Submicromolar phosphinic inhibitors of Escherichia coli aspartate transcarbamoylase

Article abstract of DOI:10.1016/j.bmcl.2008.11.115

The design, syntheses, and enzymatic activity of two submicromolar competitive inhibitors of aspartate transcarbamoylase (ATCase) are described. The phosphinate inhibitors are analogs of N-phosphonacetyl-l-aspartate (PALA) but have a reduced charge at the

Full text of DOI:10.1016/j.bmcl.2008.11.115

Bioorganic & Medicinal Chemistry Letters 19 (2009) 900–902
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Bioorganic & Medicinal Chemistry Letters
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Submicromolar phosphinic inhibitors of Escherichia coli
aspartate transcarbamoylase
Laëtitia Coudray a, Evan R. Kantrowitz b, Jean-Luc Montchamp a,
*
a Department of Chemistry, Texas Christian University, Box 298860, Fort Worth, TX 76129, USA
b Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, MA 02467-3807, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, syntheses, and enzymatic activity of two submicromolar competitive inhibitors of aspartate
Received 9 October 2008
Revised 25 November 2008
Accepted 26 November 2008
Available online 6 December 2008
transcarbamoylase (ATCase) are described. The phosphinate inhibitors are analogs of N-phosphonacetyl-
L
-aspartate (PALA) but have a reduced charge at the phosphorus moiety. The mechanistic implications are
discussed in terms of a possible cyclic transition-state during enzymatic catalysis.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Enzyme inhibitor
Aspartate transcarbamoylase
Phosphinate
Phosphonate
Since the discovery of N-phosphonacetyl-
L
-aspartate 1 (PALA) in
Analyzing the role of the charge at the phosphonate moiety of 1 is
interesting based on the proposal of an ordered cyclic transition-
state for the proton transfer from nitrogen to oxygen (Scheme 1).4
Additionally, H-phosphinate 3 could constitute a prodrug of PALA
if oxidation takes place in vivo.
1971, and its subsequent evaluation for use in cancer chemotherapy,
the inhibition of aspartate transcarbamoylase (ATCase) has received
considerable attention.1 To date, no inhibitor has been reported to be
morepotentthanPALA.2 Recently, basedoncrystallographicstudies,
Kantrowitz designed inhibitor 2, which is nearly as potent as PALA,
but is less highly charged due to the presence of an amide group in
the aspartyl moiety.3 To the best of our knowledge, inhibitors of AT-
Case, which are restricted to a monoanionic species where PALA’s
phosphonate group is located, have not been studied. Herein, we re-
port the first phosphinate inhibitors 3 and 4 of ATCase. Inhibition
with these phosphinates has mechanistic implications and may pro-
vide a new direction for the design of compounds with a reduced
charge and improved pharmacological profiles.
H-Phosphinate 3 was synthesized as shown in Scheme 2. Cin-
namyl alcohol was converted into cinnamyl-H-phosphinic acid 5
using a palladium-catalyzed allylation reaction.5 Reaction of 5 with
triethyl orthoacetate provided the protected intermediate 6 in
excellent yield.6 Ozonolysis of 6 to the aldehyde, followed by oxi-
dation7 gave 7, which was reacted without purification with
L-
aspartic acid dibenzyl ester to form amide 8. Deprotection through
catalytic hydrogenation, followed by acid hydrolysis provided 3
cleanly, and in good yield after purification by ion-exchange chro-
matography. The possible presence of PALA was ruled out based on
NMR analysis of the final product, as well as the ion-exchange
purification.
Hydroxymethyl phosphinate 4 was synthesized as shown in
Scheme 3. Hydroxymethyl-H-phosphinic acid8 was silylated and
reacted9 with the bromoacetyl derivative of aspartic acid in a
well-known Arbuzov-like reaction. Esterification with diphenyl-
diazomethane10 provided 10, which was purified by column chro-
matography over silica gel. Catalytic debenzylation provided the
desired phosphinate 4 cleanly and in quantitative yield. No ion-ex-
change purification was necessary in this case.
H
N
H
N
O
P
OH
O
P
OH
CO2H
CO2H
CONH2
CO2H
HO
HO
O
O
1 PALA
2
H
N
H
O
P
OH
O
P
OH
CO2H
CO2H
N
CO2H
CO2H
H
HO
O
O
3
4
Compounds 3 and 4, as well as PALA, were evaluated against the
catalytic subunit of ATCase which was purified from the strain/
plasmid combination pEK17/EK1104 as previously described.12
The results are shown in Table 1. Both phosphinates are weaker
* Corresponding author. Tel.: +1 817 257 6201.
E-mail address: j.montchamp@tcu.edu (J.-L. Montchamp).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.115

