Inhibition of glutamate carboxypeptidase by phosphoryl and thiophosphoryl derivatives of glutamic and 2-hydroxyglutaric acid

Article abstract of DOI:10.1080/10426500307817

Representative phosphoryl and thiophosphoryl derivatives of (S)-glutamic or (S)-2-hydroxyglutaric acid were synthesized and evaluated for their inhibitory potency against the glutamate carboxypeptidase, carboxypeptidase G (CPG). It was observed that the inhibition of CPG was highly sensitive to the individual phosphorus ligands. The most potent inhibitors were the dibasic phosphoryl and thiophosphoryl derivatives of glutamic acid and the monobasic thiophosporyl derivatives of 2-hydroxyglutaric acid.

Full text of DOI:10.1080/10426500307817

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Phosphorus, Sulfur, and Silicon and the Related
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Inhibition of Glutamate Carboxypeptidase by
Phosphoryl and Thiophosphoryl Derivatives of Glutamic
and 2-Hydroxyglutaric Acid
Haiyan Lu a , Rudy J. Ng a , Charlene C. Shieh a , Alicia R. Martinez a & Clifford E. Berkman a
a San Francisco State University , San Francisco, California, USA
Published online: 27 Oct 2010.
To cite this article: Haiyan Lu , Rudy J. Ng , Charlene C. Shieh , Alicia R. Martinez & Clifford E. Berkman (2003) Inhibition
of Glutamate Carboxypeptidase by Phosphoryl and Thiophosphoryl Derivatives of Glutamic and 2-Hydroxyglutaric Acid,
Phosphorus, Sulfur, and Silicon and the Related Elements, 178:1, 17-32, DOI: 10.1080/10426500307817
To link to this article: http://dx.doi.org/10.1080/10426500307817
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Phosphorus, Sulfur and Silicon, 2003, Vol. 178:17–32
C
Copyright 2003 Taylor & Francis
1042-6507/03 $12.00 + .00
DOI: 10.1080/10426500390170129
INHIBITION OF GLUTAMATE CARBOXYPEPTIDASE
BY PHOSPHORYL AND THIOPHOSPHORYL
DERIVATIVES OF GLUTAMIC AND
2-HYDROXYGLUTARIC ACID
Haiyan Lu, Rudy J. Ng, Charlene C. Shieh, Alicia R. Martinez,
and Clifford E. Berkman
San Francisco State University, San Francisco, California, USA
(Received May 30, 2002; accepted July 11, 2002)
Representative phosphoryl and thiophosphoryl derivatives of ( S)-
glutamic or ( S)-2-hydroxyglutaric acid were synthesized and evalu-
ated for their inhibitory potency against the glutamate carboxypepti-
dase, carboxypeptidase G (CPG). It was observed that the inhibition of
CPG was highly sensitive to the individual phosphorus ligands. The
most potent inhibitors were the dibasic phosphoryl and thiophosphoryl
derivatives of glutamic acid and the monobasic thiophosporyl deriva-
tives of 2-hydroxyglutaric acid.
Keywords: Inhibition; metallopeptidase; phosphate; phosphoramidate;
phosphoramidothionate; phosphorothionate
INTRODUCTION
Our research efforts recently have been aimed at developing po-
tent competitive inhibitors for glutamate carboxypeptidases such as
N-acetylated-alpha-linked-acidic dipeptidase (NAALADase),1 prostate-
specific membrane antigen (PSMA),2 pteroylpoly-glutamate hydrolase
(PPH),3 and carboxypeptidase G (CPG).4 The acquisition of inhibitors
This work was supported in part by the National Institutes of Health, MBRS SCORE
Program-NIGMS (grant no. S06 GM52588-04) and MBRS-RISE Program-NIGMS (grant
no. GM59298-02), a “Research Infrastructure in Minority Institutions” award from the
National Center for Research Resources with funding from the Office of Research on
Minority Health, National Institutes of Health (grant no. RR11805-02), and a GAANN
fellowship from the Department of Education (grant no. P200A70233).
Address correspondence to Clifford E. Berkman, Department of Chemistry and
Biochemistry, San Francisco State University, 1699 Holloway Avenue, San Francisco,
CA 94132, USA. E-mail: cberkman@sfsu.edu
17

18
H. Lu et al.
for such enzymes is expected to further the understanding of the bio-
logical role of these metallocarboxypeptidases as well as to serve in the
elucidation of germane active site features. In addition, inhibitors of
CPG2 (closely related to CPG) recently have been sought for the use in
inhibiting non-tumor-localized enzyme in ADEPT strategies.5
Based on preliminary evidence, alkylphosphonyl and alkylthiophos-
phonyl derivatives of glutamic and 2-hydroxyglutaric acid (1–4) have
exhibited promise as potent tetrahedral-intermediate analog inhibitors
of glutamate-liberating metallopeptidases known as glutamate car-
boxypeptidases. Although the phosphonyl motif often provides suitable
inhibitory potency toward metallopeptidases, thiophosphonyl analogs
maintain the unique property of probing enzyme active-site archi-
tecture with complementary chiral phosphorus centers.6 We observed
that, in some cases, one alkylthiophosponyl stereoisomer possesses in-
hibitory potency notably greater than either a stereoisomer of antipodal
phosphorus stereochemistry or its respective alkylphosphonyl analog.7
The basis for the enhanced inhibitory potency of such compounds, es-
pecially against zinc-metallopeptidases, presumably is due to favorable
zinc-sulfur interactions within enzyme active sites.8
Although simple N-phosphonyl and N-thiophosphonyl derivatives of
amino acids can be readily obtained from C-protected amino acids, the
corresponding derivatives of 2-hydroxyacids are more challenging due
to the lack of readily available oxygen analogs of amino acids. How-
ever, the resulting isosteric analogs of phosphonamidothionates and
phosphonamidates, such as phosphonothionates and phosphonates, are
expected not only to be similarly promising (as are metallopeptidase
inhibitors), but possess the additional benefit of being less prone to
desulfurization and hydrolysis under acidic conditions.9
The focus of the current study was to examine the effect of isos-
teric replacement of the proximal alkyl ligand carbon atom in previous
phosphonyl and thiophosphonyl inhibitors with an oxygen atom upon
glutamate carboxypeptidase inhibition. To this end, representative
FIGURE 1

