Design, Synthesis And Evaluation Of PAR-4Antagonists, GIRK1/2Activators And MrgX1Positive Allosteric Modulators

G-protein-coupled receptors (GPCRs), as the largest receptor family on the cell surface,is a central player in the signal transduction and drug discovery.40%of the modern drugstarget the GPCRs such as Olanzapine, Loratadine, Ranitidine, Tegaserod, et al. Protease-activated receptors, member of the GPCR superfamily, is a GPCR on the cell surface. Humanplatelets express two thrombin receptors PAR-1and PAR-4. In2014, the first-class PAR-1small-molecule antagonist Vorapaxar (ZontivityTM) has been approved by FDA. However, itmust not be used in patients with prior history of stroke or transient ischemic attack (TIA).Actually, the Durgs which target PAR-1receptor can usually cause bleeding events. Asanother thrombin receptor, PAR-4was identified to not affect the catalytic activity ofthrombin, and therefore is a potent therapeutic target for the treatment of thrombus. YD-3isthe sole PAR-4antagonist discovered in2000, and no other antagonists with differentchemotype were reported.G-protein-coupled inward rectifier potassium channels (GIRK) depends on G-proteincoupled receptors in modulating the wsitch on and off of potassium channel. GIRK1/2wasexpressed widely in central nervous system and is dominated in the brain, while GIRK1/4isthe main GIRK channel in heart, and are mainly expressed in atrial muscle cells. GIRKactivation plays significant role in many physiological and pathological process, it wasinvolved in excitability changes of neurons, pain perception, analgesia, drug addiction,epilepsia,Down s syndrome and Parkinson s disease. So far, the known GIRK actvators arealcohols (such as ethanol),halothane, naringen and fluoxetine-relative drugs and ML297discovered in2013. However, except for ML297, all these activators are weakly potent andunselective.MrgX1(MAS-related GPR-X1), also known as SNSR4(sensory neuron-specific G-protein-coupled receptor4), belongs to a group of4sensory neuron-specific G-proteincoupled receptors that are expressed solely in dorsal root ganglion (DRG) and trigenminalganlia (TG) small diameter primary sensory neurons, as it has not been detected in the centralnervous system (CNS) or in the rest of the body. As a result, MrgX receptors are believed toparticipate in the modulation of pain. The analgetic targeting MrgXs will avoid the side effects of current drugs targeting at prostaglandin and opoid receptors such as drugaddiction,pernicious vomiting, myalgia, diarrhea, astriction and so forth. Of these four MrgXs,MrgX1and MrgX2are the main pain modulators. As a fact, there is no positive allostericmodulators (PAM) available for MrgX1receptor.Based on the current research status of PAR-4antagonsits, GIRK1/2activators, andMrgX1PAM, in this dissertation, we conducted massive efforts of the high throughputscreening (HTS), chemical opitimization and Structure-Activity-relationship (SAR), andfinally achieved a novel selective and potent PAR-4antagonist, some selective and potentGIRK1/2activators and the first MrgX1PAM.1. Discovery of a novel PAR-4antagonist, ML354. Based on the structure of ethyl4-(1-benzyl-1H-indol-3-yl)benzoate2.2, We selected comounds which are somewhat similar to2.2and which were readily available within Vanderbilt s sample repository. Approximately160compounds were selected for single point testing and gave a novel potent and selectivePAR-4antagonist ML354(VU0099704, PAR-4IC50=140nM, PAR-1IC50~10μM). ML354is the second PAR-4antagonsit with a novel chemotype, after the first indazole compoundYD-3which was discovered in2000. The DMPK assays indicated ML354is not metaboliclystable and as a consequence it is not able to be utilized as an in vivo tool compound. With thenovel PAR-4antagonist ML354in hand, we employed iterative parallel synthesis to conductchemical optimization and multi-dimensional SAR around ML354. For the PAR-4antagonistproject,11libraries,182compounds in total, were prepared. Among of these, compound2.551(PAR-4IC50=823nM, PAR-1IC50~10Μm),2.552(PAR-4IC50=359nM, PAR-1IC50>10μM) and2.63a2(PAR-4IC50=493nM, PAR-1IC50>10μM) were identified as anovel PAR-4antagonists derived from the lead compound ML354. The