Synthesis Of Coumarin Derivatives And Their Antiparasitic And Antibacterial Activities

For the past few years, aquaculture is an important economic activity in the world. With the high-density cultivation mode and the cross-regional seedling swaps in aquaculture industry, the aquatic disease caused by pathogen or parasitic infection are increasing annually,causing serious economic losses in aquaculture industry. At present, the control of aquatic disease is mainly depending on the pesticides, veterinary drugs and human medicine. As the result of long-term usage of those drugs frequently, pathogen or parasitic resistance, the drug residue and food safety problem have occurred. Due to the advantages of the safety, low toxicity, high activity and easily degradation, botanicals seem to be an ideal drug sources.However, the use of botanicals in aquaculture industry was serious limited by its low content and high processing cost. Therefore, synthesis of botanical drugs through chemical methods may solve this problem.Base on the structrure of osthole which is known as an anti-monogenean and anti-bacteria agent. The synthesis of coumarin derivatives was carried out by structrure redesigned and modified along with screening of anthelmintic and anti-bacteria activities. The result of anthelmintic activity was further evaluated with quantitative structure-activity relationship(QSAR) base on comparative molecular field analysis(CoMFA). The compounds structure was optimized with higher anthelmintic activity. The coumarin-imidazoles hybrid compounds were evaluated its effect of the key enzyme in bacterial fatty acid synthesis pathway(FAS) to study the mechanism of anti-bacteria activity. The main results are described as follows.1. 96 coumarin derivatives were synthesized by Witting reaction, Wiiliamson etherification reaction, Claisen rearrangement reaction using 2, 4-dihydroxy benzaldehyde or phenol as the initial material. All the intermediates and products were identified by EI-MS,1H-NMR and 13C-NMR and 64 compounds are first reported.2. All the synthetic compounds were tested by in vivo anthelmintic assay against Dactylogyrus intermedius and the results showed that most of the compounds exhibit various anthelmintic activities, more than 20 compounds have even better anthelmintic activities than praziquantel among the candidates, those compounds with the alkane or alkene substituted group retained their anthelmintic activity against D. intermedius with the EC50 value of 3.00 to 13.47 mg/L. The coumarin bromide showed the anthelmintic activity with the EC50 value of 2.42 to 2.85 mg/L. The coumarin-heterocycle hybrid derivatives exhibited the anthelmintic activity with the EC50 value of 0.85-9.48 mg/L. Moreover, compound A42 has remarkable anthelmintic activity than other compounds against D. intermedius infestation with EC50 value of 0.85 mg/L.Evaluation of acute toxicity of synthesized compounds showed that those compounds with the alkane or alkene substituted group retained the toxicity to goldfish with the LC50 value of 7.88 to 27.55 mg/L, the coumarin bromide and coumarin-heterocycle hybrid derivatives showed the toxicity to goldfish with the LC50 value of 5.15-12.46 mg/L and1.21-16.27 mg/L, respectively. The therapeutic index(TI=LC50/EC50) was used to evaluate the safety of the compounds and the TI value of most of coumarin derivatives were between 2and 4.3. The comparative molecular field analysis(CoMFA) calculation method was used to evaluating the quantitative structure-activity relationship(QSAR). Based on the anthelmintic activity of CoFMA results, the stereoscopic and electrostatic field coefficient were 0.634 and0.366, respectively. The result showed that the rate of contribution of stereoscopic field is much higher than electrostatic field in anthelmintic activity CoFMA model. The acute toxicity CoFMA result showed that the stereoscopic and electrostatic field coefficient were 0.621 and0.379. To aid in visualization, the most active compound is shown with the contour maps which indicate regions in 3D space around the molecules where changes in the particular physicochemical properties are able to explain the experimental binding differences.Based on the contour maps, 8 coumarin derivatives were synthesized and screened for their anthelmintic activity and the acute toxicity. Anti-parasites results confirmed that Q1, Q2,Q6 and Q7 have the significant anthelmintic activity with the EC50 value of 0.25, 0.18, 0.26 and 0.23 mg/L, respectively. The acute toxicity data showed the four compounds with the LC50 value of 0.47, 0.51, 0.65 and 0.66 mg/L. The results confirmed that the increase of anthelmintic activity or acute toxicity are accompanied with the araise the size of stereochemical structure. Those results were consistent with the CoMFA computation and can further proves the correctness of the QSAR model.4. The antimicrobial effect of 44 derivatives against aquatic pathogenic bacteria, such as S.agalactiae, Flavobacterium cloumnare, were tested by tube double dilution method. The results showed that those compounds have the various activity against S.agalactiae,F.cloumnare, some of which displayed higher activity than norfloxacin and part of which were less active than enrofloxacin. The compound A25 showed the highest inhibition effect against F.cloumnare with the MIC and MBC value of 1.00 ?g/mL and 4.00 ?g/mL.Meanwhile, Compound A25 also showed the significant antibacterial effect against S.agalactiae with the MIC and MBC value of 2.00 ?g/mL and 8.00 ?g/m L, respectively.5. Action mechanism of coumarin-imidazoles hybrid compounds against the key enzyme in bacterial fatty acid synthesis pathway were studied. The results show that the coumarin-imidazoles hybrid compounds exhibited different levels of inhibitory activity against the rate-controlling enzymes(enoyl-ACP reductase(FabI and FabK)) in the bacterial fatty acid synthesis pathway. The results showed that compounds A25 and A30 showed a higher inhibition effect with the IC50 value of 0.40 and 0.62 ?g/mL, respectively. The results of the FabI enzyme inhibition assay showed that compounds A24 and A29 exhibited a higher inhibition effect with the IC50 value of 0.39 and 0.44 ?g/mL. QSAR results shows that the inhibition effect was influenced by R1 substitute group. The compound with more than six carbon atoms of R1 has strong inhibition on FabK. The compound which has the length of R1 substitute group is exact six carbon atoms, showed an apparent inhibitory effect on FabI and FabK, simultaneously. And the compound with less than six carbon atoms of R1 has strong inhibition on FabI. Therefore, we concluded that the coumarin derivatives has bacteriostatic effect, by preventing the synthesis of unsaturated fatty acids in bacteria.In conclusion, coumarin derivatives have great anthelmintic activity against fish monogenean and noticeable bacteriostatic effect against S.agalactiae and F.cloumnare. More importantly, several compounds have great potential for being a new class of aquaculture drugs.