Synthesis and biological evaluation of (R)- and (S)-2-(phosphonomethyl)pentanedioic acids as inhibitors of glutamate carboxypeptidase II

Article abstract of DOI:10.1016/S0957-4166(02)00412-3

Both enantiomers of 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent and selective inhibitor of glutamate carboxypeptidase II (GCP II), were successfully prepared through the resolution of racemic 2-(hydroxyphosphinoylmethyl)pentanedioic acid diben

Full text of DOI:10.1016/S0957-4166(02)00412-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1609–1614
Synthesis and biological evaluation of (R)- and
(S)-2-(phosphonomethyl)pentanedioic acids as inhibitors of
glutamate carboxypeptidase II
Dilrukshi Vitharana, Jessica E. France, David Scarpetti, George W. Bonneville, Pavel Majer and
Takashi Tsukamoto*
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA
Received 25 April 2002; accepted 28 June 2002
Abstract—Both enantiomers of 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent and selective inhibitor of glutamate
carboxypeptidase II (GCP II), were successfully prepared through the resolution of racemic 2-(hydroxyphosphinoyl-
methyl)pentanedioic acid dibenzyl ester with yohimbine and (S)-a-methylbenzylamine. The enantiomeric purity of the resolved
phosphinic acid was measured by coupling it to (−)-menthol and analyzing the resulting ester by 31P NMR. The absolute
configuration of the 2-carbon atom in the resolved phosphinic acid was determined by X-ray crystallographic studies. Optically
pure 2-PMPA was obtained by oxidation of the resolved phophinic acid to the phosphonic acid followed by catalytic
hydrogenolysis. The biological activity of each enantiomer of 2-PMPA will also be described. © 2002 Elsevier Science Ltd. All
rights reserved.
1. Introduction
value of 0.3 nM.3 The high potency of 2-PMPA can be
attributed to the strong chelation of the phosphonate
group to an active site zinc atom as well as the interac-
tion of the glutarate (pentanedioic acid) portion of the
inhibitor with the glutamate recognition site of GCP II.
2-PMPA has been extensively utilized to study the
mechanism and physiological role of GCP II as well as
the potential therapeutic effects of GCP II inhibition.
For example, 2-PMPA protects against ischemic injury
both in a neuronal culture model of stroke, and in rats
after transient middle cerebral artery occlusion.4
The metalloprotease glutamate carboxypeptidase II,
(GCP II, also known as N-acetylated alpha-linked
acidic dipeptidase, NAALADase) cleaves N-acetyl-
aspartyl-glutamate (NAAG) into N-acetyl-aspartate
(NAA) and glutamate.1 Since NAAG is believed to be
a major source of glutamate in the nervous system, the
enzyme is a target for the treatment of neurological
disorders associated with excess glutamate such as
stroke, spinal cord injury, amyotrophic lateral sclerosis
(ALS), peripheral neuropathies, chronic pain,
schizophrenia and epilepsy.2
Despite the potential therapeutic value of 2-PMPA, this
compound has never been synthesized in an enan-
tiomerically pure form. Identification of the active
enantiomer is important in understanding the enan-
tiospecificity of the enzyme as well as the elimination of
undesired pharmacological effects caused by the inac-
tive enantiomer. In this paper, we report the synthesis
and biological evaluation of both enantiomers of 2-
PMPA as inhibitors of GCP II.
CO2H
O
HO
P
CO2H
HO
2-PMPA, 1
2-(Phosphonomethyl)pentanedioic acid (2-PMPA) 1 is
the most potent known inhibitor of GCP II with a Ki
2. Results and discussion
Although a literature search on optically active phos-
phorous-containing compounds did not identify any
* Corresponding author. Tel.: +1-410-631-6762; fax: +1-410-631-6797;
e-mail: tsukamotot@guilfordpharm.com
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00412-3

1610
D. Vitharana et al. / Tetrahedron: Asymmetry 13 (2002) 1609–1614
O
OBn
OBn
method applicable to the direct resolution of 1, there
were two cases where phosphinic acids 25 and 36 have
been successfully resolved using (S)-a-methylbenzyl-
amine. Based on these results, we predicted that a
structurally analogous compound, 2-hydroxyphosphi-
noylmethyl-pentanedioic acid dibenzyl ester 4 could be
resolved in a similar manner.
a
O
P
4
O
H
O
6
Scheme 2. Reagents and conditions: (a) (−)-Menthol, EDC,
CDCl3.
