Discovery of a novel, CNS penetrant M4 PAM chemotype based on a 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core
This Letter details the discovery and subsequent optimization of a novel M4 PAM scaffold based on an 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which represents a distinct departure from the classical M4 PAM chemotypes. Optimized compounds in this series demonstrated improved M4 PAM potency on both human and rat M4 (4 to 5-fold relative to HTS hit), and displayed attractive physicochemical and DMPK profiles, including good CNS penetration (rat brain:plasma Kp = 5.3, Kp,uu = 2.4; MDCK-MDR1 (P-gp) ER = 1.1).
<p>While M4 positive allosteric modulators (PAMs) hold great therapeutic promise for schizophrenia1–4 and a number of other CNS disorders,5,6 a lack of translatable chemotypes (and very limited structural diversity within known chemotypes) has hampered the field from realizing the true therapeutic potential of selective M4 activation. The area has been dominated by differentially functionalized 3-amino–thieno[2,3-b]pyridine cores, typified by 1, or 5-amino-3,4-dimethylthieno[2,3-c]pyridazine such as 2 (Figure 1).7–16 Multiple HTS campaigns revealed similar chemotypes, with the exception of 3 and 4;17,18 however, these also failed to progress to development candidates. However, 4 (based on a 5,6-dimethyl-4-(piperidin-1-yl)thieo[2,3-d]pyrimidine core) was optimized to 5, which demonstrated exceptional CNS penetration.18 With the goal of arriving at a development candidate, we recently reported on a scaffold hopping exercise that afforded 6.19 In this Letter, we will discuss the synthesis, SAR and DMPK profiles of M4 PAMs based on a novel 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which demonstrated robust SAR beyond that of related 4–6, and holds promise for translation.</p><p>Recently, we performed a new functional M4 high-throughput screen (HTS) in 1536-well assay format against the ~100,000 compound Vanderbilt collection. Of the four structurally novel M4 PAMs that were identified, VU0619856 (7) represented an exciting new chemotype for the M4 field, and while related to 4 and 5, M4 PAM potency peaked at ~600 nM in these earlier series.18 New M4 PAM 7 was active on both rat and human (EC50s of 2565 nM and 897 nM respectively) with promising points of diversity. The initial strategy was to utilize an iterative parallel synthesis to explore the amide portion of the molecule followed by evaluation of the 3-cyano quinoline core. Would the 3-cyano quinoline be a quinazoline mimetic as in 5, or would SAR diverge?</p><p>The synthesis of analogs of M4 PAM 7, congeners 11 (secondary amides) and 12 (tertiary amides), was straightforward (Scheme 1), and employed a diverse collection of amines (benzyl, aryl/heteroaryl amines and secondary amines). Starting from commercial 4-chloro-6-fluoroquinoline-3-carbonitrile 8, an SNAr with ethyl piperidine-4-carboxylate provided 9 in 98% yield. After hydrolysis under basic conditions to provide the acid 10, a library of amides 11 and 12 were generated under standard HATU conditions.</p><p>While subtle improvement in potency was observed with a variety of substituted benzyl amines (11b–d), compounds such as 11d had high predicted hepatic clearance, CLhep (h = 19, r = 61), and low brain distribution in rat (brain:plasma Kp = 0.29, Kp,uu = 0.26). The aniline 11e, substituted with a primary sulfonamide, showed very promising hM4 potency (248 nM, 6% ACh), but later was determined to be a P-gp substrate (MDCK-MDR1 ER = 83). Analog 11g proved to be the most potent secondary amide in the series, with an hM4 PAM potency of 147 nM; however, its DMPK profile was lacking. Importantly, this new chemotype already surpassed the PAM potency floor of 4 and 5 (hM4 PAM EC50s ~ 600 nM).</p><p>As disposition and CNS penetration were low with the secondary amides, we profiled tertiary amides to assess the impact of removing one hydrogen-bond donor. As shown in Table 2, representative tertiary amide analogs 12 suffered a diminution in hM4 PAM potency relative to secondary amides 11. However, we still advanced 12d, a 648 nM M4 PAM into in vitro and in vivo DMPK profiling. PAM 12d, like analogs 11, displayed high predicted hepatic clearance, CLhep (h = 20, r = 64), but removal of the secondary amide H-bond donor improved CNS penetration (rat brain:plasma Kp = 1.3, Kp,uu = 0.68). These data led us to generate chimeras between the new 3-cyano quinoline core and the piperidinyl ethers of 5 and explore if this structural change would bring balance to hM4 PAM potency while maintaining favorable CNS penetration.