Synthesis and in vitro evaluation of aspartate transcarbamoylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.09.045

The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were sy

Full text of DOI:10.1016/j.bmc.2009.09.045

Bioorganic & Medicinal Chemistry 17 (2009) 7680–7689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of aspartate transcarbamoylase inhibitors
*
Laëtitia Coudray, Anne F. Pennebaker, Jean-Luc Montchamp
Department of Chemistry, Texas Christian University, Box 298860, Fort Worth, TX 76129, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase
(ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-
carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transi-
tion-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at
the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of
Received 17 August 2009
Revised 18 September 2009
Accepted 22 September 2009
Available online 30 September 2009
the synthesized inhibitors is more potent than N-phosphonacetyl-L-aspartate (PALA), the compounds
provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or
prodrugs.
Keywords:
Enzyme inhibitor
Aspartate transcarbamoylase
Phosphinate
Ó 2009 Elsevier Ltd. All rights reserved.
Phosphonate
1. Introduction
and because it came close to passing all clinical trials for use as a
single anticancer agent, much work has been devoted in order to
Aspartate transcarbamoylase (ATCase) catalyzes the second step
of the de novo pyrimidine biosynthesis, during which -aspartic acid
(Asp) is condensed with carbamyl phosphate (CP) to produce car-
find an even more potent and selective inhibitors of ATCase.6
Unfortunately, in spite of more than 35 years of research, PALA re-
mains the most potent ATCase inhibitor known to date.
L
bamoyl-L
-aspartate (C-Asp) and inorganic phosphate (Eq. 1).1
H
H
H
O
O
O
H
-
-
HO
H2N
CO2
CO2
-
N
CO2H
CO2H
N
CO2H
CO2H
N
CO2H
CO2H
H2N
N
CO2
CO2
ATCase
O
HO
P
H
P
P
2-
+
NH2
+ HOPO3
-2O3PO
OH
O
OH
O
OH
O
ð1Þ
O
-
CP
Asp
C-Asp
1
2
3
Inhibitors of the pyrimidine biosynthesis1 have been investi-
gated as anticancer agents, and particularly, the potent ATCase
In a communication,7 we recently reported the synthesis and
inhibitory activity of the novel phosphinates 2 and 3, which were
26- and 12-fold less potent than PALA, respectively (Ki(2) =
417 nM, Ki(3) = 193 nM), but with a reduced overall charge. These
compounds were the first submicromolar-level inhibitors of AT-
Case that did not have PALA’s phosphonic acid moiety.7
Herein, we present the full study for a series of compounds pos-
sessing various replacements for PALA’s phosphonacetyl moiety,
especially phosphinic acids which have a reduced charge around
phosphorus.
inhibitor N-phosphonacetyl-L-aspartate 1 (PALA, Ki = 16 nM) has
been evaluated in cancer chemotherapy.2 In several studies, PALA
was found to be a valuable agent, even though its current clinical
utility appears limited to combination therapies. This inhibitor
eradicated solid tumors in mice, but murine leukemias were resis-
tant.3 A combination therapy consisting of PALA and dipyridamole
showed some effectiveness on various resistant cancer cells.4
A
comprehensive summary of preclinical and clinical studies with
PALA has appeared.5 PALA displays broad spectrum antitumor
activity against solid tumors in a dose-dependent manner. Resis-
tance was due to increased ATCase activity and expression, and
PALA did not inhibit completely the de novo pyrimidine biosynthe-
sis, because of its low bioavailability (with the presence of a possi-
ble total of four negative charges, PALA does not pass through
membranes easily). Because PALA is unique in its mode of action
2. Results and discussion
2.1. Synthesis
2.1.1. Synthesis of phosphinates
Since our initial report,7 we have developed and improved upon
the synthesis of H-phosphinate 2 starting from isoprenyl-H-phos-
phinic acid 4. This is shown in Scheme 1. The key building blocks
* Corresponding author. Tel.: +1 817 257 6201; fax: + 1 817 257 5851.
E-mail address: j.montchamp@tcu.edu (J.-L. Montchamp).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.045

L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
7681
O
P
H
O
O
a or b
c
d, e
EtO
EtO
EtO
EtO
OH
HO
P
P
OEt
OEt O
4
5
6
H
O
P
H
H
O
EtO
EtO
N
CO2H
CO2H
f
g, h
N
CO2Bn
CO2Bn
HO
P
O
OEt
O
2
7
Scheme 1. Reagents and conditions: (a) H3PO2 (1.0 equiv), Pd2dba3 (0.125 mol %), xantphos (0.275 mol %), DMF, 85 °C, 13 h, sealed tube, 83%; (b) H3PO2 (1.5 equiv),
nixantphos catalyst (1.2 mol %), ACN, 85 °C, 15 h, sealed tube, 5 runs, 100%; (c) CH3C(OEt)3 (6.0 equiv), BF3ꢀOEt2 (0.2 equiv), rt, N2, 14 h, 64%; (d) O3, CH2Cl2, ꢁ78 °C, then Me2S
(6.8 equiv), ꢁ78 °C to rt, N2, 14 h; (e) NaClO2 (1.5 equiv), NaH2PO4ꢀH2O (1.5 equiv), 2-methyl-2-butene (2.0 equiv), tBuOH/H2O, 0 °C then rt, 1 h; (f)
L-aspartic acid dibenzyl
ester p-toluenesulfonate (1.0 equiv), DMAP (3.0 equiv), EDC (3.0 equiv), Et3N (1.1 equiv), rt, 17 h, N2, THF, 66% from 5; (g) H2, Pd/C, THF/H2O, 17 h; (h) Amberlite IR 120 plus,
THF/H2O, 80 °C, 15 h, 57% from 7 after ion exchange chromatography.
5 and 6 are also employed for the synthesis of other inhibitors de-
scribed in the present work. Isoprenyl-H-phosphinic acid 4 was
prepared through our own palladium-catalyzed hydrophosphiny-
lation of isoprene.8 Two methods were specifically developed for
this step: either the homogeneous reaction9 using a low palladium
loading (0.25 mol %), or the heterogeneous reaction using our reus-
able polystyrene–nixantphos catalyst10 (1 mol %). The latter meth-
od is ideal for the multigram preparation of 4, since multiple runs
can be conducted by simply filtering the catalyst at the end of one
run and adding it to another. No loss of catalytic activity was ob-
served after five runs, and the overall yield of 4 was quantitative.
Compound 4 was protected with triethyl orthoacetate/BF3ꢀOEt2
to provide the key intermediate 5, as we described previously.11
Ozonolysis of 5 followed by oxidation with NaClO2 delivers carbox-
ylic acid 6, which was used without purification to prepare amide
7. Deprotection of 7, through debenzylation then acid hydrolysis of
the acetal protecting group, produces H-phosphinate inhibitor 2. In
our original synthesis of 2,7 carboxylic acid 6 was prepared from
protected cinnamyl-H-phosphinate instead of protected isopre-
nyl-H-phosphinate 5. During the ozonolysis/oxidation sequence
of the cinnamyl group, benzoic acid is formed thus requiring the
use of larger quantities of reagents, since it also reacts to form N-
would require oxidative mechanisms such as P450, which are not
operating during assays on cell lines. Since 2 is itself a relatively
good ATCase inhibitor, if it were activated in vivo to PALA, signifi-
cant improvements in the concentration that might be achieved at
the tumor site might be observed. As a result of the special poten-
tial of 2, we decided to prepare a series of partial esters 9–11
(Scheme 2). Again, isoprenyl 5 was converted into the correspond-
ing carboxylic acid and coupled as in Scheme 1, but this time with
L-aspartic acid diethyl ester to afford 8. Acid hydrolysis of 8 was
conducted under controlled conditions to obtain an easily sepa-
rated mixture of the two compounds 9 and 10.
Debenzylation of 7 followed by acid hydrolysis under controlled
conditions (1 h at 80 °C) afforded ethyl ester 11. The syntheses of
compounds 9–11 illustrate approaches to novel PALA prodrugs,
especially since H-phosphinates can be easily converted into phos-
phonate esters (and other functionalities) through oxidative func-
tionalization (e.g., chlorination with oxalyl chloride or CCl4 and
reaction of the resulting phosphonochloridate with nucleophiles
such as amines and alcohols).13 Exploiting intermediates 7 and 8
for the purpose of PALA prodrug synthesis will be the object of fu-
ture studies.
Since the proposed ordered cyclic transition-state for the proton
transfer from nitrogen to phosphate oxygen in the reaction be-
tween Asp and CP catalyzed by ATCase (Fig. 1, X-ray structures
are generally drawn as Transition-State O-axial)14 is reasonable
and consistent with the inhibition observed with 2 and 3, some
heterocyclic analogs were synthesized next.
benzoyl-L-aspartic acid dibenzyl ester. With isoprenyl 5, acetone
is formed during ozonolysis and this cannot lead to a carboxylic
acid, not to mention the fact that acetone is easily evaporated be-
fore the NaClO2 step.
