Potent, Selective, Water Soluble, Brain-Permeable EP2 Receptor Antagonist for Use in Central Nervous System Disease Models.

Previous SAR studies around compound 1 (TG6-10-1, Figure 1B ) indicated that the acrylamide moiety is not essential for EP2 bioactivity. Thus, we developed second-generation analogs with an amide linker for SAR study. Compound 2a and 3 ( Figure 1B ) are examples of the earlier leads. These two compounds displayed high EP2 potency and selectivity and improved aqueous-solubility. However, compound 2a displayed poor DMPK properties, for example 60 min) and in vivo plasma half-life in mice (10.5 h), it showed very poor brain-to-plasma ratio of 0.02. 35 Therefore, it was not useful for proof-of concept studies in CNS disease models. The overall goal was to develop a compound with requisite brain penetration and plasma half-life. In the past, we have made several derivatives replacing the right-side ring (morpholine in 2a , tetrazole in 3 ) and learned that this ring region can be modified without substantial loss of potency at EP2 receptor. We also made limited derivatives for example, polar imidazole ring replacing the indole on the left-side ring to increase the aqueous solubility. 34 We have not made modifications on the middle phenyl ring for SAR study in the past. Therefore, we expanded our lead optimization studies with structural modification in these areas as detailed below. To explore the middle phenyl ring for structural modification and SAR studies, novel compounds 2b-c have been synthesized in a single step ( Scheme 1 ) using commercially available precursors ( 1b and 1c ) by following the synthetic method reported for 2a . 34 However, several other precursors (e.g. 6a-g, 10a-c, 13a-c ) needed for synthesis of novel derivatives were not commercially available, therefore we have synthesized here as shown in Scheme 2 . Commercially available boronic acid derivatives ( 4a-g ) have been coupled to 6-bromonicotinic acid ( 5 ) in the presence of tetrakis(triphenylphosphine)palladium (0) catalyst and a base to provide intermediates 6a-g , which were then coupled to 2-(2-methyl-1 H -indol-3-yl)ethan-1-amine ( 7 ) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.hydrochloride (EDCI.HCl) and (dimethylamino)pyridine (DMAP) to provide final compounds 8a-g . Analogously, boronic acid derivatives 4a-c on palladium catalyzed coupling with 5-bromopicolinic acid ( 9 ) resulted in intermediate derivatives 10a-c , which were further coupled to 7 to provide compounds 11a-c . Moreover, the boronic acid 4c was coupled to 2-chloropyrimidine-5-carboxylic acid ( 12a ) or 4-bromo-3-fluorobenzoic acid ( 12b ) or 4-bromo-benzoic acid ( 12c ) in the presence of palladium (II) catalyst and a base to result in intermediates 13a-c , which were similarly coupled with amine 7 to furnish final compounds 14a-c . Furthermore, we synthesized a number of derivatives with ‘NH’ as a linking moiety between the middle ring and the last ring of the molecule. As shown in Scheme 3 , the amine 7 was coupled to 2-aminopyrimidine-5-carboxylic acid ( 15 ) in presence of coupling reagent EDCI.HCl to provide intermediate amine 16 , which was further coupled to 2-bromopyridines 17a-o in the presence of tris(dibenzylideneacetone)dipalladium (0) and a base to provide final products 20a-o . Analogously, intermediate 16 was coupled to commercially available 4-bromopyridine ( 18 ) and 3-bromopyridines 19a-b to provide 20p and 20q-r respectively as final products in good yields. Maintaining the middle ring as pyrimidine, and the last ring as 4,6-dimethyl pyridine (see next section for SAR discussion), we further synthesized novel compounds with substitutions on the left side indole ring. As shown in Scheme 4 , ethyl-2-aminopyrimidine-5-carboxylate ( 21 ) on reaction with 17o in presence of palladium (II) acetate gave intermediate 22 , which on hydrolysis afforded acid precursor 23 . Requisite amines 7a-e ( SI Figure 1 ) were synthesized using the known methods, 36 , 37 and they were individually coupled with 23 in the presence of DMAP and EDCI.HCl at 50 °C to furnish the final products 24a-e ( Scheme 4 ). Similarly, compound 24f was synthesized using acid 23 and 2-trifluroindole-3-ethanolamine ( 7f ), 38 which was intern prepared form 2-vinyl-pyrrolidin-2-one ( SI Figure 1 ). Moreover, the compound 2-(2-methylpyrazolo[1,5-a]pyridin-3-yl)ethan-1-amine ( 7g ) has been synthesized starting from amino-pyridinium iodide ( SI Figure 1 ). 39 Compound 7g , and the commercially available 2-amino-1-(2-methyl-1 H -indol-3-yl)ethan-1-one ( 7h ) and 2-amino-1-(2-methyl-1 H -indol-3-yl)ethan-1-ol ( 7i ) were coupled to the acid precursor 23 in the presence of EDCI.HCl coupling reagents to provide final products 24g-i , respectively in moderate yields. All the intermediates and final products were characterized by complementary methods of NMR and LCMS. The compounds having purity > 95% were subjected to testing in bioassays as described below. Incorporation of nitrogen into the structure of small molecules often enhances aqueous solubility, intestinal permeability and other DMPK properties. 40 Therefore, we envisioned hitherto unexplored middle phenyl ring (see ring assignments in Figure 1B ) for structural modification with nitrogen heterocyclic rings for SAR studies. Recently, several groups have analyzed the physicochemical properties of CNS candidates and drugs to propose a multi-parameter optimization (MPO) approach that can be used to design novel chemical agents with CNS desirable properties. 41 – 43 Based on this MPO approach, Wager et al. proposed six physicochemical properties (MW, cLogP, cLogD, HBD, TPSA and pKa, Table 1 ) that cumulatively guide CNS activity and permeability of molecules. 43 Our previous lead compounds 2a and 3 attained desirable MPO score (>4 out of 6 maximum), however, compound 3 was found to be a CNS impermeable compound with in vivo brain-to-plasma ratio 0.02, whereas compound 2a was too short lived in vivo to determine the brain permeability. We also determined that 3 is not a substrate of efflux pumps, but it displayed low cell permeability P aap (A-B) of 0.41 × 10 −6 cm/sec in MDR1 expressed MDCK- cell monolayers with efflux ratio 0.3 ( Table 7 ). Examination of its properties ( Table 1 ) indicate that further optimization of the total polar surface area and hydrogen bond donor (HBD) properties might improve CNS permeability. 44 Moreover, compound 3 has a tetrazole ring, which is known to act as a surrogate for carboxylic acid, which could limit a passive brain permeability. 45 Therefore, we used compound 2a as a starting point for the current lead optimization studies. We previously reported compound 2a with good EP2 potency and selectivity which showed moderate aqueous solubility. 34 It has a morpholine ring on the right, phenyl ring in the middle, and 3-indole moiety on the left side ( Figure 1B ). To learn whether the middle phenyl ring can be replaced with other rings, we synthesized and tested compound 2b with a nicotinic acid ring. Compound 2b displayed 4-fold less EP2 potency than 2a , but it showed 3-fold increased aqueous solubility reinforcing the notion that nitrogen in the ring will enhance the aqueous solubility. 40 Keeping the nitrogen (nicotinic) in the middle ring, we substituted right side morpholine ring with piperidine ring ( 2c ). This resulted in 6.4-fold loss of potency in comparison to 2a . However, replacement of morpholine with a phenyl ring on the right side resulted in compound 8a , which interestingly displayed nearly equal EP2 potency to 2a , but low aqueous solubility ( Table 2 ). Incorporation of a methoxy group either at 3-position ( 8b ), or methoxy or hydroxy at 2-position ( 8c-8d ) on the phenyl ring improved the potency by 2-fold (~10 nM, Table 2 ) in comparison to 2a . Whereas, fluorine ( 8e ) at 2-position reduced the potency by 2-fold in comparison to 2a , and 4-fold in comparison to 2-methoxy derivatives 8c . Interestingly, incorporation of 4-acetamido group ( 8f ), or two methoxy groups at 3- and 5-positions ( 8g ) further enhanced the potency with K B values 4.4 and 2.9 nM respectively. To see if changing the location of nitrogen atom in the middle ring from meta-to-ortho ( see o-m positional assignments in Scheme 2 ), we synthesized derivatives 11a-c . Compound 11a was 4-fold less potent than equivalent derivative 8b . Similarly, 11b was 11-fold less potent than equivalent derivative 8g , and compound 11c was 3-fold less potent than equivalent compound 8f . These observations suggest that nitrogen atom is preferred at m -position than at o -position of the middle ring (meaning nicotinic ring is more favorable than picolinic ring) for optimal potency. Furthermore, we synthesized a pyrimidine derivative 14a and it showed 2.9-fold less potency than the corresponding nicotinic ring compound 8c , but it has the same potency as initial lead 2a in the current study. Nonetheless, nicotinic middle ring maintains high EP2 Schild potency (< 50 nM), except for 2b-c . It is important to emphasize here that derivatives 8a-g and 14a displayed enhanced solubility in simulated gastric fluid at pH 2.0, whereas compounds 11a-c have not showed