Abstract
Objective
This study aimed at synthesizing 13 series of novel derivatives with 2-phenylacrylonitrile, evaluating antitumor activity both in vivo and in vitro, and obtaining novel tubulin inhibitors.
Methods
The 13 series of 2-phenylacrylonitrile derivatives were synthesized by Knoevenagel condensation and the anti-proliferative activities were determined by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometer. Quantitative cell migration was performed using 24-well Boyden chambers. The proteins were detected by Western blotting. In vitro kinetics of microtubule assembly was measured using ELISA kit for Human β-tubulin (TUBB). Molecular docking was done by Discovery Studio (DS) 2017 Client online tool.
Results
Among the derivatives, compound 1g2a possessed strong inhibitory activity against HCT116 (IC50 = 5.9 nM) and BEL-7402 (IC50 = 7.8 nM) cells. Compound 1g2a exhibited better selective antiproliferative activities and specificities than all the positive control drugs, including taxol. Compound 1g2a inhibited proliferation of HCT116 and BEL-7402 cells by arresting them in the G2/M phase of the cell cycle, inhibited the migration of HCT116 and BEL-7402 cells and the formation of cell colonies. Compound 1g2a showed excellent tubulin polymerization inhibitory activity on HCT116 and BEL-7402 cells. The results of molecular docking analyses showed that 1g2a may inhibit tubulin to exert anticancer effects.
Conclusion
Compound 1g2a shows outstanding antitumor activity both in vivo and in vitro and has the potential to be further developed into a highly effective antitumor agent with little toxicity to normal tissues.
Keywords: 2-Phenylacrylonitrile, selective toxic effect, cell cycle arrest, apoptosis, xenograft model, tubulin inhibitor
1. INTRODUCTION
In 2019, an estimated 1,335,100 new cancer cases and 3,97,583 cancer-related deaths occurred among adolescents and young adults worldwide [1]. Many chemotherapies have been developed, but there is no ideal anticancer drug that can kill cancer cells without threatening normal human tissues. It has become a huge challenge for modern science to find targeted, low-toxicity, high-efficiency antitumor drugs [2]. Natural products play a leading role in drug discovery and many of the new drugs approved for marketing were directly or indirectly derived from natural products [3]. Thus, the structural discovery of new drugs is crucial.
Derivatives of natural products, such as combretastatin A-4 (CA-4) (Fig. 1), resveratrol (Fig. 1), and pterostilbene, are widely used as anticancer candidates [4]. These molecules all have a stilbene [also known as 1,2-diphenylethene] scaffold, which is a component of a number of biologically active natural and synthetic compounds [4, 5]. For example, CA-4 was proven to possess a wide variety of pharmacological activities, including anticancer effects [6, 7]. Recent studies have found that the 3,4,5-trimethoxyphenyl fragment of CA-4 derivatives has a prevention of tubulin polymerization effect by binding to the colchicine binding site [8]. Microtubules are a major component of the cytoskeleton consisting of α- and β-tubulin heterodimers, which play crucial roles in several cellular processes [9, 10]. It is one of the best targets for developing anti-cancer drugs. Various naturally occurring molecules are well known for their anti-tubulin effect, such as vinca (vinca binding site), paclitaxel (taxol binding site), combretastatin (colchicine binding site), colchicine (colchicine binding site) etc. Destroying the microtubule skeleton is effective in cancer chemotherapy [9, 11]. Unfortunately, in vivo, CA-4 showed a sharp decline in its anti-tubulin and cytotoxic activity due to the spontaneous isomerization of the ethylene bond from the cis-isomer to the more stable trans-isomer [12], limiting its application in clinical practice. To restrict the cis-orientation of the ethylene bond, some researchers chose to substitute the ethylene bond with rigid heterocyclic rings, such as pyridine, pyrrole, pyrazole, triazoles, imidazole, isoxazole, and azetidine [11, 13-18]. Through these attempts, compounds with good anticancer effects have been obtained, but they have not been developed into new drugs. Some studies have found that when a cyano group is introduced into the ethylene structure of stilbenes, the configuration can be fixed to cis- and some other compounds, which have a 3,4,5-trimethoxyphenyl fragment and show strong antiproliferative activity against cancer cells without toxicity to normal cells [19, 20]. The inhibitory activity of the representative compound 1 toward MGC-803 cancer cells was stronger than that of CA-4 and taxol (Fig. 1). Compounds with a 3,5-dimethoxyphenyl fragment instead of a 3,4,5-trimethoxyphenyl fragment show comparable effects against cancer cells [2]. The selectivity index (SI) of compound 2 containing a 3,5-dimethoxyphenyl fragment for HeLa cancer cells and L-02 normal cells was higher than that of taxol and resveratrol (Fig. 1). It can be inferred that a stilbene with a cyano group on the ethylene bond is likely to be a candidate for the basis of anticancer drugs with low toxicity and high efficiency. If the substituents on the A-benzene ring and B-benzene ring of the stilbene are changed, or the structure of the B-benzene ring is changed, the activity may be affected (Fig. 2).