Fused Heterocycles
19
compounds 5–12 were synthesized and evaluated for inhibitory potency
against glutamate carboxypeptidase. In line with our laboratory’s inter-
est in the inhibition of glutamate carboxypeptidases, CPG was selected
as a model enzyme for its versatility and ready availability.
Results and Discussion
Due to the significant structural similarities of the target inhibitors
5–12, the synthetic strategies for many of these compounds or their
synthetic intermediates were sustained when possible. In a few cases,
however, a different approach was necessary and therefore pursued.
Beginning with dichlorophosphite 13, phosphoroamidite intermedi-
ates 14 were obtained by a sequential one-pot reaction with glu-
tamic acid dimethyl ester and 2-cyanoethanol. Subsequent oxidization
with mCPBA or thionation with sulfur gave the phosphoramidates 15
or phosphoramidothioates 16 respectively (Scheme 1). The yield for
preparing 15a was notably improved by subjecting phosphoramidoth-
ioate 16a to oxidative desulfurization conditions using MMPP. Subse-
quent deprotection of intermediates 15 and 16 with LiOH, provided
5 and 6 respectively.
The synthesis of compounds 7a, 7b was achieved through the strat-
egy outlined in Scheme 2. Reaction of benzyl dichlorophosphite with (S)-
2-hydroxyglutarate 1710 and the corresponding alcohol in a two-step,
one-pot reaction gave phosphites 18, which were summarily subjected
to oxidation with mCPBA to give phosphate precursors 19. Deprotection
of the benzyl group by catalytic hydrogenlysis with 5% Pd/C in MeOH
afforded the phosphate monoesters 20 which were further deprotected
with LiOH, finally yielding the fully deprotected targets 7. It should
be noted that the use of dibenzyl ester of glutaric acid instead of the
dimethyl ester did not provide the phosphoric acids 7 by a one-step de-
protection through catalytic hydrogenolysis as expected. It was hypoth-
esized that the initial deprotection at the alpha-carboxylate could react
with the phosphorus center to form a cyclic pentacoordinated interme-
diate similar to that proposed for the N-phosphoryl glutamic acid.9
Methyl dichlorophosphite 13a was available from Aldrich Chemical Company
(Milwaukee, WI, U.S.A.). Propyl dichlorophosphite 13b was prepared as follows. A so-
lution of 1-propanol (7.5 mL, 100 mmol) in diethyl ether (50 mL) was added to a stirring
solution of phosphorus trichloride (9.0 mL, 100 mmol) in diethyl ether (50 mL) at 78 C.
The resulting solution was stirred for 15 min while being allowed to warm to room tem-
perature. The solution was concentrated in vacuo and purified by vacuum distillation
(20 mmHg, 109 C) to give propyl dichlorophosphite as a colorless liquid in 85.7% yield.
1H NMR: (CDCl3) 1.91–1.45 (t, 3H, J = 7.4 Hz), 1.66–1.83 (m, 2H), 4.14–4.25 (dt, 2H,
J = 6.4 and 12.9 Hz). 31P NMR: (CDCl3) 179.09.

20
H. Lu et al.
SCHEME 1
SCHEME 2
Due to the poisoning effect of sulfur on palladium catalysts, thio-
phosphoric acid derivatives 8 were prepared by an alternative ap-
proach shown in Scheme 3 in which 2-cyanoethoxy was incorporated
as the phosphorus protective group rather than benzyloxy. Thus, the
reaction of alkyl dichlorophosphites with (S)-2-hydroxyglutarate 17 and

Fused Heterocycles
21
SCHEME 3
2-cyanoethanol provided phosphites 21, which were summarily thion-
ated with sulfur to give phosphorothionates 22. Subsequent mild basic
hydrolysis with LiOH provided the phosphorothionate diesters 8.
Employing our recently developed amine-exchange reaction on
phosphoramidites,11 the respective phosphoramidate and phospho-
ramidothionate precursors 23 and 25 were prepared by the reaction
of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite with Glu(OMe)2,
followed respectively by oxidation or thionation. Deprotection of 23
or 25 with LiOH provided 9 and 10 respectively. Phosphitylation
of (S)-2-hydroxyglutarate 17 with bis(2-cyanoethyl)-N,N-diisopropyl-
phosphoramidite followed by oxidation or thionation gave the cor-
responding phosphate 24 or thiophosphate 26 respectively. Mild
hydrolysis with LiOH afforded the fully deprotected targets, 11
and 12.
In our ongoing studies with alkyl- and arylphosphonamidothionates,
we have noticed only moderate stability (t1/2 = 35–130 min) when sub-
jected to the conditions of the enzyme incubations. Knowing that these
phoshonamidothionates are prone to desulfurization under such condi-
tions, it was hypothesized that a carbon-to-oxygen isosteric replacement
would impart greater stability of compounds 6 over their alkylphos-
phonamidothionate analogs. Indeed, we observed that for over 16 h,
the phosphoramidothionates 6 were completely stable, exhibiting no
desulfurization under the conditions of the inhibition assay (pH 7.3).
Presumably, this enhanced stability is due to increased stabilization
of the phosphorus monoacid anion along with a correspondingly lower
pKa of the monoacid itself. Incidentally, it also was noted that inhibitors
5 and 7–12 were similarly stable under the assay conditions.