L. Coudray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 900–902
901
H
O
P
O
ATCase
H2N
CO2
CO2
H2N
N
CO2
CO2
O
NH2
2-
+
O
+ HOPO3
O
O
O
P
O
δ
O
H
O
CO2
CO2
O
N
H
δ
H2N
Scheme 1. ATCase-catalyzed transformation and postulated transition-state.
O
P
O
P
H
a
b
c, d
EtO
EtO
HO
Ph
Ph
Ph
OH
OEt
5
6
H
O
P
H
N
O
P
H
EtO
EtO
O
f, g
e
EtO
EtO
HO
N
COOH
COOH
COOBn
COOBn
COOH
P
EtO
O
O
OEt
7
8
3
Scheme 2. Reagents and conditions: (a) H3PO2 (2.0 equiv), Pd(OAc)2 (0.2 mol %), xantphos (0.22 mol %), DMF, 85 °C, N2, 7 h, 95%; (b) CH3C(OEt)3 (6.0 equiv), BF3ÁOEt2
(0.16 equiv), rt, N2, 24 h, 80%; (c) O3, CH2Cl2, À78 °C then Me2S (6.8 equiv), À78 °C to rt, N2, 14 h; (d) NaClO2 (1.5 equiv), NaH2PO4ÁH2O (1.5 equiv), 2-methyl-2-butene
(2.0 equiv), tBuOH/H2O, 0 °C then rt, 1 h; (e)
-aspartic acid dibenzyl ester p-toluenesulfonate (1.8 equiv), DMAP (2.5 equiv), EDCÁHCl (3.5 equiv), Et3N (2.0 equiv), THF, rt, N2,
L
16 h, 70% from 6; (f) H2, Pd/C, THF/H2O, 17 h; (g) Amberlite IR 120 plus, THF/H2O, 80 °C, 15 h, 57% from 8 after ion-exchange chromatography.
H
N
O
P
O
H
N
O
P
COOBn
COOBn
O
P
OH
c
a, b
H
OH
HO
Ph
COOH
COOH
HO
HO
O
O
Ph
9
10
4
Scheme 3. Reagents and conditions: (a) N-(bromoacetyl)-L-aspartic acid dibenzyl ester (1.0 equiv), HMDS (2.5 equiv), TMSCl (2.5 equiv), toluene, reflux, 14 h; (b) Ph2CN2,
toluene, rt, 10 min, 50% from 9; (c) H2, Pd/C, THF/H2O, 24 h, 100%.
Table 1
the phosphorus geometry for inhibition with the phosphinate
inhibitors.
Inhibition constants for PALA and phosphinates 3 and 4a
Inhibitor
Inhibition constant (Ki) (nM)b
Inhibition type
Interestingly, H-phosphinate 3 might also be oxidized in vivo to
PALA thus providing a potential prodrug of the anticancer com-
pound.6a,14 In fact, there is some precedent for the oxidation of
H-phosphinate compounds into the corresponding phosphonates:
for example, 3-aminopropyl-H-phosphinate (CGP 27492), a potent
GABAB receptor agonist, is oxidized to a significant extent when
administered to rats.6a In terms of inhibitor design, the possibility
of synthesizing cyclic phosphinate inhibitors to mimic the transi-
tion-state shown in Scheme 1 should be investigated. Even with
an initial loss in binding affinity, the resulting transition-state ana-
logs might lead to potent time-dependent inhibition.
In conclusion, we have synthesized the first phosphinate inhib-
itors of ATCase. Although the observed inhibition constants are
consistent with the enzyme initially binding to the phosphate
dianion of carbamyl phosphate, inhibitors 3 and 4 show significant
potency. In fact, these appear to be the most potent non-phospho-
nate inhibitors of ATCase. Additionally, H-phosphinate 3 could
function as an oxidatively activated PALA prodrug. Implementing
the charge reduction simultaneously in both the phosphorus moi-
ety as in 3 and 4, and in the aspartate moiety as in 2 might result in
useful inhibition while decreasing the overall charge of PALA. This,
and cyclic inhibitors to mimic the postulated transition-state, will
be the object of future studies.
PALA 1
16
2
Competitive
Competitive
Competitive
H-Phosphinate 3
Hydroxymethylphosphinate 4
417 85
193 32
a
Colorimetric assay detecting the formation of N-carbamoyl-
L
-aspartate.11 The
Escherichia coli catalytic subunit of ATCase was used.
b
Inhibition relative to carbamyl phosphate Km = 28
7 lM.
inhibitors than PALA by one to two orders of magnitude (12- to 26-
fold). This is not unexpected if ATCase binds the phosphate dianion
as suggested in Scheme 1. However, the loss in inhibition observed
with the monoanionic phosphorus moieties of 3 and 4, is perhaps
not as pronounced as what such a profound modification would
entail. For example, in the case of 3-dehydroquinate synthase
(DHQ synthase), which is an example of an enzyme exploiting its
substrate charged state for catalytic gain, a similar modification re-
sults in a three to four orders of magnitude loss in inhibition.13 Car-
boxylate 11 was synthesized (Scheme 4) and evaluated in order to
establish if simply providing a single negative charge is responsible
for the inhibition observed with 3 and 4. Compound 11 was com-
pletely inactive against ATCase, thus confirming the importance of