Fig. (1).
Structures of taxol, colchicine, CA-4, CA-4P, (Z)-3-(p-tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (1), resveratrol and (Z)-3-(4- bromophenyl)-2-(3,5-dimethoxyphenyl)acrylonitrile (2).
Fig. (2).
Design derivatives with 2-phenylacrylonitrile to achieve potential anti-proliferative activity.
More than 3800 naturally occurring halogenated compounds have been found, and many of these have good biological activity [21]. Therefore, halogen-containing groups, including trifluoromethyl or trifluoromethoxy groups, are often introduced into compounds to obtain new drugs with enhanced anticancer activity [22-24]. 1,2,3-Triazole has a high dipole moment and can form a hydrogen bond with drug targets [16]. It is an important heterocyclic structural unit distributed in a large number of biologically active molecules [25]. In recent years, natural active products and the 1,2,3-triazole molecular fragment were combined to improve antitumor activity and drug-forming properties of natural products [26]. Furthermore, the anticancer activity of target compounds can be affected by addition of a pentabasic cyclic ring, hexatomic ring, or fused ring containing nitrogen, sulfur, or no heteroatom [27-32].
In this study, we selected CA-4 as a lead compound and introduced a cyano group into the ethylene bond to fix the stilbene configuration. The A benzene ring was substituted with -OCF3, -OCH3, -CF3, or -CH3, and the B benzene ring was substituted (e.g., with amino, halogen, or alkoxy groups) or replaced (e.g., with a triazole, chalcone, or piperidine fragment). Hence, we designed and synthesized multiple series of derivatives with 2-phenylacrylonitrile (a type of stilbene with a cyano group into the ethylene bond and lack of B benzene ring) (Fig. 2). Subsequently, all 83 new compounds (Fig. S1) were screened against 11 different types of cancer cell lines. The toxicity of the compounds was also tested in normal human liver L-02 and breast MCF-10A cells. Additionally, the in vivo antitumor efficacy and mechanism of action of the most promising compound, 1g2a, was investigated.
2. MATERIALS AND METHODS
All specific materials and pharmacological experimental methods can be found in the supplementary material.
3. RESULTS AND DISCUSSION
3.1. Chemistry
According to Scheme S1 and Scheme S2 obtained 13 series of derivatives. The substituents of the target compounds synthesized by Scheme 1 and Scheme 2 are shown in Fig. (3). Before biological evaluation, the compounds (Fig. 3) were confirmed by 1H NMR and 13C NMR spectrometry as well as high-resolution mass spectrometry (supplementary material), and the Z isomer was confirmed by NOE (nuclear overhauser effect) (Fig. S2).
Scheme (1).
Reagents and conditions: (a) H2SO4, CH3OH, reflux, 4 h; (b) LiAlH4, THF, 0°C-rt, 4-6 h; (c) CH2Cl2, PBr3, 0°C-rt, 3-6 h; (d) CH3CN, TMSCN, TBAF, reflux, 4-6 h; (e) CH3OH, CH3ONa, aromatic aldehydes, 4-6 h.
Scheme (2).
Reagents and conditions: (e) CH3OH, CH3ONa, aromatic aldehydes, 4-6 h; (i) KOH, dry ethanol, stirred at rt, 12 h.
Fig. (3).
The compounds and their substituents were synthesized according to Scheme 1 and Scheme 2.
3.2. Biological Evaluation
3.2.1. In Vitro Anti-proliferative Activity and SAR Study
The anti-proliferation activities of the synthesized analogs were assessed by an in vitro MTT assay conducted on thirteen human cell lines. Their cytotoxic activities were evaluated by measuring the inhibition of net cell growth. The IC50 values are listed in Table 1 and Table S1. CA-4, CA-4P, taxol, colchicine, and resveratrol are used as positive controls.
Table 1.
In vitro anti-proliferative activity of the synthesized compounds, CA-4, CA-4P, colchicine, resveratrol and taxol against thirteen cell linesa (IC50 (µMb)).
