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Letters in Drug Design & Discovery, 2013, 10, 34-42
Synthesis and Evaluation of Anticonvulsant Activity of Some N-[(4-Chlor2-methylphenoxy)ethyl]- and N-[(4-Chlor-2-methylphenoxy)acetyl]aminoalkanols Anna M. Waszkielewicz*,1, Edward Szneler2, Marek Cega3 and Henryk Marona1 1
Department of Bioorganic Chemistry, Chair of Organic Chemistry, Jagiellonian University Medical College, 9 Medyczna Str., 30-688 Kraków, Poland; 2Faculty of Chemistry, Jagiellonian University, 3 Ingardena Str., 30-060 Kraków, Poland; 3Chair of Organic Chemistry, Jagiellonian University Medical College, 9 Medyczna Str., 30-688 Kraków, Poland Abstract: A new series of N-(4-chlor-2-methylphenoxy)ethyl- (1-6) and N-(4-chlor-2-methylphenoxy)acetylaminoalkanols (7-10) has been synthesized for evaluation of their anticonvulsant activity. Pharmacological tests included maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole seizure (ScMet) assays, as well as rotarod for neurotoxicity (TOX) and were performed in mice i.p. and rats p.o. The activity of the compounds in the group was various, and the most active compound in mice was R,S-1N-[(4-chlor-2-methylphenoxy)ethyl]aminopropan-2-ol, revealing 100% activity in MES test at 30 mg/kg b.w., 0.5 h after administration without toxicity at the same dose and time. In rats (p.o.), the most active compound was 2N-[(4-chlor-2-methylphenoxy)ethyl]amino-2-methylpropan-1-ol, revealing 25% activity in MES at 30 mg/kg b.w. 0.5 and 1 h after administration.
Keywords: Alkanolamides; Aminoalkanols; Anticonvulsant; Epilepsy; Seizures; Synthesis; MES; Rotorod. 1. INTRODUCTION According to epidemiological studies, epilepsy affects approximately 1% of human world population and 25-30% of seizures are resistant to pharmacological treatment [1]. Among mechanisms of action there are influence on inhibitory or excitatory neurotransmitter systems (GABA or glutamic and aspartic acids, respectively). Inhibition of voltage-dependent sodium or calcium channels are the mechanism of action of many anticonvulsant drugs such as carbamazepine and lamotrigine [2]. Moreover, it has been reported that anticonvulsant activity can be observed for compounds exhibiting amide bond in their structures, e.g. ameltolide, retigabine, as well as levetiracetam with its analogs brivaracetam or seletracetam [3, 4, 5] Fig. (1). Structure-activity studies in our laboratories have directed us to derive compounds exhibiting anticonvulsant activity from N-substituted derivatives of appropriate aminoalkanols. Some of them, i.e. S-(+)-2-N-[(2,6-dimethyl)phenoxyethyl]aminobutan-1-ol hydrochloride and 2-[4-(benzyloxy)benzoyl)]-2N-methylamino-1-ethanol Fig. (2), prevent maximal electroshock seizures in mice, with ED50=7.57 mg/kg b.w. and 51.8 mg/kg b.w., respectively [6, 7]. The protective indices (PI=4.55 and 2.54, respectively) in the MES test in mice are higher than that of valproate (PI=1.7) and for the isomer S it is similar to that of carbamazepine (PI=4.9). It has been reported that some amide analogues of appropriate aminoalkanols also exhibit anticonvulsant activity in the MES test [8]. *Address crrespondence to this author at the Department of Bioorganic Chemistry, Chair of Organic Chemistry, Jagiellonian University Medical College, 9 Medyczna Str., 30-688 Kraków, Poland; Tel: 0048 12620 5576; Fax: 0048 12620 5405; E-mail:
