April 2007
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bonylphthalimide 19 (4.40 g, 20.0 mmol) at 0 °C. The reaction mixture was
stirred at 0 °C for 10 min and at room temperature for 50 min, then insoluble
material was removed by filtration and 2.0 mol/l aqueous HCl was added to
the filtrate until the pH reached about 2. Then the mixture was stirred at 0 °C
for 30 min, the solution was removed and the remaining sticky solid was pu-
atives, are produced by hydrolysis at the glutarimide moiety,
the isoglutamine derivatives (4, 7) showed slightly higher ac-
tivity than the glutamine derivatives (3, 6). Interestingly, al-
though glutarimide ring opening (3, 4) decreased the activity,
further hydrolysis at distal primary amide (8) resulted in re- rified by flash column chromatography (AcOEt : AcOHꢂ30 : 1 v/v) to afford
3 (1.85 g, 6.70 mmol, 49%) as a white solid. mp 159—160 °C. 1H-NMR
covery of the activity (48% inhibition). Similarly, the activi-
ties of the phthalimide and glutarimide ring-opened metabo-
lites (6, 7) were enhanced by further hydrolysis at the distal
primary amide (10) (62%). Final hydrolysate, i.e., a-amino
acid derivatives (11—13) and phthalic acid (9) showed no or
slight activity (0—9%).
(DMSO-d6): d 13.14 (s, 1H), 7.86—7.92 (m, 4H), 7.16 (s, 1H), 6.88 (s, 1H),
4.74 (dd, Jꢂ10.7, 4.3 Hz, 1H), 2.35 (m, 1H), 2.25 (m, 1H), 2.08 (m, 2H).
MS (FAB): m/z 277 (MꢁH)ꢁ. Anal. Calcd for C13H12N2O5: C, 56.52%, H,
4.38%, N, 10.14%. Found: C, 56.37%, H, 4.39%, N, 10.07%.
Phthaloylisoglutamine (4) This compound was obtained as a white
solid (18% yield) in a manner similar to that described for the preparation of
1
3, but from isoglutamine as a starting material. mp 119—121 °C. H-NMR
The results indicate that spontaneous hydrolysis of thalido-
mide (1) may have various effects (loss, remaining, decrease
and increase) on TNF-a production-inhibitory activity. It ap-
pears that the metabolism of thalidomide (1) might not have
a dramatic effect on its TNF-a production-inhibitory activity,
because some metabolites retain the effects and increase of
the effect found in some metabolites is not drastic. Investiga-
tion of the effects of metabolism on other biological activi-
ties is under way.
(DMSO-d6): d 12.04 (s, 1H), 7.83—7.88 (m, 4H), 7.57 (s, 1H), 7.16 (s, 1H),
4.59 (dd, Jꢂ10.3, 4.3 Hz, 1H), 2.37 (m, 1H), 2.20 (m, 3H). MS (FAB): m/z
277 (MꢁH)ꢁ. Anal. Calcd for C13H12N2O5: C, 56.52%, H, 4.38%, N,
10.14%. Found: C, 56.24%, H, 4.40%, N, 10.01%.
N-tert-Butoxycarbonylglutamine Benzyl Ester (20) To a solution of
Boc-Gln-OH 14 (2.00 g, 8.12 mmol) in dry THF (50 ml) was added benzyl
bromide (1.53 g, 8.94 mmol) and TEA (911 mg, 9.00 mmol) at 0 °C. The re-
action mixture was stirred at 0 °C for 2 h and at room temperature for 18 h,
then CHCl3 was added, and whole was washed with 0.2 mol/l aqueous HCl,
sat. NaHCO3 and brine, dried over MgSO4, filtered, and concentrated. The
residue was purified by flash column chromatography (hexane : AcOEtꢂ1 : 2
to 0 : 1 v/v) to afford 20 (1.65 g, 4.91 mmol, 61%) as a white solid. 1H-NMR
(CDCl3): d 7.33—7.37 (m, 5H), 6.01 (s, 1H), 5.31 (m, 2H), 5.18 (m, 2H),
Experimental
tert-Butyl 2,6-Dioxopiperidin-3-ylcarbamate (15) To a solution of
Boc-Gln-OH 14 (10.0 g, 40.6 mmol) in dry THF (100 ml) was added 1,1ꢀ- 4.36 (m, 1H), 2.19—2.34 (m, 3H), 1.94 (m, 1H), 1.44 (s, 9H). MS (FAB):
carbonyldiimidazole (CDI) (7.78 g, 48.0 mmol), 4-dimethylaminopyridine m/z 337 (MꢁH)ꢁ.
(DMAP) (1.22 g, 10.0 mmol) and triethylamine (TEA) (4.86 g, 48.0 mmol).