22
H. Lu et al.
SCHEME 4
For the inhibitors that possessed a sulfur ligand (X) on phospho-
rus (Table I; 6, 8, 10, 12) notable differences were observed in their
31P NMR chemical shifts. While the relatively upfield chemical shifts
for inhibitors 10 and 12 at approximately 46 ppm suggest that they
TABLE I Inhibition of CPG by Phosphoryl and Thiophosphoryl Derivatives
of Glutamic and 2-Hydroxyglutaric Acid
Inhibitor
R
X
Y
31P NMRa
Ki ( M)b
9
5a
5b
10
6a
6b
11
7a
7b
12
8a
8b
H
O
O
O
S
NH
NH
NH
NH
NH
NH
O
8.66
10.11
2.1 (0.9730)
65 (0.9079)
31 (0.9589)
0.53 (0.9960)
424 (0.9950)
27 (0.9695)
170 (0.9800)
24 (0.9932)
26 (0.9990)
44 (0.9980)
4.7 (0.9823)
5.7 (0.9995)
CH3
CH3CH2CH2
H
CH3
CH3CH2CH2
H
CH3
CH3CH2CH2
H
CH3
CH3CH2CH2
8.49
46.38
S
58.61, 59.24c
56.36, 57.12c
3.93
S
O
O
O
S
O
1.80
O
0.82
O
46.52
S
S
O
59.55, 60.72
56.91, 58.32
O
aChemical shifts in CD3OD referenced to 85% H3PO4.
bCorrelation coefficient in parenthesis.
cChemical shifts in D2O referenced to 85% H3PO4.

Fused Heterocycles
23
exist as the thiolo tautomer (P-SH), the chemical shifts near 60 ppm
for inhibitors 6 and 8 suggests a preference for thiono tautomer (P S).
The latter assertion is in agreement with a similar observation reported
previously for a series of phosphonamidothionates.12
Once obtained in sufficient quantity, the individual inhibitors 5–12
were examined for inhibitory potency against CPG. It should be noted
that 6a, 6b and 8a, 8b were individually prepared as a mixture of two di-
astereomers differing only by their phosphorus configuration and their
diastereomeric ratios ranged from 1:1.1 to 1:1.3. Resolution of their
respective precursors (16a,b and 22a,b) by flash silica gel chromatog-
raphy were unsuccessful and no further attempts were made to resolve
these diastereomeric inhibitors. As such, 6a, 6b and 8a, 8b were evalu-
ated for inhibitory potency as as individual mixtures of diastereomers.
Employing the hydrolysis of methotrexate4 as the conventional mea-
sure of enzymatic activity, both Km and Vmax were initially determined
(1.3 M and 15 mol min 1 mg-protein 1 respectively). Dixon analyses
were subsequently performed to obtain Ki values for 5–12 (Table I). In
all Dixon analyses, the initial substrate (methotrexate) concentration
was 10 M and triplicate determinations were made and averaged for
each inhibitor concentration, which generally varied within a range
from 5 to 120 M depending on the potency of the individual inhibitors.
Of the series of compounds examined, the most potent inhibitor was
the phosphonamidothionate 10 (Ki = 0.53 M). Although less potent
than 10, inhibitors 8a, 8b, and 9 exhibited strong and similar potency
(Ki = 4.7, 5.7, and 2.1 M respectively) against CPG when compared
to the remaining compounds. The trend observed for inhibition results
of the phosphorus diacids 9–12 did not completely coincide with the
trends observed for the phosphoryl monoacids 5–8. The thiono analogs
10 and 12 were respectively more potent inhibitors than 9 and 11. Thus,
for these phosphoryl diacids (9–12), it was observed that substitution
of one of the oxygen ligands to phosphorus with sulfur generally en-
hanced the inhibitory potency. This observation also was in agreement
with the trend observed for the phosphoryl monoacids derivatives of
2-hydroxyglutaric acid, 7a, 7b and 8a, 8b (Y = O) but contrary to that
observed for the phosphoryl monoacids derivatives of glutamic acid, 5a,
5b and 6a, 6b (Y = NH).
Although the respective isosteric replacement of the P N linkage in
the diacids 9 and 10 (Y = NH) with that of the P O linkage found in
11 and 12 (Y = O) resulted in dramatically weaker inhibitors of CPG,
the converse was generally true for the phosphoryl monoacids 5–8. In
addition, the isosteric replacement of the P N linkage in the monoacids
5a, 5b and 6a, 6b with a P O linkage (7a, 7b and 8a, 8b) appeared to
significantly reduce the importance of the alkoxy ligand. Therefore, the

24
H. Lu et al.
NH ligand may influence the orientation of the phosphorus group in the
enzyme active site, potentially through hydrogen bonding interactions
through the amido hydrogen.
The apparent reduction in the significance of the alkoxy ligands in
7a, 7b and 8a, 8b suggests that these ligands may not be involved
in binding interactions with complimentary features within the acitve
site, thus allowing both the O P O or the S P O moieties to fully
participate in coordinating with the active site zinc ions. For these com-
pounds as well as for the diacids 9–12 which possess no alkoxy ligand,
the S P O moiety appears to provide enhanced inhibitory potency
over O P O , presumably through greater chelation of the active site
zinc ions. In contrast to the trend observed for the 7a, 7b and 8a, 8b
(Y = O), the inhibitory potency of the monoacids 5a, 5b and 6a, 6b
(Y = NH) was indeed dependent upon the length of the alkoxy ligand.
Conclusion
In summary, the procurement of a limited series of phosphoryl and
thiophosphoryl derivatives of (S)-glutamic or (S)-2-hydroxyglutaric
acid was achieved using slight modifications of previously established
methodologies. The significance of such compounds lies with their po-
tential to serve as potent inhibitors of glutamate carboxypeptidases.
Examination of the inhibitory data for these compounds with CPG re-
vealed that the inhibition was sensitive to the individual phosphorus
ligands. The most potent inhibitors were the dibasic phosphoryl (9)
and thiophosphoryl (10) derivatives of glutamic acid and the monoba-
sic thiophosphoryl derivatives of 2-hydroxyglutaric acid (8a, 8b). Even-
tual resolution of the individual phosphorus stereoisomers of 6a, 6b
and 8a, 8b may provide additional information on the effects of phos-
phorus stereochemistry upon the interactions with active site features.
Future studies involving the examination of this or an extended series of
phosphonothioic acids against more medically relevant glutamate car-
boxypeptidase such as NAALADase or PSMA are anticipated to provide
greater insight into the characteristics of those enzymes and allow for
the development of potentially therapeutic agents.
EXPERIMENTAL
Synthesis
General
All solvents used in reactions (benzene, CH2Cl2, THF), 3-
hydroxypropionitrile, diisopropylethylamine (DEA), and triethylamine