solubility enhancement in the simulated gastric fluid ( Table 2 ), suggesting nicotinic ring derivatives will have better aqueous solubility than picolinic ring derivatives. It is worth to indicate that the nitrogen in the middle ring is not absolutely essential for EP2 potency, because compound with phenyl ring, 14c has also showed high EP2 potency ( cf. 8c ), but a fluorinesubstitution on the middle phenyl ring drastically reduced the potency by 25-fold ( cf. 14b vs. 14c or 8c ) hinting that additional substitutions on the middle ring might not be tolerated for potency. Although the requisite EP2 potency is obtained for several analogs shown in Table 2 , only compound 8c has displayed good selectivity (>100-fold) against other receptors (see Table 6 and next section for selectivity discussion). Moreover, when representative compounds ( 8c, 8d, 8g, 14a ) from the Table 2 were tested for stability in mouse liver microsomes, they displayed very short half-life ( Table 5 ). Therefore, we envisioned synthesizing additional novel derivatives by keeping the middle ring as pyrimidine and modifying the third ring ( Scheme 3 ). The other key difference between these novel set of derivatives shown in Table 3 in comparison to the ones shown in Table 2 is the NH functional group, which links the third ring and the middle pyrimidine ring. We envisioned that the NH group should enhance the molecular flexibility and potentially allow us to make anionic salts. In the process, we first synthesized and tested 2-pyridyl derivative 20a . Interestingly, this compound displayed high EP2 potency with K B = 10.7 nM. Addition of methyl group ( 20b ) or a fluorine ( 20c ) at 4-position maintained high potency (K B = < 10 nM). Moving the methyl group from 4- to 6- position also maintained a high potency ( cf . 20e vs. 20b ), but similarly moving the fluorine atom to 6- position recued the potency by 16-fold (cf. 20f vs. 20c ). A methoxy group at 6- position also maintained high EP2 potency ( 20h ), whereas cyano-group reduced the potency by 23-fold ( 20g ). The acetyl group on pyridine ring whether it is at 6-,5- or 4- position ( 20j , 20l and 20k ) reduced the potency by 4-, 20-and 12-fold respectively in comparison to 20b. A compound with bulkier group 3-hydroxybutane at 6- position showed 4-fold less potency than 20b. Similarly, tert -butyl group at 4- position reduced the potency by 5-fold ( cf . 20d vs. 20b ), or 6-position reduced by 13-fold (cf. 20n vs. 20e ). A hydroxy group ( 20i ) at 6-position eliminated the potency of the molecule (K B > 1000 nM) ( Table 3 ). Incorporation of two methyl groups as in 20o did not have additive impact on the potency ( cf . 20o vs. 20b or 20e ). We also tested the 4-pyridyl derivative 20p , which showed complete loss of potency, whereas the 3-pyridyl derivative 20q regained the potency and shown nearly same potency as 20a . Addition of two methyl groups on 3-pyridyl ring ( 20r ) similar to 20o indicated 10-fold loss of potency, thus we have not subjected 3-pyridyl ring for further modification. We then synthesized a new set of derivatives ( Scheme 4 ) by modifying left side indole moiety and the two carbon linker, keeping the middle ring as pyrimidine and the right side ring as 4,6-dimethyl pyridyl ring as constant. As shown in Table 4 , a fluoro, chloro, methoxy, difluoro or dichloro derivatives 24a-e all retained high EP2 potency, whereas changing indole ring to pyrazolo-pyridine ring as in 24g reduced the potency by 32-fold in comparison to 20o . Moreover, substituting the methyl group at second position of indole with a trifluoromethyl group ( 24f ) also reduced the potency by 8-fold. Modification to the linker with ketone group next to indole ( 24h ) eliminated the potency, where as a hydroxy group as in 24i recued the potency 23-fold in comparison to 20o suggesting unsubstituted ethylene linker is optimal for high EP2 potency within the scaffold. Overall, the SAR study on the leads indicated that a number of compounds exhibit high EP2 potency. Aqueous solubility is an important property that plays crucial role in success of a drug candidate. 47 We tested several compounds for aqueous solubility by using two different methods. First, we used a kinetic assay in which we dissolved the compounds in DMSO then made serial dilutions with phosphate buffered solution (PBS) at pH 7.4, maintaining 1% DMSO in the solutions. Solubility was determined by nephelometry. 46 This assay works on the principle that when visible light is passed through a solution, part of the incident radiant energy will be scattered. The intensity of the scattered light is a function of the concentration of the dispersed phase. The nephelometric assay is used to determine either the point at which a solute begins to precipitate out of a true solution to form a suspension or the concentration at which a suspension becomes a solution when diluted further. The scattered light will remain at a constant intensity until precipitation occurs, at which point it will increase sharply. Several compounds shown in Table 2 and 3 displayed moderate solubility (100 μM aqueous solubility when determined in simulated gastric fluid (pH 2.0), suggesting that the intrinsic basic nitrogen can be explored to create salts that further increase solubility and facilitate the formulation. On this notion, compounds 20o, 24a, c, d and 24f have been converted to hydrochloride salts by stirring them in dichloromethane using dilute hydrochloride solution. Second, to ask whether the hydrochloride salt compounds ( 20o , 24a, c d and 24f ) are soluble in water, we used a thermodynamic assay (shake flask method) 48 where the compounds are shaken (2 mg/mL, at 2200 rpm) in neat water and the soluble fraction was measured against standard DMSO solutions by HPLC or LC-MS/MS quantitation methods. As shown in Table 4 , the hydrochloride salt compounds displayed millimolar level aqueous solubility in water. It is worth mentioning that the salt forms did not show increased solubility in PBS buffer at pH 7.4 by nephelometry method in comparison to their neutral compounds. This is likely due to a drop in pH in neat water, in comparison to a buffer. Indeed, the water pH was reduced from 6.4 to 3.5 when 20o.HCl was dissolved at 2 mg/mL. This observation is in line with our findings that the neutral compounds have higher solubility in simulated gastric fluid (pH 2.0) than in PBS at pH 7.4. Overall, the hydrochloride salts of compounds 20o , 24a , 24c and 24d showed 2.52, 1.16, 1.73 and 2.38 mM solubility in neat water ( Table 4 ). The structural identity among the prostanoid receptor family is rather low. EP1, EP2, EP3 and EP4 share only 20–30% structural homology. 49 In contrast, EP2 is more homologous to DP1 (44%) and IP receptors (40%). 49 In terms of cellular signaling, EP2, EP4, DP1 and IP induce cAMP mediated cell signaling, whereas EP1 promotes Ca 2+ mediated signaling and EP3 inhibits cAMP mediated signaling. Functionally, EP2 and DP1 receptors seem to promote inflammation in a variety of disease conditions, whereas EP4 seems to act as pro and anti-inflammatory, and the IP receptor seems to have cardioprotective role. 15 Thus, we determined the selectivity of several potent EP2 antagonists (K B 100-fold selectivity to DP1, then it was tested against EP4 and IP receptors. As shown in Table 6 , compounds 8a-b, 8e, 11a-b showed < 100-fold selectivity against the DP1 receptor, thus they were not tested against EP4 and IP receptors. The compounds 8c, 14a, 20a, 20c, 20e, 20h, 20o , and 24a-c displayed excellent selectivity against DP1, and subsequently against EP4 and IP receptors. However, compounds 8g and 24c displayed < 200-fold selectivity against at least one of the three (DP1, EP4, and IP) receptors suggesting selectivity will depend on the individual structure of the molecule within the scaffold. It is worth to note that we tested several compounds in the class for cytotoxicity in the C6-glioma cell line. As exemplified in Table 6 , selected compounds including 20o displayed no cytotoxicity until 50 μM indicating > 2100-fold in vitro therapeutic index (CC 50 /EP 2 K B ), except compound 8c which showed CC 50 21 μM with a therapeutic index of 2100-fold. We tested several derivatives in pooled liver microsomal fractions to determine the half-life and intrinsic clearance by the mouse liver, and to project in vivo pharmacokinetics. The starting compound for the current study, 2a was metabolized quickly in liver microsomes ( Table 5 ). To understand the potential metabolites, we incubated compound 2a in mouse liver microsomes and investigated metabolites after 5 minutes. As shown in Figure 2 , the major metabolite is the amide bond cleaved product D (MW 190 Da), which can be formed via intermediate A that would be further cleaved to generate fragments B / C (MW 158 Da) and D . The compound D can also be generated via a vinylamine F (MW 362 Da) intermediate, formation of which can be envisioned via hydroxylation at the CH 2 unit ( E ) next to 3-indole ring. Therefore, we synthesized compounds blocking the CH 2 site with a hydroxy group (e.g. 24i ). The half-life of 24i in liver microsomes increased by 3-fold (t 1/2 , 57 min) compared to its equivalent 20o , which showed 17 minutes of half-life ( Table 5 ). However, the hydroxylated derivatives displayed reduced EP2 potency compared with the equivalent compounds. ( cf . 