Discovery of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5
Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu5 negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu5 NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu5 assay, and an exemplar analog 27 was more than 60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.
<p>Glutamate is the major excitatory transmitter in the mammalian central nervous system and activates both ionotropic (iGlu) and metabotropic glutamate (mGlu) receptors. While the iGlu receptors are ligand-gated ion-channels, the mGlu receptors are a family of eight G protein-coupled receptors (GPCRs). Based on their structure, function, and effects on downstream signaling pathways, the mGlu receptors have been classified into three groups. Group I receptors (mGlu1 and mGlu5), which are found in postsynaptic locations, are coupled via Gq to the activation of phospholipase C. On the other hand, group II receptors (mGlu2-3), which are found both pre and postsynaptically, and group III receptors (mGlu4, mGlu6-8), which are found primarily in presynaptic locations, are coupled via Gi/o to the inhibition of adenylyl cyclase activity. The orthosteric glutamate binding site of the mGlu receptors is contained in the extracellular domain; however, the allosteric binding sites that have been discovered to date are found in the transmembrane domain.1,2</p><p>The design of drug-like orthosteric ligands that are highly selective for a single mGlu receptor versus the other seven mGlu family members can be quite challenging. One method for overcoming this hurdle that has proven successful for a variety of mGlu receptors has been the use of approaches focused on compounds that interact with an allosteric site.3 Among these efforts, the design and optimization of negative allosteric modulators (NAMs) of mGlu5 has been pursued extensively.4 Likewise, preclinical and clinical studies have indicated many potentially interesting therapeutic applications for mGlu5 NAMs.1–4 In fact, phase II clinical trials have been conducted with small molecule mGlu5 NAMs in fragile X syndrome (FXS),5,6 major depressive disorder (MDD),7 obsessive-compulsive disorder (OCD),8 and Parkinson's disease levodopa-induced dyskinesia (PD-LID).9,10 Regrettably, in most instances, the primary efficacy endpoints were not met;5–9 however, some secondary efficacy endpoints have been noted in both MDD7 and PD-LID.10</p><p>Small molecule mGlu5 NAM research has been a major focus of our group and recently culminated in the identification of a highly optimized mGlu5 NAM candidate for clinical evaluation, VU0424238 (1) (Fig. 1),11 a member of a series of picolinamide mGlu5 NAMs. We also recently reported on the discovery of an additional backup series of heterocyclic mGlu5 NAMs exemplified by [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide 2.12 The discovery of 2 was based on a hypothesis that an internal hydrogen bond between the amide NH and the 1-nitrogen of the heteroaryl core of 2 would help orient the molecule similarly to the postulated conformation of VU0424238 (1), where an analogous hydrogen bond between the amide NH and the picolinamide nitrogen was assumed likely. The discovery of 2 and its analogs opened the door to investigate other heterocyclic cores such as quinoline-8-carboxamides. In fact, we synthesized compound 3, a quinoline analog of 2, and verified that it was similarly potent versus mGlu5. Still, to further enhance the novelty of such new compounds and explore new SAR, we also sought to modify additional portions of the scaffold (Fig. 1). Specifically, we chose to replace the oxygen linker that had been constant across the chemotypes found in both 1 and 2 as well as other related series13,14 by exchanging it with several carbon linkers. This Letter describes our efforts toward that end.</p><p>Synthetic work on the aforementioned ether analogs had employed nucleophilic aromatic substitution (SNAr) as the means for joining the northern heteroaryl ring to the core of the scaffold.11–14 Our plan to move to a carbon linker would thus necessitate a substantially different synthetic approach (Scheme 1).15 Our first targets were a small set of ketone-linker containing analogs 11–14. 6-Bromo-8-chloroquinoline 4 was converted to vinyl analog 5 via a Suzuki cross-coupling reaction with potassium vinyltrifluoroborate.16 Ozonolysis of 5 followed by treatment with dimethyl sulfide afforded aldehyde 6. Metal-halogen exchange of 5-bromopyrimidine was carried out at low temperature, and the resultant organolithium intermediate was treated with 6