Glutamine Benzyl Ester Trifluoroacetate (21) A mixture of 20
The mixture was stirred at room temperature for 3 d, then H2O was added. (400 mg, 1.19 mmol), CH2Cl2 (6 ml) and TFA (6 ml) was stirred at room
The mixture was extracted with CHCl3 3 times, and the organic solution was temperature for 90 min. The reaction mixture was concentrated to afford 21
washed with 0.2 mol/l aqueous HCl and brine, dried over MgSO4, filtered (550 mg, quant.) as a pale yellow oil. This intermediate was used for the next
and concentrated. The residue was purified by recrystallization (CHCl3/iso- reaction without purification. MS (FAB): m/z 237 (MꢁH)ꢁ.
propyl ether) to afford 15 (8.30 g, 36.4 mmol, 90%) as white crystals. 1H-
NMR (CDCl3): d 8.22 (s, 1H), 5.36 (s, 1H), 4.31 (m, 1H), 2.79 (m, 1H),
N-(2-Benzyloxycarbonylbenzoyl)glutamine Benzyl Ester (22) This
compound was obtained as a white amorphous solid (19% yield) in a man-
2.68 (m, 1H), 2.52 (m, 1H), 1.86 (m, 1H), 1.46 (s, 9H). MS (FAB): m/z 229 ner similar to that described for the preparation of 18, but from 17 and 21 as
(MꢁH)ꢁ.
starting materials. The mobile phase solvent for flash column chromatogra-
3-Aminopiperidine-2,6-dione Hydrobromide (16) A mixture of 15 phy was AcOEt : CHCl3 : MeOHꢂ1 : 1 : 0 to 25 : 25 : 1 v/v/v. 1H-NMR
(200 mg, 0.877 mmol) and 30% HBr/AcOH (2 ml) was stirred at room tem-
perature for 30 min, then AcOEt was added until a precipitate formed. The
(CDCl3): d 7.95 (d, Jꢂ7.3 Hz, 1H), 7.56 (t, Jꢂ7.3 Hz, 1H), 7.48 (m, 2H),
7.33—7.41 (m, 10H), 6.61 (d, Jꢂ8.1 Hz, 1H), 6.37 (s, 1H), 5.30 (s, 2H),
precipitate was collected by filtration and washed with AcOEt twice to af- 5.26 (s, 1H), 5.20 (m, 2H), 4.81 (m, 1H), 2.47 (m, 1H), 2.39 (m, 1H), 2.27
1
ford 16 (180 mg, 0.861 mmol, 98%) as a white solid. H-NMR (DMSO-d6):
d 11.27 (s, 1H), 8.38 (br, 3H), 4.21 (dd, Jꢂ13.2 Hz, 5.1 Hz, 1H), 2.71 (m,
1H), 2.58 (m, 1H), 2.13 (m, 1H), 1.99 (m, 1H). MS (FAB): m/z 129
(MꢁH)ꢁ.
(m, 1H), 1.98 (m, 1H). MS (FAB): m/z 475 (MꢁH)ꢁ.
N-(2-Benzyloxycarbonylbenzoyl)isoglutamine Benzyl Ester (23) This
compound was obtained as a white solid (46% yield) in a manner similar to
that described for the preparation of 18, but from 17 and H-Glu(OBn)-
NH2·HCl as starting materials. The mobile phase solvent for flash column
chromatography was hexane : CHCl3 : MeOHꢂ1 : 1 : 0 to 20 : 20 : 1 v/v/v. 1H-
NMR (CDCl3): 7.99 (dd, Jꢂ7.7, 1.3 Hz, 1H), 7.54 (dt, Jꢂ7.7, 1.3 Hz, 1H),
7.48 (dt, Jꢂ7.7, 1.3 Hz, 1H), 7.30—7.42 (m, 11H), 7.19 (s, 1H), 6.81 (d,
Jꢂ8.0 Hz, 1H), 5.41 (s, 1H), 5.30 (m, 2H), 5.09 (m, 2H), 5.20 (m, 2H), 4.66
(ddd, Jꢂ8.0, 4.3, 4.3 Hz, 1H), 2.70 (m, 1H), 2.54 (m, 1H), 2.33 (m, 1H),
2.13 (m, 1H). MS (FAB): m/z 475 (MꢁH)ꢁ.
