Agonists of the γ-Aminobutyric Acid Type B (GABA_B) Receptor Derived from β-Hydroxy and β-Amino Difluoromethyl Ketones

Abstract

β-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA_B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of γ-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of β-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA_B receptor. Moreover, we also report that the synthesis and biological evaluation of β-amino difluoromethyl ketones provided the most potent compound across these two series.

Graphical abstract

The inhibitory neurotransmitter, γ-aminobutyric acid (GABA), reduces the excitability of neurons and assists in the regulation of other neurotransmitters, especially in the central nervous system. The two major types of GABA receptors are GABA_A and GABA_B and both are validated targets for drug discovery.¹ The GABA_A receptors are ion channels and can be controlled by three classes of pharmaceuticals, the barbiturates, the benzodiazepines, and the newer non-benzodiazepine sedatives, such as zaleplon and zopiclone.² The GABA_B receptors are metabotropic G-protein-coupled receptors, and serve as the target of the muscle relaxant, baclofen.³ Moreover, the GABA_B receptors are the focus of drug discovery efforts in muscle spasticity disorders,³ schizophrenia,⁴ pain,⁵ and gastroesophageal reflux disease (GERD).⁶ Agonists,⁷ antagonists,⁸ positive allosteric modulators,^9,10 and negative allosteric modulators¹¹ of the GABA_B receptor are known, and in 2013, the X-ray structures of this receptor in the ligand-free state and in the presence of agonists and antagonists were reported.¹²

Nearly all of the known agonists of the GABA_B receptor display the structure of GABA, and, for example, baclofen is the 3-para-chlorophenyl analogue of GABA (Figure 2).¹³ Although the pharmaceutical formulation of baclofen is a racemic mixture, the (R)-(−)-enantiomer is significantly more active. The 3-phenyl derivative is the agent, phenibut, and in a similar fashion, (R)-(−)-phenibut is the more active enantiomer.¹⁴ Also, the 2-chlorothienyl group is a surrogate for the para-chlorophenyl group of baclofen and is displayed in GABA_B agonist 1.¹⁵ Other key analogues of baclofen, which are also agonists of the GABA_B receptor, are the pyridinyl methoxy derivative 2,¹⁶ (R)-(−)-GABOB,¹⁷ and CGP44532.¹⁸ The only known agonist of the GABA_B receptor that does not display the backbone of GABA or baclofen is the difluoromethyl ketone 3.¹⁹

Figure 2.

Preparation of β-hydroxy difluoromethyl ketones 4–9 and β-amino difluoromethyl ketones 10–14. Isolated yields for the aldol and imino-aldol reactions are given in parenthesis.

The β-hydroxy difluoromethyl ketones are a distinct group of ligands of the GABA_B receptor and were first reported in 2013.¹⁹ The structure–activity relationships for the lead compound 3 in this series are that the fluorines and the β-hydroxy substituent are required for activity (Scheme 1).¹⁹ Specifically, the non-fluorinated analogue of 3 is inactive and methyl ether derivative created from methylation of the β-hydroxy substituent of 3 is also inactive. The bulky, lipophilic adamantyl group or naphthyl group is common in other active derivatives. On the other hand, the para-acetyl phenyl group of 3 tolerates many other structures such as alkyl, alkenyl, and aryl groups. Although these data are insightful, the agent 3 presents additional opportunities to identify new structure-activity relationships for agonist activity for the GABA_B receptor. In the present study, we have prepared new β-hydroxy difluoromethyl ketones and characterized the β-amino difluoromethyl ketones as another complementary scaffold.

Scheme 1.

Structure–activity relationships for compd 3 and agonist activity at the GABA_B receptor.

These unique compounds were discovered following the development of a new synthetic protocol that uses pentafluoro-gem-diols to produce difluoroenolates for aldol reactions^20,21 and was later extended to imino-aldol reactions.^22,23 The first objective was to define the role of substituents on the para-acetylphenyl group of 3. Compounds 4–6 were synthesized,²⁴ and each displays a change to the para-acetyl group on the phenyl ring (Figure 3). Compound 7 was prepared to understand if a heteroaromatic group could replace the adamantyl group. The analogue 8 bears a larger isoindolinedione to replace the para-acetyl phenyl group. Next, the β-amino difluoromethyl ketones 10–14 were synthesized using the literature methods.^22,23 The naphthyl and adamantyl groups were conserved (i.e., 10–11 and 12, respectively) and the N-phenylsulfonyl and N-tert-butylcarbamate groups were selected as substituents for the amine.

Figure 3.

Synthesis of β-amino difluoromethyl ketones 15, 16, and 17. Isolated yields for the imino-aldol reactions are given in parenthesis. See ref. 25.

