GABAB-Agonistic Activity of Certain Baclofen Homologues

Baclofen (1) is a potent and selective agonist for bicuculline-insensitive GABAB receptors and is used clinically as an antispastic and muscle relaxant agent. In the search for new bioactive chemical entities that bind specifically to GABAB receptors, we report here the synthesis of certain baclofen homologues, namely (R,S)-5-amino-3-arylpentanoic acid hydrochlorides (R,S)-1a–h as well as (R,S)-5-amino-3-methylpentanoic acid [(RS)-1i] to be evaluated as GABABR agonists. Compound 1a is an agonist to GABAB receptors with an EC50 value of 46 μM on tsA201 cells transfected with GABAB1b/GABAB2/Gqz5, being the most active congener among all the synthesized compounds.


Introduction
4-Aminobutanoic acid (GABA) is the well-known inhibitory neurotransmitter in the mammalian central nervous system where it exerts its effects through ionotropic (GABA A/C ) receptors and metabotropic (GABA B ) receptors [1]. Cloning and photoaffinity labeling experiments of the GABA B

OPEN ACCESS
receptor demonstrated two isoforms, designated GABA B 1a and GABA B 1b which dimerize with the GABA B 2 receptor subunit to produce functionally active GABA B receptors [2]. 4-Amino-3-(4chlorophenyl)butanoic acid (baclofen, 1, Figure 1) is the classical GABA B agonist and interacts with GABA B receptors stereospecifically. The GABA B agonistic activity of racemic baclofen is known to reside primarily in the R-(-)-enantiomer [3]. (R,S)-Baclofen (1) is used clinically for the treatment of spasticity associated with brain and spinal cord injuries [4], drug addiction and alcoholism [5], gastroesophageal reflux disease (GERD) [6], cancer pain [7] and overactive bladder [8]. Recently, R-(−)-baclofen is under development for the treatment of behavioral symptoms of Fragile X Disorder [9].