O
OBn
R
O
P
HO
OBn
The proton-decoupled 31P NMR spectrum of the phos-
phinate ester 6 derived from ( )-4 exhibits four peaks at
30.1, 31.3, 35.6 and 36.6 ppm in the ratio of
0.19:0.27:0.23:0.31 (Fig. 1A). The emergence of the
multiple peaks is due to the creation of a new stereo-
genic center at the phosphorous atom that results in the
formation of four diastereomers of compound 6. The
ratio of the two outer peaks to the two inner peaks
turned out to be 1:1. In theory, each pair of peaks
should correspond to two diastereomers derived from a
single enantiomer of 4. Indeed, the proton-decoupled
31P NMR spectrum of phosphinate ester 6 derived from
(R)-(+)-4 (Fig. 1B) exhibits two inner peaks while that
of (S)-(−)-4 (Fig. 1C) only exhibits two outer peaks.
These experiments clearly indicate that both of the
resolved phosphinic acids (R)-(+)-4 and (S)-(−)-4 were
obtained in enantiomerically pure form. This 31P
O
P
H
HO
OBn
O
H
O
2 (R = benzyl)
3 (R = i-propyl)
4
Racemic phosphinic acid ( )-4 was prepared by treating
2-methylene-pentanedioic acid dibenzyl ester 53 with
bis(trimethylsilyl)phosphonite (BTSP) generated in situ
from ammonium hypophosphite, chlorotrimethylsilane
and triethylamine. After extensive screening (e.g.
quinine, quinidine, cinchonine, cinchonidine and
bruicine in various solvent systems), we found that the
phosphinic acid 4 can be resolved by preparation of the
yohimbine salt in acetone/water followed by recrystal-
lization from acetone/water (Scheme 1). Treatment of
the salt with 10% sulfuric acid then liberates the phos-
phinic acid (R)-(+)-4. To obtain the other enantiomer
(S)-(−)-4, the combined mother liquors from the
yohimbine resolution were converted back to the free
phosphinic acid, which was resolved by preparing the
(S)-a-methyl benzylamine salt and recrystallizing from
acetone/water. The free acid (S)-(−)-4 was liberated
from the salt by treatment with 10% sulfuric acid.
Reiter’s group reported that neither direct quantitative
measurement of the diastereomeric purity of 3·(S)-a-
1
methylbenzylamine salt by H or 31P NMR nor their
attempts to determine the enantiomeric purity of free
1
acid 3 by chiral HPLC, H and 31P NMR were success-
ful.6 This prompted us to develop an alternative
method to measure the optical purity of the resolved
phosphinic acid 4. Thus, the phosphinic acid 4 was
coupled to (−)-menthol with EDC using CDCl3 as a
solvent in a NMR tube (Scheme 2) and the resulting
phosphinate ester 6 was subsequently analyzed by 31P
NMR spectroscopy.
Scheme 1. Reagents and conditions: (a) BTSP, CH2Cl2. (b)
Yohimbine, acetone/H2O. (c) i. Recrystallization from ace-
tone/H2O; (ii) 10% H2SO4. (d) i. 10% H2SO4; ii. (S)-a-methyl-
benzylamine, acetone/H2O.
Figure 1. Proton-decoupled 31P NMR spectra of (−)-menthol
ester 6. (A) Ester 6 derived from ( )-4. (B) Ester 6 derived
from (R)-(+)-4. (C) Ester 6 derived from (S)-(−)-4.