24i displayed about 19-fold less potency than 20o , Table 4 ). In a similar experiment, the tetrazole compound 3 did not produce any fragments even after 20 minutes of incubation in liver microsomes, suggesting the metabolic hydroxylation or cleavage also depends on the right side moiety ( Figure 2 ). The mouse liver microsomal half-life data presented in Table 5 also indicate that a fluorine atom in place of metabolically prone methyl group enhances the stability. For example, a fluoro- derivative 20c has 2-fold higher half-life in comparison to 20b , like wise 20f has 8-fold higher half-life than 20e . Regrettably, 20c is unstable in vivo, and 20f displayed less potency to EP2 receptor ( Table 3 ), therefore, these compounds are not promoted for further studies. We estimated the CNS drug-like properties for several of these compounds before synthesis using the CNS-MPO tool. 43 , 50 MPO scores indicated that several EP2 antagonists in the class as exemplified in Table 7 , display a desired MPO score of ≥ 4, but several others do not achieve the desired score > 4 derived using their physicochemical properties. Compounds ≥ 4 MPO score should have desirable CNS activity and permeability features. However, the compounds 3 while it displayed > 5 score does not have good permeability properties and does not get in to the brain ( Table 7 ). A corollary to this, the current lead compound 20o.HCl showed < 4 MPO score, yet crosses the blood-brain-barrier with in vivo brain-to-plasma ratio 0.4. Likewise, compounds 8c, 8g and 14a behaved similarly. Overall, we have seen a positive correlation between MPO score to in vivo brain to plasma ratio. Nonetheless, the MPO score does not quantitatively predict the brain permeability. We tested several compounds for permeability across MDR1 expressed MDCK cell line to investigate the potential of blood brain barrier (BBB) permeability within the class. As shown in Table 7 , we found many of these compounds have good passive permeability from the apical-to-basolateral (A-B) side as well as the basolateral-to-apical side (> 0.6 ×10 −6 cm/s) in comparison to compound 3 . and their efflux ratio is < 3, except for compound 14a which displayed ER ratio 16 indicating it may be a substrate for efflux pumps ( Table 7 ). On the other hand, the compound 20o found to show good permeability and showed similar efflux ratio in the presence and absence of an efflux pump inhibitor verapamil, confirming that it is not the substrate of efflux pumps. ( Table 7 ). Moreover, compound 20o showed 0.4 % plasma protein unbound fraction in mouse plasma proteins, but we were not able to determine its fraction unbound in the relevant brain compartment (i.e., interstitial fluid) due lack of efficient method to extract cerebrospinal fluid (CSF) from mouse brain. To determine in vivo brain-to-plasma ratio in mice, we tested several compounds whose mouse liver microsomal stability was greater than 15 min, by administering a dose of 10 mg/kg via intraperitoneal injection. Analysis of the compound concentration in the plasma and the brain tissues at 0.5, 2, and 4 h time points suggested that most of these compounds, exemplified as in Table 7 for 8c, 8g & 14a , will have a plasma half-life below 1 h, except compound 20o. A subsequent pharmacokinetic analysis on 20o.HCl with concentration analysis at 8-time points after a single i.p. injection (10 mg/kg) ( Figure 3 ) indicated that it has terminal plasma half-life 1.1 h with clearance rate of 124.1 mL/min/kg and brainto-plasma ratio of 0.4. Additional studies with oral gavage dosing (50 mg/kg, B.I.D. dosing 8h apart) indicated that the plasma half-life could be extended to 2.4 h for 20o.HCl. To determine whether the novel derivatives in this class exhibit competitive antagonism of EP2, we tested several compounds in a concentration-response manner against PGE 2 concentration effect on EP2 receptors. The Schild K B indicates the antagonist concentration required for a two-fold rightward shift in the PGE 2 concentration-response curve . Schild K B values are derived by the equation log (dr − 1) = log X B – log K B , where dr = dose ratio, i.e. the fold shift in agonist EC 50 caused by the antagonist, X B is antagonist concentration. As illustrated in Figure 4B , a linear regression of log (dr-1) on log X B with slope of unity characterizes a competitive antagonism. A smaller K B value indicates a higher inhibitory potency. As shown in Figure 4A and C , compound 20o (TG11–77 neutral and the hydrochloride salt form) induced a concentration-dependent, parallel rightward shift in the PGE 2 concentration-response curve. Schild regression analyses ( Figure 4B ) is consistent with a competitive mechanism of antagonism on EP2 with average Schild K B 9.7 nM and average slope value 1.05. The average K B value is used throughout in this manuscript for discussion and comparisons. Moreover, several other compounds synthesized in this class displayed a concentration-dependent rightward shift of PGE 2 EC 50 and with slope of unity ( SI Figure 2 ). Thus, the mechanism is competitive in general for the class of EP2 antagonists presented in this study. However, compound 20o did not show a concentration-dependent inhibition of DP1 receptor, indicating it is selective for EP2 over DP1 with selective index (DP1 K B /EP2 K B ) of 750-fold. Moreover, this compound also showed high selectivity against EP4 and IP receptors with selective index of 550-fold and >1000-fold respectively ( Table 6 ). The EP2 receptor acts as an immunomodulator with exacerbating role in chronic neurodegenerative disease such as epilepsy and Alzheimer’s disease. EP2 receptors also play an exacerbating role in chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease (colitis) and endometriosis. 51 – 53 To determine whether the new EP2 antagonists are anti-inflammatory, we tested several of these novel EP2 antagonists including 20o (TG11–77) for anti-inflammatory activity in vitro. A routine isolation of microglia from mouse brain proved to be low throughput and these primary cells behave variably depending on the animal. Thus, we created a hEP2-BV2 cell line, a mouse microglia cell line overexpressing human EP2 receptors. 54 . Upon activation of this cell line with 100 ng/mL lipopolysaccharide (LPS), mRNA levels of several proinflammatory genes including COX-2, IL-6 and IL-1β were induced. An EP2 specific agonist, ONO-AE1-259-01, at 30 nM further exacerbated the induction of these inflammatory genes. Gratifyingly, 20o blunted the upregulation of inflammatory genes COX-2, IL-1β, and IL-6 in a concentration dependent manner ( Figure 5 ). We found that EP2 activation in hEP2-BV2 cell line decreased mRNA levels of TNF-α consistent with the result we found with primary microglia. 55 Interestingly, these downregulated TNF-α levels are also reversed by the EP2 antagonist, reassuring that this compound is working by interacting with the EP2 receptor. In this experiment, neither the EP2 agonist (ONO-AE1-259-01), nor the antagonist ( 20o ) alone showed significant effects on the expression of mRNA levels of EP2 and iNOS (an oxidative stress causing enzyme) in this cell line. Overall, these data support the use of this EP2 antagonist as an anti-inflammatory agent in inflammatory- disease models. To determine whether there is a correlation between the potency of EP2 antagonists in the cAMPproduction assay and their ability to inhibit inflammatory gene expression in the BV2-hEP2 cell line, we tested four selected EP2 antagonists ( 3 , 20o , 26 (TG4–155) 56 and 27 (TG8–237) 35 ) with K B values ranging between 2 and 50 nM. The fold concentrations are calculated and presented in SI Table 2 . As shown in Figure 6 , we plotted the percent inhibition of inflammatory genes against log of multiple Schild K B values. The results show that the inhibition of cytokines was positively correlated to their respective Schild K B , suggesting the higher the potency of antagonists in the cAMP assay, the lower the concentration required to display maximum effect on the inflammatory genes ( Figure 6 ).

In conclusion, we synthesized 45 novel EP2 antagonists with good potency and selectivity. With the lead optimization following the DMPK properties on these antagonists, a novel compound 20o.HCl (named as TG11–77.HCl) was identified with acceptable brain permeability and excellent water solubility. Several compounds in this class displayed selective, competitive antagonism of EP2 receptors and increased solubility in simulated gastric fluid at pH 2.0 compared to PBS at pH 7.4. The hydrochloride salts of several compounds showed good water solubility at lower pH. Functionally, members of this class reversed inflammatory gene modulation by EP2 receptor activation, and the potency was shown to positively correlate to the anti-inflammatory actions measured following gene expression in a novel microglia cell line. Taken together, we conclude that 20o.HCl should be very useful for dosing into rodent models of CNS diseases.