Chemistry
An examination of the literature revealed that there are two common synthetic strategies, namely the Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation, which can be used to prepare the intermediate cyano esters 3a-c, 3e-h and 5i. Therefore, HWE was applied for preparation of both 3a-c and 3e-h while Knoevenagel condensation was adopted to get 5i, depending on the commercial availability of their respective starting materials. Accordingly, an allylic bromination step was required jointly with the HWE reaction to prepare compounds 3e-h, while only the HWE reaction and Knoevenagel condensation were required to prepare the cyano esters 3a-c and 5i, respectively.
The synthetic pathways which were adopted to synthesize the target compounds 1a-i are illustrated in Schemes 1-3. Thus, 3-aryl-4-chloro-2-butenoic acid ethyl esters 4a-c have been successfully produced by applying the HWE reaction on substituted acetophenones 5a-c using triethyl phosphonoacetate and sodium hydride in 1,2 dimethoxyethane following the procedure cited by Wadsworth and Emmons [13] (Scheme 1). The 13 C-NMR chemical shift differences between C-1, C-3 and in particular C-4 for the (E) and (Z)-isomers of 4a-c are consistent with the observed differences for (E) and (Z)-isomers mentioned by Allan and Tran [14].
It is noteworthy that substitution at the ortho position of the phenyl ring in 2-chloro-1-(2,4-dichlorophenyl)-1-ethanone (5b) increased the proportion of (E)-isomer in the produced diasteromeric mixture of 4-chloro-3-(2,4-dichlorophenyl)-2-butenoic acid ethyl ester (4b), which is in accordance with the findings of Jones and Maisey [15]. 3-Aryl-4-chloro-2-butenoic acid ethyl esters 4a-c (as diasteromeric mixtures) were subjected to a nucleophilic displacement of the halogen with potassium cyanide in aqueous ethanol to obtain 3-aryl-4-cyano-2-butenoic acid ethyl esters 3a-c via the trivial procedure mentioned by Ives and Sames [16]. Unfortunately, the starting materials decomposed and we did not obtain the anticipated compounds 3a-c in any detectable amounts. This troublesome nucleophilic substitution reaction was successfully achieved using a stoichiometric amount of 3-aryl-4-chloro-2-butenoic acid ethyl esters 4a-c (as diasteromeric mixtures) and tetraethylammonium cyanide (TEAC). The reaction mixture was stirred at 50 °C in acetonitrile for 18 h according to the reported procedure [17]. The crude compounds 3a-c were purified by column chromatography using the appropriate solvent system to afford mainly (E)-3a-c in 42%-66% yields. Use of a catalytic amount of TEAC instead of a stoichiometric amount to produce 3a-c led to a dramatic decrease in the yields.
(E)-3-Aryl-4-cyano-2-butenoic acid ethyl esters 3a-c are multifunctional molecules and we aimed to reduce selectively both nitrile and double bond functionalities without affecting the ester functionality to afford the title compounds (R,S)-5-amino-3-aryl-pentanoic acid hydrochlorides 1a-d. Catalytic hydrogenation is one of the most powerful methods in the arsenal of the synthetic medicinal chemistry facilitating the chemical synthesis of a myriad of bio-active molecules both in research laboratories and industrial settings. Accordingly, 3a-c were subjected to catalytic hydrogenation using a catalytic amount of PtO 2 (for 3a and 3b) or 10% Pd/C (for 3c) and concentrated hydrochloric acid in 95% ethanol on a Parr shaker apparatus under 4 bar of H 2 for 18 h at room temperature to give (R,S)-5-amino-3-aryl-pentanoic acid ethyl ester hydrochlorides 2a-c.
Synthesis of the title compounds 1e-h is portrayed in Scheme 2. The synthetic pathway was commenced with the preparation of (Z)-3-aryl-4-bromo-2-butenoic acid ethyl esters 4e-h.
Chemoselective allylic bromination of 3-aryl-2-butenoic acid ethyl esters 5e-h (as diasteromeric mixtures) was accomplished by adopting Wohl-Ziegler bromination. Compounds 5e-h and a stoichiometric amount of N-bromosuccinimide (NBS) were refluxed in carbon tetrachloride and then a catalytical amount of dibenzoyl peroxide (DBP) was added to the reaction mixture according to the method advocated by Chiefari et al. [18] to afford (Z)-3-aryl-4-bromo-2-butenoic acid ethyl esters 4e-h in moderate yields. The isolated isomers of 4e-h were assigned to be (Z)-isomers based on their 1 H-NMR spectral data.
Elaboration of 4e-h to give 3e-h was conducted using the aforementioned procedure for preparation of 3a-c. Subsequently, 3e-h were transformed to the target compounds 1e-h by adopting the same reaction sequence which was previously described for the preparation of compounds 1a-d from 3a-c.
4-Cyano-3-methyl-2-butenoic acid ethyl ester (5i, as a diasteromeric mixture) was subjected to catalytic hydrogenation using 10% Pd/C and concentrated hydrochloric acid in 95% ethanol to afford (R,S)-5-amino-3-methylpentanoic acid ethyl ester hydrochloride (4i). Without further purification, the crude 4i was hydrolyzed by reflux in 5 N hydrochloric acid to give (R,S)-5-amino-3-methylpentanoic acid hydrochloride (3i). It has to be mentioned that our attempt to obtain compound 3i in a sufficient pure form by recrystallization was unsuccessful. Accordingly, the amino functionality of 3i was derivatized with a lipophilic moiety to facilitate its purification by a simple acid-base chemical treatment.

