Synthesis,characterization and biological activities of nitrogen-containing Combretastatin A-4 derivatives

A series of new combretastatin A4 derivatives (6a-6c, 9a-12c) containing nitrogen atoms and nitrogen heterocycles were synthesized by condensation reaction, reduction reaction, hydrolysis reaction and Crutius rearrangement, and the structures were confirmed by 1H NMR, 13C NMR and Mass spectrum. The antitumor cytotoxicity of a series of newly synthesized compounds were evaluated in vitro toward the cell line of hepatoma cell with erianin as positive control. Amoung all the compound, 6b exhibit the most antitumor activity against HepG2 (Human hepatoellular carcinomas) cell line with the inhibition rate 93.85%, and the other compounds also showed moderate to potent antitumor activity. To understand the interaction of series with active binding site of receptor, docking study was performed with VEGFR-2 (Vascular Endothelial Growth Factor Receptor 2) kinase domain in complex with axitinib using Sybyl. The compound with the highest total score was 9c, reaching 8.92, which was higher than that of the positive control erianin This research highlights the therapeutic potential of novel VEGFR-2 inhibitors in treating cancers and provides a promising strategy for drug discovery. Graphical abstract Graphical abstract


Introduction
Malignant tumor is a disease that seriously endangers human health. Although it can be treated by surgery, chemotherapy and radiotherapy, the cure rate is still low. At present, numerous antitumor drugs have been studied at home and abroad, but the curative effect is not high enough and the toxic and side effects are large. It is still a research hotspot to continue to look for drugs with high efficiency and low toxicity. Due to the development of molecular biology, people have a further understanding of the molecular level mechanism of cancer occurrence and development, and found some protein molecules related to this process. Therefore, some compounds called molecular targeted antitumor drugs have been developed [1][2][3][4][5]. The standard chemotherapeutic drugs mostly take MTD (Maximum Therapeutic Dosage) as the end point of drug effect, with high toxicity [6][7][8]. Therefore, the search for high-efficiency and low toxicity antitumor drugs has always been the goal of medical scientists. Microtubules are highly dynamic components of the cytoskeleton. They are composed of tubulin heterodimers and participate in a variety of cellular functions [9]. It includes maintaining cell structure, motility, intercellular transport, cell division [10][11][12], and is responsible for the formation of mitotic spindle and the correct separation of chromosomes [13][14][15]. The biological importance and dynamic characteristics of microtubules in mitosis and cell division make them important and in-depth molecular targets of anticancer drugs. For liver cancer cells, which are rich in blood vessels, anti vascular drugs can effectively reduce the tumor load of liver cancer patients [16]. Vascular blockers in anti vascular drugs can selectively destroy the established blood vessels in the tumor to achieve anti-tumor effect [17,18].
Combretastatin A-4 (CA-4), a natural active product found in African willow in 1987 [19][20][21], can achieve its antitumor activity by inhibiting tubulin polymerization [22,23]. The anti-tumor effect of CA-4 has two mechanisms: one is to inhibit the proliferation of cancer cells and lead to apoptosis by interfering with the function of mitotic spindle [24,25]; The second is to destroy the cellular signal pathway regulated and maintained by tumor vascular endothelial cytoskeleton, resulting in the selective closure of tumor vessel [26][27][28][29]. CA-4 is a powerful anti mitotic agent with the same action site as colchicine [30,31], it's phosphate prodrug CA-4P has entered phase III clinical trial [32]. It can inhibit the polymerization and depolymerization of tubulin and the accumulation of mitotes in cells [33].
From the current series of derivatives of CA-4 and erianin, the structural modification is mostly to increase its hydrophilicity and reduce its toxic and side effects [34]. The structure-activity study showed that the benzene ring with three methoxy groups and cis-configuration was the necessary structure to maintain activity [35][36][37]. Therefore, the structural modification of CA-4 mostly focuses on B ring and the transformation of bridge carbon atom connecting two benzene rings. Due to the special physical and chemical properties of nitrogen-containing compounds, they have various biological activities such as anti-cancer and anti-bacterial activities, Moreover, nitrogen heterocyclic structure is one of the most important structural components in drugs [38]. The introduction of nitrogen can increase hydrogen bond receptors and donors and further improve the water solubility of CA-4. Therefore, we introduced nitrogen-containing groups into the CA-4 structure to improve activity and reduce toxicity.