| IC50 (μM) | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Agent | A549 | AGS | BEL-7402 | HCT-116 | HeLa | HepG-2 | L-02 | MGC-803 | MCF-7 | MCF-10A | Raji | SGC-7901 | SU-DHL-10 | |
| 1 | 1a2a | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 2 | 1a2b | >100 | 13.2±3.3 | 15.0±2.7 | 23.5±4.6 | 35.9±.2.5 | >100 | >100 | 37.4±5.8 | >100 | >100 | 49.0±7.4 | >100 | 38.8±3.7 |
| 3 | 1a2c | 65.8±8.9 | 18.6±3.0 | >100 | 20.2±1.7 | 72.3±2.4 | 20.7±1.4 | >100 | 35.2±2.8 | 55.2±2.8 | >100 | 55.1±3.2 | 56.4±9.7 | >100 |
| 4 | 1a2d | >100 | 19.6±2.9 | >100 | 25.4±6.8 | 87.3±4.1 | 24.3±3.4 | >100 | >100 | >100 | >100 | 64.9±1.5 | >100 | 73.5±17.3 |
| 5 | 1a2e | >100 | 30.8±7.9 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 6 | 1a2f | >100 | 14.6±5.9 | >100 | 43.5±3.8 | >100 | >100 | >100 | 56.7±11.0 | 59.9±2.3 | >100 | 42.7±3.4 | 77.9±12.6 | 15.1±11.6 |
| 7 | 1a2g | 71.6±24.7 | 8.7±2.1 | >100 | 57.4±7.8 | >100 | >100 | >100 | >100 | >100 | >100 | 66.6±13.3 | 89.9±5.6 | 27.3±5.3 |
| 8 | 1a2h | >100 | 69.6±15.7 | 24.1±2.8 | 8.7±1.7 | >100 | >100 | >100 | 16.8±0.7 | >100 | >100 | 67.4±31.7 | 19.7±1.5 | >100 |
| 9 | 1a2i | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 45.1±15.6 | >100 | >100 |
| 10 | 1b2a | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 11 | 1b2b | >100 | 8.2±2.6 | 3.0±1.0 | 1.1±0.3 | >100 | 15.9±5.2 | 16.1±1.0 | 1.2±0.6 | >100 | 11.3±2.3 | 17.0±4.3 | 5.4±0.2 | 60.4±0.7 |
| 12 | 1b2c | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 13 | 1b2d | >100 | 13.9±4.2 | 14.6±4.6 | 15.1±2.9 | 66.8±9.8 | 34.9±3.2 | 50.4±0.8 | 10.1±0.9 | >100 | 34.1±3.6 | 16.3±6.1 | 21.9±3.3 | 35.6±0.9 |
| 14 | 1b2e | >100 | 4.5±2.0 | 39.4±6.1 | 25.0±0.5 | 61.7±3.3 | 38.2±0.1 | 54.2±12.3 | 11.1±0.3 | >100 | >100 | 43.1±4.5 | 8.1±2.9 | 29.3±2.0 |
| 15 | 1b2f | >100 | 43.8±21.8 | 44.8±10.6 | >100 | >100 | 72.9±9.9 | >100 | 49.3±0.3 | >100 | >100 | >100 | 61.7±0.4 | >100 |
| 16 | 1b2g | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 17 | 1b2h | >100 | >100 | 1.0±0.8 | 0.3±0.2 | >100 | 42.3±18.2 | 52.0±8.4 | 3.7±1.3 | >100 | >100 | 20.1±2.3 | 7.1±2.4 | >100 |
| 18 | 1b2i | >100 | 23.9±2.7 | 13.0±1.1 | 12.3±1.6 | >100 | 23.5±4.3 | 28.5±0.8 | 10.8±2.9 | >100 | 79.6±5.7 | 23.7±2.1 | 46.0±4.9 | 53.6±7.5 |
| 19 | 1b3a | 32.6±1.1 | 15.4±0.5 | >100 | 19.3±0.1 | 38.8±3.5 | 21.5±7.6 | 35.9±1.6 | 20.7±1.5 | 25.5±0.4 | 22.7±3.5 | 56.6±5.8 | 20.9±5.1 | 59.7±0.8 |
| 20 | 1c2a | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 21 | 1c2b | >100 | 31.4±8.3 | >100 | >100 | >100 | 70.5±25.4 | >100 | 29.0±8.3 | >100 | >100 | >100 | >100 | >100 |
| 22 | 1c2c | >100 | 16.7±2.3 | >100 | 17.2±5.2 | >100 | 27.9±2.4 | 42.2±11.5 | 19.6±6.7 | >100 | >100 | 42.4±1.7 | >100 | >100 |
| 23 | 1c2d | 50.7±7.4 | 6.3±0.4 | 17.4±4.2 | >100 | >100 | 26.7±10.1 | >100 | 13.1±3.6 | >100 | >100 | 60.5±19.1 | 50.1±5.9 | >100 |
| 24 | 1c2e | >100 | 7.6±0.6 | 10.8±2.3 | 8.4±2.4 | 75.8±12.4 | 24.9±7.0 | 36.1±4.8 | 2.5±1.1 | >100 | >100 | 41.3±5.5 | 7.8±0.8 | >100 |
| 25 | 1d2a | >100 | 23.0±1.0 | >100 | >100 | >100 | >100 | >100 | 18.8±1.7 | >100 | >100 | 7.9±0.5 | >100 | 19.9±0.7 |
| 26 | 1d2b | >100 | 24.1±0.2 | 65.4±2.5 | 3.1±0.5 | >100 | >100 | >100 | 8.6±0.04 | 52.3±2.2 | >100 | 11.8±0.4 | >100 | 42.6±11.4 |
| 27 | 1e2a | >100 | 18.4±1.9 | 46.8±5.1 | 7.2±0.6 | >100 | >100 | 47.5±12.4 | 8.3±1.2 | 36.9±3.6 | 28.1±3.6 | 11.6±4.9 | 38.2±9.0 | 27.2±5.3 |
| 28 | 1f2a | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 29 | 1g2a | >100 | 0.08±0.05 | 0.007±0.001 | 0.005±0.001 | >100 | >100 | >100 | 14.0±8.4 | 0.27±0.04 | 0.02±0.001 | 0.03±0.001 | >100 | 0.03±0.01 |
| - | > 1250c | > 12820c | > 16949c | - | - | > 7.1c | - | > 2941c | - | > 2941c | ||||
| 30 | 1g2b | >100 | 7.5±0.4 | 0.03±0.01 | 1.0±0.1 | >100 | >100 | >100 | 0.12±0.03 | 0.45±0.06 | 0.05±0.001 | 0.37±0.06 | >100 | 2.4±0.1 |