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Searching for new derivatives, we have modified the structure of S-(+)-2-N-[(2,6-dimethyl)phenoxyethyl]aminobutan-1-ol in terms of the aminoalkanol structure as well as for its substituents and their position in the phenyl ring. We achieved and evaluated 2,6-dimethylphenol [9, 10], 4-chlor3-methylphenol, and 2-chlor-5-methylphenol derivatives [8], as well as cinnamic acid derivatives [11]. The most active derivatives have been subject to patent protection [12]. As a continuation of our former studies we herein report preparation and results of preliminary pharmacological studies on the expected anticonvulsant activity of some appropriate N-[(4-chlor-2-methylphenoxy)ethyl]- (1-6) and N-[(4-chlor-2-methylphenoxy)acetyl]aminoalkanols (7-9) as well as methyl N-[(4-chlor-2-methylphenoxy)acetyl]aminopropionate (10). All compounds were evaluated for their anticonvulsant activity in the MES and subcutaneous pentylenetetrazole (Metrazole, ScMet) seizures screens as well as for neurotoxicity (TOX) within the Antiepileptic Drug Development Program (ADD) at the National Institutes for Neurological Disorders and Stroke (NINDS, Bethesda, USA). 2. MATERIALS AND METHODS 2.1. Chemistry 2.1.1. Synthesis of the Tested Compounds Appropriate (4-chlor-2-methylphenoxy)ethyl- (1-6) and (4-chlor-2-methylphenoxy)acetylamines (7-10) were readily prepared according to the procedure shown in Scheme 1. Compounds 1-6 were obtained by N-alkylation of appropriate aminoalkanols using (4-chlor-2-methylphenoxy)ethyl bromide (1-6). The reaction was performed in the © 2013 Bentham Science Publishers
Synthesis and Evaluation of Anticonvulsant Activity
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
H3C O
O
O
35
CH3
NH
N H
CH3
H2N
N H
NH2
F Ameltolide
Retigabine
CH3
O
NH2
N
CH3
O
NH2
N
CH3
O
O
O Levetiracetam
O
Brivaracetam
H3C
NH2
N
F F
Seletracetam
Fig. (1). Structures of amides revealing anticonvulsant properties.
H N
O H3C
CH3
OH CH3 x HCl
S-(+)-2N-[(2,6-dimethylphenoxy)ethyl]aminobutan-1-ol hydrochloride ED50 (MES, mice, i.p.)=7.57 mg/kg b.w. PI=4.55 [6] O
O
N CH3
OH
2N-[4-(benzyloxy)benzoyl]-2N-methylaminoethan-1-ol ED50 (MES, mice, i.p.)=51.8 mg/kg b.w. PI=2.54 [7] Fig. (2). Structures of lead compounds.
presence of K2CO3 in toluene solution. The yield of alkylation was in the range 55-69%. (4-chlor-2methylphenoxy)ethyl bromide was obtained according to well known procedures as described previously [8, 13]. Compounds 7-10 were obtained through N-acylation of appropriate aminoalkanols (7-9) or alanine methyl ester hydrochloride (10), using (4-chlor-2-methylphenoxy)acetyl chloride. Two-phase system (toluene/H2O) and stoichiometric amounts of K2CO3 as a proton acceptor were used. (4-chlor-2-methylphenoxy)acetyl chloride was obt-ained according to well known procedures as described previously [8].
The purity was checked for all compounds using TLC and their structures were confirmed by spectral analyses (IR, 1 H NMR). The structures of the obtained compounds 1-6 and 7-10 are presented in Table 1 and Table 2, respectively. 2.1.2. Apparatus and Reagents Melting points were determined using a Büchi SMP-20 apparatus and are uncorrected. Analyses of C, H, N were within ± 0.4% of the theoretical values. Analytical TLC was carried out on precoated plates (silica gel, 60 F-254 Merck). Spots were visualized with UV light. The theoretical values of partition coefficient (cLogP) of the tested compounds
36 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
Waszkielewicz et al.
OH H3C
Cl 1) C2H5ONa 2) ClCH2CH2OH
2) ClCH2COOH
OH
O
OH
O H3C
H3C
O
Cl
Cl
SOCl2
PBr3 Br
O
Cl
O H3C
H3C
O
Cl
Cl aminolysis toluene/K2CO3
O
aminolysis toluene/water/K2CO3
Z Z - aminoalkanol (compounds 1-9)
H3C
Cl
Z
O H3C
or alanine methyl ester (compound 10)
Compounds 1-6
O
Cl Compounds 7-10
Scheme 1. Synthesis of the tested compounds (1-10).
