European Journal of Medicinal Chemistry 76 (2014) 20e30

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Original article

Synthesis, neuronal activity and mechanisms of action of halogenated enaminones Ivan O. Edafiogho a, *, Mohamed G. Qaddoumi b, Kethireddy V.V. Ananthalakshmi b, Oludotun A. Phillips c, Samuel B. Kombian b a b c

Department of Pharmaceutical Sciences, School of Pharmacy, University of Saint Joseph, Hartford, CT 06103, United States Department of Pharmacology and Therapeutics, Faculty of Pharmacy, Kuwait University, Kuwait Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Kuwait

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 August 2013 Received in revised form 22 January 2014 Accepted 3 February 2014 Available online 4 February 2014

Due to the excellent anticonvulsant activity of previously synthesized halogenated enaminones, more disubstituted analogs were synthesized and evaluated in vitro. The new enaminones either had no effect, depressed, or enhanced population spike (PS) amplitude in the rat hippocampus in a concentrationdependent manner. Structureeactivity relationship (SAR) analysis indicated that compounds 21 and 25 (with dibromo substituents) were equipotent, and more potent than compound 2 (with dichloro substituents), with compound 25 being the most efficacious of all tested compounds. Both diiodo derivatives 30 and 31 tested produced no significant effect on PS. For PS depression, phenyl substitution on the cyclohexenone ring produced the most efficacious compound 25. PS depressing analogues also depressed evoked excitatory postsynaptic current (EPSC) and action potential firing frequency. Removal of phenyl or methyl group from position 6 on the cyclohexenone ring of enaminone esters produced compound 28 which exhibited pro-convulsant effects. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. The mechanisms of anticonvulsant activity were the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA, and the direct suppression of action potential firing of the neurons. Published by Elsevier Masson SAS.

Keywords: Dihalogenated enaminones Neuronal activity Synthesis

1. Introduction Enaminones are synthetic compounds that consist of an amino group, joined through an alkene group to a ketone group [1e3]. Enaminones possess a range of pharmacological effects including antimalarial and anticonvulsant activities [1]. However, enaminones are largely devoid of neurotoxicity [4]. Halogenated enaminones have halogens such as fluoro, chloro, bromo, and iodo moieties incorporated in the molecules [5].

Abbreviations: aCSF, artificial cerebrospinal fluid; ADD, antiepileptic drug development; AED, antiepileptic drug; DMSO-d6, dimethyl sulfoxide-deuterated; EPSC, excitatory postsynaptic current; EPSP, excitatory postsynaptic potential; GABA, gamma aminobutyric acid; GABA-T, GABA transaminase; GAT, GABA transporters; GCeMS, gas chromatographyemass spectrometry; ND, not determined; NMR, nuclear magnetic resonance; mp, melting point; PS, population spike; SEM, standard error of the mean; TMS, tetramethylsilane; TTX, tetrodotoxin. * Corresponding author. E-mail addresses: iedafi[email protected] (I.O. Edafiogho), [email protected] (M.G. Qaddoumi), [email protected] (K.V.V. Ananthalakshmi), [email protected] (O.A. Phillips), [email protected] (S.B. Kombian). 0223-5234/$ e see front matter Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.ejmech.2014.02.002

Epilepsy is a neurological disorder characterized by the onset of spontaneous convulsant and non-convulsant seizures that result from neuronal hyperexcitability and hypersynchronous neuronal firing [6]. About 50 million people worldwide are affected by epilepsy, and it is estimated that 30% of the patients suffer from therapy-resistant epilepsy. Resistance to antiepileptic drugs (AEDs) and the side effects associated with the current AEDs are the most serious problems in the treatment of epilepsy [7e9]. Therefore, there is an urgent need to design and synthesis novel anticonvulsants for the development of more effective and safer AEDs [6]. In light of the emerging importance of enaminones as potential AEDs [5], the rationale of the current study was to synthesize and evaluate disubstituted phenylamino enaminones. Thus, we assessed the effect of modification of 2,4-dihalogenation of the phenylamino moiety of the investigated enaminones. In our current study, nine new disubstituted phenylamino enaminones were synthesized, and their anticonvulsant activity and neurotoxicity evaluated to establish if some of these enaminones might become lead compounds for further development into new AEDs.

