Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive allosteric modulators

Abstract

This Letter describes the synthesis and SAR, developed through an iterative analogue library approach, of an mGluR4 positive allosteric modulator lead based on a pyrazolo[3,4-d]pyrimidine scaffold. Despite tremendous therapeutic potential, Compound 7, VU0080421, and related congeners represent only a handful of mGluR4 positive allosteric modulators ever described.

Glutamate is the major excitatory neurotransmitter in the central nervous system, exerting its effects through both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are members of the GPCR family C, characterized by a large extracellular amino-terminal agonist binding domain. To date, eight mGluRs have been cloned, sequenced and assigned to three groups (Group I: mGluR1 and mGluR5; Group II: mGluR2 and mGluR3; Group III: mGluRs 4,6,7,8) based on their sequence homology, pharmacology and coupling to effector mechanisms.¹ The Group III mGluRs are the least explored and characterized of the mGluRs, but despite this fact, mGluR4 has garnered a great deal of attention as a therapeutic target for multiple indications.²

The reason for the slower pace of development within Group III mGluRs concerns the availability of ligands.^2,3 Most pharmacological studies employ prototypical Group III agonists such as L-(+)-2-amino-4-phosphonobutryic acid, L-AP4, 1 or functionalized carboxyphenylglycines 2, which have limited CNS penetration (Fig. 1).⁴ A major breakthrough in the field occurred when Maj and co-workers reported on the discovery of (−)-PHCCC 3, the first mGluR4 positive allosteric modulator (PAM), derived from the mGluR1 negative allosteric modulator (NAM) (−)-CPCCOEt 4.⁵ (−)-PHCCC possesses an EC₅₀ of 4.1 µM, with a 5.5-fold leftward shift of the glutamate response curve and selectivity versus mGluRs 2, 3, 5, 6, 7, 8.^5,6 However, (−)-PHCCC is a partial antagonist (30%) of mGluR1.

Figure 1.

Chemical structures of orthosteric mGluR4 agonists L-AP4 (1), functionalized phenylglycines (2) and the mGluR4 PAM (−)-PHCCC (3) which was derived from the mGluR1 NAM (−)-CPCCOEt (4).

SAR for (−)-PHCCC is very ‘flat’, with virtually any chemical modifications resulting in a complete loss of mGluR4 PAM activity (Fig. 2), a finding common with several series of mGluR PAMs.^7–10 Despite this, (−)-PHCCC has been a very important proof of concept (POC) compound demonstrating a therapeutic role for selective mGluR4 activation in Parkinson’s disease,^6,11 anxiety,¹² depression,¹³ neuroprotection¹⁴ and oncology.¹⁵

Figure 2.

Chemical modifications and the resulting ‘flat’ SAR for (−)-PHCCC.

In all of these pioneering POC experiments, PHCCC was either administered through intracerebroventricular injection (icv) or by employing toxic 50% DMSO vehicles which disrupt the blood-brain barrier, as PHCCC possesses poor physiochemical properties and limited brain penetration.^6,11–15 In order to advance this field, new mGluR4 PAMs are required with improved efficacy, physiochemical properties and novel molecular architectures. In this Letter, we describe the discovery and SAR of a novel mGluR4 PAM, based on a pyrazolo[3,4-b]pyrimidine scaffold, derived from an HTS campaign.

Our mGluR4 PAM HTS identified three pyrazolo[3,4-d]pyrimidines that afforded a concentration-dependent potentiation of an EC₂₀ of glutamate in mGluR4/Gqi5 CHO cells (Fig. 3).⁷ When HTS stocks were evaluated with full concentration-response curves, 7, 1-(2,4-diphenyl)-4-(3-methylpiperdin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine, was a stand-out compound with an EC₅₀ for potentiation of 4.6 µM while having no effect on mGluR4 in the absence of glutamate (Fig. 4).¹⁶

Figure 3.

Concentration-dependent potentiation of glutamate in mGluR4/Gqi5 CHO cells by pyrazolo[3,4-d]pyrimidine compounds 5, 6 and 7 in the high throughput screening campaign (HTS).

Figure 4.

Compound 7, potentiates mGluR4 activation by glutamate. In the absence of glutamate, 7 does not activate mGluR4. In the presence of an EC₂₀ concentration of glutamate, 7 caused a concentration-dependent potentiation of mGluR4 with an EC₅₀ for potentiation of 4.6 µM, equivalent to PHCCC, EC₅₀ ~ 4.1 µM.^5,6 Data represents the average of at least three independent determinations.

Additionally, 7 was superior to PHCCC in terms of fold-shift of the glutamate concentration-response curve. As shown in Figure 5, PHCCC induces a 5.5-fold leftward shift of the glutamate response curve with an elevation in the glutamate max, whereas 7 elicits a 27.2(±8.5)-fold shift with no increase in the glutamate max. Compound 7 was selective versus mGluR5, but was a full antagonist of mGluR1 (IC₅₀ = 2.6 µM). This finding was surprising since PHCCC was derived from a series of mGluR1 antagonists (the (−)-CPCCOEt 4 series), it was not surprising that 3 was a 30% partial antagonist of mGluR1, but 7 is structurally unrelated. It is possible that PHCCC and 7 share a common allosteric binding site on mGluR4 that is also conserved in mGluR1, but in the absence of radioligands for mGluR4, this issue will have to be addressed at a later time.

Figure 5.

