Peptidomimetic Glutathione Analogues as Novel γGT Stable GST Inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00269-3

Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a role in the onset of multidrug resistance (MDR). To evaluate the contribution of GST to this process, inhibitors are needed. Glutathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly. In this paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase mediated breakdown. The peptide bonds in GSH were replaced by isosteres, such as the 'reduced' amide (which was prepared using a novel method), N-methylamide, urethane, and methylene linkages. The in vitro evaluation of the compounds focuses on GST inhibition and stability towards γ-glutamyl-transpeptidase (γGT), the main enzyme involved in GSH breakdown. The compounds were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor. All novel GSH-analogues were shown to inhibit rat liver cytosolic GSTs. Furthermore, peptidometric changes of the γ-glutamyl-cysteine amide bond greatly improved stability towards γGT. These compounds may be useful in the design of novel in vivo applicable GST inhibitors. Copyright

Full text of DOI:10.1016/S0968-0896(01)00269-3

Bioorganic & Medicinal Chemistry 10 (2002) 195–205
Peptidomimetic Glutathione Analogues as Novel ꢀGT Stable
GST Inhibitors
Danny Burg,a,* Dmitri V. Filippov,b Ralph Hermanns,a Gijs A. van der Marel,b
Jacques H. van Boomb and Gerard J. Muldera
aDivision of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300RA,
Leiden, The Netherlands
bDivision of Bio-Organic Synthesis, Leiden Institute of Chemistry, PO Box 9502, 2300RA, Leiden University, The Netherlands
Received 25 May 2001; accepted 18 July 2001
Abstract—Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a
role in the onset of multidrug resistance (MDR). To evaluate the contribution of GST to this process, inhibitors are needed. Glu-
tathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly. In this
paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase
mediated breakdown. The peptide bonds in GSH were replaced by isosteres, such as the ‘reduced’ amide (which was prepared using
a novel method), N-methylamide, urethane, and methylene linkages. The in vitro evaluation of the compounds focuses on GST
inhibition and stability towards g-glutamyl-transpeptidase (gGT), the main enzyme involved in GSH breakdown. The compounds
were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor. All novel GSH-
analogues were shown to inhibit rat liver cytosolic GSTs. Furthermore, peptidomimetic changes of the g-glutamyl-cysteine amide
bond greatly improved stability towards gGT. These compounds may therefore be useful in the design of novel in vivo applicable
GST inhibitors. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Although the g-glutamyl-cysteine peptide bond renders
The ubiquitous tripeptide glutathione (GSH), g-l-glu-
tamyl-l-cysteinyl-glycine, is the prevalent non-protein
thiol in eukaryotic cells. GSH plays a critical role in the
cellular protection against potentially harmful electro-
philes from xenobiotic sources, or those generated by
endogenous oxidative processes.1 GSH-conjugates are
formed by nucleophilic attack of the cysteine sulphydryl
on the electrophilic center of a suitable substrate. This
process is catalyzed by glutathione-S-transferase
(GST).2 Several members of the GST-isoenzyme family
are involved in conjugation of drugs and thereby in
drug resistance. Isoenzyme selective GST inhibitors may
therefore be used to improve drug response and
decrease resistance. GSH-conjugates and their cell-
permeable esterified derivatives are effective competitive
inhibitors of GST.3,4 Unfortunately, the inhibitory
action of GSH-conjugates is severely limited by their
sensitivity towards peptidase-mediated breakdown.
it resistant to most a-peptidases, GSH is rapidly degra-
ded by g-glutamyl transpeptidase (gGT). Cleavage by
this enzyme, which has particularly high activity in the
kidney, prevents the use of GSH conjugates in vivo.
Being potential drug candidates, effort has been put into
the design of new glutathione analogues as GST inhibi-
tors. Several modifications have been made to the GSH
peptide backbone, mostly by replacing the amino acid
residues by other naturally occurring or synthetic ana-
logues.5ꢀ9 Recently, most of the known structural mod-
ifications of the glutathione molecule have been
reviewed.10 In recent years, our laboratory has been
involved in the development of GSH analogues for the
functional mapping and inhibition of GST isoenzymes.
This resulted in the development of non-thiol containing
S-alkyl-glutathione analogues, in which the cysteinyl-
glycine part was replaced by d-aminoadipic acid. These
compounds showed improved stability towards g-GT
and exhibited in vivo inhibition of some GST iso-
enzymes, although inhibition was limited to a and m class
GST isoenzymes.11ꢀ15 In this study, new compounds
*Corresponding author. Tel.: +31-71-527-6227; fax; +31-71-527-
6292; e-mail: d.burg@lacdr.leidenuniv.nl
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00269-3