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We had reported that anticonvulsant enaminones are very stable compounds at room temperature [10,11], and previous investigations indicated that cyclized enaminones were more potent as anticonvulsants than the acyclic analogs. The NH proton was mandatory for anticonvulsant activity, and that enaminones were very promising as potential AEDs [12e20]. Initial evaluations of halogenated enaminones revealed some important analogs of which the most potent analog 20 was investigated for mechanisms of anticonvulsant activity [21e23]. We have investigated further in the current work the halogenated enaminones with more emphasis on disubstituted phenyl enaminones. We hereby report our findings that the 2,4-dibromophenyl enaminones are potent and efficacious anticonvulsant agents. 2. Chemistry The general synthetic routes for the halogenated enaminones is shown in Scheme 1. Three independent routes of synthesis were employed in unequivocal synthesis of the cyclized 4hydroxycyclohex-3-en-2-oxo-1-oates which were important intermediates for obtaining the desired halogenated enaminone esters. The alkyl acrylate was reacted with alkyl acetoacetate in the presence of freshly prepared sodium alkoxide to obtain the 4hydroxycyclohex-3-en-2-oxo-1-oate which existed as tautomers. The second synthetic route involved the Michael addition reaction of the alkylidene ketone and dialkyl malonate in sodium alkoxide to give the 4-hydroxycyclohex-3-en-2-oxo-1-oate. The third route involved the reaction of the alkylidene ketone with dialkyl malonate under mild conditions to give the uncyclized Michael adduct which was cyclized in the presence of sodium alkoxide to 4hydroxycyclohex-3-en-2-oxo-1-oate. The condensation of 4hydroxycyclohex-3-en-2-oxo-1-oates with appropriate amino compounds yielded the halogenated enaminones as in Scheme 1, and in Table 1. The halogenated enaminone derivatives (22, 23, 26 and 27) lacking the ester functionality were prepared from the commercially available b-diketo compounds, namely, 4,4dimethylcyclohexane-1,3-dione, 5,5-dimethylcyclohexane-1,3dione, cyclohexane-1,3-dione and 5-methylcyclohexane-1,3dione. The chiral enaminones were synthesized by non-

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stereoselective methods and evaluated pharmacologically as racemates. The synthesized compounds were stable solids and purified by recrystallization from suitable solvents. 1H nuclear magnetic resonance (NMR) spectra of the synthesized enaminones were in CDCl3 or DMSO-d6 using TMS as an internal standard. The 13C NMR spectra of representative compounds 2, 21, 25, 26 and 31, were also performed. Elemental analyses were performed for all new enaminones (21e22, 25e31). Calculated log of partition coefficient (C log P) values for the new compounds were performed using ChemDrawÒ Ultra version 8.0, 2003, Cambridge Soft Corporation. 3. Pharmacology All experiments in this study were carried out on male SpragueeDawley rats (100e150 g). Coronal slices (350 mm thick) containing hippocampi were sliced in ice-cold artificial cerebrospinal fluid (aCSF). Slices were perfused fully submerged at 2e3 mL/min with aCSF (29e31
C) that was bubbled with 95% O2/5% CO2). Tungsten bipolar stimulating electrodes were placed in the dendritic layer of area CA1 while recording glass electrodes were placed in the cell body layer to record field population spikes (PS) (Fig. 1). All drugs were applied by bath perfusion. PS magnitude was measured as the absolute amplitude from peak to trough and used as a measure of neuronal excitation. All values are presented as mean  SEM and P < 0.05 was taken as being statistically significant. 4. Results and discussion The halogenated enaminones 1e31 were synthesized from bhydroxyketo starting materials, according to methods reported previously [1,3,5]. The general synthesis is shown in Scheme 1. The condensation of alkyl acrylate with alkyl acetoacetate, or alkyl vinyl ketone with dialkyl malonate gave the b-hydroxyketo intermediates, which were reacted with appropriate halogen substituted-phenylamines to obtain the halogenated enaminones. Since compound 2 was completely characterized in this study (mp, UV, IR, 1H NMR, 13C NMR, and MS), its synthesis was markedly different from reported literature [5].

Scheme 1. Synthesis of halogenated enaminones. Reagents and conditions: (i) Na/MeOH for methyl esters; and Na/EtOH for ethyl esters, reflux, 2e4 h, (ii) Potassium carbonate, (iii) appropriate amino compound (NH2R4).

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Table 1 Structure, and anticonvulsant activity of halogenated enaminones.

Cpd.

R1

R2

R3

R4

MP (
C)

ADDa

1 2d 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20d 21d 22d 23 24 25d 26d 27d 28 29 30 31

eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOCH3 eCOOC2H5 eCOOC2H5 eCOOCH3 eCOOC2H5 Geminal CH3 H eCOOC2H5 eCOOCH3 H eCOOCH3 eCOOC2H5 H eCOOCH3 eCOOC2H5

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H CH3 Ph Ph H CH3 H CH3 Ph Ph

H H H H H H H H H H H H H H H H H H H H H H CH3 H H H H H H H H

2,4-dichlorophenyl 2,4-dichlorophenyl 2,5-dichlorophenyl 2,5-dichlorophenyl 3,4-dichlorophenyl 3,4-dichlorophenyl 2,6-dichlorophenyl 2,6-dichlorophenyl 3,5-dichlorophenyl 3,5-dichlorophenyl 2,3,4-trichlorophenyl 2,3,4-trichlorophenyl 2,4,5-trichlorophenyl 2,4,5-trichlorophenyl 3,4,5-trichlorophenyl 3,4,5-trichlorophenyl 2,4,6-trichlorophenyl 2,4,6-trichlorophenyl 4-chlorophenyl 4-bromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-dibromophenyl 2,4-diiodophenyl 2,4-diiodophenyl