902
L. Coudray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 900–902
H
H
N
a
b
BnO
N
CO2Bn
CO2Bn
HO
CO2H
CO2H
O
O
O
O
O
O
BnO
OH
11
Scheme 4. Reagents and conditions: (a)
78% from 6; (b) H2, Pd/C, THF/H2O, 24 h, 83%.
L
-aspartic acid dibenzyl ester p-toluenesulfonate (1.0 equiv), DMAP (2.5 equiv), EDCÁHCl (2.5 equiv), Et3N (1.1 equiv), THF, rt, N2, 5 h,
Biophys. Res. Commun. 1987, 142, 893; (f) White, J. C.; Hines, L. H. Biochem.
Pharmacol. 1984, 33, 3645.
Acknowledgments
5. (a) Bravo-Altamirano, K.; Montchamp, J.-L. Org. Lett. 2006, 8, 4169; (b) Bravo-
Altamirano, K.; Montchamp, J.-L. Org. Synth. 2008, 85, 96.
We thank the National Institute of General Medical Sciences/
NIH (R01 GM067610 for L.C. and J.L.M., and 5RO1 GM026237 for
E.R.K.) for the financial support of this research.
6. Triethyl orthoformate has been used occasionally to protect H-phosphinic
acids: (a) Froestl, W.; Mickel, S. J.; Hall, R. G.; von Sprecher, G.; 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 Stano, A.;
Mucha, A.; Kafarski, P. Synth. Commun. 1999, 29, 4269. However, triethyl
orthoacetate has not been previously used for this purpose and we are planning
a full study on the scope and utility of this H-phosphinic acid protection in the
near future: Coudray, L.; Montchamp, J.-L., unpublished results.
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