Synthesis and characterization of an N-acylsulfonamide inhibitor of human asparagine synthetase

Article abstract of DOI:10.1021/ol034212n

(Matrix presented) The synthesis of N-acylsulfonamide 6, which is an analogue of β-aspartyl-AMP, is described. This compound appears to be the first and only potent inhibitor of human asparagine synthetase that has been described to date. The N-acylsulfon

Full text of DOI:10.1021/ol034212n

ORGANIC
LETTERS
2003
Vol. 5, No. 12
2033-2036
Synthesis and Characterization of an
N-Acylsulfonamide Inhibitor of Human
Asparagine Synthetase†
Lukasz Koroniak, Mihai Ciustea, Jemy A. Gutierrez, and Nigel G. J. Richards*
Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611
richards@qtp.ufl.edu
Received February 5, 2003
ABSTRACT
The synthesis of N-acylsulfonamide 6, which is an analogue of â-aspartyl-AMP, is described. This compound appears to be the first and only
potent inhibitor of human asparagine synthetase that has been described to date. The N-acylsulfonamide 6 exhibits slow-onset inhibition
kinetics, with a K * of 728 nM. Preparation and characterization of two additional N-acylsulfonamide analogues has also demonstrated the
i
importance of hydrogen-bonding interactions in the recognition of the AS inhibitor with the enzyme. These observations provide the basis for
the discovery of new compounds with application in the treatment of drug-resistant leukemia.
In humans, asparagine is biosynthesized from aspartic acid
by asparagine synthetase (AS), a glutamine-dependent Ntn
amidotransferase,1 in an ATP-dependent reaction for which
the cellular nitrogen source is glutamine.2 Several lines of
evidence suggest that overexpression of asparagine synthetase
(AS) in human T-cells results in metabolic changes that
underpin the appearance of drug-resistant forms of acute
lymphoblastic leukemia (ALL).3 Hence, it is widely believed
that, in combination with other drugs, potent AS inhibitors
will provide new clinical approaches for the treatment of
ALL and other solid tumors. The failure of large-scale
screening studies to identify compounds that act as potent
and selective AS inhibitors,4 and a lack of routine access to
large amounts of the human enzyme, have significantly
hindered efforts to solve this important problem in medicinal
chemistry. Our recent determination of the three-dimensional
structure of the glutamine-dependent AS present in Escheri-
chia coli (AS-B)5 and demonstration that adenylated sulfox-
imine 1 (Figure 1)6 inhibits AS-B,7 however, have set the
stage for rational strategies for discovering AS inhibitors.
The likely synthetic difficulty of routinely obtaining
simplified analogues of 1 in large amounts prompted us to
explore whether stable analogues of â-aspartyl-AMP
(âAspAMP) 2 (Figure 1), which is an intermediate in the
AS-catalyzed reaction,8 would function as inhibitors of the
human enzyme. Our initial work focused on the âAspAMP
analogue 3 (Figure 1) that could be obtained using a novel
† Portions of the work described in this paper were presented at the 223rd
National Meeting of the American Chemical Society, Orlando, FL, April
2002 (MEDI-143).
(1) (a) Zalkin, H.; Smith, J. L. AdV. Enzymol. Relat. Areas Mol. Biol.
1998, 72, 87-144. (b) Smith, J. L. Biochem. Soc. Trans. 1995, 23, 894-
898.
(2) Richards, N. G. J.; Schuster, S. M. AdV. Enzymol. Relat. Areas Mol.
Biol. 1998, 72, 145-198.
(3) (a) Aslanian, A. M.; Fletcher, B. S.; Kilberg, M. S. Biochem. J. 2001,
357, 321-328. (b) Hutson, R. G.; Kitoh, T.; Moraga-Amador, D. A.; Cosic,
S.; Schuster, S. M.; Kilberg, M. S. Am. J. Physiol. 1997, 272, C1691-
C1699. (c) Chakrabarti, R.; Schuster, S. M. J. Ped. Hematol./Oncol. 1997,
4, 597-611.
(4) (a) Cooney, D. A.; Driscoll, J. S.; Milman, H. A.; Jayaram, H. N.;
Davis, R. D. Canc. Treat. Rep. 1976, 60, 1493-1557. (b) Cooney, D. A.;
Jones, M. T.; Milman, H. A.; Young, D. M.; Jayaram, H. N. Int. J. Biochem.
1980, 11, 519-539.
(5) Larsen, T. M.; Boehlein, S. K.; Schuster, S. M.; Richards, N. G. J.;
Thoden, J. B.; Holden, H. M.; Rayment, I. Biochemistry 1999, 38, 16146-
16157. Erratum: Biochemistry 2000, 39, 7330-7330.
(6) Koizumi, M.; Hiratake, J.; Nakatsu, T.; Kato, H.; Oda, J. J. Am. Chem.
Soc. 1999, 121, 5799-5800.
(7) Boehlein, S. K.; Nakatsu, T.; Hiratake, J.; Thirumoorthy, R.; Stewart,
J. D.; Richards, N. G. J.; Schuster, S. M. Biochemistry 2001, 41, 11168-
11175.
10.1021/ol034212n CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/09/2003