153e155 153e155 190e192 160e162 160e161 171e173 205e210 186e189 212e215 191e193 205e207 165e167 200e201 183e184 181e183 168e170 204e213 190e197 161e163 188e190 155e157 156e161 144e147 196e198 220e222 150e153 128e130 128e130 191e192 234e238 190e192

1b 1b 1b 3b 1b 2b 3b 3b 3b 3b 3b 2b 1b 3b 1b 2b 3b 3b 1b 1b 1 NDc NDc NDc 1 NDc 2 NDc NDc NDc NDc

a Anticonvulsant Drug Development classification: 1 ¼ active dichloro-(2: 25%) > diiodo-(31: þ4% e inactive) > diiodo-(30: þ8% e inactive). Removal of the phenyl or methyl substituent from position 6 on the cyclohexenone ring of enaminone esters (28: þ35%) resulted in a pro-convulsant compound. Therefore, six halogenated enaminones 2, 21e22, 25e27 were anticonvulsant, two analogs 30e31 were inactive, and two other compounds 28e29 were pro-convulsant. For all the compounds that depressed the PS amplitude, concentrations beyond 10 mM i.e. 100 mM produced either a decrease in the amount of depression (see doseeresponse curve in Fig. 2A for compound 25) or even caused an enhancement in PS amplitude i.e. pro-convulsant effects. As such, the concentration of 10 mM was selected to further characterize the effects of the anticonvulsant enaminones.

Fig. 2. Effects of selected enaminones on population spike amplitude. (A) shows sample PS in control, 6 min after application of Cpd 25 and 10 min after washing out the compound. Below the traces is the concentrationeresponse curve for Cpd 25. Note that at 100 mM, the PS depressant effect is almost reversed. (B) Concentration-response curves for Cpds 2 and 21. (C) Summary bar graph showing the effects of several enaminones at 10 mM on the PS amplitude.

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We further investigated the effects of compounds 21 and 25 (the most potent and efficacious analogs) on excitatory postsynaptic currents (EPSCs), action potential firing and postsynaptic neuronal excitability by recording whole-cell responses in the CA1 pyramidal cells using voltage and current clamp recording modes as appropriate. In voltage clamp mode, cells were held around their resting membrane potential (60 mV) and excitatory postsynaptic currents (EPSCs) were evoked by afferent stimulation and recorded (Fig. 3A and B inserts). At 10 mM, compound 25 depressed the EPSC amplitude by 31.2  5.7% (n ¼ 6, P < 0.05, Fig. 3A and C) while compound 21 depressed it by 47.5  14.2% (n ¼ 5, P < 0.05; Fig. 3B and C) effects that, unlike those on PS, did not show appreciable recovery following 15e20 min washout of the compounds (Fig. 3A and B). At this same concentration, compound 21 depressed action potential firing frequency by 76.3  11.9% (n ¼ 4, P < 0.05) while compound 25 depressed it by 39.2  4.5% (n ¼ 4; P < 0.05, Fig. 4). To further investigate if their mechanisms of action were similar to

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those reported earlier [21], we examined if the effects of compounds 21 and 25 were dependent on GABAergic mechanisms. Bath application of a saturating concentration of vigabatrin (500 mM) depressed the evoked EPSC amplitude by 39.3  15.2% (n ¼ 4, P < 0.05). In the presence of vigabatrin, compound 21 was no longer able to depress EPSC amplitude (3.9  18.6%, P > 0.05, Fig. 5A and B). In another set of experiments, vigabatrin (500 mM) again depressed the evoked EPSC by 31.4  7.2% (n ¼ 7, P < 0.05). Here again, vigabatrin (500 mM) was chosen to saturate the response. In its presence, compound 25 (10 mM) was still able to depress the evoked EPSC amplitude by 40.0  7.1% (n ¼ 7, P < 0.05 compared to effect in presence of vigabatrin alone, Fig. 5C). To confirm a role for GABA, we blocked GABAB receptors with a selective antagonist, CGP55845 (1 mM) and asked if these compounds still depressed EPSC amplitude. CGP55845 predictably enhanced the EPSC amplitude by 41.6  4.8% (n ¼ 5, P < 0.05). At the peak of this CGP55845 effect, compound 25 was no longer able to depress EPSC amplitude (16.6  10.1%, P > 0.05, Fig. 6A and B). In a similar experiment, the ability of compound 21 to depress EPSC amplitude was also blocked by CGP55845 (16.6  13.7%, n ¼ 4, P > 0.05, Fig. 6C and D). 4.3. Mechanisms of action of halogenated enaminones By comparison to other enaminones and even a clinically available anticonvulsant agent (vigabatrin), compounds 21 and 25

Fig. 3. Effects of Cpds 21 and 25 on evoked whole cell excitatory postsynaptic currents recorded in pyramidal cells of the hippocampus. (A) Timeeeffect plot of 10 mM Cpd 25. On top are sample EPSCs taken in control, in the presence of Cpd 25 and following washout. (B) Timeeeffect plot for 10 mM Cpd 21 as in (A). (C) Summary bar graph showing the relatively long acting effects of Cpds 21 and 25.