Fused Heterocycles
25
(TEA) were freshly distilled prior to use. All other reagents were used
as supplied unless otherwise stated. Liquid (flash) chromatography was
1
carried out using silica gel 60 (230–400 mesh). H, 13C, and 31P NMR
spectra were recorded on a Bruker DRX 300 MHz NMR Spectrome-
ter. 1H NMR chemical shifts are relative to TMS = 0.00 ppm), CDCl3
( = 7.26 ppm), or CD3OD ( = 4.87 and 3.31 ppm). 13C NMR chemical
shifts are relative to CD3OD = 49.15 ppm or CDCl3 ( = 77.23). 31P
NMR chemical shifts in CDCl3, CD3OD, or D2O were externally refer-
enced to 85% H3PO4 ( = 0.00 ppm) in CDCl3, CD3OD, and D2O respec-
tively. Combustion analyses were performed by Quantitative Technolo-
gies Inc. (Whitehouse, NJ). High resolution mass spectra (FAB) were
performed by the University of Notre Dame Mass Spectrometry Facility
(Notre Dame, IN).
General procedure for 2-cyanoethyl alkyl [methyl (1-carbomethoxy-
butanoate)] phosphoramidothionates (16). A solution of H-Glu(OMe)-
OMe (0.58 g, 3.29 mmol) in THF (25 mL) was added dropwise to a solu-
tion of alkyldichlorophosphite (3.31 mmol) and diisopropylethylamine
(0.69 mL, 3.96 mmol) in THF (25 mL) at 78 C. After removal of exter-
nal cooling, stirring was continued for 20 min. The solution was filtered
and concentrated in vacuo. The reaction mixture was dissolved in THF
(25 mL) at 78 C. To this was added a solution of 3-hydroxypropionitrile
(0.25 mL, 3.29 mmol) and diisopropylethylamine (0.69 mL, 3.96 mmol)
in THF (25 mL). The resulting solution was stirred for 20 min while
being allowed to warm to room temperature. The solution was filtered
and sulfur (0.22 g, 6.96 mmol) was added. The solution was stirred at
room temperature for 2 h, filtered, concentrated in vacuo, and purified
by silica gel column chromatography to give a pale yellow oil 16a (hex-
ane:ethyl acetate:dichloromethane 1:1:1, v:v:v, Rf = 0.28) or 16b (hex-
ane:ethyl acetate:dichloromethane 2:1:1, v:v:v, Rf = 0.22). 16a: 1H NMR
(CDCl3) 1.89–2.02 (m, 1H), 2.12–2.25 (m, 1H), 2.37–2.54 (m, 2H), 2.70–
2.82 (m, 2H), 3.63–3.84 (m, 9H), 4.02–4.29 (m, 3H); 31P NMR (CDCl3)
74.10, 74.59 (1.3:1 diastereomers); Anal. Calcd. for C11H19N2O6PS:
C, 39.05; H, 5.66; N, 8.28. Found: C, 38.94; H, 5.54; N, 8.12. 16b:
1H NMR (CDCl3) 0.94–1.01 (m, 3H), 1.67–1.75 (m, 2H), 1.91–2.02
(m, 1H), 2.10–2.20 (m, 1H), 2.43–2.52 (m, 2H), 2.74–2.81 (m, 2H), 3.72
(s, 3H), 3.78 and 3.80 (s, 3H), 3.97–4.02 (m, 2H), 4.03–4.16 (m, 1H), 4.17–
4.24 (m, 2H); 31P NMR (CDCl3) 72.17, 72.62 (1.3:1 diastereomers);
Anal. Calcd. for C13H23N2O6PS: C, 42.62; H, 6.33; N, 7.65. Found: C,
42.54; H, 6.18; N, 7.46.
2-Cyanoethyl methyl [methyl (1-carbomethoxybutanoate)] phospho-
ramidate (15a). Dichloromethane was neutralized with basic alu-
minum oxide and dried over warm molecular sieves prior to use. MMPP

