Novel Cyclic Phosphinic Acids as GABA_C ρ Receptor Antagonists: Design, Synthesis, and Pharmacology

Abstract

Understanding the role of GABA_C receptors in the central nervous system is limited due to a lack of specific ligands. Novel γ-aminobutyric acid (GABA) analogues based on 3-(aminomethyl)-1-oxo-1-hydroxy-phospholane 17 and 3-(guanido)-1-oxo-1-hydroxy-phospholane 19 were investigated to obtain selective GABA_C receptor antagonists. A compound of high potency (19, K_B = 10 μM) and selectivity (greater than 100 times at ρ₁ GABA_C receptors as compared to α₁β₂γ_2L GABA_A and GABA_B(1b,2) receptors) was obtained. The cyclic phosphinic acids (17 and 19) are novel lead agents for developing into more potent and selective GABA_C receptor antagonists with increased lipophilicity for future in vivo studies.

γ-Aminobutyric acid 1 (GABA 1, Figure 1) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS) and is essential for the overall balance between neuronal excitation and inhibition.^1,2 GABA influences neurons via three major classes of receptors that are grouped on the basis of their subunit composition, gating properties, and pharmacological profiles: GABA_A, GABA_B, and GABA_C (GABA ρ) receptors. GABA_A and GABA_C receptors are ionotropic receptors, belonging to the Cys loop family of ligand-gated ion channels, which also incorporates nicotinic acetylcholine, strychnine-sensitive glycine, serotonin type 3, and some invertebrate anionic glutamate receptors.^1,2 Both GABA_A and GABA_C receptors are chloride channels that mediate fast synaptic inhibition when activated by GABA. In contrast, GABA_B receptors are members of the family 3 class metabotropic receptors; these receptors couple via G proteins (G_i/o) to interact with inwardly rectifying potassium (GIRK) and voltage-gated calcium channels, mediating slow, longer lasting synaptic inhibition by increasing potassium and decreasing calcium conductances.³

Figure 1.

Structures of GABA (1), compounds active at GABA receptors (2−7), and synthesized novel 3-AMOHP (17) and 3-GOHP (19).

The ionotropic GABA_A receptors are transmembrane protein complexes composed of five heteropentameric subunits. So far, 16 human GABA_A receptor subunits have been identified, and they have been classified into α (α₁−α₆), β (β₁−β₃), γ (γ₁−γ₃), δ, ε, π, and θ. Although a wide range of different GABA_A receptor combinations exist in vivo, the most common is the α₁β₂γ₂ combination, constituting approximately 18% of all GABA_A receptors in the human brain.⁴ The GABA_B receptors consist of heterodimers, which are composed of two subunits, a ligand-binding subunit (GABA_B1), and a signal transduction subunit (GABA_B2).³

The GABA_C receptor has distinct pharmacology, physiology, and subunit composition to that of GABA_A and GABA_B receptors.^1,5,6 Consisting of homo-oligomeric or pseudohomo-oligomeric pentameric compositions of ρ subunits (ρ₁, ρ₂, and ρ₃ subunits) in mammals,^5,7 the receptors are highly expressed in many parts of the brain, including the superior colliculus,⁸ cerebellum,⁹ hippocampus (high ρ₂ subunit expression),¹⁰ and, most prominently, the retina (high ρ₁ subunit expression).¹¹

The first selective GABA_C receptor antagonist developed was 1,2,5,6-tetrahydropyridine-4-yl-methyl-phosphinic acid 3 (TPMPA 3, Figure 1).¹² TPMPA 3 has been shown to enhance memory in chicks,¹³ inhibit myopia development in chicks,¹⁴ modulate the sleep-waking behavior in rats,¹⁵ and exert influence on the lateral nucleus of the amygdala (LA).¹⁶ However, TPMPA 3 probably does not cross the blood brain barrier, and there have been no reports delineating the CNS effects of TPMPA 3 upon systemic administration.