SAR indicated C-5electron-withdrawing group, C-2methanol pharmacophore and C-3hydrogen bond receptorsare necessary in terms of maintaining the compounds PAR-4antagonism activitiy. ML354discovered by HTS efforts and these three novel selective and potent PAR-4antagonistsderived from ML354can be ultilized as tool compounds to study the therapeutical benefit ofPAR-4antagonism for thrombosis treatment in the preclinical stage, especially the Tier1invitro research step. At the meantime, they provided a novel chemotype for further PAR-4antagonist discovery.2. Discovery of selective and potent GIRK1/2activators. We employed iterativeparallel synthesis to conduct chemical optimization and SAR around ML297, the firstselective and potent GIRK1/2activator discovered in2013. For the GIRK1/2activator project,11libraries,172compound in total, were prepared. The activity assessment identifiedN-benzyl and chemically different N-trifluoroethyl is validated as the substituent of N-phenylof ML297series, and for the first time, we found the “molecule switches” on the C-3positionof the pyrazole core, which modulates the GIRK pharmacology mode (GIRK activation orGIRK inhibition). At the same time, a novel activation factor C-3cyclopropyl was discovered,which provided selective GIRK1/2activators. Subsequently, we combined the newlydiscovered activation factor C-3cyclpropyl and the old activation factor C-3methyl withinML297, which finally afforded selective and potent GIRK1/2activators3.19.1,3.19.2,3.19.4and3.19.20, and also the submicromolar-level GIRK1/2and GIRK1/4dual inhibitors3.13.9,3.17.11,3.17.7and3.17.6. In addition, a novel molecular switch on the cyclopropyl ring wasidentified. When methyl group was located at C-2of the ring, the GIRK activation mode wasswitched on, while when at C-1position, the GIRK inhibition mode was swithched on. Thisimportant finding directs our future chemical optimization efforts, and facilitate us to obtainGIRK tool compound (activators or inhibitors) efficiently.Since fluorine atom are incorporated into drugs to develop activity, improve metabolicstability and biovailability, improve selectivity against target protein and so forth, We choseto employ two fluorine atoms at C-2postion of the cylopropyl. Unexpectedly, the addition offluorine led to the highly potent GIRK1/2and GIRK1/4dual inhibitors3.23.8, which is avaluable tool compound to inhibit GIRK1/4to understand the role of GIRK1/4in arrhythmiassuch as atrial fibrillation. We applied the N-benzyl and N-trifluoroethyl to replace N-phenyl,which were identified a favorable substituent for the N-phenyl through the library3.3. As weexpected, five more selective and potent GIRK1/2activators3.27.2,3.27.3,3.27.12,3.29.2and3.29.3were obtained.The selectivity and (or) activitiy of novel GIRK1/2activators is prior to the leadcompound ML297. They are valuable tool compounds for the treatment of epilepsy, Down’ssyndrome, Parkinson’s disease, et al.3. Disvoery of the first potent and selective MrgX1PAM. We firstly conducted HTSof the NIH Molecular Library Small-Molecule Repository (MLSMR) compound collectioncontaining>300,000compounds. The THS campaign identified four compoundsSID4258711, SID4262381, SID3665540and SID3607659from a sulfonamide benzamideseries. Secondly, we chose the most potent hit SID4258711as the lead compound andemployed the iterative parallel synthesis strategy to conduct the chemical optimizationoffering40analogs, which finally afforded the first potent and selective MrgX1PAM ML382 (compound4.431, MrgX1EC50=109nM).ML382was further profiled in a battery of Tier1in vitro DMPK assays. The highintrinsic clearance indicated ML382is unstable to oxidative metabolism, and was predictedto display high clearance in human and rat (Human CLHEP:15.7mL/min/kg, Rat CLHEP:65.7mL/min/kg). In addition, the protein binding of ML382was evaluated and was shown to havemoderate free fraction in rat (1.7%) and low free fraction (0.4%) in human. Hence, ML297possesses favorable free fraction in rat plasma protein binding; howerver, instability in ratliver microsomes indicates the utility of ML382as an in vivo probe will be limited to non-oral dosing regimens (intraperitoneal, subcutaneous, or intrathecal).