Inhibition of glyoxalase I: The first low-nanomolar tight-binding inhibitors

Article abstract of DOI:10.1021/jm900382u

A series of rational modifications to the structure of known S-(N-aryl-N-hydroxycarbamoyl)glutathione-based glyoxalase I inhibitors culminated in the discovery of the first single-digit nanomolar inhibitor. This study makes available key information about possible means to address the issues of metabolic instability, low potency, and synthetic complexicity that have plagued the area of glyoxalase I inhibition. Knowledge garnered from this study has implications in the design of inhibitors with higher conformational definition and lower peptidic character.

Full text of DOI:10.1021/jm900382u

4650 J. Med. Chem. 2009, 52, 4650–4656
DOI: 10.1021/jm900382u
Inhibition of Glyoxalase I: The First Low-Nanomolar Tight-Binding Inhibitors
Swati S. More† and Robert Vince*
Center for Drug Design, Academic Health Center, and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota,
8-123A Weaver Densford Hall, 308 Harvard Street SE, Minneapolis, Minnesota 55455. †Current address: Department of Biopharmaceutical
Sciences, University of California, San Francisco, San Francisco, CA 94158. Phone: 415-514-4363. E-mail: swati.more@ucsf.edu.
Received April 22, 2009
A series of rational modifications to the structure of known S-(N-aryl-N-hydroxycarbamoyl)glu-
tathione-based glyoxalase I inhibitors culminated in the discovery of the first single-digit nanomolar
inhibitor. This study makes available key information about possible means to address the issues of
metabolic instability, low potency, and synthetic complexicity that have plagued the area of glyoxalase I
inhibition. Knowledge garnered from this study has implications in the design of inhibitors with higher
conformational definition and lower peptidic character.
Introduction
γ-glu-cys bond and thus the rapid loss of Glx-I inhibitory
activity. A solution to this problem was found in the replace-
ment of this labile linkage by a robust urea isostere, leading to a
metabolically stable analogue (2, Figure 2), which retains the
Glx-I inhibitory potency of the parent compound.6 On a
separate note, Murthy and Creighton exploited successfully
the Zn2þ-chelating ability of the hydroxamate function in their
design of enediol(ate) transition state analogue inhibitors of
Glx-I, such as the S-(N-Aryl-N-hydroxycarbamoyl) deriva-
tives of glutathione (e.g., 3, Figure 2).7 The structural similarity
borne by compounds such as 3 to glutathione made them
function as substrates of γ-GT, making them extremely labile.
Furthermore, Crieghton et al. have shown that the similarity of
the thiohydroxamates to S-lactoyl glutathione make com-
pounds like 3 function as substrates of Glx-II.8,9 We reported
recently the union of these two distinct strategies represented
by 4 (Figure 2), the urea-isostere analogue of 3, that is resis-
tant to γ-GT-mediated breakdown.10 We next decided to
focus our efforts on simplifying the structural scaffolding of
these hydroxamate inhibitors from a synthetic viewpoint. The
growing synthetic complexicity of these systems due to the
thiohydroxamate linkage, in addition to its potential inst-
ability, also made the replacement of this link particularly
attractive.
The present study was aimed, at least at the outset, to
identify isosteric replacements for the thiohydroxamate link-
age that would improve the facility of the synthesis of the
resultant analogues while retaining their potency against Glx-
I and stability toward γ-GT.
Structural Iteration I. Replacement of Sulfur by Methylene.
Rationale. We turned our attention first to the replacement
of the thiocarbamate linkage in inhibitors like 3 and 4. The
X-ray crystal structure of a hydroxamate ligand bound to
Glx-I, as described by Cameron and Mannervik, was not
suggestive of any specific conformational, electronic, or
spatial elements of recognition contributed by the sulfur
atom.11 Replacement of this sulfur atom by a carbon would
remove the thioester structural motif, potentially rendering
the resultant carbo-analogue resistant to Glx-II. Ly et al.
have demonstrated the utility of such an approach in the
Glycolysis results in a number of electrophilic carbonyl
compounds such as glyoxal, methylglyoxal (MG), and 3-
deoxyglucosone, which are capable of forming adducts with
proteins, lipids, and nucleic acids. Such adducts, collectively
known as advanced glycation end-products (AGEs),1 are
inducers of apoptosis. Several mechanisms exist to protect
the cellular components against such “carbonyl stress”, one of
them being the glyoxalase system thatconverts methylglyoxal,
the simplest of the R-ketoaldehydes, to D-lactate. Depicted in
Figure 1, the system is composed of two enzymes; glyoxalase I
(Glx-I, EC 4.4.1.5) andglyoxalase II (Glx-II, EC 3.1.2.6). Glx-
I mediates the enolization of the hemithioacetal of glutathione
with glyoxal to an enediol(ate). This process amounts to a net
transfer of a hydride from the methine carbon of the hemi-
acetal of methylglyoxal and glutathione to the ketone carbo-
nyl, thereby resulting in the formation of S-D-lactoyl
glutathione. Glx-II then hydrolyzes this labile thioester to
D-lactate, regenerating glutathione.2 A feedback mechanism
exists between glycolysis and detoxification systems such as
the glyoxase system. The proper function of this feedback
mechanism is essential to the prevention of apoptosis.