Recently, we have synthesized a series of conformationally restricted 3-aminocyclopentane/cyclopentene alkylphosphinic acids from a structure−function study of various aminocyclopentane/cyclopentene phosphinic acid analogues of GABA, and these conformationally restricted alkyl phosphinic acids are highly potent and selective for GABA_C receptors.¹⁷ (S)-4-ACPBPA 6 is the most potent and selective GABA_C receptor antagonist of this series. Interestingly, (S)-4-ACPBPA 6 is a conformationally restricted analogue of the orally active GABA_B/C receptor antagonist 3-aminopropyl-n-butylphosphinic acid 7 (CGP36742 or SGS742) (GABA_B, GABA_C, and GABA_A: IC₅₀ = 38, 62, and 508 μM, respectively). CGP36742 or SGS742 reached phase II clinical trials, enhancing memory in mildly memory-impaired subjects.¹⁸ However, because CGP36742 (SGS742) is approximately equipotent on GABA_B as it is on GABA_C receptors, it is not clear whether the pharmacological effects of CGP36742 are due to its GABA_B or GABA_C activity. In continuation of our efforts to identify the role of GABA_C receptors in the CNS, we discovered that the selective GABA_C receptor antagonists, cis- and trans-(3-aminocyclopentanyl)-n-butylphosphinic acid (cis-3-ACPBPA 4 and trans-3-ACPBPA 5; Figure 1), inhibit myopia progression in chicks and facilitate learning and memory in the Morris Water Maze task in rats.¹⁹

The discovery of potent and selective GABA_C antagonists emphasizes some important pharmacological differences between these GABA receptor families. Thus, GABA_C receptors may be of clinical and pharmacological interest as potential therapeutic targets for myopia, in enhancing cognition and managing memory-related disorders, and its presence in the amygdala might be an alternative target for the development of antianxiety drugs.¹⁶

To date, the structural manipulations made in developing GABA analogues for the GABA_C receptor have mainly been confined to the carboxylic acid end of the molecule or restricting the conformations of the flexible GABA backbone. As the structure−activity-relationship (SAR) profile of selective GABA_C receptor ligands is limited, there is a need to develop more structurally diverse GABA_C receptor ligands to understand the physiological role of these receptors. During the course of our ongoing research, we envisioned a novel template for the GABA_C receptor by modifying the terminal nitrogen to incorporate a guanidino functional group, which is known to act at GABA receptors²⁰ along with restraining the phosphinic acid moiety (bioisostere of the carboxylic acid). Therefore, in the present study, we report the design, synthesis, and pharmacological evaluation of synthetically challenging cyclic phosphinic acid analogues as GABA_C receptor antagonists. The cyclic phosphinic acid nucleus has not been previously used for GABA analogues, and only one paper has described similar cyclic phosphinic acid derivatives as glutamate receptor agonists.²¹

The synthesis of 3-(aminomethyl)-1-oxo-1-hydroxy-phospholane 17 (3-AMOHP) is depicted in Scheme 1. The intermediate isopropoxy-1-oxophosphorinan-4-one 12 was prepared in five steps from water-free hypophosphoric acid, with few modifications as previously described by Verkade et al.²² Hypophosphoric acid was treated with commercially available tetraisopropyl orthosilicate to afford isopropyl phosphinate 8, which was treated with methyl acrylate to provide diester 9. The diester 9 was treated with methyl acrylate in isopropanol to afford triester 10, which was cyclized in the presence of base to afford compound 11. The selective hydrolysis of compound 11 is a key step to afford intermediate isopropoxy-1-oxophosphorinan-4-one 12. The selectivity relies on the difference in reactivity of the carboxylic and phosphinic esters. We investigated several different reaction conditions (such as different concentrations of HCl in isopropanol at room/elevated temperature), and the best condition, which gave a reasonable yield of the intermediate 12, was the treatment of the compound 11 with 3 N hydrochloric acid in the presence of isopropanol.

Scheme 1. Synthesis of Compounds 15−17.

Reagents and conditions: (a) (i-PrO)₄Si, CH₃CN, reflux for 2 h, 86%. (b) CH₂=CHCOOCH₃, NaOMe, room temperature for 24 h, 89%. (c) CH₂=CHCOOCH₃, NaOPrⁱ, i-PrOH, room temperature, 95%. (d) n-BuLi, THF, 0 °C to reflux for 4 h, 86% or KOtBu, toluene, 0 °C for 5 h, 83%. (e) 3 N HCl, i-PrOH, reflux for 12 h, 49%. (f) Tl(NO₃)₃·3H₂O, CH₂Cl₃, room temperature for 48 h, 86%. (g) ClCOOCH₂CH₃, Et₃N, THF, −10 °C for 1 h; NaBH₄ 0−5 °C, 4 h, 79%. (h) 3 N HCl, reflux for 8 h, 60%. (i) 3 N HCl, reflux for 5 h, 68%. (j) DEAD, 1 M NH₃ in benzene, PPh₃, THF, 12 h; 3 N HCl, reflux for 3 h, 55%.