26
H. Lu et al.
(0.31 g, 5.05 mmol) was added to a solution of the phosphoramidothion-
ate 16a (0.15 g, 4.55 mmol) in CH2Cl2 (10 mL). The solution was refluxed
4 h, filtered, concentrated in vacuo, and purified by silica gel column
chromatography (dichloromethane:acetone 2:1, v:v, Rf = 0.20) to give a
pale yellow oil 15a. 1H NMR (CDCl3) 1.99–2.05 (m, 1H), 2.11–2.24 (m,
1H), 2.38–2.57 (m, 2H), 2.73–2.85 (m, 2H), 3.41–3.54 (m, 1H), 3.71–3.83
(m, 9H), 3.88–4.02 (m, 1H), 4.16–4.28 (m, 2H); 31P NMR (CDCl3) 9.54,
9.62 (1.2:1 diastereomers); Anal. Calcd. for C11H19N2O7P: C, 41.00; H,
5.94; N, 8.69. Found: C, 40.97; H, 5.78; N, 8.48.
2-Cyanoethyl propyl [methyl (1-carbomethoxybutanoate)] phospho-
ramidate (15b). The procedure to prepare 15b was identical to those
described for the synthesis of compound 16b with the exception that
the intermediate phosphoramidite 14b was intercepted with mCBPA
rather than sulfur. The resulting reaction mixture was stirred at room
temperature for 15 min, concentrated in vacuo, and redissolved in
CH2Cl2 (20 mL). The solution was extracted twice with 10% wt. sodium
carbonate, dried over MgSO4, filtered, concentrated in vacuo, and puri-
fied by silica gel column chromatography (dichloromethane:acetone 3:1,
v:v, Rf = 0.28) to give a clear yellowish oil 15b. 1H NMR (CDCl3) 0.95–
1.02 (m, 3H), 1.65–1.76 (m, 2H), 1.89–2.01 (m, 1H), 2.09–2.19 (m, 1H),
2.37–2.54 (m, 2H), 2.70–2.83 (m, 2H), 3.29–3.39 (t, 1H), 3.70 (s, 3H),
3.78 (s, 3H), 3.91–4.06 (m, 3H), 4.14–4.31 (m, 2H); 31P NMR (CDCl3)
8.25, 8.35 (1.3:1 diastereomers); Anal. Calcd. for C13H23N2O7P: C,
44.57; H, 6.62; N, 8.00. Found: C, 45.17; H, 6.61; N, 7.51.
General procedure for benzyl alkyl [methyl (1-carbomethoxy-
butanoate)] phosphates (19). Into
a flask charged with benzyl
dichlorophosphite (645 mg, 3.09 mmol) in anhydrous ether (40.0 mL)
was added a solution of (S)-dimethyl 2-hydroxyglutarate (436 mg,
2.47 mmol) and triethylamine (0.43 mL, 3.09 mmol) in anhydrous ether
(15.0 mL) via syringe at 70 C under an argon atmosphere. The result-
ing solution was stirred 0.5 h. A solution of the corresponding alcohol
(6.18 mmol) and TEA (1.2 mL, 6.18 mmol) in anhydrous ether (15.0 mL)
was added dropwise to the reaction mixture ( 70 C), which was then
allowed to warm to ambient temperature. The intermediate phosphite
18 was oxidized by the addition of mCPBA (832 mg, 4.94 mmol). The so-
lution was stirred an additional 1 h, filtered, concentrated in vacuo, and
Benzyldichlorophosphite was prepared as follows: A solution of benzyl alcohol
(100 mmol) in diethyl ether (50 mL) was added to a stirring solution of phosphorous
trichloride (9.0 mL, 100 mmol) in diethyl ether (50 mL) at 78 C. The resulting solution
was stirred for 15 min while being allowed to warm to room temperature. The solution
was concentrated in vacuo and purified by vacuum distillation to give benzyldichlorophos-
phite as a colorless liquid.

Fused Heterocycles
27
purified by flash chromatography to give 19a (59%) or 19b (62%) [ether:
1
methanol 100:1 v/v (19a: Rf = 0.32. 19b: Rf = 0.28)]. 19a: H NMR
(CDCl3) 2.04–2.35 (m, 2H), 2.36–2.66 (m, 2H), 3.67 (d, J = 5.4 Hz,
3H), 3.73 and 3.75 (s, 3H), 3.76 and 3.78 (s, 3H), 4.84–4.93 (m, 1H),
5.11 (d, J = 17.7 Hz, 1H), 5.14 (dd, J = 17.1 Hz, J = 0.8 Hz, 1H),
7.27–7.66 (m, 5H); 31P NMR (CDCl3) 0.29; Anal. Calcd. for C15H21O8P:
1
C, 50.00; H, 5.83; Found: C, 49.68; H, 5.65. 19b: H NMR (CDCl3)
0.93 and 0.92 (t, J = 7.5 Hz, 3H), 1.60–1.74 (m, 2H), 2.10–2.31 (m,
2H), 2.38–2.51 (m, 2H), 3.65 and 3.67 (s, 3H), 3.73 and 3.74 (s, 3H),
3.96–4.09 (m, 2H), 4.81–4.92 (m, 1H), 5.10 (d, J = 16.2 Hz, 1H), 5.13
(dd, J = 15.3 Hz, J = 0.9 Hz, 1H), 7.26–7.55 (m, 5H); 31P NMR (CDCl3)
0.63; Anal. Calcd. for C17H25O8P: C, 52.51; H, 6.44; Found: C, 50.86;
H, 6.36.
General procedure for 2-cyanoethyl alkyl [methyl (1-carbomethoxy-
butanoate)] phosphorothionates (22). A solution of (S)-dimethyl
2-hydroxyglutarate (0.53 g, 3.0 mmol) and triethylamine (0.40 g,
4.0 mmol) in anhydrous ether (20.0 mL) was added via syringe to a
stirring solution of alkyl dichlorophosphite (3.9 mmol) in anhydrous
ether (60 ml) at 70 C under an argon atmosphere. The resulting so-
lution was stirred for 20 min at 70 C, followed by the addition of a
solution of 3-hydroxypropionitrile (0.34 g, 4.8 mmol) and triethylamine
(0.51 g, 5.0 mmol) in anhydrous ether (20.0 ml). The solution was stirred
an additional 20 min and warmed to the room temperature. Sulfur
(0.16 g, 4.78 mmol) was added and the reaction mixture was stirred
overnight. Subsequent filtration and concentration in vacuo afforded
a yellow oil, which was purified by flash chromatography to give the
ester 22a (49%, ether:hexane 2.5:1, v/v, Rf = 0.19) and 22b (52%, ethyl
1
acetate:hexane 3:1, v/v, Rf = 0.41). 22a: H NMR (CDCl3) 2.14–2.30
(dm, 2H), 2.44–2.52 (m, 2H), 2.74–2.84 (m, 2H), 3.69 (s, 3H), 3.79 (s,
3H), 3.79 and 3.84 (d, J = 12.0 Hz, 3H), 4.23–4.38 (m, 2H), 4.96–5.04
(m, 1H); 31P NMR (CDCl3) 70.34, 70.76 (1.3:1 diastereomers); Anal.
Calcd. for C11H18NO7PS: C, 38.94; H, 5.31; N: 4.13; Found: C, 39.23; H,
5.19; N: 4.12. 22b: 1H NMR (CDCl3) 0.97 (t, J = 7.2 Hz, 3H), 1.57–1.78
(m, 2H), 2.08–2.34 (dm, 2H), 2.41–2.58 (m, 2H), 2.72–2.88 (m, 2H), 3.69
(s, 3H), 3.78 (s, 3H), 4.02–4.15 (m, 2H), 4.21–4.38 (m, 2H), 4.95–5.05
(m, 1H); 31P NMR (CDCl3) 68.65, 69.16 (1.1:1 diastereomers); Anal.
Calcd. for C13H22NO7PS: C, 42.51; H, 5.99; N: 3.81; Found: C, 43.07; H,
5.94; N: 3.70.
O,O-Bis(2-cyanoethyl) methyl 1-carbomethoxybutanoate phospho-
ramidate (23) and phosphate (24). 1H-tetrazole (0.08 g, 1.2 mmol) dis-
solved in 6 ml acetonitrile was added to a solution of bis(2-cyanoethyl)-
N0,N0-diisopropylphosphoramidite (0.27 g, 1.0 mmol), L-glutamic acid