were calculated by means of Pallas 3.1.1.2 program, using PrologP option. 1H NMR spectra for compounds 1-5 and 7-8 were recorded on a Bruker spectrometer 500 MHz, using signal from TMS in CDCl3 as internal standards. The 1 H NMR spectra for compounds 6 and 9-10 were recorded in CDCl3 with a Varian Mercury-VX 300 NMR spectrometer at 29˚C. Chemical shifts were referenced against solvent lock signal. Standard Varian pulse sequences were used for 2D experiments. The IR spectra were recorded on a Jasco FT/IR 410 spectrometer (KBr pellets). The reagents were purchased from Alfa Aesar GmbH&Co KG (Karlsruhe, Germany). The aminoalkanols as well as alanine methyl ester hydrochloride used for
synthesis of compounds 1-4 and 6-10 were racemic (Tables 1 and 2). All solvents were commercially available materials of reagent grade. 2.1.3. General Procedure for Synthesis of 1-6 0.01 mole of an appropriate aminoalkanol was added to a solution of 0.01 mole of (4-chlor-2-methylphenoxy)ethyl bromide in 30 cm3 of toluene and the reaction mixture was refluxed in the presence of 0.01 mole K2 CO3 for 6 h. Inorganic salts were filtered off from the hot mixture and washed with hot toluene (5 cm3). The solvent was distilled off from the filtrate under reduced pressure. After addition of n-heptane to the residue, the mixture was refluxed and
Synthesis and Evaluation of Anticonvulsant Activity
Table 1.
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
37
Chemical Structures of the Synthesized N-[(4-Chlor-2-methylphenoxy)ethyl]aminoalkanols (1-6). Z
O H3C
Cl Compound
Z
Configuration
OH
1
H N
CH3
H N
2
R,S
R,S OH CH3
3
OH
R,S
H N
CH3
H N
4
R,S OH CH3
H N
5
CH3
-
OH CH3
6
OH H N
cooled. The crystals formed were collected by filtration and dried. Recrystallization was performed from n-heptane. 2.1.4. General Procedure for Synthesis of 7-10 A mixture of 0.01 mole of appropriate aminoalkanols or alanine methyl ester hydrochloride with 0.025 mole K2CO 3 in 15 cm3 of water and 15 cm3 of toluene was cooled to 10-12 °C. After cooling a solution of 0.011 mole (4-chlor-2methylphenoxy)acetyl chloride in 30 cm3 of dry toluene was added in vigorous stirring at 10-12 °C for 0.5 h. Then the reaction mixture was heated and then left to cool down. The precipitated amides deposit was filtered off, stirred with a 10% solution of NaHCO3, and after drying 7-10 and 13 were recrystallized from a mixture of toluene-hexane (1:1). 2.1.5. Physicochemical Properties of the Synthesized Compounds 2.1.5.1. R,S-1N-[(4-chlor-2-methylphenoxy)ethyl]aminopropan-2-ol (1) Yield 69%; M=243.74; C12H18NO2Cl; M.p.: 74-76 °C; Ccalc/Cfound 59.14/59.07, Hcalc/Hfound 7.44/7.82, Ncalc/Nfound 5.75.5.66; Rf = 0.35 (CHCl3 /CH3OH 4:1); cLogP=2.25; IR (KBr, cm-1) v: 3274, 3119, 2964, 2955, 2920, 2849, 1497, 1253, 1190, 1132, 966, 787; 1H-NMR: ( ppm) 7.12 (s, 1H, H-3); 7.08 (d, J=8.0, 1H, H-5); 6.72 (d, J=8.7, 1H, H-6);
R,S
