Novel Crtn Inhibitors With Skeleton Of Oxygenic Benzcyclohexane By Blocking Staphyloxanthin(STX) Against Mrsas:Design,Synthesis And Pharmacological Evaluation

Staphylococcus aureus(S.aureus),a type of Gram-positive bacterial,causes a series of diseases,resulting in significant morbidity and mortality and health care.The tolerance and persistence of S.aureus evolve rapidly under the antibiotic exposure,and change into the methicillin-resistant S.aureus(MRSA),known as "superbug".In recent years,more methicillin-resistant S.aureus(MRSA)strains were identified,which have captured almost all"antibio-defenses" established.To decelerate the spread of antibiotic resistance,it is extremely urgent to facilitate the next class of chemotherapeutics with novel mechanisms of action.Virulence factors are products of the pathogen and modulated by host susceptibility and resistance,which affect the host-pathogen relationship by either damaging the host or circumventing and evading the host immune system and strengthen the growth and survivability of S.aureus under stressful environment.Hence,targeting bacterial virulence factors is a rational approach to combat pathogenesis without exerting immediate life-or-death pressure on bacterium.The golden pigment of staphyloxanthin(STX)is a membrane-bound carotenoid virulence factor of S.aureus,whose structure contains numerous conjugated double bonds to resist the reactive oxygen species(ROS)produced by neutrophils and macrophages,is deemed as an important virulence factor with antioxidant properties.Therefore,it is a viable application to treat MRSA infections by the intervention of STX biosynthesis.The STX biosynthesis genes are organized in an operon crtOPQMN,and CrtN is one of the most important pathways.A previous study by our group revealed that compound 43(NTF),an FDA-approved antifungal agent,was capable of blocking the STX biosynthesis pathway of three MRSA strains by inhibiting CrtN.Based on the structure of 43,a series of new benzocycloalkane derivatives was synthesized by replacing the naphthyl of NTF,and showed remarkable pigment inhibitory activities.In particular,benzocycloalkane derivative 44 displayed excellent bioactivity in vitro and in vivo.However,low water solubility,high hERG inhibition and administration at high dosages in vivo limited further development of 44.Herein,our work focus on the design,synthesis and evaluation of a CrtN inhibitor with new scaffolds,which not only demonstrate excellent activity against MRSA in vitro and in vivo at lower dosages,but also overcome the disadvantages of water solubility and hERG inhibition.Benzocycloalkane derivative 46 exhibited more significant potency(IC50= 1.9 nM)than 44(IC50 =4.1 nM)in a previous report,but was blocked by poor water solubility(0.69 mg/mL)and strong hERG inhibition(IC50= 3.2 ?M,Table 4),which was valuable for further evaluation as a lead compound.Primarily,one general strategy of structural modification was utilized to promote the solubility in this study,due to its conciseness and efficiency.Additionally,in order to improve its water solubility and maintain its potency with fewer changes simultaneously,it is rational to insert a hydrophilic oxygen into the cycloalkane of 46.According to these reasons,we quickly synthesized new derivative 60 by replacing benzocycloalkane with a chroman segment,which exhibited excellent pigment inhibitory activity(IC50=4.6 ± 0.2 nM)and improved water solubility(13.2 mg/mL,about 20 fold increase),and the strategy of scaffold hopping is obviously responsible.Then,we proposed that anchoring core structures and introducing comprehensive modifications in other parts of the structure of 60 were the logical process to decrease hERG inhibition and maintain pigment inhibitory activity and solubility of derivatives.Initial studies involved exploration around the substitution of the terminal carbon of propylene and systemic incorporating hydrogen,cycloalkyls,heteroaryls and substituted aryls in region A in the structure of 46.38 novel chroman derivatives(47-84)were designed and synthesized for pigment inhibitory activities against S.aureus Newman.Among these compounds,it was shown that there were 9 derivatives displayed the potency at single nanomolar level.According to these results,the clear structure-activity relationship(SAR)was displayed,which aided us to screen these compounds at the first time.Derivative 67 were selected to further investigate due to its best pigment inhibitory activity(IC50 = 3.8 ± 0.1 nM),whose terminal carbon was incorporated by para-biphenyl group.According to these,we anchored the para-biphenyl group on the structure of 67 and started to change its' N-methyl group and the allyl linker.Next,10 novel derivatives(85-94)were designed and synthesized and evaluated the pigment inhibition.The results were displayed that methyl group was removed or converted into an ethyl or isopropyl group(85-87),the pigment inhibition activities abated(IC50>1000 nM).Elimination of the double bonds(89,92 and 93)led to a loss of pigment inhibitory activities(IC50>1000 nM).Subsequently,the introduction of an additional methyl moiety deprived the activities of derivatives(90,91,IC50>1000 nM).Finally,the introduction of a branched methyl on a vinyl linker resulted in a decrease in pigment inhibitory activity,while the vinyl linker was replaced with multiconjugated polyene without any substitution,related derivatives 88 and 94(pigment inhibition:IC50 = 3.0 ± 0.4 nM and IC50 = 3.3±0.3 nM,respectively)evenly displayed the most potency