196
D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
were designed that closely resemble GSH-conjugates,
but in which native peptide bonds were replaced by
peptidomimetics. These modified amides were expected
to be less sensitive towards peptidase mediated break-
down. To increase inhibitory potency towards GST, the
cysteine sulphydryl was conjugated to the diuretic drug
ethacrynic acid (EA). The rationale behind this is that
the GSH–ethacrynic acid conjugate is a potent inhibitor
of most GST isoenzymes.16,17 These compounds might
therefore yield new lead compounds towards peptidase-
stable GST inhibitors. Figure 1 shows the GSH-con-
jugate mimics synthesized for this purpose. Structure I
depicts the native GSH conjugate, while compounds II
and III indicate N-methylated tripeptides. In compound
IV, an ethylene group replaces the cysteinyl-glycine
amide, thereby introducing more flexibility to the pep-
tide backbone. Amine V is a ‘reduced’ isostere; it lacks
the carbonyl function and is therefore also expected to
be protonated in physiological solutions. Synthesis of
the peptide portion of the urethane VI has recently been
described.18 This GSH analogue was shown to be a
competitive inhibitor of gGT, and its ethacrynic acid
conjugate was therefore of much interest.
II was prepared by incorporating the N-methylamino
acid sarcosine instead of the C-terminal glycine. The
protected dipeptide, 2, was obtained in high yield by
condensing Fmoc-Cys(Acm)-OH with H-Src-OtBu,
using DIC/HOBt as coupling agent. After removal of
the N-terminal Fmoc (3), coupling to the unprotected
side-chain carboxyl of Boc-Glu-OtBu was achieved via
the same coupling method giving compound 8. The
Acm protecting group was removed with iodine in dry
methanol.18 The symmetrical disulfide, obtained by this
oxidation was then treated with acid to remove Boc and
tert-butyl protecting groups. In a one-pot reaction, the
disulfide was reduced with tri-n-butylphosphine and
coupled with EA, to give II. The resulting compound
was epimeric at Ca of the ethacrynic acid moiety. Dur-
ing disulfide reduction and subsequent EA conjugation
Results and Discussion
Chemistry
The reference compound I, was prepared by Michael
addition of the a,b-unsaturated ketone moiety of etha-
crynic acid (EA) to the cysteinyl-thiol of glutathione in
a slightly basic water/ethanol mixture as has been pre-
viously described.17 The product was homogeneic on
HPLC and all spectroscopic data corresponded with
literature values.
Figure 1. Structure of the glutathione–ethacrynic acid conjugate (I)
and new peptidomimetic analogues. I=g-Glu[Cys(EA)-Gly-OH]-OH
(GS-EA), II=g-Glu[Cys(EA)-Src-OH]-OH, III=g-Glu[MeCys-Gly-
OH]-OH, IV=g-Glu[2-amino-1-mercapto(S-EA)-6-hexanoic acid]-OH,
V=H-Glu[É(CH2NH)-Cys(EA)-Gly-OH]-OH, VI=H-Glo[Cys(EA)-
Gly-OH]-OH.
The synthetic route towards the N-methylated glu-
tathione derivatives is shown in Scheme 1. Compound
Scheme 1. Synthesis of N-methylated GSH-mimics. Conditions: (i). DBU, DCM; (ii). nitrobenzenesulfonylchloride, DiPEA, DCM; (iii). triphenyl-
phosphine, DIAD, MeOH, THF, 0 ꢁC; (iv). thiophenol, DiPEA, DMF; (v). 30, HOBt, DIC, DCM; (vi). I2, MeOH; (vii). TFA, 1% H2O; (viii). Tri-
n-butylphosphine, nPrOH/H2O, ethacrynic acid.

