Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 621
5.59 (s, 1 H), 6.22 (s, 1 H), 7.29-7.36 (m, 10 H); MS (methane,
DCI) m/z 325 (M+ + 1).
mL/min, 20 × 10 mL fractions. HPLC analysis, Zorbax C8
column, isocratic water-methanol (4:1), showed the product
(tR ) 1.6 min) was contained in fractions 7-10. These fractions
were combined and lyophilized to afford 3 (0.44 g, 38%): 1H
NMR (300 MHz, D2O) δ 1.91 (ddd, J ) 17.4, 15.5, 7.7 Hz, 1
H), 2.13 (dd, J ) 18.0, 15.5, 6.1 Hz, 1 H), 2.71-2.92 (m, 2 H)
3.01-3.15 (m, 1 H); MS (methane, DCI-) m/z 211 (M+ - 1).
Anal. Calcd for C5H9O7P‚2H2O: C, 24.2; H. 5.28. Found: C,
24.2; H, 5.34.
Diben zyl 2-[[Bis(ben zyloxy)p h osp h or yl]m eth yl]p en t-
a n ed ioa te (2a ). To a solution of dibenzyl phosphite (0.53 g,
2.02 mmol) in CH2Cl2 (20 mL) at 0 °C was added trimethyl-
aluminum (1.01 mL, 2.0 M solution in hexanes, 2.02 mmol).
After 20 min a solution of 1a (0.66 g, 2.02 mmol) in CH2Cl2 (5
mL) was added. The cooling bath was removed, and stirring
at room-temperature continued for 16 h. The reaction was
quenched by the slow addition of 1 N hydrochloric acid (20
mL). After stirring for 10 min the phases were separated, and
the aqueous phase was extracted with CH2Cl2 (2 × 10 mL).
The organic phases were combined, dried (MgSO4), and
concentrated. Flash chromatography over silica gel (3 × 18
cm) with a hexanes-EtOAc (4:1 f 1:1) gradient gave 2a as a
Ben zyl 3-(Hyd r oxyp h osp h in yl)p r op ion a te (5). Hypo-
phosphorous acid (1.82 g, 13.8 mmol, 50% aqueous solution)
and triethylamine (0.97 g, 13.8 mmol) were mixed and dried
by azeotropic removal of toluene (3 × 20 mL) in vacuo at 50
°C. The residue was dissolved in dry CH2Cl2 (50 mL) and
cooled to 0 °C. Triethylamine (2.4 g, 24.2 mmol) and chloro-
trimethylsilane (2.56 g, 23.6 mmol) were added followed after
5 min by benzyl acrylate (0.32 g, 1.98 mmol) in CH2Cl2 (5 mL).
The mixture was stirred for 24 h at room temperature and
then filtered. The filtrate was washed with 1 N hydrochloric
acid (10 mL) and water (10 mL), dried (MgSO4), and concen-
1
clear oil (0.58 g, 48%): TLC Rf 0.50 (1:1 hexanes-EtOAc); H
NMR (300 MHz, CDCl3) δ 1.80-1.99 (m, 3 H), 2.25-2.35 (m,
3 H), 2.78-2.90 (m, 1 H), 4.88-5.01 (m, 6 H), 5.05 (s, 2 H),
7.23-7.35 (m, 20 H); MS (methane, DCI) m/z 587 (M+ + 1).
Anal. Calcd for C34H35O7P: C, 69.61; H, 6.01. Found: C, 69.21;
H, 6.00.
1
trated to afford 5 in quantitative yield: H NMR (300 MHz,
CDCl3) δ 2.05 (dquintet, J ) 1.8, 7.7 Hz, 2 H), 2.66 (quintet,
J ) 7.7 Hz, 2 H), 5.12 (s, 2 H), 7.25-7.38 (m, 5 H), 10.89 (brs,
1 H).
2-(P h osp h on om eth yl)p en ta n ed ioic Acid (3). To a solu-
tion of 2a (2.89 g, 4.93 mmol) in methanol (10 mL) was added
10% palladium on carbon (0.26 g, 5 mol %). After shaking at
40 psi for 24 h, the mixture was filtered through Celite and
the filter cake washed with methanol. The combined filtrate
was concentrated in vacuo to afford 3 in quantitative yield.