Our interest in H-phosphinate 2 was twofold: (1) the P–H ana-
log has a reduced charge (it is less hydrophilic than PALA), so high-
er concentrations in cells could potentially be attained, improving
upon PALA’s bioavailability; and (2) this compound may be oxi-
dized in vivo to PALA thus generating an even more potent inhib-
itor. There are indications that H-phosphinic acids can be oxidized
in vivo to the corresponding phosphonic acids.12 However, this
The simplest heterocycle 14 was synthesized from compound
12 as shown in Scheme 3. We have previously described the prep-
aration of 12 (although the bis-isoprenyl analog would be better,
we have not investigated this possibility).15 Double ozonolysis of
12 followed by treatment with dimethylsulfide provided the corre-
sponding dialdehyde15 (along with benzaldehyde) which was
H
H
O
P
H
O
P
H
H
N
O
N
CO2Et
CO2Et
N
CO2Et
CO2Et
EtO
EtO
O
EtO
EtO
CO2Et
CO2Et
d
EtO
HO
a, b, c
+
P
P
O
O
OEt
O
OEt
5
8
9
10
H
H
O
O
P
EtO
EtO
N
CO2Bn
CO2Bn
N
CO2H
CO2H
e, f
P
EtO
OEt
O
H
O
7
11
Scheme 2. Reagents and conditions: (a) O3, CH2Cl2, ꢁ78 °C then Me2S (6.8 equiv), ꢁ78 °C to rt, N2, 14 h; (b) NaClO2 (1.5 equiv), NaH2PO4ꢀH2O (1.5 equiv), 2-methyl-2-butene
(2.0 equiv), tBuOH/H2O, 0 °C then rt, 1 h; (c) -aspartic acid diethyl ester hydrochloride (1.0 equiv), DMAP (3.0 equiv), EDC (3.0 equiv), Et3N (1.1 equiv), rt, 17 h, N2, THF, 50%
from 5; (d) Amberlite IR 120 plus, THF/H2O, 80 °C, 5 h, 37% for 9 and 60% for 10; (e) H2, Pd/C, THF/H2O, 23 h; (f) Amberlite IR 120 plus, THF/H2O, 80 °C, 1 h, 59% from 7.
L

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L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
H
O
P
O
P
O
P
H
N
N
CO2Bn
CO2Bn
O
NH2
O
CO2Bn
CO2Bn
δ
δ
a, b
Br
H
H
HO
Ph
P
H
OH
O
O
O
O
O
CO2
CO2
O
CO2
CO2
+
O
HO
O
N
H
O
N
H
Ph
δ
δ
H2N
O
15
16
17
Transition-State O-axial
Transition-State O-equatorial
O
N
O
N
Figure 1. Postulated transition-state.
O
O
CO2Bn
CO2Bn
CO2H
CO2H
d
P
c
P
O
HO
immediately reacted under reductive amination conditions to pro-
duce compound 13. Debenzylation then afforded heterocycle 14.
The more elaborate ‘cyclo-PALA’ heterocycle 19 was synthe-
sized according to Scheme 4. The known hydroxymethyl-H-phos-
Ph
Ph
18
19
-aspartic acid dibenzyl
Scheme 4. Reagents and conditions: (a) N-(bromoacetyl)-
L
phinic acid 1516 undergoes
a
sila-Arbuzov reaction17 with
ester 16 (1.0 equiv), HMDS (2.5 equiv), TMSCl (2.5 equiv), toluene, reflux, 14 h; (b)
Ph2CN2, toluene, 50% from 15; (c) DIAD (1.0 equiv), Ph3P (1.0 equiv), THF, 30 min, rt;
(d) H2, Pd/C, THF/H2O, 24 h, 44% from 17.
bromide 16, and the crude mixture is esterified with diphenyldia-
zomethane7,18 to afford 17 (hydrogenolysis of 17 gives hydroxy-
methyl inhibitor 3 in quantitative yield). Intermediate 17 was
cyclized using Mitsunobu conditions19 to produce crude 18, which
was hydrogenolyzed directly to the five-membered amide 19.
The six-membered ‘cyclo-PALA’ 27 was then synthesized as
shown in Scheme 5. Isoprenyl-H-phosphinic acid 4 reacted with
bromide 16 under sila-Arbuzov conditions,17 and the resulting
crude phosphinic acid was esterified to 20, using BnBr/Ag2O.20
Ozonolysis and reduction with sodium borohydride afforded inter-
mediate 21. Since hydroxymethyl 3 was a potent inhibitor of AT-
Case, 21 was hydrogenolyzed to the homologous hydroxyethyl
22 for evaluation.
Compound 20 was converted into 23 using an ozonolysis/oxida-
tion/esterification sequence in 63% overall yield. The esterification
of the intermediate carboxylic acid was conducted with Ph2CN2
to facilitate purification. Hydrogenolysis of 23 afforded compound
24.
To prepare 27, compound 21 was cyclized using Mitsunobu
conditions19 as for the five-membered amide, except this time,
some vinyl phosphinate 25 also formed. In order to separate 26
from the impurities and 25, the crude mixture was ozonolyzed be-
fore chromatography. Finally, compound 26 was hydrogenolyzed
to deliver heterocyclic amide 27.
silver oxide20 and dibenzylphosphate to afford intermediate 35.
Hydrogenolysis then provided phosphate 36 in quantitative yield.
The simple two-step sequence thus produced 36 in outstanding
yield from 16.
2.1.3. Synthesis of carboxylic analogs
Some compounds where the phosphonate moiety of PALA is re-
placed with carboxylates were synthesized as shown in Scheme 8.
Di-tert-butyl malonate was alkylated with bromide 16 under stan-
dard conditions to afford 37. Hydrolysis of the tert-butyl ester
groups, followed by hydrogenolysis produced malonate 38. Esteri-
fication of malonic acid monobenzyl ester using EDC afforded
perbenzylated intermediate 39. Again hydrogenolysis provided
carboxylate 40.7 Finally, homocarboxylate 42 was synthesized
from succinic anhydride through reaction with 30, followed by
esterification with Ph2CN2.18 This was done so that intermediate
41 can be purified easily. Hydrogenolysis then afforded homocarb-
oxylate 42.
18
2.2. Biological evaluation
With a large number of potential inhibitors now at hand, eval-
uation against ATCase could be determined. The compounds were
evaluated against the catalytic subunit of Escherichia coli ATCase,
which was purified from the strain/plasmid combination pEK17/
EK1104 as previously described by Kantrowitz and co-workers24
The colorimetric assay also developed by Kantrowitz and co-work-
2.1.2. Synthesis of bi-substrate/reactive intermediate analogs
PALA behaves as a bi-substrate analog. In spite of the many
compounds that have been synthesized over the years,6 several rel-
atively simple bi-substrate analogs have never been investigated.
In this section, we describe three of these analogs. The first two tar-
gets were carbamate 32 and urea 34 (Scheme 6).
The phosphonate moiety for each was prepared through the
reaction of dibenzylphosphite with paraformaldehyde to form
28,21 or with 1,3,5-tribenzylhexahydro-1,3,5-triazine to form
29.22 Amine salt 30 was converted to the corresponding isocyanate
using triphosgene, as reported in the literature.23 Addition of 28
gave carbamate 31, whereas addition of 29 gave urea 33. In both
cases, the yield was moderate because isocyanate formation also
leads to the symmetrical urea derived from 30.23
ers,25 was used to detect the formation of N-carbamoyl-
and inhibition was determined relative to carbamyl phosphate
(Km = 28 M). Under these conditions, PALA 1 has an inhibition
L-aspartate,
l
constant of Ki = 16 nM.7 The results for the phosphorus-containing
compounds are collected in Table 1. Compounds 9–11 (Scheme 2)
were not tested against ATCase since they are expected to be inac-
tive on the purified enzyme. The partial esters of H-phosphinate 2
were prepared as possible prodrugs of H-phosphinate 2, which it-
self is a potential in vivo, oxidatively-activated prodrug of PALA 1.
Phosphorus-free carboxylate compounds 38, 40, and 42 were
synthesized because similar carboxylate-containing compounds
significantly inhibited the enzyme 3-dehydroquinate synthase
The third bi-substrate analog is phosphate 36 (Scheme 7) in
which the position of the carbon and oxygen has been inverted
compared to carbamate 32. Bromide 16 was phosphorylated using
Ph
O
CO2H
CO2H
O
CO2Bn
CO2Bn
O
c
a, b
P
N
P
N
P
HO
BnO
BnO
Ph
12
13
14
L-aspartic acid dibenzyl ester p-toluenesulfonate (3.0 equiv),
Scheme 3. Reagents and conditions: (a) O3, CH2Cl2, ꢁ78 °C then Me2S (12.0 equiv), ꢁ78 °C to rt, N2, 14 h; (b)
NaCNBH3 (7.7 equiv), Et3N (3.0 equiv), AcOH to pH 4–6, rt, 4 h, ACN, 54%; (c) H2, Pd/C, THF/H2O, 24 h, 85%.

L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
7683
H
N
O
P
H
N
HO
O
P
OH
O
P
H
N
O
P
HO
CO2Bn
CO2Bn
a, b
c, d, e
f
CO2H
CO2H
HO
H
CO2Bn
CO2Bn
OBn O
O
OBn O
21
22
4
20
O
P
i, c, j
c, d, g, h
CONHAsp-Bn
OBn
25
O
H
N
CONHAsp-Bn
O
P
OH
HO2C
O
O
P
CO2H
CO2H
Ph2CHO2C
O
CO2H
f
f
O
CO2Bn
CO2Bn
P
N
O
HO
P
N
CO2H
OBn
BnO
24
26
27
23
Scheme 5. Reagents and conditions: (a) N-(bromoacetyl)-L-aspartic acid dibenzyl ester 16 (1.0 equiv), HMDS (2.5 equiv), TMSCl (2.5 equiv), toluene, reflux, 14 h; (b) BnBr
(1.2 equiv), Ag2O (0.6 equiv), CHCl3, reflux, N2, 2 h, 59% from 4; (c) O3, CH2Cl2, ꢁ78 °C; (d) Me2S (7.0 equiv), ꢁ78 °C to rt, N2, 14 h; (e) NaBH4 (1.9 equiv), MeOH, 0 °C, 1 h, 61%
from 20; (f) H2, Pd/C, THF/H2O, 24 h, 100% for 22, 94% for 24, 100% for 27; (g) NaClO2 (1.5 equiv), NaH2PO4ꢀH2O (1.5 equiv), 2-methyl-2-butene (2.0 equiv), tBuOH/H2O, 0 °C
then rt, 1 h; (h) Ph2CN2, toluene, rt, 63% from 20; (i) DIAD (1.0 equiv), Ph3P (1.0 equiv), THF (0.05 M), 30 min, rt; (j) Me2S (7.0 equiv), ꢁ78 °C to rt, N2, 14 h, 33% from 21.