A hallmark of rapid tumor growth is increased glycolysis,
which is compensated duly in tumor cells through overexpres-
sion of the glyoxalase enzyme system.3 Obstruction of the
glyoxalase system in cancerous cells would lead to the induc-
tion of apoptosis in such cells because of the excessive
intracellular accumulation of methylglyoxal. Our laboratory
was the first to propose the inhibition of Glx-I as a means to
induce apoptosis in cancerous cells and to pioneer the ex-
ploration of the S-alkyl derivatives of glutathione as compe-
titive inhibitors of Glx-I, leading ultimately to a potent Glx-I
inhibitor, S-p-bromobenzyl glutatione (1, PBBG,a Figure 2).4,5
Unfortunately, 1 was recognized as a substrate by γ-glutamyl-
transpeptidase (γ-GT), resulting in a rapid cleavage of its
*To whom correspondence should be addressed. Phone: 612-624-
9911. Fax: 612-624-0139. E-mail: vince001@umn.edu.
a Abbreviations: Glx-I, glyoxalase I; Glx-II, glyoxalase II; γ-GT, γ-
glutamyltranspeptidase; PBBG, S-p-bromobenzyl glutathione.
r
pubs.acs.org/jmc
Published on Web 07/17/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4651
Scheme 1. Synthesis of Hydroxamate-Based Glyoxalase I
Inhibitor 5a
Figure 1. The glyoxalase system.
a Reagents and conditions: (a) HCl H2N-Gly-OtBu, EDC, HOBt,
3
NMM, CH2Cl2, 91%; (b) 4 N HCl/dioxane, 0 °C, 99%; (c) 16, EDC,
HOBt, NMM, CH2Cl2, 84%; (d) H2, 10% Pd/C, CH3OH, 86%; (e) 18,
EDC, CH2Cl2, 52%; (f) TFA, CH2Cl2 (1:1), 76%; (g) TMSCl, CH3OH,
97%; (h) Boc2O, NaHCO3, dioxane/H2O (2:1), 91%; (i) tBuOH, DCC,
DMAP, 94%; (j) LiOH, THF/H2O (3:1), 84%; (k) 5% Rh-C,
Figure 2. Developments in the design of glyoxalase I inhibitors.
NH2NH2 H2O, THF, 71%.
3
inhibition of Glx-I.12 In addition, the appropriate residue
that bears the hydroxamate appendage in the carbo-analo-
gues would be glutamic acid, creating a point of synthetic
entry. We therefore set forth to synthesize 5 (the carbo-
analogue of 3), which embodies this change.
attempted but it gave the coupled product 11 as an insepar-
able mixture with DCU. Finally, this problem was overcome
by utilizing a combination of EDC and a 5-fold excess of
amine 18 at 0-4 °C to give the pure product 11 in 61% yield.
Global deprotection of hydroxamate 11 with trifluoroacetic
acid gave the target 5 as its CF3COOH salt.
Synthesis of 5. Compound 5 was approached through the
synthesis of a suitably protected tripeptide backbone (Glu-
Glu-Gly), followed by coupling of the γ-carboxylic acid of
the central glutamic acid to an aryl-hydroxylamine. As
shown in Scheme 1, synthesis of compound 5 began with
the coupling of commercially available N-Boc-5-benzyl ester
of glutamic acid (6) with glycine tert-butyl ester to give
dipeptide 7. Selective Boc-deprotection over the tert-butyl
ester13 (Hruby’s conditions) and the coupling of the resulting
dipeptide amine 8 with N-Boc-1-tert-butyl ester of glutamic
acid (16) afforded the tripeptide 9. Compound 16 in turn was
prepared from L-glutamic acid through a four-step sequence.
Finally, hydrogenolysis of the side chain benzyl ester of the
tripeptide 9 provided acid 10.
p-Bromophenylhydroxylamine (18, an unstable solid) was
synthesized by a Rh-catalyzed transfer hydrogenation
(“diimide reduction”) of 4-bromonitrobenzene (17).14 Re-
sults of attempted coupling of 18 with acid 10 under varying
reaction temperatures were suggestive of appreciable degra-
dation of hydroxylamine 18 at room temperature, especially
in the presence of tertiary amines. The precise chemical
nature of the degradation, however, was intractable. Hence
our next attempts utilized low reaction temperatures with the
absence of any tertiary amine base. DCC coupling at 0-4 °C
for 6 h, which does not require any tertiary amine base, was
Evaluation of 5 and Resulting Implications. Compound 5
was evaluated for its ability to inhibit yeast glyoxalase I,
along with compounds 1-4 for comparison. The isomeriza-
tion process leading to the formation of S-D-lactoyl glu-
tathione is accompanied by an increase in the absorption at
240 nm. This increase in UV absorption for the first 6 min
was utilized as a gauge to calculate initial rate of the enzy-
matic reaction. Concentrations of the hemimercaptal formed
by equilibration of methylglyoxal and GSH in the presence
and absence of an inhibitor were calculated using the pre-
viously determined dissociation constant of 3.1 ꢀ 10-3 M.5
As seen in Table 1, 5 carries a spectacular increase in potency
over 3 and 4. With an inhibition constant of 6.17 ( 1.64 nM,
it effectively is the most potent inhibitor of Glx-I known to
date and is 1000-fold more potent than the parent thiocar-
boxamate analogue 3. Kinetic experiments indicate that 5 is a
tight-binding inhibitor of Glx-I.