D. Vitharana et al. / Tetrahedron: Asymmetry 13 (2002) 1609–1614
1611
NMR-based approach served as an accurate, reliable,
Table 1. Biological activity of (RS)-1, (R)-1, and (S)-1
and convenient method for measuring the enantiomeric
purity of 4 and may be applicable to a wide range of
optically active phosphinic acids.
Compound
GCP II inhibition Cell culture ischemia assay
IC50 (nM)
EC50 (nM)
The stereochemistry at C-2 of phosphinic acids 4 was
established by X-ray structure analysis of (−)-4·1-
adamantanamine salt7 as shown in Fig. 2. The X-ray
analysis revealed that (−)-4 is the (S)-enantiomer of the
phosphinic acid.
(RS)-1
(R)-1
(S)-1
0.3
30
0.1
1.2
70
0.2
The enantiomers of 2-PMPA, (R)-1 and (S)-1, were
obtained by oxidation of the resolved phosphinic acids
(R)-4 and (S)-4 with sodium periodate followed by
catalytic hydrogenolysis (Scheme 3).
group reported that GCP II-catalyzed hydrolysis of
NAAG is specific to N-acetyl-aspartyl- -glutamate and
that N-acetyl-aspartyl- -glutamate is neither substrate
L
D
nor inhibitor of GCP II.8 Their finding is in a good
agreement with our results with the two enantiomers of
2-PMPA. We also assessed the protective effects of
(RS)-1, (R)-1, and (S)-1 in a tissue culture model of
cerebral ischemia. As shown in Table 1, (RS)-1 and
(S)-1 exhibited a strong neuroprotection with median
effective concentration (EC50) values of 1.2 and 0.2 nM,
respectively. Compound (R)-1 exhibited much weaker
potency in this model with an EC50 value of 70 nM.
The good correlation between GCP II inhibitory and
neuroprotective potencies of (R)-1 (less potent in both
assays) and (S)-1 (more potent in both assays) provides
further experimental evidence that the neuroprotective
effect of these compounds is due to their ability to
inhibit GCP II.
The biological activity of (RS)-1, (R)-1, and (S)-1 are
summarized in Table 1. GCP II inhibitory assay
revealed that (S)-1 is slightly more potent (IC50=0.1
nM) than (RS)-1 (IC50=0.3 nM) while (R)-1 has 100-
fold lower potency (IC50=30 nM). These biological
results demonstrate that the potent inhibition of GCP
II by 2-PMPA is enantiospecific for the (S)-enantiomer,
which has stereochemistry corresponding to
L-gluta-
mate of the endogenous substrate NAAG. Serval’s
3. Conclusions
Both enantiomers of 2-hydroxyphosphinoylmethyl-pen-
tanedioic acid dibenzyl ester (R)-(+)-4 and (S)-(−)-4
have been successfully obtained by fractional recrystal-
lization of the corresponding yohimbine and (S)-a-
methylbenzylamine salts. A facile method to measure
the optical purity of 4 allowed us to demonstrate that
both enantiomers were obtained in optically pure
forms. Both enantiomers of 2-PMPA have been suc-
cessfully synthesized from (R)-(+)-4 and (S)-(−)-4 and
evaluated in GCP II assay. The active enantiomer (S)-1
should serve as a more appropriate probe in under-
standing the physiological role of GCP II as well as the
full therapeutic potential of GCP II inhibition.
4. Experimental
4.1. General
Figure 2. Crystal structure of (S)-(−)-4·1-adamantanamine
salt.
1H, 13C, and 31P NMR spectra were recorded on a
Bruker DRX-400 instrument at 400, 100, and 162
MHz, respectively. Seeds used in crystallization of
yohimbine and (S)-a-methylbenzylamine salts were
obtained from preliminary small-scale experiments. Ele-
mental analyses were obtained from Atlantic Micro-
labs, Norcross, GA and optical rotations were obtained
from Robertson Microlit Laboratories, Inc., Madison,
3
NJ. N - Acetyl -
L
- aspartyl -
L
- [3,4 - H]glutamate
Scheme 3. Reagents and conditions: (a) NaIO4, CH3CN/H2O.
(b) H2, Pd/C, H2O.
