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(CDCl3) d: 1.94 (m, 4H), 2.34 (m, 2H), 2.68 (m, 2H),
3.67 (m, 6H), 3.94 (m, 1H), 4.26 (m, 2H), 4.56 (m,
1H), 7.22 (m, 7H), 7.60 (m, 2H), 7.77 (d, J = 7.7 Hz,
2H). 13C NMR (CDCl3) d: 27.68, 28.80, 29.54, 29.75,
29.84, 48.15, 48.25, 51.86, 52.59, 53.03, 65.55, 100.21,
120.22, 124.88, 124.94, 125.21, 125.26, 127.35, 128.06,
128.12, 128.17, 131.77, 132.77, 141.71, 141.78, 143.33,
173.21, 173.37. 31P NMR (CDCl3) d: 31.77, 32.33. Anal.
Calcd for C30H30ClF3NO6P: C, 57.75; H, 4.85; N, 2.24
N. Found: C, 57.76; H, 4.56; N, 2.11.
(m, 2H), 3.30 (m, 1H), 7.08 (d, J = 8.4 Hz, 2H), 7.15
(d, J = 8.4 Hz, 2H). 13C NMR (D2O) d: 30.20, 31.87,
33.46, 33.62, 33.69, 35.26, 57.89, 129.71, 131.00,
132.19, 142.88, 143.12, 169.68, 183.30, 183.34, 184.50.
31P NMR (D2O) d 26.23. FAB-HRMS: Calcd for
C13H14ClLi2NO6P (MÀLi)À 360.0567. Found 360.0542.
4.1.38. N-{Hydroxy[2-(3,4-dichloro)phenylethyl]phosphin-
yl}-L-glutamic acid trilithium salt (2d). Yield: 84%. H
1
NMR (D2O) d: 1.72 (m, 4H), 2.16 (m, 2H), 2.70 (m,
2H), 3.43 (m, 1H), 7.15 (d, J = 6.3 Hz, 1H), 7.40 (m,
2H). 13C NMR (D2O) d: 28.91, 30.42, 32.01, 32.54,
32.61, 34.14, 45.66, 56.78, 128.23, 128.89, 130.10,
130.37, 131.43, 143.71, 143.95, 182.09, 183.24. 31P
NMR (D2O) d: 26.16. FAB-HRMS: Calcd for
C13H13Cl2Li2NO6P (MÀLi)À 394.0178. Found 394.0158.
4.1.34. N-{9-Fluorenylmethoxy[2-(2,4-dichloro)phenyleth-
yl]phosphinyl}-L-glutamic acid dimethyl ester (14d). Chro-
matography conditions: a step gradient of 50% and 75%
1
ethyl acetate/hexanes. Yield: 26%. H NMR (CDCl3) d:
1.97 (m, 4H), 2.35 (m, 2H), 2.82 (m, 2H), 3.68 (m, 6H),
3.97 (m, 1H), 4.26 (m, 2H), 4.51 (m, 1H), 7.18 (m, 2H),
7.37 (m, 5H), 7.62 (m, 2H), 7.77 (d, J = 7.1 Hz, 2H). 13C
NMR (CDCl3) d: 26.27, 26.31, 27.10, 27.18, 28.79,
28.92, 29.57, 29.62, 29.77, 29.82, 48.12, 48.22, 48.32,
51.77, 51.82, 52.53, 52.99, 53.09, 65.61, 65.70, 120.19,
120.22, 125.02, 125.06, 125.27, 125.35, 127.31, 127.51,
128.00, 128.04, 128.07, 128.11, 129.52, 131.22, 133.07,
133.10, 134.56, 137.20, 141.56, 141.61, 141.66, 141.73,
143.33, 143.54, 143.87, 143.98, 173.20, 173.31, 173.93.
31P NMR (CDCl3) d: 32.20, 32.79. Anal. Calcd for
C30H30ClF3NO6P: C, 58.99; H, 5.12; N, 2.37. Found: C,
59.05; H, 5.02; N, 2.41.
4.1.39. N-{Hydroxy[2-(4-chloro-3-trifluromethyl)phenyleth-
yl]phosphinyl}-L-glutamic acid trilithium salt (10a). Yield:
97%. 1H NMR (D2O) d: 1.68 (m, 4H), 2.06 (m, 2H), 2.67
(m, 2H), 3.37 (m, 1H), 7.35 (m, 2H), 7.55 (s, 1H). 13C
NMR (D2O) d: 30.19, 31.61, 33.20, 33.65, 35.29, 57.92,
128.78, 132.60, 134.49, 143.75, 183.34, 184.50. 31P NMR
(D2O) d: 25.75. FAB-HRMS: Calcd for C14H13ClF3Li2-
NO6P (MÀLi)À 428.0441. Found 428.0432.