D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
197
a tri-n-butylphosphine–ethacrynic acid adduct was
formed, as determined by LC–MS. This side-product
was easily removed by reversed-phase HPLC. An iden-
tical adduct was also formed during the preparation of
compounds III, V and VI.
Glu-OtBu, as described by Ouwerkerk et al.13 The adi-
pic acid a-carboxyl was reacted with isobutyl-
chloroformate to furnish a mixed anhydride, which was
then reduced with NaBH4 to give alcohol 11.23 Mitsu-
nobu reaction with thioacetic acid transformed the
dipeptide-alcohol into the thioacetate 12. The thioace-
tate and the ethyl ester were cleaved simultaneously
with aq NaOH in dioxane/MeOH. This was followed by
conjugation of the free sulfhydryl with EA and sub-
sequent acidolytic cleavage of Boc and tBu groups.
Compound IV was obtained after purification by
Sephadex LH20 gelfiltration.
N-Methylamide III was prepared by selective methyla-
tion of the cysteine amine. The liberated amino-group
of cysteinyl-glycine was protected with o-nitrobenzene-
sulfonylchloride (4!5) to make the amine proton suffi-
ciently acidic for Mitsunobu transformation.19 Reaction
of sulfonamide 5 with triphenylphosphine/DIAD and
methanol gave 6 in high yield. The N-terminal o-nitro-
benzylsulfonyl was cleaved with thiophenol and DiPEA
in DMF (!7).20 The N-methylated dipeptide could now
be condensed with protected glutamic acid. Using DIC/
HOBt as coupling agents, the N-methylated tripeptide 9
was obtained in moderate yields (60%). Because the
coupling of N-methylated amino acids has often shown
to be troublesome,21 an attempt was made to improve
the coupling efficiency. The use of DiPEA/PyBroP (an
halogenated homologue of PyBOP22) as coupling
reagent was not succesful, as this resulted in even lower
yields (45%). Compound III was obtained after the
same deprotection and conjugation steps as performed
for compound II.
Reduced peptide isosteres are usually obtained after
reductive amination of a peptide with a suitable peptide-
aldehyde.24 Although reduction of glutamic acid to the
corresponding alcohol 13 proceeded in high yields
(Scheme 3), subsequent oxidation to the aldehyde
proved more difficult. Dess–Martin oxidation of the
pure alcohol gave complex product-mixtures. No pure
glutamic-aldehyde could be obtained via this method. In
another attempt, the Weinreb amide of glutamic acid
was prepared.25 Reduction of this compound with
LiAlH4 also gave complex mixtures from which the
product could not be isolated. A new route towards
the reduced peptide isostere therefore had to be
investigated.
Synthesis of compound IV is shown in Scheme 2. Boc-
Glu[-2-aminoadipic acid-OEt]-OtBu, 10, was prepared
by condensing d-aminoadipic acid ethyl ester with Boc-
This method is depicted in Scheme 3. In analogy with
the methylated compound III, condensation of the glu-
tamic alcohol 13 with the oNs-protected dipeptide 5
under Mitsunobu conditions gave the desired reduced
peptide isostere 14. After facile cleavage of the o-nitro-
benzenesulfonyl protecting-group (!15), subsequent
removal of Acm by iodine treatment led to complex
mixtures of products on TLC. The free amine was
therefore Boc-protected (!16) to prevent possible side-
reactions and to facilitate purification. Indeed, the Acm
group could now easily be removed to yield the sym-
metrical disulfide. After disulfide reduction, EA con-
jugation and TFA treatment compound V was isolated
by Sephadex LH20 gelfiltration.
The reaction sequence towards urethane VI is depicted
in Scheme 4. The peptide portion of this compound was
prepared by a method adapted from Calcagni et al.17
Boc-Ser(OH)-OtBu was acylated to yield its nitro phe-
nylcarbonate (25), which was condensed with the amino
group of H-Cys(Acm)-Gly-OtBu (4). The obtained ure-
thane-based mimetic was then deprotected and con-
Scheme 2. Methylene replacement of the cysteinyl–glycine peptide
bond. Conditions: (i). 29, TMS-Cl, EtOH; (ii). BocGlu(OSu)OtBu,
DiPEA, DMF; (iii). NMM, ethylchloroformate, NaBH4, MeOH,
THF; (iv). Triphenyl phosphine, DIAD, thioacetic acid, THF, 0 ꢁC;
(v). aq NaOH, dioxane, MeOH, ethacrynic acid.
Scheme 3. Preparation of the reduced isostere. Conditions: (i). 30, isobutylchloroformate, triethylamine, NaBH4, THF/H2O; (ii). triphenylpho-
sphine, DIAD, 5, THF; (iii). thiophenol, DiPEA, DMF; (iv). Boc2O, DiPEA, MeCN; (v). I2, MeOH; (vi). TFA, 1%H2O; (vii). Tri-n-butylpho-
sphine, nPrOH/H2O, ethacrynic acid.