28
H. Lu et al.
dimethyl ester hydrochloride (0.25 g, 1.2 mmol) or (S)-dimethyl
2-hydroxyglutarate (0.21 g, 1.2 mmol), and diisopropylethylamine
(0.26 g, 2.0 mmol) in THF (1.5 mL) under an argon atmosphere at
room temperature. The mixture was stirred for 0.5 h, followed by the
addition of tert-butyl hydroperoxide (0.18 g, 2.0 mmol), and stirred an-
other 3 h. After filtration of the resulting reaction mixture, the solvent
was removed under reduced pressure. Purification by flash chromatog-
raphy provided 23 (ethyl acetate, Rf = 0.16) and 24 (ethyl acetate,
Rf = 0.36). 23: 1H NMR (CDCl3)
1.90–2.20 (dm, 2H), 2.36–2.49
(m, 2H), 2.70–2.82 (m, 4H), 3.40–3.62 (m, 1H), 3.70 (s, 3H), 3.78 (s,
3H), 3.86–4.02 (m, 1H), 4.17–4.31 (m, 4H); 31P NMR (CDCl3) 7.54;
Anal. Calcd. for C13H20N3O7P: C, 43.21; H, 5.54; N: 11.63; Found: C,
1
45.18; H, 5.56; N: 11.52. 24: H NMR (CDCl3) 2.12–2.39 (dm, 2H),
2.41–2.59 (m, 2H), 2.73–2.88 (m, 4H), 3.71 (s, 3H), 3.79 (s, 3H), 4.32
(dt, J = 8.1 Hz, J = 6.2 Hz, 2H), 4.41 (dt, J = 8.4 Hz, J = 6.2 Hz, 2H),
4.96 (dt, J = 3.3 Hz, J = 4.5 Hz, 1H); 31P NMR (CDCl3)
2.35; Anal.
Calcd. for C13H19N2O8P: C, 43.09; H, 5.25; N: 6.63; Found: C, 42.84; H,
5.08; N: 7.94.
O,O-Bis(2-cyanoethyl) methyl 1-carbomethoxybutanoate phospho-
ramidothionate (25) and phosphorothionate (26). To a solution of
bis(2-cyanoethyl)-N0,N0-diisopropylphosphoramidite (0.27 g, 1.0 mmol),
L-glutamic acid dimethyl ester hydrochloride (0.25 g, 1.2 mmol) with
diisopropylethylamine (0.26 g, 2.0 mmol) in THF or (S)-dimethyl
2-hydroxyglutarate (0.21 g, 1.2 mmol) in THF (1.5 mL) was added a so-
lution of 1H-tetrazole (0.08 g, 1.2 mmol) in acetonitrile (6 mL) dropwise
under argon at room temperature for 30 min. Sulfur (0.06 g, 2 mmol)
was then added and the reaction mixture was stirred overnight. Evap-
oration of solvent under reduced pressure followed by purification by
flash chromatography gave 25 ( ether, Rf = 0.21) or 26 (hexane:ether
1:9, Rf = 0.42). 25: 1H NMR (CDCl3) 1.72–1.85 (m, 1H), 1.95–2.11 (m,
1H), 2.31–2.38 (m, 2H), 2.61–2.70 (m, 4H), 3.59 (s, 3H), 3.67 (s, 3H),
3.80–4.05 (m, 1H), 4.07–4.16 (m, 4H); 31P NMR (CDCl3) 71.88; Anal.
Calcd. for C13H20N3O6PS: C, 41.38; H, 5.31; N: 11.14; Found: C, 41.41;
H, 5.21; N: 11.01. 26: 1H NMR (CDCl3) 2.06–2.15 (m, 1H), 2.17–2.29
(m, 1H), 2.33–2.48 (m, 2H), 2.72–2.84 (m, 4H), 3.63 (s, 3H), 3.73 (s, 3H),
4.21–4.34 (m, 4H), 4.91–4.99 (m, 1H); 31P NMR (CDCl3) 67.66; Anal.
Calcd. for C13H19N2O7PS: C, 41.27; H, 5.03; N: 7.41; Found: C, 41.18;
H, 4.94; N: 7.56.
General Procedure for 5, 6, 8, 9, 10, 11, 12
Phosphoramidate 15 or 23, phosphoramidothionate 16 or 25, phos-
phorothionates 22 or 26, or phosphate 24 (0.5 mmol) was dissolved in

