Design, synthesis, and bioactivity of novel inhibitors of E. coli aspartate transcarbamoylase

Article abstract of DOI:10.1016/j.bmcl.2006.12.050

A series of inhibitors of the aspartate transcarbamoylase, an enzyme involved in pyrimidine nucleotide biosynthesis, has been synthesized. These inhibitors are analogues of a highly potent inhibitor of this enzyme, N-phosphonacetyl-l-aspartate (PALA). Analogues have been synthesized with modifications at the α- and β-carboxylates as well as at the aspartate moiety. The ability of these compounds to inhibit the enzyme was evaluated. These studies, with functional group modified PALA derivatives, showed that amide groups can be a useful substitute of the carboxylate in order to reduce the charge on the molecule, and indicate that the relative position of the functional group in the β-position is more critical than the nature of the functional group. Some of the molecules synthesized here are potent inhibitors of the enzyme.

Full text of DOI:10.1016/j.bmcl.2006.12.050

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2086–2090
Design, synthesis, and bioactivity of novel inhibitors
of E. coli aspartate transcarbamoylase
Joby Eldo, Sabrina Heng and Evan R. Kantrowitz*
Boston College, Department of Chemistry, Merkert Chemistry Center, Chestnut Hill, MA 02467, USA
Received 30 October 2006; revised 12 December 2006; accepted 13 December 2006
Available online 21 December 2006
Abstract—A series of inhibitors of the aspartate transcarbamoylase, an enzyme involved in pyrimidine nucleotide biosynthesis, has
been synthesized. These inhibitors are analogues of a highly potent inhibitor of this enzyme, N-phosphonacetyl-L-aspartate (PALA).
Analogues have been synthesized with modifications at the a- and b-carboxylates as well as at the aspartate moiety. The ability of
these compounds to inhibit the enzyme was evaluated. These studies, with functional group modified PALA derivatives, showed that
amide groups can be a useful substitute of the carboxylate in order to reduce the charge on the molecule, and indicate that the rel-
ative position of the functional group in the b-position is more critical than the nature of the functional group. Some of the mol-
ecules synthesized here are potent inhibitors of the enzyme.
Ó 2007 Elsevier Ltd. All rights reserved.
In mammals aspartate transcarbamoylase (ATCase) is a
portion of a multifunctional enzyme (CAD)1 which is re-
quired for de novo pyrimidine nucleotide biosynthesis.
The ATCase portion of CAD catalyzes the second
step in pyrimidine nucleotide biosynthesis, the reaction
between carbamoyl phosphate and L-aspartate to give
N-carbamoyl-L-aspartate and inorganic phosphate.2
ATCase has become a target for the development of
anti-proliferative drugs and inhibitors of ATCase are
considered as potential anti-tumor agents, since the
levels of ATCase have been shown to be elevated in
cancer cells.3
No structural data are available for CAD or the ATCase
portion of CAD, however the amino acid sequences of
the mammalian and Escherichia coli enzymes are 43%
identical, and therefore the structure of the E. coli
enzyme has often been used as a model for the structure
of the ATCase portion of CAD.12 All of the side chains
important for catalytic activity in the E. coli enzyme are
also present in the mammalian enzyme. Finally, PALA
has been shown to be an effective inhibitor of both the
mammalian and the E. coli enzymes.4,6
Numerous analogues of PALA have been reported,
unfortunately none of them are as potent as PALA.13–21
Functional group modifications of PALA, without sig-
nificant perturbations to the core structure, may be a
promising method to design new inhibitors for
ATCase. Although several research groups have been
interested in the chemical and biological consequences
of the modification of the carboxyl groups in the aspar-
tic acid portion of PALA by other functional groups like
phosphonic22,23 or polyethyleneglycol monomethylether
groups for a better prodrug,24 few studies have been per-
formed to evaluate the effect of replacing the carboxyl-
ate moieties of PALA. This study describes the
synthesis of a series of PALA analogues with modifica-
tions on the aspartate unit and determines how effective
these compounds are in binding to and inhibiting the en-
zyme. Functional group modifications reported here in-
clude the introduction of alcohol and amide groups
N-(Phosphonacetyl)-L-aspartate (PALA), a bi-substrate
analogue of ATCase,4 is a potent inhibitor which shows
strong anti-proliferative5,6 and anti-tumor7,8 activities in
cell culture. It has been examined in clinical trials as a
possible anti-proliferation agent, however, a consider-
able drop in its effectiveness was observed.9 This is prob-
ably due to the difficult translocation of PALA into
cells,10 since the highly ionic nature of PALA makes it
difficult for it to diffuse through the lipid bilayer of the
cell membrane.11
Keywords: Allosteric enzyme; Bi-substrate analogue; Aspartate
carbamoyltransferase.
*
Corresponding author. Tel.: +1 617 552 4558; e-mail:
evan.kantrowitz@bc.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.050