Fig. 4. Effects of Cpds 21 and 25 on action potential firing frequency. (A) Sample voltage traces with action potential firing following 75 ms step current injection in pyramidal neurons in control, presence of Cpd 21 and after washing out. (B) Summary bar graph showing the effects of 10 mM each of Cpds 21 and 25.

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Fig. 5. Blocking GABA transaminase enzyme with vigabatrin completely occludes Cpd 21 effects while partly occluding those of Cpd 25. (A) Sample traces taken from B. (B) Timeeeffect plot of the effects of vigabatrin and Cpd 21 on EPSC amplitude. To the left is a summary bar graph of this effect. (C) Simple plot as in B but with Cpd 25. Note that Cpd 25 still had a significant effect, albeit smaller, in the presence of vigabatrin.

were the most potent analogs of the series synthesized and compound 25 was the most efficacious. Both compounds were class 1 anticonvulsants in the in vivo model, and depressed PS amplitude in the rat hippocampus in vitro. Accordingly, they were chosen as lead compounds that can be developed into promising AEDs. It is planned to carry out detailed pharmacological screening with compounds 21 and 25 to determine how the two compounds affect the spontaneous burst that usually accompanies multiple spikes. The two compounds caused significant depression of PS amplitude in the rat hippocampus. In addition, both compounds significantly depressed EPSC amplitude recorded in the CA1 pyramidal cells and significantly depressed the action potential firing frequency of the pyramidal neurons. These enaminones depress synaptic excitation by a gamma-aminobutyric acid (GABA)-mediated mechanism involving GABAB receptors on glutamate terminals leading to decrease in EPSC amplitude [1,21,26]. Therefore, one mechanism of anticonvulsant action of the halogenated enaminones is to reversibly suppress the glutamate-mediated EPSCs, most likely by enhancing extracellular GABA levels rather than direct activation of GABAB receptors [23]. We earlier reported that enaminone 20 interacted with adrenergic receptors, ultimately leading to GABAB receptor-mediated synaptic depression. Here, we show that compounds 21 and 25 depress PS and EPSC by a GABAergic mechanism. Since enaminones have been reported not to interact with GABAB receptors [27], the dihalogenated enaminones in this series most likely also produced their synaptic depressant effect by enhancing

Fig. 6. Effects of Cpds 21 and 25 are blocked by a GABAB receptor antagonist, CGP55845. (A) Sample traces taken from B. (B) Timeeeffect plot of the effects of CGP and Cpd 25 on EPSC amplitude. (C) Simple plot as in B but with Cpd 21. (D) Summary bar graph showing the blockade of Cpds 21 and 25 effects by CGP.

extracellular GABA levels rather than by direct interaction with GABAB receptors. The increase in extracellular GABA levels may be through one or several mechanisms including inhibition of GABA transporters (GAT); inhibition of the GABA transaminase (GABA-T) enzyme or through interaction with a-2 adrenoceptors [1]. Compound 25, being more efficacious, may produce this effect by several mechanisms since profound inhibition of GABA transaminase with 500 mM vigabatrin only partially occluded its effect. The second mechanism of action is a direct effect on neuronal excitability independent of synaptic transmission. These dihalogenated enaminones suppress compound action potential amplitudes (in PS) and action potential firing frequency of single pyramidal neurons [22]. These non-synaptic effects of halogenated enaminones were through the inhibition of tetrodotoxin (TTX)sensitive sodium channel currents involved in action potential firing as previously reported [22].

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5. Conclusion Enaminones are a class of compounds that have been reported to possess anticonvulsant activity, and the enaminone pharmacophore appears to provide the potential for anticonvulsant activity in in vivo and in vitro models [28]. Enaminones, when evaluated in the rat provided broad protection in both test models, i.e. MES and scPTZ. However, it was also noted that the protection was greater by the intraperitoneal (ip) route than orally, indicating differences in absorption and distribution by different routes of administration [26]. The mechanism of resistance to AEDs is currently the subject of intense investigations with suggestions for a radical rethink of antiepileptic therapy to include antiepileptogenic agents [29]. Furthermore, pharmacokinetic and enzyme induction studies are required to fully characterize these new and promising anticonvulsant compounds and to determine if they possess potential for further development into clinical trials [30]. In this paper, we report the synthesis and neuronal evaluation of halogenated enaminones in vitro. Not all enaminones in this series are anticonvulsant even at low concentrations. There was no direct correlation between C log P values and anticonvulsant activity of the halogenated enaminones. Phenyl and methyl substitution on position 6 of the cyclohexenone ring of enaminone esters yielded analogs with actions consistent with anticonvulsant potential. Dibromo substitution on the phenylamino ring yielded the most potent and most efficacious analogs with anticonvulsant activity. Enaminones with dibromo substitution on the phenylamino ring and phenyl substitution on position 6 of the cyclohexenone ring hold the greatest potential for development as anticonvulsant agents. The mechanisms of anticonvulsant action of the halogenated enaminones include the indirect suppression of excitatory synaptic transmission by enhancing extracellular GABA; and the direct suppression of action potential firing of pyramidal neurons in the hippocampus, a seizure relevant brain region of the CNS. 6. Experimental section