O
P
O
P
O
P
BnO
BnO
a
b
BnO
BnO
BnO
BnO
NHBn
OH
H
28
29
O
CO2H
CO2H
O
CO2Bn
CO2Bn
O
P
TsOH•H2N
CO2Bn
CO2Bn
e
e
c, d
O
P
HO
HO
CO2H
BnO
BnO
O
N
H
O
N
H
30
31
32
O
O
P
O
CO2Bn
CO2Bn
HO
HO
CO2H
O
P
c, f
N
H
N
H
BnO
BnO
30
N
N
H
Bn
33
34
Scheme 6. Reagents and conditions: (a) (CH2O)n (1.0 equiv), Et3N (0.11 equiv), 50 °C, 45 min then 90 °C, 1.5 h, 61%; (b) 1,3,5-tribenzylhexahydro-1,3,5-triazine (0.33 equiv),
toluene, reflux, 14 h, 73%; (c) 30 (1.2 equiv), Et3N (4.8 equiv), CH2Cl2, triphosgene (0.6 equiv), ꢁ15 °C, 2 h then rt, 1 h; (d) hydroxymethyl-phosphonic acid dibenzyl ester 28
(1.0 equiv), rt, 17 h, 50% from 30; (e) H2, Pd/C, THF/H2O, 24 h, 100% for both 32 and 34; (f) 29 (1.0 equiv), rt, 17 h, 43% from 30.
O
O
P
O
P
H
HO
HO
H
a
b
BnO
BnO
BnO
BnO
N
CO2H
CO2H
P
N
CO2Bn
CO2Bn
OH
O
O
O
O
35
36
Scheme 7. Reagents and conditions: (a) N-(bromoacetyl)-L-aspartic acid dibenzyl ester 16 (1.0 equiv), Ag2O (1.0 equiv), CH3CN, 60 °C, 19 h, 94%; (b) H2, Pd/C, THF/H2O, 24 h,
100%.
(DHQS),26 and replacing a phosphonate with carboxylate(s) has
significant implications for the design of prodrugs.27 DHQS was
established as an example of an enzyme which exploits a phos-
phate residue in its substrate to achieve catalytic efficiency.28 In-
deed, the same is postulated for ATCase ( Fig. 1). The enzyme
DHQS is a prime example of the elegant and efficient way nature
has evolved catalysis. Scheme 9 shows the mechanistic analogy be-
tween ATCase and DHQS.
With ATCase, nucleophilic attack of carbamyl phosphate by
aspartate forms the tetrahedral intermediate A. Since a dianionic
phosphate is one of the strongest base in nature, one of the phos-
phate oxygen can intramolecularly abstract the proton on the
nitrogen atom (transition state B). Full proton transfer leads to
reactive intermediate C, wherein the nitrogen is now neutral and
the phosphate moiety is monoprotonated. Since a phosphate
monoanion is now a much better leaving group, collapse of C
would lead to inorganic phosphate dianion D and carbamoyl aspar-
tate C-Asp. In the established sequence for DHQS, the molecular
events are strikingly similar: the bound phosphate dianion in the
substrate is oxidized to E with a tightly bound NAD cofactor.28
One of the strongly basic phosphate oxygens then attacks the
now acidic proton in
tonated, phosphate G becomes a good leaving group, which col-
lapses to produce ,b-unsaturated ketone and inorganic
a of the ketone (transition-state F). Once pro-
a
H
phosphate dianion D. Thus, the two mechanisms would share
striking similarities, particularly as far as the role of the phosphate
group is concerned. In the case of DHQS, the transformation E to H
is well supported experimentally.28,29 For ATCase, computational
studies and X-ray crystallography also support of the proposed
cyclic transition-state (Fig. 1).14 In general, PALA’s amide carbonyl
oxygen is represented as being axial (Fig. 1, Transition-State O-ax-
ial), gas-phase modeling places PALA’s amide oxygen in the equa-
torial position so the phosphate oxygen is hydrogen-bonded to the
amide NH. It is also interesting to note that in the enzyme active
site, there does not seem to be residues that are binding to NH2
of carbamyl phosphate CP other than lysine 267.

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L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
H
H
HO2C
HO2C
t-BuO2C
t-BuO2C
N
CO2H
CO2H
N
CO2Bn
CO2Bn
b, c
a
t-BuO2C
CO2t-Bu
O
O
37
38
H
N
CO2H
CO2H
H
HO2C
c
c
d
N
CO2Bn
CO2Bn
BnO2C
CO2H
BnO2C
O
O
39
40
O
H
Ph
O
H
HO2C
N
CO2H
CO2H
e, f
N
CO2Bn
CO2Bn
O
O
Ph
O
O
O
41
42
Scheme 8. Reagents and conditions: (a) NaH (1.0 equiv), THF, rt, 5 min then N-(bromoacetyl)-
L
-aspartic acid dibenzyl ester 16 (0.77 equiv), 89%; (b) TFA, CH2Cl2, 0 °C, 15 h;
-aspartic acid dibenzyl ester p-toluenesulfonate 30 (1.0 equiv), EDCꢀHCl (2.5 equiv), DMAP
L-aspartic acid dibenzyl ester p-toluenesulfonate 30 (1.0 equiv), Et3N (6.0 equiv), THF, rt, 4 h; (f) Ph2CN2, THF, rt, 33% from
(c) H2, Pd/C, THF/H2O, 24 h, 95% from 37; 83% from 39; 99% from 41; (d)
L
(2.5 equiv), Et3N (1.1 equiv), THF, rt, 5 h, 78%; (e)
succinic anhydride.
Table 1
aInhibition constants for the synthesized inhibitors
Entry
1
Inhibitor
Compound number
Inhibition constant (Ki)b
(lM)
Ki/Ki(PALA)
O
NHAsp
1
0.016
1
HO
P
OH
O
NHAsp
O
2
3
4
5
2
0.42
26
12
—
H
P
OH
O
O
P
HO
NHAsp
3
0.19
OH
O
O
P
14
19
Inactive
Inactive
N Asp
HO
O
O
—
P
N
Asp
HO
O
HO
NHAsp
NHAsp
6
7
22
24
27
32
34
36
2.5c
3.6
156c
225
63
P
OH
O
O
HO2C
P
OH
O
O
N
O
8
1.0
P
Asp
HO
O
O
HO
P
9
Inactive
2.1
—
O
NHAsp
HO
O
O
HO
P
10
11
131
13
N
H
NHAsp
NHAsp
HO
O
HO
P
0.21
O
HO
O
a
b
c
Colorimetric assay detecting the formation of N-carbamoyl-
Inhibition relative to carbamyl phosphate Km = 28
Compound 22 was an uncompetitive inhibitor.
L
-aspartate.25 The E. coli catalytic subunit of ATCase was used.
lM. Unless otherwise noted, all active compounds were competitive inhibitors.

L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
7685
O
P
O
P
O
P
O
P
O
X
X
X
δ
O
O
O
+
H2N
O
H
O
H
O
H
N
H
Asp
O
OH
O
O
O
δ
O
N
Asp
N
Asp
N Asp
Y
Y
Y
H
H
H
A
B
D
C-Asp
C
X = O-, Y = NH2 (O-axial), or X = NH2, Y = O- (O equatorial)
O
P
O
P
O
P
O
P
δ
O
H
H
H
O
O
O
O
+
O
O
OH
O
O
O
O
O
δ
O
O
O
E
F
D
H
G
Scheme 9. Mechanistic analogy between ATCase and 3-dehydroquinate synthase (DHQS).
In the inhibition of DHQS, some dicarboxylates were very po-
tent (nanomolar-level) inhibitors and therefore good mimics of
the phosphate moiety.26 Even the simplest monocarboxylate ana-
log displayed an inhibition constant comparable to the Michaelis
constant (Km) of the substrate (DAHP). However, none of the car-
boxylates 38, 40, and 42 displayed any significant inhibition
against ATCase. Since compound 40 is inactive, whereas H-phosph-
inate 2 is a good inhibitor, this clearly points to a geometrical ef-
fect. In the case of DHQS, the monoanion needs to be aligned
with the hydrogen to be abstracted, and this remains possible be-
cause it is an in-plane reaction. On the other hand, with ATCase,
the deprotonation requires a tetrahedral anionic moiety because
the amide group in 38, 40, and 42 prevents the carboxylate from
adopting the required ‘perpendicular’ geometry. The potency of
species (2 and 3) constrained to the phosphorus monoanion, also
provides support for this mechanism. ATCase would bind to the
monoanion, which is then deprotonated by an arginine residue14
(Arg-54 in the E. coli enzyme). At the end of the sequence, inorganic
phosphate can regenerate the basic residue (Arg) and leave the ac-
tive site as monobasic phosphate. DHQS also has a single arginine
residue29 (Arg-130), which could act as a general base to deproto-
nate the phosphate monoanion once it is initially bound from
solution.
None of the newly synthesized inhibitors were more potent
than PALA 1 (Table 1). As discussed in our communication,7 re-
duced-charge compounds 2 and 3 (entries 2 and 3, respectively)
are interesting, at least mechanistically, and their activity is consis-
tent with the proposed intramolecular proton transfer14 depicted
in Figure 1. On the other hand the simple heterocycle 14 (entry
4) is totally inactive, and this is not surprising since the carbonyl
amide of PALA is expected to be very important for binding affinity
and to mimic the developing negative charge in the transition-
state (Fig. 1). The five-membered ring cyclo-PALA 19 (entry 5) is
also inactive. This might, seemingly, be more surprising. But inter-
estingly, the six-membered homolog 27 (entry 8) shows significant
inhibition [Km(CP)/Ki(27) = 28] especially when compared to 19.
