Methylphosphinic Research Articles

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K+-stimulated release of [3H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA(B) and GABA(C) receptors. The involvement of GABA(A) receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K+-evoked taurine release. Furthermore, several GABA(B) receptor effectors were able to inhibit K+-stimulated taurine release in the adults, while the GABA(C) receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds.

Functional and pharmacological properties of GABAρ1Δ51 receptors

Gamma-aminobutyrate is the main inhibitory neurotransmitter in the vertebrate brain, and the gamma-aminobutyric acid (GABA) receptor subunit GABArho1delta51 is an alternatively spliced form of the GABArho1 receptor that was recently isolated from human retina cDNA libraries. The rho1delta51 receptor subunit lacks 17 amino acids in the extracellular N-terminal domain and, when expressed in Xenopus oocytes, forms functional homomeric GABA receptors. Unexpectedly, even after a such a big deletion, the fundamental properties of the deleted variant receptors are very similar to those of the complete GABArho1 receptors. For example, both types of receptors are bicuculline resistant, desensitize very little, and are negatively modulated by Zn2+ and positively modulated by La3+. In spite of such similarities, the GABArho1delta51 receptors are more sensitive to GABA, to the specific GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and to Zn2+, than the complete GABArho1 receptors. The GABArho1delta51 receptors extend the variety of inhibitory receptors in the retina. Their functional significance still remains to be determined.

Syntheses of new racemicNG-(1-iminoethyl)phosphalysine derivatives as potential inhibitors of nitric oxide synthase

The efficient syntheses of three new racemic NG-(1-iminoethyl)phosphalysine derivatives are reported where the lysine carboxylate group is systematically replaced by phosphonic acid, 4, methyl phosphinic acid, 5, and phosphinic acid, 6. These compounds were evaluated as potential inhibitors of the three isoforms of human nitric oxide synthase. © 2000 John Wiley & Sons, Inc. Heteroatom Chem 11:505–511, 2000

The 'ABC' of GABA receptors: a brief review.

1. In the mammalian central nervous system, GABA is the main inhibitory neurotransmitter. GABA is a highly flexible molecule and, thus, can exist in many low-energy conformations. Conformationally restricted analogues of GABA have been used to help identify three major GABA receptors, termed GABAA, GABAB and GABAC receptors. 2. GABAA and GABAC receptors are members of a super-family of transmitter-gated ion channels that include nicotinic acetylcholine, strychnine-sensitive glycine and 5HT3 receptors. GABAA receptors are hetero-oligomeric Cl- channels that are selectively blocked by the alkaloid bicuculline and modulated by steroids, barbiturates and benzodiazepines. To date, 16 human GABAA receptor cDNA have been cloned. 3. GABAB receptors are seven transmembrane receptors that are coupled to G-proteins and activate second messenger systems and Ca2+ and K+ ion channels. To date, three GABAB receptor proteins have been cloned and these resemble metabotropic glutamate receptors. GABAB receptors are hetero-oligomeric receptors made up of a mixture of a combination of the subunits. These receptors are selectively activated by (-)-baclofen and CCGP27492 and are blocked by phaclofen, the phosphonic acid analogue of baclofen. 4. In contrast, GABAC receptors represent a relatively simple form of transmitter-gated Cl- channel made up of a single type of protein subunit. Two human GABAC receptor cDNA have been cloned. These receptors are not blocked by bicuculline nor are they modulated by steroids, barbiturates or benzodiazepines. Instead, GABAC receptors are selectively activated by the conformationally restricted analogues of GABA in the folded conformation cis-4-aminocrotonic acid and (1s,2R)-2-(aminomethyl)-1-carboxycyclopropane. (1,2,5,6-Tetrahydropyridine-4-yl)methylphosphinic acid, a methylphosphinic acid analogue of GABA in a partially folded conformation, is a selective antagonist at GABAC receptors.

GABA(A) and GABA(C) receptors have contrasting effects on excitability in superior colliculus.

We have recently found that GABA(C) receptor subunit transcripts are expressed in the superficial layers of rat superior colliculus (SC). In the present study we used immunocytochemistry to demonstrate the presence of GABA(C) receptors in rat SC at protein level. We also investigated in acute rat brain slices the effect of GABA(A) and GABA(C) receptor agonists and antagonists on stimulus-evoked extracellular field potentials in SC. Electrical stimulation of the SC optic layer induced a biphasic, early and late, potential in the adjacent superficial layer. The late component was completely inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione or CoCl(2), indicating that it was generated by postsynaptic activation. Muscimol, a potent GABA(A) and GABA(C) receptor agonist, strongly attenuated this postsynaptic potential at concentrations >10 microM. In contrast, the GABA(C) receptor agonist cis-aminocrotonic acid, as well as muscimol at lower concentrations (0.1-1 microM) increased the postsynaptic potential. This increase was blocked by (1,2,5, 6-tetrahydropyridine-4-yl)methylphosphinic acid, a novel competitive antagonist of GABA(C) receptors. Our findings demonstrate the presence of functional GABA(C) receptors in SC and suggest a disinhibitory role of these receptors in SC neuronal circuitry.

