Abstract
This letter describes the further lead optimization of the VU0486321 series of mGlu1 positive allosteric modulators (PAMs), focused on addressing the recurrent issue of plasma instability of the phthalimide moiety. Here, we evaluated a number of phthalimide bioisosteres, and ultimately identified isoindolinones as the ideal replacement that effectively address plasma instability, while maintaining acceptable mGlu1 PAM potency, DMPK profile, CNS penetration and mGluR selectivity.
Keywords: mGlu1, Metabotropic glutamate receptor, Positive allosteric modulator (PAM), Schizophrenia, Structure-Activity Relationship (SAR)
Graphical abstract
Recent genetic data implicating GRM1 in schizophrenia1–3 and studies showing that the adverse affect liabilities of group I metabotropic glutamate receptors (mGluRs) is mediated by mGlu5 and not mGlu1,4 have rekindled interest in the development of mGlu1 positive allosteric modulators (PAMs).3–6 However, early mGlu1 PAMs lacked the DMPK profiles to serve as robust in vivo tool compounds (Figure 1).7
Figure 1.
Structures of representative mGlu1 PAMs 1–5.
In response, our lab has focused efforts on developing novel mGlu1 PAMs,3–6 with an intent to discover the ideal in vivo tool compound to then validate the target for multiple neuropsychiatric disorders. Within the phthalimide-based VU0486321 series of mGlu1 PAMs, most optimization parameters could be effectively addressed (potency, disposition, CNS penetration), except for reocurring and variable in vitro plasma instability due to hydrolysis of the phthalimide moiety.3–6,8 In this Letter, we explore phthalimide biosiosteres and other replacements, ultimately identifying isoindolinones as the ideal substitute, and the discovery of VU0487351, a potent, selective, CNS penetrant and stable mGlu1 PAM tool compound.
The optimization plan targeted expansion of the phthalimide moiety, saturated analogs and sequential deletion of carbonyl moieties to provide isoindolionones and isoindolines (Figure 2) to deliver diverse analogs 6. The chemistry required is straight forward, as shown in Scheme 1, requiring only three steps from commercial materials to access final compounds 9, 13 and 16. Nucleophilic aromatic substitution between 7 and different amines produced intermediates 8, which where hydrogenated and submitted to amide coupling with 3-methyl-2-furoic acid to give analogs 9. Functionalized p-amino nitroarenes/heteroarenes 10 were condensed with various anhydrides to afford analogs 11. The nitro group was reduced to the aniline 12 via hydrogenation conditions, and final analogs 6 were afforded by standard amide coupling conditions with a diverse array of heterocyclic carboxylic acids to provide analogs 13. Alternatively, 7 could be employed to displace benzylic bromides 14, which upon heating formed the γ-lactams 15. Then, nitro reduction and acylation would yield the diversely functionalized isoindolinones 16.9
Figure 2.
Chemical optimization plan to replace the phthalimide moiety of 5 with novel analogs 6.
Scheme 1.
Reagents and conditions: (a) K2CO3, ACN, 60 °C, 43–89%, (b) H2, Pd/C, EtOH, rt, 94–99-%; (c) heterocyclic carboxylic acids, HATU, DCM, r.t., 39–98%; (d) aryl anhydrides, AcOH, reflux, 64–94%; (e) K2CO3, DMF, μW, 150 °C, 15 min, 29–92%.
Initially, we evaluated analogs of 3, and the SAR was intriguing. Expansion of the phthalimide moiety to an isoquinoline dione 17 (Figure 2) afforded a potent mGlu1 PAM (EC50 = 242 nM, pEC50 = 6.65±0.09, 105% Glu Max)10 with a clean CYP profile, but high intrinsic clearance (13.5 mL/min/kg and 54 mL/min/kg for human and rat, respectively) and was found to be unstable in both rat and human plasma. Further expansion to the 7-membered benzo[d]azepine core 18, led to a significant loss of activity (EC50 = 3.5 μM, pEC50 = 5.44±0.08, 95% Glu Max). Interestingly, a 3,4-dihydroquinolinone congener 19, was a modest mGlu1 PAM (EC50 = 1.29 μM, pEC50 = 5.88±0.09, 107% Glu Max) with supprahepatic clearance; however, 19 was stable in human and rat plasma, and suggested lactams may be productive in engendering plasma stability.
