Design And Synthesis Of A-naphthoflavone Chimera Derivatives Able To Eliminate CYP1B1-mediated Drug Resistance Via Targeted CYP1B1 Degradation

ObjectiveAs a disease with high morbidity and mortality worldwide,tumors are a serious threat to human health and become one of the important social problems faced by countries all over the world.A large number of studies have found that the occurrence and development of tumors are closely related to the overexpression of some oncoproteins.The development of small molecule inhibitors for these oncoproteins has made great progress in the targeted therapy of tumors.Extrahepatic cytochrome P450 1B1(CYP1B1)is a drug metabolizing enzyme that is overexpressed in tumor cells.Its metabolism accelerates the inactivation of various anticancer drugs such as paclitaxel,docetaxel and doxorubicin.CYP1B1 has now become a potential target for reversing drug resistance and cancer treatment.As we all know,CYP1B1 protein inhibits anti-cancer drugs by maximizing the use of drug receptors,but it often causes adverse reactions due to excessive drug concentration.On the contrary,the proteolytic targeting chimera(PROTACs)as an emerging technology has a significant effect on the degradation of the protein of interest(POI),and the dosage is low.PROTACs is bifunctional molecules containing a POI ligand,an E3 ligase ligand and a linke bridging POI and E3 ligase ligands,as a result,POI is ubiquitinated and then degraded by the ubiquitin-proteasome system.PROTACs molecules do not need to be combined with POI for a long time,allowing use of low doses.This degradation process is similar to a catalytic reaction.PROTACs can be recycled and reused to degrade POI,not requiring for the equivalent amount of PROTAC.Therefore,the establishment of effective PROTACs molecules is practicable to achieve the degradation effect of CYP1B1,In this study,we make the first attempt to degrade CYP1B1 through a PROTACs technology and overcome CYP1B1-mediated drug resistance,which provides a new direction for eliminating drug resistance caused by CYP1B1 expression.MethodsIn this study,a series of PROTACs were synthesized by using α-Naphthoflavone(α-ANF)derivatives as CYP1B1 ligands and linker-coupled thalidomide derivatives as E3 ligase ligands.Firstly,2 was synthesized from 1,5-dihydroxynaphthalene(1)by a sevenstep reaction,including selective methoxylation,selective aldehylation,Grignard reaction,oxidation and selective demethylation.After benzoylation,rearrangement,cyclization or Claisen Schmidt condensation,basic cyclization,oxidation and esterification,2 was used as starting materials to obtain alkynyl substituted α-ANF derivatives 5,8c,13.Thalidomide substituted by carbon chain with different length and azide was coupled with 8c containing alkynyl group by a copper-catalyzed click reaction to form four PROTACs 21a-d with a respective two-carbon chain,a five-carbon chain,a sixcarbon chain and an eight-carbon chain.Adopting a copper-catalyzed or ruthenium-catalyzed click reaction,thalidomide substituted by carbon chain or water-soluble polyethylene glycol chain with the best carbon chain length and azide reacted with 5,8c,13 containing alkynyl group,generating 12 PROTACs 21c-26c,30o-35o,respectively.Then EROD assay in vitro was used to determine the inhibitory activity and selectivity(IC50)of PROTACs against CYP1B1 and CYP1A2;Western blotting(WB)was used to test the degradation activity of PROTACs on CYP1B1;MTT cytotoxicity assay was used to determine the reversal of drug resistance(IC50 value)of cancer cells to docetaxel(IC50);Finally,the water solubility of PROTACs was determined using a HPLC method.Collectively,biological evaluation,and molecular docking simulation were used to explore the effects of the connection sites of ligands,length,properties of the connection chain and a coupling mode on the CYP1B1 degradation and result reversal resistance of PROTACs,and the PROCTs with the best ability to reverse CYP1B1 resistance and degrade CYP1B1 was analyzed.ResultsFifteen new α-ANF based PROTACs 21a-21d,22c-26c and 30o-35o,were synthesized in 14.0%,13.9%,13.7%,14.0%,25.5%,7.5%,7.2%,12.5%,12.5%,3.3%,13.4%,25.2%,7.6%,5.9%,11.6%,3.2%overall yields,respectively.EROD analysis showed that PROTACs 21c and 25c had the best inhibitory activity towards CYP1B1(IC50 were 72.4±2.4μM and 35.7±0.7μM,respectively);MTT cytotoxicity assay in vitro showed that all PROTACs could eliminate docetaxel resistance of DU145/CY cells to varying degrees,and PROTACs 21c,22c,24c and 25c had the best ability to reverse drug resistance of DU 145/CY cells towards docetaxel,with IC50 values of 5.54 μM,3.28 μM,3.20 μM and 4.50μM,respectively.The IC50 of PROTACs is comparable to the parental DU145 cells(IC50=3.0μM).The reversal ability of water-soluble polyethylene glycol chain coupled PROTACs was lower than that of PROTACs with the corresponding carbon chains.WB showed that all PROTACs had different degrees of degradation ability to CYP1B1.Among them,the PROTAC substituted at the position 3 of the C ring has weak degradability.The degradation ability of water-soluble polyethylene glycol chain PROTACs to CYP1B1 was weaker than that of the corresponding carbon-chain coupled PROTACs.The ability of 25c to CYP1B1 in reversing drug resistance of DU 145/CY cells towards docetaxel was the strongest,followed by 21c,and CYP1B1 degradation appeared in a concentration-and time-dependent manner.Water solubility testes showed that 23c,25c and 21c of PROTACs had the poor water solubility,which were 2.5533 μg/ml,1.9159μg/ml and 1.5428 μg/ml,respectively.The water solubility of PROTACs was greatly improved(more than 89 times)by replacing carbon chain with water-soluble polyethylene glycol chain.ConclusionReversal of CYP1B1 mediated resistance is due to the fact that PROTACs effect CYP1B1 degradation rather than inhibition.All the ANF-based conjugates have the ability of CYP1B1 degradation and thus reverse drug resistance toward doxetaxel with a varying degree.All the modifications of α-ANF via introducing linker-coupled thalidomide derivatives can reduce its inhibitory activity to CYP1B1.C-3’ on the B ring of α-ANF is the most effective connection site;inserting oxygen atom into a carbon chain can greatly improve the water solubility of PROTACs molecules,but simply adding oxygen atom into the corresponding carbon chain will obviously change the structure of PROTACs,thus affecting the potency of PROTACs in CYP1B1 degradation and resultant drug resitance reversal.