The stability of 5 toward γ-glutamyltranspeptidase
mediated cleavage was then determined next by its incuba-
tion at 37 °C with equine kidney γ-glutamyltranspeptidase
(γ-GT, Sigma) at pH = 8.4 (Supporting Information, Figure
S1).6 Inhibitor 5 underwent facile breakdown at its γ-Glu-
Glu bond with incubated with γ-GT, indicating that this

4652 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
More and Vince
Scheme 3. Synthesis of the Glyoxalase I Inhibitor 26a
Table 1. Inhibitory Constants (Ki) of Glyoxalase I Inhibitors 1-5
Ki ( SEM (μM)
inhibitor
(experimental)
(from literature)
1
2
3
4
5
6.28 ( 1.45
12.8 ( 3.23
9.14 ( 1.306
15.5 ( 3.106
1.20 ( 0.207
2.19 ( 0.5711
1.15 ( 0.46
2.19 ( 0.5711
0.00617 ( 0.00164
Scheme 2. Synthesis of the Glyoxalase I Inhibitor 19a
a Reagents and conditions: (a) allyl bromide, benzene, reflux, 92%;
(b) 4 N HCl/dioxane, 96%; (c) 38, CDI, NMM, CH2Cl2, 82%;
(d) (PPh3)4Pd, morpholine, CH2Cl2; (e) HCl H2N-Gly-OtBu, EDC,
3
HOBt, NMM, CH2Cl2, 76% over two steps; (f) H2, 10% Pd/C, CH3OH,
89%; (g) 18, EDC, CH2Cl2, 64%; (h) TFA, CH2Cl2 (1:1), 78%; (i) PIDA,
EtOAc/CH3CN/H2O (2:2:1), 67%; (j) CbzCl, KOH, K2CO3, THF/H2O
(3:1), 94%; (k) tBuOH, DCC, DMAP, CH2Cl2, 84%; (l) H2, 10% Pd/C,
CH3OH, 94%.
a Reagents and conditions: (a) TMSCl, CH3OH, 98%; (b) Boc2O,
NaHCO3, dioxane-H2O (2:1), 86%; (c) HCl H2N-Gly-OtBu, EDC,
3
HOBt, NMM, CH2Cl2, 88%; (d) 4 N HCl/dioxane, 0 °C, 95%; (e) 16,
EDC, HOBt, NMM, CH2Cl2, 34%; (f) LiOH, THF/H2O (1:1), 91%;
(g) 18, EDC, CH2Cl2, 62%; (h) TFA, CH2Cl2 (1:1), 79%.
followed by suitable functionalization as described in our
previous report.16 The allyl ester function of 29 was cleaved
in the presence of the benzyl ester with (Ph3P)4Pd, and the
resultant carboxylic acid was coupled to H-Gly-OtBu giving
31. Hydrogenolysis of 31 and subsequent coupling to 18
afforded 33, which was subjected to global deprotection with
breakdown should necessarily be the subject of the next
structural iteration (Supporting Information, Figure S1).
Structural Iteration II. Building Resistance toward γ-GT. It
is known that most proteases do not recognize unnatural
enantiomers of amino acids at the cleavage sites. We de-
signed compound 19 (Scheme 2), which bears a D-glu residue
at the site of cleavage. This compound was approached
through a synthetic route similar to that for 5 (Scheme 2).
The δ-methyl ester of Boc-D-glutamic acid was utilized as the
starting material.
Upon evaluation for Glx-I-inhibitory properties, the po-
tency of 19 was found to be 1.66 ( 0.67 μM, about 1000-fold
lower than its L-counterpart, 5. Inhibitor 19 was indeed
found to be resistant toward breakdown by γ-GT
(Supporting Information, Figure S1). It is thus evident that
the stereochemical requirements at the central residue for
recognition at the active site of Glx-I are stringent; in other
words, the potency of 5 most likely is a result, in part, of a
well-defined conformation.
Toward the end of imparting γ-GT resistance to 5, we also
employed our previously described strategy of substituting
the labile link by a ureide isostere, leading to compound 26.
The synthesis of 26, as shown in Scheme 3, begins from 6 and
proceeds via the dipeptide 29, the ureide formed by the union
of 28 and the diaminopropanoic acid derivative 38.15
The compound 38 was in turn formed through the
PIDA-mediated Hoffman degradation of the asparagine 34
TFA to afford 26 CF3COOH. Compound 26 was found to
3
retain the strong inhibitory properties of 5; it inhibits Glx-I
with a Ki of 32.6 ( 4.17 nM. In addition, 26 was found to be
expectedly stable toward γ-GT mediated breakdown
(Supporting Information, Figure S1).
Structural Iteration III. Stabilizing the Hydroxamate
Linkage. Having addressed successfully the problem of
γ-GT mediated breakdown, we next turned our attention
to the instability of the hydroxamate itself. We and others
have observed that this linkage itself is susceptible to un-
catalyzed hydrolytic breakdown, caused in part due to the
fact that the anilino nitrogen of the hydroxamate is a good
leaving group.17,18 This problem has previously been ad-
dressed by Michaelides et al. through reversal of the elements
in relation with the scaffold, leading to “retro hydroxa-
mates”, which were found to have superior stability toward
hydrolytic breakdown.19 We decided to examine the effect of
such a substitution on 5, thusly leading to compound 39
(Figure 3).
Synthesis of 39. Our first attempts at the synthesis of the
retrohydroxamate bearing residue were focused on the alky-
lation of N-p-bromobenzoyl-O-benzyl hydroxylamine with
derivatives of homoserine (Scheme 4).20 The allyl ester of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4653
Scheme 5. Attempted Reductive Amination of 46a
a Reagents and conditions: (a) DMP, CH2Cl2, 82%; (b) HCl H2-
3
NOBn, Et3N, CH2Cl2; (c) NaCNBH3, CH3COOH; (d) NaBH4,
CH3OH; (e) NaBH(OAc)3.
Figure 3. Design of retrohydroxamate 39 based on Michaelides’
concept.