4,06 (t, J=5.1, 2H, O-CH2-CH2-NH); 3.89-3.79 (m, 1H, C*HOH); 3.15-3.02 (m, 2H, O-CH2-CH2-NH); 2.85 (dd, J=12.1, J=3.1, 1H, NH-CHH-C*HOHCH3); 2.72 (bs, 2H, OH, NH); 2.52 (dd, J=12.2, J=9.6, 1H, NH-CHH-C*HOH); 2.20 (s, 3H, CH3-Ar); 1.16 (d, J=6.2, 3H, CH3). 2.1.5.2. R,S-2N-[(4-chlor-2-methylphenoxy)ethyl]aminopropan-1-ol (2) Yield 65%; M=243.74; C12H18NO2Cl; M.p.: 84-86 °C; Ccalc/Cfound 59.14/58.87, Hcalc/Hfound 7.44/6.89, Ncalc/Nfound 5.75/5.43; Rf = 0.38 (CHCl3/CH3OH 4:1); cLogP=2.25; IR (KBr, cm-1) v: 3294, 3158, 2963, 2919, 2839, 2363, 1496, 1458, 1382, 1295, 1246, 1192, 1133, 1044; 1H-NMR: ( ppm) 7.10 (s, 1H, H-3); 7.09 (d, J=8.2, 1H, H-5); 6.72 (d, J=8.9, 1H, H-6); 4.06 (t, J=4.6, 2H, O-CH2-CH2-NH); 3.63 (dd, J=10.6, J=3.8, 1H, CHHOH); 3.31 (dd, J=10.8, J=7.1, 1H, CHHOH); 3.19-3.13 (m, 1H, N-CHH); 3.00-2.85 (m, 2H, N-C*H, NCHH) 2.42 (bs, 2H, OH, NH); 2.19 (s, 3H, CH3-Ar); 1.11 (d, J=6.4, 3H, N-C*H-CH3). 2.1.5.3. R,S-1N-[(4-chlor-2-methylphenoxy)ethyl]aminobutan-2-ol (3) Yield 60%; M=257.77; C13H20NO2Cl; M.p.: 72-74 °C; Ccalc/Cfound 60.58/60.34, Hcalc/Hfound 7.82/7.37, Ncalc/Nfound 5.43/5.04; Rf = 0.45 (CHCl3/CH3OH 4:1); cLogP=2.59; IR (KBr, cm-1) v: 3272, 3189, 2974, 2930, 2879, 2836, 2797,
38 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
Table 2.
Waszkielewicz et al.
Chemical Structures of the Synthesized N-[(4-Chlor-2-metylofenoksy)acetylo]aminoalkanols and –aminopropionic acid methyl ester (7-10). Z
O H3C
O
Cl Compound
Z
7
Configuration R,S
OH H N
8
CH3 R,S
OH H N
9
CH3
OH
R,S
O
R,S
H N
10 H N
O
CH3
CH3
2736, 1495, 1483, 1250, 1191, 1136; 1H-NMR: ( ppm) 7.11 (s, 1H, H-3); 7.08 (d, J=7.9, 1H, H-5); 6.72 (d, J=8.9, 1H, H6); 4.05 (t, J=5.13, 2H, O-CH2-CH2-NH); 3.61-3.53 (m, 1H, C*HOH); 3.10-3.00 (m, 2H, O-CH2-CH2-NH); 2.83 (dd, J=12.1, J=3.1, 1H, NH-CHH-C*HOHCH3); 2.58 (bs, 2H, OH, NH); 2.52 (dd, J=12.2, J=9.2, 1H, NH-CHH-C*HOH); 2.19 (s, 3H, CH3-Ar); 1.52-1.42 (m, 2H, CH2-CH3); 0.97 (t, J=7.4, 3H, CH2-CH3). 2.1.5.4. R,S-2N-[(4-chlor-2-methylphenoxy)ethyl]aminobutan-1-ol (4) Yield 70%; M=257.77; C13H20NO2Cl; M.p.: 68-70 °C; Ccalc/Cfound 60.58/60.46, Hcalc/Hfound 7.82/7.77, Ncalc/Nfound 5.43/5.25; Rf = 0.49 (CHCl3/CH3OH 4:1); cLogP=2.59; IR (KBr, cm-1) v: 3257, 3113, 2971, 2932, 2874, 2846, 2826, 2348, 1493, 1374, 1251; 1H-NMR: ( ppm) 7.11 (s, 1H, H3); 7.08 (d, J=7.4, 1H, H-5); 6.72 (d, J=8.9, 1H, H-6); 4.103.99 (m, 2H, O-CH2-CH2-NH); 3.65 (dd, J=10.8, J=3.8, 1H, CHHOH); 3.34 (dd, J=10.8, J=6.7, 1H, CHHOH); 3.17-3.11 (m, 1H, O-CH2-CHH-NH); 3.01-2.94 (m, 1H, O-CH2-CHHNH); 2.70-2.62 (m, 1H, C*H-NH); 2.42 (bs, 2H, OH, NH); 2.19 (s, 3H, CH3-Ar); 1.60-1.42 (m, 2H, CH2-CH3); 0.95 (t, J=7.4, 3H, CH2-CH3). 2.1.5.5. 2N-[(4-chlor-2-methylphenoxy)ethyl]amino-2-methylpropan-1-ol (5) Yield 55%; M=257.77; C13H20NO2Cl; M.p.: 122-123 °C; Ccalc/Cfound 60.58/60.69, Hcalc/Hfound 7.82/8.13, Ncalc/Nfound 5.43/5.42; Rf = 0.46 (CH3OH); cLogP=2.81; IR (KBr, cm-1 ) v: 3428, 3273, 3097, 2961, 2932, 2875, 2822, 2759, 1494, 1250, 1072, 796; 1H-NMR: ( ppm) 7.10-7.01 (m, 2H, NH, H-3, H-5 Ar); 6.71 (d, J=8.0, 1H, H-6 Ar); 4.03 (t, J=5.3, 2H O-CH2-CH2-); 3.33 (s, 2H, CH2-OH); 2.94 (t, J=5.3 2H OCH2-CH2-); 2.19 (s, 4H, CH3Ar, OH); 1.12 (s, 6H, C(CH3)2).