a-(2-Benzyloxycarbonylbenzamido)glutarimide (18) To a solution of
phthalic acid monobenzyl ester 17 (300 mg, 1.17 mmol) in dry THF (50 ml)
was added methyl chloroformate (144 mg, 1.50 mmol) and TEA (485 mg,
4.80 mmol) at 0 °C. The mixture was stirred at 0 °C for 15 min, and 16
(500 mg, 2.40 mmol) was added. The reaction mixture was stirred at 0 °C for
45 min and room temperature for 4 h, then AcOEt was added, and the mix-
ture was washed with 0.2 mol/l aqueous HCl, brine, dried over MgSO4, fil-
tered, and concentrated. The residue was purified by flash column chro-
N-(2-Benzyloxycarbonylbenzoyl)glutamic Acid Dibenzyl Ester (24)
matography (hexane : AcOEtꢂ2 : 3 to CHCl3 : acetoneꢂ1 : 1 v/v) to afford This compound was obtained as a white solid (5.4% yield) in a manner simi-
1
18 (160 mg, 0.437 mmol, 37%) as a white solid. H-NMR (CDCl3): d 7.99
(d, Jꢂ7.7 Hz, 1H), 7.82 (s, 1H), 7.58 (t, Jꢂ7.6 Hz, 1H), 7.52 (d, Jꢂ7.6 Hz,
1H), 7.50 (t, Jꢂ7.6 Hz, 1H), 7.33—7.42 (m, 5H), 6.54 (d, Jꢂ4.7 Hz, 1H),
lar to that described for the preparation of 18, but from 17 and H-Glu(OBn)-
OBn·Tos-OH as starting materials. The mobile phase solvent for flash col-
1
umn chromatography hexane : AcOEtꢂ1 : 1 v/v. H-NMR (CDCl3): 7.92 (d,
5.30 (d, Jꢂ5.3 Hz, 2H), 4.31 (ddd, Jꢂ12.8, 4.7, 4.7 Hz, 1H), 2.61—2.80 (m, Jꢂ8.1 Hz, 1H), 7.43—7.53 (m, 3H), 7.28—7.38 (m, 15H), 6.53 (d,
3H), 1.72 (m, 1H). MS (FAB): m/z 367 (MꢁH)ꢁ.
Jꢂ7.3 Hz, 1H), 5.25 (m, 2H), 5.17 (m, 2H), 5.08 (s, 2H), 4.77 (m, 1H),
2.39—2.54 (m, 2H), 2.30 (m, 1H), 2.09 (m, 1H). MS (FAB): m/z 566
(MꢁH)ꢁ.
N-(2-Carboxybenzoyl)glutamine (6) This compound was obtained as a
white amorphous solid (quantitative yield) in a manner similar to that de-
a-(2-Carboxybenzamido)glutarimide (2) To a solution of 18 (300 mg,
0.820 mmol) in dry 1,4-dioxane (15 ml) was added 10% Pd–C (60 mg). The
mixture was stirred at room temperature under an H2 atmosphere for 2 h,
then filtered through Celite, and the filtrate was concentrated. The residue
was purified by flash column chromatography (CHCl3 : MeOHꢂ20 : 1 to 6 : 1 scribed for the preparation of 2, but from 22 as a starting material. mp 98—
v/v) to afford 2 (180 mg, 0.652 mmol, 80%) as a white powder. mp 86— 100 °C. 1H-NMR (DMSO-d6): d 8.14 (d, Jꢂ7.7 Hz, 1H), 7.24 (d, Jꢂ7.7 Hz,
88 °C. 1H-NMR (DMSO-d6): d 12.93 (s, 1H), 10.81 (s, 1H), 8.61 (d, 1H), 7.08 (t, Jꢂ7.7 Hz, 1H), 7.01 (t, Jꢂ7.7 Hz, 1H), 6.95 (d, Jꢂ7.7 Hz, 1H),
Jꢂ8.4 Hz, 1H), 7.77 (d, Jꢂ7.8 Hz, 1H), 7.60 (t, Jꢂ7.8 Hz, 1H), 7.51 (t,
Jꢂ7.8 Hz, 1H), 7.46 (d, Jꢂ7.8 Hz, 1H), 4.69 (ddd, Jꢂ17.1, 8.4, 8.4 Hz, 1H), 1.53 (m, 1H), 1.36 (m, 1H). MS (FAB): m/z 295 (MꢁH)ꢁ.
2.75 (m, 1H), 2.53 (m, 1H), 2.00 (m, 2H). MS (FAB): m/z 276 (MꢁH)ꢁ.
N-(2-Carboxybenzoyl)isoglutamine (7) This compound was obtained
Anal. Calcd for C13H12N2O5: C, 56.52%, H, 4.38%, N, 10.14%. Found: C, as a white amorphous solid (98% yield) in a manner similar to that described
6.69 (s, 1H), 6.28 (s, 1H), 3.83 (ddd, Jꢂ7.7, 7.7, 4.7 Hz, 1H), 1.71 (m, 2H),
56.50%, H, 4.53%, N, 10.03%.
Phthaloylglutamine (3) To a solution of glutamine (2.00 g, 13.7 mmol)
in 0.55 mol/l aqueous Na2CO3 (30 ml, 16.4 mmol) was added N-ethoxycar- Jꢂ7.9 Hz, 1H), 7.58 (t, Jꢂ7.9 Hz, 1H), 7.51 (t, Jꢂ7.7 Hz, 1H), 7.42 (d,
for the preparation of 2, but from 23 as a starting material. mp 90—93 °C.
1H-NMR (DMSO-d6): d 12.85 (br s, 2H), 8.50 (d, Jꢂ7.9 Hz, 1H), 7.82 (d,