Again, electron density at the amide carbonyl should be critical
in the mimicry of the transition-state: amide resonance (or dipolar
interaction) in a five-membered lactam is conformationally disfa-
vored, thus decreasing electron density at the carbonyl oxygen.
In six-membered 27, the carbonyl is more electron-rich than in
19, because resonance better contributes to the negative charge
on oxygen to mimic the postulated transition-state (Fig. 1). Thus
the drastic difference between 27 and 19 points to clear stereoelec-
tronic effects. We surmise that the transition-state looks more like
O-equatorial (Fig. 1), which would be bound to threonine 55 and
histidine 134 in the E. coli enzyme.
Hydroxyethyl 22 (entry 6) shows significant inhibitory activity,
but it is still 13-times less potent than hydroxymethyl 3, and it is
no longer a competitive inhibitor, but instead it is uncompetitive.
The inhibition displayed by the phosphinate-acetate 24 is interest-
ing when compared to its reduced analog 22. Although the inhibi-
tion constants are comparable, carboxylate 24 is once again a
competitive inhibitor. The six-membered cyclo-PALA 27 (entry 8)
is more active than either 22 or 24 even if only slightly (a micro-
molar-level inhibitor). The six-membered cyclic amide 27 also sup-
ports the sequence of events A to C-Asp and a cyclic transition-
state ( Fig. 1), and is more tightly bound than the substrate CP
(Scheme 9).
The last three entries in Table 1 (entries 9–11) are particularly
revealing. Compounds 32, 34, and 38 were designed as bi-substrate
analogs, related to PALA 1. Carbamate 32 (entry 9) is mimicking the
departure of phosphate in the transition-state. However, this com-
pound does not inhibit ATCase at all. Carbamate 32 is likely a fail-
ure because of the reduced electron density on the carbonyl
oxygen (carbamate vs amide). In fact, the activity of urea 34 (entry
10) is confirming this possibility: an electron-rich carbonyl oxygen
must be present (i.e., ureas are better hydrogen bond acceptors,
and their carbonyl oxygen is more basic than the analogous carba-
mates). Most revealing perhaps, is the potent inhibition observed
with phosphate 36 (entry 11). The difference between entry 9
and entry 11 is a switch between the carbon and oxygen in the
phosphorus-containing moiety. While 36 is not as potent as PALA
(13-fold weaker), its extreme ease of synthesis makes it a candi-
date for the preparation of esters in order to optimize its bioavail-
ability and its potency further supports the above discussion of
carbamate 32 and urea 34.
For transition-state mimicry in the case of ATCase, the question
remains as to which protonation state of the phosphate analog is
optimum. Transition-state B might look like the starting material
A (an early transition-state) and thus have a charge around the
phosphorus closer to 2, or it could be more like the monoanionic
product D (a late transition-state) with a charge closer to 1
(Scheme 9). Yet, if the transition-state were nearly symmetrical,
the ideal state for the phosphate mimic would then be 1.5 negative
charges. Although not as potent as PALA 1 by one order of magni-
tude, inhibitors 2 and 3 clearly support a mechanism in which the
phosphate charge is reduced from two to one in the transition-
state.
Overall, the results observed with our compounds support the
need for: (1) a tetrahedral, at least monoanionic, phosphate mimic
(1, 2, 3, 22, 27); and (2) significant electronic density on the amide
carbonyl oxygen along with proper spatial orientation (19 vs 27,
and 32 vs 36, e.g.). In the case of PALA, both the monoanionic

7686
L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
and dianionic phosphonate group are accessible, and the amide
oxygen has high electron density. This suggests that N-methyl-
PALA might be an improved inhibitor, and this will be explored
in the near future (compounds like N-methyl-PALA have not been
described in the literature). The preparation of cyclic transition-
state analogs is challenging because although phosphorus monoa-
nions can be good inhibitors, the transition-state has a higher neg-
ative charge (anywhere between 2 and 1), and in addition, an
additional functional group mimicking the amino group in the tet-
rahedral intermediate needs to be perpendicular to the plane of the
ring.
4.2. General procedures
4.2.1. Procedure A
Neat H-phosphinic acid 15 or 4 (4.6 mmol, 1.0 equiv) was
placed under vacuum in a dry two-neck flask for 10 min then
placed under N2 and dissolved in distilled toluene (0.3 M, 16 mL).
N-(bromoacetyl)-L-aspartic acid dibenzyl ester 16 (2.0 g, 4.6 mmol,
1.0 equiv), HMDS (2.4 mL, 11.5 mmol, 2.5 equiv) and TMSCl
(1.5 mL, 11.5 mmol, 2.5 equiv) were added at room temperature
under N2. The reaction mixture was refluxed for 14 h under N2.
The solution was cooled down, quenched by adding methanol
(5 mL) and concentrated in vacuo.
3. Conclusion
4.2.2. Procedure B
To a solution of hydroxyl 17 or 21 (1.0 equiv) in THF (0.05 M)
was added triphenylphosphine (1.0 equiv) and diisopropyl azodi-
carboxylate (1.0 equiv) at room temperature, under N2. After
30 min at room temperature, the crude mixture was quenched
with water and concentrated in vacuo.
The present work provides a number of new compounds for the
inhibition of ATCase, and a better understanding of PALA’s activity.
Potential prodrugs of (or related to) PALA could be accessible
through either in vivo metabolic oxidation of H-phosphinate 2, bet-
ter translocation of reduced-charge compounds such as 2 and 3, or
oxidative esterification of intermediates related to 9–11. While
none of the described compounds showed an inhibition of ATCase
superior to that of PALA, the results provide guidelines for the de-
sign of new inhibitors. Compounds 2, 3, and 36 are all submicrom-
olar inhibitors with functionalities significantly differing from the
phosphonate moiety in PALA. The described compounds also shed
some light on the mechanism of the ATCase-catalyzed transforma-
tion. PALA itself, while appearing more like a bi-substrate analog,
may indeed behave like a transition analog as Stark originally sug-
gested. Although our work (like all other prior works) has failed to
identify an inhibitor that is more potent than PALA in vitro, several
research avenues remain to be explored. Two directions are identi-
fied as: (1) the development of PALA-related prodrugs through the
masking of charges especially around the phosphorus atom in or-
der to improve PALA’s bioavailability; and (2) the design of cyclic
or conformationally restricted transition-state analogs. In spite of
the 38 years that have elapsed since Stark’s discovery of PALA,
much remains to be investigated for the design of potent in vitro
and in vivo inhibitors of ATCase, and ultimately to improve upon
PALA’s anticancer activity. The potential in vivo oxidation of H-
phosphinic acid 2 to produce PALA remains to be established.
4.2.3. Procedure C
To a solution of ester (0.6 g) in a mixture THF/H2O (2:1, v/v,
4.5 mL) was added 10 wt % Pd/C (0.30 g). The reaction was placed
in a hydrogenator under 48 psi H2 at room temperature. After
24 h, the reaction mixture was filtered through CeliteÒ and concen-
trated in vacuo. The resulting oil was dissolved in water (20 mL)
and washed with ethyl acetate (3 ꢂ 10 mL). The aqueous layer
was concentrated to afford the acid.
4.2.4. Procedure D
To triphosgene (0.36 g, 1.2 mmol, 0.6 equiv) in CH2Cl2 (9.0 mL)
at ꢁ15 °C was added over 2 h a solution of
L-aspartic acid dibenzyl
ester p-toluenesulfonate 30 (1.16 g, 2.4 mmol, 1.2 equiv) and tri-
ethylamine (1.3 mL, 9.6 mmol, 4.8 equiv) in CH2Cl2 (8.0 mL) under
N2. After the addition, the reaction mixture was allowed to warm
to room temperature over 1 h. Then a solution of ester 28 or 29
(2.0 mmol, 1.0 equiv) in CH2Cl2 (10.0 mL) was added dropwise at
room temperature. After 17 h at room temperature, the reaction
mixture was concentrated in vacuo. The resulting oil was diluted
in ethyl acetate (10 mL) and filtered through a cotton plug. The fil-
trate was concentrated in vacuo and the resulting oil was purified
by chromatography over silica gel.
4. Experimental section
4.1. General information
4.3. Synthesis of the ‘cyclo-PALA’ heterocycle 19 (Scheme 4)
4.3.1. (N-Acetyl-L-aspartic acid dibenzyl ester)-hydroxymethyl-
phosphinic acid benzhydryl ester (17)
Concentrated hypophosphorous acid was obtained by rotary
evaporation (0.5 mmHg) of the 50 wt % aqueous solution at room
temperature for 20–30 min before reaction. Caution: overdrying
H3PO2 may result in the formation of a yellow solid of high phosphorus
content that could be pyrophoric. Tetrahydrofuran (THF) was freshly
distilled from sodium benzophenone ketyl under N2. Toluene, t-
amyl alcohol, acetonitrile (ACN) and dichloromethane (CH2Cl2)
were freshly distilled from CaH2 under N2. Dimethylformamide
(DMF) over 4 Å sieves and pyridine over 3 Å were used. Triethyl-
amine (Et3N) was distilled from CaH2 and stored over 4 Å sieves.
Absolute ethanol, methanol, t-butanol (tBuOH) and chloroform
were used as received. 1H NMR spectra were recorded on a 300-
MHz spectrometer. Chemical shifts for 1H NMR spectra are re-
ported (in parts per million) relative to internal tetramethylsilane
(Me4Si, d = 0.00 ppm) with CDCl3 or H2O (d = 4.75 ppm) in D2O.