Scheme 6. Synthesis of 51 and Its Conversion to 53a
Scheme 4. Alkylation of 41 with Homoserine Electrophilesa
a Reagents and conditions: (a) CbzCl, DIPEA, CH2Cl2, 85%; (b)
NaH, DMF, 53, 89%; (c) 4 N HCl/dioxane, 95%; (d) 18, EDC, HOBt,
NMM, CH2Cl2.
a Reagents and conditions: (a) Boc2O, NaOH, H2O/CH3CN (1:1); (b)
allyl bromide, DMF, 60% over two steps; (c) MsCl, Et3N, CH2Cl2; (d)
LiBr, acetone, 94% over two steps; (e) 41, NaH, DMF; (f) NaI, acetone,
reflux, 89%.
Scheme 7. Synthesis and Unsuccessful Selective Hydrogena-
tion of 57a
Boc-homoserine, 42, was elaborated into the corresponding
bromide 43 and mesylate 44 derivatives. Treatment of the
sodium imidate of 41 with these electrophiles led to 45, whose
δ-CH2 1H resonances appeared at 4.20-4.50 ppm (COSY
assignment) with a corresponding 13C resonance at 153.0
ppm (HMBC assignment). These values are not in agreement
with the expected N-alkylated product, but rather, it seemed
that predominant O-alkylation had occurred. We also at-
tempted to introduce the retrohydroxamate through reduc-
tive amination of glutamic acid semialdehyde 46.21 Although
we could form the oxime 47, we found impossible the
reduction of 47 to 48; NaBH4 and NaCNBH3 led to intrac-
tible mixtures while Na(OAc)BH3 failed to react with 47
(Scheme 5).
Reasoning that the formation of imidate 45 was enhanced
by the electron-withdrawing nature of the bromobenzoyl
substitutent, we attempted to indirectly incorporate the
retrohydroxamate through the corresponding Cbz-deriva-
tive. As shown in Scheme 6, such an alkylation indeed
occurred on the nitrogen. However, the deprotected residue
51 underwent predominantly a Cbz-migration to 53 when its
acylation was attempted, with no trace of the required
dipeptide 52 detected.
To bypass this migration, we synthesized the dipetide 54
instead, which underwent substitution with 49 to afford 55
(Scheme 7). Elaboration of 55 to the tripeptide 57 was also
found to proceed uneventfully. Unfortunately, we could not
cleave selectively the Cbz-protective group of 57, with 58
being the predominant product. This persisted even when an
inhibitor (CHCl3) was added to the reaction. In the latter
a Reagents and conditions: (a) 4 N HCl/dioxane, 95%; (b) 16,
PyAOP, Et3N, CH2Cl2, 82%; (c) NaH, DMF, 49, 76%; (d) Pd(PPh3)4,
morpholine, CH2Cl2, 94%; (e) HCl H2N-Gly-OtBu, EDC, HOBt,
3
NMM, CH2Cl2, 72%; (f) H2, 10% Pd/C, CH3OH, 88%; (g) H2, 10%
Pd/C, 10% CHCl3 in CH3OH.
case, the reaction was indeed retarded but the product
composition was unchanged.
The amine tripeptide 58 obtained in Scheme 6 was exploited
for possible oxidation to the desired hydroxylamine 65.

4654 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
More and Vince
Scheme 8. Direct Oxidation of Primary Amine: A Successful
Approach for the Synthesis of Retrohydroxamate Inhibitor 39a
Compound 39 was found to indeed inhibit Glx-I and was a
tight-binding inhibitor with potency in the nanomolar range
(Ki = 123 ( 5.36 nM). Compound 39 was, in addition, found
to resist uncatalyzed hydrolytic cleavage, validating the
rationale for its design (Supporting Information, Figure S1).
Discussion
This study has lent, perhaps through sheer serendipity, the
first low-nanomolar inhibitor of glyoxalase I (inhibitor 5). We
have also been able to examine the effect of various modifica-
tions of 5 on potency as well as stability. Our previously
reported ureide-isostere strategy was found to be applicable to
5, conferring on this novel class of inhibitors the much needed
stability toward γ-GT mediated breakdown. Reversal of the
atom-order in the hydroxamate function led to the identifica-
tion of the lead inhibitor 39, that possesses stability toward
hydrolytic breakdown. Of much interest is the loss of potency
observed when the configuration about the R-carbon of the
central residue was inverted because this is indicative of a well-
defined conformation being necessary for recognition of
inhibitors such as 5 at the active site of Glx-I. It may be
worthwhile at this juncture to the design and synthesize
constrained molecules in order to delineate the optimal con-
formation.
The peptidic nature of inhibitors as well as low potency has
precluded further research in the area of glyoxalase I inhibi-
tion for a number of years. It is expected that the information
garnered from the studies in this report would be useful
toward the design and synthesis of inhibitors with higher
conformational definition and lower peptidic character,
which translate to higher potency and more favorable phar-
macokinetics.
a Reagents and conditions: (a) PIDA, THF/H2O (2:1), 76%; (b)
CbzCl, K2CO3, THF/H2O (3:1), 81%; (c) HCl H2N-Gly-OtBu, EDC,
3
HOBt, NMM, CH2Cl2, 88%; (d) 4 N HCl/dioxane, 0 C; (e) 16, EDC,
HOBt, NMM, CH2Cl2, 76% over two steps; (f) H2, 10% Pd/C, CH3OH,
96%; (g) benzoylperoxide, carbonate buffer/CH2Cl2 (1:1); (h) 4-bromo-
benzoyl chloride, carbonate buffer/CH2Cl2, 71% over two steps; (i) 10%
NH4OH/CH3OH, 82%; (j) TFA, CH2Cl2 (1:1), 70%.
