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(80 : 20) (B), (90 : 10) (C), (95 : 5) (D), (98 : 2) (E), n-butanol/AcOH/H2O (4 :
1 : 5, organic phase) (BAW) (F), n-butanol/pyridine/H2O (65 : 35 : 65, or-
ganic phase) (BPW) (G), phenol saturated with H2O (H).
Preparation of Boc-Amino Acids and Removal of Boc Group The
Boc group was introduced by the method of Schwyzer et al.7) using Boc-
azide and was removed with HCl/dioxane in the typical manner.
keep the temperature below Ϫ15 °C. BZA (10 mmol) in THF (4 ml) was
added in small portions and the reaction mixture was stirred at Ϫ15 °C for
30 min and at room temperature for 1 h. The solution was concentrated in
vacuo and the residue was dissolved in EtOAc (20 ml). This solution was
washed with 20 ml portions of 1 M HCl, saturated NaHCO3 solution and sat-
urated NaCl solution, then dried with anhydrous MgSO4, filtered and con-
centrated in vacuo. The residue was crystallized with EtOAc/hexane.
Pharmacology. Preliminary Evaluation of Anticonvulsant Activity
and Neurotoxicity in Phase I Identification (ASP) The synthesized com-
pounds 2, 4, 5, 7, 9—11, 14—16 were tested in mice after ip administration.
Tests of MES, sc PTZ and Tox were performed according to the method
given by Krall et al.11) as described in the previous paper.1) The results quali-
fying the compounds into class I, II or III of ASP are given in Table 1.
Quantitative Assessment of Anticonvulsant Activity in Phase VIb
Quantification (ASP) Three compounds (7, 11, 15) were examined in rats
after oral administration using the method of Krall et al.11) described in the
previous paper.1) The results are given in Table 3.
Determination of Anticonvulsant Activity of Compound 15 This
compound was administered ip in adult male Swiss mice. Pilocarpine and
strychnine were injected ip. Bicuculline and PTZ were administred sc.
Kainic acid, NMDA and AMPA were applied intracerebroventricularly (icv).
All convulsants were administered at the dose inducing seizures in 97% of
tested animals. A detailed description of the methods was published previ-
ously.12—14) The results are given in Table 4.
Acetylation (2S,4R)-Ac-Pro(4-OH)-BZA (4) and (RS)-Ac-Pip-BZA
(14) were obtained by acetylation of HClϫ(2S,4R)-Pro(4-OH)-BZA or HCl
ϫ(RS)-Pip-BZA with acetic anhydride in methylene chloride at room tem-
perature for 4 h. Then the solution was concentrated in vacuo, the residue
was crystallized with EtOAc/hexane and dried in vacuo. (RS)-Ac-ϽGlu-OH
(6) and (RS)-Ac-ϽGlu-OBzl (9) were obtained by the acetylation of (RS)-
ϽGlu-OH and (RS)-ϽGlu-OBzl with boiling acetic anhydride for 15 min.
Then the solution was concentrated in vacuo and several times concentrated
with MeOH until the acetic acid smell disappeared and then it was dried in
vacuo over solid NaOH.
Cbz-Tau-OH This compound was obtained by the modified Bergmann
et al. method.8) Tau-OH was dissolved in 4 M NaOH. The solution was
cooled to 0°C and stirred with an excess of Cbz-Cl chloride for 2 h and at
room temperature overnight. The mixture was then extracted 3 times with
ethyl ether. The alkaline solution was acidified to pH 6.0 and concentrated.
The residue was extracted with dimethylformamide (DMF), the solution was
filtered and concentrated in vacuo to a small volume. A four times volume
of ethyl ether was added into this solution and the mixture was left for 12 h
in refrigerator. The obtained crystals were filtered, dissolved in dry DMF,
treated with ethyl ether and again left to crystallize. Crystals of Cbz-Tau-
ONa were dried in vacuo, dissolved in H2O and filtered on a column with
Amberlite IRC 50 in the Hϩ form. The filtrate was concentrated in vacuo,
then concentrated several times with MeOH and benzene and dried in vacuo.
The obtained raw product was used without further purification.
Cbz-Tau-Cl Raw Cbz-Tau-OH was dissolved in dry benzene and an ex-
cess of thionyl chloride was added. The mixture was stirred overnight at
room temperature and then boiled for 1 h. This solution was concentrated in
vacuo and the oily residue was dissolved in dry benzene. The solution was
filtered, concentrated in vacuo, treated with hexane and left for several hours
in a refrigerator. The obtained crystalline raw material was dried in vacuo.
Cbz-Tau-BZA Raw Cbz-Tau-Cl (0.97 g, 3.4 mmol) was dissolved in
ethyl ether (20 ml) and benzylamine (0.72 g, 6.8 mmol) in ethyl ether was
added. This solution was stirred at room temperature for 6 h, then filtered
and concentrated in vacuo. The oily residue was dissolved in ethyl ether
(20 ml) and washed with 20 ml portions of 2 M HCl, saturated NaHCO3 solu-
tion and saturated NaCl solution. The ethereal solution was dried with anhy-
drous MgSO4 and concentrated in vacuo. The obtained product was purified
by CC. Physicochemical data are given in Tables 1 and 2 .
Acknowledgements This investigation was supported in part by the
State Committee for Scientific Research (Grant 4 PO5F 014 13).
References
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N-Methylation (R)-Pro-OH and HClϫ(2S,4R)-Pro-(4-OH)-BZA were
N-methylated using formaldehyde solution and catalytical hydrogenation ac-
cording to the method of Moore.9)
(RS)-ϽGlu-OBzl Benzyl ester group was introduced by the procedure
of Wang et al.10) Physicochemical data are given in Tables 1 and 2.
Preparation of Benzylamides A BZA group was introduced by the fol-
lowing procedure: An amino acid or its derivative with a free carboxyl func-
tion (10 mmol) was dissolved in dry tetrahydrofuran (THF) or a mixture of
THF/DMF (40 ml). Then N-methylmorpholine (NMM) (10 mmol) was
added and the mixture was stirred under nitrogen and chilled to Ϫ15 °C.
Isobutyl chloroformate (10 mmol) in THF (4 ml) was added dropwise to
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