| 31 | 8a | >100 | >100 | 13.2±2.7 | 56.3±8.4 | >100 | >100 | >100 | >100 | 17.6±6.5 | 11.3±2.3 | 1.2±0.5 | >100 | 1.3±0.1 |
| CA-4 | >100 | 3.4±0.01 | 2.0±0.03 | 0.16±0.001 | >100 | >100 | 1.1±0.03 | 0.06±0.02 | 0.2±0.1 | 3.2±1.2 | 0.18±0.001 | 0.30±0.001 | 1.1±0.1 | |
| - | - | - | > 6.8c | - | - | > 18.3c | > 14.6d | > 5.8c | > 3.6c | - | ||||
| CA-4P | >100 | 4.7±0.2 | 5.4±2.0 | 0.5±0.2 | >100 | >100 | 2.6±0.6 | 0.03±0.02 | 0.4±0.2 | 14.5±1.3 | 0.42±0.01 | 0.1±0.03 | 0.88±0.07 | |
| - | - | - | > 4.5c | - | - | > 89c | > 30.3d | > 6.2c | > 22.2c | > 3.0c | ||||
| Taxol | 0.4±0.02 | 0.02±0.01 | 0.09±0.02 | 0.03±0.01 | 12.9±0.4 | 0.9±0.03 | >100 | 0.06±0.01 | 0.08±0.01 | 0.18±0.02 | 0.26±0.02 | >100 | 0.18±0.03 | |
| > 227c | > 5000c | > 1111c | > 3333c | > 7.7c | > 106c | > 16666c | > 2.2d | > 384c | - | > 555c | ||||
| Colchicine | 6.5±1.5 | 0.1±0.03 | 0.02±0.001 | 0.01±0.001 | 1.8±0.1 | 1.7±0.4 | 0.4±0.02 | 0.13±0.01 | 0.6±0.3 | 1.4±0.3 | 0.3±0.1 | 0.1±0.04 | 0.29±0.001 | |
| - | - | > 16.7c | > 29.3c | - | - | > 3.6c | > 2.3d | > 1.3c | > 4.2c | > 1.6c | ||||
| Resveratrol | >100 | >100 | 42.5±5.4 | 16.7±3.6 | >100 | >100 | 12.8±0.2 | >100 | 72.4±9.9 | >100 | >100 | >100 | >100 | |
Note:aCytotoxicity, as IC50 for each cell line, refers to the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.
bData represent the mean values of three independent determinations.
cSI (selectivity index), IC50 (L-02) / IC50 (cancer cell)
dSI (selectivity index), IC50 (MCF-10A) / IC50 (MCF-7)
-: not suitable for calculation
Introduction of the -OCF3 group at the p-position of benzene ring A yielded 1a2a-1a2u. When the p-position of the B-ring was substituted by -Cl (1a2c) or -Br (1a2d), the compounds showed antiproliferation activity against 10 tumor cell lines. Moreover, this series of compounds showed very weak inhibitory activities against both L-02 and MCF-10A normal cells. When the B-ring was substituted with an alkyl group, compound 1a2g showed the strongest antiproliferation activity against AGS cells (IC50 = 8.7 µmol/L). The antiproliferative activity against AGS cells was related to the length of the alkyl substituent chain at the p-position on the B-ring. When the B-ring was replaced by an alkoxy group, only 1a2j (p-OCH3) and 1a2m (p-OCH2CH2CH3) showed weak inhibition of proliferation of a few tumor cell lines (Table S1). However, there was some enhancement in antiproliferative activity when the B-ring was substituted with a nitrogen-containing group. For instance, compound 1a2h [p-N(CH3)2] showed strong antiproliferation activity against HCT116 cells (IC50 = 8.7 µmol/L). When the B-ring was substituted with a nitrogen-containing group, the antiproliferative activity decreased with the increase of carbon chain length connected to the nitrogen atom (1a2h-i and 1a2s-u). When the B-ring was changed to a furan (1a3a), thiophene (1a3b), or naphthalene (1a3c) ring, only compound 1a3b showed weak antiproliferative activity against a few kinds of cancer cells. However, when the B-ring was replaced with a chalcone fragment obtaining compounds 8a and 8c, they showed good antiproliferation activities against Raji and SU-DHL-10 cells, with IC50 values ~1 µmol/L. The antiproliferation effect of 8d against these two types of cells was also quite obvious. However, there was almost no antiproliferation activity when the B-ring was replaced with triazole (7a-7c) or indole (9). In summary, we found that when the B-ring was replaced by a larger ring fragment or possessed a larger substituent (other than chalcone), the synthesized compounds showed decreased or no antiproliferative activity. On the basis of the above experimental results, we made further modifications.