The phospholane-1-oxide carboxylic acid 13 was synthesized from keto phosphinane-1-oxide 12 via ring contraction along with oxidation reaction in one step.²³ This observation may be of synthetic utility for five-membered heterocycle formation; as to the authors knowledge, it is the first demonstration of direct one-step access to heterocyclic five-membered carboxylic acid from heterocyclic six-membered ketone. The carboxylic acid 13 was reduced to primary alcohol 14 using NaBH₄ through in situ mixed carbonic−carboxylic acid anhydride. Finally, the amine was generated via a one pot Mitsunobu−Staudinger reaction, followed by phosphinate ester hydrolysis using aqueous HCl and the crude product purified via ion-exchange chromatography and recrystallization to give the free amine 17 (Scheme 1). Compounds 15 and 16 were prepared by hydrolysis of corresponding phosphinate esters using aqueous HCl.

The syntheses of 3-(amino)-1-oxo-1-hydroxy-phospholane (3-AOHP) 18 and 3-(guanido)-1-oxo-1-hydroxy-phospholane (3-GOHP) 19 are depicted in Scheme 2. Modified Curtius rearrangement of the acid 13 and hydrolysis of the intermediate isocyanates with aqueous HCl afforded the amine 18. Compound 19 was prepared by guanylation of amine 18 using formamidinesulfinic acid in the presence of a base.

Scheme 2. Synthesis of Compounds 18 and 19.

Reagents and conditions: (a) ClCOOCH₂CH₃, Et₃N, acetone, −10 °C for 1 h. (b) NaN₃, H₂O, 2 h. (c) Toluene, reflux for 2 h. (d) 3 N HCl, reflux for 3 h, 48%. (e) Formamidinesulfinic acid, NaOH, H₂O, room temperature for 12 h, 76%.

The functional characterization of the cyclic phosphinic acids at GABA receptors was performed using two-electrode voltage clamp on recombinant human GABA receptors expressed in Xenopus oocytes.^17,24 The cyclic phosphinic acids (15−19) were evaluated for activity alone and in the presence of GABA on GABA_A (α₁β₂γ_2L), GABA_B (1b/2), and GABA_C (ρ₁ and ρ₂) receptors to determine whether they behave as agonists, antagonists, or modulators. It has previously been observed that orientations of the acid, backbone, and amine group are important for antagonist activity at GABA receptors, but the distance between acid−amine counterparts and the use of alkyl phosphinic acids as a bioisostere of a carboxylic acid are also an important criterion to develop selective GABA_C antagonists.¹⁷

As shown in Table 1, we have examined the effects of cyclic phosphinic acids (15−19) on GABA_A, GABA_B, and GABA_C receptors. Compounds 15 and 16, which lack the terminal nitrogen showed no effects at 100 μM on all three GABA receptors. It proved that both the acid and the amino counterparts are important for the ligand's affinity at these receptors. 3-AMOHP 17 was found to be a potent antagonist (IC₅₀ = 19.91 μM) at ρ₁ GABA_C receptors, inactive (at 600 μM) at α₁β₂γ_2L GABA_A receptors, and a weak agonist at GABA_B(1b/2) receptors. Figure 2A shows the effect of 3-AMOHP 17 on GABA_B(1b/2) receptors expressed in Xenopus oocytes. 3-AMOHP 17 shows activation itself at 100 (33.22 ± 4.65%) and 600 μM (45.17 ± 5.23%), indicating its weak agonist nature at GABA_B(1b/2) receptors. 3-GOHP 19 was developed based on the observed activities of 4-guanidinobutanoic acid 2 (4-GBA), which is an antagonist at GABA_A and GABA_C receptors with no effects on GABA_B receptors.²⁰ ω-Guanidino acids are known to act like GABA at GABA receptors, indicating that guanidino acids behave as though the guanidino group is equivalent to the amino functionality with a more basic nature.²⁰ 3-GOHP 19 is a potent and selective antagonist (K_B = 10.0 ± 1.6 μM) at the ρ₁ GABA_C receptor. 3-GOHP 19 has reduced activity (29% inhibition at 600 μM) at α₁β₂γ_2L GABA_A receptors and was inactive (at 600 μM) at GABA_B(1b/2) receptors, indicating that substituting the amine functionality with a guanidino moiety is well tolerated at the GABA_C receptor ligand-binding site. 3-AOHP 18 was inactive at all three GABA receptors, which indicates that the distance between the acidic group and the terminal nitrogen of GABA receptor ligands appears to be important for ligand affinity at these receptors. Figure 2B shows the inhibitory concentration−response curves for the active analogues (TPMPA 3, 3-AMOHP 17, and 3-GOHP 19) against GABA (1 μM) at ρ₁ GABA_C receptors. The active compounds were further tested at human ρ₂ GABA_C receptors. Figure 2C shows the inhibitory concentration−response curves for the TPMPA 3 (IC₅₀ = 22.09 μM), 3-AMOHP 17 (IC₅₀ = 57.13 μM), and 3-GOHP 19 (IC₅₀ = 51.31 μM) against GABA (1 μM) at ρ₂ GABA_C receptors. Both compounds (17 and 19) are moderately potent antagonists at ρ₂ GABA_C receptors. In addition, 3-GOHP 19 caused a parallel rightward shift of the GABA concentration−response curves in the presence of three antagonist concentrations, indicating its competitive nature (Figure 2D; K_B = 10.0 ± 1.6 μM).