4.1.40. N-{Hydroxy[2-(2,4-dichloro)phenylethyl]phosphin-
yl}-L-glutamic acid trilithium salt (10b). Yield: 82%. H
1
NMR (D2O) d: 1.70 (m, 4H), 2.12 (m, 2H), 2.74 (m, 2H),
3.42 (m, 1H), 7.16 (m, 2H), 7.31 (s, 1H). 13C NMR (D2O)
d: 28.56, 29.84, 31.44, 33.80, 35.27, 128.62, 130.12,
132.53, 132.99, 135.17, 140.24, 140.48, 183.23, 184.57. 31P
NMR (D2O) d: 25.83. FAB-HRMS: Calcd for
C13H13Cl2Li2NO6P (MÀLi)À 394.0178. Found 394.0163.
4.1.35. N-{9-Fluorenylmethoxy[2-(4-trifluoromethyl)phen-
ylethyl]phosphinyl}-L-glutamic acid dimethyl ester (14e).
Chromatography conditions: 75% EtOAc/hexanes,
Rf = 0.43. Yield: 38%. 1H NMR (CDCl3) d: 1.96 (m, 4H),
2.34 (m, 2H), 2.76 (m, 2H), 3.65 (m, 6H), 4.96 (m, 1H),
4.23 (m, 2H), 4.54 (m, 1H), 7.22 (m, 2H), 7.37 (m, 4H),
7.59 (m, 4H), 7.78 (m, 2H). 13C NMR (CDCl3) d: 28.22,
28.27, 28.32, 28.37, 28.84, 29.51, 29.58, 29.64, 29.74,
29.83, 30.51, 30.58, 48.16,48.26, 48.36, 51.79, 51.84,
52.56, 53.02, 53.08, 65.53, 65.62, 120.18, 120.23, 120.27,
124.94, 125.00, 125.22, 125.28, 125.67, 125.72,
127.33,128.03, 128.07, 128.10, 128.15, 128.57,128.62,
141.66, 141.78,143.33, 143.56, 143.88, 144.00, 173.34,
173.96. 31P NMR (CDCl3) d: 32.20, 32.78. Anal. Calcd
for C30H31F3NO6P: C, 61.12; H, 5.30; N, 2.38. Found: C,
60.68; H, 5.08; N, 2.69.
4.1.41. N-{Hydroxy[2-(4-trifluoromethyl)phenylethyl]phos-
phinyl}-L-glutamic acid trilithium salt (10c). Yield: 74%.
1H NMR (D2O) d: 1.79 (m, 4H), 2.17 (m, 2H), 2.77 (m,
2H), 3.42 (m, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.56 (d,
J = 7.8 Hz, 2H). 13C NMR (D2O) d: 30.83, 31.73,
33.33, 33.76, 33.83, 35.40, 58.05, 126.78, 127.67, 128.56,
130.02, 148.82, 149.06, 183.43, 184.56. 31P NMR (D2O)
d: 25.92. FAB-HRMS: Calcd for C14H14F3Li2NO6P
(MÀLi)À 394.0831. Found 394.0809.
4.2. PSMA inhibition assay
4.1.36. General procedure for 2-phenylethylphosphonami-
dates (2c–d, 10a–c). A solution of 80 mg (0.14 mmol, 1.0
equiv) of 14 in 0.8 mL MeOH was prepared. While stir-
ring, a solution of 21 mg (0.51 mmol, 3.8 equiv) of
LiOHÁH2O dissolved in 0.5 mL water was added. After
stirring the mixture at rt for 3.5 h, the solvent was re-
moved by rotary evaporation. The crude material was
resuspended in MeOH and was filtered through a
0.2 lm Whatman teflon membrane. The filtrate was rota-
ry evaporated and resuspended in 4 mL water. The mix-
ture was washed with CH2Cl2 (3· 2 mL). The aqueous
was concentrated under reduced pressure and was dried
under vacuum to give the product as a light yellow solid.
Inhibition studies were performed as described previously
with only minor modifications16 and are briefly presented
here. Working solutions of the substrate (N-[4-(phenyl-
azo)benzoyl]-glutamyl-c-glutamic acid, PABGcG) and
all inhibitors were made in Tris buffer (50 mM, pH 7.4)
containing 150 mM NaCl. Working solutions of purified
PSMA16 were appropriately diluted in Tris buffer
(50 mM, pH 7.4) to provide no more than 15% conversion
of substrate to product in the absence of inhibitor. A typ-
ical incubation mixture (final volume 250 lL) was pre-
pared by the addition of either 25 lL of an inhibitor
solution or 25 lL Tris buffer (50 mM, pH 7.4) to 175 lL
Tris buffer (50 mM, pH 7.4) in a test tube. A volume of
25 lL of the PSMA working solution was added to the
above solution. The enzymatic reaction was initiated by
the addition of 25 lL PABGcG (10 or 20 lM). In all
4.1.37. N-{Hydroxy[2-(4-chloro)phenylethyl]phosphinyl}-
L-glutamic acid trilithium salt (2c). Yield: quantitative.
1H NMR (D2O) d: 1.63 (m, 4H), 2.05 (m, 2H), 2.57