J. Eldo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2086–2090
2087
instead of the carboxylates at the a- and b-positions of
the aspartate moiety, along with the replacement of
the entire aspartate moiety with other amino acids such
as aminomalonate, threonine, tyrosine and serine. In
this communication, we report the design, synthesis,
and inhibitory ability of this unique class of structurally
modified PALA analogues (Fig. 1), and describe aspects
of their structure–activity relationship.
or b-positions of the aspartate moiety and then an amide
was introduced in the a- or b-positions of the aspartate
moiety.
The b-hydroxy or homoserine derivative (2) was synthe-
sized from commercially available b-benzyl-L-aspartate
(10) in six steps (Scheme 1). After protecting the a-car-
boxylic acid as the tert-butylester, chloroacetylation
was performed on 11 to give the amide 12 in 68% yield.
The reaction of chloroacetyl L-aspartate with triethyl
phosphite under reflux conditions afforded the corre-
sponding phosphonate ester (13) in good yield. Then
A series of functional group modified PALA molecules
(1–4) were synthesized according to Schemes 1–3. Ini-
tially, a hydroxy group was introduced in the a- and/
Figure 1. Structure of PALA, functional group modified analogues of PALA (1–9).
Scheme 1. Reagents and conditions: (a) H2SO4, isobutylene, dioxane, 20 h, 74%; (b) chloroacetic anhydride (2 equiv), pyridine (5 equiv), CH2Cl2,
4 h, 68%; (c) P(OEt)3, 150 °C, 8 h, 97%; (d) 10% Pd/C, H2, EtOH, overnight, 92%; (e) i—ethyl chloroformate (1.1 equiv), Et3N (1.1 equiv), À17 °C,
40 min; ii—NaBH4 (3.5 equiv), THF/H2O (4:1 v/v), 5 h, 59%; (f) TFA, CH2Cl2, 3 h; (g) i—TMSBr (6 equiv), CH3CN, 0 °C to rt, overnight; ii—H2O,
1 h, 85%.
Scheme 2. Reagents and conditions: (a) LiAlH4 (3 equiv), THF, 0 °C, then reflux for 30 min, 80%; (b) di-tert-butyl dicarbonate (1.3 equiv), MeOH/
t-BuOH (1:1, v/v), 24 h, 18 (92%); 19 (85%); (c) BnBr (3.7 equiv), KOH (3.7 equiv), DMF, 4 h, 20 (59%); 21 (52%); (d) TFA, CH2Cl2, 3 h, 22 (83%);
23 (89%); (e) phosphonoacetic acid, Et3N (1 equiv), DCC (1.1 equiv), HOBt (1 equiv), CH2Cl2, THF, overnight, 24 (74%); 25 (76%); (f) i—TMSBr
(5.6 equiv), CH3CN, 0 °C to rt, overnight, ii—H2O, 1 h, 98%; (g) 10% Pd/C, H2, EtOH, overnight, 8 (90%); 4 (77%).