GABA B Agonistic Activity
We have previously described a robust pharmacological assay of heterodimeric GABA B R1b/GABA B R2 receptors co-expressed with the chimeric G protein Gαq-z5 in tsA201 cells (a transformed HEK293 cell line). Co-expression of Gαq-z5 convert the endogenous coupling to the Gαi/o signaling pathway to the Gq pathway, which generally leads to more robust assays measured as increases in inositol phosphates or intracellular calcium levels [22]. We have previously shown that the pharmacological profiles of a range of standard agonists using this assay correlate well with other assays using either cell lines with recombinant receptor expression or tissues with endogenous GABA B R expression. Furthermore, we have shown that the GABA B R antagonists 2-OH-saclofen and CGP35348 can antagonize agonist responses in this assay [23,24]. Finally, like other groups [25], we have not found any pharmacological differences of orthosteric ligands between GABA B R1a and GABA B R1b subunits co-expressed with GABA B R2 using this assay [23]. The assay is thus suitable for characterization of orthosteric GABA B R ligands, and in the present study we have characterized the synthesized ligands on GABA B R1b/GABA B R2 receptors co-expressed with the chimeric G protein Gαq-z5 in tsA201 cells measuring responses as increases in intracellular calcium measured by the calcium sensitive fluorescent probe Fluo-4.
The GABA B agonistic activity of the synthesized compounds 1a-i is summarized in Table 1. Compounds 1a, 1e and 1f are active as GABA B R agonists (EC 50 value 46-170 μM, Figure 2) whereas compounds 1b, 1c, 1d, 1g, 1h and 1i (EC 50 > 300 μM) are considered inactive as GABA B R agonists in the GABA B R subtype used in our assay.  The curves are representative for the average pharmacological profile of the agonists. The Ca 2+ measurement assays were performed as described in the materials and methods section.
Regarding the structure-activity relationship in the synthesized series 1a-i, it has to be mentioned that mono-substitution on the aromatic moiety attached to the 3-position of the DAVA backbone with a halogen, especially a para-chloro, is optimum for GABA B R agonistic activity. The synthesized compounds which evoked GABA B R agonistic activity have the following decreasing order of activity: 1a > 1e > 1f. On the other hand, substitution in the para-position of the aromatic moiety in the three position of the DAVA backbone with methoxy, methyl or no substitution led to loss of GABA B R agonistic activity (EC 50 > 300 μM). These results are comparable with the previously published results of GABA B agonists [26]. The lack of GABA B R agonistic activity of compound 1b bearing a 2,4-dichloro aromatic moiety in the three position of the DAVA backbone could be attributed to steric reasons which affect the interaction of 1b with the binding regions of GABA B receptors. In addition, replacement of the aryl moiety in the three position of the DAVA backbone with a methyl group, i.e., compound 1i, led to a loss of GABA B R agonistic activity. Compounds 1b, 1c, 1d, 1g, 1h and 1i which showed EC 50 > 300 μM as GABA B R agonists were evaluated as GABA B R antagonists at 1 mM concentration against 10 μM GABA, but none of these compounds were effective as GABA B R antagonists.

General
Melting points were determined using a capillary melting point apparatus (Gallenkamp, Sanyo) and are uncorrected. Infrared (IR) spectra were recorded as thin layer films (for oils) or as pellets (for solids) with BIO-RAD spectrometer and values are represented in cm −1 . NMR ( 1 H-NMR and 13 C-NMR) spectra were recorded on a Bruker AC 250 spectrometer (at 250 MHz for 1 H-NMR and 63 MHz for 13 C-NMR) and chemical shift values were recorded in ppm on the δ scale. All samples were measured at room temperature. The 1 H-NMR data are presented as follows: Chemical shifts, multiplicity, number of protons, assignment. Column chromatography was carried out on silica gel 60 (0.063-0.200 mm) obtained from Merck. Elemental analyses were performed by the microanalytical section of the Institute of Inorganic Chemistry, University of Würzburg, Würzburg, Germany.

General Procedure for the Preparation of 3-Aryl-4-chloro-2-butenoic Acid Ethyl Esters 4a-c
Triethyl phosphonoacetate (2.92 g, 13 mmol) was added dropwise to a cold (5-10 °C) stirred slurry of 60% sodium hydride (0.52 g, 13 mmol) in dry 1,2 dimethoxyethane (20 mL). After complete addition, the reaction mixture was stirred at ambient temperature for 30 min or until gas evolution ceased. A solution of the appropriate ketone 5a-c (10 mmol) in dry 1,2 dimethoxyethane (10 mL) was then added dropwise to the resulting solution. The reaction mixture was heated under stirring at 50 °C for 18 h. The reaction mixture was cooled to room temperature, poured into water (100 mL) and extracted with diethyl ether (3 × 50 mL). The organic extract was dried (Na 2 SO 4 ), filtered and evaporated under vacuum to afford viscous oils which were purified by column chromatography using petroleum ether (40-60 °C): Diethyl ether (9:1) to give compounds 4a-c in 40%-88% yields as pale yellow viscous oils.

General Procedure for the Preparation of 3-Aryl-2-butenoic Acid Ethyl Esters 5e-h
To a cold (5-10 °C) solution of potassium t-butoxide (1.46 g, 13 mmol) in dry tetrahydrofuran (20 mL) was added dropwise triethyl phosphonoacetate (2.92 g, 13 mmol). The resulting solution was stirred at room temperature for 30 min. A solution of the appropriate ketone 6e-h (10 mmol) in dry tetrahydrofuran (10 mL) was added dropwise to the resulting solution. The reaction mixture was refluxed under stirring for 18 h. The reaction mixture was concentrated under vacuum, diluted with water (100 mL) and extracted with diethyl ether (3 × 50 mL). The combined organic extracts were dried (Na 2 SO 4 ), filtered and evaporated under reduced pressure to give viscous oils which were purified by column chromatography using petroleum ether (40-60 °C): Diethyl ether (9:1) to afford compounds 5e-h in 75%-91% yields as pale yellow viscous oils.