Scheme 1a
a Key: (a) R1R2CHCH2CO2H, EDCI, HOBt, DMAP, CH3CN,
0 °C to rt; (b) TFA/H2O, rt; (c) Pd black, H2, 5% TFA/MeOH, rt.
Figure 1.
prepared from 2′,3′-isopropylideneadenosine and sulfamyl
chloride12 by slight modification of literature conditions.13
The adenosinesulfamate derivative 7 was then acylated with
appropriately protected carboxylic acid derivatives to give
the N-acylsulfamate derivatives 8-10 in 63-80% isolated
yield after chromatographic purification. Removal of the
acetonide protecting groups was then accomplished by
treatment with aqueous TFA, giving the diols 11-13 in 80-
89% isolated yield. Removal of the benzyl protecting groups,
however, proved particularly problematic for diols 12 and
13, with many standard methods leading to extensive
decomposition of the product N-acylsulfonamides. We
therefore employed short reaction times and determined that
5% TFA in MeOH increased the rate of Pd-catalyzed
hydrogenolysis of the N-benzyloxycarbonyl protecting group.
This procedure reproducibly gave the target N-acylsulfon-
amides 4-6 as their hygroscopic trifluoroacetate salts in 85-
90% yield. Prior to their use in our enzyme assays, all of
these compounds were subjected to chromatography on
Mitsubishi HPSS20 resin11a and their purity checked using
reversed-phase HPLC.14
synthetic procedure for the “one-pot” preparation of N-
acylphosphoramidates.9 Although 3 was found to be an
inhibitor of AS-B, technical limitations in preparing the
compound caused us to undertake the synthesis and char-
acterization of alternate âAspAMP analogues so as to
identify inhibitors containing functional moieties that were
more “drug-like” in structure.10 As a consequence of our
efforts in this area, we now report the preparation of a series
of N-acylsulfonamides 4-6 together with their ability to
inhibit the synthetase activity of recombinant, wild-type,
human AS. These experiments clearly show that sulfonamide
derivative 6 is the first inhibitor of the human enzyme to be
described and reveal the importance of the R-carboxyl and
R-amino groups in the recognition and/or binding of this
compound by AS.
Our synthesis and purification of N-acylsulfonamides 4-6
(Scheme 1) was based on literature procedures for the
preparation of isoleucyl- and tyrosyl-tRNA synthetase inhibi-
tors.11 Thus, our route employed the 2′,3′-protected adeno-
sinesulfamate 7 as starting material, which could be readily
Since our goal was to obtain clinically useful AS inhibitors,
we also developed a baculovirus-based expression system
so as to gain access to large quantities of human AS.15 This
procedure gave wild-type enzyme in multi-milligram amounts,
which exhibited ammonia-dependent synthetase activity16
comparable to that reported previously for recombinant,
human AS obtained using a yeast-based expression system.17
It is therefore unlikely that the additional C-terminal residues
(8) (a) Boehlein, S. K.; Stewart, J. D.; Walworth, E. S.; Thirumoorthy,
R.; Richards, N. G. J.; Schuster, S. M. Biochemistry 1998, 37, 13230-
13238. (b) Luehr, C. A.; Schuster, S. M. Arch. Biochem. Biophys.1985,
237, 335-346.
(9) Ding, Y.; Wang, J.; Schuster, S. M.; Richards, N. G. J. J. Org. Chem.
2002, 67, 4372-4375.
(10) (a) Hann, M. M.; Leach, A. R.; Harper, G. J. Chem. Inf. Comput.
Sci. 2001, 41, 856-864. (b) Teague, S. J.; Davis, A. M.; Leeson, P. D.;
Oprea, T. Angew. Chem., Int. Ed. 1999, 38, 3743-3748. (c) Lipinski, C.
A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. AdV. Drug DeliVery ReV.
1997, 23, 3-25.
(11) (a) Brown, P.; Richardson, C. M.; Mensah, L. M.; O’Hanlon, P. J.;
Osborne, N. F.; Pope, A. J.; Walker, G. Bioorg. Med. Chem. 1999, 7, 2473-
2485. (b) Brown, M. J. B.; Mensah, L. M.; Doyle, M. L.; Broom, N. J. P.;
Osbourne, N.; Forrest, A. K.; Richardson, C. M.; O’Hanlon, P. J.; Pope,
A. J. Biochemistry 2000, 39, 6003-6011. (c) Forrest, A. K.; Jarvest, R. L.;
Mensah, L. M.; O’Hanlon, P. J.; Pope, A. J.; Sheppard, R. J. Bioorg. Med.
Chem. Lett. 2000, 10, 1871-1874.
(12) Peterson, E. M.; Brownell, J.; Vince, R. J. Med. Chem. 1992, 35,
3991-4000.
(13) (a) Heacock, D.; Forsyth, C.; Shiba, K.; Musier-Forsyth, K. Bioorg.
Chem. 1996, 24, 273-289. (b) Jenkins, I. D.; Verheyden, P. H.; Moffatt,
J. G. J. Am. Chem. Soc. 1976, 98, 3346-3357.
(14) All compounds prepared in this study exhibited spectroscopic
properties consistent with their proposed structures. Full experimental details
are included in the Supporting Information.
2034
Org. Lett., Vol. 5, No. 12, 2003

