exploration of their anticonvulsant activity against maximal electroshock, picrotoxin- induced clonic convulsions and bicuculline-induced clonic convulsions.
Synthesis and Anticonvulsant Activity of Some 1-Substituted-2-oxopyrrolidine Derivatives, II. ⊗ Abdulrahman M. Al-Obaid,1 Hussein I. El-Subbagh,*1 Othman A. Al-Shabanah,2 and Mohamed M. Elmazar2 Departments of 1Pharmaceutical Chemistry and 2Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh-11451, Saudi Arabia.
In the present study, as an attempt to locate new antiepileptic agent(s) with less side effects as well as toxicity, a new series of N-substituted-2-oxopyrrolidine derivatives was synthesized as GABA prodrugs and evaluated for their anticonvulsant activity adopting
various
screening
models.
Compound
N-(4-flourobenzyl)-2-oxo-1-
pyrrolidineacetamide (14) proved to possess a potent broad spectrum anticonvulsant activity with wide safety margin, compared with valproic acid. Compound 14 is more potent (ED50 = 0.43 vs 0.71 mmol/kg for valproate) and has a higher protective index against convulsions (PI = 2.81 vs 1.4-2.36 for valproate). Compound 14 with doses up to 0.5 and 1.0 g/kg, i.p., did not produce mortality within 24 h after administration. Compounds N-(4-methoxybenzyl)-2-oxo-1-pyrrolidineacetamide (15), N-(phenylethyl)-2oxo-1-pyrrolidineacetamide (16) and N-[2-(4-fluorophenyl)ethyl]-2-oxo-1-pyrrolidineacetamide (17) are also among the potent derivatives explored in this investigation. The finding that compounds 14-16 protect against Bicuculline-induced convulsions, confirms the rational behind the design of the present series of compounds as GABA prodrugs.
Introduction Epilepsy has been found to have point prevalence rates in the range of 4-10/1000 in the general population.1 The majority (60-70%) of these cases occur without clear ⊗
For Part I, see reference 13.
1
etiology. Despite this, anticonvulsant drugs are estimated to be useful in treating 90% of all epileptic patients. However, all currently approved anticonvulsant agents have doserelated toxicity and idiosyncratic side effects.2 Additionally, many antiepileptic drugs induce xenobiotic-metabolizing liver enzymes resulting in complex and undesirable side effects. Major medical breakthroughs in non-pharmacological therapies for the treatment of epilepsy in the near future seem remote, that is why, the search for new antiepileptic drugs with lower toxicity and fewer side effects continues.3,4 GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain, which controls the excitability of many central nervous system (CNS) pathways. The principal mode of action for this neurotransmitter occurs by modulation of the GABA chloride ion channel complex. 5,7 Epilepsy has been reported to be associated with a decrease in CNS-GABA concentration.8 This observation suggests that the increase of CNS-GABA levels may be useful in the treatment of such neuropsychatiric disorder. However, attempts to use GABA in clinical trials failed due to the extremely high doses required to force the drug across the blood brain barrier (BBB). Numerous GABA derivatives including their alkyl, steroid ester,9,10 Schiff's bases and isonicotinoyl GABA11 have been synthesized to facilitate their uptake into the brain. Another developed approach was the use of 2pyrrolidinone and its acyl derivatives12 as GABA prodrugs. The lipophilicity of these derivatives increases their ability to cross the BBB then hydrolyse to release GABA. A recent study performed at our laboratory described the anticonvulsant activity of a series of 2-pyrrolidinones (γ-butyrolactams) aas a piracetam (1) analogs.13 The type of substituents used to prepare this piracetam analogs was selected to determine the electronic effect of the phenyl group on activity and as an attempt to identify novel lead
2
compound(s) for future development as anticonvulsants. The results obtained from this latter study allowed us to identify five lead compounds (2-6) representing a new class of anticonvulsant agents producing 83, 83, 83, 50 and 83% protection, respectively, against pentylenetetrazole (PTZ)-induced convulsion in mice at a dose of 100 mg/kg (Figure 1). Based on our recent findings,13 in addition to certain reports14 revealing that compounds of the type 2-6 are good candidates for future development as antiepileptic agents, our objective is to determine the scope and limitations of activity for the 2-oxopyrrolidine derivatives, specially compound 2 (R = benzyl), through additional structural modifications in the benzyl moiety area through its replacement with phenethylamine carrying various substituents or heteroaromatic or cycloalkylamine functions in the hope to obtain more potent compounds with minimal side effects. Chemistry 2-Oxopyrrolidine (7)13 is alkylated using ethyl bromoacetate to yield the starting material ethyl 2-oxo-1-pyrrolyidineacetate (8). Treatment of 8 with n. propylamine, n. butylamine, cyclohexylamine and cyclohexylmethylamine afforded the targets 9-12. Further utilization of 8 is attained through its reaction with a variety of benzylamines and phenethylamines as well as pyridylamines to give compounds 13-21. Reacting 8 with a collection of five and six membered heterocyclic nuclei carrying either methylamine or ethylamine residue produced compounds 22-28 (Scheme 1, Table 1). Results and Discussion Measurements of sedative and anti-PTZ-induced convulsions. The results of the preliminary screening of neurological deficits and modulation of PTZ-induced convulsions of compounds 9-28 are shown in Table 1. Intraperitoneal (ip) administration
3
of 1.5 mmol/kg of compound 26 produced 100% mortality within 5-7 min. Compound 22 also produced 40% mortality. Evaluation of minimal neurological deficits using chimney test 25 min. after administration of 1.5 mmol/kg of the different synthesized analogs revealed the following: Compounds 19 and 27 produced the lowest sedative effect, where only 20% and 25% of the animals, respectively, failed to climb up the glass tube backward within 30 sec. Compounds 10, 13 and 21 produced sedation in 40% of animals, while compounds 14, 16 and 28 produced 60% - 67% sedation. Compounds 22, 24 and 25 were more sedative, producing 80% - 86% effect. Administration of the other eight compounds (9, 11, 12, 15, 17, 18, 20 and 23) resulted in 100% sedation. The chimney test has been designed to detect minimal neurological deficit (e.g. impairment of motor coordination, sedation, catalepsy) and in this respect similar to and as sensitive as the rotarod test.16 When PTZ (70 mg/kg, sc) was given 30 min. after administration of each agent (1.5 mmol/kg, ip), compounds 9, 18, 20, 23 and 28 failed to protect mice against PTZinduced seizure threshold. Two of these compounds (compounds 9 and 20), however, potentiated PTZ-induced convulsions, and therefore considered proconvulsants. Nine compounds (10, 11, 13, 19, 21, 22, 24, 25, and 27) produced minimal protection (13% 43%) against PTZ-induced seizures. Only five compounds (12 and 14-17) produced 83% - 100% protection against PTZ-induced convulsions. These five compounds, therefore were chosen for further investigations. Protection against PTZ-induced seizure threshold is an indication for the potential anticonvulsant activity of these compounds particularly 17 against absence seizures.