Proton NMR spectra were recorded in solvent in DMSO- d 6 /CDCl 3 on Varian and Inova-400 (400 MHz). Thin layer chromatography was performed on pre-coated, aluminum-backed plates (silica gel 60 F 254 , 0.25 mm thickness) from EM Science and was visualized by UV lamp, PMA solution and ninhydrin. Chemicals and drugs: PGE 2 , BW245C, iloprost, and rolipram were purchased from Cayman Chemical. LPS was purchased form Sigma-Aldrich. ONO-AE1-259-01 was generously provided by ONO Pharmaceuticals (Osaka, Japan). Column chromatography was performed with silica gel cartridges on Teledyne-ISCO instrument. Agilent LC-MS was used to determine the mass and purity of the products. LC-MS conditions: Mobile phase A: methanol (0.1% acetic acid); mobile phase B: water (0.1% acetic acid); column: ZORBAX Eclipse XDB C18 5μM, 4.6 × 150 mm. Gradient B 80% at 0 min, linearly decreased to 5% by 7 min, and then linear increase to 40% by 12 min; UV wavelength = 254 nm; flow rate = 1 mL/min. Furthermore, purity of several key compounds is determined by Water’s HPLC instrument. HPLC Conditions: Mobile phase A: water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); column: XBridge C18 5μM, 4.6 × 150 mm; gradient: 10% B at 0 min, increased linearly to 90% by 10 min, then decreased to 10% by 12 min; UV wavelength = 230 nm; flow rate = 1 mL/min. Compounds with >95% purity by HPLC were tested in cellular bioassays and DMPK properties. Compounds 7a 57 , 7b 58 , 7e 59 were reported in the literature and the characterization data for these derivatives was in good agreement with the literature data. The compound 2-(2-(trifluoromethyl)-1 H -indol-3-yl)ethan-1-amine ( 7f ) was synthesized as reported before 60 , 61 and 2-(2-methylpyrazolo[1,5-a]pyridin-3-yl)ethan-1-amine ( 7g ) was synthesized following the literature procedure. 62 2-Amino-1-(2-methyl-1 H -indol-3-yl)ethan-1-one ( 7h ), and 2-amino-1-(2-methyl-1 H -indol-3-yl)ethan-1-ol ( 7i ) were commercially available. To a solution of commercially available acid 1b or 1c (0.4 mmol, 1 equiv.) and 7 (70 mg, 0.4 mmol, 1 equiv.) in mixture of dichloromethane and N,N- dimethylformamide (3 mL, 5:1) was added DMAP (catalytic amount, 2 mg) followed by EDCI.HCl (114 mg, 0.59 mmol, 1.3 equiv.) and the reaction mixture was stirred at room temperature for 10 h. Organic solvent was evaporated and reaction mixture was added a saturated solution of ammonium chloride (5 mL) and extracted with ethyl acetate (3 × 10 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (5 mL) followed by brine solution (5 mL), dried over sodium sulfate and concentrated to dryness. The crude material was purified on silica gel chromatography using 60–70% ethyl acetate in hexanes to get the required product 2b or 2c ( Scheme 1 ). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.69 (s, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.40 (t, J = 5.6 Hz, 1H), 7.95 (dd, J = 8.9, 2.4 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 6.97 – 6.85 (m, 2H), 6.81 (d, J = 9.0 Hz, 1H), 3.70 – 3.63 (m, 4H), 3.58 – 3.47 (m, 4H), 3.37 (q, J = 7.1 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H), 2.27 (s, 3H); LCMS (ESI): >97% purity. MS m/z , 365 [M + H] + ; HPLC purity: 99.7%. 1 H NMR (400 MHz, CDCl 3 ): δ 8.36 (d, J = 2.3 Hz, 1H), 8.08 (s, 1H), 7.74 (dd, J = 9.0, 2.5 Hz, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.28 – 7.22 (m, 1H), 7.11 – 7.08 (m, 2H), 6.54 (d, J = 9.0 Hz, 1H), 6.02 (t, J = 5.6 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 3.57 (d, J = 5.0 Hz, 4H), 2.99 (t, J = 6.6 Hz, 2H), 2.32 (s, 3H), 1.67− 1.56 (m, 6H); LCMS (ESI): >97% purity. MS m/z 363 [M + H] + ; HPLC purity: 98.9%. A solution of boronic acid ( 4a-g ) (4.1 mmol, 1 equiv.) and bromo-acid, 5 (4.1 mmol, 1 equiv.) in tetrahydrofuran or toluene and water (6:1) were loaded in to a sealed tube. To this solution, 1M Na 2 CO 3 (8.2 mmol, 2 equiv.) was added and purged with nitrogen for 10 min. Then, Pd(PPh 3 ) 4 (0.2 mmol, 0.05 equiv.) catalyst was added to the reaction mixture, sealed and heated to 100 °C for 12 h. Reaction mixture was cooled to room temperature and solvent was evaporated under vacuum. The residue was washed with dichloromethane to remove organic impurities. Then, aqueous layer was acidified to pH 2 with concentrated HCl to result in white precipitate, which was filtered and dried under vacuum to provide the intermediates ( 6a , 6b 63 , 6c, 6d 64 , 6e 65 , 6f and 6g ). 1 H NMR (400 MHz, DMSO- d 6 ): δ 13.36 (s, 1H), 9.24 – 9.03 (m, 1H), 8.35 – 8.31 (m, 1H), 8.17 (dd, J = 8.2, 1.3 Hz, 2H), 8.14 – 8.09 (m, 1H), 7.54 – 7.52 (m, 3H). LCMS (ESI): >95% purity; MS m/z , 198 [M - H] + . 1 H NMR (300 MHz, DMSO- d 6 ): δ 12.52 (s, 1H), 9.14 (d, J = 2.2 Hz, 1H), 8.46 (dd, J = 8.4, 2.2 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.79 (dd, J = 7.7, 1.7 Hz, 1H), 7.59 – 7.47 (m, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.12 (dd, J = 11.5, 4.2 Hz, 1H), 3.87 (s, 3H). LCMS (ESI): >95% purity; MS m/z , 228 [M - H] + . 1 H NMR (300 MHz DMSO- d 6 ): δ 13.32 (s, 1H), 10.17 (s, 1H), 9.08 (s, 1H), 8.26 (d, J = 6.5 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 8.02 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 2.06 (s, 3H). LCMS (ESI): >97% purity; MS m/z , 255 [M - H] + . 1 H NMR (300 MHz, DMSO- d 6 ): δ 13.31 (bs, 1H), 9.11 (d, J = 2.2 Hz, 1H), 8.29 (dd, J = 8.3, 2.2 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 2.3 Hz, 2H), 6.61 (t, J = 2.2 Hz, 1H), 3.81 (s, 6H). LCMS (ESI): >95% purity; MS m/z , 258 [M - H] + . To a solution of 25a-e (2 mmol, 1 equiv.) in acetonitrile (20 mL) was added cyclopropyl methyl ketone (4 mmol, 2 equiv.) and refluxed for 24 h. Then, reaction mixtures were cooled to room temperature. Solids precipitated were filtered to obtain corresponding hydrochloride salts of 7a-e . To a suspension of these salts in dichloromethane was added 50% ammonium hydroxide solution (1.2 equiv.) and stirred for 3 h at room temperature. Organic layer was extracted, dried over sodium sulfate and concentrated to dryness to get the amines 7a-e (see SI Figure 1 ). Corresponding references were provided in the general experimental section for reported compounds and data for 7c and 7d are shown below. 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.48 (s, 1H), 8.15 (bs, 3H), 7.21 – 7.15 (m, 1H), 6.88 – 6.79 (m, 1H), 2.98 – 2.82 (m, 4H), 2.35 (s, 3H); LCMS (ESI): >95% purity. MS m/z, 211 [(M – HCl) + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.46 (s, 1H), 8.13 (bs, 3H) 7.58 (s, 1H), 7.14 (s, 1H), 3.02− 2.81 (m, 4H), 2.37 (s, 3H); LCMS (ESI): >95% purity. MS m/z, 243 [(M – HCl) + H] + . To a solution of 6a-g (0.5 mmol, 1 equiv.) and 2-(2-methyl-1 H -indol-3-yl)ethan-1-amine ( 7 ) (0.5 mmol, 1 equiv.) in N,N- dimethylformamide and dichloromethane (1:1) was added DMAP (catalytic amount) followed by EDCI.HCl (0.65 mmol, 1.3 equiv.) and the reaction mixture was stirred at room temperature for 10 h. Then, dichloromethane was evaporated and the crude reaction mixture was added a saturated solution of ammonium chloride (15 mL) and extracted with ethyl acetate (15 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (15 mL) followed by brine solution (15 mL). Combined organic layer was dried over sodium sulfate, concentrated to dryness. The crude was purified on silica gel chromatography using 50–70% ethyl acetate in hexanes to get the required products ( 8a-g ). 1 H NMR (400 MHz, CDCl 3 ): δ 8.85 (dt, J = 2.4, 0.8 Hz, 1H), 8.07 – 8.03 (m, 1H), 8.01 – 7.96 (m, 2H), 7.90 (s, 1H), 7.76 – 7.71 (m, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 – 7.41 (m, 3H), 7.31 (dd, J = 4.5, 4.0 Hz, 1H), 7.18 – 7.07 (m, 2H), 6.22 (t, J = 6.4 Hz, 1H), 3.76 (dd, J = 6.4, 3.4 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.40 (s, 3H); LCMS (ESI):. LCMS (ESI): >97% purity; MS m/z , 356 [M + H] + ; HPLC purity: 99.4%. 