Next, we introduced an -OCF3 group at the ortho (o)- or meta (m)- position of the A benzene ring. The compounds in which the m-position of the A-ring was substituted with -OCF3 and a halogen was introduced into the B-ring (1b2b-1b2d) generally showed good antiproliferative activity. Among them, the introduction of -F resulted in compound 1b2b, which had potent activity against HCT116, MGC-803, BEL-7402, SGC-7901, and AGS cells with IC50 values of 1.1, 1.2, 3.0, 5.4, and 8.2 µmol/L, respectively. Introduction of a methyl group at the p-position of the B-ring yielded compound 1b2e, which had good inhibitory activity of proliferation of AGS (IC50 = 4.5 µmol/L) and SGC-7901 (IC50 = 8.1 µmol/L) cells. The antiproliferative activities of compounds 1b2e-1b2g decreased with the extension of the carbon chain when an alkyl group was introduced at the p-position of the B-ring. However, when introducing an alkoxy group at the p-position of the B-ring, the synthesized compounds (1b2j-1b2k) had little antiproliferative activity. When introducing a nitrogen-containing group at the p-position of the B-ring or replacing the B-ring with a heterocycle, the compounds (1b2h, 1b2i, and 1b3a) showed strong inhibitory effects on the proliferation of a variety of cancer cells. Among these compounds, 1b2h [p-N(CH3)2] exerted potent antiproliferative activity against BEL-7402 (IC50 = 1.0 µmol/L), HCT116 (IC50 = 0.3 µmol/L), MGC-803 (IC50 = 3.7 µmol/L), and SGC-7901 (IC50 = 7.1 µmol/L) cells. When introducing -OCF3 at the o-position of the A-ring and changing the p-position substituent of the B-ring, the compounds produced (1c2a-1c2e) showed various degrees of antiproliferative activity against cancer cells. In conclusion, when the p-position of the B-ring was replaced by a nitrogen atom-containing group and the m-position of the A-ring was substituted by -OCF3, the compounds showed better inhibitory activity against tumor proliferation.
Introduction of -N(CH3)2, -N(CH2CH3)2, or an oxygen heteroatom substituent at the p-position of the B-ring and replacement of the A-ring with -CH3 or -OCH3 resulted in seven compounds (1d2a-1d2b, 1e2a-1e2c, and 1g2a-1g2b). When introducing -CH3 at the p-position of the A-ring, the resulting compounds 1d2a [-N(CH3)2] and 1d2b [-N(CH2CH3)2] showed good antiproliferative activity against some tumor cell lines, especially 1d2b, whose IC50 value against HCT116 cells was 3.1 µmol/L. Replacing the A-ring with 3,4,5-OCH3 and introducing -N(CH3)2 or -N(CH2CH3)2 at the p-position of the B-ring yielded 1g2a and 1g2b, respectively, which exerted excellent antiproliferative activity against AGS, BEL-7402, HCT116, and other cancer cell lines. Compound 1g2a displayed the most potent antiproliferative activity against AGS (IC50 = 0.08 µmol/L), BEL-7402 (IC50 = 7.8 nmol/L), HCT116 (IC50 = 5.9 nmol/L), Raji (IC50 = 0.03 µmol/L), and SU-DHL-10 (IC50 = 0.03 µmol/L) cells of all the compounds tested in this study. The inhibitory effect of compound 1g2a on the proliferation of BEL-7402, HCT116, Raji, and SU-DHL-10 cells was stronger than that of the five positive control agents used in this study, including taxol. Moreover, 1g2a demonstrated little cytotoxicity to L-02 cells. Compound 1g2b also exerted strong inhibition of proliferation, but it was slightly weaker in effect than 1g2a.
Introduction of a -CF3 group at the para (p)-position of benzene ring A resulted in compounds 1f2a-1f2w and 1f3a-1f3d. As shown in Table S1, most of the compounds exhibited low antiproliferative activity. Surprisingly, however, 1f2w (containing -CF3 on the side of the B benzene ring) displayed strong antiproliferative activities against Raji (IC50 = 48 nmol/L) and SU-DHL-10 (IC50 = 22 nmol/L) cells, but no antiproliferative activity against other tested cells.
On the basis of the above results, we summarize various structure-activity relationships: 1. When the A-ring was replaced by -OCF3, the antiproliferative activity sequence of the compounds was m-OCF3 > o-OCF3 > p-OCF3. When the meta-position of A-ring was replaced by -OCF3, the anti-proliferative activity sequence of B-ring substituents was p-heteroatom > p-halogen > p-alkyl. 2. When the p-position of the A-ring was substituted with -OCH3 and the p-position of the B-ring was substituted with nitrogen-containing groups, the antiproliferative activity sequence of the compounds was p-N(CH3)2 > p-N(CH2CH3)2. 3. When the A-ring was replaced by 3,4,5-OCH3 and the p-position of the B-ring was substituted with nitrogenous groups, the antiproliferative activity sequence of the compounds was p-N(CH3)2 > p-N(CH2CH3)2.
