,
A.
,
Kopelyanskiy
,
D.
,
Kaiser
,
M.
,
Yli-Kauhaluoma
,
J.
,
Oksman-Caldentey
,
K.M.
et al (
2016
)
Abietane-type diterpenoid amides with highly potent and selective activity against Leishmania donovani and Trypanosoma cruzi
.
J. Nat. Prod.
79
,
362
368
6
Mustufa
,
M.A.
,
Aslam
,
A.
,
Oze
,
C.
,
Hashmi
,
I.A.
,
Naqvi
,
N.U.H.
,
Ozturk
,
M.
et al (
2017
)
Phenacyl group containing amide derivative of dehydroabietylamine exhibiting enhanced cytotoxic activity against PLC and MCF7 cancer cell lines
.
Med. Chem. Res.
26
,
1367
1376
7
Zhao
,
F.Y.
,
Wang
,
W.F.
,
Lu
,
W.
,
Xu
,
L.
,
Yang
,
S.L.
,
Cai
,
X.M.
et al (
2018
)
High anticancer potency on tumor cells of dehydroabietylamine Schiff-base derivatives and a copper(II) complex
.
Eur. J. Med. Chem.
146
,
451
459
8
Liu
,
H.F.
,
Ma
,
J.
,
Li
,
Y.G.
,
Yue
,
K.X.
,
Li
,
L.R.
,
Xi
,
Z.Q.
et al (
2019
)
Polyamine-Based Pt(IV) prodrugs as substrates for polyamine transporters preferentially accumulate in cancer metastases as DNA and polyamine metabolism dual-targeted antimetastatic agents
.
J. Med. Chem.
62
,
11324
11334
9
Qiao
,
X.
,
Ma
,
Z.Y.
,
Xie
,
C.Z.
,
Xue
,
F.
,
Zhang
,
Y.W.
,
Xu
,
J.Y.
et al (
2011
)
Study on potential antitumor mechanism of a novel Schiff Base copper(II) complex: Synthesis, crystal structure, DNA binding, cytotoxicity and apoptosis induction activity
.
J. Inorg. Biochem.
105
,
728
737
10
Banerjee
,
A.
,
Guha
,
A.
,
Adhikary
,
J.
,
Khan
,
A.
,
Manna
,
K.
,
Dey
,
S.
et al (
2013
)
Dinuclear cobalt(II) complexes of Schiff-base compartmental ligands: syntheses, crystal structure and bio-relevant catalytic activities
.
Polyhedron.
60
,
102
109
11
Arjmand
,
F.
and
Aziz
,
M.
(
2009
)
Synthesis and characterization of dinuclear macrocyclic cobalt(II), copper(II) and zinc(II) complexes derived from 2,2,2,2-S,S[bis(bis-N,N-2-thiobenzimidazolyloxalato-1,2-ethane)]: DNA binding and cleavage studies
.
Eur. J. Med. Chem.
44
,
834
844
12
Zhang
,
Z.L.
,
Yu
,
P.
,
Gou
,
Y.
,
Zhang
,
J.Z.
,
Li
,
S.H.
,
Cai
,
M.L.
et al (
2019
)
Novel brain-tumor-inhibiting copper(II) compound based on a human serum albumin (HSA)-cell penetrating peptide conjugate
.
J. Med. Chem.
62
,
10630
10644
13
Kreppel
,
A.
,
Blank
,
I.D.
and
Ochsenfeld
,
C.
(
2018
)
Base-Independent DNA base-excision repair of 8-Oxoguanine
.
J. Am. Chem. Soc.
140
,
4522
4526
14
Hassoon
,
A.A.
,
Harrison
,
R.G.
,
Nawar
,
N.
,
Smith
,
S.J.
and
Mostafa
,
M.M.
(
2020
)
Synthesis, single crystal X-ray, spectroscopic characterization and biological activities of Mn2+, Co2+, Ni2+ and Fe3+ complexes
.
J. Mol. Struct.
1203
,
127240
15
Garoufis
,
A.
,
Hadjikakou
,
S.K.
and
Hadjiliadis
,
N.
(
2009
)
Palladium coordination compounds as antiviral, antifungal, antimicrobial and antitumor agents
.
Coord. Chem. Rev.
253
,
1384
1397
16
Naeimi
,
H.