1 H NMR (400 MHz, CDCl 3 ): δ 8.85 (dd, J = 2.3, 0.7 Hz, 1H), 8.04 (dd, J = 8.3, 2.3 Hz, 1H), 7.92 (s, 1H), 7.74 (dd, J = 8.3, 0.8 Hz, 1H), 7.60 – 7.57 (m, 1H), 7.57 – 7.53 (m, 2H), 7.39 (t, J = 7.9 Hz, 1H), 7.32 – 7.28 (m, 1H), 7.18 – 7.07 (m, 2H), 7.00 (dd, J = 8.2, 2.6 Hz, 1H), 6.23 (t, J = 6.0 Hz, 1H), 3.89 (s, 3H), 3.75 (dd, J = 6.4, 3.6 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.39 (s, 3H); LCMS (ESI): LCMS (ESI): >97% purity; MS m/z , 386 [M + H] + ; HPLC purity: 96.8%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 9.06 – 9.02 (m, 1H), 8.82 (t, J = 5.6 Hz, 1H), 8.19 – 8.15 (m, 1H), 7.96 – 7.92 (m, 1H), 7.81 – 7.76 (m, 1H), 7.52 – 7.40 (m, 2H), 7.25 – 7.15 (m, 2H), 7.11 – 7.05 (m, 1H), 7.01 – 6.89 (m, 2H), 3.85 (bs, 3H), 3.44 (dd, J = 7.4, 3.6 Hz, 2H), 2.91 (t, J = 7.7 Hz, 2H), 2.32 (s, 3H); LCMS (ESI): >99% purity; MS m/z , 386 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 13.75 (s, 1H), 10.74 (s, 1H), 9.01 (d, J = 1.3 Hz, 1H), 8.89 (t, J = 5.6 Hz, 1H), 8.39 – 8.28 (m, 2H), 8.07 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 7.00 – 6.89 (m, 4H), 3.49 – 3.41 (m, 2H), 2.92 (t, J = 7.3 Hz, 2H), 2.32 (s, 3H); LCMS (ESI): LCMS (ESI): >97% purity; MS m/z , 372 [M + H] + ; HPLC purity: 97.1%. 1 H NMR (400 MHz, CDCl 3 ): δ 8.89 (d, J = 1.6 Hz, 1H), 8.05 – 7.96 (m, 2H), 7.88 (s, 1H), 7.84 – 7.80 (m, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.44 – 7.37 (m, 1H), 7.31 – 7.25 (m, 2H), 7.19 – 7.07 (m, 3H), 6.21 (bs, 1H), 3.77 (q, J = 6.4 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.39 (s, 3H); LCMS (ESI): >99% purity; MS m/z , 374.0 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.03 (d, J = 1.6 Hz, 1H), 8.79 (t, J = 5.7 Hz, 1H), 8.22 (dd, J = 8.4, 2.3 Hz, 1H), 8.11 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.01 – 6.89 (m, 2H), 3.49 – 3.38 (m, 2H), 2.96 – 2.86 (m, 2H), 2.32 (s, 3H), 2.08 (s, 3H); LCMS (ESI): LCMS (ESI): >95% purity; m/z , 413 [M + H] + ; HPLC purity: 95.8%. 1 H NMR (400 MHz, CDCl 3 ): δ 8.84 (s, 1H), 8.02 (dt, J = 8.3, 2.1 Hz, 1H), 7.92 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.30 (d, J = 7.9 Hz, 1H), 7.18 – 7.05 (m, 4H), 6.55 (q, J = 2.0 Hz, 1H), 6.23 (t, J = 4.8 Hz, 1H), 3.86 (d, J = 1.8 Hz, 6H), 3.77 (q, J = 6.3 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H), 2.39 (d, J = 1.5 Hz, 3H). LCMS (ESI): >99% purity; MS m/z , 416.2 [M + H] + . A solution of boronic acid ( 4a-c ) (4.1 mmol, 1 equiv.) and bromo-acid 9 (4.1 mmol, 1 equiv.) in tetrahydrofuran or toluene and water (6:1) were loaded in to a sealed tube. To this solution, 1M Na 2 CO 3 (8.2 mmol, 2 equiv.) was added and purged with nitrogen for 10 min. Then, Pd(PPh 3 ) 4 (0.2 mmol, 0.05 equiv.) catalyst was added to the reaction mixture, sealed and heated to 100 °C for 12 h. Reaction mixture was cooled to room temperature and solvent was evaporated under vacuum. The residue was washed with dichloromethane to remove organic impurities. Then, aqueous layer was acidified to pH 2 with concentrated HCl to result in white precipitate, which was filtered and dried under vacuum to provide the intermediate s 10a 66 , 10b 66 and 10c ). Often these compounds used for next reaction without purification. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.18 (s, 1H), 9.01 (s, 1H), 8.24 (d, J = 11 Hz, 1H), 8.08 (d, J = 11 Hz, 1H), 7.84 – 7.70 (m, 4 H), 2.08 (s, 3H). LCMS (ESI): >95% purity; MS m/z , 255 [M - H] + . To a solution of 10a-c (0.5 mmol, 1 equiv.) and 2-(2-methyl-1 H -indol-3-yl)ethan-1-amine ( 7 ) (0.5 mmol, 1 equiv.) in N,N- dimethylformamide and dichloromethane (1:1) was added DMAP (catalytic amount) followed by EDCI.HCl (0.65 mmol, 1.3 equiv.) and the reaction mixture was stirred at room temperature for 10 h. Then, dichloromethane was evaporated and the crude reaction mixture was added a saturated solution of ammonium chloride (15 mL) and extracted with ethyl acetate (15 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (15 mL) followed by brine solution (15 mL). Combined organic layer was dried over sodium sulfate, concentrated to dryness. The crude was purified on silica gel chromatography using 50–70% ethyl acetate in hexanes to get the required products ( 11a-c ). 1 H NMR (400 MHz, CDCl 3 ): δ 8.68 (d, J = 2.2 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.17 (t, J = 5.6 Hz, 1H), 8.04 (s, 1H), 8.00 – 7.95 (m, 1H), 7.57 (d, J = 7.4 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.18 – 7.05 (m, 4H), 6.97 (dd, J = 8.3, 2.5 Hz, 1H), 3.86 (s, 3H), 3.72 – 3.75 (m, 2H), 3.04 (t, J = 6.9 Hz, 2H), 2.37 (s, 3H); LCMS (ESI): > 98% purity; MS m/z , 386 [M + H] + . 1 H NMR (400 MHz, CDCl 3 ): δ 8.69 (d, J = 6.8 Hz, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.16 (t, J = 5.9 Hz, 1H), 8.01 – 7.97 (m, 1H), 7.86 (s, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.31 – 7.27 (m, 1H), 7.16 – 7.06 (m, 2H), 6.71 (d, J = 2.2 Hz, 2H), 6.53 (t, J = 2.2 Hz, 1H), 3.86 (s, 6H), 3.74 (q, J = 6.8 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.39 (s, 3H); LCMS (ESI):LCMS (ESI): >97% purity; MS m/z , 416 [M + H] + ; HPLC purity: 97.6%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.69 (s, 1H), 10.14 (s, 1H), 8.87 (d, J = 2.1 Hz, 1H), 8.81 (t, J = 5.9 Hz, 1H), 8.20 (dd, J = 7.4, 3.1 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.66 – 7.78 (m, 4H), 7.58 – 7.43 (m, 1H), 7.20 (d, J = 7.7 Hz, 1H), 6.96 – 6.63 (m, 2H), 3.33 – 3.46 (m, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.29 (s, 3H), 2.04 (s, 3H); LCMS (ESI): LCMS (ESI): >97% purity; MS m/z , 413 [M + H] + . A solution of boronic acid, 4c (4.1 mmol, 1 equiv.) and with 12a-c (4.1 mmol, 1 equiv.) in dioxane and water (6:1) were loaded in to a sealed tube. To this solution, 1M Na 2 CO 3 (8.2 mmol, 2 equiv.) was added and purged with nitrogen for 10 min. Then, Pd(dppf)Cl 2 (0.2 mmol, 0.05 equiv.) catalyst was added to the reaction mixture, sealed and heated to 120 °C for 12 h. Reaction mixture was cooled to room temperature and solvent was evaporated under vacuum. The residue was washed with dichloromethane to remove organic impurities. Then, aqueous layer was acidified to pH 2 with concentrated HCl to result in white precipitate, which was filtered and dried under vacuum to provide the intermediates ( 13a , 67 , 68 13b 69 – 71 and 13c 72 ). To a solution of 13a-c (0.5 mmol, 1 equiv.) and 2-(2-methyl-1 H -indol-3-yl)ethan-1-amine ( 7 ) (0.5 mmol, 1 equiv.) in N,N- dimethylformamide and dichloromethane (1:1) was added DMAP (catalytic amount) followed by EDCI.HCl (0.65 mmol, 1.3 equiv.) and the reaction mixture was stirred at room temperature for 10 h. Then, dichloromethane was evaporated and the crude reaction mixture was added a saturated solution of ammonium chloride (15 mL) and extracted with ethyl acetate (15 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (15 mL) followed by brine solution (15 mL). Combined organic layer was dried over sodium sulfate, concentrated to dryness. The crude was purified on silica gel chromatography using 50–70% ethyl acetate in hexanes to get the required products ( 14a-c ). 1 H NMR (400 MHz, CDCl 3 ): δ 9.00 (s, 2H), 7.92 (s, 1H), 7.74 (dd, J = 7.6, 1.6 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.49 – 7.43 (m, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.17 – 6.99 (m, 4H), 6.25 (s, 1H), 3.86 (s, 3H), 3.75 (q, J = 6.4 Hz, 2H), 3.07 (t, J = 6.5 Hz, 2H), 2.37 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 387 [M + H] + . 