Fused Heterocycles
29
methanol (2.5 mL) to which was added aqueous LiOH (2.5 mL, 1.0 M).
The resulting solution was stirred at room temperature for 16 h and
then filtered. The solvent was evaporated in vacuo to give the corre-
sponding crude products. The residue was resuspended in anhydrous
methanol, filtered (0.45 M Teflon membrane), and concentrated in
vacuo to give the trilithium salt of the desired product as a white
solid.
Methyl hydrogen (1-carboxybutanoic acid) phosphoramidate (5a).
1H NMR (CD3OD) 1.72–1.98 (m, 2H), 2.10–2.29 (m, 2H), 3.42 (d,
J = 10.8 Hz, 3H), 3.40–3.61 (m, 1H); 13C NMR (CD3OD) 33.86, 35.52,
52.28 (d, J = 4.6 Hz), 58.26, 182.37 (d, J = 3.4 Hz), 183.40; 31P NMR
(CD3OD) 10.11; FABHRMS (M-Li) calcd. 252.0437, found 252.0460
for C6H9Li2NO7P.
1
Propyl hydrogen (1-carboxybutanoic acid) phosphoramidate (5b). H
NMR (CD3OD) 0.88 (t, J = 7.5 Hz, 3H), 1.46–1.58 (m, 2H), 1.81–2.02
(m, 2H), 2.13–2.41 (m, 2H), 3.48–3.60 (m, 1H), 3.61–3.72 (m, 2H); 13C
NMR (CD3OD) 10.98, 25.37, 33.91, 35.52, 58.22, 67.28, 182.48, 183.51;
31P NMR (CD3OD) 8.49; FABHRMS (M-Li) calcd. 280.0750, found
280.0759 for C8H13Li2NO7P.
Methyl hydrogen (1-carboxybutanoic acid) phosphoramidothionate
1
(6a). H NMR (CD3OD) 1.58–1.82 (m, 2H), 1.98–2.12 (m, 2H), 3.31
and 3.34 (d, J = 13.2 Hz, 3H), 3.42–3.58 (m, 1H); 13C NMR (CD3OD)
33.65 (d, J = 11.1 Hz), 35.46 (d, J = 6.2 Hz), 52.68 (dd, J = 4.2 Hz,
J = 17.1 Hz), 58.24, 181.90 (dd, J = 4.9 Hz, J = 11.8 Hz), 183.43 (d,
J = 14.0 Hz); 31P NMR (CD3OD) 60.90, (D2O) 58.61, 59.24 (1:1.3 di-
astereomers); FABHRMS (M-Li) calcd. 268.0209, found 268.0214 for
C6H9Li2NO6PS.
Propyl hydrogen (1-carboxybutanoic acid) phosphoramidothionate
1
(6b). H NMR (CD3OD) 0.87 and 0.88 (t, J = 7.2 Hz, 3H), 1.42–1.60
(m, 2H), 1.72–1.93 (m, 2H), 2.08–2.30 (m, 2H), 3.58–3.80 (m, 3H); 13C
NMR (CD3OD) 11.05, 25.08 (d, J = 8.8 Hz), 33.78 (d, J = 4.7 Hz), 35.44
(d, J = 11.5 Hz), 58.22, 67.87 (dd, J = 6.1 Hz, J = 12.3 Hz), 181.90 (dd,
J = 5.9 Hz, J = 20.1 Hz), 183.46 (d, J = 19.3 Hz); 31P NMR (CD3OD)
58.88, 58.92, (D2O) 56.36, 57.12 (1:1.3 diastereomers); FABHRMS
(M-Li) calcd. 296.0522, found 296.0535 for C8H13Li2NO6PS.
Methyl hydrogen (1-carboxybutanoic acid) phosphorothionate (8a).
1H NMR (CD3OD) 1.92–2.03 (m, 2H), 2.16–2.26 (m, 2H), 3.45 and 3.46
(d, J = 13.6 Hz, 3H), 4.52–4.63 (m, 1H); 13C NMR (CD3OD) 32.29 (d,
J = 35.4 Hz), 35.03 (d, J = 53.1 Hz), 53.50 (d, J = 22.8 Hz), 78.62 (d, J =
112.5 Hz), 179.73 and 179.86, 182.95 and 183.05; 31P NMR (CD3OD)

30
H. Lu et al.
59.55, 60.72 (1.3:1 diastereomers); FABHRMS (M-Li) calcd.
269.0049, found 269.0035 for C6H8Li2O7PS.
Propyl hydrogen (1-carboxybutanoic acid) phosphorothionate (8b).
1H NMR (CD3OD) 0.90 and 0.91 (t, J = 7.4 Hz, 3H), 1.53–1.66 (m,
2H), 2.02–2.17 (m, 2H), 2.22–2.38 (m, 2H), 3.78–3.89 (m, 2H), 4.60–4.77
(m, 1H); 13C NMR (CD3OD) 10.87, 24.98 (d, J = 34.5 Hz), 32.24 and
32.43 (d, J = 25.8 Hz), 34.92 and 35.16, 68.70 and 68.87 (d, J = 24.9 Hz),
78.21 and 78.80 (d, J = 23.4 Hz), 179.67 and 179.86, 182.89 and 183.01;
31P NMR (CD3OD) 56.91, 58.32 (1:1.1 diastereomers); FABHRMS
(M-Li) calcd. 297.0362, found 297.0356 for C8H12Li2O7PS.
1
N-(1-Carboxybutanoic acid) phosphoramidic acid (9). H NMR
(CD3OD) 1.66–1.74 (m, 2H), 1.94–2.05 (m, 2H), 3.35–3.40 (m, 1H);
13C NMR (CD3OD) 29.87, 34.07, 57.25, 181.97, 183.21; 31P NMR
(CD3OD) 8.66; FABHRMS (M-Li) calcd. 244.0362, found 244.0364
for C5H6Li3NO7P.
1
N-(1-Carboxybutanoic acid) phosphoramidothionic acid (10). H
NMR (CD3OD) 1.83–1.99 (m, 2H), 2.14–2.36 (m, 2H), 3.65–3.73 (m,
1H); 13C NMR (CD3OD) 30.29, 33.80, 57.53, 182.14, 183.66; 31P NMR
(CD3OD) 46.38; FABHRMS (M-Li) calcd. 260.0134, found 260.0107
for C5H6Li3NO6PS.
1
O-(1-Carboxybutanoic acid) phosphoric acid (11). H NMR (CD3OD)
1.72–1.93 (m, 2H), 1.96–2.08 (m, 1H), 2.12–2.24 (m, 1H), 4.19–4.30 (m,
1H); 13C NMR (CD3OD) 28.44, 30.61, 71.71, 178.28, 180.91; 31P NMR
(CD3OD) 3.93; FABHRMS (M-Li) calcd. 245.0202, found 245.0221
for C5H5Li3O8P.
1
O-(1-Carboxybutanoic acid) phosphorothionic acid (12). H NMR
(CD3OD) 1.95–2.19 (dm, 2H), 2.29–2.33 (m, 2H), 4.50–4.56 (m, 1H);
13C NMR (CD3OD) 31.43 (d, J = 4.0 Hz), 33.90, 76.06 (d, J = 5.7 Hz),
180.70 (d, J = 4.9 Hz), 182.72; 31P NMR (300 MHz, CD3OD) 46.52;
FABHRMS (M-Li) calcd. 260.9974, found 260.9967 for C5H5Li3O7PS.
General Procedure for Alkyl Hydrogen
(1-Carboxybutanoic Acid) Phosphates (7)
A mixture of 19 (0.33 mmol) and palladium on carbon (5%, 26 mg)
in methanol (12 ml) was stirred for 3 h under a hydrogen atmosphere.
The resulting reaction mixture was filtered and concentrated in vacuo
to give 20 as a yellow oil. Intermediate 20 was dissolved in methanol
(2 mL) to which was added aqueous lithium hydroxide (2 mL, 1.0 M).
The resulting solution was stirred at room temperature for 16 h, filtered,
and the solvent evaporated in vacuo to give a white residue. The residue