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parts per million (ppm) downfield from tetramethylsilane (TMS) as an internal reference or DMSO-d6 (d ¼ 2.5; 39.7) as solvent. Chemical structures of newly synthesized compounds were assessed by 1H NMR and elemental analyses. The melting point was determined on a Stuart Scientific melting point apparatus (SMP1, UK). Elemental analyses were performed on an Elementar Vario Micro Cube CHN Analyzer (Elementar, Germany). Elemental analysis (C, H, N) was used to confirm the purity of all newly synthesized compounds (>95%). Analyses indicated by the symbols of the elements or functions were within 0.4 of theoretical values. 6.3. General procedure for the synthesis of halogenated enaminones The following procedure is typical for the synthesis of the halogenated enaminones, starting with the preparation of the intermediate b-hydroxyketo compound that is condensed with appropriate amino compounds carrying halogen substituents to give the products. 6.3.1. Ethyl 4-hydroxy-6-methyl-2-oxocyclohex-3-en-1-oate To a freshly prepared solution of sodium (17.8 g, 0.77 g-atom) in absolute ethanol (350 mL) was added ethyl acetoacetate (100.2 g, 0.77 mol) over 30 min and the mixture stirred on an ice bath for an additional 30 min. Ethyl crotonate (96%, 100 mL, 0.77 mol) was added dropwise and the mixture stirred at room temperature for an additional 30 min. After refluxing for 2 h, the reaction mixture was cooled and the white precipitate which formed from the reaction mixture was collected, dissolved in a minimum amount of cold water, acidified with 2 M sulfuric acid (480 mL), and extracted with dichloromethane (2  900 mL). The dichloromethane extract was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated using rotary evaporator, and the residue recrystallized from toluene to yield 56 g (40%): white crystals; mp 95e98
C (mixed mp with authentic sample); 1H NMR (CDCl3) d 1.03 (3H, d, J ¼ 6 Hz, CH3CH), 2.50 (2H, m, CH2), 3.20 (2H, m, 2  CH), 3.70 (3H, t, J ¼ 7 Hz, OCH2CH3), 4.08 (2H, q, J ¼ 7 Hz, OCH2CH3), 5.03 (1H, s, ]CH); IR (CHCl3 solution) 3150 (br), 3020 (CH), 1730, 1660, 1610 cm1.

6.1. Chemicals The starting materials 1,3-cyclohexanedione, 5-methyl-1,3cyclohex-anedione, 5,5-dimethyl-1,3-cyclohexanedione, and 4,4dimethyl-1,3-cyclohexanedione; and common reagents including 2,4-dibromoaniline, 2,4-diiodoaniline, dichloromethane and sodium metal; and solvents including ethyl acetate, petroleum ether, methanol, absolute ethanol and diethyl ether were obtained from SigmaeAldrich (United States). 6.2. Materials and methods The mass spectra were recorded on a Thermo Scientific DFS High Resolution Gas Chromatography/Mass Spectrometer (DFS GCeMS). One crystal of the sample is loaded to the direct inlet probe, heated from room temperature to 350
C at the rate of 30
C/minute and inserted to the ion source with electron impact ionization setup. The Infrared spectra of solids (KBr) were recorded on JASCO FT-IR6300 (JASCO, Japan) spectrometer. The 1H NMR spectra in DMSO-d6 using tetramethylsilane (TMS) as the internal standard, were recorded on a Bruker DPX 400 MHz NMR spectrometer. Chemical shifts (d scale) are reported in parts per million (ppm) relative to residual TMS. Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublet), and br m (broad multiplet). Coupling constants (J) are given in (Hz). In addition, the 13C NMR spectrum of representative compounds 2, 21, 25, 26 and 31, were recorded on Bruker Avance II 600 NMR spectrometer. Chemical shifts of protons and carbons are reported in