CNS actions of a drug including sedative and anticonvulsant activity depend on its lipophilicity which enable the compound to cross BBB and reach the cellular site of action.
4
The calculated lipophilicity, Log (p) of the present series of compounds (9-28) is shown in Table 1. Compounds 9-28 with a broad structural variations showed a wide range of Log (p) of !2.11 to 0.8. When the series is subdivided into four subgroups of closely related compounds such as 9-12; 13-15; 16-18 and 23-25 the change in Log (p) was narrowed. Increasing Log (p) value from !0.67 for compound 9 to 0.49 for compound 12 was correlated with an increase in the anticonvulsant activity from 0% to 100%, respectively. The sedative activity, however, was 100% for compounds 9, 11 and 12 and 40% for compound 10 (Figure 2A). The same pattern was shown for compounds 16-18, where increasing Log (p) from 0.27 for compound 18 to 0.68 for compound 17, is associated with an increase in the anticonvulsant activity from 0% to 100%, respectively, with a slight change of the sedative effect (Figure 2C). On the other hand, compound 13-15 showed virtually the same range of Log (p) as that of 16-17; increasing Log (p) from 0.12 for compound 15 to 0.8 for compound 13 decreased the anticonvulsant activity from 100% to 20% and sedative activity from 100% to 40%, respectively (Figure 2B). In another subgroup (23-25), however, increasing Log (p) from !2.11 for compound 25 to !0.76 for compound 23 decreased the anticonvulsant activity from 40% to 0% but increased sedation from 80% to 100%, respectively (Figure 2D). Structure Activity Relationship (SAR). The lead compound 2, with benzyl moiety, protected 83% of the animals against PTZ-induced convulsions when given at 0.43 mmol/kg, i.p. This dose increased thiopental sleeping time from 18.2 min to 47.2 min.13 From Table 1, the following SAR can be deduced after administration of equimolar doses of compounds 9-28. All of these compounds share the same 2-oxo-1pyrrolidineacetamide basic skeleton and only differ in the N-substituted area (see Table 1):
5
Π
Compound 9 with aliphatic n.propyl side chain did not protect the animals against PTZ-induced convulsions with a proconvulsant and high sedative potential, while compound 10 with aliphatic n.butyl side chain, the sedation is decreased to 40% and PTZ-induced convulsions was protected in 40% of animals when compared with 9.
Π
Changing the aromatic benzyl moiety in 2 with its saturated analoge (cyclohexylmethyl) as in 12, maintained the anticonvulsant activity but increased sedation. Comparing the activity of compound 11 (R = cyclohexyl) with that of 12, a reduction in the anticonvulsant potency with increased sedation was observed in 11.
Π
The presence of a phenethyl group as in compound 16, which is one carbon atom more than that of the lead compound 2, slightly decreased the anticonvulsant activity and slightly increased sedation. Introduction of 3,4-dimethoxy groups to the aromatic ring of compound 16 as in 18 abolished the anticonvulsant activity and increased sedation.
Π
Introduction of 4-methoxy function to the aromatic ring of the lead compound 2, as in 15 did not alter the anticonvulsant activity but increased sedation.
Π
Introduction of a chlorine atom at the 4-position of the aromatic ring of the lead compound 2, as in 13 decreased the anticonvulsant activity, while introducing a fluorine atom in the same position as in 14 maintained the high anticonvulsant activity with a slight increase in sedation when compared with the lead compound 2. Introduction of a fluorine atom in the 4-position of compound 16 as in 17 increased both anticonvulsant
and sedative activities.
6
Compound 17
(R = 4-fluorophenylethyl) was also more sedative than compound 14 (R = 4-fluorophenyl), which confirms the earlier observation that compound 16 was more sedative than the lead compound 2. Both pairs differ only in the linking chain which changed from methyl (2, 14) to ethyl group (16 and 17). Π
Replacement of the phenyl ring in 2 by a pyridine function in such a manner that the heterocyclic nitrogen becomes in two different positions, as in 19 (R = 2-pyridyl) and 20 (R = 3-pyridyl), showed that: Compound 19 had lower anticonvulsant activity, while compound 20 was highly sedative with a proconvulsant potential. Increasing the linking chain of 19 by one carbon atom produced 21 with no change in the anticonvulsant activity and increased its sedation. Compound 21, however, was less effective when compared with its phenyl counterpart 16. In general, replacing the phenyl ring with its pyridine bioisostere decreased the anticonvulsant activity.
Π
Replacement of the phenyl ring of 2 by a 4-piperidino moiety (22) decreased the anticonvulsant activity, while replacement of the phenethyl residue of 16 by Nethylpiperidino function (23) rendered the compound rather toxic with 40% mortality.
Π
Replacement of the phenyl ring of 16 with N-morpholino (24) or N-piperazino (25) functions decreased the anticonvulsant potency to nearly half and slightly increased sedation.
Π
Decreasing the size of the 6 membered N-piperidino ring (23) to the 5 membered N-pyrrolidino analoge (26) increased mortality from 40% to 100%.
Π
Replacement of the phenyl group in 2 and 16, respectively, by the N-substituted
7
(ethyl or methyl) pyrrolidine analoge (27 and 28) showed minimal anticonvulsant activity. From the previous structure activity correlations, it could be concluded that: Changing the aromatic residue of the lead compound 2 by an aliphatic side chain as in 9-11, or by a cycloalkyl heterocyclic five membered (27) or six membered (22) ring, as well as, introduction of 4-chloro function (13) led to compounds with decreased anticonvulsant activity. Compounds 16-18, 21, 23-26 and 28 which differ from the lead compound 2 by having one carbon atom extra in the linking chain and various substituents, exhibited lower anticonvulsant activity, except compounds 16 and 17 which showed promising anticonvulsant potential. Compounds, 12 and 14-17 were chosen for further exploration of their anticonvulsant activity against maximal electroshock, picrotoxininduced clonic convulsions and bicuculline-induced clonic convulsions. These compounds showed 80% or more protection against PTZ-induced convulsion with equi or less sedative potential. Determination of the median effective (ED50) and median sedative (TD50) doses as well as the protective index (PI) of compounds 12 and 14-17. Compound 14 is the most active member of the group (ED50 = 0.43 mmol/kg) followed by 12 and 17 (0.65 and 0.70 mmol/kg, respectively) and compound 15 (1.14 mmol/kg). Compound 16, however was the least effective. The protective index (PI = TD50/ED50) of compound 14 was the highest (2.81) and that of 12 and 17 was 1.32 and 1.34, respectively, while that of 15 was only 1.00. The PI gives an idea about the safety margin of these compounds, the higher the PI, the more possibility that the compound will show anticonvulsant activity 17 with minimal neurological deficits (sedation), (Table 2).