In 1978, Naegeli and Keller-Schierlein22 reported an elegant
total synthesis of D-ferrichrome by an indirect oxidation of
primary amine to hydroxylamine.23 Their indirect oxidation
featured the conversion of primary amines to imines followed
by oxidation of imines to oxaziridines. Aminolysis of oxazir-
idines afforded hydroxylamines, which led to the successful
synthesis of D-ferrichrome. This indirect oxidation though an
attractive approach for obtaining our reverse-amide hydro-
xamate scaffold was unsuccessful in our hands. However,
another promising alternative was to use direct oxidation
protocol of Nemchik and Phanstiel24,25 that utilizes benzoyl
peroxide as an oxidant.
The tripeptide amine 58 required for this transformation
was obtained in large scale starting with L-glutamine (59,
Scheme 8). Hoffman rearrangement of primary amide in
glutamine26 resulted in amine 60, which was subjected to
Cbz-protection. The acid 61 thus obtained was coupled to
glycine tert-butyl ester, resulting in dipeptide 62. N-Boc-de-
protection followed by coupling of dipeptide 62 with Boc-Glu-
tert-butyl ester 27 offered the tripetide 64. Hydrogenolysis of
Cbz-group gave the primary amine 58, which was subjected to
BPO-mediated oxidation.27,28 Indeed, this protocol gave the
desired O-benzoylhydroxylamine tripeptide 65 along with
∼5% yield of benzoylated derivative of the primary amine
58 due to its acylation with BPO. The tripeptide hydroxy-
lamine 65 was subjected without isolation to acylation with
p-bromobenzoyl bromide, resulting in tripeptide 66. Benzoyl
ester functionality in 66 was hydrolyzed with ammonium
hydroxide, resulting in hydroxamate 67.27 Finally, concomi-
tant deprotection of N-Boc and tert-butyl functional groups
gave the target compound 39 as its trifluoroacetate salt.
Experimental Section
TFA H2N-γ-Glu[-Glu(CON(OH)-p-bromophenyl)-Gly-OH]-
3
OH (5). The protected tripeptide 19 (100 mg, 0.14 mmol) was
added to a mixture of TFA and CH2Cl2 (1:1 v/v, 6 mL) and
stirred at room temperature for 4 h. Concentration of the
reaction mixture and trituration of the residue with ether and
EtOAc afforded a light-brown solid. Recrystallization of this
solid by dissolution into 50% ethanol and cooling in ice gave
analytically pure 5 (65 mg, 76% yield). Rf 0.50 (butanol/acetic
1
acid/H2O, 12:5:3); [R]D -15.4 (c 0.5, 1N HCl). H NMR (300
MHz, CD3OD/CF3COOD) δ 7.58-7.32 (bd, 4H, Ar), 4.41 (m,
1H, R-CH:Glu), 3.96-3.74 (m, 3H, R-CH:Glu, CH2:Gly),
2.81-2.62 (m, 2H, γ-CH2:Glu), 2.54-2.39 (m, 2H, γ-CH2:Glu),
2.20-1.94 (m, 4H, β-CH2:Glu). 13C NMR (75 MHz, CD3OD/
CF3COOD) δ 173.9, 173.8, 172.9, 171.8, 167.4 (C(dO), 138.4,
131.8, 123.6, 119.2 (CAr), 54.2, 53.6 (R-C:Glu), 42.3 (CH2:Gly),
32.8, 30.6, 28.7, 27.5 (β-C:Glu, γ-C:Glu). ESI-HRMS m/z
503.0767 (MþH)þ; C18H23BrN4O8 þ Hþ requires 503.0778.
Reverse phase HPLC was run on Varian Microsorb column
(C18, 5 μm, 4.6 mm ꢀ 250 mm) using two solvent systems with
0.5 mL/min flow rate and detected at 254 nm. Solvent system 1:
0.04 M TEAB (triethylammonium bicarbonate) in water/70%
acetonitrile in water=1/1, tR=6.96 min, purity=98.6%. Solvent
system 2: 0.04 M TEAB in water/70% acetonitrile in water=
20-100% B linear, tR=13.12 min, purity=99.5%.
TFA NH2-γ-Glu[-D-Glu(CON(OH)-p-bromophenyl))-Gly-OH]-
3
OH (19). The protected tripeptide 20 (100 mg, 0.14 mmol) was
added to a 50% solution of TFA in CH2Cl2. After 4 h at room
temperature, the reaction mixture was evaporated to dryness and
triturated with EtOAc and ether to get a white solid. Recrystalliza-
tion of this solid from 50% ethanol gave analytically pure 6 (68 mg,
79% yield). Rf 0.50 (butanol/acetic acid/H2O, 12:5:3); [R]D þ26.4
(c 0.25, 1N HCl). 1H NMR (300 MHz, CD3OD/CF3COOD)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4655
δ 7.58-7.32 (bd, 4H, Ar), 4.41 (m, 1H, R-CH:Glu), 3.96-3.74 (m,
3H, R-CH:Glu, CH2:Gly), 2.81-2.62 (m, 2H, γ-CH2:Glu), 2.54-
2.39 (m, 2H, γ-CH2:Glu), 2.20-1.94 (m, 4H, β-CH2:Glu). 13C
NMR (75 MHz, CD3OD/CF3COOD) δ 173.9, 173.8, 172.9,
171.8, 167.4 (C(dO), 138.4, 131.8, 123.6, 119.2 (CAr), 54.2, 53.6
(R-C:Glu), 42.3 (CH2:Gly), 32.8, 30.6, 28.7, 27.5 (β-C:Glu, γ-C:
Glu). ESI HRMS m/z 503.0754 (M þ H)þ; C18H23BrN4O8 þ Hþ
requires 503.0778. HPLC purity (detected at 254 nm) = 99.4%,
Reverse phase HPLC was run on Varian Microsorb column (C18, 5
μm, 4.6 mm ꢀ 250 mm) using two solvent systems with 0.5 mL/min
flow rate and detected at 254 nm. Solvent system 1: 0.04 M TEAB
(triethylammonium bicarbonate) in water/70% acetonitrile in
water=1/1, tR=5.31 min, purity=99.4%. Solvent system 2: 0.04
M TEAB in water/70% acetonitrile in water=20-100% B linear,
tR=13.42 min, purity=98.99%.
polymerization product and then diluted with distilled water.