In summary, compound 1g2a, in which the A-ring was replaced by 3,4,5-OCH3 and the p-position of the B-ring was substituted with -N(CH3)2, exerted the most potent antiproliferative effect against many kinds of cancer cell line, but little cytotoxicity toward L-02 cells, and thus showed good and selective anticancer activity.
As shown in Table 1 and S2, the selectivity of compound 1g2a for BEL-7402, HCT116, Raji, and SU-DHL-10 cells was 12,820-, 16,949-, 2,941- and 2,941-fold that for L-02 cells, respectively, much higher than that of any of the positive control agents. Therefore, it was chosen for further biological studies.
3.2.2. Compound 1g2a Induces Cell Cycle Arrest with a Change in the Expression of Cyclin A, Cyclin B1, Cyclin D1 and Cyclin E1
Cell cycle dysregulation, uncontrolled mitosis, is an important cause of proliferation of cancer cells [33]. Tubulin-destabilizing agents block the cell cycle in the G2/M phase due to microtubule depolymerization and disruption of the cytoskeleton [34]. As shown in Fig. (4A), 1g2a increased the percentage of the HCT116 cell population in the G2/M phase of the cell cycle in a concentration-dependent manner from 33.25% to 65.74%, compared with cells incubated in DMSO as a vehicle control. At 0.1 μM, 1g2a showed greater inhibition than taxol. The effects of compound 1g2a on BEL-7402 cells were similar to those on HCT116 cells (Fig. 4B). In L-02 normal cells, the population in the G2/M phase of the cell cycle was slightly increased in 1g2a-treated cells compared with the vehicle group; however, from 0.001 to 0.1 μM 1g2a, there was almost no effect on the cell cycle (Fig. 4C). The above results showed that compound 1g2a caused cell cycle arrest at the G2/M phase in HCT116 and BEL-7402 cells but had no significant effect on L-02 cells, which might be one of the possible mechanisms for its selective cytotoxicity.
Fig. (4).
Compound 1g2a induced G2/M arrest in HCT116 and BEL-7402 cancer cells. Cells were incubated with varying concentrations of 1g2a (0.001, 0.01, 0.1 μM), 0.1% DMSO (control), 0.1 μM taxol and the percentage histograms of cell cycle distribution results in (A) HCT116 cells; (B) BEL-7402 cells; (C) L-02 cells. **p < 0.01 compared to control group; *p < 0.05 compared to control group; ##p < 0.01 compared to 0.1 μM taxol treatment group.
Entry to the S phase of the cell cycle requires the accumulation of cell-cycle activation-related cyclins [26, 35]. As shown in Fig. (5A-C), in HCT116 and BEL-7402 cells, the 1g2a treatment groups had lower expression of cyclin A, cyclin D1, and cyclin E1 than the vehicle group. The reduced expression was dose-dependent. The effect on the expression of cyclin D1 and cyclin E1 by 1g2a was much greater than that of taxol treatment at 0.1 μM; the cyclin A level by 1g2a was similar to that of taxol treatment at 0.1 μM. Cyclin B1 plays an important role in the transition from interphase to the mitotic phase and governs cell cycle progression by enhancing cell-cycle distribution in the G2/M fraction [36]. It has been reported that expression of cyclin B1 is increased by exposure to anticancer agents in BEL-7402 cells [20]. In our experiment, 1g2a treatment induced increased expression of cyclin B1 compared with treatment with vehicle, and cyclin B1 was upregulated in a concentration-dependent manner on 1g2a treatment of HCT116 and BEL-7402 cells. The cyclin B1 level on 1g2a treatment was similar to that in the taxol treatment group at 0.1 μM in both cancer cell lines. However, in the same conditions, 1g2a hardly changed the expression of the four kinds of cyclin in normal L-02 cells (Fig. 5A and D). These results suggest that the downregulation of cyclins A, D1, and E1 and the upregulation of cyclin B1 are related to the decrease in cell proliferation induced by 1g2a treatment of HCT116 and BEL-7402 cancer cells.
Fig. (5).
Compound 1g2a induced G2/M arrest in HCT116 and BEL-7402 cancer cells. (A) Western blotting analysis on the effect of 1g2a on the G2/M regulatory proteins. The histograms of the density ratio of cyclin A, B1, D1 and E1 to β-actin of (B) HCT116 cells; (C) BEL-7402 cells; (D) L-02 normal cells. Data represented as mean ± SD of independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group; #p < 0.05 and ###p < 0.001 compared to 0.1 μM taxol treatment group at same concentration treatment.