6.3.2. Ethyl 4-(20 ,40 -dibromophenylamino)cyclohex-3-en-6-methyl2-oxo-1-oate (21) To a solution of ethyl 4-hydroxy-6-methyl-2-oxocyclohex-3-en1-oate (1 g, 5 mmol) in 80 mL of absolute ethanol was added a solution of 2,4-dibromoaniline (98%, 1.28 g, 5 mmol) in 50 mL of absolute ethanol and the reaction mixture refluxed with stirring for 8 h. The reaction mixture was evaporated using rotary evaporator, and the residue recrystallized from ethyl acetate/petroleum ether bp 60e80
C to obtain 0.60 g of enaminone 21, 28% yield. 1H NMR (DMSO, 400 MHz): d (ppm) d, 3H, 1.00, J ¼ 1 Hz, 6-methyl group), 1.19 (3H, t, J ¼ 2 Hz, OCH2CH3), 2.33e2.35 (4H, m, cyclohexenone ring), 4.10 (2H, q, J ¼ 2 Hz, OCH2CH3), 4.66 (1H, s, ]CH), 7.30e8.01 (3H, m, C6H3 phenyl ring); 8.94 (1H, bs, NH). 13C NMR (DMSO-d6): d 191.31, 171.21, 163.38, 136.91, 135.79, 132.26, 131.04, 122.51, 120.43, 97.43, 60.52, 60.42, 34.87, 32.35, 19.68, 14.61. IR (KBr pellet) 1470, 1518, 1562, 1598 enaminone system, 3224 NH; UV lmax EtOH 95% ¼ 296 nm; MS 431 (Mþ). Anal. (C16H17NO3Br2) C, H, N, O. 6.3.3. Ethyl 4-(2,4-dichlorophenylamino)-6-methyl-2-oxocyclohex3-en-1-oate (2) (0.82 g, 82.8% yield), mp 150e155
C. 1H NMR (DMSO-d6, 600 MHz): d 1.00 (d, 3H, J ¼ 6.1 Hz, CH3), 1.19 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 2.36e2.58 (m, 3H, cyclohexene), 3.04 (d, 1H, J ¼ 11.4 Hz, cyclohexene H), 4.10 (q, 2H, J ¼ 7.1 Hz, CO2CH2CH3), 4.71 (s, 1H, ]CH), 7.40 (d, 1H, J ¼ 2.3 Hz, phenyl H), 7.49 (dd, 1H, J ¼ 2.4 Hz, 8.6 Hz, phenyl H), 7.77 (d, 1H, J ¼ 2.3 Hz, phenyl H), 8.96 (s, 1H, NH). 13C NMR (DMSO-d6): d 191.41, 171.20, 163.33, 135.01,

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131.98, 131.47, 130.41, 130.23, 128.80, 97.53, 60.51, 60.42, 34.88, 32.35, 19.67, 14.61. IR (KBr pellet, cm1): n 3452, 1735, 1600, 1567. UV EtOH lmax ¼ 296 nm; MS 341.1 (Mþ). Anal. (C16H17Cl2NO3) C, H, N, O. 6.3.4. 3-(2,4-dibromophenylamino)-6,6-dimethylcyclohex-2-enone (22) (0.22 g, 31.5% yield), mp 156e161
C. 1H NMR (DMSO-d6, 600 MHz): d 1.00 (s, 6H, cyclohexene 2(CH3)), 1.76 (t, 2H, J ¼ 6.2 Hz, cyclohexene CH2), 2.50e2.54 (m, 2H, cyclohexene CH2, overlaps with the DMSO signal), 4.57 (s, 1H, ]CH), 7.31 (d, 1H, J ¼ 8.5 Hz, phenyl H), 7.62 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 7.98 (d, 1H, J ¼ 2.2 Hz, phenyl H), 8.60 (s, 1H, NH). IR (KBr pellet, cm1): n 3444, 1611, 1597, 1561, 1527. UV EtOH lmax 296 nm. MS 373.0 (Mþ). Anal. (C14H15Br2NO) C, H, N, O. 6.3.5. Methyl 4-(2,4-dibromophenylamino)-2-oxo-6phenylcyclohex-3-en-1-oate (25) (1.26 g, 25.6% yield), mp 220e228
C. 1H NMR (DMSO-d6, 600 MHz): d 2.62 (dd, 1H, J ¼ 4.2 Hz, 16.3 Hz, cyclohexene H), 2.97 (dd, 1H, J ¼ 12.9 Hz, 16.4 Hz, cyclohexene H), 3.39 (s, 3H, CO2CH3), 3.56 (tt, 1H, J ¼ 4.2 Hz, 12.4 Hz, cyclohexene H), 3.82 (d, 1H, J ¼ 12.5 Hz, cyclohexene H), 4.77 (s, 1H, ]CH), 7.23e7.41 (m, 6 H, phenyl H), 7.67 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 8.04 (d, 1H, J ¼ 2.2 Hz, phenyl H), 9.05 (s, 1H, NH). 13C NMR (DMSO-d6): d 190.80, 191.77, 170.95, 163.11, 142.03, 136.84, 136.81, 135.35, 132.33, 130.95, 129.00, 128.92, 127.85, 127.58, 122.40, 120.50, 97.26, 59.24, 59.22, 51.70, 51.67, 43.37, 35.30, 21.23. IR (KBr pellet, cm-1): n 3446, 1737, 1592, 1532.UV EtOH lmax 296 nm MS 479.0 (Mþ). Anal. (C20H17Br2NO3) C, H, N, O. 6.3.6. 3-(2,4-dibromophenylamino)cyclohex-2-enone (26) (1.55 g, 90% yield), mp 184e188
C. 1H NMR (DMSO-d6, 600 MHz): d 1.86e1.92 (m, 2H, cyclohexene H), 2.14 (t, 2H, J ¼ 6.4 Hz, cyclohexene CH2), 2.48e2.51 (m, 2H, cyclohexene CH2, overlaps with the DMSO signal), 4.66 (s, 1H, ]CH), 7.30 (d, 1H, J ¼ 8.5 Hz, phenyl H), 7.63 (dd, 1H, J ¼ 2.2 Hz, 8.5 Hz, phenyl H), 7.99 (d, 1H, J ¼ 2.2 Hz, phenyl H), 8.69 (s, 1H, NH). 13C NMR (DMSO-d6): d 195.91, 163.50, 137.30, 135.72, 132.14, 130.90, 122.40, 119.95, 99.06, 36.84, 28.30, 21.99. IR (KBr pellet, cm1): n 3230, 1607, 1589, 1556, 1508. UV EtOH lmax ¼ 296 nm MS 344.9 (Mþ). Anal. (C12H11Br2NO): C, H, N, O. 6.3.7. Methyl 4-(20 ,40 -dibromophenylamino)cyclohex-3-en-6methyl-2-oxo-1-oate (27) 1.02 g, 49% yield. 1H NMR (DMSO, 400 MHz): d (ppm) 0.98e1.00, (3H, d, J ¼ 1 Hz, 6-methyl group); 2.36e2.57 (4H, m, cyclohexenone ring); 3.61 (3H, s, OCH3), 4.66 (1H, s, ¼CH proton); 7.31e8.01 (3H, m, phenyl ring); 8.96 (1H, s, NH). MS 416.9 (Mþ). Anal. (C15H15NO3Br2) C, H, N, O. 6.3.8. Ethyl 4-(20 ,40 -dibromophenylamino)-2-oxocyclohex-3-en-1oate (28) 0.75 g, 36% yield. 1H NMR (DMSO, 400 MHz): d (ppm) 1.14e1.16 (3H, t, J ¼ 2 Hz, OCH2CH3); 1.85e2.58 (5H, m, cyclohexenone ring); 4.06 (2H, q, J ¼ 2 Hz OCH2CH3), 4.64 (1H, s, ]CH), 7.29e8.00 (3H, m, phenyl protons); 8.89 (1H, bs, amino group). MS 416.9 (Mþ). Anal. (C15H15NO3Br2) C, H, N, O. 6.3.9. 3-(20 ,40 -dibromophenylamino)-5-methylcyclohex-2-en-1one (29) 1.04 g, 58% yield. 1H NMR (DMSO, 400 MHz): d (ppm) 1.02e1.04 (3H, d, J ¼ 2 Hz, 6-methyl protons); 1.89e2.54 (5H, m, cyclohexenone protons); 4.65 (!H, s, ]CH); 7.29e8.00 (3H, m, phenyl

