Synthesis and Biological Activity of Allosteric Modulators of GABAB Receptors Part 3. 3-(2,6- Bis-iso-propyl-4-hydroxyphenyl)propanols

Uma série de seis derivados do propan-1-ol 2,2-disubistituído 3-[3,5-di-iso-propil-4hydroxifenil] foi preparada para avaliação como moduladores alostéricos de receptores GABA B . A maior atividade (EC 50 30 μM) encontrada na série foi para os análogos do dimetil, mas os compostos isopropilfenil foram, de maneira geral, mais fracos que os compostos t-butílicos. A metilação do grupo fenólico levou à uma perda de atividade.


Introduction
Following a random mass screening, Urwyler et al. 1 recently reported that 2,6-di-tert-butyl-4-(3-hydroxyphenyl)-2,2-dimethylpropionaldehyde, CGP 13501, (1)  acted as a positive allosteric modulator for GABA B receptors, 2,3 and its reduction product, the corresponding alcohol CGP 7930 (2) was found to be even more potent.Because of our interest in such modulators, 2,4,5 we speculated that the latter could be regarded as a hybrid of propofol (3)  and hydroxybutyric acid (GHB, 4).Propofol 6,7 is a shortacting hypnotic agent (GABA A modulator), effective for induction and maintenance of anaesthesia when admi-nistered intravenously either as repeated bolus injections or by continuous infusion.GHB is best known as a drug of abuse, 8 and is a GABA analogue.Accordingly, we have recently synthesized a series of compounds modifying the structure 2, all of which acted as positive modulators at GABA B receptors. 9The most active was the cyclohexyl analogue (5), which was 2 to 3 times as active as the lead compound (2).Our working hypothesis for the lead compound (2) is that it acts through the 3-hydroxyphenylpropyl moiety, with the hydroxyphenyl group corresponding to the carboxyl group of GHB, in addition to providing hydrophobic properties that would facilitate transport across cell membranes.Since our previous work 9 was concerned mainly with an examination of the effect of changing the hydrophobicity and steric bulk near the hydroxyl end of the molecule, we now report the preparation of some variants at the phenolic portion of the molecule; at this stage little is known of the pharmacokinetics of these compounds, and their therapeutic potential is uncertain.The basic synthetic approach continues to be basically that of Urweyler et al. 1
Since we suspected that these compounds were GABA mimics, it was desirable to synthesise at least one example of a primary amine, and the reductive amination method of Borch et al. 10 was chosen.
Reaction of the aldehyde 7b with ammonium acetate and sodium cyanoborohydride, 10 gave a good yield, not of the desired primary amine 10, but of the secondary amine 11.We suspect that the initially formed 10 reacted with 7b faster than with ammonium acetate, leading to the new intermediate 13, reduction of which gave the observed product 11, as shown in Scheme 2.
The subtle difference in the decreased activating effect of isopropyl groups compared to tert-butyl groups was noted when the reaction of ether 6 with 3methylbutan-2-one failed to occur to any significant extent under basic conditions.However, the use of trifluoroacetic acid in refluxing dichloromethane allowed efficient reaction of the ether with ketones, and indeed also gave cleaner reaction products with aldehydes, leading to products 7a-7c.
The requirement for an acidic or hydrogen-bonding centre on the phenyl group was probed by methylation of 8a in this series to give 14, but attempt to synthesize methyl ethers from tertiary butyl derivatives failed.
In order to further examine the suspected importance of the bulkiness of the aryl substituents in these compounds, the synthesis of methyl analogues was investigated.However, as reported in the literature, 11  base catalysed condensation of 2,6-dimethylphenol with formaldehyde in basic methanol gave 4,4'-dihydroxy-3,5,3',5'-tetramethylphenylmethane 15 instead of the desired 2,6-dimethyl-4-methoxymethylphenol 16, and this area was not pursued further.

Biological evaluation
As described previously, 4,12,13 the compounds prepared above were examined for their pharmacological effects in enhancing baclofen-induced hyperpolarizing responses at GABA B post-synaptic receptors, and as potentiators of baclofen actions in reducing electricallyevoked release of [ 3 H]-GABA or [ 3 H]-glutamate at presynaptic receptors, mediated via GABA B auto or heteroreceptors, respectively, in rat neocortical slice preparations. 14,15Baclofen is a classical selective agonist at GABA B receptor sites, 16,17 and is commonly used in rat brain preparations to stimulate these receptors to induce a neuronal response.Herein (Table 1) we summarise the results of the effects of the test compounds in modulating baclofen mediated function using grease gap recording in rat neocortical slices. 12,13The responses induced by baclofen are dose-dependent, with baclofen generating an EC 50 value of approximately 10 μM. 12 The EC 50 value for the agonist baclofen is calculated as the concentration of baclofen inducing 50% of the maximum hyperpolarizing response.Using this fixed concentration of baclofen at 10 μM, the concentration-response curves of differing concentrations of the test compounds are subsequently constructed.From these curves, the EC 50 values of the test compounds in potentiating the baclofen response are measured, the values representing the concentration of the compound needed to induce 50% of the maximal potentiated responses.
All the new 3-(hydroxyphenyl)propanol derivatives had relatively low activity with EC 50 typically around 30-100 μM.As with the t-butyl compounds reported earlier, 9 this may be a reflection of their extremely low solubility even in DMSO/water mixtures, leading to partial precipitation during the testing procedure: real activities possibly may be somewhat greater.The methyl ethers were of very low activity, and their solubility was even lower.The tert-butyl derivatives reported previously 9 appeared to be two to three times more active than the isopropyl series, but still several orders of magnitude lower than that of the N-(phenylpropyl)-1arylethylamines. 2

Conclusion
Generally, it can be concluded that 3-(4-hydroxy-3,5dialkylphenyl)propanols represent a new distinct classes of GABA B receptor modulators, limited at this stage by very poor solubility.The 3,5-di i-propyl analogues are less active than the corresponding t-butyl compounds.However, these GABA B modulators may still represent a novel therapeutic strategy for the treatment of various neurological and pathological diseases mentioned previously, without the side effects of full GABA B receptor agonists.

Experimental
All solvents used were freshly distilled and dried according to the methods of Perrin and Armarego. 18elting points were determined on a Reichert hot stage microscope and are uncorrected. 1H (300 MHz) and 13 C (75.5 MHz) NMR spectra were recorded on a Gemini Varian 300 spectrometer in deuteriochloroform with tetramethylsilane as internal standard, unless otherwise stated.Infrared spectra were recorded on a Thermonicolet (Nexus 670) FT-infrared spectrophotometer, measured as films or KBr disks.Mass spectra and high resolution mass spectra were recorded on a Kratos MS25RF spectrometer.