In 2018, we reported a procedure in which unactivated imines bearing N-benzyl, aryl, or alkyl group reacted with difluoroenolates generated from pentafluoro-gem-diols in the presence of magnesium salts.²⁵ This process enables the creation of additional β-amino difluoromethyl ketones displaying a N-benzyl group to complement those bearing N-phenylsulfonyl and N-tert-butylcarbamate group (Figure 4). The naphthyl derivative 15 completes the series from 10 and 11, and compound 16 and 17 were prepared to obtained additional structure-activity data.²⁵

Figure 4.

Active site residues (gray) of the GABA_B receptor from docking studies with: A) (R)-3 (GlideScore = −10.080) (magenta) and B) (S)-3 (GlideScore = −9.606) (purple). (R)-Baclofen (GlideScore = −11.063) is also shown in each image (green). Amino acids are depicted as tubes and ligands are represented as ball-and-stick. H-bonds and aromatic π–π stacking are shown as black and cyan dashed lines, respectively.

Compounds 4–17 were tested for agonist activity at the GABA_A and GABA_B receptors according to the previously reported procedures.¹⁹ The screen for agonist activity at the GABA_B receptor is conducted in a HEK293/Cre-luc cell line expressing both subunits of the human GABA_B receptor. Activation of the GABA_B receptor accesses an endogenous signaling pathway and results in the inhibition of cAMP production. In this assay, forskolin is applied to stimulate cAMP production, and inhibition of the forskolin-stimulated cAMP production is measured (assays are performed in triplicate). All of the compounds that display activity at the GABA_B receptor are also screened for agonist activity at the GABA_A receptor using a whole-cell voltage-clamp method with HEK293 cells transfected with the α₁β₂γ₂ subunits of the rat GABA_A receptor. The pharmaceutical, baclofen, is a racemic mixture and selective agonist for the GABA_B receptor; however, most of its pharmacological activity results from the (−)-baclofen enantiomer.¹⁹ In a similar fashion, the lead compound 3 also has more agonist activity from a single enantiomer (i.e., the (+)-3-enantiomer); however, an absolute stereochemical assignment of secondary alcohol was not reported.¹⁹ The fluorinated compounds 4–17 were all tested as racemic mixtures. The β-hydroxy difluoromethyl ketones 4–8 do not display any observable activity, even at concentrations up to 100 μM. These results demonstrate the low tolerance for changes to the para-acetyl phenyl group, especially as the replacement of the acetyl with an ethyl group produces an inactive compound (i.e., compound 4). On the other hand, some structural variation is allowed because our prior studies validated that the styrene 9 displays activity of 40 μM compared to the lead compound 3 at 24.9 μM at the GABA_B receptor.¹⁹ The β-amino difluoromethyl ketone 11 was characterized as the most active agent across the entire difluoromethyl ketone series. The β-amino difluoromethyl ketone 11 displays EC₅₀ = 19.4 μM, and, notably, when the N-Boc group in 11 is replaced with N-SO₂Ph in 10 or with N-Bn in 15, agonist activity decreases to EC₅₀ = 32.7 μM or EC₅₀ = 91 μM, respectively. All three of these molecules display no activity at the GABA_A receptor, which correlates well with the β-hydroxy difluoromethyl ketones, which also preferential for the GABA_B receptor (over the GABA_A receptor). The other β-amino analogues 12–14 and 16 were inactive at the GABA_B receptor. The para-chlorophenyl derivative 17 displays moderate activity at the GABA_B receptor.

Additional biological characterizations of compounds 3 and 11 were performed using a radio-ligand binding assay screen with cloned neurotransmitter receptors and transporters (Table 2).²⁶ Difluoromethyl ketones are known in the literature to display activity at many targets,^27,28 so these screens provide an important perspective on the relative role of these ligand for the GABA_B receptor over other neurotransmitter receptors. Adrenoreceptors and biogenic amine, dopamine, opioid, histamine, muscarinic, serotonin, and sigma receptors were examined, and interestingly, both 3 and 11 display less than 30% inhibition and are considered inactive in all of these assays. These data strengthen the knowledge of the preference of this class of compounds (i.e., β-hydroxy difluoromethyl ketone and β-amino difluoromethyl ketone) for the GABA_B receptor.

Table 2.

Radio-ligand binding assay screen for compds 3 and 11 in cloned receptors and transporters. Both 3 and 11 are inactive in all assays.