The acidic impurities were removed by passing the diluted
distillate through Amberlite-400 resin (carbonate form, pre-
pared by stirring 450 mL of chloride form of resin with an
aqueous solution containing 40 g of Na2CO3). The methyl
glyoxal solution thus obtained after filtering off the resin was
standardized by the method of Friedemann.29 Enzyme assays
were performed (30 °C, 0.05 M phosphate buffer (pH 6.6)) using
a thermostatted Beckman DU 7400 spectrophotometer. A fresh
GSH solution was prepared, on the day of the assays, using
distilled, deionized water. For each assay, the cell contained a
total volume of 3.0 mL, which was no more than 6.0 mM with
respect to methylglyoxal and 1.3 mM with respect to GSH.
Sufficient amounts of glyoxalase I, in the presence of 0.1%
bovine serum albumin (Sigma) as a stabilizing agent, were
employed to give an easily measurable initial rate, which was
followed by an increase in absorption at 240 nm. Stock inhibitor
solutions were prepared in the distilled water and adjusted, when
necessary, to pH 6.6. Methylglyoxal, GSH, and buffer (and
inhibitor) were added to a cuvette and allowed to stand for 6 min
in the thermostatted compartment of the spectrophotometer to
allow complete hemimercaptal equilibration. Hemimercaptal
substrate concentrations were calculated from the concentra-
tions of GSH and methylglyoxal added, using a value of 3.1 ꢀ
10-3 M for the dissociation constant of the hemimercaptal at pH
6.6. Data were analyzed using an Enzyme kinetics module of
Sigmaplot 9.0 from Systat Software Inc.
γ-Glutamyltranspeptidase Assay. The stability of glyoxalase I
inhibitors toward γ-GT mediated degradation was determined
by incubating 100 μL of 10 mM solutions of these inhibitors
(dissolved in 200 mM of 2-amino-2-methyl-1,3-propanediol
buffer at pH 8.5) with 10 μL of 0.54 mg/100 μL equine kidney
γ-glutamyltranspeptidase in the above buffer in presence of
20 μL of acceptor dipeptide gly gly (40 mM in the above buffer).
At selected time intervals, a compound from each tube was
spotted on silica gel TLC plate during its incubation at 37 °C.
Thin layer chromatography of these samples was performed
on silica gel TLC plates and compared with the authentic
predicted degradation products (solvent system: (6:2.5:1.5)
butanol/acetic acid/water). Visual detection of TLC spots was
performed under UV and also by iodine and fluorescamine
staining solution.
TFA NH2-γ-Gla[-Glu(CON(OH)-p-bromophenyl)Gly-OH]-
3
OH (26). A mixture of 36 (100 mg, 0.14 mmol) and TFA-
CH2Cl2 (1:1 v/v, 6 mL) was stirred at room temperature for 4 h.
After concentration, the residue was triturated with ether and
EtOAc to get a white residue. Recrystallization by first dissol-
ving in 0.1 N HCl at rt and then cooling in ice/salt gave
analytically pure 7 (66 mg, 78% yield) as a feathery solid. Rf
0.55 (butanol/acetic acid/H2O, 12:5:3); [R]D -19.2 (c 0.5, 1 N
HCl). 1H NMR (300 MHz, CD3OD/CF3COOD) δ 7.59 (d, J=
9.0, 2H, Ar), 7.49 (d, J=8.7 Hz, 2H, Ar), 4.60 (q, J=4.8, 8.4 Hz,
1H, R-CH:Glu), 4.48 (q, J=4.8, 7.8 Hz, 1H, R-CH:Dap), 4.17
(m, 2H, CH2:Gly), 3.68-3.62, 3.41-3.34 (2m, 2H, β-CH2:
Dap), 3.24-2.98 (m, 2H, β-CH2:Glu). 13C NMR (75 MHz,
CD3OD/CF3COOD) δ 174.5, 172.6, 170.5, 159.2, 157.1
(C(dO)), 140.6 (CArNOH), 131.5 (CAr: ortho to Br), 121.1
(CArBr), 117.7 (CAr: ortho to NOH), 52.4 (R-C:Glu), 49.1 (R-C:
Dap), 44.3 (β-C:Dap), 42.6 (CH2:Gly), 31.5 (β-C:Glu). ESI-
HRMS m/z 504.0740 (M þ H)þ; C17H22BrN5O8 þ Hþ requires
504.0730. Reverse phase HPLC was run on Varian Microsorb
column (C18, 5 μm, 4.6 mm ꢀ 250 mm) using two solvent
systems with 0.5 mL/min flow rate and detected at 254 nm.
Solvent system 1: 0.04
bicarbonate) in water/70% acetonitrile in water = 1/1, tR
M TEAB (triethylammonium
=
7.00 min, purity=96.06%. Solvent system 2: 0.04 M TEAB in
water/70% acetonitrile in water=20-100% B linear, tR=14.67
min, purity=95.90%.