</p><p>Synthesis of the targeted chimera ligands 14 was facile (Scheme 2). Once again, commercial 4-chloro-6-fluoroquinoline-3-carbonitrile 8 underwent an SNAr reaction with 4-hydroxypiperdine to provide 13 in 90% yield. Then, a Mitsunobu reaction with various functionalized phenols and heteroaryl alcohols delivered analogs 14 in yields ranging from 10–80%.</p><p>As shown in Table 3, these chimeric ligands proved to possess hM4 PAM activity, but SAR was steep with few active PAMs. Of these, 14f (VU6009003) stood out as a potent hM4 PAM (EC50 = 197 nM, pEC50 = 6.78 ± 0.17, ACh Max = 79± 10). Prior to deeper characterization of 14f, we elected to survey the necessity of the 3-cyano quinolone core, and assess if deletion of the 3-CN moiety, or additional functionalization of the quinoline core would afford hM4 PAMs for further evaluation. To assess this possibility, analogs 17 were readily prepared in two steps from commercial 15 and 5-fluoro-2-methlypyridine via an SNAr reaction to provide, after Boc deprotection 16. A second SNAr reaction with various 4-halo quinolines produced analogs 17.</p><p>Deletion of the 3-CN moiety as in 17a led to a greater than 10-fold loss in hM4 PAM potency, highlighting the necessity of this novel moiety. Moreover, this result suggests that the 3-CN moiety in this core is not bioequivalent to the nitrogen of the quinazoline-based hM4 PAMs represented by 5. Replacement of the 6-fluoro moiety with either a 6-Cl atom (17b, hM4 PAM EC50 = 905 nM) results in a ~4-fold loss of potency, while replacement with a methyl group (17c, hM4 PAM EC50 = 3018 nM) results in a ~15-fold loss in hM4 PAM potency relative to 14f. Of these analogs 17, only a 6,8-difluorocongener 17e, proved of equivalent potency to 14f.</p><p>Thus, 14f (VU6009003) emerged from this effort as a novel M4 PAM worthy of further profiling. PAM 14f was potent at both human M4 (197 nM, 80% ACh Max) and rat M4 (437 nM, 74% ACh Max) with very modest species difference in PAM potency, and a marked improvement over the HTS hit 7. In terms of physicochemical properties, 14f was a low molecular weight compound (MW =362) with an attractive xLogP (2.83) for a CNS agent (Table 5). In a microsomal intrinsic clearance assay, 14f displayed moderate clearance in both species (predicted human CLhep = 15 mL/min/kg, predicted rat CLhep = 52 mL/min/kg) with moderate fraction unbound in human plasma (fu = 0.024), rat plasma (fu = 0.031) and rat brain (fu = 0.014). In our standard rat plasma:brain level IV cassette paradigm, 14f showed excellent CNS penetration with a rat brain:plasma partitioning coefficient (Kp) of 5.3, and when corrected for fraction unbound in plasma and brain, a Kp,uu of 2.4. We also assessed human P-gp efflux potential in a MDCK-MDR1 transwell assay, and found no P-gp liability (ER = 1.1) coupled with high passive permeability (Papp = 28 × 10−6 cm/s). In vivo, 14f displayed lower clearance (CLp =36 mL/min/kg) in rat (IV PK cassette, 0.2 mg/kg) than predicted (CLhep = 52 mL/min/kg), with a short t1/2 (1.7 hours) and moderate volume (Vss = 3.2 L/kg). In a dog IV PK cassette study (0.2 mg/kg), 14f displayed clearance (CLp = 41 mL/min/kg) at hepatic blood flow with a t1/2 of 1.6 hours (likely as a consequence of moderate-to-high volume, Vss = 4.0 L/kg); however, 14f did display a reasonable IVIVC with predicted hepatic clearance in dog (CLhep = 27 mL/min/kg) and good fraction unbound (fu = 0.045). While attractive overall in terms of novel chemotype, favorable CNS MPO score (>5),21 fraction unbound and CNS penetration, the disposition of 14f remains to be addressed (after a deeper understanding of metabolic soft spots).</p><p>In summary, an HTS campaign identified a new M4 PAM chemotype 7, related to 4 and 5, but displaying distinct SAR, improved M4 PAM functional potency and enhanced CNS penetration. The discovery of new M4 PAM chemical matter is of critical importance to enable translation to the clinic, as the field has been largely dominated by limited chemotypes 1 and 2, fraught with development challenges. Metabolite identification studies and further optimization efforts are underway and will be reported in due course.</p>