1 H NMR (400 MHz, CDCl 3 ): δ 7.85 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.44 −7.26 (m, 4H), 7.22 (d, J = 6.3 Hz, 2H), 7.17 – 7.06 (m, 2H), 7.05 – 6.95 (m, 2H), 6.16 (t, J = 5.3 Hz, 1H), 3.76 (s, 3H), 3.74 – 3.67 (m, 2H), 3.04 (t, J = 6.6 Hz, 2H), 2.38 (s, 3H); LCMS (ESI): >98% purity; MS m/z , 403 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.70 (s, 1H), 8.60 (t, J = 5.9 Hz, 1H), 7.87 – 7.79 (m, 2H), 7.56 – 7.49 (m, 2H), 7.46 (d, J = 7.5 Hz, 1H), 7.37 – 7.28 (m, 2H), 7.22 – 7.17 (m, 1H), 7.11 (dd, J = 5.5, 4.6 Hz, 1H), 7.05 – 6.98 (m, 1H), 6.98 – 6.86 (m, 2H), 3.75 (d, J = 1.9 Hz, 3H), 3.43 – 3.34 (m, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.29 (d, J = 1.9 Hz, 3H); LCMS (ESI): >97% purity; MS m/z , 385 [M + H] + ; HPLC purity: 96%. To a solution of commercially available acid 15 (3 g, 21 mmol, 1 equiv.) in N,N- dimethylformamide (20 mL) was added DMAP (0.78 g, 6.3 mmol, 0.3 equiv.) followed by EDCI.HCl (5.35 g, 28 mmol, 1.3 equiv.) and stirred at room temperature for 10 minutes. Then, 2-(2-methyl-1 H -indol-3-yl)ethan-1-amine ( 7 ) was added to the reaction mixture and stirred at room temperature for 24 h. Reaction mixture was added saturated solution of ammonium chloride (5 mL) and extracted with ethyl acetate (3 × 10 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (5 mL) followed by brine solution. Combined organic layer was dried over sodium sulfate, concentrated to dryness. The crude material was purified on silica gel chromatography using 4–6% methanol in dichloromethane to get the required product 16 as solid (Yield: 59%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.69 (s, 1H), 8.61 (s, 2H), 8.39 (t, J = 5.6 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.22 – 7.17 (m, 1H), 7.16 (s, 2H), 6.97 – 6.85 (m, 2H), 3.41 – 3.27 (m, 2H), 2.82 (t, J = 7.4 Hz, 2H), 2.27 (s, 3H); LCMS (ESI): > 98% purity. MS m/z , 296 [M + H] + . To a solution of 16 (0.5 mmol, 1 equiv.) and 2-bromopyridines ( 17a-o or 18 or 19a-b) (0.5 mmol, 1 equiv.) in dioxane was added Cs 2 CO 3 (1.0 mmol, 2 equiv.). The solution was purged with nitrogen for 10 minutes. Then, Xantphos (0.05 mmol, 0.1 equiv.) was added followed by Pd 2 (dba) 3 catalyst (0.05 mmol, 0.1 equiv.) and heated to 100 °C for 12–18 h. Reaction mixture was cooled to room temperature and added water (10 mL). Resultant solid was filtered and purified on silica gel chromatography using 3–5% methanol in dichloromethane to get the required products 20a-r . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 10.29 (s, 1H), 8.88 (s, 2H), 8.66 (t, J = 5.4 Hz, 1H), 8.29 (d, J = 4.9 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.79 – 7.72 (m, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.06 – 6.99 (m, 1H), 6.97 – 6.86 (m, 2H), 3.35− 3.42 (m, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 373 [M + H] + ; HPLC purity: 98.1%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 10.22 (s, 1H), 8.92 – 8.89 (m, 2H), 8.67 (t, J = 5.7 Hz, 1H), 8.18 (d, J = 5.0 Hz, 1H), 8.08 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.26 – 7.19 (m, 1H), 7.01 – 6.87 (m, 3H), 3.45 – 3.38 (m, 2H), 2.89 (t, J = 7.3 Hz, 2H), 2.34 (s, 3H), 2.32 (s, 3H); LCMS (ESI): > 95% purity; MS m/z, 387 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 10.70 (s, 1H), 8.95 (s, 2H), 8.72 (t, J = 8 Hz 1H), 8.37 – 8.31 (m, 1H), 8.20 – 8.13 (m, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.03 – 6.89 (m, 3H), 3.45− 3.38 (m, 2H), 2.89 (t, J = 6.7 Hz, 2H), 2.31 (d, J = 2.0 Hz, 3H); LCMS (ESI): >97% purity; MS m/z , 391 [M + H] + ; HPLC purity: 98.1%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 10.23 (s, 1H), 8.89 (d, J = 2.8 Hz, 2H), 8.62 (t, J = 5.6 Hz, 1H), 8.27 – 8.30 (m, 1H), 8.21 – 8.17 (m, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.20 (dd, J = 7.8, 0.8 Hz, 1H), 7.05 (dd, J = 5.3, 1.8 Hz, 1H), 6.97 – 6.85 (m, 2H), 3.42 – 3.33 (m, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H), 1.29 – 1.24 (m, 9H); LCMS (ESI): >97% purity; MS m/z , 429 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 10.17 (s, 1H), 8.90 (d, J = 2.1 Hz, 2H), 8.67 (t, J = 5.3 Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.00 – 6.88 (m, 3H), 3.45− 3.38 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H), 2.41 (s, 3H), 2.31 (s, 3H); LCMS (ESI): >98% purity; MS m/z , 387 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 10.61 (s, 1H), 8.95 – 8.91 (m, 2H), 8.73 (t, J = 5.7 Hz, 1H), 8.22 – 8.17 (m, 1H), 7.97 (q, J = 8.4 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.25 – 7.20 (m, 1H), 7.01 – 6.89 (m, 2H), 6.77 (dd, J = 7.8, 2.4 Hz, 1H), 3.45 – 3.37 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H), 2.31 (s, 3H); LCMS (ESI): >97% purity. MS m/z , 391 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.89 (s, 1H), 10.74 (s, 1H), 8.95 (s, 2H), 8.74 (t, J = 4.8 Hz, 1H), 8.60 – 8.54 (m, 1H), 8.02 (t, J = 8.1 Hz, 1H), 7.71 – 7.61 (m, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.00 – 6.89 (m, 2H), 3.42 (dd, J = 12.5, 6.2 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H), 2.31 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 398 [M + H] + ; HPLC purity: 97%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.74 (s, 1H), 10.07 (s, 1H), 8.91 (s, 2H), 8.68 (t, J = 5.7 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.01 – 6.87 (m, 2H), 6.46 (d, J = 7.8 Hz, 1H), 3.85 (s, 3H), 3.45 – 3.37 (m, 2H), 2.89 (t, J = 7.1 Hz, 2H), 2.32 (s, 3H); LCMS (ESI): >96% purity; MS m/z , 403 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 12.01 (s, 1H), 10.74 (s, 2H), 8.95 (s, 2H), 8.74 (t, J = 5.5 Hz, 1H), 7.50 – 7.40 (m, 2H), 7.23 (d, J = 8.2 Hz, 1H), 7.01 – 6.87 (m, 2H), 6.41 (bs, 1H), 5.98 (d, J = 8.8 Hz, 1H), 3.46 – 3.38 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H), 2.31 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 389 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 10.49 (s, 1H), 8.91 (s, 2H), 8.73 – 8.63 (m, 1H), 8.41 (d, J = 9.2 Hz, 1H), 7.96 (t, J = 7.2 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.98 – 6.82 (m, 2H), 3.39 (dd, J = 13.5, 6.7 Hz, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.60 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 415 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 10.61 (s, 1H), 8.92 (s, 2H), 8.71 – 8.60 (m, 2H), 8.49 (d, J = 5.1 Hz, 1H), 7.54 – 7.37 (m, 2H), 7.20 (d, J = 7.7 Hz, 1H), 6.92 (dd, J , 14.1, 7.1 Hz, 2H), 3.39 (dd, J = 13.9, 6.5 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 2.60 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 415 [M + H] + ; HPLC purity: 96.2%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.87 (s, 1H), 10.72 (s, 1H), 8.94 (s, 2H), 8.88 (d, J = 2.4 Hz, 1H), 8.72 (t, J = 5.7 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.27 (dd, J = 8.9, 2.3 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 6.92 (dt, J = 14.6, 7.0 Hz, 2H), 3.39 (dd, J = 13.4, 6.6 Hz, 2H), 2.87 (t, J = 7.3 Hz, 2H), 2.54 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >98% purity; MS m/z , 415 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 10.06 (s, 1H), 8.88 (s, 2H), 8.65 ( t , J = 5.7 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.74 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.20 (dd, J = 7.7, 3.2 Hz, 2H), 6.91 (ddd, J = 14.7, 13.6, 6.2 Hz, 2H), 5.07 (s, 1H), 3.38 (dd, J = 13.5, 7.0 Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H), 1.87 – 1.58 (m, 2H), 1.37 (s, 3H), 0.64 (t, J = 7.4 Hz, 3H); LCMS (ESI): >98% purity; MS m/z , 445 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s, 1H), 9.87 (s, 1H), 8.88 (d, J = 6.0 Hz, 2H), 8.64 (t, J = 5.6 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.20 (d, J = 7.3 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 7.02 – 6.80 (m, 2H), 3.43 – 3.34 (m, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.29 (s, 3H), 1.30 – 1.25 (m, 9H); LCMS (ESI): >97% purity; MS m/z , 429 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.74 (s, 1H), 10.08 (s, 1H), 8.90 (s, 2H), 8.65 (t, J = 5.5 Hz, 1H), 7.92 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 6.95 (dt, J = 20.4, 7.3 Hz, 2H), 6.76 (s, 1H), 3.45 – 3.37 (m, 2H), 2.89 (t, J = 7.3 Hz, 2H), 2.37 (s, 3H), 2.32 (s, 3H), 2.30 (s, 3H); LCMS (ESI): >95% purity. MS m/z , 401 [M + H] + ; HPLC purity: 96.4%. To a solution of 20o (500 mg, 1.25 mmol, 1 equiv.) in dichloromethane (5 mL) was added 4M HCl in dioxane (0.62 mL, 2.5 mmol, 2 equiv.) at 0 °C and allowed to stir at room temperature for 12 h. The precipitated solid was filtered and washed with dichloromethane (5 mL) followed by ethyl acetate (5 mL) and dried to get the required salt, 20o.HCl (Yield: 86%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.95 (s, 1H), 10.79 (s, 1H), 9.13 (s, 2H), 8.99 (t, J = 5.3 Hz, 1H), 7.61 (s, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.18 (s, 1H), 7.00 – 6.88 (m, 2H), 3.66 (bs, 1H), 3.44 (q, J = 6.7 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H), 2.62 (s, 3H), 2.47 (s, 3H), 2.32 (s, 3H); LCMS (ESI): >97% purity. MS m/z , 401 [(M – HCl) + H] + ; HPLC purity: 99%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.78 (d, J = 2.3 Hz, 1H), 9.34 – 9.29 (m, 4H), 9.22 – 9.14 (m, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.08 – 7.01 (m, 2H), 6.97 – 6.82 (m, 2H), 3.46 – 3.40 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.29 (d, J = 2.0 Hz, 3H); LCMS (ESI): > 97% purity. MS m/z , 373 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.70 (s, 1H), 10.26 (s, 1H), 8.91 (s, 1H), 8.88 (s, 2H), 8.62 (t, J = 5.5 Hz, 1H), 8.26 – 8.18 (m, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.35 (dd, J = 8.2, 4.8 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.00 – 6.87 (m, 2H), 3.48 – 3.35 (m, 2H), 2.89 (t, J = 7.3 Hz, 2H), 2.32 (s, 3H); LCMS (ESI): >97% purity. MS m/z , 373 [M + H] + ; HPLC purity: 97.6%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.73 (s, 1H), 9.44 (s, 1H), 8.75 (s, 2H), 8.55 (t, J = 5.