Design,Synthesis,and Action Mechanism Of Anti-Plant Viral Molecules Derived From The Natural Product Phrymarolin Ⅱ

Natural products are a treasure for the discovery and development of antiviral agents.Furofuran lignans,a common type of natural products,are widely distributed in nature and exhibit diverse biological activities.However,due to their limited natural resources,complex structure,research and development of their agricultural biological activities have been restricted.Natural product phrymarolin Ⅱ,a kind of furofuran lignan isolated from the plant Phryma leptostachya L.,exhibits potential agricultural activities.In this study,118target compounds（series I-IV）were synthesized based on the natural product phrymarolin Ⅱ as the lead compound.We first investigated the concise total synthesis method and found their anti-TMV activities of phrymarolin Ⅱ and its derivatives（series I）.Then,a series of deacetate phrymarolin Ⅱ and its derivatives with anti-TMV activities（series Ⅱ）were designed and synthesized by the strategy of structure simplification with the lead compound phrymarolin Ⅱ;Two series of arylγ-butyrolactone compounds with anti-TMV activities（series ⅡI and IV）were designed and synthesized by the strategy of structural diversification based on the key synthetic intermediates of series Ⅱ;The antiviral mechanism of the highly active compounds in series Ⅱ,ⅡI,and IV was explored.The main research results are as follows:（1）Using sesamin as a crude material,14 natural products including（±）-phrymarolin Ⅱ and its derivatives（series I）were synthesized in 9 steps.The key reactions included Zn-mediated Barbier type allylation reaction and a Cu-catalyzed O-arylation Chan-Lam-Evans cross-coupling reaction.The structure of（±）-phrymarolin Ⅱ and its derivatives were confirmed by ¹H NMR,¹³C NMR and HRMS.The bioactivity test showed that many of the target compounds exhibited good anti-TMV activity.（±）-Phrymarolin Ⅱ（I-1）exhibited good inactivation,protection and curative activities against TMV at 500μg/m L（77.4%,59.2%and 49.3%）,which was comparable to the positive control ningnanmycin（75.5%,59.4%and 42.1%）.Therefore,the natural product phrymarolin Ⅱ can be further studied as an antiviral candidate compound or lead compound.（2）In order to simplify the synthesis,we designed and synthesized 37 deacetated phrymarolin Ⅱ derivatives（series Ⅱ）usingβ-vinyl-γ-butylactone and aromatic aldehydes as crude materials through structural diversification modification in 4-5 steps.The results of the anti-TMV activity assay showed that many of the target compounds had good antiviral activity against TMV.The inactivation activity of deacetate phrymarolin Ⅱ（I-a1）(EC₅₀=175.5μg/m L)was slightly better than that of the lead compound phrymarolin Ⅱ(EC₅₀=191.2μg/m L).These results indicated that the acetyl-ester group（-OAc）at the C-1 position of the 3,7-dioxabicyclo[3.3.0]octane had little effect against TMV.Compound Ⅱ-a24showed the best anti-TMV activity,and its inactivation,protection and curative activities were 89.3%,54.0%and 46.7%,respectively,at 500μg/m L,which were better than those of the positive controls,ningnanmycin and ribavirin.At the same time,the preliminary antiviral mechanism indicated that compound Ⅱ-a24 may destroy the integrity of virus particles.Compound Ⅱ-a24 could bind to TMV coat protein（K_d=1.721μM）which indicated that the target of compound Ⅱ-a24 may be TMV-CP.（3）It was found that the key intermediate of series Ⅱ,γ-butyrolactone containing benzyl alcohol moiety（3-3a and 3-3b）,had good anti-TMV activity（73.7%and 74.4%）.We further used this compound as the lead compound to design and synthesize 68 arylγ-butyrolactone compounds（series ⅡI and IV）through the structural diversification modification strategy.The results of anti-TMV activity showed that many of the target compounds had good antiviral activity against TMV.Among them,compound ⅡI-a7showed the best anti-TMV activity at a concentration of 500μg/m L,and its anti-TMV inactivation,protection and curative activities were 87.8%,71.7%and 67.7%,respectively,which were better than the positive controls ribavirin and ningnanmycin.In addition,the two diastereomers of compound ⅡI-a7 showed different anti-TMV activity,the syn-isomer（syn-ⅡI-a7）(EC₅₀=120.7μg/m L)was comparable to ⅡI-a7(EC₅₀=128.7μg/m L)and was superior to the anti-isomer（anti-ⅡI-a7）(EC₅₀=152.4μg/m L).The preliminary antiviral mechanism showed that compound syn-ⅡI-a7 had an inhibitory effect on TMV-GFP in tobacco.q RT-PCR results showed that the expression of TMV-GFP in tobacco leaves was significantly decreased under the treatment of compound syn-ⅡI-a7.At concentrations of 100μM and 200μM,the expression levels were 0.67 and 0.23 times that of the control group,respectively.The pigment content（chlorophyll a,b,a+b and carotenoids）in tobacco leaves increased with the increase of the concentration of compound syn-ⅡI-a7,which proved that the compound had an inhibitory effect on the replication of TMV and could reduce the damage of TMV to tobacco leaves.Compound syn-ⅡI-a7 may destroy the integrity of virus particles.Further ITC titration experiments showed that syn-ⅡI-a7 showed a strong binding ability to TMV-CP（K_d=1.426μM）,which was better than that of ribavirin（K_d=1.699μM）.Therefore,we hypothesized that compound syn-ⅡI-a7 may act on TMV CP,thereby inhibiting TMV infection of plants.In conclusion,we have developed a highly efficient total synthesis route for the furofuran lignan phrymarolin Ⅱ and its derivatives（series I）.The deacetate phrymarolin Ⅱ derivatives（series Ⅱ）and arylγ-butylactone compounds（series ⅡI and IV）were designed and synthesized by structural simplification and structural diversification,and a number of anti-TMV compounds were found.The mechanism of their action of selected compound Ⅱ-a24 and syn-ⅡI-a7 was preliminarily investigated.The target of these two compounds maybe all acted on TMV-CP.These findings of this study offer potential or leading compounds for the development of novel anti-TMV agents,and establish a robust theoretical basis for the creation of new anti-plant virus agents,which holds significant implications in ensuring crop safety.