13C NMR spectra were recorded at 75 MHz and in some cases at
100 MHz. Chemical shifts for 13C NMR spectra are reported (in
parts per million) relative to CDCl3 (d = 77.0 ppm). 31P NMR spectra
were recorded at 121 MHz, and chemical shifts reported (in parts
per million) relative to external 85% phosphoric acid (d = 0.0 ppm).
Procedure A. The resulting oil was dissolved in distilled ACN
18
(10 mL) and treated with Ph2CN2 (1.0 M in toluene) until com-
plete esterification (persistence of the pink color and 31P NMR
monitoring, 2 singlets at 48 ppm) at room temperature under N2.
After removal of the solvent in vacuo, the resulting oil was dis-
solved in ethyl acetate (50 mL) and washed with 10% aqueous tar-
taric acid (1 ꢂ 10 mL) and brine (2 ꢂ 10 mL). The organic layer was
dried over MgSO4 and concentrated in vacuo. The resulting oil was
purified by chromatography over silica gel (hexanes/ethyl acetate,
3:7, v/v) to afford the ester 17 (1.4 g, 50% yield). 1H NMR (300 MHz,
CDCl3) d: 7.65 (d, J = 7.0 Hz, 0.5H, NH), 7.50 (d, J = 7.0 Hz, 0.5H, NH),
7.26–7.36 (m, 20H, CHarom.), 6.56 (d, J = 9.0 Hz, 1H, Ph2CH–), 5.01–
5.15 (m, 4H, Bn –CH2–), 4.85 (m, 1H, N–CH–), 3.70–3.84 (m, 2H, P–
CH2–OH), 2.74–3.05 (m, 4H, P–CH2–CO & CH–CH2–CO2Bn); 13C
NMR (75.45 MHz, CDCl3) d: 170.8 & 170.6, 170.4 & 170.2, 165.7
(2 d, JPCC = 3.0 Hz), 140.6 (2C), 135.5, 135.2, 128.9, 128.8, 128.7,
128.7, 128.6, 128.5, 127.3, 127.2, 78.8 (d, JPOC = 6.0 Hz), 67.9, 67.2
& 67.1, 60.0 (d, JPC = 108.5 Hz), 49.5 & 49.3, 37.0 (2 d, JPC = 77.5 Hz),
36.3; 31P NMR (121.47 MHz, CDCl3) d: 48.03 (s, 50%), 48.00 (s, 50%);

L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
7687
HRMS (ESI+) calcd for C34H35NO8P, ([M+H]+) 616.2100, found
616.2103.
tion mixture was diluted with ethyl acetate (50 mL) and washed
with brine (1 ꢂ 20 mL), the aqueous layer was extracted with ethyl
acetate (2 ꢂ 50 mL). The combined organic layer was dried over
MgSO4 and concentrated in vacuo. The resulting oil was purified
by chromatography over silica gel (hexanes/ethyl acetate, 1:9, v/v
to ethyl acetate/methanol 98:2, v/v) to afford the ester 21
(1.09 g, 57% yield). 1H NMR (300 MHz, CDCl3) d: 8.03 (d,
J = 8.5 Hz, 0.5H, NH), 7.59 (d, J = 8.0 Hz, 0.5H, NH), 7.26–7.36 (m,
15H, CHarom.), 5.03–5.15 (m, 6H, Bn –CH2–), 4.86–4.98 (m, 1H, N–
CH–), 3.77–3.99 (m, 2H, –CH2–OH), 2.80–3.18 (m, 4H, N–CH–CH2
4.3.2. 2-(3-Benzhydryloxy-3,5-dioxo-3k5-[1,3]azaphospholidin-
1-yl)-succinic acid dibenzyl ester (18)
Procedure B. The resulting oil was purified by chromatography
over silica gel (hexanes/ethyl acetate, 6:4 to 4:6, v/v) to afford
the mixture of the ester 18 and triphenylphosphine oxide
(0.614 g). 1H NMR (300 MHz, CDCl3) d: 7.26–7.40 (m, 20H, aro
CH), 6.58 (d, J = 9.0 Hz, 1H, Ph2CH–), 5.12 (s, 2H, Bn –CH2–), 5.06
(s, 2H, Bn –CH2–), 4.45–3.56 (m, 1H, N–CH–), 2.04–3.15 (m, 6H,
P–CH2–N & P–CH2–CO & CH–CH2–CO2Bn); 31P NMR (121.47 MHz,
CDCl3) d: 46.25 (s, 22%, 1 diastereoisomer), 46.16 (s, 62%, 1 diaste-
reoisomer), 30.51 (s, 16%, Ph3PO).
&
P–CH2–CO), 1.94–2.38 (m, 2H, P–CH2–CH2OH); 13C NMR
(75.45 MHz, CDCl3) d: 170.8 & 170.4, 170.7, 165.4, 136.4 (d,
JPOCC = 6.0 Hz), 135.6, 135.6, 135.3 (2 s), 128.9 (2C), 128.8 (2C),
128.7 (2C), 128.6 (4C), 128.3, 128.2, 67.9 & 67.8, 67.1, 66.7 &
66.6 (d, JPOC = 9.5 Hz), 56.4, 49.4 & 49.1, 39.2 (d, JPC = 75.5 Hz),
36.6 & 36.4, 31.5 (d, JPC = 95.0 Hz) & 31.2 (d, JPC = 95.5 Hz); 31P
NMR (121.47 MHz, CDCl3) d: 52.05 (s, 51%), 51.69 (s, 49%); HRMS
(EI) calcd for C29H32NO8P, ([M]+) 553.1866, found 553.1875.
4.3.3. 2-(3-Hydroxy-3,5-dioxo-3k5-[1,3]azaphospholidin-1-yl)-
succinic acid (19)
Procedure C. Yellow oil (0.183 g, 44% yield from hydroxyl 17). 1H
NMR (300 MHz, D2O) d: 5.00 (t, J = 7.0 Hz, 1H, N–CH–), 3.31 (d,
JHCP = 11.5 Hz, 2H, P–CH2–N), 2.93 (dd, J = 17.0 Hz, J = 6.5 Hz, 1H,
N–CH–CH2–), 2.72 (dd, J = 17.0 Hz, J = 6.5 Hz, 1H, N–CH–CH2–),
2.43 (d, JHCP = 13.0 Hz, 2H, P–CH2–CO); 13C NMR (75.45 MHz,
D2O) d: 174.3, 172.6 (d, JPCC = 12.0 Hz), 172.3, 53.1 & 53.0, 48.3
(d, JPC = 88.5 Hz), 33.2, 32.8 (d, JPC = 88.5 Hz); 31P NMR
(121.47 MHz, D2O) d: 33.84 (s); HRMS (ESI+) calcd for C7H11O7NP,
([M+H]+) 252.0273, found 252.0266.
4.4.3. 2-(4-Benzyloxy-2,4-dioxo-4k5-[1,4]azaphosphinan-1-yl)-
succinic acid dibenzyl ester (26)
Procedure B. The resulting oil was dissolved in dichloromethane
(25 mL). After saturation of the solution at ꢁ78 °C by ozone, Me2S
(1.7 mL, 23.2 mmol, 7.0 equiv) was added under N2 at ꢁ78 °C. The
reaction mixture was allowed to warm up to room temperature
overnight then the solution was concentrated in vacuo. The result-
ing oil was purified by chromatography over silica gel (hexanes/
ethyl acetate, 3:7, v/v) to afford the ester 26 as a lightly yellow
oil (1.2 g, 33% yield). 1H NMR (CDCl3, 300 MHz) d: 7.25–7.35 (m,
15H, CHarom.), 4.93–5.17 (m, 7H, Bn –CH2– & N–CH–), 3.42–3.74
(m, 2H, P–CH2–CH2–N), 2.83–3.14 (m, 4H, P–CH2–CO & N–CH–
CH2–), 1.93–2.21 (m, 2H, P–CH2–CH2–N); 13C NMR (CDCl3,
75.45 MHz) d: 170.7, 169.3 & 169.2, 165.9 (d, JPCC = 5.0 Hz) &
165.8 (d, JPCC = 5.0 Hz), 135.8, 135.5, 135.2, 129.0 (2C), 128.9 (3C),
128.7 (3C), 128.6 (2C), 128.4 (2C), 67.9 & 67.8, 67.2, 67.0 (d,
4.4. Synthesis of the six-membered ‘cyclo-PALA’ 27 (Scheme 5)
4.4.1. (N-Acetyl-L-aspartic acid dibenzyl ester)-(3-methyl-but-
2-enyl)-phosphinic acid benzyl ester (20)
Procedure A. The resulting oil was dissolved in ethyl acetate
(50 mL) and washed with brine (1 ꢂ 30 mL), the aqueous layer
was extracted with ethyl acetate (2 ꢂ 50 mL). The combined or-
ganic layers were dried over MgSO4 and concentrated in vacuo.