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 7.00 – 6.88 (m, 2H), 3.43 – 3.35 (m, 2H), 2.87 (t, J = 7.3 Hz, 2H), 2.42 (s, 3H), 2.35 (s, 3H), 2.31 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 401[M + H] + ; HPLC purity: 97.3%. To a solution of 21 (250 mg, 1.6 mmol, 1 equiv.) and 17o (35 mg, 1.6 mmol, 1 equiv.) in dioxane (5 mL) were added Cs 2 CO 3 (1.1 g, 3.26 mmol, 2 equiv.) followed by BINAP (100 g, 0.16 mmol, 0.1 equiv.). Reaction mixture was purged with nitrogen for 10 minutes and added Pd(OAc) 2 (36 mg, 0.16 mmol, 0.1 equiv.) and heated to 100 °C for 48 h. Reaction mixture was cooled to room temperature and added water and filtered the solid, which was purified on column chromatography using 30–40% ethyl acetate in hexanes to get the required compound 22 . 1 H NMR (400 MHz, CDCl 3 ): δ 11.75 (s, 1H), 9.12 (s, 2H), 8.62 (s, 1H), 6.84 (s, 1H), 4.50 – 4.28 (m, 2H), 2.70 (s, 3H), 2.54 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H); LCMS (ESI): > 94% purity. MS m/z , 273 [M + H] + . To a solution of 22 (100 mg, 0.38 mmol, 1 equiv.) in tetrahydrofuran and water (7:3, 5 mL) was added LiOH.H 2 O (46 mg, 1.14 mmol, 3 equiv.) and heated to 60 °C for 12 h. Reaction mixture was brought to room temperature and acidified with 1N HCl and extracted with ethyl acetate (20 mL). Organic layer was concentrated to dryness to obtain the required acid 23 . 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.54 (s, 1H), 8.77 (s, 2H), 7.92 (s, 1H), 6.67 (s, 1H), 2.31 (s, 3H), 2.25 (s, 3H); LCMS (ESI): > 96% purity; MS m/z , 243 [M - H] + . To a solution of 23 (0.61 mmol, 1 equiv.) and compound 7a-i (0.61 mmol, 1 equiv.) in N,N- dimethylformamide (5 mL) was added DMAP (catalytic amount) followed by EDCI.HCl (0.78 mmol, 1.3 equiv.) and the reaction mixture was stirred at 50 °C for 24–48 h. Reaction mixture was brought to room temperature and added saturated solution of ammonium chloride (10 mL) and extracted with ethyl acetate (10 mL). Organic layer was separated and washed with saturated solution of sodium bicarbonate (10 mL) followed by brine solution (10 mL). Combined organic layer was dried over sodium sulfate, concentrated to dryness. The crude was purified on silica gel chromatography using 5–7% methanol in dichloromethane to get the required products ( 24a-i ). 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.85 (s, 1H), 10.06 (s, 1H), 8.88 (s, 2H), 8.63 (t, J = 5.8 Hz, 1H), 7.91 (s, 1H), 7.22 – 7.17 (m, 2H), 6.83 – 6.74 (m, 2H), 3.43 – 3.35 (m, 2H), 2.85 (t, J = 7.3 Hz, 2H), 2.36 (s, 3H), 2.31 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >96% purity; MS m/z , 419 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.97 (s, 1H), 10.09 (s, 1H), 8.89 (s, 2H), 8.63 (t, J = 4.8 Hz, 1H), 7.92 (s, 1H), 7.48 (s, 1H), 7.23 (d, J = 8.5 Hz, 1 H) 7.08 – 6.88 (m, 1H), 6.76 (s, 1H), 3.44 – 3.37 (m, 2H), 2.91 – 2.82 (m, 2H), 2.36 (s, 3H), 2.31 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >95% purity; MS m/z , 435 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.32 (s, 1H), 10.09 (s, 1H), 8.88 (s, 2H), 8.62 (t, J = 5.4 Hz, 1H), 7.91 (s, 1H), 7.11 (dd, J = 9.6, 1.8 Hz, 1H), 6.82 (t, J = 10.5 Hz, 1H), 6.76 (s, 1H), 3.44 – 3.36 (m, 2H), 2.86 (t, J = 7.0 Hz, 2H), 2.37 (s, 3H), 2.32 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 437 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.29 (s, 1H), 10.04 (s, 1H), 8.82 (s, 2H), 8.56 (t, J = 6.1 Hz, 1H), 7.92 (s, 1H), 7.45 (s, 1H), 7.09 (s, 1H), 6.73 (s, 1H), 3.36 (dd, J = 12.0, 5.3 Hz, 2H), 2.85 – 2.81 (m, 2H), 2.69 (s, 3H), 2.33 (s, 3H), 2.26 (s, 3H); LCMS (ESI): >95% purity; MS m/z , 469 [M + H] + ; HPLC purity: 95.5%. 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.57 (s, 1H), 10.07(s, 1H), 8.89 (s, 2H), 8.64 (t, J = 5.6 Hz, 1H), 7.91 (s, 1H), 7.11 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 6.76 (s, 1H), 6.61 (dd, J = 8.6, 2.3 Hz, 1H), 3.71 (s, 3H), 3.46 – 3.35 (m, 2H), 2.85 (t, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.29 (bs, 6H); LCMS (ESI): >96% purity; MS m/z , 431 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.97 (s, 1H), 10.07 (s, 1H), 8.86 (bs, 2H), 8.71 (t, J = 5.7 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.32 – 7.25 (m, 1H), 7.16 – 7.08 (m, 1H), 6.76 (s, 1H), 3.53 – 3.44 (m, 2H), 3.11 (t, J = 6.8 Hz, 2H), 2.36 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >97% purity; MS m/z , 455 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.03 (s, 1H), 8.81 (s, 2H), 8.59 (t, J = 5.6 Hz, 1H), 8.45 (dd, J = 7.0, 0.8 Hz, 1H), 7.86 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.13 – 6.97 (m, 1H), 6.77 – 6.64 (m, 2H), 3.42 – 3.37 (m, 2H), 2.87 (t, J = 6.1 Hz, 2H), 2.33 (s, 3H), 2.30 (s, 3H), 2.25 (s, 3H); LCMS (ESI): >96% purity; MS m/z , 402 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 11.97 (s, 1H), 10.10 (s, 1H), 8.97 (s, 2H), 8.82 (t, J = 6.6 Hz, 1H), 8.05 – 7.95 (m, 1H), 7.91 (s, 1H), 7.46 – 7.33 (m, 1H), 7.21 – 7.10 (m, 2H), 6.74 (s, 1H), 4.72 – 4.53 (m, 2H), 2.71 (s, 3H), 2.34 (s, 3H), 2.27 (s, 3H); LCMS (ESI): >98% purity; MS m /z, 415 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.77 (s, 1H), 10.06 (s, 1H), 8.63 (t, J = 8 Hz, 1H), 8.90 (s, 2H), 7.91 (s, 1H), 7.65 (d, J = 8 Hz, 1H), 7.26 – 7.20 (m, 1H), 7.00 – 6.88 (m, 2H), 6.76 (s, 1H), 5.21 (d, J = 3.3 Hz, 1H), 5.09 – 5.03 (t, J = 8.2 Hz, 1H), 3.66 – 3.44 (m, 2H), 2.36 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H); LCMS (ESI): >96% purity; MS m/z , 417 [M + H] + . The rat C6 glioma (C6G) cells stably expressing human DP1, EP2, EP4, or IP receptors were created in the laboratory 19 , 30 , 73 and grown in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin (Invitrogen), and 0.5 μg/mL G418 (Invitrogen). Intracellular cAMP was measured with a cell-based homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) method (Cisbio Bioassays), as previously described. 