Development Of Novel Pyrimidinylthiourea Multifunctional Inhibitors Against AD,and Disulfide FBPase Covalent Inhibitors For The Treatment Of Type ? Diabetes Mellitus

This dissertation involves the discovery and development of new candidate drugs for two chronic degenerative diseases,as follows:Part 1 presents the development of novel pyrimidinylthiourea multifunctional inhibitors against AD.Alzheimer's disease(AD)is a complex neurodegenerative disorder,caused by multiple factors and involving multiple molecular pathogenesis.Patients with AD are often featured by memory loss,cognitive impairments,and impaired thinking.Brain atrophy,senile plaques(SP)formed by A? aggregation,and neurofibrillary tangles(NFT)formed by highly phosphorylated tau protein are the main pathological features of AD.Although the pathological characteristics of AD are relatively obvious,the complicated and changeable molecular pathogenesis results in traditional single-target anti-AD drugs failing to fully respond to the current new situation of clinical AD treatment.At present,the main pathogenesis of AD involves impaired cholinergic function,abnormal homeostasis of biometals,and oxidative stress.Thus,development of multifunctional(multi-target)drug by rational design strategy is an effective and comprehensive therapy for AD,as well as the hotspots in the field of AD therapeutic drugs development.However,the existing multi-target drug design strategies are mainly based on the reasonable assembly of different anti-AD pharmacophores which relies excessively on the structure of limited known bioactive compounds,resulting in little breakthroughs in terms of efficacy or mechanism.Therefore,discovering efficacy fragment with new skeleton plays a key role in the development of multifunctional anti-AD drugs.Our group have been committed to developing the multitarget-directed ligands with new skeleton against AD,and in previous work,we designed and synthesized a series of nonfused pyrimidinylthiourea derivatives as multifunctional agents against AD,including AChE inhibitory activity,specific metal-chelating ability,significant antioxidant effects,using "fragment-based drug design" strategy.In particular,compound 5r exhibited displayed excellent anti-AD bioactivity in vitro and in vivo.However,administration at high dosages in vivo(200 mg/kg)limited further development of 5r.Notablely,the multifunctional anti-AD activity of these derivatives resulted from the pyrimidinethiourea core,a new fragment which has not been reported by others,thus,it could be identified as the potential anti-AD pharmacophore.But the single-structured pyrimidinethiourea pharmacophore generated insufficient efficacy and excessive doses in vivo.In view of the good extensibility of pyrimidinethiourea structure,moderate size of the skeletal space,and clear structure-activity relationship(SAR),it is very suitable for the development of multifunctional agents.Therefore,this part aims to introduce another anti-AD pharmacophore into the pyrimidinethiourea structure by rational multitarget-directed ligands strategy,and greatly improve efficacy in vitro and in vivo of these pyrimidinethiourea derivatives while reducing the dosage.Monoamine oxidases(MAOs)are mitochondria enzyme that are responsible for the oxidative deamination of endogenous and exogenous monoamine substances.The high expression levels of MAO-B in the brain of AD could cause the excessive oxidative deamination of neurotransmitters such as dopamine,resulting in the increased production of hydrogen peroxide to promote AD progression,and the decreased levels of dopamine,5-HT and other neurotransmitters in brain,causing the neuropsychological symptoms in patients with AD.Therefore,MAO-B inhibitors,as an important class of anti-AD clinical research drugs,can produce dual beneficial effect on AD by reducing oxidative stress and rebuilding the activity of monoaminergic neurotransmission.The key pharmacophore propargylamine of MAO-B inhibitors is highly consistent with the substituted amine methyl fragment of the pyrimidinethiourea derivatives in structure,thus,it was rational to introduce propargylamine into the structure of pyrimidinethiourea derivatives though the pharmacophore fusion strategy.According to these reasons,we quickly synthesized a new propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivative A2,which exhibited potent inhibitory activity both toward AChE(IC50?0.324 ?M)and MAO-B(IC50?1.427 ?M)and the molecular-protein docking simulations showed that A2 interacts with AChE and MAO-B in higher affinity.Then,with A2 as the lead compound,the substituents at the N-atom of propargylamine and linker between propargylamine and imidazole ring was modified and optimized,generating a series of novel propargylamine-modified pyrimidinylthiourea derivatives(A1-16),which were evaluated the ChEs&MAOs inhibitory and antioxidant activities in vitro.Among these derivatives,compound A2 displayed good selective inhibitory activity against AChE and MAO-B,and desirable balance between multifunctional efficacy.Further experiments in vitro show that A2 demonstrated good copper chelating property,effective inhibitory activity against Cu2+-induced A?1-42 aggregation,and moderate neuroprotection.What's more,A2·HCl was capable of ameliorating scopolamine-induced cognitive impairment in mice at a dose of 30 mg/kg,which was nearly 7 times lower than compound 5r·HCl.As mentioned above,introducing the MAO-B inhibitors'pharmacophore into the structure effectively improve the anti-AD efficacy of pyrimidinethiourea derivatives,and the strategy of pharmacophore fusion is obviously responsible.However,the relatively low blood-brain barrier(BBB)permeability(Pe=3.16×10-6 cm s-1)of A2 limited the further