TFA NH2-γ-Glu[-Dab(N-(p-bromobenzoyl)-N0-hydroxyl)-Gly-
3
OH]-OH (39). Protected tripeptide 68 (100 mg, 0.14 mmol) was
treated with a mixture of TFA and CH2Cl2 (1:1 v/v, 6 mL) at room
temperature for 4 h. The reaction mixture was then concentrated
and the residue obtained was triturated with ether and EtOAc to
afford a white solid. Recrystallization of the TFA salt from 0.1 N
HCl aqueous gave analytically pure 8 (60 mg, 70% yield). Rf 0.60
(butanol/acetic acid/H2O, 12:5:3); [R]D -12.6 (c 0.44, 1 N HCl).
1H NMR (300 MHz, CD3OD/CF3COOD) δ 7.59 (d, J=9.0, 2H,
Ar), 7.49 (d, J=8.7 Hz, 2H, Ar), 4.43 (m, 1H, R-CH:Glu), 4.17-
3.89 (m, 3H, R-CH:Dab, CH2:Gly), 3.44 (m, 2H, γ-CH2:Dab),
2.55-1.89 (m, 6H, β-CH2:Dab, β-CH2:Glu, γ-CH2:Glu). 13C
NMR (75 MHz, CD3OD/CF3COOD) δ 173.5, 173.1, 172.6,
170.3, 167.2 (C(dO)), 139.7, 130.8, 123.7, 119.4 (CAr), 54.1, 52.4
(R-C:Glu, R-C:Dab), 46.4 (γ-C:Dab), 42.4 (CH2:Gly), 31.9 (γ-C:
Glu), 28.7, 23.2 (β-C:Glu, β-C:Dab). ESI-HRMS m/z 503.0751
(M þ H)þ; C18H23BrN4O8 þ Hþ requires 503.0772. Reverse
phase HPLC was run on Varian Microsorb column (C18, 5 μm,
4.6 mm ꢀ 250 mm) using two solvent systems with 0.5 mL/min
flow rate and detected at 254 nm. Solvent system 1: 0.04 M TEAB
(triethylammonium bicarbonate) in water/70% acetonitrile in
water=1/1, tR=5.45 min, purity=96.65%. Solvent system 2:
0.04 M TEAB in water/70% acetonitrile in water=20 - 100% B
linear, tR=6.86 min, purity=95.07%.
Acknowledgment. This work was supported financially by
a grant from the Center for Drug Design, Academic Health
Center, University of Minnesota. We thank Jay Brownell for
his guidance while performing the glyoxalase I enzyme ki-
netics assays.
Supporting Information Available: Experimental details for
the synthesis of all new compounds and thin-layer chromato-
grams for the γ-glutamyltranspeptidase assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Ramasamy, R; Vannucci, S. J.; Yan, S. S.; Herold, K; Yan, S. F.;
Schmidt, A. M. Advanced glycation end products and RAGE: a
common thread in aging, diabetes, neurodegeneration, and inflam-
mation. Glycobiology 2005, 15, 16R–28R.
(2) Thornalley, P. J. The glyoxalase system: new developments to-
wards functional characterization of a metabolic pathway funda-
mental to biological life. Biochem. J. 1990, 269, 1–11.
(3) Antognelli, C.; Baldracchini, F.; Talesa, V. N.; Costantini, E.;
Zucchi, A.; Mearini, E. Overexpression of glyoxalase system
enzymes in human kidney tumor. Cancer J. 2006, 12, 222–228.
(4) Vince, R.; Wadd, W. B. Glyoxalase Inhibitors as Potential Antic-
ancer Agents. Biochem. Biophys. Res. Commun. 1969, 35, 593–598.
(5) Vince, R.; Daluge, S.; Wadd, W. B. Inhibition of Glyoxalase I by S-
Substituted Glutathiones. J. Med. Chem. 1971, 14, 402–404.
(6) Vince, R.; Brownell, J.; Akella, L. B. Synthesis and Activity of γ-(L-γ-
azaglutamyl)-S-(p-bromobenzyl)-L-cysteinylglycine: A Metabolically
Glyoxalase I Enzyme Kinetics Assay. The glyoxalase I inhibi-
tors were tested for their ability to inhibit yeast glyoxalase I in an
enzyme kinetic assay. Yeast glyoxalase I and the substrate
methylglyoxal were purchased from Sigma Chemical Co. The
commercial 40% methylglyoxal solution was distilled to remove

4656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
More and Vince
Stable Inhibitor of Glyoxalase I. Bioorg. Med. Chem. Lett. 1999, 9,
853–856.
(7) Murthy, N. S. R. K.; Bakeris, T.; Kavarana, M. J.; Hamilton, D. S.;
Lan, Y.; Creighton, D. J. S-(N-Aryl-N-hydroxycarbamoyl)glu-
tathione Derivatives Are Tight-Binding Inhibitors of Glyoxalase
I and Slow Substrates for Glyoxalase II. J. Med. Chem. 1994, 37,
2161–2166.
(8) Hamilton, D. S.; Creighton, D. J. Inhibition of Glyoxalase I by The
Enediol Mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione.
The Possible Basis of a Tumor-Selective Anticancer Strategy. J.
Biol. Chem. 1992, 267, 24933–24936.
(9) Zheng, Z. B.; Creighton, D. J. Bivalent Transition-State Analogue
Inhibitors of Human Glyoxalase I. Org. Lett. 2003, 5, 4855–4858.
(10) More, S. S.; Vince, R. A Metabolically Stable Tight-binding
Transition-State Inhibitor of Glyoxalase-I. Bioorg. Med. Chem.
Lett. 2006, 16, 6039–6042.