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J. Eldo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2086–2090
these compounds to inhibit ATCase. The synthesis and
detailed studies of the a-amide derivative (1) have been
reported26 and 1 showed nanomolar level inhibition of
ATCase. The b-amide derivative (3) was synthesized
by a shorter route from commercially available starting
material, tert-butyl L-asparaginate (28) (Scheme 3).
Coupling of tert-butyl L-asparaginate with phosphono-
acetic acid under standard amino acid coupling condi-
tions afforded the phosphonate (30), which upon
sequential deprotection of the tert-butyl and the phos-
phonate esters resulted in the b-amide analogue (3) of
PALA.
Scheme 3. Reagents and conditions: (a) Et3N (1 equiv), DCC
(1.1 equiv), HOBt (1 equiv), CH2Cl2, THF, overnight, 85%; (b) 4 N
HCl, dioxane, 0 °C to rt, 6 h, 100%; (c) i—TMSBr (5.6 equiv),
CH3CN, 0 °C to rt, overnight; ii—H2O, 1 h, 81%.
All other structurally modified analogues were
synthesized in three step reactions, except for (8), using
similar synthetic routes (Scheme 4).14 The malonate
alcohol derivative (8) was synthesized according to a
similar method adopted for the synthesis of inhibitor 4
(Scheme 2).
the b-benzyl-protecting group was removed using
hydrogenolysis and the acid obtained (14) was further
reduced to the alcohol (15) using NaBH4. The deprotec-
tion of tert-butyl and phosphonate esters of the alcohol
provided the homoserine derivative of PALA (2).
Different protected amino acid or alcohol starting
materials, either purchased (33a) or prepared (33b–d),
were reacted with phosphonoacetic acid to give the cor-
responding amino acid phosphonate derivatives (34a–
d). The phosphonate esters were first deprotected under
TMSBr conditions, and then the alkyl esters were
deprotected with LiOH to provide the derivatives
(5–7,9).
The bis hydroxy analogue of PALA (4) was synthesized
by a different synthetic route starting from diethyl L-as-
partate (Scheme 2). Reduction of ethyl ester of 16 using
LiAlH4 gave the corresponding bis alcohol (17).25 After
the sequential protection of the amino group as Boc and
the hydroxy group as the benzyl ether, the Boc was
cleaved with TFA and the resulting amine (23) was cou-
pled with phosphonoacetic acid to afford the phospho-
nate ester (25) in 76% yield. Final deprotection of the
phosphonate ester and benzyl ether under TMSBr con-
ditions and hydrogenolysis, respectively, afforded the
bis hydroxy analogue 4.
The ability of these compounds to inhibit ATCase
(IC50) (1–9) was evaluated against the catalytic subunit
of E. coli aspartate transcarbamoylase (ATCase) by a
colorimetric determination of the amount of N-car-
bamoyl-L-aspartate formed.27 The results obtained are
summarized in Table 1. These functional group modifi-
cations have a very large impact on the ability of these
compounds to inhibit the enzyme. Compounds 1 and 3
The introduction of an amide group in the a- or b-posi-
tions resulted in significant changes in the ability of
Scheme 4. Reagents and conditions: (a) SOCl2, CH3OH, 0 °C to rt, overnight, 33b (96%); 33c (92%); 33d (86%); (b) phosphonoacetic acid, Et3N
(1 equiv), DIC (1.1 equiv), HOBt (1 equiv), CH2Cl2, THF, overnight, 34a (73%); 34b (38%); 34c (83%); 34d (73%); (c) i—TMSBr (5.6 equiv),
CH3CN, 0 °C to rt, overnight, ii—H2O, rt, 1 h, 35a (70%); 35b (93%); 35c (95%); 35d (90%); (d) LiOH, CH3OH, rt, 6 h, 5 (98%); 6 (98%); 7 (95%); 9
(96%).

J. Eldo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2086–2090
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Table 1. IC50 values of inhibitors 1–9 and PALA against the catalytic subunit of ATCase
R1
R3
O
P
NH
O
R2 NH
OH
OH
OH
P
O
O
OH
Compounds 5-9
Compounds 1-4
Compound
R1
R2
R3
IC50 (lM)
PALA
COOH
COOH
CH2OH
CONH2
CH2OH
COOH
CONH2
COOH
COOH
CH2OH
0.055
0.087
3.900
0.225
2900
1
2
3
4
5
6
7
8
9
CH(COOH)2
CH(COOH)CH2OH
43.50
30.00
42.00
12500
63.00
CH(COOH)CH(OH)CH3
CH(CH2OH)2
CH(COOH)CH2–p-(C6H4)OH
showed inhibition at the nanomolar level, which is very
close to the inhibition observed for PALA. Compari-
son of inhibitor 1 with PALA indicates that the amide
modification in the a-position does not affect the
inhibition significantly. Among the amide analogues,
the a-amide showed more than twofold better inhibi-
tion than the b-amide, indicating that the b-carboxylate
has more influence than the a-carboxylic group in the
binding of the inhibitor. Though the analogues with
an amide group did not make any dramatic changes
in the observed inhibition as compared to PALA,
introduction of the alcohol functionality resulted in
analogues that were had substantially reduced ability
to inhibit the enzyme. The mono alcohol 2 showed a
100-fold weaker inhibition compared to PALA,
whereas the di-alcohol 4 only inhibited in the millimo-
lar range. All the structurally modified compounds, ex-
cept for 8, exhibited inhibition at the micromolar level.
Inhibitor 5, which has one methylene unit less than
PALA, showed an approximate 103-fold decrease in
ability to inhibit the enzyme. A comparison of the
IC50 values of inhibitors 5, 6, and 7 (see Table 1) indi-
cates that the conversion of the carboxylate group into
a primary or secondary alcohol does not have a signif-
icant influence on the inhibition, and also indicates that
the relative position of the b-carboxylate moiety plays
a crucial role in binding.
Acknowledgment
This work was supported by Grant GM26237 from the
National Institutes of Health.
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In summary, we described the synthesis of a series of
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modifications. Also studied was the effect of these mod-
ifications on the ability of these analogues to inhibit the
activity in ATCase. These studies with functional group
modified PALA derivatives showed that amide groups
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these analogues indicated that the methylene unit in
the b-position is more critical than the functional group
itself. Some of the newly synthesized molecules are po-
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