protons); 8.68 (1H, bs, amino proton). MS 358.9 (Mþ). Anal. (C13H12NOBr2) C, H, N, O. 6.3.10. Methyl 4-(2,4-diiodophenylamino)-2-oxo-6phenylcyclohex-3-en-1-oate (30) (0.25 g, yield 31.5%), mp 234e238
C. 1H NMR (DMSO-d6, 600 MHz): d 2.60 (dd, 1H, J ¼ 4.0 Hz, 16.5 Hz, cyclohexene H), 2.94 (m, 1H, J ¼ 12.7 Hz, 16.2 Hz, cyclohexene H), 3.39 (s, 3H, CO2CH3), 3.51e3.59 (m, 1H, cyclohexene H), 3.80 (d, 1H, J ¼ 12.5 Hz, cyclohexene H), 4.67 (s, 1H, ]CH), 7.12 (d, 1H, J ¼ 8.3 Hz, phenyl H), 7.21e 7.40 (m, 5 H, phenyl H), 7.81 (dd, 1H, J ¼ 1.9 Hz, 8.3 Hz, phenyl H), 8.31 (d, 1H, J ¼ 1.9 Hz, phenyl H), 8.99 (br. s, 1H, NH). IR (KBr pellet, cm1): n 3188, 1736, 1587, 1570, 1530. UV lmax ¼ 298 nm. MS 573.0 (Mþ). Anal. (C20H17I2NO3) C, H, N, O. 6.3.11. Ethyl 4-(2,4-diiodophenylamino)-2-oxo-6-phenylcyclohex3-en-1-oate (31) (0.10 g, yield 6%), mp 190e192
C 1H NMR (DMSO-d6, 600 MHz): d 0.90 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 2.59 (dd, 1H, J ¼ 4.0 Hz, 16.6 Hz, cyclohexene H), 2.94 (dd, 1H, J ¼ 12.0 Hz, 16.4 Hz, cyclohexene H), 3.55 (tt, 1H, J ¼ 4.2 Hz, 12.3 Hz, cyclohexene H), 3.75 (d, 1H, J ¼ 12.5 Hz, cyclohexene H), 3.85 (q, 1H, J ¼ 7.1 Hz, CO2CH2CH3), 4.67 (s, 1H, ]CH), 7.12 (d, 1H, J ¼ 8.3 Hz, phenyl H), 7.23e7.40 (m, 5H, phenyl H), 7.81 (dd, 1H, J ¼ 1.9 Hz, 8.2 Hz, phenyl H), 8.31 (d, 1H, J ¼ 1.9 Hz, phenyl H), 8.98 (br. s, 1H, NH). 13C NMR (DMSO-d6): d 190.68, 170.37, 163.11, 147.08, 141.92, 140.53, 138.76, 130.59, 128.91, 127.97, 127.56, 101.04, 97.10, 94.16, 60.08, 59.32, 35.38, 14.34. IR (KBr pellet, cm-1): n 3256, 1736, 1607, 1582, 1547, 1508; UV EtOH. lmax ¼ 299 nm; MS 587.0 (Mþ). Anal. (C21H19I2NO3) C, H, N, O. 6.4. Anticonvulsant evaluation in vivo The preliminary evaluations for the anticonvulsant activity of the halogenated enaminones 1e20 were performed in vivo by the Antiepileptic Drug Development (ADD) Program of the Anticonvulsant Screening Program of the National Institute of Neurological Disorders and Stroke (NINDS). Phase 1 testing procedures were performed in male Carworth Farms no 1 (CF1) mice with tests consisting of maximal electroshock (MES), subcutaneous pentylenetetrazole (Metrazol) seizure threshold to assess anticonvulsant activity, and the rotorod test to assess neurological toxicity. Compounds were either dissolved or suspended in 30% polyethylene glycol 400 and were administered by intraperitoneal injection at three dosage levels (30, 100, and 300 mg/kg). Anticonvulsant activity and neurotoxicity were noted 30 min and 4 h after administration. The results of the anticonvulsant activity for compounds 1e20 were reported previously [5] and included in this study for comparison. 6.5. Anticonvulsant evaluation in vitro 6.5.1. General electrophysiology methodology Extracellular electrophysiological experiments were performed in coronal hippocampal slices generated from rats (100e150 g) using previously published techniques and methods [19,21,22]. Briefly, male SpragueeDawley rats were deeply anesthetized with halothane and killed by quick decapitation. The brains were quickly removed and placed in ice cold (4
C) artificial cerebrospinal fluid (aCSF) bubbled continuously with 95% O2 and 5% CO2 (Carbogen). Using Leica VT 1000S (Leica Microsystems, Wetzlar, Germany) or OTS-4000 (Electron Microscopy Sciences, Hatfield, PA, USA) tissue slicers, 350 mm thick coronal slices of the forebrain containing the hippocampus were cut from a block of brain tissue in ice cold aCSF. Prior to recording, slices were incubated for 1 h in aCSF which was continuously bubbled with carbogen at room temperature. Slices