19 , 30 The assay is based on generation of a strong FRET signal upon the interaction of two molecules, an anti-cAMP antibody coupled to a FRET donor (Cryptate) and cAMP coupled to a FRET acceptor (d2). Endogenous cAMP produced by cells competes with labeled cAMP for binding to the cAMP antibody and thus reduces the FRET signal. Cells stably expressing human DP1, EP2, EP4, or IP receptors were seeded into 384-well plates in 30 μL complete medium (4,000 cells/well) and grown overnight. The medium was carefully withdrawn and 10 μL Hanks’ Buffered Salt Solution (HBSS) (Hyclone) containing 20 μM rolipram was added into the wells to block phosphodiesterases. The cells were incubated at room temperature for 0.5–1 h and then treated with vehicle or test compound for 10 min before addition of increasing concentrations of appropriate agonist: BW245C for DP1, PGE 2 for EP2 and EP4, or iloprost for IP. The cells were incubated at room temperature for 40 min, then lysed in 10 μL lysis buffer containing the FRET acceptor cAMP-d2 and 1 min later another 10 μL lysis buffer with anti-cAMP-Cryptate was added. After 60–90 min incubation at room temperature, the FRET signal was measured by an Envision 2103 Multilabel Plate Reader (PerkinElmer Life Sciences) with a laser excitation at 337 nm and dual emissions at 665 nm and 590 nm for d2 and Cryptate (50 μs delay), respectively. The FRET signal was expressed as: F665/F590 × 10 4 . Stable BV2-hEP2 microglia cells were created in the lab 54 and were grown overnight on poly-D-lysine coated 12 well plates at 200,000 cells per well in culture media. The cells were exposed to the test compounds 20o or others (0.3 μM or 1 μM) for 1 h, and EP2 selective agonist ONO-AE1-259-01 (30 nM) for an additional hour and subsequently LPS (100 ng/mL) for 2 h. All compounds were dissolved in DMSO and diluted in media just prior to cell treatment. Following incubation, media was removed from the wells and the cells were subjected to RNA extraction and purification using Trizol and the Zymo Research Quick-RNA miniprep kit according to the manufacturer’s protocol (Genesee Scientific). First-strand cDNA synthesis, qRT-PCR and analysis was performed using the primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a single internal control for relative quantification to determine whether EP2 activation modulates expression of inflammatory mediators in BV2-hEP2 microglia ( Figure 5 , 6 ). PCR gene expression data are presented as the mean fold change of each gene of interest in the compound treated groups compared to vehicle.

Neuroinflammation has emerged as a key feature that exacerbates chronic neurodegenerative pathology in several central nervous system diseases including epilepsy, Alzheimer’s disease (AD) and Parkinson’s disease. 1 – 4 Neuroinflammation is also an early event that plays a deleterious role in status epilepticus and traumatic brain injury. 5 , 6 Therefore, anti-inflammatory agents should offer a benefit to patients with these central nervous system (CNS) diseases. Production of inflammatory cytokines (interleukins) and chemokines, activation of glia, and induction of cyclooxygenase-2 are the early features of neuroinflammation. 7 , 8 Thus, there is an excellent opportunity for therapeutic discovery targeting the mediators of neuroinflammatory signaling. A significant body of evidence suggests that neuroinflammation precedes the appearance of amyloid-β plaques and neurofibrillary tangles, which themselves precede clinical signs of AD in animal models and patients. 9 Among the neuroinflammatory inducers, COX-2 has been very well studied thus far in AD patients by using small molecule inhibitors of COX-2. 10 – 12 Although retrospective epidemiological studies using non-selective COX-2 inhibitors suggested that chronic use of COX-2 inhibitors might prevent or delay the onset of Alzheimer’s disease in patients, prospective studies concluded that selective COX-2 drugs do not provide a clear benefit for Alzheimer’s patients. 13 Reconciliation of the epidemiologic studies with prospective clinical trial results might be found in the clinical trial design, in which the treatment of patients was initiated when the disease is already at advanced stages in terms of classical pathological markers β-amyloid plaques and neurofibrillary tangles, or insufficient duration of treatment. The prevailing hypothesis emerging from clinical trials of a COX-2 drug is that treatment of patients starting at the prodromal stage of disease could offer benefit, but not treatment starting at late stages of the disease. Testing this proof of concept requires administration of a drug for several months (in animals) to years (in human) before clinical dementia symptoms are diagnosed. Regrettably, the COX-2 inhibitors cause adverse cardiovascular effects upon chronic use, by blunting PGI 2 synthesis. 14 , 15 Two selective COX-2 inhibitors rofecoxib (Vioxx) and valdecoxib (Bextra) were withdrawn from the USA market, but a less selective COX-2 inhibitor, celecoxib (Celebrex), is in the clinic with black-box warning for thrombotic cardiotoxicity. 16 Thus, future anti-inflammatory therapy within the COX-2 pathway should be targeted through a specific proinflammatory prostanoid synthase or a receptor downstream of COX-2, rather than blocking the entire COX-2 signaling pathways. 17 , 18 COX-2 catalyzes the synthesis of five prostanoids, PGD 2 , PGE 2 , PGF 2 , PGI 2 and TXA 2 . These prostanoids activate nine receptors, DP1, DP2, EP1, EP2, EP3, EP4, FP, IP and TP respectively. Each of these receptors can play protective as well as deleterious roles in CNS and peripheral pathophysiologies. 17 , 18 The EP2 receptor has emerged as a predominantly proinflammatory receptor that mediates much of the COX-2 driven inflammatory consequences. 19 , 20 When activated by PGE 2 , EP2 receptors stimulate adenylate cyclase resulting in elevation of cytoplasmic cAMP concentration, which initiates downstream events via cell signaling pathways mediated by protein kinase A or exchange protein activated by cAMP (Epac). 17 , 18 Genetic ablation of the EP2 receptor reduces oxidative damage and amyloid-β burden in a mouse model of AD. 21 EP2 deletion also attenuates neurotoxicity and reduces α-synuclein aggregation in a mouse model of PD. 22 Moreover, EP2 deletion improves motor strength and survival of the ALS mouse. 23 Furthermore, PGE 2 signaling via EP2 receptor increases injury in models of cerebral ischemia, 24 and mice lacking EP2 receptors have shown less cerebral oxidative damage produced by the activation of innate immunity. 25 EP2 activation promotes inflammation and neurotoxicity in models of status epilepticus induced by pilocarpine and diisopropylfluorophosphate (DFP). 19 , 26 , 27 In vitro, microglia cultures from mice lacking EP2 have shown enhanced amyloid-β phagocytosis and are less sensitive to amyloid-β induced neurotoxicity. 28 Although these results strongly support the notion that pharmacological inhibition of EP2 should be explored in preclinical and clinical setting, the currently available EP2 antagonists have sub-optimal drug-like features such as low in vivo metabolic stability, in vivo plasma half-life and aqueous solubility for chronic dosing and proof-of-concept testing in preclinical AD models. Initially Pfizer, 29 our laboratory 30 , 31 and recently Amgen 32 identified four distinct classes of EP2 antagonists ( Figure 1A ). Only the Amgen compounds seems to be useful for chronic dosing, but the derivatives developed in Pfizer and in our laboratory displayed too low liver metabolic stability, plasma half-life, or brain permeability to be useful in chronic CNS disease models such as Alzheimer’s disease. We initially identified a cinnamic amide compound TG4–155 ( Figure 1A ) from high-throughput screening as a potent EP2 antagonist. 30 Structure activity relationship study of this compound resulted in the first generation brain-permeable lead compound 1 (TG6-10-1, Figure 1B ), which displays brain-to-plasma ratio of 1.7, but only 10-fold selectivity for EP2 over DP1, low aqueous solubility (27 μM) and moderate plasma half-life (1.7 h). 33 Thus, developing a selective, aqueous-soluble compound with enhanced plasma half-life while maintaining potency and brain permeability is the main goal of our project. In the present study, we report further lead-optimization studies that led to the synthesis of several second-generation analogs and show that improvements are made in terms of selectivity, aqueous solubility and in vivo plasma half-life. Moreover, we developed a compound 20o.HCl (named TG11–77.HCl), which is water-soluble (2.52 mM), yet crosses the blood-brain-barrier with brain-to-plasma ratio of 0.4. Furthermore, several novel compounds in this class, including 20o.HCl exhibited anti-inflammatory activity in EP2-expressing microglia-derived BV2 cell cultures in vitro. Therefore, compound 20o.HCl and other derivatives in this class are now suitable candidates for dosing into - disease models such as AD.