Table 1. Pharmacological Data.

	human GABAC receptors IC50 (95% CI)/KB (μM)
compd	ρ1	ρ2	human GABAB (1b/2) receptors EC50 (μM) or % inhibition	human α1β2γ2L GABAA receptors KB (μM) or % inhibition
TPMPA (3)	IC50 = 2.22 (1.32−6.09) μM	IC50 = 22.09 (18.87−25.86) μM	EC50 = ∼500 μMa	KB = 320 μMa
	KB = 2.1 μMa	KB = 14.9 μMb
(S)-4-ACPBPA (6)	KB = 4.97 μM c		22.5 ± 1.9%c,d	24.2 ± 1.7%c,e
CGP36742 (7)	IC50 = 62 μMf		IC50 = 38 μMf	IC50 = 508 μMf
15	inactive at 100 μM		inactive at 100 μM	inactive at 100 μM
16	inactive at 100 μM		inactive at 100 μM	inactive at 100 μM
17	IC50 = 19.91 (16.79−23.60) μM	IC50 = 57.13 (54.92−63.39) μM	weak agonist g	inactive at 600 μM
18	inactive at 600 μM		9.76 ± 4.6% h	inactive at 600 μM
19	IC50 = 29.74 (26.64−35.42) μM	IC50 = 51.31 (47.30−55.40) μM	inactive at 600 μM	29.21 ± 2.3% e
	KB = 10 ± 1.6 μMi

^aData from ref (12).

^bData from ref (24).

^cData from ref (17).

^dPercentage inhibition by 300 μM compound of the current produced by a submaximal does of GABA (3 μM, EC₅₀). Data are the means ± SEMs (n = 3−5 oocytes).

^ePercentage inhibition by 600 μM compound of the current produced by a submaximal does of GABA (30 μM, EC₅₀). Data are the means ± SEMs (n = 3 oocytes).

^fData from ref (18).

^gFigure 2A showing the weak agonist effects of 3-AMOHP 17.

^hPercentage inhibition by 600 μM compound of the current produced by a submaximal does of GABA (3 μM, EC₅₀). Data are the means ± SEMs (n = 3 oocytes).

ⁱThe K_B value is the mean ± SEM.

Figure 2.

(A) Sample current trace showing the effects of 3-AMOHP 17 on human GABA_B(1b/2) receptors coexpressed with GIRK1/4 channels in Xenopus oocytes using 45 mM K⁺ buffer (forward hatched bar). 3-AMOHP 17 had no effect at 10 μM (black bar) but activated GABA_B receptors at 100 (green bar) and 600 μM (purple bar) in a concentration-dependent manner, indicating weak agonist effects at this receptor. (B) Inhibitory concentration−response curves for TPMPA 3 (red dot, n = 4), 3-AMOHP 17 (green square, n = 3), and 3-GOHP 19 (blue triangle, n = 4) against GABA (1 μM) at human ρ₁ GABA receptors expressed in Xenopus oocytes. Data are the means ± SEMs (n = 3−4 oocytes). (C) Inhibitory concentration−response curves for TPMPA 3 (red dot, n = 3), 3-AMOHP 17 (green square, n = 4), and 3-GOHP 19 (blue triangle, n = 4) against GABA (1 μM) at human ρ₂ GABA receptors expressed in Xenopus oocytes. Data are the means ± SEMs (n = 3−4 oocytes). (D) Concentration−response curves of GABA alone (red dot, n = 3) and GABA in the presence of 10 (green triangle, n = 4), 30 (blue square, n = 3), and 100 μM (⧫, n = 4) 19 at human ρ1 GABAC receptors expressed in Xenopus oocytes. Data are the means ± SEMs (n = 3−4 oocytes).