3.2.3. Compound 1g2a Induces Cancer Cell Apoptosis
To determine whether compound 1g2a could induce apoptosis, vehicle-, 1g2a-, and taxol-treated HCT116 and BEL-7402 cancer cell lines and normal L-02 cells were stained with FITC-annexin V and PI. As shown in Fig. (6A), incubation with 1g2a (0.001, 0.01, or 0.1 μM) for 12 h induced a concentration-dependent increase in the percentage of total apoptotic (early and late stage) HCT116 cells from 2.6% to 35.3%. Treatment with 0.1 μM 1g2a resulted in a slightly increased number of total apoptotic cells (35.3%) compared with the taxol treatment group (26.8%), and the proportion in both was obviously higher than that in the vehicle treatment group (2.6%). The effect of compound 1g2a on apoptosis of BEL-7402 cells was very similar to that on HCT116 cells (Fig. 6B). However, compound 1g2a and taxol induced almost no apoptosis in normal L-02 cells (Fig. 6C).
Fig. (6).
Apoptosis induction after treatment for 12 h with 0.1% DMSO (control), 0.1 μM taxol, 0.001 μM of 1g2a, 0.01 μM of 1g2a, 0.1 μM 1g2a, and the total apoptosis results in (A) HCT116 cells; (B) BEL-7402 cells; (C) L-02 cells.
3.2.4. Compound 1g2a Inhibited HCT116 and BEL-7402 Cells Migration
Aggressive tumors have a strong ability to proliferate and migrate, and in many cases, cell mobility affects cell proliferation [2, 20]. As shown in Fig. (7A-E), compared with the control group, compound 1g2a inhibited the migration of HCT116 and BEL-7402 cells in a concentration-dependent manner. At the same concentration of 0.1 μM, the inhibitory effect of 1g2a on the migration of HCT116 and BEL-7402 cancer cells was significantly stronger than that of taxol. However, 1g2a and taxol induced almost no migration in normal L-02 cells (Fig. 7C and F).
Fig. (7).
Transwell assay of compound 1g2a showing inhibition of HCT116 and BEL-7402 cells migrations without effecting the migration of normal L-02 cells. The images of stained (A) HCT116, (B) BEL-7402, and (C) L-02 cells adhering in the lower layer of insert of transwell with phase-contrast microscopy (200× magnification). The relative migration ratio of (D) HCT116 cells; (E) BEL-7402 cells; (F) L-02 cells. Data expressed as mean±SD (n≥3). ***p < 0.001 compared to control group; ##p < 0.01 compared to 0.1 μM taxol treatment group.
3.2.5. Compound 1g2a Inhibited HCT116 and BEL-7402 Cells Colony Formation
The abnormal proliferation of cancer cells results in malignant tumor. Colony formation assay directly shows the proliferation ability of a single cancer cell to turn into a cell colony in vitro [20, 37]. As shown in Fig. (8A-B), compared with the control group, with the increase of 1g2a incubation concentration (0.001-0.1 µM), the concentration dependence of HCT116 and BEL-7402 cells on colony formation decreased. At the same concentration of 0.1 µM, the colony-forming ability of 1g2a on HCT116 cancer cells was significantly stronger than that of taxol. However, 1g2a expressed little effect on the colony formation of normal L-02 cells (Fig. 8C).
Fig. (8).
Compound 1g2a inhibited the colony-forming ability of two cancer cells but had little effect on normal cells. (A) HCT116 cancer cells; (B) BEL-7402 cancer cells; (C) L-02 normal cells. Each part is divided into the images of stained colonies under phase-contrast microscopy and the statistical results of the number of colonies per dish. Values are represented as mean±SD (n≥3). *p < 0.05 and ***p < 0.001 compared to control group; ##p < 0.01 compared to 0.1 μM taxol treatment group.
3.2.6. Compound 1g2a Inhibited HCT116 and BEL-7402 Cells Tubulin Polymerization
Compound 1g2a and taxol, as a positive control, were tested for their ability to inhibit tubulin polymerization at 1, 0.1 and 0.01 µM concentrations using ELISA assay for β-tubulin (Table 2). Compound 1g2a (56.6%; 68.38%) showed significant ability to inhibit tubulin polymerization compared to the taxol (29.58%; 46.04%) on BEL-7402 and HCT116 cells at 1 µM concentration, respectively, and also (49.86%) better than that of taxol (20.08%) at 0.1 µM concentration on BEL-7402 cells. The above results indicated that 1g2a can selectively inhibit the colony formation of cancer cells.
Table 2.
Compound 1g2a inhibits the polymerization of β-microtubulin.
| Cpd. No. | IC50 ±SD (µM) | Tubulin % inhibition a | IC50 ±SD (µM) | Tubulin % inhibition a |
|---|---|---|---|---|
| BEL-7402 | HCT116 | |||
| 1g2a (1µM) | 0.007±0.001 | 56.6±5.46***, ## | 0.005±0.001 | 68.38±0.03***, ## |
| 1g2a (0.1µM) | 49.86±6.66***, ## | 26.11±1.02***, # | ||
| 1g2a (0.01µM) | 18.38±5.90* | 26.02±0.29*** | ||
| Taxol (1µM) | 0.09±0.02 | 29.58±4.88** | 0.03±0.01 | 46.04±4.89*** |
| Taxol (0.1µM) | 20.08±5.34** | 36.45±6.07*** | ||
| Taxol (0.01µM) | 14.96±2.65** | 19.07±6.27** | ||
| Vehicle | 0 | 0 |
Note: a tubulin inhibition of BEL-7402, HCT116 cells treated with compound 1g2a and taxol compared to control untreated cells.