(NAA[3H]G) was obtained from Perkin–Elmer Life Sci-
ences Inc., Boston, MA.

1612
D. Vitharana et al. / Tetrahedron: Asymmetry 13 (2002) 1609–1614
4.2. Synthesis of ( )-2-hydroxyphosphinoylmethyl-pen-
tanedioic acid dibenzyl ester ( )-4
with 10% H2SO4 (2×615 mL) and water (2×615 mL).
The organic layer was concentrated by rotary evapora-
tion at 40°C to give (R)-(+)-4 as a thick, pale yellow
1
To a suspension of ammonium hypophosphite (229 g,
2.76 mol) in dichloromethane (4400 mL) were added
chlorotrimethylsilane (791 g, 7.28 mol) and triethyl-
amine (671 g, 6.63 mol) maintaining the temperature
below 10°C. The reaction mixture was allowed to stir
at 0–10°C for 30 min. A solution of 53 (179 g, 0.55
mol) in dichloromethane (200 mL) was added keeping
the temperature below 10°C. The reaction mixture was
allowed to warm to 17–22°C and stirred for 18–20 h,
and then quenched by careful addition of 3N HCl
(2000 mL) ensuring that the temperature did not
exceed 25°C. The organic layer was washed with 3N
HCl (4×1000 mL) and water (2×1000 mL). The
organic layer was concentrated on a rotary evaporator
at 40°C to give ( )-4 as a viscous colorless oil (173 g,
80%): 1H NMR (CDCl3) l 1.8–1.9 (m, 1H), 2.0–2.1
(m, 2H), 2.1–2.3 (m, 1H), 2.3–2.4 (m, 2H), 2.8–3.0 (m,
1H), 5.07 (s, 2H), 5.11 (s, 2H), 6.62 (brs, 1H), 7.12 (d,
J=565 Hz, 1H), 7.1–7.3 (m, 10H); 13C NMR (CDCl3)
l 28.5 (d, J=12.1 Hz) 31.6 (d, J=94.4 Hz), 31.7, 38.5
(d, J=1.8 Hz), 66.8, 67.4, 128.7 (2 C), 128.7, 128.7 (2
C), 128.8, 129.0 (2 C), 129.0 (2 C), 135.8, 136.2, 172.6,
173.9 (d, J=6.2 Hz): 31P NMR (CDCl3) l 36.0 (dm,
J=578 Hz). Anal. calcd for C20H23O6P: C, 61.54; H,
5.94. Found: C, 61.72; H, 5.98%.
oil (56.9 g, 88%): H and 31P NMR spectral data were
identical to those of ( )-4; [h]2D0=+2.9 (c 1.0, CHCl3).
Anal. calcd for C20H23O6P·0.1H2O: C, 61.25; H, 5.96.
Found: C, 60.93; H, 5.99%.
4.4. Preparation of (S)-(−)-4
4.4.1. Recovery of 4 from the mother liquors. The
mother liquors and washings from Sections 4.3.1 and
4.3.2 were combined and concentrated to dryness by
rotary evaporation at 40°C to give 241 g of the
yohimbine salt. The residue was dissolved in
dichloromethane (3000 mL) and the solution was
washed with 10% H2SO4 (3×1000 mL) and water (2×
1000 mL). The organic layer was concentrated to dry-
ness by rotary evaporation at 40°C to give the free
phosphinic acid 4 as a viscous orange oil (116 g, 92%
recovery).
4.4.2. Formation of (S)-a-methylbenzylamine salt. A
solution of the above phosphinic acid 4 (107 g, 0.27
mol) and (S)-a-methylbenzylamine (34.9 mL, 0.27
mol) in acetone (267 mL) was heated to reflux then
was cooled to 40°C and seeded with enantiomerically
pure (S)-a-methylbenzylamine salt. Cooling was
resumed to 25°C and acetone (800 mL) was added.
The reaction mixture was cooled to 0–5°C and held
for 1–2 h. The solid product was washed with cold
acetone (2×214 mL) and dried under vacuum at 17–
22°C to give the (S)-a-methylbenzylamine salt as an
off-white solid (124 g, 89%).