Antagonism by (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid of synaptic transmission in the neonatal rat spinal cord in vitro: an electrophysiological study

The effect of the novel GABA C receptor antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) on synaptic transmission and GABA-mediated responses was investigated with electrophysiological recordings from the in vitro spinal cord preparation of the neonatal rat. Bath-applied TPMPA (10 μM) had no effect on spinal reflexes evoked by dorsal root stimulation, on ventral root polarization level or amplitude of ventral root depolarizations induced by exogenously applied GABA (0.5 mM). TPMPA significantly attenuated the depressant action of GABA on spinal reflexes without changing responses induced by the GABA A receptor agonist isoguvacine (50 μM) or the GABA B receptor agonist baclofen (0.5–2 μM). Following block of GABA A receptors by bicuculline (20 μM) and of glycine receptors by strychnine (1 μM), regular bursting activity recorded from ventral roots developed spontaneously and persisted unchanged for many hours. This bursting pattern, which is generated at the level of the interneuronal network, was significantly slowed down by TPMPA, which also increased the duration of individual bursts and the number of intraburst oscillations. These results suggest that in the neonatal rat spinal cord some functional GABA C receptors exist: their role was clearly unmasked following pharmacological block of GABA A (and glycine) receptors. Under these conditions GABA C receptors appeared to contribute to the excitation of spinal interneurons supporting rhythmic bursting activity.

Piperidinyl-3-phosphinic acids as novel uptake inhibitors of the neurotransmitter γ-aminobutyric acid (GABA)

Piperidinyl-3-phosphinic acid 2, piperidinyl-3-methylphosphinic acid 3 and N-(4,4-diphenyl-3-butenyl)-piperidinyl-3-phosphinic acid 4 have been synthesized as bioisosteres of the corresponding amino carboxylic acids, which are potent and specific GABA-uptake inhibitors. The novel amino phosphinic acids were tested for their GABA-uptake inhibitory activity and 2 and 4 were identified as the first phosphinic acid based GABA-uptake inhibitors. The methylphosphinic acid 3 was found to be inactive.

Syntheses of Methylphosphinic Acids using Tandem Pudovik/Abramov-Barton/McCombie Reactions

Methylphosphinic acids are very effective and potent carboxylic acid bioisosteres. We have developed a novel mild and eficient method for the syntheses of (sec-alky1)methylphosphinic acids based on tandem Abramov addition of readily available ethyl methylphosphinate, 2, to a ketone, la-lg, followed by a modified Barton-McCombie deoxygenation procedure. The resulting esters, 4a-4g. are readily hydrolyzed. The method gives high yields even for sterically demanding ketones. Using this method we have synthesized several new biologically interesting methylphosphinic acids.

Syntheses and Gaba Receptor Binding Properties of 1-, 2- and 3-Hydroxy Aminobutyl Phosphinic Acids

Novel racemic 1-hydroxy-, 2-hydroxy- and 3-hydroxy aminobutyl-phosphinic and metylphosphinic acids were synthesized. The acids are bioisosteres of the corresponding carboxylic acids, some of which are GABA, antagonists. The novel hydroxylated phosphinic and methylphosphinic acids were evaluated for their GABA, and GABA, receptor binding properties using rat brain synaptosomes and were also tested for GABA activity in a guinea pig ileum model. All the compounds tested were inactive.

Preparation of Novel Piperidine Phosphinic Acid Analogues of the Inhibitory Neurotransmitter γ-Aminobutyric Acid (GABA)

Novel pipendine phosphinic, methylphosphinic and phosphonic acids were synthesized (5a-5c and 6 in the figure). The acids are bioisosteres of the corresponding carboxylic acid isonipecotic, which is a GABAA agonist.

Syntheses and GABA receptor binding properties of 4-amino-1-, 2-, and 3-hydroxybutylphosphinic acids

Novel racemic 4-amino-1-, 2-, and 3-hydroxybutylphosphinic acids and the corresponding 4-amino-1-, 2-, and 3-hydroxybutyl methylphosphinic acids have been synthesized. The phosphinic acid groups are bioisosteres of the carboxylic acid group, and some of these hydroxy amino acids are GABA B antagonists. The novel phosphinic acids were evaluated for their GABA A and GABA B receptor binding properties using rat brain synaptosomes and were also tested for GABAergic activity in a guinea pig ileum model. None of the phosphinic acids tested were found to be active.