have any significant impact on the activity of the enzyme
relative to that of wild-type human AS. We also note that
this baculovirus-based expression system offers significant
advantages for the obtaining human AS for the assay of
clinically useful inhibitors, including simple scale-up for
large-scale protein production.
tance of the ionized R-amino- and R-carboxylate groups to
the interaction of N-acylsulfonamide 6 with the enzyme.
Importantly, control experiments established that the pyro-
phosphate reagent is not affected by the presence of
N-acylsulfonamides 4-6.
Analysis of the steady-state kinetics of AS inhibition by
N-acylsulfonamide 6 was undertaken to determine the
mechanism by which the inhibitor exerted its effects. The
data were best fit (Figure 2) using the following equation,
corresponding to slow-onset inhibition19
With the target N-acylsulfonamide analogues 4-6 in hand,
we examined their ability to inhibit the ammonia-dependent
activity of the recombinant, C-terminally tagged human AS
using similar conditions to those previously used in charac-
terizing inhibition of E. coli AS-B by the adenylated
sulfoximine 1.7 Hence, progress curves were generated by
incubating the purified enzyme (4 µg) in reaction mixtures
containing 100 mM NH4Cl, 0.5 mM ATP, 10 mM aspartate,
and 10 mM MgCl2 in 100 mM EPPS, pH 8, together with
the inhibitor (0-50 µM) (1 mL total volume). Since
asparagine and PPi are formed in a 1:1 stoichiometric ratio
in the enzyme-catalyzed reaction, the synthetase activity of
human AS under these conditions was determined by
spectrophotometric monitoring of PPi production.18 The
results showed that compound 6 did inhibit the recombinant
human enzyme (Figure 2), presumably by binding within
[PPi] ) Vsst + [(Vo - Vss)/k][1 - exp(-kt)]
(1)
where Vo is the initial velocity of the reaction, Vss is the
velocity at large t, and k is a parameter that depends on the
inhibitor concentration. Further experiments demonstrated
that elevated levels of ATP decreased the ability of 6 to
inhibit recombinant human AS (see the Supporting Informa-
tion). This implies that ATP and 6 compete for the free
enzyme, an observation consistent with the assumption that
6 binds within the C-terminal, synthetase site of AS. The
inhibition mechanism is consistent with the following model
for which the variation of k with inhibitor concentration is
given by (see the Supporting Information):
k ) k6 + k5(I/Ki)/[1 + [ATP]/Ka + I/Ki]
(2)
Analysis of the progress curves using eq 1 then gave values
of k as a function of the concentration of 6, and a replot of
(15) Briefly, the gene encoding human AS was obtained and modified
so that the expressed enzyme would contain additional residues at the
C-terminus corresponding to a c-myc tag. The resulting construct was then
cloned into a pBAC-1 transfer vector (Novagen), which includes a
C-terminal histidine tag for protein purification, so that expression of human
AS is under control of the polh promoter allowing high levels of protein
expression during late phases of viral infection. Recombination of pBAC-1
with the BacVector-3000 construct (Novagen) was then used successfully
to obtain a baculovirus capable of replicating and infecting conditioned
Sf9 cells, which also contained the gene encoding human AS. Using standard
procedures, we expressed human AS in conditioned Sf9 cells, the recom-
binant enzyme being purified by chromatography on a preequilibrated Ni-
Agarose column (see the Supporting Information). Additional details of
this expression system can be found in the following references: (a) Farrell,
P. J.; Lu, M. L.; Prevost, J.; Brown, C.; Behie, L.; Iatrou, K. Biotechnol.
Bioeng. 1998, 60, 656-663. (b) Lenhard, T.; Reilander, H. Biochem.
Biophys. Res. Commun. 1997, 238, 823-830.
(16) An important limitation of the baculovirus-based expression protocol
is that oxidation of the N-terminal cysteine residue can take place during
protein isolation and purification. Since the thiolate side chain of Cys-1 is
required for glutamine-dependent activity, our samples of wild-type human
AS cannot usually employ glutamine as a nitrogen source. The active site
that catalyzes the formation of the âAspAMP intermediate 2 and its
subsequent reaction with ammonia is unaffected by oxidation of Cys-1,
and therefore, recombinant human AS can be used in assaying for AS
inhibitors with clinical utility.
Figure 2. Progress curves showing the effect of N-acylsulfonamide
6 on PPi production in the ammonia-dependent synthetase reaction
catalyzed by recombinant, C-terminally tagged human AS. Key:
[6] ) 0 µM, filled circles; [6] ) 5 µM, open circles; [6] ) 10 µM,
filled squares; [6] ) 25 µM, open squares; [6] ) 50 µM, filled
triangles. Solid lines represent the theoretical curve computed from
eq 1 that best fit the experimental data, and error bars correspond
to the standard deviation of the [PPi] concentration measured at a
given time point.
the C-terminal, synthetase site of human AS in a fashion
similar to the âAspAMP intermediate 2 that is formed in
the catalytic mechanism of asparagine production. Under
similar experimental conditions, we observed that sulfon-
amides 4 and 5 were only capable of inhibiting the
recombinant human AS at 100-fold greater concentrations
(data not shown). These results therefore show the impor-
(17) (a) Sheng, S.; Moraga-Amador, D. A.; Van Heeke, G.; Schuster, S.
M. Prot. Exp. Purif. 1992, 3, 337-346. (b) Van Heeke, G.; Schuster, S.
M. Prot. Engng. 1990, 3, 739-744. (c) Sheng, S.; Moraga-Amador, D. A.;
Van Heeke, G.; Allison, R. D.; Richards, N. G. J.; Schuster, S. M. J. Biol.
Chem. 1993, 268, 16771-16780.
Org. Lett., Vol. 5, No. 12, 2003
2035