8
Effect of compounds 12 and 14-17 against maximal electroshock (MES). The dose used in this test was the one that produced maximal protection against PTZ-induced convulsions (Table 2). Compounds 14-16 produced 80-100% protection against MESinduced tonic extension seizure which suggests that they also has the ability to prevent the spread of seizure discharge through neural tissue as well as their ability to raise the seizure threshold.17 Compound 17 protected 50% of the animals against MES and produced 33% mortality within 10 min. after administration, while compound 12 was inactive in this test. Therefore, compounds 14-16 and possibly compound 17 may have a broad anticonvulsant activity similar to that of the anticonvulsant drug valproate. Compound 12, however, may have a selective anticonvulsant activity against absence seizures similar to the anticonvulsant drug ethosuximide. Compound 9 and 20 which increased the frequency of PTZ-induced seizures (proconvulsants), an effect which commonly observed with phenytoin, were also tested against MES-induced tonic seizures. Both compounds 9 and 20, however, were inactive against MES-induced seizures when used in a dose of 1.5 mmol/kg, 30 min. prior to the electric shock. Effect of compounds 12 and 14-17 against picrotoxin (Pic)-induced convulsions. Picrotoxin is a chloride-channel blocker producing clonic convulsions. Protection against Pic-induced convulsions indicates that the test substance may act by increasing chloride influx via brain chloride channels.17 Compounds 14 and 16 were effective in this test, while compounds 12 and 15 were inactive. Compound 17, however, was not tested because 20% of the animals died within 10 min. while the remaining animals showed rigidity and tremors for 40 min before administration of picrotoxin (Table 2).
9
Effect of compounds 12 and 14-17 against bicuculline (Bic)-induced convulsions. Bicuculline in a GABAA antagonist when given to mice, it induces clonic convulsions. Agents that protect against Bic-induced convulsions would be expected to act as a direct GABAA agonist or indirectly through enhancing GABA synthesis or its release as a brain inhibitory neurotransmitter. Compound 16 produced 100% protection against Bic-induced convulsions, while compounds 14 and 15 produced 40% and 80% protection, respectively (Table 2). Conclusion From the present results, it can be concluded that, compound 14 [N-(4flourobenzyl)-2-oxo-1-pyrrolidineacetamide] may have potent broad spectrum anticonvulsant activity with wide safety margin, compared with the established broad spectrum antiepileptic drug valproate.
17,18
Compound 14 is more potent (ED50 = 0.43 vs
0.71 mmol/kg for valproate) and have a higher protective index (PI = 2.81 vs 1.4-2.36 for valproate). Preliminary acute toxicity of compound 14 with doses of 0.5 and 1.0 g/kg, i.p, (equivalent to about 5 and 10 times the ED50, respectively) revealed that both doses did not produce mortality within 24 h after administration. With the high dose (1.0 g/kg), animals lost the writing reflex (hypnosis), within 15 min after administration for a period of 60 min, and then regained consciousness. The 0.5 g/kg dose only produced slight sedation without loss of writing reflex. Observing those animals for 7 days after a single administration, unfortunately, all animals in both groups died on the 7th day, which warrant further toxicological investigations. Compound 17 also shows broad-spectrum activity, but it was less potent than compound 14. It is twice as active against seizure threshold than seizure speed. Compound 17, however, produced 20-33% mortality in mice
10
within 40 min. of administration of only 1.5 mmol/Kg. Compound 12, however, exhibits a selective anticonvulsant activity against seizure threshold. The finding that compounds 14-16 protect against Bic-induced convulsions, confirms the rational behind the chemical design of the present series of synthetic compounds as GABA prodrugs. Experimental Section Unless otherwise specified all chemicals were commercial grade, used without further purification and were obtained from Aldrich Chemical Co. (Milwaukee, WI). Solvents used for extraction were dried over MgSO4, filtered and removed on a rotary evaporator. Elemental analyses were performed at College of Pharmacy, King Saud University, Central Laboratory. 1H and
13
C NMR spectra were obtained in CDCl3 at
300 MHz, Brucker instrument, with TMS as an internal standard. Thin layer and flash chromatography utilized E. Merck silica gel (230-400 mesh). Preparative centrifugal thin layer chromatography was performed on a Harrison model 7924A chromatotron using Analtech silica gel GF rotors. The synthesis of compound 8 was previously reported in ref. 13. General procedure for the preparation of N-Substituted-2-oxo-1-pyrrolidineacetamides (9-28). Ethyl 2-oxo-1-pyrrolidinacetate (8, 5.0 g, 0.03 mol) and the appropriate amine (0.04 mol) were heated under reflux, neat, at 100ΕC with stirring for 4-6 h. The reaction mixture was cooled and chromatographed on a silica gel column using the suitable solvent system. N-(n. Propyl)-2-oxo-1-pyrrolidineacetamide (9). The crude product was chromatographed on silica (CHCl3 ) to give 9 in 70% yield: 1H NMR (CDCl3) δ 0.87-0.94
11
(t, 3H, J = 7.2 Hz, CH3 (CH2)2-), 1.45-1.57 (m, 2H, J = 7.2 Hz, CH3CH2-), 2.04-2.14 (m, 2H, J = 7.2 Hz, CH2-pyrrolidone), 2.39-2.46 (t, 2H, J = 7.2 Hz, CH2CO-pyrrolidone), 3.15-3.23 (q, 2H, J = 7.2 Hz, CH3CH2CH2-), 3.50-3.56 (t, 2H, J = 7.2 Hz, CH2NCO), 3.91 (s, 2H, NCH2CO), 6.47 (brs, 1H, NH).
13
C: 11.24, 17.91, 22.61, 30.38, 41.09,
47.18, 48.45, 168.06, 176.0. Anal. (C9H16N2O2) C, H, N. N-(n. Butyl)-2-oxo-1-pyrrolidineacetamide (10). The crude product was chromatographed on silica (CHCl3) to give 10 in 82% yield: 1H NMR (CDCl3) δ 0.820.94 (t, 3H, J = 7.1 Hz, CH3 (CH2)3-), 1.19-1.36 (m, 2H, J = 7.1 Hz, CH3CH2 (CH2)2-), 1.38-1.51 (m, 2H, J = 7.1 Hz, CH3CH2CH2), 1.89 (brs, 1H, NH), 1.99-2.12 (m, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.33-2.45 (t, 2H, J = 7.2 Hz, CH2CO-pyrrolidone), 3.14-3.24 (q, 2H, J = 7.1 Hz, CH3 (CH2)2 CH2-),3.44-3.54 (t, 2H, J = 7.2 Hz, CH2NCO), 3.86 (s, 2H, NCH2CO).