in the other scaffold.12 compounds(53?56?57?58?59?60?63?66?67?81?88?94)were screened to test the solubility and hERG safety profiles due to their pigment inhibitory activity(IC50<10 nM),among which derivatives 67 and 88 were selected for further investigations due to their potent pigment inhibitory activity(IC50<3.5 nM),weak hERG inhibition(IC50>10 ?M).In CrtN enzymes inhibition assay,these two derivatives displayed excellent enzymatic inhibitory activity of CrtN at submicromolar concentrations.In the assay of pigment inhibitory activities against MRSA,four MRSA strain were introduced to investigate the potency of selected compounds,including two community acquired MRSAs,USA400 MW2 and USA300 LAC,along with other two hospital-acquired MRSAs,Mu50(vancomycin-intermediate resistance,VISA/MRSA),and NRS271(linezolid-resistance,LRSA/MRSA).Pigment inhibition results of these MRSA strains are shown that either 67 or 88 showed excellent inhibition in these four MRSA strains,especially,derivative 88 presented better inhibitory activities in Mu50(IC50=0.36 ± 0.1 nM)and NRS271(IC50=0.4±0.1 nM),Consequently,we chose derivative 88 as candidate compound for further evaluation due to its potency.Meanwhile,we conducted identical HPLC experiments at 286 nm for the analysis of 4,4'-diapophytoene(the product of CrtM and the substrate of CrtN),and confirm derivative 88 targeting the enzyme CrtN.Next,four in vitro assays were built for evaluating the effect of derivative 88 incubating with MRSA under the immune clearance.The results confirmed that derivative 88 sensitized S.aureus to immune clearance in human blood without affecting the growth of S.aureus strains.Different from previous assay in vivo,in this study seven different regimens established in vivo,including MOCK,two control groups,and four different drug regimens,for comprehensively evaluating the effect of 88 in vivo,which still displayed a broad and significantly potent antibacterial spectrum against pigmented S.aureus Newman,Mu50(VISA/MRSA)and NRS271(LRSA/MRSA),especially,88 treatment with low-dosage cases(0.1 mg b.i.d.)possessed excellent inhibitory activities compared to vancomycin in the hearts and livers in NRS271,99%decrease.To further improve the solubility,salt type selection was set up,ten different acids were introduced to salify with 88 free base,Notably,phosphate 88b represented remarkable improvement of solubility(12.9 mg/mL).All results proved that both derivative 88 have the potential to be developed as good antibacterial candidates targeting virulence factors.A proof-of-concept study had demonstrated that the strategy about inserting a hydrophilic oxygen into the cycloalkane of 46 was feasible to improve the solubility.However,the continuous introduction of lipophilic groups into the scaffolds descended the solubility,while these groups helpfully improved hERG inhibitory activity.In this study,we established the strategy of the replacement the hydrophobic carbon with two hydrophilic oxygen atoms on the skeleton of lead compound 46 for improving more water solubility and extended the type of the skeleton of pigment inhibition,which owned the novel skeleton of 1,4-benzodioxan.According to the research strategies similar to chroman derivative 88,46 1,4-benzodioxan derivatives(A01-A46)were synthesized with the systematic study of structure and activity relationship,along with the investigation of pigment inhibitory activities against S.aureus Newman in vitro.According to the SAR,A05,A06,A37,A38 were picked out for further evaluation of therapeutic potency in vitro,including hERG inhibitory activity,solubility,CrtN inhibitory activities and pigment inhibitory activities against USA300 LAC,USA400 MW,Mu50 and NRS271 due to their excellent activities(IC50<4 nM).Furthermore,compound A37 with better druggability was selected as candidate to verify the antivirulence effects in vitro,including sensitizing S.aureus strains to killing by H2O2 and human whole blood,without the influence of the bacterial and fungi growth of S.aureus.In this study,through the introduction of vancomycin and linezolid as positive controls and the change of dosages(0.4 mg b.i.d.vs 0.1 mg b.i.d.)and administration(pretreatment vs normal treatment),in vivo evaluations of candidate compound A37 were further optimized and supplemented against S.aureus Newman,Mu50(VISA/MRSA)and NRS271(LRSA/MRSA),which was the same as the in vivo assay of candidate compound 88.Pharmacological results showed that:(1)most of compound-treatment groups had more beneficial effects on lowering abscess formation than Mock controls.(2)A37-treatment with low-dosage in normal treatment group displayed comparable effect with the two positive groups(linezolid and vancomycin)in heart in Mu50 and NRS271,even more effective than the two positive groups in liver in Newman.Altogether,these data demonstrate that A37 may be a good candidate to combat MRSA,VISA and LRSA infections.The innovation in our work focused on the following three aspects:(1)We established the strategy about inserting the hydrophilic oxygen into the cycloalkane of leading compound 46,which formed the novel targeting CrtN inhibition with the skeletons of either chroman or 1,4-benzodioxan against the pigment of MRSAs.(2)Overcoming the defects of benzocycloalkane inhibition and elevated the water solubility and improved the hERG inhibition of candidate compounds.(3)To optimize the model of pharmacodynamic evaluation,we introduced into multidrug MRSAs as tested bacteria and increased the linezolid and vancomycin.