2.1.5.6. R,S-2N-[(4-chlor-2-methylphenoxy)ethyl]amino-1phenylethan-1-ol (6) Yield 57%; M=305.80; C17H20NO2Cl; M.p.: 119-121 °C; Ccalc/Cfound 66.71/66.99, Hcalc/Hfound 6.59/6.56, Ncalc/Nfound 4.58/4.58; Rf = 0.56 (CH3OH); cLogP=3.40; IR (KBr, cm-1 ) v: 3313, 3149, 3061, 3034, 2950, 2935, 2920, 2890, 2836, 2361, 2342, 1497, 1257; 1H-NMR: ( ppm) 7.37-7.28 (m, 4H, H-Ar); 7.25-7.20 (m, 1H, H-Ar); 7.19 (d, J=2.3, 1H, H3); 7.16 (dd, J=8.7, J=2.3, 1H, H-5); 6.92 (d, J=8.7, 1H, H6); 5.29 (bs, 1H, OH); 4.64 (t, J=6.3, 1H, CH-OH); 4.00 (t, J=5.4, 2H, O-CH2); 2.92 (t, J=5.4, 2H, N-CH2); 2.72 (d, J=6.3, 2H, N-CH2-CH); 2.10 (s, 3H, CH3-Ar); 1.90 (bs, 1H, NH). 2.1.5.7. R,S-1N-[(4-chlor-2-methylphenoxy)acetyl]aminopropan-2-ol (7) Yield 64%; M=257.72; C12H16NO3Cl; M.p.: 76-78 °C; Ccalc/Cfound 55.93/56.03, Hcalc/Hfound 6.26/6.48, Ncalc/Nfound 5.43/5.24; Rf = 0.86 (CH3OH); cLogP=1.32; IR (KBr, cm-1 ) v: 3426, 3330, 2972, 2919, 2875, 1651, 1248, 1192, 1134, 805; 1H-NMR: ( ppm) 7.16-7.10 (m, 2H, H-3, H-5 Ar); 6.97 (bs, 1H, NH); 6.69 (d, J=8.5, 1H, H-6 Ar); 4.49 (s, 2H, O-CH2-CO); 4.02-3.92 (m, 1H, NH-CH2-CHOH); 3.60-3.52 (m, 1H, NH-CHH); 3.25-3.16 (m, 1H, NH-CHH); 2.26 (s, 3H, CH3Ar); 2.10 (bs, 1H, OH); 1.21 (d, J=6.4, 3H, CHCH3). 2.1.5.8. R,S-1N-[(4-chlor-2-methylphenoxy)acetyl]aminobutan-2-ol (8) Yield 60%; M=271.75; C13H18NO3Cl; M.p.: 78-80 °C; Ccalc/Cfound 57.46/57.45, Hcalc/Hfound 6.68/7.01, Ncalc/Nfound 5.15/5.24; Rf = 0.88 (CH3OH); cLogP=2.02; IR (KBr, cm-1 ) v: 3429, 3326, 2963, 2936, 2917, 2878, 1549, 1245, 803; 1H-
Synthesis and Evaluation of Anticonvulsant Activity
Table 3.
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
39
Anticonvulsant Activity of the Tested Compounds (1-10) (Mice, i.p.).
Compd.
Dose
MES a)
ScMet a)
Neurotoxicity b)
ASP class c)
[mg/kg b.w.]