13
C: 13.66, 17.96, 19.97, 30.38, 31.46, 39.18, 47.38, 48.48, 168.04,
176.0. Anal. (C10H18N2O2) C, H, N. N-(Cyclohexyl)-2-oxo-1-pyrrolidineacetamide (11). The crude product was purified on silica gel column (CH2Cl2) to afford 11 in 85% yield: 1H NMR (CDCl3) δ 1.02-1.20 (m, 3H, J = 10.0, 12.0 Hz, cyclohexyl), 1.23-1.42 (m, 2H, J = 10.0, 12.0 Hz, cyclohexyl), 14.8-1.73 (m, 3H, J = 10.0, 12.0 Hz, cyclohexyl), 1.75-1.94 (m, 2H, cyclohexyl), 1.96-2.14 (t, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.30-2.48 (t, 2H, J = 7.9 Hz, CH2CO-pyrrolidone), 3.39-3.56 (t, 2H, J = 7.9 Hz, CH2NCO), 3.62-3.77 (m, 1H, cyclohexyl), 3.84 (s, 2H, -NCH2CO), 6.0 (brs, 1H, NH). 13C: 18.02, 24.67, 25.42, 30.45, 32.89, 47.47, 48.23, 48.40, 167.09, 175.94. Anal. (C12H20N2O2) C, H, N. N-(Cyclohexylmethyl)-2-oxo-1-pyrrolidineacetamide (12). The crude product was chromatographed on silica (CH2Cl2) to give 12 in 74% yield: 1H NMR (CDCl3 ) δ
12
0.74-0.77 (m, 3H, J = 10.0, 12.0 Hz, cyclohexyl), 1.00-1.02 (m, 2H, J = 10.0 Hz, cyclohexyl), 1.05-1.08 (m, 3H, J = 12.0 Hz, cyclohexyl), 1.52-1.56 (m, 3H, J = 10.0, 12.0 Hz, cyclohexyl), 1.92-1.96 (m, 2H, J = 7.8 Hz, CH2-pyrrolidone), 2.25-2.29 (t, 2H, J = 7.8 Hz, CH2CO-pyrrolidone), 2.89-2.92 (t, 2H, J = 6.9 Hz, CH2NH), 3.37-3.41 (t, 2H, J = 7.8 Hz, CH2NCO), 3.78 (s, 2H, NCH2CO), 6.8 (brs, 1H, NH). 13C: 17.60, 25.45, 26.02, 30.18, 30.44, 37.43, 45.31, 46.35, 48.12, 167.82, 175.71. Anal. (C13H22N2O2) C, H, N. N-(4-Chlorobenzyl)-2-oxo-1-pyrrolidineacetamide (13). The crude gummy product was chromatographed on silica gel column using EtOAc as eluent to give 13 in 82% yield: 1H NMR (CDCl3) δ 1.89-1.97 (m, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.17-2.20 (t, 2H, J = 7.9 Hz, CH2CO-pyrrolidone), 3.30-3.50 (t, 2H, J = 7.9 Hz, -CH2NCO-), 3.83 (s, 2H, -NCH2CO-), 4.32 (d, 2H, J = 5.8 Hz, CH2ph), 6.90 (brs, 1H, NH), 7.07-7.17 (m, 4H, ArH). 13C: 18.17, 30.76, 42.90, 46.81, 48.85, 128.97, 129.35, 133.32, 137.21, 168.57, 176.48. Anal. (C13H15ClN2O2) C, H, N. N-(4-Fluorobenzyl)-2-oxo-1-pyrrolidineacetamide (14). The crude oily product was chromatographed on silica gel column using EtOAC to afford 14 in 68% yield: 1H NMR (CDCl3) δ 1.93-1.95 (m, 2H, J = 8.0 Hz, CH2-pyrrolidone), 2.17-2.19 (t, 2H, J = 8.0 Hz, CH2CO-pyrrolidone), 3.38-3.41 (t, 2H, J = 8.0 Hz, CH2NCO), 3.81 (s, 2H, NCH2CO), 4.23 (d, 2H, 5.6 Hz, CH2ph), 6.88-6.90 (m, 2H, J = 12.0 Hz, ArH), 7.10-7.13 (m, 2H, J = 12.0 Hz, ArH), 7.39 (brs, 1H, NH). 13C: 17.56, 30.11, 42.25, 46.21, 48.19, 114.95, 115.16, 128.98, 133.81, 167.88, 175.81. Anal. (C13H15FN2O2) C, H, N. N-(4-Methoxybenzyl)-2-oxo-1-pyrrolidineacetamide (15). The obtained oily product was chromatographed on silica gel column (CHCl3) to give 15 in 81% yield: 1H NMR (CDCl3) δ 1.93-2.01 (m, 2H, J = 8.0 Hz, CH2-pyrrolidone), 2.24-2.28 (t, 2H, J =
13
8.0 Hz, CH2CO-pyrrolidone), 3.42-3.45 (t, 2H, J = 8.0 Hz, CH2NCO), 3.73 (s, 3H, CH3O), 3.85 (s, 2H, NCH2CO), 4.27 (d, 2H, J = 5.6 Hz, CH2ph), 6.79 (d, 2H, J = 8.0 Hz, ArH), 6.99 (brs, 1H, NH), 7.16 (d, 2H, J = 8.0 Hz, ArH). 13C: 17.69, 30.20, 42.63, 46.59, 48.29, 55.10, 113.78, 128.81, 130.01, 158.72, 167.79, 175.86. Anal. (C14H18N2O3) C, H, N. N-(Phenylethyl)-2-oxo-1-pyrrolidineacetamide (16). The crude product was chromatographed on silica gel column (CHCl3) to give 16 in 78% yield: 1H NMR (CDCl3) δ 1.91-1.97 (m, 2H, CH2-pyrrolidone), 2.28-2.31 (m, 2H, CH2CO-pyrrolidone), 2.76-2.79 (m, 2H, CH2NCO-), 3.32 (m, 2H, CH2), 3.44-3.48 (m, 2H, CH2), 3.82 (s, 2H, NCH2CO-), 6.49 (br s, 1H, NH), 7.14-7.2 (m, 3H, ArH), 7.24-7.28 (m, 2H, ArH). 13C: 19.33, 31.82, 36.88, 41.85, 48.46, 49.79, 111.09, 127.99, 130.06, 130.24, 140.20, 169.59, 177.48. Anal. (C14H18N2O2) C, H, N. N-[2-(4-Fluorophenyl)ethyl]-2-oxo-1-pyrrolidineacetamide (17). The crude product was chromatographed on silica gel column (CH2Cl2) to afford 17 in 65% yield: 1H NMR (CDCl3) δ 1.94-2.04 (m, 2H, J = 8.0 Hz, CH2-pyrrolidone), 2.31 (t, 2H, J = 8.0 Hz, CH2CO-pyrrolidone), 2.73-2.78 (t, 2H, J = 8.0 Hz, CH2NCO), 3.37-3.48 (m, 4H, 2CH2), 3.82 (s, 2H, NCH2CO), 6.45 (brs, 1H, NH), 6.93-6.99 (m, 2H, ArH), 7.09-7.15 (m, 2H, ArH). 13C: 18.27, 30.71, 35.04, 40.84, 47.59, 48.79, 115.56, 115.84, 130.59, 134.71, 168.55, 176.37. Anal. (C14H17FN2O2) C, H, N. N-[2-(3,4-Dimethoxyphenyl)ethyl]-2-oxo-1-pyrrolidineacetamide (18). The crude oily product was chromatographed on silica gel column using CH2Cl2 as eluant to give 18 in 86% yield: 1H NMR (CDCl3) δ 1.83-1.86 (m, 2H, CH2-pyrrolidone), 2.19-2.20 (m, 2H, CH2CO-pyrrolidone), 2.61-2.62 (m, 2H, CH2NCO-), 2.83 (brs, 1H, NH), 3.27-
14
3.32 (m, 4H, 2CH2), 3.69-3.73 (m, 8H, CH2 & 2CH3O-), 6.58-6.75 (m, 3H, ArH). 13C: 18.05, 30.61, 35.24, 40.89, 46.82, 48.52, 56.11, 56.15, 111.56, 112.25, 120.90, 131.61, 147.81, 149.15, 168.34, 176.23. Anal. (C16H22N2O4) C, H, N. N-(2-Pyridylmethyl)-2-oxo-1-pyrrolidineacetamide (19). The obtained gum was chromatographed on silica (CHCl3) to produce 19 in 56% yield: 1H NMR (CDCl3) δ 1.94-2.16 (m, 2H, J = 8.0 Hz, CH2 -pyrrolidone), 2.35-2.48 (t, 2H, J = 8.0 Hz, CH2COpyrrolidone), 3.44-3.57 (t, 2H, J = 8.0 Hz, CH2NCO), 3.99 (s, 2H, -NCH2CO-), 4.52 (d, 2H, J = 4.8 Hz, CH2NH), 7.10-7.31 (m, 3H, ArH & NH), 7.55-7.72 (m, 1H, ArH), 8.438.55 (m, 1H, ArH).