k versus [6] then yielded values for Ki, k5, and k6 of 21 µM,
3.6 × 10-2 s-1, and 1.3 × 10-3 s-1, respectively. The overall
Acknowledgment. We thank the Chiles Endowment
Biomedical Research Program of the Florida Department of
Health for funding. Useful discussions with W. Wallace
Cleland (University of WisconsinsMadison) and Pamela
Brown (GlaxoSmithKline) concerning details of slow-onset
inhibition kinetics and selected synthetic procedures, respec-
tively, are gratefully acknowledged. We also thank Professor
Michael Kilberg (University of Florida) who generously
provided the gene encoding human AS.
dissociation constant (K* ) for the slow-onset inhibitor 6 is
i
therefore 728 nM.19 While still not sub-nanomolar in potency,
we note that 6 inhibits human AS at a level that is 1000-
fold greater than for any compound yet reported, and hence,
this N-acylsulfonamide represents a useful lead compound
for future studies in this area.
In summary, we have synthesized and characterized the
first inhibitor of human AS, and have obtained important
information for the rational discovery of more potent
compounds that can be employed clinically to treat drug-
resistant leukemia. Efforts to discover nonionizable moieties
that can be substituted for the amino and carboxylate
substituents in 6 are underway in our laboratory, and the
results will be reported in due course.
Supporting Information Available: Synthetic procedures
and spectroscopic characterization of compounds 4-6 and
8-13, a brief description of procedures used to obtain
recombinant, wild-type human AS, and progress curves for
the kinetics of AS inhibition by N-acylsulfonamide 6. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(18) Boehlein S. K.; Richards N. G. J.; Schuster S. M. J. Biol. Chem.
1994, 269, 26789-26795.
(19) Morrison, J. F.; Walsh, C. T. AdV. Enzymol. Relat. Areas Mol. Biol.
1988, 61, 201-301.
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