Purification of 3 was carried out by HPLC: semipreparative
C8 column, linear gradient of water and methanol (0% f 20%)
over 20 min, 10 mL/min, 20 × 10 mL fractions. HPLC
analysis, Zorbax C8 column, isocratic water-methanol (4:1),
showed the product (tR ) 1.6 min) was contained in fractions
7-10. These fractions were combined and lyophilized to afford
Dib en zyl 2-[[[2-(Ben zylca r b oxy)et h yl]h yd r oxyp h os-
p h in oyl]m eth yl]p en ta n ed ioa te (6). Compound 5 (0.82 g,
3.63 mmol) was dried by azeotropic removal of toluene (3 ×
20 mL) in vacuo at 50 °C. The residue was dissolved in dry
CH2Cl2 (50 mL) and cooled to 0 °C. Triethylamine (1.5 g, 14.6
mmol) and chlorotrimethylsilane (1.34 mL, 12.4 mmol) were
added followed after 5 min by compound 1a (1.28 g, 3.94 mmol)
in CH2Cl2 (5 mL). The mixture was stirred for 24 h at room
temperature and then washed with 1 N hydrochloric acid (10
mL) and water (10 mL), dried (MgSO4), and concentrated.
Flash chromatography over silica gel (3 × 18 cm) with a
hexane-EtOAc (4:1 f 1:1) gradient gave 6 as a clear oil (0.74
g, 37%): TLC Rf 0.1 (1:1 hexanes-EtOAc); 1H NMR (300 MHz,
CDCl3) δ 1.70-1.84 (m, 1 H), 1.84-2.05 (m, 4 H), 2.12-2.35
(m, 3 H), 2.51-2.66 (m, 2 H), 2.76-2.94 (m, 1 H), 5.00-5.17
(m, 6 H), 7.20-7.40 (m, 15 H); MS (methane, DCI) m/z 553
(M+ + 1).
1
3 (0.62 g, 55%): H NMR (300 MHz, D2O) δ 1.75-2.21 (m, 4
H), 2.39-2.49 (m, 2 H), 2.66-2.83 (m, 1 H); MS (methane,
DCI-) m/z 225 (M+ - 1). Anal. Calcd for C6H11O7P‚1.25H2O:
C, 28.98; H, 5.47. Found: C, 28.98; H, 5.45.
Diben zyl Ita con a te (1b). A solution of itaconic acid (1.23
g, 9.45 mmol), triethylamine (18.9 mmol, 1.92 g), and benzyl
bromide (23.62 mmol, 4.04 g) in toluene (30 mL) was heated
at 80 °C for 16 h. After cooling to room temperature, the
solution was diluted with ether (100 mL), washed with 1 N
hydrochloric acid (20 mL) and water (20 mL), dried (MgSO4),
and concentrated. Flash chromatography over silica gel (3 ×
18 cm) with a hexane-EtOAc (20:1 f 10:1) gradient gave 2b
as a clear oil (1.84 g, 63%): TLC Rf 0.28 (9:1 hexanes-EtOAc);
1H NMR (300 MHz, CDCl3) δ 3.41 (s, 2 H), 5.09 (s, 2 H), 5.17
(s, 2 H), 5.73 (s, 1 H), 6.39 (s, 1 H), 7.23-7.36 (m, 10 H); MS
(methane, DCI) m/z 311 (M+ + 1).