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were carefully trimmed of most cortical and midbrain tissue and suspended on a nylon mesh in a 500 mL capacity recording chamber. Bath temperature was tightly maintained at 29e31
C to ensure that changes in responses were not due to variation in temperature. Slices were perfused at a flow rate of 2e3 mL/min with aCSF that was bubbled with carbogen. An extracellular field recording glass electrode filled with 3M NaCl was placed in the stratum pyramidale of area CA1 for recording and bipolar stimulating electrodes were placed in the stratum radiatum near area CA1 to activate Schaffer collateral/commissural fibers afferents (see setup in Fig. 2). The composition of the aCSF used for dissection, storage, PS and whole cell recordings was (in mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.23 NaHPO4, 25 NaHCO3 and 10 D-glucose [19,21,22]. For PS, each stored trace was an average of five successively triggered responses elicited at 10 s intervals. The amplitude of the PS was measured from the peak of the positive going wave to the tip of the negative going wave in both control and drug application. The data were expressed as mean and standard error. For whole-cell recording, blind patches were made by glass electrodes filled with gluconate-based internal recording solution with the following composition (in mM): K-gluconate (135), NaCl (8), EGTA (0.2), HEPES (10), Mg-ATP (2) and GTP (0.2). pH and osmolarity were adjusted to 7.3 (with KOH) and 270e280 mOsm respectively. The patch electrodes had tip resistance between 3 and 10 MU. The extracellular perfusing solution was identical to that used for the extracellular recording. Recordings were performed on pyramidal cells of the hippocampus which had resting membrane potentials ranging from 55 to 63 mV. In voltage clamp experiments, they were held around rest (60 mV). Excitatory postsynaptic currents (EPSCs) were recorded by activating the same afferents as in extracellular recording above. Two successive responses (30 s apart) were averaged and stored for off line analysis. For action potential recording, these were done in current clamp mode by applying increasing step depolarizing and hyperpolarizing currents and recording the corresponding potentials. For these recordings, no averaging was done. Authors contribution to the manuscript

[4]

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

All authors have contributed equally to the manuscript. [19]

Conflict of interest [20]

All authors have no conflict of interest. [21]

Acknowledgment The research project was supported by Kuwait University Grant number PR01/08, and the Instrument Grants GS01/01, GS01/03 and GS01/05 awarded to the Science Analytical Facilities (SAF).

[22]

[23]

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.02.002.

[24]

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