(11) Cameron, A. D.; Ridderstrom, M.; Olin, B.; Kavarana, M. J.;
Creighton, D. J.; Mannervik, B. Reaction Mechanism of Glyox-
alase I Explored by an X-ray Crystallographic Analysis of the
Human Enzyme in Complex with a Transition State Analogue.
Biochemistry 1999, 38, 13480–13490.
(12) Ly, H. D.; Clugston, S. L.; Sampson, P. B.; Honek, J. F. Syntheses
and Kinetic Evaluation of Hydroxamate-Based Peptide Inhibitors
of Glyoxalase I. Bioorg. Med. Chem. Lett. 1998, 8, 705–710.
(13) Han, G.; Tamaki, M.; Hruby, V. J. Fast, Efficient and Selective
Deprotection of the tert-Butoxycarbonyl (Boc) Group using HCl/
dioxane (4 M). J. Pept. Res. 2001, 58, 338–341.
Rio, M. C.; Basset, P.; Yiotakis, A.; Dive, V. Phosphinic Pseudo-
Tripeptides as Potent Inhibitors of Matrix Metalloproteinases: A
Structure-Activity Study. J. Med. Chem. 1999, 42, 2610–2620.
(19) Michaelides, M. R.; Dellaria, J. F.; Gong, J.; Holms, J. H.; Bouska,
J. J.; Stacey, J.; Wada, C.; Heyman, H. R.; Curtin, M. L.; Guo, Y.;
Goodfellow, C. L.; Elmore, I. B.; Albert, D. H.; Magoc, T. J.;
Marcotte, P. A.; Morgan, D. W.; Davidsen, S. K. Biaryl Ether
Retrohydroxamates as Potent, Long-lived, Orally Bioavailable
MMP Inhibitors. Bioorg. Med. Chem. Lett. 2001, 11, 1553–1556.
(20) Carrasco, M. R.; Brown, R. T. A Versatile Set of Aminooxy Amino
Acids for the Synthesis of Neoglycopeptides. J. Org. Chem. 2003,
68, 8853–8858.
(21) Filira, F.; Biondi, B.; Biondi, L.; Giannini, E.; Gobbo, M.; Negri,
L.; Rocchi, R. Opioid Peptides: Synthesis and Biological Properties
of [(Nγ-Glucosyl,Nγ-methoxy)-R,γ-diamino-(S)-butanoyl]4-del-
torphin-1-neoglycopeptide and Related Analogues. Org. Biomol.
Chem. 2003, 1, 3059–3063.
(22) Naegeli, H. U.; Keller-Schierlein, W. Stoffwechselprodukte Von
Mikroorganismen. Eine Neue Synthese des Ferrichroms: Enantio-
Ferrichrom. Helv. Chim. Acta 1978, 61, 2088–2095.
(23) Lin, Y.; Miller, M. J. Practical Synthesis of Hydroxamate-Derived
Siderophore Components by an Indirect Oxidation Method and
Syntheses of a DIG-Siderophore Conjugate and a Biotin-Side-
rophore Conjugate. J. Org. Chem. 1999, 64, 7451–7458.
(24) Nemchik, A.; Badescu, V.; Phanstiel, O. N-(Benzoyloxy)amines:
An Investigation of Their Thermal Stability, Synthesis, and In-
corporation into Novel Peptide Constructs. Tetrahedron 2003, 59,
4315–4325.
(14) Oxley, P. W.; Adger, B. M.; Sasse, M. J.; Forth, M. A. N-Acetyl-N-
phenylhydroxylamine via Catalytic Transfer Hydrogenation of
Nitrobenzene Using Hydrazine and Rhodium on Carbon. Org.
Synth. 1989, 67, 187–192.
(25) Wang, Q. X.; King, J.; Phanstiel, O. An Improved Synthesis of O-
Benzoyl Protected Hydroxamates. J. Org. Chem. 1997, 62, 8104–
8108.
(15) Zhang, X.; Rodrigues, J.; Evans, L.; Hinkle, B.; Ballantyne, L.;
Pena, M. Formation of Urea Dipeptides from Carbonyldiimida-
zole: Application toward the Protease Inhibitors GE 20372 and
MAPI. J. Org. Chem. 1997, 62, 6420–6423.
(16) More, S. S.; Vince, R. Design, Synthesis and Biological Evaluation
of Glutathione Peptidomimetics as Components of Anti-Parkin-
son Prodrugs. J. Med. Chem. 2008, 51, 4581–4588.
(26) Andruszkiewicz, R.; Rozkiewicz, D. An improved preparation of
N2-tert-butoxycarbonyl- and N2-benzyloxy-carbonyl-(S)-2,4-dia-
minobutanoic acids. Synth. Commun. 2004, 34, 1049–1056.
(27) Nemchik, A.; Badescu, V.; Phanstiel, O. N-(Benzoyloxy)amines:
An Investigation of Their Thermal Stability, Synthesis, and In-
corporation into Novel Peptide Constructs. Tetrahedron 2003, 59,
4315–4325.
(17) Suzuki, T.; Miyata, N. Rational Design of Non-Hydroxamate
Histone Deacetylase Inhibitors. Mini-Rev. Med. Chem. 2006, 6,
515–526.
(28) Wang, Q. X.; King, J.; Phanstiel, O. An Improved Synthesis of O-
Benzoyl Protected Hydroxamates. J. Org. Chem. 1997, 62, 8104–
8108.
(18) Vassiliou, S.; Mucha, A.; Cuniasse, P.; Georgiadis, D.; Lucet-
(29) Friedemann, T. E. The Action of Alkali and Hydrogen Peroxide on
Levannier, K.; Beau, F.; Kannan, R.; Murphy, G.; Knaeuper, V.;
Glyoxals. J. Biol. Chem. 1927, 73, 331–334.