Structure-Based Design,Synthesis And Activity Evaluation Of Novel Sulfamoybenzamide-Typed HBV Capsid Assembly Modulators

Hepatitis B virus is a kind of hepatotropic double-stranded DNA virus,which is mainly transmitted through mother-to-child,blood,sexual contact and iatrogenic pathways.According to the statistics of World Health Organization,there are about 296 million hepatitis patients worldwide and 820,000 deaths annually from HBV infection.China is one of the countries with high incidence of hepatitis B,with about 30 million hepatitis patients.The available drugs for the treatment of HBV mainly include interferon and nucleos(t)ide analogues.However,interferon can cause bone marrow suppression during the treatment,while the virus can easily develop resistance to nucleos(t)ide analogues and relapse after drug withdrawal.Neither of two categories can cure hepatitis B completely.Therefore,it is imminent to develop HBV therapeutic drugs with improved potency and low toxicity.The development of small molecule inhibitors aiming at critical targets is a hot topic in drug research and development.The capsid protein plays important role in multiple stages of HBV replication,including the encapsidation and reverse transcription of pgRNA,the synthesis of rcDNA and the formation of virus particles.Noteworthily,the capsid protein is also associated with the genetic modification of cccDNA.The concealment of cccDNA makes it persistent and stable throughout the replication cycle and difficult to eradicate hepatitis B.Long-term treatment of NAs can prevent viral rebirth to some extent,but cccDNA can reemerge after cessation of the treatment.The capsid protein is involved in the protein modification of cccDNA and drugs that affect capsid assembly can exhaust free capsid protein,influence the structure and function of cccDNA,and hinder the reverse transcription.Wherefore,the development of capsid assembly modulators is expected to clear cccDNA and cure hepatitis B.At present,dozens of different structural HBV capsid assembly modulators are in preclinical and clinical studies,such as heteroaryldihydropyrimidines,sulfamoybenzamides,phenylpropenamides and pyridazones.According to the mechanisms,different structures of capsid assembly modulators are divided into two categories.The class Ⅰ capsid assembly modulators,represented by heteroaryldihydropyrimidines,inhibit the proliferation of virus by promoting the aggregation of core proteins and forming abnormal non-capsid structures.The class Ⅱcapsid assembly modulators,represented by sulfamoybenzamides,interfere with the encapsidation of pgRNA to form empty capsids without viral genome.The sulfamoybenzamide derivative NVR3-778 is a capsid assembly modulator in clinical phase Ⅱ stage.However,its antiviral activity is still at the micromolal level(HepG2.2.15:EC50=0.40 μM,CC50=14.5 μM)and the poor water solubility(pH=2.0,0.003 mg/mL)restricts its clinical application.In this thesis,we took NVR3-778 as the lead compound for drug design,hoping to obtain compounds with better activity and improved water solubility.The crystal structure of the capsid in complex with NVR3-778 shows that the compound is accommodated in the dimer-dimer interface.In addition to NVR3-778,the binding pocket can accommodate small molecules of multiple structures.Heteroaryldihydropyrimidine HAP_R01(HepG2.2.15:EC 50=6.4 nM)is advantageous by a 6-position carboxyl group,which forms strong bidentate interaction with the backbone nitrogen and the sidechain oxygen of Ser141 in the solvent-exposed region.The overlay of HAP_R01 and NVR3-778 binding pockets shows that the dihydropyrimidine 6-position side chain of the HAP_R01 is also solvent accessible.With the thorough study on the structure and function of the capsid,researchers found that there are hot spot residues(Asn136,Ala137 and Ile139)associated with capsid disassembly in the solvent-exposed region.Furthermore,Asn136 contains hydrogen bond donors and acceptors on the side chain.Based on the above,we can design drugs by molecular hybridization strategy.In the second chapter of this thesis,we designed and synthesized 22 carboxylcontaining compounds by using molecular hybridization strategy,hoping to improve the antiviral activity and water solubility.The activity test showed that only compound I-5a-7 had an inhibitory effect on HBV DNA(HepDES19:EC50=2.80 ±0.24 μM,CC50>100 μM).The result of water solubility showed that the water solubility of I-5a-7 was better than that of NVR3-778 under three pH conditions.After activity analysis,we speculated that the introduction of carboxyl group made the sulfonamide side chain incompatible with the surrounding amino acid environment in the solvent-exposed region,leading to the loss of activity.Based on the phenomenon that compounds containing carboxyl acid possessed lower antiviral activity,we replaced carboxylic acid with boronic acid which could be considered as a bioisostere of carboxylic acid and synthesized 27 boronic acid-bearing sulfamoybenzamide derivatives in the third chapter.These compounds were evaluated for their anti-HBV activity and cytotoxicity against HepDES19 cell line.Compound I-4h(EC50=0.83±0.33 μM,CC50=19.40 ± 5.03 μM)displayed roughly comparable anti-HBV activity to NVR3-778(EC50=0.73 ± 0.20 μM,CC50=23.40 ±6.97 μM).Four bioactive compounds(I-4i,I-4k,I-4h and I-41)were selected to be reevaluated for their anti-HBV activity and cytotoxicity against HepAD38 cell line,the result showed that all four compounds exhibited low micromolar inhibitory activity against HBV DNA(EC50:0.47～1.14 μM),consistent with the activity against HepDES 19 cells,which confirmed that compounds containing boronic acid showed stable cellular activity.We verified the mechanism of I-4h by size exclusion chromatography and quantitative experiments on capsids and encapsidated pgRNA.The results showed that I-4h belongs to class II capsid assembly modulators.The molecular dynamic simulation of I-4h indicated that I-4h formed hydrogen bonds with Leu140 and hot spot residue Asn136 in the solvent-exposed region,which verified the design.The result of water solubility test showed that I-4h was better than NVR3-778 under three pH conditions.The in vitro stability test of I-4h was carried out,and it was found that I-4h was very stable in human plasma at 37℃(T1/2=170.8 min),but metabolized rapidly in human liver microsomes(T1/2=8.6 min).Subsequently,the pharmacokinetic study of I-4h was performed in SD rats,and the result showed that the clearance of oral administration is much greater than that of intravenous administration,and the bioavailability was only 2.85%.We speculated that I-4h may have a noticeable liver extraction rate and first-pass effect.In this thesis,we took NVR3-778 as the lead compound,designed and synthesized novel sulfamoybenzamide HBV capsid assembly modulators containing carboxyl acid by using structure-based rational design and molecular hybridization strategy.And we synthesized a series of compounds containing boronic acid in order to improve the antiviral activity by using strategy of bioisosteric replacement.Among them,I-4h was worthy to further study because of the outstanding activity and improved water solubility compared with NVR3-778.