in situ to yield secondary alcohol 7. Oxidation of 7 was accomplished with manganese oxide to provide ketone 8. A palladium-catalyzed reaction with zinc cyanide gave nitrile 9, and subsequent acidic hydrolysis with heating gave the penultimate acid 10. Formation of amide analogs 11–14 was accomplished with phosphorous oxychloride in pyridine, conditions that we have employed successfully in the past.11–14</p><p>Monofluoromethylene linker analogs 20–21 and difluoromethylene linker analogs 26–36 were prepared from intermediate 8 (Scheme 2). Suzuki cross-coupling reaction with potassium vinyltrifluoroborate as before afforded vinyl intermediate 15. Reduction of the ketone was accomplished with sodium borohydride to provide alcohol 16, which was treated with diethylaminosulfur trifluoride (DAST) to yield 17. Ozonolysis of 17 followed by treatment with dimethyl sulfide afforded aldehyde 18, which was subsequently oxidized to penultimate acid 19 with sodium chlorite. Conversion of 19 to amides 20–21 was carried out as previously described. Preparation of difluoromethylene linker analogs 26–36 was analogous; however, the route sequence was modified such that conversion of ketone 8 to difluoromethylene intermediate 22 was the initial step in the synthesis.</p><p>Synthesis of analogs with methoxy (47–48) and methyl (51–52) substituents at the methylene linker employed an alternative method for joining the northern heteroaryl ring to the quinoline core (Scheme 3). Here, a Suzuki cross-coupling reaction between potassium vinyltrifluoroborate and 5-bromopyrimidine 37 was used to access vinyl intermediate 38. Bromination of the olefin, followed by treatment with 1,8-diazabicycloundec-7-ene (DBU), gave vinyl bromide 39. Meanwhile, a palladium-catalyzed coupling of methyl 6-bromoquinoline-8-carboxylate 40 with bis(pinacolato)diboron provided boronic ester 41.17 A Suzuki coupling between 39 and 41 was used to join the northern pyrimidine ring to the quinoline core and gave intermediate 42. Ozonolysis of 42 followed by treatment with dimethyl sulfide afforded ketone 43, which was reduced to alcohol 44 with sodium borohydride. Alcohol 44 was methylated with potassium bis(trimethylsilyl) amide and methyl iodide to yield 45. Treatment of 45 with aqueous lithium hydroxide gave penultimate acid 46, which was converted to analogs 47 and 48 as previously described. Intermediate 42 was also subjected to a palladium catalyzed hydrogenation to afford reduced intermediate 49, which was subsequently converted to analogs 51 and 52 utilizing previously outlined chemistry.</p><p>Ketone linker analogs 11–14 were the first compounds from this work to be evaluated in our functional assay that measures the inhibition of Ca2+ mobilization induced by an EC80 concentration of glutamate in HEK293A cells expressing rat mGlu5 (Table 1).18 The heteroaryl amides were chosen as these had proven effective for engendering mGlu5 potency in other similar chemotypes.11,12,14 Interestingly, in this series, substitution of the pyridin-2-yl amide proved critical for potency; both 12 and 13 were more than 10-fold more active than 11. The most potent analog in this set was 4-methylthiazol-2-yl amide 14.</p><p>With the knowledge that ketones can be metabolically unstable, we decided to study that possibility with 14. Incubation of 14 in rat whole blood at 37 °C for one hour showed near complete conversion to a metabolite thought to be the result of ketone reduction (Fig. 2). To verify this result, the secondary alcohol 53 was prepared through sodium borohydride reduction of 14. Indeed, alcohol 53 was confirmed as the major metabolite from the rat whole blood stability study. To determine if alcohol 53 might be an active metabolite, it was also tested in the mGlu5 functional assay; however, it was only a weak antagonist (IC50 > 10 μM). Finally, to verify if this metabolic event occurred in vivo, a rat pharmacokinetics (PK) study was conducted with 14 using intravenous (IV) dosing (Table 2). Not surprisingly, compound 14 was consumed quite rapidly with a corresponding appearance of metabolite 53 in the plasma. With such data in hand, we were keen to evaluate non-ketone carbon linkers.