Based on these data, we then explored a diverse array of phthalimide replacements 20, with varying degrees of success (Table 1). Representative examples include 20a and 20b, the direct, saturated (both cis- and trans-isomers) of 3, which proved to be inactive. The hydrolyzed product of 3, 20c, was synthesized and found to be active (EC50 = 930 nM, pEC50 = 6.03±0.14, 108% Glu Max); however, 20c was not CNS penetrant and displayed poor disposition, yet an ‘active’ in vitro metabolite. Other benzoates, with diverse functional groups in place of the carboxylic acid were all inactive. Isoindolinone 20e was active, but the regioisomeric congener 20g was inactive, and both regioisomeric isoindolines, 20f and 20h were active. 20l, a regiosiomer of 19 was potent (EC50 = 780 nM, pEC50 = 6.11±0.12, 94% Glu Max) as well. Profiling of all the active analogs 20 in our in vitro DMPK assays quickly led us to focus on the isoindolinone 17e (EC50 = 3.72 μM, pEC50 = 5.43±0.19, 91% Glu Max), as it displayed complete stability in rat and human liver microsomes with low intrinsic clearance. Now, the focus was to improve mGlu1 PAM potency.
Table 1.
Structures and activities for analogs 20.
| |||
|---|---|---|---|
| Cpd | Het | hmGlu1 EC50 (μM)a [% Glu Max ±SEM] | mGlu1 pEC50 (±SEM) |
| 20a |
|
>10 [-] | >5 |
| 20b |
|
>10 [-] | >5 |
| 20c |
|
0.93 [108±7] | 6.03±0.14 |
| 20d |
|
>10 [-] | >5 |
| 20e |
|
3.72 [91±9] | 5.43±0.19 |
| 20f |
|
1.76 [95±23] | 5.75±0.43 |
| 20g |
|
>10 [-] | >5 |
| 20h |
|
5.71 [82±8] | 5.24±0.14 |
| 20i |
|
9.55 [78±2] | 5.02±0.09 |
| 20j |
|
3.62 [99±9] | 5.44±0.14 |
| 20k |
|
2.61 [93±4] | 5.58±0.07 |
| 20l |
|
0.781 [94±4] | 6.11±0.12 |
| 20m |
|
2.47 [107±5] | 5.88±0.09 |
Calcium mobilization mGlu1 assays, values are average of three (n=3) independent experiments performed in triplicate.
For the next iteration of parallel synthesis, we surveyed functionalized isoindolinone congeners 21, of the mGlu1 PAM 3 (Table 2). Gratifyingly, substituents on the isoindolinone phenyl ring increased potency by >10-fold in some cases, providing mGlu1 PAMs with nanomolar potency.
Table 2.
Structures and activities for analogs 21.
| |||
|---|---|---|---|
| Cpd | R | hmGlu1 EC50 (μM)a [% Glu Max ±SEM] | mGlu1 pEC50 (±SEM) |
| 21a | 4-Cl | 0.211 [88±4] | 6.75±0.07 |
| 21b | 5-Cl | >10 [58±1] | >5 |
| 21c | 7-Cl | 0.484 [108±4] | 6.31±0.1 |
| 21d | 4-Me | 1.07 [89±3] | 5.96±0.09 |
| 21e | 4-F | 0.878 [82±4] | 6.05±0.11 |
| 21f | 4-Br | 0.391 [99±4] | 6.41±0.10 |
| 21g | 4-CF3 | 1.13 [105±5] | 5.94±0.09 |
| 21h | 4-CN | 2.09 [107±9] | 5.67±0.17 |
| 21i | 4-OMe | >10 [-] | >5 |
| 21j | 4-Aza | >10 [-] | >5 |
| 21k | 7-Aza | >10 [-] | >5 |
| 21l | 4,7-Aza | >10 [-] | >5 |
| 21m | 3,3-diMe | 6.93 [76±3] | 5.15±0.09 |
Calcium mobilization mGlu1 assays, values are average of three (n=3) independent experiments performed in triplicate.
Evaluation of analogs 21 in our in vitro and in vivo battery of DMPK assays quickly identified 21a (VU0487351),3–6,11 as an exceptional compound. First, and in contrast to 3–5, 21a was hydrolytically stable in rat and human liver microsomes and displayed moderate intrinsic clearance (rat CLhep 54.1 mL/min/kg and human CLhep 8.53 mL/min/kg). Moreover, 21a had a clean CYP profile (IC50s > 30μM against 3A4, 1A2, 2C9 and 2D6), was inactive at mGlu4 (EC50 > 10 μM) and was found to be highly CNS penetrant (Kp = 1.36, Cn plasma 193 nM and Cn brain 269 nM). In vivo, 21a displayed favorable rat PK, with a low clearance (CLp 11.1 mL/min/kg), high distribution volume (VD 3.36 L/kg) and good half-life (t1/2 = 93 min, MRT = 302 min). Therefore, the isoindolinone moiety solved the plasma instability issue, as well as affording a favorable in vitro and in vivo DMPK profile. The only blemish for 21a was high protein binding (>99% in human and rat plasma, as well as rat brain homogenate binding), which was true for all analogs 21 (fu <0.01). Therefore, we elected to pursue a matrix library strategy to identify new isoindolinone-based mGlu1 PAMs that maintained the overall profile of 21a, while improving fraction unbound.