Results and discussion
Chemistry Studies have shown that the benzene ring with three methoxy groups is a necessary structure to maintain activity [39]. Therefore, the structural transformation of CA-4 mostly focuses on the transformation of B ring and bridge ring connecting two benzene rings. Moreover, cis-structure is usually necessary for biological activity. Therefore, we fixed its cis conformation by introducing a cyclic pyrazolone structure on the bridge carbon, so that it showed good biological activity in anti-tumor. In addition, compounds containing nitrogen heterocycles often have excellent biological activities because they contain active hydrogen atoms and can form hydrogen bonds with action targets.
Series I includes 4 compounds 6a-6d, which were synthesized according to Scheme 1. Acrylic acid (3) was prepared from substituted benzaldehyde (2) and substituted phenylacetic acid (1) by Perkin reaction in the presence of acetic anhydride and triethylamine. The intermediate (3) was esterified by using ethanol as solvent and p-toluenesulfonic acid for activation to prepare acrylate (4). Except that the compound (5) needed to be coupled with 3-Chloro-1-(N,N-dimethyl)propylamine and then further condensed with hydrazine hydrate, other compounds are formed by the direct reaction of intermediate (4) with hydrazine hydrate in n-butanol solution. Using benzaldehyde and phenylacetic acid substituted by different functional groups as raw materials, four different Combretastatin A-4 analogues containing pyrazolone heterocycles were synthesized.
Series II includes 8 compounds 9a-12c. Target molecules (9a-12c) were synthesized as shown in Scheme 2. The initial raw material was the same as that in Scheme 1. Firstly, benzaldehyde (2) and phenylacetic acid (1) were used to generate acrylic acid intermediate (3), then the double bond of intermediate (3) was reduced by hydrogen in dichloromethane solution. In the third step, intermediate (7) reacted with diphenylphosphoryl azide to produce boc protected amino intermediate (8) through Crutius rearrangement in tertbutanol solution. The boc group of Intermediate (8) was removed with trifluoroacetic acid to obtain target amino compound (9). In formaldehyde and formic acid solution, compound 9 was heated and refluxed to obtain target compound 10 containing tetrahydroisoquinoline structure. Compound 10 was reduced by palladium carbon in ethanol solution to obtain ring opening product 11. In addition, the

Antitumor activities
Most of the compounds in series I showed good anti proliferative activity of hepatoma cell HepG2, while the anti proliferative activity of hepatoma cell in series II was poor, which was lower than that of the positive control erianin. For the compounds in series I (Table 1), the activity of compounds containing hydroxyl groups in the B ring is much higher than that of compounds without hydroxyl groups. However, when the para hydroxyl groups in the benzene ring are replaced by N, N-dimethylpropylamine, their activity is greatly reduced. Furthermore, when comparing compound 6a, 6b and compound 6c, 6d, we found that the position of the carbanyl group substituent also has a great impact on the activity. When the carbonyl group is close to the benzene ring containing three methoxyl groups (6a, 6b), its activity is higher than that near the other benzene ring (6c, 6d).
For compounds in series II (Table 2), their antiproliferative activity on hepatoma cells was much lower than that in series I and positive control, This may be because some compounds in series II do not form a ring structure and can not fix the cis conformation of two benzene rings on the same side. As for the activity of hydroxyl substituted compounds, we got similar results as series I, the activity of compounds with hydroxyl substitution is higher than that of compounds without hydroxyl substitution. However, the effect of the position of substituent on the activity is opposite to that of series I, When methylamine substituents are on the side of the B ring (11、12c), they usually show higher biological activity than that on the side of the A ring (12a、12b), Amino substituted compounds also show the same rule as methylamine substituted compounds.

Molecular docking study
The ability of CA-4 to destroy and inhibit tumor blood vessels is related to its inhibition of vascular endothelial cell receptors. CA-4 binds to VEGFR-2 to activate downstream signaling pathways that inhibit tumor cell growth and migration by affecting angiogenesis, vascular development, and vascular permeability. For a further study of the compounds which interact within the active site of VEGFR-2, molecular docking simulation was performed to explore the binding affinity of the receptor protein and ligand.
In the present study, H-bonding and free-binding energy say, total score were considered for the analysis. Compounds 6a-6d and 9a-12c which were docked into the active binding cavity of VEGFR-2 (PDB code: 4AG8) and the results were summarized in Table 3. Among them, the compound with the highest score is 9c, which is lower than the original ligand axitinib but higher than the positive control erianin. As Fig. 1 showed, both original ligand and compound 9c exhibited similar interactions with VAL 848, VAL 916, LEU 1035 and ASP 1046. This also explains the 9c score is relatively high. As shown in Fig. 2, hydroxyl forms hydrogen bond with ASP 1046, which play an important role in the interaction between 9c and the VEGFR-2.