13
C: 18.02, 30.37, 44.20, 46.83, 48.29, 121.92, 122.40, 136.79,
149.02, 156.02, 168.01, 175.94. Anal. (C12H15N3O2) C, H, N. N-(3-Pyridylmethyl)-2-oxo-1-pyrrolidineacetamide (20). The obtained grum product was charged to a silica gel column and eluted with 5% MeOH/CHCl3 to give 20 in 42% yield: 1H NMR (CDCl3) δ 1.99-2.11 (m, 2H, J = 7.9, CH2-pyrrolidone), 2.27-2.41 (t, 2H, J = 7.9 Hz, CH2CO-pyrrolidone), 3.46-3.58 (t, 2H, J = 7.9 Hz, CH2NCO), 3.94 (s, 2H, NCHCO), 4.41 (d, 2H, J = 6.0 Hz, CH2NH), 7.21-7.29 (m, 1H, ArH), 7.46 (brs, 1H, NH), 7.62 (m, 1H, ArH), 8.40-8.56 (m, 2H, ArH). 13C: 17.82, 30.30, 40.73, 46.78, 48.52, 123.52, 133.86, 135.56, 148.56, 148.91, 168.30, 176.14. Anal. (C12H15N3O2) C, H, N. N-[2-(2-Pyridyl)ethyl]-2-oxo-1-pyrrolidineacetamide (21). The crude product was chromatographed on silica (5% MeOH/CHCl3) to produce 21 in 62% yield: 1H NMR (CDCl3) δ 1.95-2.10 (m, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.35-2.44 (t, 2H, J = 7.9 Hz, CH2CO-pyrrolidone), 2.88-3.01 (t, 2H, J = 5.8 Hz, NHCH2CH2), 3.34-3.46 (t, 2H, J = 7.9 Hz, CH2NCO), 3.59-3.67 (q, 2H, J = 5.8 Hz, NHCH2CH2), 3.88 (s, 2H, NCH2CO), 7.05-7.17 (m, 3H, ArH & NH), 7.54-7.69 (m, 1H, ArH), 8.43-8.5 (m, 1H, ArH). 13C:
15
17.94, 30.33, 36.54, 38.55, 46.94, 48.16, 121.69, 123.51, 136.73, 148.99, 159..52, 167.78, 175.69. Anal. (C13H17N3O2) C, H, N. N-(4-Piperidinomethyl)2-oxo-1-pyrrolidineacetamide (22). The obtained oily product was purified using silica gel column (5% MeOH/CHCl3) to afford 22 in 52% yield: 1H NMR (CDCl3) δ 0.9-1.10 (m, 2H, CH2), 1.59-1.61 (m, 2H, CH2), 1.92-1.94 (m, 2H, CH2), 2.25-2.43 (m, 3H), 2.86-2.96 (m, 3H), 3.35, 3.36 (m, 3H), 3.65-3.98 (m, 2H, CH2), 3.77 (s, 2H, CH2), 4.34 (brs, 1H, NH), 7.26 (brs, 1H, NH). 13C: 18.21, 30.40, 36.32, 42.21, 44.56, 44.73, 45.03, 46.72, 48.43, 48.80, 168.72, 176.41. Anal. (C12H21N3O2) C, H, N. N-[2-(1-Piperidino)ethyl]-2-oxo-1-pyrrolidineacetamide (23). The crude oily product was chromatographed on silica gel column (5% MeOH/CHCl3) to give 23 in 63% yield: 1H NMR (CDCl3) δ 1.37-1.60 (m, 6H, CH2), 2.03-2.13 (m, 2H, J = 7.9 Hz, CH2pyrrolidone), 2.33-2.47 (m, 8H, CH2), 3.27-3.36 (m, 2H, J = 5.8 Hz, NHCH2CH2), 3.443.53 (t, 2H, J = 7.9 Hz, CH2NCO), 3.91 (s, 2H, NCH2CO), 6.69 (brs, 1H, NH). 13C: 18.09, 24.19, 25.89, 30.41, 35.79, 46.83, 48.27, 54.23, 56.91, 167.78, 175.73. Anal. (C13H23N3O2) C, H, N. N-[2-(1-Morpholino)ethyl]-2-oxo-1-pyrrolidineacetamide (24). The obtained product was chromatographed on silica gel column (5% MeOH/CHCl3) to produce 24 in 76% yield: 1H NMR (CDCl3) δ 2.01-2.12 (m, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.382.50 (m, 8H, CH2), 3.29-3.35 (q, 2H, J = 5.8 Hz, NHCH2CH2), 3.45-3.52 (t, 2H, J = 7.9 Hz, CH2NCO), 3.63-3.70 (m, 4H, N(CH2)2-morpholine), 3.89 (s, 2H, NCH2CO), 6.54 (brs, 1H, NH).