1
2
3
4
5
6
7
8
9
10
a)
0.5 h
4h
0.5 h
4h
0.5 h
4h
3
–
|
|
|
–
|
10
–
|
|
|
–
|
30
1/1
–
–
–
–
–
100
3/3
1/3
–
–
7/8
–
300
1/1
|
|
–
4/4
1/1
30
–
–
–
–
–
–
100
3/3
–
–
–
4/8
–
300
|
|
|
|
4/4
–
30
–
–
–
–
1/4
–
100
3/3
–
–
–
7/8
–
300
|
|
|
|
4/4
|
30
–
–
–
–
–
–
100
3/3
–
–
–
8/8
–
300
|
|
|
|
4/4
|
30
–
–
–
–
–
–
100
3/3
–
–
–
–
–
300
|
|
–
|
4/4
|
30
–
–
–
–
–
–
100
–
–
–
–
–
–
300
–
–
–
–
1/4
–
30
–
–
–
–
–
–
100
–
–
–
–
–
–
300
1/1
–
1/1
–
4/4
–
30
–
–
–
–
–
–
100
–
–
–
–
–
–
300
1/1
–
1/1
–
4/4
–
30
–
–
–
–
–
–
100
–
–
–
–
2/8
–
300
–
–
–
–
4/4
1/2
30
–
–
–
–
–
–
100
–
–
–
–
1/8
–
300
–
–
–
–
–
–
1
1
4
1
1
3
2
2
3
3
b)
Number of animals protected / number of animals tested; number of animals exhibiting toxicity / number of animals tested in the rotorod test; c) ASP classification: 1 – anticonvulsant activity at doses 100 mg/kg or less; 2 – anticonvulsant activity at doses greater than 100 mg/kg; 3 – compound inactive at 300 mg/kg; 4 – compound either active or inactive but toxic at doses of 30 mg/kg; – indicates that the compound was not active or toxic in the particular case; | indicates that the compound was not tested in the particular case.
NMR: ( ppm) 7.13-7.08 (m, 2H, H-3, H-5 Ar); 7.01 (bs, 1H, NH); 6.6 (d, J=8.5, 1H, H-6 Ar); 4,45 (s, 2H, O-CH2 CO); 3.70-3.54 (m, 2H, NH-CHH-CHOH); 3.24-3.15 (m, 1H, NH-CHH-CHOH); 2.58 (s, 1H, OH); 2.23 (s, 3H, CH3Ar); 1.54-1.44 (m, 2H, -CH2-CH3); 0.95 (d, J=7.5, 3H, CH2-CH3).
2.1.5.9. R,S-2N-[(4-chlor-2-methylphenoxy)acetyl]amino-1phenylethan-1-ol (9) Yield 59%; M=319.79; C17H18NO3Cl; M.p.: 111-112 °C; Ccalc/Cfound 63.85/64.07, Hcalc/Hfound 5.67/5.52, Ncalc/Nfound 4.38/4.36; Rf = 0.90 (CH3OH); cLogP=2.76; IR (KBr, cm-1 ) v: 3379, 3288, 3084, 3061, 3027, 2989, 2954, 2925, 2866,
40 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
Table 4.
Anticonvulsant Activity of the Tested Compounds (2, 5) (Rats, p.o.).
Compound
2
Test
Time [h] 0.25
0.5
1.0
2.0
4.0
a)
30
–
–
–
–
–
b)
30
–
–
–
–
–
a)
30
–
1/4
1/4
–
–
b)
30
–
–
–
–
–
MES TOX
Dose [mg/kg b.w.]
MES TOX
5
Waszkielewicz et al.
a)
Number of animals protected / number of animals tested; b) number of animals exhibiting toxicity / number of animals tested in the rotorod test; – indicates that the compound was not active or toxic in the particular case.