Molecular Dynamics simulations
In order to understand the activity of compound 9c inside VEGFR-2, the docked solution of the VEGFR-2-compound 9c complexwas subjected to molecular dynamics with Gromacs to access the stability of the compound predicted binding mode inside protein. The root mean square devia-  tion (RMSD) from the initial conformation is a central criterion used to evaluate the transformation of the protein system. The RMSD value for the system versus the simulation time was illustrated in Fig. 3. As the result shows, VEGFR-2 complexed with the compound reached the stable state after 10 ns, which was around 0.4 nm. It was revealed that the binding of protein to ligand is relatively stable and compound 9c did not restrict the transformation of the binding site of VEGFR-2.

ADME properties
In the R&D process for new drugs, ADME played significant roles in drug-likeness. ADME prediction was performed on all compounds. As showen in Table 4. The results showed that the Log P values of the compound 11 was 3.26, indicating that it has a reasonable absorbency.
Meanwhile, all compounds were considered to have a good solubility in the body.

Conclusion
In this study, a series of novel CA-4 derivatives containing nitrogen heterocycles and nitrogen atoms were designed and synthesized, ADME prediction was performed, and the results showed that all compounds had good bioavailability.
The antiproliferative activities of all compounds against hepatoma HepG2 cells in vitro were determined by CCK-8 assay. Most compounds show moderate to potent activity, the activity of compound 6b was higher than that of the positive control with inhibition rate 93.85%. In addition, in order to study the interaction between the compound and the target, we also carried out molecular docking study, the docking score of compound 9c was 8.92, higher than that of positive control erianin. In order to explore the abnormal activity of compound 9c, Molecular dynamics simulation of the docking pose of compound 9c and VEGFR-2 complex was also carried out, and the results showed that the binding between ligand and protein was relatively stable. Although the docking results are not absolutely consistent with the in vitro activity test results, it provides a basis for us to find new antitumor drugs.
To as solution of 3,4,5-trimethoxyphenylacetic acid (44.2 mmol) and p-methoxybenzaldehyde (44.2 mmol) in 15 mL acetic anhydride (44.2 mmol) was added 15 mL triethylamine, the mixture was refluxed tat 140°C for 6 h, after the complete reaction, the reaction solution was cooled to room temperature, poured into ice water bath, adjusted the pH to acid with hydrochloric acid, and stirred for 2 h, the precipitate yellow solid was filtered. The solid was redissolve in 10% sodium hydroxide solution, washed with ethyl acetate, separated the organic layer and retain the aqueous phase. Adjust the pH of aqueous phase to 3~4 with 2 mL dilute hydrochloric acid. The solid was precipitated, filtered and recrystallized with ethyl acetate to obtain compound (3a) as yellow solid, yield: 78%. 1  To a solution of 3-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl) acrylic acid (14.52 mmol) in dry ethanol (60 mL) was added p-toluenesulfonic acid (2.90 mmol), the reaction mixture was stired at 90°C for 12 h, then cooled to room temperature, extracted with ethyl acetate, dried with anhydrous sodium sulfate. Filterd and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (dichloromethane: methanol = 20:1) to obtain white solid with a yield of 62%. 1   Ethyl (E)-2-(4-(3-(dimethylamino)propoxy)phenyl)-3-(3,4,5-trimethoxyphenyl) acrylate. (5) A solution of compound 4 (11.16 mmol) in acetone (40 mL) was added K 2 CO 3 (44.64 mmol), the reaction mixture was stirred at room temperature for 0.5 h, then N, N-dimethylchlorpropylamine (1.49 g, 11.28 mmol) was added, and the reaction was refluxed for 5 h. The completion of the reaction was monitored by TLC. Then it was concentrated, 50 mL of water was poured into the reaction solution and the pH was adjusted to 4-5 by acetic acid. The mixture was extracted with EA (40 mL × 3), and the organic phases were combined and dried with anhydrous Na 2 SO 4 . After filtering to remove Na 2 SO 4 , the filtrate was evaporated to yield a solid product, which was recrystallized with EtOH to obtain white solid, yield: 80%. 1    To the suspension of 7a (8.66 mmol) in anhydrous tert butyl alcohol (60 mL), triethylamine (9.53 mmol) and DPPA (9.53 mmol) was added to result in a solution, it was stirred at 85°C for 10 h, the reaction was monitored by TLC. The solution was then cooled to room temperature, saturated sodium carbonate solution was added to quench the reaction, Add ethyl acetate for extraction, evaporate the solvent under reduced pressure, and perform column chromatography on silica gel (petroleum ether: ethyl acetate = 2:1) to obtain white solid, yield: 74%. 1  To a solution of 8a (1.2 mmol) in DCM (5 mL), was added TFA (4.79 mmol) slowly at ambient temperature, stir it at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1) after completion of reaction, 15% aqueous solution of NaOH was used to alkaline the mixture to pH 7-8, the ethyl acetate was added, the organic layer was separated, and aqueous layer was extracted twice with ethyl acetate. The combined organic extracts were dried with Na 2 SO4, filtered, and evaporated. The residue was purified by column chromatography on silica (dichloromethane: methanol = 12:1), to obtain light yellow oil, yield: 80%. 1 H NMR (501 MHz, Chloroform-d) δ 7.09 (d, J = 7.0 Hz, 2H), 6.83 (d, J = 6.9 Hz, 2H), 6.57 (s, 2H), The reaction flask was evacuated and flushed with N 2 prior to addition of 10% Pd/C (40 mg). Tetrahydrofuran (5 mL) was added to the flask followed by a solution of substrate (0.58 mmol). The reaction flask was evacuated and backfilled with N 2 (×2) prior to evacuation and and stirred in a hydrogen atmosphere. After stirring overnight, the reaction mixture filtered through a bed of Celite. The Celite was washed with ethyl acetate, and the solvent removed under reduced pressure. The resulting residue was purified by column chromatography (Dichloromethane: methanol = 12:1) to afford the desired compound 11 as colorless oil, yield: 69%. 1  Antitumor activities (CCK-8 assay) Using erianin as positive control, the antitumor activities of 12 compounds in HepG2 cells were evaluated. The minimum inhibitory concentration (MIC) is expressed in mmol/ ml. Logarithmic growth cells were taken for experiment. The cells were digested, counted, made into cell suspension and inoculated in 96 well plate (100 μL/well), incubated in a 5% CO 2 incubator at 37°C for 24 h; Medium containing 10 μM of test substance was added to each well, and negative control group and blank group were set up at the same time; The plates were placed in an incubator for 72 h, and the cell morphology of each group was observed under a microscope. 10 μL CCK8 solution was added to each well, and the cells were incubated in the incubator for another 4 h. The absorbance value was measured at 450 nm, and the proliferation inhibition rate was calculated.