GABA B receptors as potential targets for drugs able to prevent excessive excitatory amino acid transmission in the spinal cord

The effects of GABA B receptor activation on the Ca 2+-dependent depolarization-induced overflow of endogenous glutamic acid and γ-aminobutyric acid (GABA) was studied in rat spinal cord nerve terminals exposed in superfusion to 15 mM KCl. The GABA B receptor agonist (−)-baclofen inhibited the K +-evoked overflow of glutamate (EC 50=0.098 μM) but was almost inactive against that of GABA. The overflow of both transmitters could be quite similarly inhibited by two other GABA B receptor agonists, 3-APPA (3-aminopropylphosphonous acid; EC 50=0.087 and 0.050 μM in the case of GABA and glutamate, respectively) and CGP 44532 (3-amino-2( S)-hydroxypropyl)methylphosphinic acid; EC 50=0.81 and 0.50 μM). The GABA B receptor antagonist CGP 35348 [3-amino-propyl(diethoxymethyl)phosphinic acid] blocked the effect of 3-APPA (1 μM) at the autoreceptors (IC 50≃1 μM), but not at the heteroreceptors. In contrast, the effects of 3-APPA at both autoreceptors and heteroreceptors could be similarly prevented by another GABA B receptor antagonist, CGP 52432 [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl) phosphinic acid (IC 50≃10 μM). The data suggest that, in the spinal cord, GABA B autoreceptors on GABA-releasing terminals differ pharmacologically from GABA B heteroreceptors on glutamatergic terminals. Selective GABA B receptor ligands may be helpful for conditions characterized by excessive glutamatergic transmission in the spinal cord.

GABA C receptor antagonists differentiate between human ρ1 and ρ2 receptors expressed in Xenopus oocytes

The selective GABA C receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), is eight times more potent against human recombinant ρ1 receptors than ρ2 receptors expressed in Xenopus oocytes. (3-Aminopropyl)methylphosphinic acid (CGP35024), the methylphosphinic acid analogue of GABA, and [( E)-3-aminopropen-1-yl]methylphosphinic acid (CGP44530), an open chain analogue of TPMPA, were five and four times, respectively, more potent as antagonists of ρ1 receptors than as antagonists of ρ2 receptors. Isoguvacine was a weak partial agonist at both ρ1 and ρ2 receptors with intrinsic activities (calculated as a percentage of the maximum whole cell current produced by a maximum dose of GABA) of 45 and 68%, respectively, of the maximum response produced by GABA. In agreement with other workers, it was found that imidazole-4-acetic acid was a partial agonist at both ρ1 and ρ2 receptors, showing higher intrinsic activity at ρ2 than at ρ1 receptors. The ρ1 receptor antagonist, trans-4-amino-2-methylbut-2-enoic acid (2-MeTACA), was a partial agonist at ρ2 receptors with an intrinsic activity of 34%. 2-MeTACA may be useful in differentiating between homo-oligomeric ρ1 and ρ2 receptors in native systems. These studies reveal significant differences in the antagonist profile of human recombinant ρ1 and ρ2 GABA C receptors.

Syntheses of novel piperidin-4-ylphosphinic acid, and piperidin-4-ylphosphonic acid analogues of the inhibitory neurotransmitter 4-aminobutyric acid (GABA)

Piperidin-4-ylphosphinic acid, methyl(piperidin-4-yl)phosphinic acid and piperidin-4-ylphosphonic acid analogues of the GABAA agonist piperidin-4-ylcarboxylic acid (isonipecotic acid) were synthesised. The acid groups were introduced using a sequential Pudovik addition followed by a Barton deoxygenation procedure and finally followed by acidic hydrolysis. The mild and efficient procedure gave the target amino acids in good yields.

The determination of magnesium in human blood plasma by 31P magnetic resonance spectroscopy using a macrocyclic reporter ligand

