Synthesis And Biological Activities Of Dehydroabietylamine Derivatives

Rosin is a mixture of various resin acids, small quantity of fatty acids and neutral substances.The main components of rosin are resin acids, which account to over90%of the total. Rosin isan important forestry resource of our country, and the output occupies the first place in the world.Deep-processing products of rosin were used not only in traditional industrial areas such aspapermaking, painting, soap, printing ink, but also in biomedicine field.Dehydroabietylamine which has three-ring phenanthrene skeleton in molecule is one ofimportant modified products of rosin, and it is also main components of disproportionated rosinamine. In this paper, a series of amide derivatives was synthesized through reaction ofdehydroabietylamine and (substituted) benzoyl chlorides. Functional groups such as carbonylgroup, nitro group, oximido group, hydroxyl group, etc. were introduced on B ring and C ringthrough oxidation, nitration, oximation and reduction. Carboxymethyl was introduced intodehydroabietylamine by modification of amino group. The novel dehydroabietylaminederivatives were characterized by IR,1H NMR,13C NMR, MS, and HRMS. Anticancer activities,androgen receptor(AR) binding activities, blocking/agitating activities to α1A-adrenoceptor,inhibitory activities on Cdc25B phosphatase, inhibitory activities against KDR and antibacterialactivities of dehydroabietylamine derivatives were tested. The structure-activity relationship werestudied and summarized. Dehydroabietylamine4-hydroxysalicylidene Schiff base complexesenwrapped with β-cyclodextrin were prepared by saturation solution method. Structure andanticancer activity of the inclusion complexes were studied. Through induction-arrangement ofdata, the conclusions were drawn as followings:(1) Dehydroabietylamine was purified by salt forming method, through reaction ofdisproportionated rosin amine and p-toluene sulphonic acid with yield of63.7%. Analogues ofN-benzoyl-dehydroabietylamine (2a²d) were synthesized through reaction ofdehydroabietylamine and (substituted) benzoyl chlorides with yield of68.4⁷2.8%.(2) Nitro group was introduced in the12th position and analogues of N-benzoyl-12-nitrodehydroabietylamine (3a³d) were obtained through nitration on ring C ofdehydroabietylamine derivatives using copper nitrate-acetic anhydride system with yield of69.6⁷4.8%. Copper nitrate-acetic anhydride system used in this experiment has characteristicsof moderate reaction and good selectivity, compared with the traditional HNO₃-H₂SO₄mixedacid system.(3) Chromic anhydride absorbed on100²00mesh macroporous silicon gel was prepared asa CrO₃/SiO₂solid-supported oxidizing reagent. Synthesis of N-benzoyl-dehydroabietylamine-7-one (4a) via selective oxidation of N-benzoyl-dehydroabietylamine (2a) by CrO₃/SiO₂solid-supported oxidizing reagent was studied. Effect of solvent species, solvent quantity,reaction temperature, loading amount and oxidant dosage on the yield was investigated. Experimental results indicated that analogues of N-benzoyl-dehydroabietylamine-7-one(4a⁴e) can be obtained with yield of53.0⁶0.2%through introduction of carbonyl group in the7th position of ring B by oxidation of2.57mmol analogues of N-benzoyl-dehydroabietylamine(2a²d,3a) in the present of2equimolar amount of CrO₃/SiO₂(loading amount2.0mmol/g) in50mL cyclohexane under the reflux temperature for8h. Compared with the traditionalCrO₃/CH3COOH liquid system, post-treatment process of products is more simple usingCrO₃/SiO₂solid-supported oxidizing reagent. It has a certain significance in environmentalprotection that these is not production of chromium waste liquid during the preparation ofCrO₃/SiO₂solid-supported oxidizing reagent and separation and purification of products.(4) Analogues of N-benzoyl-dehydroabietylamine-7-oxime (5a⁵d) were obtainedthrough oximation of carbonyl group in the7th position of ring B using hydroxylaminehydrochloride with yield of80.6⁸5.3%. N-benzoyl-dehydroabietylamine-7-ol (6a) and N-(2-chlorobenzoyl)-dehydroabietylamine-7-ol (6c) were obtained through reduction of carbonylgroup in the7th position of ring B using sodium borohydride with yield of84.3%and83.2%respectively..(5) N, N-dicarboxymethyl dehydroabietylamine (7) and N-carboxymethyldehydroabietylamine (potassium salt)(8) were obtained with yield of58.3%and53.0%respectively under alkaline conditions, using dehydroabietylamine and chloroacetic acid as rawmaterials. Regulation of pH value is the key technique in this experiment.