Synthesis of 4-Cyano-3-methyl-2-butenoic Acid Ethyl Ester (5i)
A mixture of cyanoacetic acid (6i, 4.51 g, 53 mmol), ethyl acetoacetate (6.51 g, 50 mmol), ammonium acetate (0.77 g, 10 mmol) and acetic acid (1.58 g, 1.5 mL, 26.3 mmol) in benzene (15 mL) was refluxed for 8 h using a Dean-Stark apparatus. The reaction mixture was evaporated under reduced pressure, water (10 mL) was added to the residue and extracted with diethyl ether (3 × 15 mL). The organic layer was separated, dried (Na 2 SO 4 ) and evaporated under vacuum. The residue was distilled under vacuum to yield 5.2 g (68%) of 5i as a colorless oil b.p. 100-102 °C/5 mm (lit. [19] 130 °C/20 mm) with E/Z ratio = 1.7 as detected by 1  3.1.9. Synthesis of (R,S)-5-Benzyloxycarbonylamino-3-methylpentanoic Acid (2i) To a solution of 4-cyano-3-methyl-2-butenoic acid ethyl ester (5i, 0.77 g, 5 mmol) in 95% ethanol (25 mL) was added concentrated hydrochloric acid (1 mL) and 10% Pd/C (0.26 g). The reaction mixture was hydrogenated on a Parr shaker apparatus under 4 bar of H 2 for 18 h at room temperature. The catalyst was removed by filtration and the solvent was evaporated under vacuum to give (RS)-5-amino-3-methyl-pentanoic acid ethyl ester hydrochloride (4i) which was dissolved in 5 N GABA B R1b-pcDNA3.1, 3.5 μg GABA B R2-pcDNA3.1 and 0.7μg Gαq-z5-pcDNA using SuperFect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany). The day after transfection, cells were split into one poly-D-lysine coated 96-well black-walled-clear-bottomed tissue culture plates in the same medium as mentioned above and incubated overnight. The following day the measurement of intracellular calcium was performed as follows. The media was exchanged with Hanks balanced saline solution containing 1 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, 2.5 mM probencid and 4 μM Fluo-4AM (pH = 7.4). The cells were incubated for 1 h at 37 °C in a humidified 5% CO 2 incubator. Cells were then washed twice with the same buffer without Fluo-4AM and finally 100 μL of the buffer was left in the wells. The cell plate was then transferred to the NovoStar (BMG Labtechnologies, Offenburg, Germany) and the basal fluorescence level was adjusted to ~10,000 fluorescence units (FU) using excitation/emission wavelengths of 485-520 nm, respectively. Fluorescence readings were measured for 45 s after addition of ligand and response was calculated as peak response minus basal level. Inactive compounds were also tested as antagonists. Twenty min after application of ligand, 10 μM GABA was added to the well and fluorescence was measured as above.

Data Analysis
All data analysis has been carried out using GraphPad Prism version 6.0c for Mac OS X (GraphPad Software, San Diego, CA, USA). Concentration-response curves have been fitted by non-linear regression using the equation for sigmoidal concentration-response function: R = R min + (R max − R min )/(1 + 10 ^ (logEC 50 − X)) in which X is the logarithm of the agonist concentration, R is the response, R max is the maximal response, R min is the minimal response and EC 50 is the concentration giving half maximum response. All experiments were performed in triplicate and the results are given as mean pEC 50 ± S.E.M of 3-4 experiments.

Conclusions
Synthesis and GABA B R agonistic activity of certain amino acids 1a-i as homologues of the clinically used drug, baclofen (1), are reported. The presence of an aryl moiety in position three of the DAVA backbone is essential for GABA B R agonistic activity as replacement of this aryl moiety with a methyl group gave compound 1i which is devoid of GABA B R agonistic activity. Additionally, the substitution pattern of this aryl moiety plays an important role in the exhibited GABA B R agonistic activity. Thus, mono-substitution on the aromatic moiety attached to the three position of the DAVA backbone with a halogen, especially para-chloro (compound 1a), is optimum for GABA B R agonistic activity. Compound 1a showed GABA B R agonistic activity with EC 50 = 46 μM, being the most active congener in the whole synthesized series.