To the resulting oil in chloroform (75 mL) was added benzyl bro-
mide (4.3 mL, 35.9 mmol, 1.2 equiv). The mixture was refluxed
and silver oxide(I) was added in portions every 30 min (4.0 g,
17.0 mmol, 0.6 equiv, in 4 portions) under N2. After cooling down,
the suspension was filtered through CeliteÒ and the solvent was re-
moved in vacuo. The resulting oil was purified by chromatography
over silica gel (hexanes/ethyl acetate, 6:4, v/v to ethyl acetate
100%) to afford the ester 20 (10.2 g, 59% yield). 1H NMR
(300 MHz, CDCl3) d: 7.59 (d, J = 8.0 Hz, 1H, NH), 7.25–7.35 (m,
15H, CHarom.), 4.98–5.14 (m, 7H, Bn –CH2– & –CH@), 4.89–4.95
(m, 1H, N–CH), 2.78 (d, JHCP = 14.5 Hz, 2H, P–CH2–CH@), 2.58–
3.08 (m, 4H, N–CH–CH2– & P–CH2–CO), 1.72 (d, J = 5.0 Hz, 3H,
CH3–C@), 1.59 (m, 3H, CH3–C@); 13C NMR (75.45 MHz, CDCl3) d:
170.5 (2s), 170.3, 164.9, 138.4 (d, JPCCC = 14.0 Hz), 136.5, 135.6,
135.3, 128.9 (2C), 128.8 (2C), 128.7 (4C), 128.6 (2C), 128.3, 128.2,
JPOC = 7.5 Hz)
& 66.9 (d, JPOC = 6.5 Hz), 57.6 & 57.1, 43.9 (d,
JPCC = 26.0 Hz), 36.9 (d, JPC = 74.5 Hz) & 36.8 (d, JPC = 73.5 Hz), 35.0
(2 s), 27.5 (d, JPC = 94.5 Hz) & 27.3 (d, JPC = 95.5 Hz); 31P NMR
(CDCl3, 121.47 MHz) d: 49.23 (s, 55%), 48.77 (s, 45%); HRMS (CI,
NH3) calcd for C29H31NO7P, ([M+H]+) 536.1838, found 536.1829.
4.4.4. 2-(4-Hydroxy-2,4-dioxo-4k5-[1,4]azaphosphinan-1-yl)-
succinic acid (27)
Procedure C. Orange oil (0.30 g, 64% yield). 1H NMR (D2O,
300 MHz) d: 4.88 (t, J = 8.0 Hz, 1H, N–CH–), 3.65 (t, J = 6.5 Hz, 1H,
P–CH2–CH2–N), 3.57 (t, J = 6.5 Hz, 1H, P–CH2–CH2–N), 2.99 (dd,
JHCP = 17.0 Hz, J = 6.5 Hz, 1H, N–CH–CH2–), 2.69–2.83 (m, 3H, N–
CH–CH2– & P–CH2–CO), 1.87–1.96 (m, 2H, P–CH2–CH2–N); 13C
NMR (D2O, 100.6 MHz) d: 174.7, 173.0, 170.4, 57.0, 44.3 (d,
JPCC = 5.0 Hz), 38.1 (d, JPC = 74.0 Hz), 33.9, 27.5 (d, JPC = 95.5 Hz);
31P NMR (D2O, 121.47 MHz) d: 40.56 (s); HRMS (ESI+) calcd for
C8H13NO7P, ([M+H]+) 266.0429, found 266.0427.
111.8 (d, JPCC = 10.0 Hz), 67.8 (2 s), 67.1, 67.0
& 66.8 (2d,
JPOC = 7.0 Hz), 49.2, 37.0 (2 d, JPC = 75.5 Hz), 36.5 & 36.4, 30.2 &
30.0 (2d, JPC = 94.0 Hz), 26.1 (d, JPCCCC = 3.0 Hz), 18.3; 31P NMR
(121.47 MHz, CDCl3) d: 50.26 (s, 52%), 49.78 (s, 48%); HRMS (EI)
calcd for C32H36NO7P, ([M]+) 577.2229, found 577.2235.
4.5. Synthesis of the carbamate and urea analogs (32 and 34,
Scheme 6)
4.4.2. (N-Acetyl-
phosphinic acid benzyl ester (21)
L
-aspartic acid dibenzyl ester)-hydroxyethyl-
4.5.1. 2-(Bis-benzyloxy-phosphorylmethoxycarbonylamino)-
succinic acid dibenzyl ester (31)
After saturation of a solution of the ester 20 (2.0 g, 3.47 mmol,
1.0 equiv) in CH2Cl2 (0.2 M, 17.0 mL) at ꢁ78 °C by ozone, Me2S
(1.8 mL, 24.3 mmol, 7.0 equiv) was added under N2 at ꢁ78 °C.
The reaction mixture was allowed to warm up to room tempera-
ture overnight then the solution was concentrated in vacuo. The
resulting oil was dissolved in methanol (0.2 M, 17 mL) and treated
by sodium borohydride in portions (0.20 g, 5.2 mmol, 1.5 equiv) at
0 °C. The reaction was followed by TLC, after completion the reac-
Procedure D. Purification by chromatography over silica gel
(hexanes/ethyl acetate, 8:2 to 6:4,v/v) to afford the ester 31 as a
light yellow oil (0.625 g, 50% yield). 1H NMR (CDCl3, 300 MHz) d:
7.24–7.39 (m, 20H, CHarom.), 5.88 (d, J = 8.0 Hz, 1H, NH), 5.00–
5.12 (m, 8H, Bn –CH2–), 4.57–4.62 (m, 1H, N–CH–CH2), 4.27–4.42
(m, 2H, P–CH2–O), 3.05 (dd, J = 17.0 Hz, J = 4.0 Hz, 1H, –CH2–
CO2Bn), 2.83 (dd, J = 17.0 Hz, J = 5.0 Hz, 1H, –CH2–CO2Bn); 13C
NMR (CDCl3, 75.45 MHz) d: 170.7, 170.3, 155.1 (d, JPCOC = 10.0 Hz),

7688
L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
136.0 (d, JPOCC = 5.5 Hz, 2C), 135.5, 135.3, 128.9 (10C), 128.8 (2C),
128.7 (2C), 128.6 (2C), 128.3 (4C), 68.5 (d, JPOC = 6.5 Hz,), 68.4 (d,
JPOC = 6.0 Hz), 68.9, 67.2, 58.3 (d, JPC = 170.0 Hz), 50.9, 36.8; 31P
NMR (CDCl3, 121.47 MHz) d: 21.49 (s); HRMS (EI) calcd for
C34H34NO9P, ([M]+) 631.1971, found 631.1947.
121.47 MHz) d: ꢁ0.685 (s); HRMS (EI) calcd for C34H34NO9P,
([M]+) 631.1971, found 631.1979.
4.6.2. N-(2-Hydroxy-acetyl)-L-aspartic acid-phosphate (36)
Procedure C. Orange oil (0.357 g, 100% yield). 1H NMR (D2O,
300 MHz) d: 4.69 (t, J = 6.0 Hz, 1H, N–CH–CH2), 4.25 (d,
JHCOP = 7.0 Hz, 2H, P–O–CH2–CO), 2.83 (m, 2H, N–CH–CH2–); 13C
NMR (D2O, 75.45 MHz) d: 174.5, 173.8, 171.4 (d, JPOCC = 9.0 Hz),
63.8 (d, JPOC = 5.0 Hz), 48.7, 35.5; 31P NMR (D2O, 121.47 MHz) d:
0.40 (s); HRMS (ESI+) calcd for C6H10NO9P, ([M+H]+) 272.0173,
found 272.0171.
4.5.2. 2-[3-Benzyl-3-(bis-benzyloxy-phosphorylmethyl)-
ureido]-succinic acid dibenzyl ester (33)
Procedure D. Purification by chromatography over silica gel
(hexanes/ethyl acetate, 7:3, v/v) to afford the ester 33 as a light yel-
low oil (0.620 g, 43% yield). 1H NMR (CDCl3, 300 MHz) d: 7.12–7.38
(m, 25H, CHarom.), 6.13 (br s, 1H, NH), 4.92–5.10 (m, 8H, O–CH2-Ph),
4.77–4.82 (m, 1H, N–CH–CH2–), 4.52 (d, J = 16.0 Hz, 1H, N–CH2-Ph),
4.40 (d, J = 16.0 Hz, 1H, N–CH2-Ph), 3.59 (m, 2H, P–CH2–N), 2.99
(dd, J = 17.0 Hz, J = 5.0 Hz, 1H, N–CH–CH2–), 2.84 (dd, J = 17.0 Hz,
J = 5.0 Hz, 1H, N–CH–CH2–); 13C NMR (CDCl3, 75.45 MHz) d:
171.4, 170.8, 157.8, 136.6, 136.3, 136.2, 135.7 (d, JPOCC = 8.5 Hz,
2C), 129.0 (2C), 128.9 (4C), 128.8 (5C), 128.5 (10C), 128.5 (2C),
127.8 (4C), 68.3 (d, JPOC = 7.5 Hz, 2C), 67.5, 66.9, 51.0 (d,
JPCNC = 18.5 Hz), 50.9, 42.9 (d, JPCNC = 160.0 Hz), 37.2; 31P NMR
(CDCl3, 121.47 MHz) d: 25.00 (s); HRMS (EI) calcd for C41H41N2O8P,
([M]+) 720.2601, found 720.2616.
4.7. Biological assay
Before testing, each compound was neutralized to pH 7.0 with
1.0 M aqueous NaOH then concentrated to dryness. For the com-
pounds containing a phosphorus atom, the phosphorus assay30
was used to determine accurate concentrations, and dilute solu-
tions were prepared from this standardized solution. All com-
pounds tested were at least 97% pure as determined by 1H and
31P NMR. The potential oxidation of 2 into PALA could not be de-
tected over a period of two weeks (31P NMR monitoring).