13
C: 18.07, 30.40, 35.55, 46.98, 48.37, 53.27, 56.83, 66.91, 167.91,
175.79. Anal. (C12H21N3O3) C, H, N.
16
N-[2-(1-Piperazino)ethyl]-2-oxo-1-pyrrolidineacetamide (25). The obtained crude gum was purified by column chromatography (5% MeOH/CHCl3) to give 25 in 41% yield: 1 H NMR (CDCl3) δ 2.01-2.12 (m, 2H, CH2-pyrrolidone), 2.35-2.49 (m, 8H, CH2), 2.81-2.90 (m, 4H, CH2-piperazine), 3.25-3.35 (q, 2H, J = 5.8 Hz, NHCH2CH2), 3.44-3.53 (t, 2H, J = 7.9 Hz, CH2NCO), 3.90 (s, 2H, NCH2O), 6.58 (brs, 1H, NH). 13C: 18.06, 30.41, 35.63, 45.92, 46.91, 48.34, 53.88, 56.86, 167.83, 175.77. Anal. (C12H22N4O2) C, H, N. N-[2-(1-Pyrrolidino)ethyl]-2-oxo-1-pyrrolidineacetamide (26). The crude oily product was chromatographed on silica (10% MeOH/CHCl3) to produce 26 in 74% yield: 1
H NMR (CDCl3) δ 1.71-1.80 (m, 4H, CH2-pyrrolidine), 1.98-2.10 (m, 2H, J = 7.9 Hz,
CH2-pyrrolidone), 2.36-2.43 (t, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.45-2.54 (m, 4H, CH2-pyrrolidone), 2.54-2.61 (t, 2H, J = 5.8 Hz, NHCH2CH2), 3.29-3.37 (q, 2H, J = 5.8 Hz, NH-CH2CH2), 3.44-3.52 (t, 2H, J = 7.9 Hz, CH2NCO), 3.89 (s, 2H, NCH2CO), 6.65 (brs, 1H, NH). 13C: 18.01, 23.45, 30.41, 37.89, 46.77, 48.31, 53.81, 54.47, 167.9, 175.8. Anal. (C12H21N3O2) C, H, N. N-(1-Ethylpyrrolidin-2-yl-methyl)-2-oxo-1-pyrrolidineacetamide (27). The obtained gum was chromatographed on silica (10% MeOH/CHCl3) to give 27 in 60% yield: 1H NMR (CDCl3) δ 1.09-1.16 (t, 3H, J = 7.2 Hz, CH3CH2), 1.49-1.61 (m, 1H, pyrrolidine), 1.68-1.79 (m, 2H, pyrrolidine), 1.83-1.94 (m, 1H, pyrrolidine), 2.03-2.15 (m, 2H, J = 7.9 Hz, CH2-pyrrolidone), 2.18-2.34 (m, 2H, NHCH2-pyrrolidine), 2.40-2.48 (t, 2H, J = 7.9 Hz, CH2CO), 2.62-2.90 (m, 4H, pyrrolidine & NH), 3.13-3.25 (m, 2H, J = 7.2 Hz, CH3CH2-), 3.45-3.55 (t, 2H, J = 7.9 Hz, CH2NCO), 3.85-4.03 (q, 2H, J = 16.0 Hz,
17
NCH2CO). 13C: 13.44, 17.94, 22.84, 28.01, 30.32, 40.25, 46.80, 48.32, 48.52, 53.41, 62.89, 168.28, 175.79. Anal. (C13H23N3O2) C, H, N. N-[2-(1-Methylpyrrolidin-2-yl)ethyl]-2-oxo-1-pyrrolidineacetamide (28). The crude product was chromatographed on silica (10% MeOH/CHCl3) to afford 28 in 69% yield: 1H NMR (CDCl3) δ 1.48-1.56 (m, 2H, CH2 ), 1.62-1.69 (m, 2H, CH2), 1.81-1.86 (m, 1H), 1.98-2.17 (m, 5H), 2.23 (s, 3H, CH3), 2.34-2.37 (m, 2H), 2.94-2.98 (m, 2H), 3.16-3.17 (m, 1H), 3.33-3.44 (m, 3H), 3.78-3.88 (m, 2H).
13
C: 18.38, 22.83, 29.61,
30.91, 31.16, 37.31, 41.14, 47.44, 48.84, 57.60, 65.19, 168.43, 176.25. Anal. (C13H23N3O2) C, H, N.
Pharmacology. Male and female mice (NMRI: Harlan-Winkelman, Borchen, Germany) weighing (23-29 g), bred and maintained under controlled conditions of temperature (25°C) and relative humidity (~ 50%) at the Animal facility of College of Pharmacy, King Saud University, Riyadh, S.A. Animals were allowed food and water ad libitum. A preliminary experiment was designed to evaluate the potential anticonvulsant and neurotoxic effects of all compounds. Anticonvulsant activity was measured by the sc pentylenetetrazol (PTZ) seizure threshold test, and minimal neurotoxicity was evaluated by the chimney test. For all compounds, a dose of 1.5 mmol/kg dissolved in 0.5 % carboxymethyl cellulose (CMC) was used and given ip as 10 ml/kg. Compounds which produced 100% protection against PTZ-induced seizure threshold were chosen for further investigations. Lower doses of those compounds were tried against PTZ-induced seizure threshold and minimal neurotoxicity test to calculate ED50 and TD50, respectively. The lowest dose that produced 100% protection against PTZ-induced convulsions was tested
18
against maximal electroshock seizure (MES), sc picrotoxin (Pic)-induced clonic convulsions and sc bicuculline (Bic)-induced clonic convulsions. sc PTZ seizure threshold test: Compounds were injected ip in groups of 5-8 animals. After 30 min, PTZ at 70 mg/kg (0.7 % solution in saline) was applied sc in a loose fold of skin on the back of the neck. The animals were observed for 30 min. The number of animals protected (those showing an absence of a single 5-sec. episode of clonic spasms; threshold seizure)19 was recorded and compared with the control (dosed only with PTZ, which produced at least one convulsive episode in each animal). The total number of convulsive episodes was also counted for each group to detect compounds with proconvulsive potentials. The dose of PTZ used was found to be the minimal dose that induced 100% convulsion in NMRI mice in our laboratory. Minimal neurotoxicity (chimney test)20: At 15 and 25 min. after ip administration of each compound, the inability of mice to climb up backwards in a glass tube of 25 cm length and 3 cm inner diameter within 30 sec. was recorded and taken as a measure of neurological deficits. Normal mice climb up in 5-10 sec. Maximal Electroshock Seizure (MES) test17: Animals were given the compound ip, and 30 min. later were subjected to a 60-HZ alternating current, 50 mA for 0.2 sec. delivered through ear electrodes (moistened with saline) by means of ECT UNIT (UGO Basile, Varese, Italy). The animals were restrained by hand and released immediately following electrical stimulation to permit observation of the seizure throughout its entire course. Abolition of the hind-limb tonic extensor component is taken as the endpoint for this test. Absence of this component in each group was recorded.
19
sc Pic-induced convulsions: Animals were given the compound ip, and 30 min. later were injected sc with 3.15 mg/kg picrotoxin (0.032 % solution in normal saline). The mice were placed in isolation cages and observed for the presence or absence of a clonic seizure for 45 min.17 The dose of picrotoxin used was found to be the minimal dose that induced 100% convulsion in NMRI mice in our laboratory. sc Bic-induced convulsions: Animals were given the compound ip, and 30 min later were injected sc with 1.0 mg/kg (0.03% solution in normal saline containing 1.0 ml 0.1N HCl, and diluted 1:3 in normal saline just before use). The mice were placed in isolation cages and observed for the presence or absence of a clonic seizure for a period of 30 min.17 The dose of bicuculline used was found to be the minimal dose that induced 100% convulsion in NMRI mice in our laboratory. Lipophilicity, Log (p) calculations: Estimation of the logarithm of the partition coefficient, Log (p), [n. octanol/water] was calculated using the fragmentation method of Ghose and Crippen.21 Statistical calculations: The values of ED50, TD50, and their 95% confidence limits were calculated according to Litchfield and Wilcoxon15. Acknowledgement. We are pleased to acknowledge the financial support of King Abdulaziz City for Science and Technology, Grant No. LG-1-14. The technical assistants of Mr. A.F. Ramadan, Mr. T.M. Al-Hadeiah and Mr. Tanvir A. Butt are greatly appreciated.