1657, 1548, 1491, 1250; 1H-NMR: ( ppm) 7.37-.10 (m, 7H Ar); 6.9 (bs, 1H, CO-NH); 6.3 (d, J=8.5, 1H, H-6 Ar); 4.884.84 (m, 1H, CH-OH); 4.43 (s, 2H, O-CH2-CO); 3.83-3.75 (m, 1H, NH-CHH); 3.50-3.43 (m, 1H, NH-CHH); 2.86 (bs, 1H, OH); 2.18 (s, 3H, CH3Ar). 2.1.5.10. R,S-2N-[(4-chlor-2-methylphenoxy)acetyl]-aminopropionic acid methyl ester (10) Yield 69%; M=285.73; C13H16NO4Cl; M.p.: 103-105 °C; Ccalc/Cfound 54.65/54.74, Hcalc/Hfound 5.64/5.54, Ncalc/Nfound 4.90/4.87; Rf = 0.86 (CHCl3/CH3OH 4:1); cLogP=2.22; IR (KBr, cm-1) v: 3350, 3071, 3031, 2994, 2983, 2955, 2859, 1739, 1657, 1542, 1248, 806; 1H-NMR: ( ppm) 7.21-7.10 (m, 3H, NH, H-3, H-5 Ar); 6.70 (d, J=8.5, 1H, H-6 Ar); 4.72-4.63 (m, 1H, NH-CH-COOCH3); 4.47 (s, 2H, O-CH2 CO); 3.78 (s, 3H, COOCH3); 2.30 (s, 3H, CH3Ar); 1.47 (d, J=7.2, 3H, NH-CH-CH3). 3. PHARMACOLOGY 3.1. Anticonvulsant Assays Anticonvulsant activity and neurological toxicity assays were performed within the Antiepileptic Drug Development Program, Epilepsy Branch, National Institutes of Neurological and Communicative Disorders and Stroke, National Institute of Health in Rockville, USA [14]. Compounds were administered as suspensions in 0.5% methylcellulose at three dosage levels (30, 100 and 300 mg/kg b.w.) intraperitoneally (i.p.) into mice and, if anticonvulsant activity was satisfactory, (30 mg/kg b.w.) peroral (p.o.) into rats. This qualitative assay used one to eight animals and included three tests: maximal electroshock seizure (MES) for grand mal seizures, subcutaneous pentylenetetrazol (ScMet) for petit mal seizures, and neurotoxicity (rotorod, TOX), noted at 30 min and 4 h after administration. The seizures were induced by 60 Hz alternating current at 50 mA (mice) or 150 mA into rats delivered for 0.2 s via corneal electrodes. A drop of 0.9% NaCl solution was placed into each eye prior to applying the electrodes. Protection in the MES test was defined as the abolition of the hindlimb tonic extension component of the seizure. The ScMet test was performed by administration 85 mg/kg of pentylenetetrazole dissolved in 0.9% NaCl solution into the posterior midline of mice. A minimal time of 30 min subsequent to subcutaneous administration of pentylenetetrazole (ScMet) was used for seizure detection. A failure to
observe even a threshold seizure (a single episode of clonic spasm of at least 5 s in duration) was regarded as protection. Neurotoxicity was measured in mice and/or in rats by the rotorod test. The animal was placed on a 1 inch diameter knurled plastic rod rotating at 6 rpm. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of the three trials. 3.2. Anticonvulsant Activity Results The synthesized compounds 1-10 have been subject to screening in mice, i.p. The results are presented in Table 3. Compounds 2 and 5 have been advanced to assays in rats, p.o. The results are presented in Table 4. It can be seen from Table 3 that among the tested aminoalkanols 1-6, five compounds exhibit anticonvulsant activity in at least 100 mg/kg b.w. which defines four of them (1-2, and 4-5) as class 1 ASP. Among the tested amides 7-10, only two of them (7 and 8) exhibit anticonvulsant activity at the lowest dose 300 mg/kg b.w. Therefore, it can be generalized for the 4-chlor-2-methylphenol derivatives that aminoalkanols (1-6) exhibit stronger activity than alkanolamides (7-10). Moreover, compounds 2-5 reveal 100% activity at 100 mg/kg b.w. in mice, i.p., while within the alkanolamide group (7-10) the most active were compounds 7 and 8, revealing 100% neuroprotection at 300 mg/kg b.w. It was also exhibiting 100% neurotoxicity at the same dose and time. The most promising compound among the synthesized groups were 1, revealing 100% activity at 30 mg/kg b.w. (mice, i.p.), as well as 5, revealing 100% activity at dose 100 mg/kg b.w. in mice, i.p., 0.5 h after administration, without neurotoxicity at the same dose and time. Compound 5 was advanced to the assay in rats, p.o. Table 4, where it proved 25% activity 0.5 and 1 h after p.o. administration of 30 mg/kg b.w., without neurotoxicity through the 4 h period after administration. Another compound that was advanced to evaluation in rats, p.o. was 2, however, without activity nor toxicity at 30 mg/kg b.w. 3.3. Structure-activity relationship Structure-anticonvulsant activity relationship studies in the group of N-(4-chlor-2-methylphenoxy)ethyl- and N-(4chlor-2-methylphenoxy)acetylaminoalkanols indicate beneficial role of the amine moiety compared to amide, since
Synthesis and Evaluation of Anticonvulsant Activity