4.3. Preparation of (R)-(+)-4
4.3.1. Formation of yohimbine salt. A mixture of ( )-4
(213 g, 0.55 mol) and yohimbine (193 g, 0.55 mol) in
acetone (1070 mL) was heated to reflux (55–57°C) and
water (120 mL) was added at reflux until a solution
was obtained. The resulting solution was cooled to
45°C and a seed of enantiomerically pure yohimbine
salt was added. The mixture was cooled to 25°C over
one hour and acetone (3200 mL) was added. The
mixture was cooled to 5°C and held for 2 h. The
solids were isolated by filtration, washed with cold
acetone (426 mL×2), and dried under vacuum at 17–
22°C to give the yohimbine salt as a white crystalline
solid (169 g, 41%).
4.4.3. Recrystallization of the (S)-a-methylbenzylamine
salt. The above (S)-a-methylbenzylamine salt (76.0 g,
0.15 mol) was heated in acetone (380 mL) and water
(28 mL) was added to reflux, maintaining reflux to
achieve dissolution. After cooling to 40–45°C, the
solution was seeded with enantiomerically pure (S)-a-
methylbenzylamine salt. Cooling was resumed to 25°C
and acetone (1100 mL) was added. The mixture was
cooled to 0–5°C and held for 2 h. The product was
filtered, washed with acetone (2×152 mL), dried under
vacuum at 17–22°C to give the (S)-a-methylbenzyl-
amine salt as a white crystalline solid (63.2 g, 83%).
4.3.2. Recrystallization of yohimbine salt. The yohim-
bine salt (136 g, 0.18 mol) was heated in acetone (679
mL) to reflux (55–57°C) and water (140 mL) was
added, maintaining reflux until
a
solution was
attained. The resulting solution was cooled to 45°C
and a seed of enantiomerically pure yohimbine salt
was added. The mixture was cooled to 25°C over 1 h
and acetone (2000 mL) was added. The mixture was
cooled to 0–5°C and held for 2 h. The solids were
isolated by filtration, washed with cold acetone (2×270
mL), dried under vacuum at 17–22°C to give the
yohimbine salt as a white crystalline solid (122 g,
89%).
4.4.4. Liberation of (S)-(−)-4 from the (S)-a-methylben-
zylamine salt. A mixture of the (S)-a-methylbenzyl-
amine salt (62.0 g, 0.12 mol), dichloromethane (620
mL), and 2N HCl (300 mL) was stirred for 15 min.
The organic layer was washed with 2N HCl (300 mL×
2) and water (300 mL×2). The organic layer was con-
centrated to dryness on a rotary evaporator at 40°C to
give (S)-(−)-4 as a viscous pale yellow oil (47.6 g,
1
4.3.3. Liberation of (R)-(+)-4 from the yohimbine salt.
The recrystallized yohimbine salt (123 g, 0.17 mol) in
dichloromethane (1200 mL) and 10% H2SO4 (615 mL)
was stirred for 15 min. The organic layer was washed
100%): H and 31P NMR spectral data were identical
to those of ( )-4; [h]D20=−2.3 (c 1.0, CHCl3). Anal.
calcd for C20H23O6P0.1H2O: C, 61.25; H 5.96. Found:
C, 60.97; H, 5.99%.

D. Vitharana et al. / Tetrahedron: Asymmetry 13 (2002) 1609–1614
1613
1
4.5. Determination of enantiomeric purity of (R)-(+)-4
and (S)-(−)-4. Preparation and proton-decoupled 31P
NMR analysis of (−)-menthol ester 6
solid (0.201 g, 80%): H NMR (D2O) l 1.75-1.95 (m,
3H), 2.08 (dt, J=9.1, 16.3 Hz, 1H), 2.36 (t, J=7.3 Hz,
2H), 2.60-2.75 (m, 1H); 13C NMR (D2O) l 28.3 (d,
J=13.8 Hz), 29.4 (d, J=136.8 Hz), 31.4, 40.0 (d, J=2.3
Hz), 177.7, 179.1; 31P NMR (D2O) l 26.8; [h]2D0=−5.9
(c 1.0, water). Anal. calcd for C6H11O7P0.6H2O: C,
30.42; H, 5.19. Found: C, 30.11; H, 4.90%.