To delineate the key interactions responsible for differences in binding affinity, the structures of TPMPA 3, (S)-4-ACPBPA 6, and each stereoisomer of 3-AMOHP 17, 3-AOHP 18, and 3-GOHP 19 were flexibly docked into the ligand-binding site of a ρ₁ GABA_C homology model (see the Supporting Information for details). Figures 3B,C shows the S- and R-stereoisomers of both 3-AMOHP 17 and 3-GOHP 19. These compounds are predicted to bind similarly to TPMPA 3 and (S)-4-ACPBPA 6 (Figure 3A). The binding affinity is largely ascribed to various electrostatic interactions,²⁵⁻²⁸ including (i) a salt bridge interaction between the phosphinic acid and the Arg104,²⁵ (ii) a salt bridge interaction between the basic amine/guanidino and the Glu196,²⁵ (iii) hydrogen bond contacts to Ser168 and Thr244 and to the backbone carbonyl of Tyr198,^26,27 and (iv) a cation···π attraction between the basic amine and the Tyr247, an experimentally determined interaction.²⁸ While similar binding energies are predicted for the structures of 3-AMOHP 17 and 3-GOHP 19 (see the Supporting Information), the larger guanidino group of 3-GOHP 19 incurs a slightly higher ligand strain penalty (∼3−4 kcal/mol) than 3-AMOHP 17, which may account for it is slightly decreased activity on ρ₁ GABA_C activity. The complete inactivity of 3-AOHP 18 on ρ₁ GABA_C is most likely due to the fact that neither the S- nor the R-stereoisomer can optimally span the width of the binding site (Figure 3D); therefore, they are unable to interact with Arg104 and Glu196 simultaneously, amino acids known to be important for GABA binding. Furthermore, they cannot establish a favorable cation···π attraction with Tyr247. Among all ligands docked, the largest difference is seen with (S)-4-ACPBPA 6, which additionally inserts a butyl chain into a hydrophobic pocket enclosed by Tyr198, Leu166, and Arg158 (Cα, Cβ, Cγ, and Cδ atoms) (Figure 3A). This moiety potentially liberates thermodynamically unfavorable waters²⁹ and highlights a region for further lead optimization. In conclusion, the synthesis of novel cyclic phosphinic acid template has been developed. The activity of these compounds has been investigated at the three major GABA receptor families, and 3-AMOHP 17 and 3-GOHP 19 (ω-guanidino acid) are discovered as potent and selective GABA_C receptor antagonists. This is first demonstration of a ω-guanidino acid as a selective GABA_C receptor antagonist. These results offer new knowledge of the architecture of GABA_C receptor ligand's for studies regarding the binding site and receptor flexibility. In future studies, it might be useful to generate complete SARs of this novel template for developing into more potent and selective GABA_C receptor antagonists.

Figure 3.

View of the ρ₁ GABA_C ligand-binding site with predicted binding modes. (A) TPMPA 3 (green carbons) and (S)-4-ACPBPA 6 (yellow carbons). (B) Stereoisomer of 3-AMOHP 17: (R)-3-AMOHP (green carbons) and (S)-3-AMOHP (yellow carbons). (C) Stereoisomer of 3-GOHP 19: (R)-3-GOHP (green carbons) and (S)-3-GOHP (yellow carbons). (D) Stereoisomer of 3-AOHP 18: (R)-3-AOHP (green carbons) and (S)-3-AOHP (yellow carbons).

Supporting Information Available

Synthetic experimental procedures, analytical and spectral characterization data of all synthesized compounds including elemental analyses, and details of molecular modeling and pharmacology. This material is available free of charge via the Internet at http://pubs.acs.org.