2-[[(2-Ca r b oxye t h yl)h yd r oxyp h osp h in oyl]m e t h yl]-
p en ta n ed ioic Acid (7). The procedure used for the prepara-
tion of 3 was followed using 6 (0.58 g, 1.06 mmol) in methanol
(10 mL) with 10% palladium on carbon (0.11 g, 10 mol %) to
afford 0.31 g of compound 7. Purification of 7 was carried out
by HPLC: semipreparative C8 column, linear gradient of water
and methanol (0% f 20%) over 20 min, 10 mL/min, 20 × 10
mL fractions. HPLC analysis, Zorbax C8 column, isocratic
water-methanol (4:1), showed the product (tR ) 1.9 min) was
contained in fractions 12-15. These fractions were combined
Dib en zyl 2-[[Bis(b en zyloxy)p h osp h or yl]m et h yl]su c-
cin a te (2b). To a solution of dibenzyl phosphite (3.04 g, 11.6
mmol) in THF (20 mL) at 0 °C was added n-butyllithium (11.6
mmol, 4.6 mL, 2.5 M solution in hexanes) followed after 10
min by trimethylaluminum (11.6 mmol, 5.8 mL, 2.0 M solution
in hexanes). After 20 min a solution of 1b (3.0 g, 9.7 mmol)
in THF (5 mL) was added. The cooling bath was removed,
and the resulting solution stirred at room temperature for 16
h. The reaction was quenched by the slow addition of 1 N
hydrochloric acid (20 mL). After stirring for 10 min the phases
were separated, and the aqueous phase was extracted with
ethyl acetate (2 × 10 mL). The organic phases were combined,
dried (MgSO4), and concentrated. Flash chromatography over
silica gel (3 × 18 cm) with a hexane-EtOAc (4:1 f 1:1)
gradient gave 2b as a clear oil (2.6 g, 55%): TLC Rf 0.39 (1:1
1
and lyophilized to afford 7 (0.17 g, 56%): H NMR (300 MHz,
MeOD) δ 1.82-2.08 (m, 5 H), 2.14-2.27 (m, 1 H), 2.31-2.43
(m, 2 H), 2.51-2.63 (m, 2 H), 2.74-2.91 (m, 1 H); MS (FAB)
m/z 283 (M+).
NAALADa se Assa y. NAALADase activity was assayed as
was previously described.22 In brief, the assay measured the
amount of [3H]Glu liberated from [3H]NAAG in 50 mM Tris-
Cl buffer in 15 min at 37 °C using 30-50 µg of synaptosomal
protein; substrate and product were resolved by anion-
exchange liquid chromatography. Duplicate assays were
always performed so that no more than 20% of the NAAG was
digested, representing the linear range of peptidase activity.
Quis (100 µM) was included in parallel assay tubes to confirm
the specificity of measurements.
1
hexanes-EtOAc); H NMR (300 MHz, CDCl3) δ 2.08 (ddd, J
) 17.6, 15.5, 8.2 Hz, 1 H), 2.35 (ddd, J ) 19.2, 15.5, 5.5 Hz, 1
H), 2.83 (d, J ) 6.5 Hz, 2 H), 3.10-3.27 (m, 1 H), 4.82-5.06
(m, 8 H), 7.22-7.37 (m, 20 H); MS (methane, DCI) m/z 573
(M+ + 1). Anal. Calcd for C33H33O7P: C, 69.22; H, 5.81.
Found: C, 69.28; H, 5.86.
Ack n ow led gm en t. The authors would like to thank
Dr. Andrew Shaw for helpful discussions during the
course of this work.
2-(P h osp h on om eth yl)su ccin ic Acid (4). The procedure
used for the preparation of 3 was followed using 2b (1.4 g,
2.44 mmol) in methanol (10 mL) with 10% palladium on carbon
(0.25 g, 5 mol %) to afford 4 (0.55 g). Purification of 4 was
carried out by HPLC: semipreparative C8 column, linear
gradient of water and methanol (0% f 20%) over 20 min, 10
Refer en ces
(1) Coyle, J . T.; Stauch-Slusher, B.; Tsai, G.; Rothstein, J .; Meyer-
hoff, J . L.; Simmons, M.; Blakely, R. D. N-Acetyl-Aspartyl
Glutamate: Recent Developments. In Excitatory Amino Acids;
Meldrum, B. S., Moroni, F., Simon, R. P., Woods, J . H., Eds.;
Raven Press: New York, 1991; pp 69-77.