Adrenoreceptors	α1A, α1B, α1Δ, α2A, β1, β2, β3
Biogenic amine transporters	DAT, NET, SERT
Dopamine receptors	D1, D2, D3, D4, D5
Opioid receptors	DOR, KOR, MOR
Histamine receptors	H1, H2, H3, H4
Muscarinic receptors	M1, M2, M3, M4, MS
Serotonin receptors	5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7
Sigma receptors	rSigma1, rSigma2

In order to integrate the new structure–activity relationships with the prior data, both enantiomers of the lead compound 3 were docked into the (R)-baclofen-bound GABA_B receptor complex (PDB ID: 4MS4),¹² using the Induced Fit Docking protocol of the Schrӧdinger small molecule drug discovery suite.²⁴ The difluoromethyl ketone 3 is hydrated in the model, because difluoromethyl ketones are known to exist in the hydrated (gem-diol) form at physiological pH.¹⁹ The respective gem-diol, in turn, occupies a similar site as the carboxylate of baclofen. Hydrogen-bonding is conserved with Ser130 and Gly171 (as observed with GABA-bound X-ray structure, PDB-ID:4MS3) and the gem-diol maintains a close proximity to the Ser153 (Figure 5). The β-hydroxy substituent in both the (R)-3 and (S)-3 alcohols cannot occupy the same site as the amino group of baclofen due to the shorter intramolecular distance from the difluoromethyl ketone group. However, the para-acetylphenyl group of both the (R)-3 and (S)-3 compounds employs a similar site as the para-chlorophenyl group of baclofen and maintains key interactions with the Trp278 residue. Lastly, docking studies present potential docking poses for 3 that accommodate the bulky adamantyl group within the ligand binding site of the GABA_B receptor.

Figure 5.

Active site residues (gray) of the GABA_B receptor from docking studies with: A) (S)-10 (GlideScore = −11.037) (magenta) and B) (S)-11 (GlideScore = −10.592) (magenta). (R)-Baclofen is also shown in each image (green). Amino acids are depicted as tubes and ligands are represented as ball-and-stick. H-bonds and aromatic π–π stacking are shown as black and cyan dashed lines, respectively.

We attempted to dock both enantiomers of 10 and 11 into the GABA_B receptor using both SP and XP Induced Fit Docking, and the methods gave similar results, so we report the XP ones. The sulfonamide group of (S)-10 is engaged in hydrogen-bonding with Ser130 whereas the gem-diol of (S)-11 is hydrogen-bonded to the Ser130 and Gly151, and both these functional groups lie in close proximity to Ser153 (Figure 5). The phenyl group attached to the stereogenic carbon in (S)-10 occupies the same site as the amino group of baclofen. By contrast, the naphthalene ring of (S)-11 occupies this site. Moreover, the naphthyl group of (S)-10 is in the site occupied by the para-chlorophenyl group of baclofen, whereas for (S)-11 this site is occupied by its phenyl group (Figure 6). (R)-10 failed to dock into the binding site in several trials with different grid settings. The docked pose of (R)-11 (GlideScore = −8.500) is provided in the supplementary information. Hence, we predict that the (S)-enantiomers are the more active for 10 and 11.

The difluoromethyl ketones represent the newest class of agonists of the GABA_B receptor, and further investigations through synthesis, biological evaluation, and computational analysis have not only revealed that enhancements in potency are possible but also that the structure–activity relationships are distinct from other classes of agonists that display the structure of GABA. The critical nature of the substituent on the aromatic group of the β-hydroxy difluoromethyl ketone was characterized. Also, the identification of the β-amino difluoromethyl ketones as potent agonists of the GABA_B receptor is another significant advance.

Figure 1.

Structures of some known agonists of the GABA_B receptor.

Table 1.

GABA_A and GABA_B assay data for baclofen and compds 3–17.

compd	GABAB EC50 (μM)a	GABAA EC50 (μM)a
GABAc	0.53 ± 0.33	2.30 ± 0.59
(±)-baclofenc	1.7 ± 0.10	>100
(±)-3b	24.9 ± 1.30	>100
(−)-3b	37.8 ± 0.78	ndc
(+)-3b	15.9 ± 1.84	nd
4	>100d	nd
5	>100d	nd
6	>100d	nd
7	>100	nd
8	>100	nd
9c	40 ± 3.59	>100
10	32.7 ± 1.44	>100
11	19.4 ± 7.69	>100
12	>100	nd
13	>100	nd
14	>100	nd
15	91 ± 5.7	>100
16	>100	nd
17	57.4 ± 2.2	>100

^aValues are given with the standard error.

^bRef 19.

^cnd is not determined.

^dCytotoxic in assay at 50 μM and 100 μM doses.

Highlights.

• β-Hydroxy difluoromethyl ketones have distinct SAR data at the GABA_B receptor

• β-Amino difluoromethyl ketones are now characterized as GABA_B agonists

• Docking studies at the GABA_B receptor suggest similar binding modes to baclofen