The ligand 1,4,7-triazacyclononane-1,4,7-tris(methylene methylphosphinic acid), NOTMP, was used to measure free Mg II levels in blood plasma by 31P MRS. Separate resonances were observed for the free ligand and the Mg II complex and the ratio of their resonance areas was used to evaluate the free, ionized Mg II concentration, [Mg] free. The Ca II and the Zn II complexes gave rise to separate resonances in the 31P spectrum in an aqueous sample. In human blood plasma samples, however, these resonances were never observed thus excluding the interference of these metal ions. Heparin, up to 150 units/ml, had no influence on the Mg-NOTMP equilibrium. The 31P MRS methodology was applied to twenty human blood plasma samples. Total Mg II ([Mg] total), as measured by atomic absorption spectroscopy, averaged 0.85±0.12 mM while free ionized Mg II ([Mg] free) measured by 31P MRS was 0.66±0.09 mM. The 31P MRS method gave inherently larger values for free ionized Mg II than that reported by ion-selective electrodes (ISE). This was traced to a redistribution of existing plasma Mg II species after the addition of about 2 mM of NOTMP. Calculations using existing thermodynamic data show that the ionized Mg II concentration (iMg) and the concentration of Mg II weakly complexed to small anions (Mg comp) both drop after the addition of NOTMP, with Mg comp dropping to negligible levels. Thus, the 31P MRS method appears to be less sensitive to variations in the concentration of weakly binding anions (bicarbonate, carbonate, chloride, lactate, phosphate, etc.) than the ISE method. Our data indicates that the difference between Mg total, as measured by atomic absorption spectroscopy, and Mg free, as measured by 31P MRS, provides an direct estimate of the protein bound Mg II fraction.

Unsaturated phosphinic analogues of γ-aminobutyric acid as GABA C receptor antagonists

The phosphinic and methylphosphinic analogues of γ-aminobutyric acid (GABA) are potent GABA C receptor antagonists but are even more potent as GABA B receptor agonists. Conformationally restricted unsaturated phosphinic and methylphosphinic analogues of GABA and some potent GABA B receptor phosphonoamino acid antagonists were tested on GABA C receptors in Xenopus oocytes expressing human retinal ϱ 1 mRNA. 3-Aminopropyl- n-butyl-phosphinic acid (CGP36742), an orally active GABA B receptor antagonist, was found to be a moderately potent GABA C receptor antagonist (IC 50 = 62 μM). The unsaturated methylphosphinic and phosphinic analogues of GABA were competitive antagonists of the GABA C receptors, the order of potency being [( E)-3-aminopropen-1-yl]methyl-phosphinic acid (CGP44530, IC 50 = 5.53 μM) > [( E)-3-aminopropen-1-yl]phosphinic acid (CGP38593, IC 50 = 7.68 μM) > [( Z)-3-aminopropen-1-yl]methylphosphinic acid (CGP70523, IC 50 = 38.94 μM) > [( Z)-3-aminopropen-1-yl]phosphinic acid (CGP70522, IC 50 > 100 μM). This order of potency differs from that reported for these compounds as GABA B receptor agonists, where the phosphinic acids are more potent than the corresponding methylphosphinic acids.

DHPMP: a novel group I specific metabotropic glutamate receptor agonist

The synthesis and preliminary pharmacological evaluation of (RS)-amino(3,5-dihydroxyphenyl)methylphosphinic acid (DHPMP) is reported. DHPMP has been identified as possessing Group I specific metabotropic agonist activity.

The first selective antagonist for a GABA C receptor

(1,2,5,6-Tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA, 1), which is synthesized in six steps from 3-butyn-1-ol, is the first γ-aminobutyric acid (GABA) receptor antagonist that shows strong selectivity for mammalian GABA C receptors.

Phosphinic acid analogues of GABA. 1. New potent and selective GABAB agonists.

The antispastic agent and muscle relaxant baclofen 1 is a potent and selective agonist for bicuculline-insensitive GABAB receptors. For many years efforts to obtain superior GABAB agonists were unsuccessful. We describe the syntheses and biological properties of two new series of GABAB agonists, the best compounds of which are more potent than baclofen in vitro and in vivo. They were obtained by replacing the carboxylic acid group of GABA or baclofen derivatives with either the phosphinic acid or the methylphosphinic acid residue. Surprisingly, ethyl- and higher alkylphosphinic acid derivatives of GABA yielded novel GABAB antagonists, which are described in part 2 of this series. Structure-activity relationships of the novel GABAB agonists are discussed with respect to their affinities to GABAB receptors as well as to their effects in many functional tests in vitro and in vivo providing new muscle relaxant drugs with significantly improved side effect profiles.

Preparation of alkyl- and aryl-amino[2H2]methylphosphinic acids

Diethyl chloro[2H2]methylphosphonate upon successive treatment with POCl3 and Grignard reagents (RMgX) gave alkyl(chloro[2H2]methyl)phosphinates. Phenyl(chloro[2H2]methyl)phosphinate was prepared by deuteriolysis of the α-silylated α-phosphonylated carbanion 11 with D2O. After conversion into trifluoroethyl phosphinic esters, the alkyl- and phenyl-(chloro[2H2]methyl)phosphinates were converted into azides and then reduced and hydrogenated into alkyl- and phenyl-(amino[2H2]methyl)phosphinic acids.