investigation against AD.In general,high liposoluble compound easily penetrate BBB,on the basis of this reason,we replaced the imidazole ring with higher liposoluble benzimidazole,generating lead compound B4.PAMPA experiment and enzymes inhibition tests in vitro showed that B4 not only overcome the disadvantage of BBB permeability(Pe=7.67×10-6 cm s-1),but also demonstrate improved inhibitory activities against AChE and MAO-B.Similarly,we performed comprehensive modifications in other parts of the structure of B4,including analogous aza-aromatic rings,the propargylamine substitution site,substituents at the N-atom of propargylamine,and linker between propargylamine and imidazole ring,and synthesized a series of novel benzoheterocycle-contained and propargylamine-modified pyrimidinylthiourea derivatives(B1-20),which were evaluated the ChEs and MAOs inhibitory activities,antioxidant ability,and BBB permeability in vitro.In particular,compound B3 produced about 10-fold AChE inhibitory potent improvement(IC50?0.032 ?M)with respect to compound A2,and MAO-B inhibitory activity(IC50=2.117 ?M),antioxidant ability,selective Cu2+ chelating property,inhibitory activity against Cu2+ induced A?1-42 aggregation were well maintained.Animal studies showed that compound B3·HCl could inhibit the cerebral AChE&MAO-B activities at the same dose(100 mg/kg),showing a good dual target efficacy balance,and alleviate scopolamine induced cognitive impairment in mice at 10 mg/kg.Combined with good water solubility(12.12 mg/mL of B3·HCl)and oral bioavailability(F=45.55%),these findings demonstrated that compound B3 may be a potent brain permeable multifunctional candidate for the treatment of AD.Part 2 presents development of disulfide FBPase covalent inhibitors for the treatment of type ? diabetes mellitus.Type ? diabetes mellitus(T2DM)is chronic degenerative metabolic disorder,characterizcd by increased blood glucose level.Impaired insulin sensitivity,relative insulin insufficiency,and increased endogenous glucose production(EGP)are main pathogenesis of T2DM.Most of the current anti-diabetic drugs target insulin sensitivity and secretion,often accompanied by side effects such as weight gain,hypoglycemia,and gastrointestinal disturbances.Therefore,the key to the research and development of diabetes drugs is to find new disease-related targets,and through the regulation of new targets to achieve the best blood glucose control and fewer side effects.Fructose-1,6-bisphosphatase(FBPase)is a key rate-limiting enzyme that catalyzes the second to last step in the GNG pathway.Inhibition of FBPase can effectively block GNG,reduce endogenous glucose production,and achieve hypoglycemic effects.FBPase inhibitors have been considered as a novel pharmacological approach towards the management of elevated blood glucose levels and a research hotspot in the field of T2DM therapeutic drugs development,as they are devoid of hypoglycemia and combat with the key factor responsible for hyperglycemia.Our collaborative group identified a new allosteric site(C128)on FBPase,which could affect the bind affinity of substrate FBP with FBPase.Covalently modifying C128 can inhibit the catalytic activity of FBPase and achieve hypoglycemic effects.We screened ten known cysteine covalent drugs from self-built old drug entity library(including 1400 old drugs)via an enzymatic assay to explore the novel FBPase inhibitors that covalently bind to the C128 site in this part.Disulfiram,a drug approved for relieving alcohol dependence,was identified as a good FBPase inhibitor(IC50?1.78 ?M).Based on the structure of disulfiram,60 disulfiram analogues(C1-60)was designed and synthesized to investigate their FBPase inhibitory activities and obvious SAR was obtained.Among these compounds,C30 showed a good inhibitory activity against FBPase(IC50?0.22 ?M).Further studies in vivo showed that C30 could significantly inhibite FBPase and block gluconeogenesis pathway,along with the improvement of glucose tolerance in ICR mice and diabetic db/db mice.The mechanism study including amino acid mutation assay,UV-visible spectroscopy experiments,proteome analysis,and molecular docking,verified that C30 covalently modified the C128 site and suppressed the FBPase activity through the N125-S124-S123 allosteric pathway.In summary,C30 has the potential to be a novel FBPase covalent inhibitor for diabetes therapy,and can be considered as an good lead structure for the treatment of T2DM.The innovation in our work focused on two aspects,as follows:(1)Base on the structure of multifunctional pyrimidinethiourea fragment previously discovered in our group,a new generation multifunctional inhibitor targeting AChE and MAO-B with skeleton of propargylamine-pyrimidinethiourea were developed by pharmacophore fusion strategy and drugability improved strategy.Among them,compound B3 not only exhibited good multi-target efficacy balance and pharmacokinetic properties in vivo,but also produced at least 20-fold improvement in anti-AD efficacy in vivo with respect to compound 5r,a single-target multifunctional AChE inhibitor.This work provides solid theoretical guidance and structural basis for the development of a new generation of multi-target(AChE&MAO-B)anti-AD drug candidates.(2)Disulfiram was found to have FBPase inhibitory activity firstly,and with it as a lead structure,a new generation of disulfide covalent inhibitors targeting FBPase was developed.Optimized compound C30 exhibited good FBPase inhibition by covalently modifying the C128 site,and antidiabetic efficacy in vivo and in vivo.This work provides solid theoretical guidance and structural basis for the development of new drug candidates for the treatment of T2DM targeting FBPase in a novel mechanism(covalent allosteric).