,
Rabiei
,
K.
and
Salimi
,
F.
(
2007
)
Rapid efficient and facile synthesis and characterization of novel Schiff bases and their complexes with transition metal ions
.
Dyes Pigments
75
,
294
297
17
Borisova
,
N.E.
,
Reshetova
,
M.D.
and
Ustynyuk
,
Y.A.
(
2007
)
Metal-free methods in the synthesis of macrocyclic schiff bases
.
Chem. Rev.
107
,
46
79
18
Shokrollahi
,
A.
,
Ghaedi
,
M.
,
Montazerozohori
,
M.
,
Kianfar
,
A.H.
,
Ghaedi
,
H.
,
Khanjari
,
N.
et al (
2011
)
Spectrophotometric study of complexation between some new N2O2-Schiff bases and some transition metal ions in nonaqueous solvents
.
J. Chem.
8
,
495
506
19
Malik
,
B.A.
,
Maurya
,
R.C.
,
Mir
,
J.M.
and
Shah
,
F.F.
(
2016
)
Cobalt(II) bactericidal and heat resistant complexes of ONS donor schiff base ligands: synthesis and combined DFT-experimental characterization
.
Int. J. Curr. Res. Chem. Pharm. Sci.
3
,
50
72
20
Al-Shemary
,
R.K.
,
Al-khazraji
,
A.M.A.
and
Niseaf
,
A.N.
(
2016
)
Preparation, spectroscopic study of schiff base ligand complexes with some metal ions and evaluation of antibacterial activity
.
J. Pharm. Innov.
5
,
81
86
21
Putta
,
V.P.R.K.
,
Gujjarappa
,
R.
,
Tyagi
,
U.
,
Pujar
,
P.P.
and
Malakar
,
C.C.
(
2019
)
A metal and base-free domino protocol for the synthesis of 1,3-benzoselenazines, 1,3-benzothiazines and related scaffolds
.
Org. Biomol. Chem.
17
,
2516
2528
22
Mohammadizadeh
,
F.
,
Falahati
,
P.S.K.
,
Rezaei
,
A.
,
Mohamadi
,
M.
,
Hajizadeh
,
M.R.
,
Mirzaei
,
M.R.
et al (
2018
)
The cytotoxicity effects of a novel Cu complex on MCF-7 human breast cancerous cells
.
Biometals
31
,
233
242
23
Gouda
,
A.M.
,
El-Ghamry
,
H.A.
,
Bawazeer
,
T.M.
,
Farghaly
,
T.A.
,
Abdalla
,
A.N.
and
Aslam
,
A.
(
2018
)
Antitumor activity of pyrrolizines and their Cu(II) complexes: design, synthesis and cytotoxic screening with potential apoptosis-inducing activity
.
Eur. J. Med. Chem.
145
,
350
359
24
Bernhardt
,
P.V.
,
Lawrance
,
G.A.
,
McCleverty
,
J.A.
and
Meyer
,
T.J.
(
2003
) Cobalt. In
Comprehensive Coordination Chemistry II
,
6
,
1
45
,
Elsevier Science, Amsterdam
25
Dimiza
,
F.
,
Papadopoulos
,
A.N.
and
Tangoulis
,
V.
(
2010
)
Biological evaluation of non-steroidal anti-inflammatory drugs-cobalt(II) complexes
.
Dalton Trans.
39
,
4517
4528
26
Liu
,
Z.C.
,
Wang
,
B.D.
,
Wang
,
B.Q.
,
Yang
,
Z.Y.
,
Li
,
T.R.
and
Li
,
Y.
(
2010
)
Crystal structures, DNA-binding and cytotoxic activities studies of Cu(II) complexes with 2-oxo-quinoline-3-carbaldehyde Schiff-bases
.
Eur. J. Med. Chem.
45
,
5353
5361
27
Jorge
,
S.G.
,
Elena
,
B.G.M.
,
Eric
,
L.R.
and
Lena
,
R.A.
(
2017
)
Genotoxic assessment of the copper chelated compounds Casiopeinas: clues about their mechanisms of action
.
J. Inorg. Biochem.
166
,
68
75
28
Loganathan
,
R.
,
Ganeshpandian
,
M.
,
Bhuvanesh
,
N.S.P.
,
Palaniandavar
,
M.
,
Muruganantham
,
A.