Analogs 22 surveyed SAR for three regions: the central phenyl core, alternative heterocyclic amides and substitutions on the isoindolinone phenyl ring (Table 3). The removal of the hydrogen in the central ring (22a–c) led to a similar SAR tendency as the 2-Cl analogs, with 22b having a comparable potency to 21a, suggesting that an asymmetric substitution pattern in the central ring is not necessary. Other substitutions, such as 2,3-dichloro (22d–f) and 3-fluoro (22g–i) caused a dramatic loss in potency. This trend shows how variable and steep the SAR can be in this scaffold, as the 3-fluoro substitution has been an excellent modification to achieve potent and selective mGlu1 PAM activity in our phthalimide scaffold. As we assessed the replacement of the furan with different heterocycles in the isoindolinone scaffold, a substantial loss in potency was found when using 3-substituted picolinamides, and only the 4-Cl substituted 22h and 22k (direct comparators to 21a ) maintained mGlu1PAM potency of around 1μM; while in the thiazole (22p–r) congeners the 4-Cl substituted (22q) was most potent of the group (EC50 = 950 nM). However, these attempts to engender improved free fraction in the isoindolinone series failed, and SAR was steep.
Table 3.
Structures and activities for analogs 22.
| |||||
|---|---|---|---|---|---|
| Cpd | R1 | R2 | Het | hmGlu1 EC50 (μM)a [% Glu Max ±SEM] | mGlu1 pEC50 (±SEM) |
| 22a | H | H |
|
1.56 [94±12] | 5.81±0.31 |
| 22b | H | 4-Cl |
|
0.245 [99±3] | 6.61±0.11 |
| 22c | H | 7-Cl |
|
0.353 [101±3] | 6.45±0.11 |
| 22d | 2,3-diCl | H |
|
>10 [-] | >5 |
| 22e | 2,3-diCl | 4-Cl |
|
>10 [-] | >5 |
| 22f | 2,3-diCl | 7-Cl |
|
6.52 [81±6] | 5.18±0.09 |
| 22g | 3-F | H |
|
>10 [-] | >5 |
| 22h | 3-F | 4-Cl |
|
1.26 [86±6] | 5.90±0.15 |
| 22i | 3-F | 7-Cl |
|
>10 [-] | >5 |
| 22j | 2-Cl | H |
|
>10 [-] | >5 |
| 22k | 2-Cl | 4-Cl |
|
1.36 [97±6] | 5.84±0.14 |
| 22l | 2-Cl | 7-Cl |
|
>10 [78±2] | >5 |
| 22m | 2-Cl | H |
|
>10 [-] | >5 |
| 22n | 2-Cl | 4-Cl |
|
1.27 [93±5] | 5.89±0.12 |
| 22o | 2-Cl | 7-Cl |
|
>10 [51±3] | >5 |
| 22p | 2-Cl | H |
|
>10 [33±6] | >5 |
| 22q | 2-Cl | 4-Cl |
|
0.954 [66±4] | 6.02±0.12 |
| 22r | 2-Cl | 7-Cl |
|
4.69 [69±2] | 5.32±0.73 |
Calcium mobilization mGlu1 assays, values are average of three (n=3) independent experiments performed in triplicate.
In conclusion, the continued optimization of the VU0486321 series of mGlu1 PAMs led us to pursue alternatives for the phthalimide moiety, plagued with unpredictable plasma instability issues. Here we identified isoindolinones as biosiosteres for the phthalimide group. mGlu1 PAMs such as 21a, that retained mGlu1 PAM potency and mGluR selectivity, while also affording plasma stability, attractive in vitro and in vivo PK profiles and high CNS penetration (Kp = 1.36). Together, with VU0487351 (21a) and VU6004909 (5), the field now has mGlu1 PAMs that can serve as robust in vivo tool compounds to further dissect the therapeutic potential of selective mGlu1 activation.
Figure 3.
Homologated analogs 14–16 of 3 that retained mGlu1 PAM activity.
Acknowledgments
We thank William K. Warren, Jr. and the William K. Warren Foundation who funded the William K. Warren, Jr. Chair in Medicine (to C.W.L.). P.M.G. would like to acknowledge the VISP program for its support. This work was funded by the William K. Warren, Jr. Chair in Medicine and the NIH (U54MH084659).
Footnotes
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References
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