An NMR tube was charged with 4 (3.1 mg, 0.008
mmol), CDCl3 (0.6 mL), (−)-menthol (0.21 g, 1.3 mmol)
and EDC (4.0 mg, 0.02 mmol). After 3–5 h, the sample
was analyzed by proton-decoupled 31P NMR (see Fig.
1).
4.9. Synthesis of (S)-2-(phosphonomethyl)pentanedioic
acid (S)-1
4.6. Preparation of 1-adamantanamine salt of (S)-(−)-
47
The (S)-enantiomer (S)-1 was synthesized as described
above for (R)-1 except starting with (S)-4: 1H, 13C
and 31P NMR spectral data were identical to those
of (R)-1; [h]2D0=+6.1 (c 1.0, water). Anal. calcd for
C6H11O7P0.6H2O: C, 30.42; H, 5.19. Found: C, 30.13;
H, 4.88%.
Phosphinic acid (S)-(−)-4 (1.17 g, 3.0 mmol) from
Section 4.4.4 was dissolved in diethyl ether (10 mL) and
treated with a solution of 1-adamantylamine (0.454 g,
3.0 mmol) in diethyl ether (10 mL). The resulting
precipitate was collected, washed with ether and dried
under vacuum to give (S)-(−)-4·1-adamantanamine salt
as a white solid (1.50 g, 92%): mp 174–176°C; [h]2D0=
−0.6 (c 1.0, MeOH). Anal. calcd for C30H40NO6P-
0.2H2O: C, 66.09; H 7.47; N, 2.57. Found: C, 65.73; H,
7.49; N, 2.53%. The salt was recrystallized from etha-
nol/water prior to the X-ray analysis.
4.10. Biological evaluation of (RS)-1, (R)-1 and (S)-1
The GCP II assay was carried out as outlined
previously9
with
modifications.
Radiolabeled
NAA[3H]G (30 nM, 15 Ci/mmol) and inhibitors (10
pM to 100 mM) were incubated with purified recombi-
nant GCP II10 (20 pM) in Tris buffer (pH 7.6, 50 mM)
containing CoCl2 (1 mM) in a total volume of 50 mL at
37°C for 15 min. The reaction was terminated with
phosphate buffer (0.1 M, pH 7.4, 50 mL) and the
material was applied to a strong anion exchange mini
column (AG 1-X8 anion exchange resin, 200–400 mesh;
formate form). [3H]Glutamate was eluted with formate
(1.0 M) while unreacted NAA[3H]G remained bound to
the column. The eluate was transferred to a solid
scintillator coated plate and dried. The radioactivity
was measured with a top scintillation counter. Tissue
culture model of cerebral ischemia was performed as
previously described.4
4.7. X-Ray structure analysis of (S)-(−)-4·1-adaman-
tanamine salt
Measurement of the crystal structure of (S)-(−)-4·1-
adamantanamine salt was performed on a Rigaku
RAXIS-RAPID
diffractometer
with
graphite-
,
monochromated Cu Ka (u=1.54182 A) irradiation.
Crystallographic data (excluding structure factors) for
the structure in this paper has been deposited with the
Cambridge Crystallographic Data Centre as supple-
mentary publication number CCDC 179558. Copies of
the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
[fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.
ac.uk].
Acknowledgements
4.8. Synthesis of (R)-2-(phosphonomethyl)pentanedioic
acid (R)-1
The optical resolution of 4 was co-developed with
Oxford Asymmetry International. X-Ray structure
analysis of (S)-(−)-4·1-adamantanamine salt was per-
formed by Rigaku MSC. The authors thank Dr.
Camilo Rojas, Ms. Juliet M. Flanary and Mr. Ajit G.