,
Ghosh
,
S.K.
et al (
2017
)
DNA and protein binding, double-strand DNA cleavage and cytotoxicity of mixed ligand copper(II) complexes of the antibacterial drug nalidixic acid
.
Inorg. Biochem.
174
,
1
13
29
Campero-Peredo
,
C.
,
Bravo-Gómez
,
M.E.
,
Hernández-Ojeda
,
S.L.
,
Olguin-Reyes
,
S.R.
,
Espinosa-Aguirre
,
J.J.
and
Ruiz-Azuara
,
L.
(
2016
)
Effect of [Cu(4,7-dimethyl-1,10-phenanthroline)(acetylace-tonato)]NO3, Casiopeína III-Ea, on the activity of cytochrome P450
.
Toxicol In Vitro
33
,
16
22
30
Lu
,
W.
,
Yang
,
S.L.
,
Xu
,
L.
,
Xu
,
Y.Y.
and
Chi
,
X.W.
(
2016
)
Crystal structure and magnetic properties of salicylaldehyde schiff base binuclear copper(II) complex
.
Chem. Reag.
38
,
716
720
31
Flack
,
H.D.
,
Sadki
,
M.
,
Thompson
,
A.L.
and
Watkin
,
D.J.
(
2011
)
Absolute structure determination using crystals
.
Acta Crystallogr. Sect. A.
67
,
21
34
32
Holló
,
B.
,
Rodić
,
M.
,
Vojinović-Ješić
,
L.S.
,
Živković-Radovanović
,
V.
,
Vučković
,
G.
,
Leovac
,
V.M.
et al (
2014
)
Crystal structure, thermal behavior, and microbiological activity of a thiosemicarbazide-type ligand and its cobalt complexes
.
J. Therm. Anal. Calorim.
116
,
655
662
33
González
,
M.A.
(
2014
)
Synthetic derivatives of aromatic abietane diterpenoids and their biological activities
.
Eur. J. Med. Chem.
87
,
834
842
34
Zhao
,
F.Y.
,
Lu
,
W.
,
Su
,
F.
,
Xu
,
L.
,
Jiang
,
D.
,
Sun
,
X.
et al (
2018
)
Synthesis and potential antineoplastic activity of dehydroabietylamine imidazole derivatives
.
MedChemComm.
9
,
2091
2099
35
Chen
,
C.N.
,
Wu
,
C.L.
and
Lin
,
J.K.
(
2007
)
Apoptosis of human melanoma cells induced by the novel compounds propolin A and propolin B from Taiwenese propolis
.
Cancer Lett.
245
,
218
231
36
Khacha-ananda
,
S.
,
Tragoolpua
,
K.
,
Chantawannakul
,
P.
and
Tragoolpua
,
Y.
(
2016
)
Propolis extracts from the northern region of Thailand suppress cancer cell growth through induction of apoptosis pathways
.
Invest. New. Drugs.
34
,
707
722
37
Li
,
Z.
,
Wang
,
J.
,
Zhou
,
Y.
and
Liu
,
H.
(
2014
)
Lead compound optimization strategy (3)-Structure modification strategies for improving water solubility
.
Acta Pharm. Sin.
49
,
1238
1247
38
Sarfraz
,
R.M.
,
Bashir
,
S.
,
Mahmood
,
A.
,
Ahsan
,
H.
,
Riaz
,
H.
,
Raza
,
H.
et al (
2017
)
Application of various polymers and polymers based techniques used to improve solubility of water soluble drugs: a review
.
Acta Pol Pharm.
74
,
347
356
39
Hu
,
M.
,
Zhao
,
J.X.
,
Ai
,
X.Z.
,
Budanovic
,
M.
,
Mu
,
J.
,
Webster
,
R.D.
et al (
2016
)
Near infrared light-mediated photoactivation of cytotoxic Re(I) complexes by using lanthanidedoped upconversion nanoparticles
.
Dalton Trans.
45
,
14101
14108
40
Gao
,
E.J.
,
Wang
,
K.H.
,
Zhu
,
M.C.
and
Liu
,
L.
(
2010
)
Hairpin-shaped tetranuclear palladium(II) complex: Synthesis, crystal structure, DNA binding and cytotoxicity activity studies
.
Eur. J. Med. Chem.
45
,
2784
2790