Molecular docking
Molecular docking studies were performed to assess the interactions of hits with VEGFR-2 protein. Surflex Dock within SYBYL-X 2.0 program package was used to perform the molecular docking procedure. The crystal structure of tubulin was obtained from the protein data bank (PDB ID: 4AG8). The protein crystal structure was hydrogenated and water molecules were removed, and Amber FF99 atomic charges was assigned to the protein atoms. All compounds were also treated with energy minimization. Then, the treated protein was docked with small molecules. Discovery Studio (version 3.5) was used to observe 2D diagram of the compounds interacting with the surrounding amino acid residues.

Molecular dynamics simulations
GROMACS software package was used to simulate the protein. The charmm36 force field was used for protein and small lig. Select TIP3P dominant water model and set periodic boundary conditions. The workflow of molecular dynamics simulation includes four steps: energy minimization, NVT equilibrium, NPT equilibrium and production dynamics simulation. Firstly, the systems were constrained to minimize the energy of water molecules by 5000 steps steepest descent; Then, maintaining the constraints, 50,000 step NVT ensemble simulation was carried out for the whole system. The temperature was 298 K, and the time step was 2 fs; Then 50,000 step NPT ensemble simulation was carried out for the whole system, the temperature was 298k, and the time step was 2 fs; Finally, the molecular dynamics simulation of the system was carried out in the NPT ensemble for 200 ns. with a time step of 2 fs. The relevant parameters were analyzed by the module of GROMACS software package.

ADME prediction
Although there are millions of active compounds, few of them can be used as medicine. In addition to technical problems, insufficient efficacy and safety may be the main reason, which is mainly related to absorption, distribution, metabolism and excretion (ADME). The oral bioavailability of a chemical compound can be predicted by the calculation of certain physicochemical properties. The physicochemical properties of the selected compounds including molecular weight (MW), hydrogen bond receptor (HBA), hydrogen bond donor (HBD), number of rotatable bonds, log of the octanol/water partition coefficient (logP), log of the aqueous solubility (logs), topological polar surface area (TPSA) was calculated using ADMETlab 2.0 website (https://admetmesh.scbdd.com/).