20
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Shoravon, S.D. Epidemiology, Classification, Natural History and Genetics of Epilepsy. Lancet 1990, 336, 93-96.
(2)
Bordie, M.J. Established Anticonvulsants and Treatment of Refractory Epilepsy. Lancet 1990, 336, 350-354.
(3)
Meldrum, B.S, Porter, R.J., eDS. In New Anticonvulsant Drugs. Current Problems in Epilepsy 4; John Libby: London, 1986.
(4)
Flaherty, P.T., Greenwood, T.D., Manheim, A.L., Wolfe, J.F. Synthesis and Evaluation of N-(Phenylacetyl)-trifluoromethansulfonamides as Anticonvulsant Agents. J. Med. Chem. 1996, 39, 1509-1513.
(5)
Jacobsen, E.J., Tenbrink, R.E., Stelzer, L.S., Belonga, K.L. High-Affinity Partial Agonist Imidazo[1,5-a]quinoxaline Amides, Carbamates and Ureas at the 8Aminobutyric Acid/Benzodiazepine Receptor Complex. J. Med. Chem., 1996, 39, 158-175.
(6)
Reddy, P.A., Woodward, K.E., McIlheran, S.M., Hsiang, C. H., Latifi, T.N., Covey, D.F. Synthesis and Anticonvulsat Activities of 3,3-Dialkyl- and 3-Alkyl-3benzyl-2-piperidinones (δ-Valerolactams) and Hexahydro-2H-azepin-2-ones (εCaprolactams). J. Med. Chem. 1997, 40, 44-49.
(7)
Monn, J.A., Valli, M.J., Massey, S.M., Wright, R.A., Salhoff, C.R., Johnson, B.G., Rhodes, G.A., Tizzano, J.P., Schoepp, D.D. Design, Synthesis and Pharmacological Characterization of (+)-2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylic Acid: A Potent, Selective and Orally Active Group 2 Metabotropic
21
Glutamate Receptor Agonist Possessing Anticonvulsant and Anxiolytic Properties. J. Med. Chem. 1997, 40, 528-537. (8)
Larsen, P.K., Frouland, B., Jorgensen, F.S., Schousboe, A. GABAA Receptor Agonists, Partial Agonists, and Antagonists. Design and Therapeutic Prospects. J. Med. Chem. 1994, 37, 2489-2505.
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Jacob, J.N., Schashoua, V.E., Campbell, A.A, Baldessorini, R.J. 8-Aminobutyric Acid Esters, Synthesis, Brain Uptake and Pharmacological Properties of its Lipid Esters. J. Med. Chem., 1985, 28, 106-110.
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Jacob, J.N., Hesse, G.W., Shashoua, V.E. 8-Aminobutyric Acid Esters, Synthesis, Brain Uptake and Pharmacological Properties of C–18 Lipid Esters with Varying Degree of Unsaturation. J. Med. Chem. 1987, 30, 1573-1578.
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Kaplan, J.P., Raizon, B.M., Desarmenien, M., Feltz, P. Headly, P.M., Worms, P., Lloyd, K., Bartholini, G. New anticonvulsants: Schiff bases of gammaaminobutyric acid and gamma-aminobutyramide. J. Med. Chem. 1980, 23, 702-704.
(12)
Sasaki, H., Mori, J., Nakamura, Synthesis and Anticonvulsant Activity of 1-Acyl2-Pyrrolidinone Derivatives. J. Med. Chem. 1991, 34, 628-630.
(13)
El-Subbagh, H.I., Nasr, M.A., Abdelal, A.M., Gineinah, M.M., El-Kashef, H.A. Synthesis and Anticonvulsant Activity of Some 1-Substituted-2-oxopyrrolidine Derivatives. Med. Chem. Res. 1994, 4, 335-345.
(14)
Bardel, P., Bolanos, A., Kohn, H. Synthesis and Anticonvulsant Activity of αAcetamido-N-benzylacetamide Derivatives Containing an Electron-deficient αheteroaromatic substituent. J. Med. Chem. 1994, 37, 4567.
22
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Litchfield, J.T., Jr., Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 1949, 96, 99-113.
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Löscher, W., Nau, H. Pharmacological evaluation of various metabolites and analogues of valproic acid. Neuropharmacology, 1985, 24, 427-435.
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White, H.S., Woodhead, J.H., Franklin, M.R., Swinyard, E.A., Wolf, H.H. In Antiepileptic Drugs, 4th ed.; Levy, R.H., Mattson, R.H. Meldrum, B.S., Eds, Raven: New York, 1995, pp 99-121.
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Elmazar, M.M.A., Hauck, R.-S., Nau, H. Anticonvulsant and neurotoxic activities of twelve analogues of valproic acid. J. Pharm. Sci., 1993, 82, 1255-1258.
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Swinyard, E.A., Woodhead, J.H. In Antiepileptic Drugs, 2nd ed.; Woodbury, D.M., Penny, J.K., Pippenger, C.E., Eds, Raven Press: New York, 1982, pp 111126.
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Boissier, J.-R., Tardy, J. Diverres, J.-C. Une nouvelle mèthode simple pour explorerl’action ‘tranquillisante”: le test de la cheminèe. Med. Exp. Basel, 1960, 3, 81-84.
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Ghose, A.K., Crippen, G.M. Atomic physicochemical parameters for three dimensional structure directed quantitative structure activity relationship. 2. Modeling dispersive and hydrophobic interactions. J. Chem. Inf. Comput. Sci., 1987, 27, 21-35.
23
Figure Captions
Figure 1: Structure and anticonvulsant activity of the lead compounds 1-6. Compounds were given ip (n = 6), 15 min. prior to sc injection of PTZ (65 mg/kg).
Figure 2: Correlation of lipophilicity, Log (p), with anticonvulsant (PTZ) and neurotoxicity (Chim) at a dose of 1.5 mmol/kg of 4 subgroups (A-D) of 1-substituted-2-oxopyrrolidine derivatives (numbered in bold italic).