Both PHCCC (3) and 7 shift the glutamate agonist response curves to the left 5.5- and 27.2 (±8.5)-fold, respectively at 30 µM (EC₅₀ shifts from 7.5 ± 1.6 µM to 317 ± 100 nM). Interestingly, PHCCC increases maximal response whereas 7 affords no increase in the glutamate max, akin to other mGluR5 PAMs reported from our labs.⁷

With a bona fide novel mGluR4 PAM lead in hand, we employed an iterative analogue library synthesis approach to rapidly evaluate SAR for 7. Despite HTS stocks of 7 confirming structure and purity, re-synthesis of 7 (VU0080241) resulted in a compound with slightly lower potency and a less robust fold shift (EC₅₀ ~ 5 uM, fold shift 11.8±3.3, Table 1); however, VU0080241 still represents an improvement over PHCCC. In light of the ‘flat’ SAR observed by ourselves and others in the field with PHCCC,^5–7 we initially synthesized multidimensional diversity libraries to determine the scope and breadth of the SAR. However, classical synthetic approaches to pyrazolo[3,4-d]pyrimidines yields were typically moderate (<50%) with prolonged reaction times at high temperatures (multiple steps requiring >48 h at reflux).¹⁷ As recently reported, we have developed a rapid, high yielding and general microwave-assisted approach to access diverse pyrazolo[3,4-d]pyrimidines, and this expedited protocol was employed to produce analogues of VU0080421 (Scheme 1).¹⁸ The synthesis began by reacting 2-(ethoxymethylene)malononitrile 8 with seven arylhydrazines under microwave irradiation to deliver 5-amino-1-aryl-1H-pyrazole-4-carbonitriles 9. The nitrile analogues 9 were then hydrolyzed with H₂SO₄ to produce the corresponding 5-amino-1-aryl-1H-pyrazole-4-carboxamides 10. A microwave-assisted condensation employing carboxamides 10 in neat formamide smoothly afforded the corresponding analogous 1-aryl-1H-pyrazolo[3,4-d]pyrimidines 11. MAOS conditions (POCl₃, DMF, 120 °C, 45 min, mw) were used for the conversion to the chloro congeners, which were then subjected to a microwave-assisted S_NAr reaction to provide the final analogues 12 based on HTS lead 7, VU0080421.¹⁹ In short order, over 126 analogues 12 (7 arylhydrazines × 18 amines) were prepared and evaluated as mGluR4 PAMs (Fig. 6).

Table 1.

Glutamate fold shifts by VU0080421 analogues 12.

Compound	NR1R2	Ar	Fold shifta
(−)-PHCCC, 3			5.5
7 VU0080241			27.2 11.8
12a			9.9
12b			3.5
12c			2.7
12d			2.4

^aFold shifts represent the average of at least three independent experiments performed in quadruplicate at 30 µM.

Scheme 1.

Reagents and conditions: (a) ArNHNH₂, EtOH, mw, 105 °C, 10 min, 54–77%; (b) i) H₂SO₄, 0°C, ii) H₂NCHO, mw, 200 °C, 20 min, 48–91%; (c) i) POCl₃, DMF, mw, 120 °C, 45 min, ii) HNR¹R², DMF, PS-DIEA, mw, 90 °C, 20 min, 84–99%.^18,19

Figure 6.

Monomers employed in the diversity library of pyrazolo[3,4-d]pyrimidines 12 (7 arylhydrazines × 18 amines) based on HTS lead 7, VU0080421. Monomers in boxes indicate that they produced analogues 12 that potentiated an EC₂₀ of glutamate in mGluR4/Gqi5 CHO cells.

As in the case of PHCCC, analogues 12 of VU0080241 were uniformly inactive on mGluR4 with only the parent HTS lead 7, the re-synthesized VU0080421 and four other analogues (12a–12d) showing any activity as mGluR4 PAMs. SAR for this series was ‘flat’ with only a 3.9% active rate. With one exception (12c, containing a 2-chlorophenyl group), the 2,4-dimethylphenyl moiety (6, 7, 12a, 12b, 12d) was required for activity, and little diversity was tolerated with respect to the nature of the NR₁R₂ moiety. As shown in Table 1, analogues 12 lost efficacy, EC₅₀s ≥ 10 µM, but provided robust leftward shifts of the glutamate response curve 2.4- to 9.9-fold. One explanation for the ‘flat’ SAR is that the allosteric binding sites which VU0080421 and (−)-PHCCC occupy are very shallow, similar to the second, non-MPEP, allosteric binding site on mGluR5 that CPPHA occupies.^8,9 In addition, in vitro DMPK studies identified stability issues with VU0080241. Importantly, VU0080241 was found to be unstable in fortified liver microsome preparations, with only 9% of the parent compound remaining after 90 minutes.

Despite the disappointing SAR and microsomal instability, VU0080421 (7) represents a significant advance in the mGluR4 PAM field. VU0080421 (7) possesses a large 11.8- to 27.2-fold shift, the largest we have observed for an mGluR PAM, and it does not contain the oxime or amide NH moieties that are speculated to contribute to the observed lack of brain penetration for PHCCC in vehicles other than DMSO. Moreover, VU0080421 represents a novel chemotype for a PAM of mGluRs and is one of only a handful of reported PAMs of mGluR4. Further refinements to VU0080241 and related series of mGluR4 PAMs are in progress and will be reported in due course.