(6) Antitumor activities of dehydroabietylamine derivatives (2a,2c,2d,3a,3c,3d,4a,4cand4d) against PC-3(human prostate carcinoma cell line) and Hey-1B (human ovariancarcinoma cell line) cells by the MTT assay were investigated. IC50values of compounds onPC-3and Hey-1B cell lines were5.7⁸4.9μg/mL and11.3¹29.8μg/mL respectively. The7thposition on ring B and the12th position on ring C of dehydroabietylamine derivatives might bevery important active sites, which have great influence on antitumor activities by structureactivity relationship study. Antitumor activities of some compounds against PC-3and Hey-1Bcell lines were enhanced through the introduction of carbonyl group in the7th position of ring Band nitro group in the12th position of ring C. For instance, IC50values on PC-3and Hey-1B ofN-benzoyl-dehydroabietylamine-7-one (4a) which has a carbonyl group in the7th position onring B are only9.1%and26.0%of its parent compound N-benzoyl-dehydroabietylamine (2a).IC50values on PC-3and Hey-1B of N-benzoyl-12-nitrodehydroabietylamine (3a) which has anitro group in the12th position on ring C are only13.1%and18.3%of its parent compound2a.(7) Cytotoxicity of some dehydroabietylamine derivatives against HepG2(humanhepatocarcinoma cell line) and L02(human hepatocyte cell line) cells were tested by the MTTassay. The results showed that, N-(4-nitrobenzoyl)-12-nitrodehydroabietylamine (3d) and N-benzoyl-dehydroabietylamine-7-one (4a) show high cytotoxicity on HepG2cells, but show lowcytotoxicity on L02. (8) Androgen receptor (AR) binding activities of dehydroabietylamine derivatives weretested by ligand-receptor competitive binding assay in vitro. It has been found that N-benzoyl-dehydroabietylamine-7-one (4a) present a certain binding activity with AR with IC50value of83.8nmol/L at an initial concentration of10μmol/L.(9) Blocking/agitating activities to α1A-adrenoceptor of compounds were tested usingCalcium mobilization assays. The results showed that N-benzoyl-dehydroabietylamine-7-oxime (5a) has25%antagonistic effect on α1A-adrenoceptor, and N-(2-chlorobenzoyl)-dehydroabietylamine-7-one (4c), N-(4-nitrobenzoyl)-dehydroabietylamine-7-one (4d) andN-benzoyl-12-nitrodehydroabietylamine-7-one (4e) have45⁵0%antagonistic effect onα1A-adrenoceptor at the concentration of10μmol/L.(10) Inhibitory activity of compounds against kinase insert domain containing receptor(KDR) was studied using homogeneous time resolved fluorescence (HTRF) technology. It hasbeen found that the tested compounds present a certain inhibitory activity against KDR with theinhibition rates of38.27⁹9.34%at the concentration of4×10-4mol/L. Among these compounds,N-benzoyl-dehydroabietylamine-7-one (4a), N-(4-nitrobenzoyl)-dehydroabietylamine-7-one (4d), dehydroabietylamine3,4-dihydroxy-salicylidene Schiff base (LIN110), N-(2-hydroxy)-benzyl-dehydroabietylamine hydrochloride (LIN402), N-(5-nitro-2-hydroxy)-benzyl-dehydroabietylamine hydrochloride (LIN404),12,14-dinitro dehydroabietylamine4-hydroxy-benzaldehyde Schiff base (LIN609) and12-nitro-dehydroabietylamine5-nitro-salicylaldehyde Schiff base (LIN804) show relatively high inhibition rates that are more than90%. The inhibition rates of N-(4-nitrobenzoyl)-dehydroabietylamine (2d), N-(4-nitrobenzoyl)-12-nitrodehydroabietylamine (3d),12,14-dinitro dehydroabietylamine-indole-3-carboxaldehyde Schiff base (LIN608) and4-hydroxy-salicylidene-12-amino-acetyl-dehydroabietylamine Schiff base (LIN702) were80⁹0%.(11) Antibacterial activities of dehydroabietylamine derivatives were studied by filter papermethod. The results showed that N-(4-chlorobenzoyl)-dehydroabietylamine-7-one (4b), N-(4-nitrobenzoyl)-dehydroabietylamine-7-one (4d) and N-(4-chlorobenzoyl)-dehydroabietylamine-7-oxime (5b) have strong inhibition effect on the growth ofStaphylococcus aureus, compound5b has strong inhibition effect on the growth of Bacillussubtilis, compound4b and5b have strong inhibition effect on the growth of Escherichia coli.(12)2D-QSAR was studied using IC50values of25dehydroabietylamine derivatives onHey-1B in order to predict the structure reformation method for high antitumor activity. Theresults showed that increase of lipid solubility, aromatic ring structure or refractive index ofcompounds were beneficial to activity enhancement of compounds against Hey-1B cells.(13) Dehydroabietylamine4-hydroxy-salicylidene Schiff base complexes enwrapped withβ-cyclodextrin were prepared by saturation solution method. The inclusion complexes wereidentified by infrared spectrum, differential scanning calorimetry (DSC) and ultraviolet spectrum.The antitumor activities of the dehydroabietylamine4-hydroxy-salicylidene Schiff base and the inclusion complexes were studied. The results showed that, An1:1molar ratiodehydroabietylamine4-hydroxy-salicylidene Schiff base complexes enwrapped withβ-cyclodextrin could be formed. The solubility of dehydroabietylamine4-hydroxy-salicylideneSchiff base in mixture solvent (DMSO/H₂O=1/1, volume ratio) was increased in3.4folds bythe formation of inclusion complex with β-cyclodextrin. The inhibitory ratio of thedehydroabietylamine4-hydroxy-salicylidene Schiff base and the inclusion complexes onHey-1B (human ovarian carcinoma cell line) were50.34%and41.65%at the concentration of10μg/mL.