aminoalkanol group (1-6) seems more active than the alkanolamides (7-9) and appropriate amide derivative of aminoacid ester (10). Among aminoalkanols 1-6 and alkanolamides 7-9, compound 8 as the amide analog of 3, exhibits lower activity and lower toxicity, and its lower lipophilicity (cLogP=2.59 and 2.02 for compounds 3 and 8, respectively) supposedly has influence on this fact. Change of aminoalkanol moiety into alkanolamide in case of compound 9 (the amide analog of compound 6), did not improve anticonvulsant profile of 6, moreover, it caused increase in toxicity from the dose 300 mg/kg b.w. (in 6, mice, i.p.) to 100 (in 9, mice, i.p.). Therefore, it can be stated that within the synthesized group the amine moiety is superior to amide. Compound 10 is a methyl ester of Nsubstituted alanine and it did not prove active but neurotoxic, revealing that amino acid ester moiety is not beneficial in this case. Comparing 4-chlor-2-methylphenol analog (4) of S-(+)2-N-[(2,6-dimethyl)phenoxyethyl]aminobutan-1-ol hydrochloride [6], the new derivative was less active. The 100% activity was observed at 100 mg/kg b.w. with neurotoxicity at the same dose. Therefore, change in location of the substituent from position 2,6 to 2,4 together with change of one methyl substituent into chlorine (in position 4) in the phenyl ring is not beneficial in case of anticonvulsant activity in this group. It is worth to take into account values of the calculated partition coefficient (cLogP) as an important factor influencing capability of crossing the blood-brain barrier. The most promising compound in this group (5) exhibits cLogP=2.81, which is in the upper range of the observed values 1.32-3.40. However, the most lipophilic compound 6 (cLogP=3.40) was not active in the used experiments and the least lipophilic compound 7 (cLogP=1.32) was active only in 300 mg/kg b.w. (mice, i.p.) with concomitant neurotoxicity. Among the synthesized ten new substances, the most promising compound was 2N-[(4-chlor-2-methylphenoxy)ethylamino-2-methylpropan-1-ol (5). It contains modification of S-(+)-2-N-[(2,6-dimethylphenoxy)ethyl]aminobutan-1-ol hydrochloride [6] in terms of substituent and its location in the phenyl ring, as well as the aminoalkanol moiety. Moreover, compound 5 was evaluated as base and the lead compound was a salt, which may have impact on many aspects such as lipophilicity and bioavailability. As a summary, modification of aminoalkanol as well as change of a substituent and its location in the phenyl ring have impact on anticonvulsant activity in the synthesized group. The presented results confirm that lipophilicity is an important but not the only factor influencing on the anticonvulsant activity and neurotoxicity.
Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1
However, its activity was not superior to the lead compounds. Since four of the synthesized compounds are described as class 1 ASP, there exist premises for further modifications of the lead compounds, in terms of aminoalkanol moieties, as well as substituents and their location within the phenyl group. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The authors would like to thank Prof. James P. Stables and prof. Jeff Jiang for providing the results of pharmacological assays through the ADD program at National Institute of Neurological Disorders and Stroke (Bethesda, USA), as well as Prof. Katarzyna KieKononowicz for coordination of the cooperation with the Faculty of Pharmacy, Jagiellonian University Medical College. This project is supported by the program K/ZDS/003328. REFERENCES [1] [2] [3]
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CONCLUSIONS In conclusion, we synthesized ten new N-(4-chlor-2methylphenoxy)ethyl- (1-6) and N-(4-chlor-2-methylphenoxy)acetylaminoalkanols (7-9) and one appropriate amino acid derivative (10) and evaluated their activity in the MES, ScMet, and TOX assays within the Antiepileptic Drug Development Program. The most active was 2N-[(4-chlor-2methylphenoxy)ethyl]amino-2-methylpropan-1-ol (5).
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Received: July 18, 2012
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Stables, J. P.; Kupferberg, H. J. In Molecular and Cellular Targets for Anti-epileptic Drugs; Avanzini, G.; Regesta, P.; Tanganelli, A.; Avoli, M., Eds.; John Libbey & Company Ltd: London, 1997; Vol. 23129, pp. 191-198.
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Revised: October 03, 2012
Accepted: October 08, 2012