***p < 0.001 compared to control group; **p < 0.01 compared to control group; *p < 0.05 compared to control group.
##p < 0.01 compared to 1 μM taxol treatment group; #p < 0.05 compared to 0.1 μM taxol treatment group.
3.2.7. Compound 1g2a Tightly Bound to Tubulin
Since compound 1g2a showed potent anticancer activity, we further investigated the binding pattern of 1g2a to tubulin (PDB code: 1SA0) [38]. CA-4 was used as a positive control. The docking results are shown in Fig. (9). Compound 1g2a interacted with several amino acid residues of the tubulin colchicine binding site to form abundant chemical bonds, including Glu183, Gly143, Gly144, Gln11, and Glu71 on the α chain of tubulin and Leu248 and Thr353 on the β chain of tubulin (Fig. 9A). In the case of CA-4, the diagram effectively displays these structural features, highlighting several binding regions on the β subunit but not with the α subunit (Fig. 9B-C). When the 1g2a and CA-4 structures were simultaneously docked to the tubulin colchicine binding site, we found that the structures of 1g2a were in the middle of the tubulin α chain and β chain. However, CA-4 showed different results (Fig. 9C), which may account for why 1g2a showed strong anticancer activity and a high selective index [19].
Fig. (9).
Molecular modeling of compounds in complex with tubulin (PDB: 1SA0). Shown is the proposed binding mode and interaction between tubulin and selected compounds. (A) 1g2a, (B) CA-4. (C) Superposition of 1g2a and CA-4 in the tubulin. The compounds are shown in a stick model with carbon atoms in green (1g2a) and pink-red (CA-4).
CONCLUSION
In this study, 83 novel small molecules with 2- phenylacrylonitrile were designed, synthesized, and biologically evaluated. Many of them displayed significantly enhanced antiproliferative efficacy against a panel of cancer cell lines. Among these molecules, compound 1g2a, with 3,4,5-OCH3 introduced on the A-ring and an -N(CH3)2 group at the p-position of the B-ring, exerted the most potent antiproliferative activity on many kinds of cancer cell line, but showed little cytotoxicity toward L-02 normal cells (i.e., it showed selective anticancer activity). The SI of 1g2a for BEL-7402, HCT116, Raji, and SU-DHL-10 cells compared with normal L-02 cells was 12,820, 16,949, 2,941, and 2,941, respectively, much higher than the values for CA-4, resveratrol, colchicine, and taxol. Compound 1g2a arrested BEL-7402 and HCT116 cells in the G2/M phase of the cell cycle and induced apoptosis in a dose-dependent manner. Mechanistic studies suggested that the blockage in the G2/M phase was associated with the downregulation of cyclin A, D1, and E1 and the upregulation of cyclin B1. Molecular modeling suggested that the selectivity of 1g2a to inhibit only cancer cell proliferation was achieved by penetrating deep into the hydrophobic pocket of tubulin and binding between the α- and β-subunits of tubulin. Compound 1g2a inhibited the migration levels of BEL-7402 and HCT116 cells to reduce the colony formation abilities of BEL-7402 and HCT116 cells in a concentration-dependent manner and significantly inhibited β-tubulin polymerization of HCT116 and BEL-7402 cells.
Furthermore, in vivo, compound 1g2a significantly suppressed tumor volume and reduced its weights by 66% (♀) and 78% (♂) at a dose of 25 mg/kg/day (iv) in an HCT116 colon cancer xenograft model in mice without affecting the mouse body weight (Fig. S3). Although taxol also inhibited the growth of tumors in the same treatment conditions, the body weight of the mice decreased significantly. Moreover, through a molecular ADMET forecast study, it was found that 1g2a is water soluble, shows good intestinal absorption, has no inhibitory effect on CYP2D6, and has good PPB possibility (Table S3).
In summary, the novel tubulin inhibitor, compound 1g2a, showed outstanding antitumor activity both in vivo and in vitro and has the potential to be developed into a promising effective anticancer agent with little toxicity to normal tissues.
ACKNOWLEDGEMENTS
We would like to thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was supported by the National Natural Science Foundation of China and the Jilin Scientific and Technological Development Program.
LIST OF ABBREVIATIONS
- DS
Discovery Studio
- CA-4
Combret Astatin A-4
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
Not applicable.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The data and supportive information are available within the article.
FUNDING
The study has been funded by the Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was supported by the National Natural Science Foundation of China (No. 81260226) and the Jilin Scientific and Technological Development Program (No. YDZJ202201ZYTS559).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s website along with the published article.
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Supplementary Materials
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Data Availability Statement
The data and supportive information are available within the article.