24
Table 1. In vivo screening of compounds 9-28 for their minimal nurological deficits (chimney test) and their influence on pentylenetetrazol (PTZ)-induced clonic convulsions. O
H N
N
(CH2)n-R
O n Compda
R
Log (p)
% Protectionb (PTZ)
TCc
Chimneyd test (%)
Notes
9
n. propyl
0
! 0.67
00
11/5
100
10
n. butyl
0
!0.25
20
5/5
40
–
11
cylcohexyl
0
0.06
40
3/5
100
–
12
cyclohexyl
1
0.49
100
0/5
100e
Anticonvulsant
13
4-chlorophenyl
1
0.8
20
0/5
40e
–
14
4-flourophenyl
1
0.4
100
0/5
60e
Anticonvulsant
15
4-methoxyphenyl
1
0.12
100
0/5
100e
Anticonvulsant
16
phenyl
2
0.52
83
1/6
67e
Anticonvulsant
17
4-flourophenyl
2
0.68
100
1/6
100e
Anticonvulsant
18
3,4-dimethoxyphenyl
2
0.27
00
6/5
100
–
19
2-pyridyl
1
!0.67
40
6/5
20
–
20
3-pyridyl
1
!1.09
00
12/5
100
21
2-pyridyl
2
!0.56
40
7/5
40
–
22
4-piperidyl
1
!1.42
40
3/5
80
–
23
1-piperidyl
2
!0.76
00
3/3
100
40% Mortality
24
1-morpholine
2
!1.89
43
4/7
86
–
25
1-piperazine
2
!2.11
40
3/5
80
–
26
1-pyrrolidine
2
!1.17
–
–
–
100% Mortality
27
1-ethylpyrrolidin-2-yl
1
!0.86
13
9/8
25
–
28
1-methylpyrrolidin-2-yl
2
!1.09
00
5/5
60
–
a
Proconvulsant
Proconvulsant
1.5 mmol/kg of each compound, ip, 30 min. before PTZ in groups of 5-8 NMRI male or female mice. b70 mg/kg of PTZ in saline, sc, % protection is recorded. cTotal convulsive episodes / no. of animals in the group. d Chimney test was caried out 15 and 25 min. after administration of each compound, % failure was recorded at 25 min. eCompounds were chosen for further investigations (See Table 2).
25
Table 2: Detailed investigation of the anticonvulsant potential of the promising compounds in NMRI mice Compd 12
dose (mmol/kg)
PTZa
Chimneya
MESb
Pic.c
Bic.d
1.5 1.0 0.5
100% 100% 20% ED 50=0.65f (0.42-0.99)
80% 60% 20% TD50=0.86f (0.54-1.37)
0% -
0%
-
1.5 1.0 0.5
100% 100% 60% ED 50=0.43f (0.24-0.76)
60% 40% 20% TD50=1.21f (0.59-2.50)
100% 0%
67%
40% -
1.5 1.0 0.5
100% 20% 0% ED 50=1.14f (0.85-1.52)
100% 20% 0% TD50=1.14f
80% -
0% -
80% -
(0.85-1.52)
PI= 1.32e (95% C.L)
14 PI= 2.81e (95% C.L)
15 PI= 1.00e (95% C.L)
16
1.5
83%
67%
100%
80%
100%
17
1.5 1.0 0.5
100% 80% 25% ED 50=0.70f (0.42-1.15)
100% 40% 0% TD50=0.94f (0.54-1.63)
50%g -
-g -
-
PI= 1.34e (95% C.L.)
a PTZ and Chimney : see footnote of Table 1. b MES : Maximal electroshock test ( see experimental section), and % of animals protected is recorded cPic.: Picrotoxin, 3.15 mg/kg sc, given 30 min after the test substance, and the % of animals protected is recorded. dBic.: Bicuculline, 1 mg/kg, sc, given 30 min after the test substance, and the % of animals protected is recorded. ePI : Protective Index = TD50 / ED50. fED50 and TD50: Median effective and sedative doses, respectively, and their 95% confidence limit (C.L) are calculated 15 according to Litchfield and Wilcoxon. g33% of the animals died before subjected to MES; and 20% died
before giving picrotoxin, the remaining animals showed rigidity and tremors for 40 min., therefore were not injected with picrotoxin or bicuculline.
26
O
H N
N
R
mmol/kg
% Protection (PTZ)
0.50 0.43 0.43 0.30 0.35 0.37
00.0 83.0 83.0 83.0 50.0 83.0
O 1, R = H 2, R = benzyl 3, R = 4-methylphenyl 4, R = 4-methoxyphenyl 5, R = 4-nitrobenzylideneamino 6, R = N-(n. butyl)thioureido
27
A 100
B 100
80
80
12
14
Effect (%)
Efeect (%)
13
60
11
40
9 10
20
Chim
-0.5
0.0
0 0.0
0.5
15
40
PTZ
20 0 -1.0
60
PTZ Chim 0.2
0.4
Log (p)
D 100
80
80 17
60
Effect (%)
Effect (%)
C 100
18 16
40 20
60
20
PTZ
0.4
0.6 Log (p)
28
0.8
0.8
25
24
23
40
Chim 0 0.2
0.6
Log (p)
0 -2.5
PTZ Chim -2.0
-1.5 Log (p)
-1.0
-0.5
Scheme 1a O
O N H
H N
N O
7
Alkyl or cycloalkyl
9-12 iii
i, ii
O
O N
iv
COOEt
H N
N O
8
(CH2)n-Phenyl or substituted phenyl
13-18 v vi
O
H N
N O
O
H N
N
(CH2)n-five membered Heterocycles
O
26-28
(CH2)n-six membered Heterocycles
19-25
a
(i) t-BuOK, toluene, 60°C; (ii) BrCH2COOEt, toluene, r.t.; (iii) alkyl or cycloalkylamines, neat, 100°C; (iv) Substituted benzyl or phenethylamines, neat, 100°C; (v) Six membered heterocyclic methyl or ethylamines, neat, 100; (vi) Five membered heterocyclic methyl or ethylamines, neat, 100°C.
29
Elemental Analyses of compounds 9-28. Compounds 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd. Calc. Fd.
C
H
N
58.67 58.82 60.58 60.62 64.26 64.29 65.52 65.50 58.54 58.70 62.39 62.48 64.11 64.32 68.27 68.29 63.62 63.69 62.73 62.75 61.79 61.83 61.79 61.87 63.14 63.20 60.26 60.40 61.63 61.74 56.45 56.56 56.67 56.83 60.23 60.19 61.63 61.70 61.63 61.65
8.75 8.82 9.15 9.30 8.99 9.01 9.30 9.39 5.67 5.82 6.04 6.23 6.92 7.05 7.37 7.40 6.48 6.56 7.24 7.30 6.48 6.52 6.48 6.56 6.93 7.03 8.84 8.90 9.15 9.32 8.29 8.42 8.72 8.80 8.85 8.92 9.15 9.22 9.15 9.20
15.21 15.43 14.13 14.26 12.49 12.52 11.75 11.85 10.50 10.67 11.19 11.26 10.68 10.75 11.37 11.42 10.60 10.73 9.14 9.20 18.01 18.15 18.01 18.25 17.00 16.98 17.56 17.69 16.59 16.60 16.46 16.50 22.03 22.00 17.56 17.59 16.59 16.65 16.59 16.68
30