The assay was performed with E. coli His-tagged C3 ATCase pro-
vided by Professor Evan Kantrowitz (Boston College).24 The assay
was performed as described by Professor Evan Kantrowitz25 at
room temperature, in the presence of 0.1 M Tris–acetate pH 8.3,
4.5.3. 2-Phosphonomethyl-oxycarbonylamino-succinic acid
(32)
Procedure C. Orange oil (0.26 g, 100% yield). 1H NMR (D2O,
300 MHz) d: 4.43 (t, J = 6.0 Hz, N–CH–CH2), 4.05 (d, JHCP = 8.5 Hz,
2H, P–CH2–O), 2.80 (d, J = 6.0 Hz, 2H, –CH2–CO2H); 13C NMR
(D2O, 75.45 MHz) d: 170.7, 174.5, 157.6, 60.3 (d, JPC = 159.0 Hz),
50.6, 36.0; 31P NMR (D2O, 121.47 MHz) d: 17.54 (s); HRMS (ESI+)
calcd for C6H11NO9P, ([M+H]+) 272.0171, found 272.0176.
75 ng/mL His-tagged C3 ATCase and 10 mM
was measured with four carbamyl phosphate concentrations (20,
40, 60, 80 M) and five inhibitors concentrations (0, 0.5, 1.0, 3.0,
6.0 M) to determine the inhibition constant (Ki) and the mode
L-aspartate. Kinetic
l
l
of inhibition.
4.5.4. 2-(3-Phosphonomethyl-ureido)-succinic acid (34)
Procedure C. Orange oil (0.133 g, 100% yield). 1H NMR (D2O,
300 MHz) d: 4.49 (t, J = 5.5 Hz, 1H, N–CH–CH2–), 3.27 (d,
JHCP = 11.5 Hz, 2H, P–CH2–N), 2.79 (d, J = 5.5 Hz, 2H, N–CH–CH2–);
13C NMR (D2O, 75.45 MHz) d: 175.5, 174.9, 159.3, 49.8, 37.1 (d,
JPCNC = 150.0 Hz), 36.4; 31P NMR (D2O, 121.47 MHz) d: 20.20 (s);
HRMS (ESI+) calcd for C6H12N2O8P, ([M+H]+) 271.0331, found
271.0323.
Acknowledgments
We thank the National Institute of General Medical Sciences/
NIH (R01 GM067610 for LC and JLM), and TCU-SERC (AFP) for the
financial support of this research. We also thank Professor Evan
R. Kantrowitz (Boston College) for providing the enzyme used in
the assays.
Supplementary data
4.6. Synthesis of the phosphate analog 36 (Scheme 7)
Supplementary data (Additional experimental details (including
the preparations of 2, 3, 9, 10, 11, 14, 22, 24, 28, 29, 38, 40, and 42),
spectral data, and copies of NMR spectra) associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.bmc.2009.09.045.
4.6.1. Dibenzyl-(N-(2-hydroxy-acetyl)-L-aspartic acid dibenzyl
ester)-phosphate (35)
To
a
mixture of dibenzylphosphate (0.278 g, 1.0 mmol.,
-aspartic acid dibenzyl ester 16
1.0 equiv), N-(bromoacetyl)-
L
(0.434 g, 1.0 mmol, 1.0 equiv) in acetonitrile (0.1 M, 10 mL) was
added silver(I) oxide (0.231 g, 1.0 mmol, 1.0 equiv). The reaction
mixture was heated at 60 °C under N2 for 16 h. After cooling down,
the suspension was filtered thought CeliteÒ and concentrated in
vacuo. The resulting oil was diluted by CH2Cl2 (50 mL) and washed
with water (3 ꢂ 10 mL). The organic layer was dried over MgSO4
and concentrated in vacuo. The resulting oil was purified by chro-
matography over silica gel (hexanes/ethyl acetate, 6:4, v/v) to af-
ford the ester 35 as a lightly yellow oil (0.595 g, 94% yield). 1H
NMR (CDCl3, 300 MHz) d: 7.26–7.34 (m, 20H, CHarom.), 5.13 (s,
2H, Bn –CH2–), 5.02–5.07 (m, 6H, Bn –CH2–), 4.91 & 4.88 (2 t,
J = 4.5 Hz, 1H, N–CH–CH2), 4.39 (d, JHCOP = 7.0 Hz, 2H, P–O–CH2–
CO), 3.05 (dd, J = 17.0 Hz, J = 4.5 Hz, 1H, N–CH–CH2–), 2.85 (dd,
J = 17.0 Hz, J = 4.5 Hz, 1H, N–CH–CH2–); 13C NMR (CDCl3,
75.45 MHz) d: 170.6, 170.0, 167.0 (d, JPOCC = 9.0 Hz), 135.3 (d,
JPOCC = 6.5 Hz, 2C), 135.2, 135.0, 128.8, 128.7 (4C), 128.6 (4C),
128.5 (2C), 128.4, 128.3, 128.2 (4C), 69.9 (d, JPOC = 5.0 Hz, 2C),
67.6, 66.9, 65.1 (d, JPOC = 6.0 Hz), 48.3, 36.1; 31P NMR (CDCl3,
References and notes
1. Reviews of inhibitors of pyrimidine biosynthesis as chemotherapeutic agents:
(a) Christopherson, R. I.; Lyons, S. D. Med. Res. Rev. 1990, 10, 505; (b) Kensler, T.
W.; Cooney, D. A. Adv. Pharmacol. Chemother. 1981, 18, 273.
2. (a) Collins, K. D.; Stark, G. A. J. Biol. Chem. 1971, 246, 6599; (b) Grem, J. L.; King,
S. A.; O’Dwyer, P. J.; Leyland-Jones, B. Cancer Res. 1988, 48, 4441; (c) Fleming, R.
A.; Capizzi, R. L.; Muss, H. B.; Smith, S.; Fernandes, D. J.; Homesley, H.; Loggie, B.
W.; Case, L. D.; Morris, R.; Russell, G. B.; Richards, F. Clin. Cancer Res. 1996, 2,
1107.
3. Johnson, R. K.; Inouye, T.; Goldin, A.; Stark, G. R. Cancer Res. 1976, 36, 2720.
4. Chan, T. C. K.; Young, B.; King, M. E.; Taetle, R.; Howell, S. B. Cancer Treatment
Rep. 1985, 69, 425.
5. Grem, J. L.; King, S. A.; O’Dwyer, P. J.; Leyland-Jones, B. Cancer Res. 1988, 48,
4441.
6. (a) Eldo, J.; Heng, S.; Kantrowitz, E. R. Bioorg. Med. Chem. Lett. 2007, 17, 2086;
(b) Eldo, J.; Cardia, J. P.; O’Day, E. M.; Xia, J.; Tsuruta, H.; Kantrowitz, E. R. J. Med.
Chem. 2006, 49, 5932; (c) Pfund, E.; Lequeux, T.; Masson, S.; Vazeux, M.; Cordi,
A.; Pierre, A.; Serre, V.; Hervé, G. Bioorg. Med. Chem. 2005, 13, 4921; (d) Grison,
C.; Coutrot, P.; Comoy, C.; Balas, L.; Joliez, S.; Lavecchia, G.; Oliger, P.; Penverne,
B.; Serre, V.; Hervé, G. Eur. J. Med. Chem. 2004, 39, 333; (e) Gagnard, V.; Leydet,
A.; Le Mellay, V.; Aubenque, M.; Morère, A.; Montero, J.-L. Eur. J. Med. Chem.

L. Coudray et al. / Bioorg. Med. Chem. 17 (2009) 7680–7689
7689
2003, 38, 883; (f) Folhr, A.; Aemissegger, A.; Hilvert, D. J. Med. Chem. 1999, 42,
2633; (g) Ben-Bari, M.; Dewynter, G.; Aymard, C.; Jei, T.; Montero, J.-L.
Phosphorus, Sulfur Silicon Relat. Elem. 1995, 105, 129; (h) Laing, N.; Chan, W. W.-
C.; Hutchinson, D. W.; Oberg, B. FEBS Lett. 1990, 260, 206; (i) Lindell, S. D.;
Turner, R. M. Tetrahedron Lett. 1990, 31, 5381; (j) Dutta, P. L.; Foye, W. O. J.
Pharm. Sci. 1990, 79, 447; (k) Farrington, G. K.; Kumar, A.; Wedler, F. C. J. Med.
Chem. 1985, 28, 1668; (l) Kafarski, P.; Lejczak, B.; Mastalerz, P.; Dus, D.;
Radzikowski, C. J. Med. Chem. 1985, 28, 1555; (m) Goodson, J. J.; Wharton, C. J.;
Wrigglesworth, R. J. Chem. Soc., Perkin Trans. 1 1980, 2721.
14. (a) Wang, J.; Eldo, J.; Kantrowitz, E. R. J. Mol. Biol. 2007, 371, 1261; (b) Heng, S.;
Stieglitz, K. A.; Eldo, J.; Xia, J.; Cardia, J. P.; Kantrowitz, E. R. Biochemistry 2006,
45, 10062; (c) Huang, J.; Lipscomb, W. N. Biochemistry 2006, 45, 346; (d)
Kosman, R. P.; Gouaux, J. E.; Lipscomb, W. N. Proteins: Struct. Funct. Genet. 1993,
15, 147; (e) Gouaux, J. E.; Krause, K. L.; Lipscomb, W. N. Biochem. Biophys. Res.
Commun. 1987, 142, 893; (f) White, J. C.; Hines, L. H. Biochem. Pharmacol. 1984,
33, 3645.
15. Coudray, L.; Bravo-Altamirano, K.; Montchamp, J.-L. Org. Lett. 2008, 10, 1123.
16. Cristau, H.-J.; Herve, A.; Virieux, D. Tetrahedron 2004, 60, 877.
17. Examples of related sila-Arbuzov reactions: (a) Rosenthal, A. F.; Gringauz, A.;
Vargas, L. A. J. Chem. Soc., Chem. Commun. 1976, 384; (b) Thottathil, J. K.;
Przybyla, C. A.; Moniot, J. L. Tetrahedron Lett. 1984, 25, 4737; (c) Boyd, E. A.;
Regan, A. C.; James, K. Tetrahedron Lett. 1994, 35, 4223; (d) Boyd, E. A.; Corless,
M.; James, K.; Regan, A. C. Tetrahedron Lett. 1990, 31, 2933; (e) Malachowski, W.