Thomas for their contributions in the biological evalua-
tion of 2-PMPA. We thank Mr. Douglas E. Wilkinson
for his assistance in the proton-decoupled 31P NMR.
To a solution of (R)-4 (2.06 g, 5.12 mmol) in CH3CN–
H2O (12 mL, 1:1 by volume) was added sodium perio-
date (1.37 g, 6.41 mmol) at rt and the mixture was
stirred at 50°C for 3 h. The reaction mixture was then
concentrated in vacuo. The residual material was dis-
solved in EtOAc (100 mL) and washed with saturated
aq. KHSO4 (75 mL), 0.2 M Na2S2O3 (100 mL), H2O
(75 mL), and brine (75 mL), respectively. The organic
layer was dried over MgSO4 and the solvent was
removed in vacuo. The residue was purified by column
chromatography (hexanes/EtOAc/AcOH/MeOH, 1/1/
0.01/0.01) to afford (R)-7 as a colorless oil (1.14 g,
References
1
57%): H NMR (CD3OD) l 1.72–2.21 (m, 4H), 2.28–
1. Carter, R. E.; Coyle, J. T. In Handbook of Proteolytic
Enzymes; Barrett, A. J.; Rawlings, N. D.; Woessner, J.
F., Eds.; Academic: New York, 1998; pp. 1434–1437.
2. Whelan, J. Drug Discovery 2000, 5, 171–172.
3. Jackson, P. F.; Cole, D. C.; Slusher, B. S.; Stetz, S. L.;
Ross, L. E.; Donzanti, B. A.; Trainor, D. A. J. Med.
Chem. 1996, 39, 619–622.
2.38 (m, 2H), 2.76–2.93 (m, 1H), 5.02–5.14 (m, 4H),
7.24–7.39 (m, 10H). A solution of (R)-7 (0.455g, 1.12
mmol) in H2O (50 mL) was shaken under hydrogen (30
psi) in the presence of 10% Pd/C (80 mg) for 20 h. The
catalyst was removed by filtration and the filtrate was
concentrated to afford (R)-1 as a hygroscopic white

1614
D. Vitharana et al. / Tetrahedron: Asymmetry 13 (2002) 1609–1614
7. Karanewsky, D. S.; Badia, M. C.; Cushman, D. W.;
4. Slusher, B. S.; Vornov, J. J.; Thomas, A. G.; Hurn, P. D.;
DeForrest, J. M.; Dejneka, T.; Loots, M. J.; Perri, M. G.;
Petrillo, E. W., Jr.; Powell, J. R. J. Med. Chem. 1988, 31,
204–212.
Harukuni, I.; Bhardwaj, A.; Traystman, R. J.; Robinson,
M. B.; Britton, P.; Lu, X. C.; Tortella, F. C.; Wozniak,
K. M.; Yudkoff, M.; Potter, B. M.; Jackson, P. F. Nat.
Med. 1999, 5, 1396–1402.
8. Serval, V.; Galli, T.; Cheramy, A.; Glowinski, J.;
Lavielle, S. J. Pharmacol. Exp. Ther. 1992, 260, 1093–
1100.
9. Robinson, M. B.; Blakely, R. D.; Couto, R.; Coyle, J. T.
J. Biol. Chem. 1987, 262, 14498–14506.
10. Barinka, C.; Rinnova´, M.; Sa´cha, P.; Rojas, C.; Majer,
P.; Slusher, B. S.; Konvalinka, J. J. Neurochem. 2002, 80,
477–487.
5. Caldwell, C. G.; Sahoo, S. P.; Polo, S. A.; Eversole, R.
R.; Lanza, T. J.; Mills, S. G.; Niezwiecki, L.; Izquierdo-
Martin, M.; Chang, B. C.; Harrison, R. K.; Kuo, D. W.;
Lin, T.-Y.; Stein, R. L.; Durette, P. L.; Hagmann, W. K.
Bioorg. Med. Chem. Lett. 1996, 6, 323–328.
6. Reiter, L. A.; Jones, B. P. J. Org. Chem. 1997, 62,
2808–2812.