Fused Heterocycles
31
was resuspended in anhydrous methanol, filtered (0.2 m Teflon mem-
brane), and concentrated in vacuo to give the trilithium salt of the de-
sired product 7 as a white solid. 7a: 1H NMR (CD3OD) 1.95–2.06 (m,
2H), 2.10–2.28 (m, 2H), 3.50 (d, J = 10.9 Hz, 3H), 4.36–4.42 (m, 1H); 13
C
NMR (CD3OD) 32.39 (d, J = 16.5 Hz), 34.97, 53.33 (d, J = 21.0 Hz),
77.86 (d, J = 22.2 Hz), 179.81, 182.78; 31P NMR (CD3OD)
1.80;
FABHRMS (M-Li) calcd. 253.0277, found 253.0294 for C6H8Li2O8P.
1
7b: H NMR (CD3OD) 0.85 (t, J = 7.2 Hz, 3H), 1.47–1.59 (m, 2H),
1.98–2.03 (m, 2H), 2.04–2.27 (m, 2H), 3.70–3.80 (m, 2H), 4.37–4.43 (m,
1H); 13C NMR (CD3OD) 10.83, 25.22 (d, J = 32.4 Hz), 32.48, 34.96,
68.33 (d, J = 19.2 Hz), 77.75 (d, J = 21.9 Hz), 179.81, 182.82; 31P NMR
(CD3OD) 0.82; FABHRMS (M-Li) calcd. 281.0590, found 281.0582
for C8H12Li2O8P.
Enzyme Inhibition
General
Glutamate carboxypeptidase (carboxypeptidase G from Pseudo-
monas sp. strain ATCC 25301, C 4053) and methotrexate were obtained
from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of
the highest purity and purchased from commercial sources.
Determination of Km and Vmax. Each incubation mixture (final vol-
ume 0.25 mL) was prepared by the addition of 200 L TRIS buffer
(50 mM, pH 7.3) to 25 L methotrexate (concentration varied from
10 to 100 M) dissolved in this same buffer. The enzymatic reac-
tion was initiated by the addition of 25 L of an enzyme solution
(0.17–0.21 g protein/mL buffer). The reaction was allowed to proceed
for one minute with constant shaking at 30 C and was terminated by
the addition of 100 L methanolic TFA (1% trifluoroacetic acid by vol-
ume in methanol) followed by vortexing and centrifugation (7000 g).
A 100 L aliquot of the resulting supernatant was subsequently
quantified by HPLC. Methotrexate and its hydrolytic product (4-(N-
[2,4-diamino-6-pteridinylmethyl]-N-methylamino)benzoic acid) were
separated and quantified with an analytical reversed phase HPLC col-
umn (4.6 150 mm, Sphereclone 5u ODS(2), Phenomenex, Torrence,
CA) with a mobile phase of CH3OH/[potassium phosphate, 50 mM,
pH 6.8] (22:78. v:v). At a flow rate of 0.9 mL/min, methotrexate and its
hydrolytic product were detected at 304 nm with retention times of 3.8
and 7.7 min respectively. For the determination of Km and Vmax careful
time-course studies (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 min) were performed
to define the linear portion of the initial rate (0-2 min) for the enzymatic
reaction. Under the assay conditions described above, it was noted that

32
H. Lu et al.
the initial substrate concentration was not substantially depleted dur-
ing the time course of the incubation (e.g., approximately 10% conver-
sion to product was observed for incubations with the lowest substrate
concentration, 1 M).
Inhibition assay procedures. A typical incubation mixture (final vol-
ume 0.25 mL) was prepared by the addition of 200 L TRIS buffer
(50 mM, pH 7.3) to either a 25 L mixture of both methotrexate and
inhibitor in buffer or 25 L of a buffered solution of methotrexate alone.
The enzymatic reaction was initiated by the addition of 25 L of an en-
zyme solution (0.17–0.21 g protein/mL buffer). In all cases, the final
concentration of methotrexate was 10 M while the inhibitor concen-
tration varied from 7.5 to 120 M. The reaction was allowed to proceed
for one minute with constant shaking at 30 C and was terminated by
the addition of 100 L methanolic TFA (1% trifluoroacetic acid by vol-
ume in methanol) followed by vortexing and centrifugation (7000 g).
A 100 L aliquot of the resulting supernatant was then quantified by
HPLC as described above.
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