P.; Coward, J. K. J. Org. Chem. 1994, 59, 7625; (f) Reck, F.; Marmor, S.; Fisher, S.;
Wuonola, M. A. Bioorg. Med. Chem. Lett. 2001, 11, 1451; (g) An, H.; Wang, T.;
Maier, M. A.; Manoharan, M.; Ross, B. S.; Cook, P. D. J. Org. Chem. 2001, 66, 2789;
(h) Ribière, P.; Altamirano-Bravo, K.; Antczak, M. I.; Hawkins, J. D.; Montchamp,
J.-L. J. Org. Chem. 2005, 70, 4064.
7. Coudray, L.; Kantrowitz, E. R.; Montchamp, J.-L. Bioorg. Med. Chem. Lett. 2009,
19, 900.
8. Bravo-Altamirano, K.; Abrunhosa-Thomas, I.; Montchamp, J.-L. J. Org. Chem.
2008, 73, 2292.
9. Deprèle, S.; Montchamp, J.-L. J. Am. Chem. Soc. 2002, 124, 9386.
10. Deprèle, S.; Montchamp, J.-L. Org. Lett. 2004, 6, 3805.
11. Coudray, L.; Montchamp, J.-L. Eur. J. Org. Chem. 2009, 4646.
12. The in vivo oxidation of H-phosphinates has some precedent: (a) Froestl, W.;
Mickel, S. J.; Hall, R. G.; von Sprecher, G.; Diel, P. J.; Strub, D.; Baumann, P. A.;
Brugger, F.; Gentsch, C.; Jaekel, J.; Olpe, H.-R.; Rihs, G.; Vassout, A.; Waldmeier,
P. C.; Bittiger, H. J. Med. Chem. 1995, 38, 3297; (b) Howson, W.; Mistry, J.;
Broekman, M.; Hills, J. M. Bioorg. Med. Chem. Lett. 1993, 3, 515; (c) Magnin, D.
R., ; Dickson, J. K., Jr.; Logan, J. V.; Lawrence, R. M.; Chen, Y.; Sulsky, R. B.;
Ciosek, C. P., Jr.; Biller, S. A.; Harrity, T. W.; Jolibois, K. G.; Kunselman, L. K.; Rich,
L. C.; Slusarchyk, D. A. J. Med. Chem. 1995, 38, 2596; The related in vivo
oxidation of triethylphosphine is also known: (d) Shaw, C. F., III Chem. Rev.
1999, 99, 2589.
18. (a) Manthey, M. K.; Huang, D. T. C.; Bubb, W. A.; Christopherson, R. I. J. Med.
Chem. 1998, 41, 4550; (b) Smith, L. I.; Howard, K. L. Org. Synth. Coll. Vol. 1955, 3,
351; (c) Javed, M. I.; Brewer, M. Org. Synth. 2008, 85, 189.
19. (a) But, T. Y. S.; Toy, P. H. Chem. Asian J. 2007, 2, 1340; (b) Hughes, D. L. Org.
React. 1992, 42, 335.
20. Furata, T.; Torigai, H.; Osawa, T.; Iwamura, M. J. Chem. Soc., Perkin Trans. 1 1993,
3139.
13. Functional group interconversion of RP(O)(OR0)H. Reviews on the
phosphinylidene [P(O)H] group: (a) Stawinski, J.; Kraszewski, A. Acc. Chem.
Res. 2002, 35, 952; (b) Yudelevich, V. I.; Sokolov, L. B.; Ionin, B. I. Russ. Chem.
Rev. 1980, 49, 46; (c) Nifant’ev, E. E. Russ. Chem. Rev. 1978, 47, 835; General
reviews: (d) Frank, A. W. Chem. Rev. 1961, 60, 389; (e)Methoden der Organischen
Chemie (Houben-Weyl); Sasse, K., Ed.; Thieme: Stuttgart, 1964. Band XII/1, pp
294–337; (f)Methoden der Organischen Chemie (Houben-Weyl); Regitz, M., Ed.;
Thieme: Stuttgart, 1982; Vol. E2, (g) Frank, A. W.. In Organic Phosphorus
Compounds; Kosolapoff, G. M., Maier, L., Eds.; Wiley: New York, 1972; Vol. 4,.
21. (BnO)2P(O)CH2OH synthesis: (a) Krecmerova, M.; Hrebabecky, H.; Holy, A.
Collect. Czech. Chem. Commun. 1990, 55, 2521; (b) Czollner, L.; Kuszmann, J.;
Vasella, A. Helv. Chim. Acta 1990, 73, 1338.
22. (BnO)2P(O)CH2NHBn is
a new compound. For the reaction of 1,3,5-
tribenzylhexahydro-1,3,5-triazine with other H-phosphonate diesters, see:
(a) Oberhauser, T.; Meduna, V. Tetrahedron 1996, 52, 7691; (b) Lewis, R. T.;
Motherwell, W. B. Tetrahedron 1992, 48, 1465; (c) Afarinkia, K.; Rees, C. W.;
Cadogan, J. I. G. Tetrahedron 1990, 46, 7175; (d) Ratcliffe, R. W.; Christensen, B.
G. Tetrahedron Lett. 1973, 4645.
Chapter 10 For
a
few representative examples, see: RP(O)(OR1)H to
23. Kozikowski, A. P.; Nan, F.; Conti, P.; Zhang, J.; Ramadan, E.; Bzdega, T.;
Wroblewska, B.; Neale, J. H.; Pshenichkin, S.; Wroblewski, J. T. J. Med. Chem.
2001, 44, 298.
24. Nowlan, S. F.; Kantrowitz, E. R. J. Biol. Chem. 1985, 260, 14712.
25. Pastra-Landis, S. C.; Foote, J.; Kantrowitz, E. R. Anal. Biochem. 1981, 118, 358.
26. Tian, F.; Montchamp, J.-L.; Frost, J. W. J. Org. Chem. 1996, 61, 7373.
27. Reviews: (a) He, G.-X.; Krise, J. P.; Oliyai, R. Prodrugs: Challenges Rewards 2007,
5, 923; (b) Krise, J. P.; Stella, V. J. Adv. Drug Deliv. Rev. 1996, 19, 287; (c)
Bundgaard, H. Drugs Future 1991, 16, 443.
28. (a) Montchamp, J.-L.; Frost, J. W. J. Am. Chem. Soc. 1997, 119, 7645; (b)
Widlanski, T. S.; Bender, S. L.; Knowles, J. R. J. Am. Chem. Soc. 1987, 109, 1873;
(c) Widlanski, T.; Bender, S. L.; Knowles, J. R. J. Am. Chem. Soc. 1989, 111, 2299;
(d) Bender, S. L.; Widlanski, T.; Knowles, J. R. Biochemistry 1989, 28, 7560; (e)
Widlanski, T.; Bender, S. L.; Knowles, J. R. Biochemistry 1989, 28, 7572; (f)
Knowles, J. R. Aldrichim. Acta 1989, 22, 59.
RP(O)(OR1)Cl: (h) Sampson, N. A.; Bartlett, P. A. J. Org. Chem. 1988, 53, 4500;
RP(O)(OR1)H to RP(O)(OR1)OR2 (i) Coudray, L.; Abrunhosa-Thomas, I.;
Montchamp, J.-L. Tetrahedron Lett. 2007, 48, 6505; (j) Bravo-Altamirano, K.;
Montchamp, J.-L. Tetrahedron Lett. 2007, 48, 5755. and references cited therein;
(k) Gouault-Bironneau, S.; Deprèle, S.; Sutor, A.; Montchamp, J.-L. Org. Lett.
2005, 7, 5909. RP(O)(OR1)H to RP(O)(OR1)NR2 R3; (l) See Ref.13h.; (m) Mucha,
A.; Kafarski, P.; Plenat, F.; Cristau, H.-J. Tetrahedron 1994, 50, 12743;
RP(O)(OR1)H to RP(O)(OR1)SR2: (n) Benschop, H. P.; Plattenburg, D. H. J. M. J.
Chem. Soc., Chem. Commun. 1970, 1098; RP(O)(OR1)H to RP(S)(OR1)OH: (o)
Belabassi, Y.; Gushwa, A. F.; Richards, A. F.; Montchamp, J.-L. Phosphorus, Sulfur
Silicon Relat. Elem. 2008, 183, 2214; (p) Selvam, C.; Goudet, C.; Oueslati, N.; Pin,
J.-P.; Acher, F. C. J. Med. Chem. 2007, 50, 4656; (q) Gautier, A.; Garipova, G.;
Salcedo, C.; Balieu, S.; Piettre, S. R. Angew. Chem., Int. Ed. 2004, 43, 5963; (r)
Swierczek, K.; Peters, J. W.; Hengge, A. C. Tetrahedron 2003, 59, 595; (s) Reiff, L.
P.; Aaron, H. S. J. Am. Chem. Soc. 1970, 92, 5275; RP(O)(OR1)H to RP(Se)(OR1)OH:
(t) Rachon, J.; Cholewinski, G.; Witt, D. Chem. Commun. 2005, 2692;
RP(O)(OR1)H to RP(O)(OR1)BH3: (u) Barral, K.; Priet, S.; Sire, J.; Neyts, J.;
Balzarini, S.; Canard, B.; Alvarez, K. J. Med. Chem. 2006, 49, 7799; (y) See also
29. Elisabeth, P.; Carpenter, E. P.; Hawkins, A. R.; Frost, J. W.; Brown, K. A. Nature
1998, 394, 299.
30. Ames, B. N. Methods Enzymol. 1966, 8, 115.
Ref.13o
.