</p><p>Table 3 presents the SAR for four new carbon linkers in the context of the 5-fluoropyridin-2-yl and 4-methylthiazol-2-yl amides, groups that had demonstrated activity in this series and other favorable properties in related historical series.11,12,14 Both the monofluoromethylene analogs 20–21 and difluoromethylene analogs 26–27 demonstrated potency on par with ketone comparators 13 and 14 (Table 1). On the other hand, larger substituents on the methylene carbon in the form of methoxy (47–48) and methyl (51–52) negatively impacted mGlu5 activity. Analogs 20–21 and 47–52 were tested as racemates. With the most potent compound (27) bearing the difluoromethylene linker, we prepared and tested several additional amide derivatives with that linker (Table 4). SAR here showed little tolerance for further substitution as all new pyridin-2-yl analogs (28–35) were less potent than 5-fluoropyridin-2-yl derivative 26. Likewise, increasing the size of the 4-substituent on the thiazol-2-yl amide from methyl (27) to cyclopropyl (36) led to a 3.5-fold drop in mGlu5 potency.</p><p>We have previously demonstrated that an unsubstituted pyrimidin-5-yl ether moiety is a potential site for aldehyde oxidase (AO) and/or xanthine oxidase (XO)-mediated metabolism.11,20–22 Since predicting human pharmacokinetics for compounds that are largely metabolized by AO and/or XO can be challenging due to their variable expression across preclinical species,20,23 we were interested in profiling some of these new compounds to understand what fraction of their metabolism was mediated by P450 versus non-P450 mechanisms. Thus, we evaluated 21 and 27 in metabolic stability assays in rat and human liver S9 hepatic fractions both with and without the addition of NADPH (Table 5).24 Since NADPH is required for cytochrome P450-mediated oxidations, experiments that lack NADPH provide an assessment of non-P450-mediated metabolism. Whereas VU0424238 (1) was metabolized almost exclusively by AO/XO,20
21 and 27 demonstrated a mixture of P450- and non-P450-mediated metabolism. Interestingly, while 21 exhibited a higher percentage of P450-mediated metabolism in rat S9 than in human S9 hepatic fractions, the profile for 27 was similar across species. Such results highlight how subtle the SAR can be for P450 versus non-P450-mediated metabolism. Of note, while these experiments do not conclusively identify AO/XO as the specific mechanism for the non-P450 mediated component of metabolism, our prior studies with related unsubstituted pyrimidin-5-yl ethers20–22 suggest AO/XO involvement.</p><p>Since analog 27 was the most potent analog in this series, we chose to profile that compound further (Table 6). Thus, selectivity was assessed versus the other seven mGlu receptors utilizing fold-shift experiments that assess the effect of 10 μM 27 on the concentration response curves (CRC) of an orthosteric agonist in cell lines expressing each individual mGlu receptor.25 Compound 27 was inactive versus the other seven mGlu receptor subtypes, indicating a selectivity for mGlu5 of at least 60-fold. An analogous fold-shift experiment was conducted in HEK293A cells expressing human mGlu5, and not surprisingly, 10 μM 27 fully blocked the glutamate CRC in that cell line. This result was consistent with prior studies on compounds from related chemotypes where the difference between rat and human mGlu5 activity was negligible.11,14 The extent to which 27 was bound to proteins was also assessed, and the compound was highly bound in both rat (fu = 0.015) and human (fu = 0.014) plasma.26 Finally, a P450 inhibition profile was determined for 27, and the compound was a moderate inhibitor of CYP3A4 and CYP2C9 and a potent inhibitor of CYP1A2.26</p><p>When compared to clinical candidate VU0424238 (1) or backup analog 2, compound 27 does not offer as attractive of an overall profile (Table 6), and significant room for optimization within this series remains. Nonetheless, the work described herein demonstrates that a variety of single carbon linkers, including both sp2 and sp3 hybridized carbons, can be accessed from common intermediates and function as competent isosteric replacements for diaryl ethers. In addition, the utility of the quinoline core is consistent with our previously published work utilizing nitrogen-containing fused heterocycles12 as replacements for the picolinamide core of our lead series. Finally, it is worth noting that such trends may be somewhat general with the potential to translate to other chemotypes designed to interact with a variety of targets.</p>