198
D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
corrected for the spontaneous breakdown. Figure 2 depicts
the hydrolysis of the various GS-EA mimics by gGT.
As expected, the native glutathione–ethacrynic acid
conjugate (I) was cleaved rapidly. The chosen condi-
tions resulted in complete degradation of the GSH
backbone within 20 min (t1=2=8 min). Compounds II
and IV, each having an unmodified g-glutamyl peptide
bond, are also very sensitive towards gGT mediated
cleavage. For these compounds, t1=2 was approximately
equal to the half-life of GS-EA.
The g-glutamyl modified compounds (III, V, and VI) all
showed greatly improved stability towards gGT. N-
methylated compound III was hydrolyzed by gGT,
albeit much slower than unmodified g-glutamyl amides.
After prolonged exposure to gGT, urethane VI also
underwent minor hydrolysis. The reduced peptide iso-
stere V was completely insensitive towards g-glutamyl
transpeptidase.
Scheme 4. Urethane isostere synthesis. Conditions: (i). 31, bis(4-
nitrophenyl)carbonate, DiPEA, DMF; (ii). 4, dioxane, 80 ꢁC; (iii). I2,
MeOH; (iv). TFA, 1%H2O; (v). Tri-n-butylphosphine, nPrOH/H2O,
ethacrynic acid.
jugated to EA as was reported for the previously
described compounds.
Inhibition of rat liver cytosolic GSTs
Rat liver cytosol (S100), containing cytosolic GSTs, was
obtained after ultracentrifugation of a rat liver homo-
genate. GST inhibition was tested at six concentrations
of the GS-EA mimics by a method adapted from Habig
et al.,26 modified for a 96-wells plate assay. Inhibition
profiles of the various inhibitors are shown in Figure 3.
Biological evaluation
ꢀ-Glutamyl transpeptidase mediated breakdown. Stabi-
lity towards hydrolysis of the g-glutamyl-cysteine amide
by gGT was determined by incubating GS-EA analo-
gues in the presence of the purified enzyme. Breakdown
of the ethacrynic acid conjugates was determined by
HPLC-analysis. All compounds were slightly unstable:
After prolonged incubation (3 h) at 37 ꢁC in Tris–HCl
buffer, pH=7.4, an unidentified peak was observed in
the chromatograms, resulting from instability of the
ethacrynic acid group (estimated breakdown t1=2>10 h).
Rat liver cytosol contains mainly a (1-1, 2-2 and 1-2)
and m (3-3, 4-4, and 3-4) class GSTs. All peptidomi-
metics were able to inhibit GSH-conjugation by S100
GSTs, indicating that the compounds can be accom-
modated in the active sites of one or several isoenzymes.
The unmodified glutathione conjugate (I) seemed to be
the best GST inhibitor, inhibiting virtually all GSTs in the
cytosol. Inhibition profiles indicate that most compounds
reach a plateau at higher inhibitor concentration, which
suggests that not all enzymes are inhibited equally. Dis-
turbing the moieties involved in H-bonding has distinct
consequences for enzyme–inhibitor interactions. The
This highly polar compound rapidly eluted from the
column and did not interfere with peaks of the GSH
analogues or their gGT-degraded products. As all GST
inhibition studies are conducted using fresh solutions
and lasted only 5 min, no loss of activity is expected as a
result of this breakdown. All degradation data were
Figure 3. Inhibition of rat liver cytosolic GSTs (S100) by GS-EA
mimics. Inhibition of GST mediated CDNB-conjugation was tested at
pH=6.5, in the presence of non-limiting concentrations of GSH (1
mM) and CDNB (1 mM). The shown data are averages of three
separate determinations. For clarity, error-bars were not included.
Standard deviations did not exceed 15% of the indicated averages.
Open markers with dotted lines indicate gGT sensitive compounds (see
Fig. 2), closed markers with closed lines are gGT stabilized structures.
Figure 2. g-Glutamyltranspeptidase mediated hydrolysis of GSH ana-
logues. Peptide isosteres were incubated with 0.25 mg/ml bovine g-
glutamyltranspeptidase in 0.1 M Tris–HCl buffer pH=7.4. At the
appropriate time point a sample (100 mL) was taken, heat inactivated
and stored on ice until HPLC analysis. Disappearance of the indicated
compounds was determined by RP-HPLC. Open markers indicate
gGT sensitive compounds, closed markers are gGT stabilized.

D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
199
GSH-binding site in GST is very tightly suited to
accommodate GSH.27 The binding capacity of the
highly polar GSH-backbone is dictated by its electro-
static complementarity with the enzyme. Certain pepti-
domimetic changes to the GSH backbone can therefore
result in drastically altered inhibition patterns.
synthetase, and members of the MRP membrane-trans-
porter family, are also inhibited by GSH-con-
jugates.29ꢀ31 Because of the multitude of glutathione-
dependent enzymes, g-glutamyltranspeptidase stable
GSH analogues might be interesting as research tools in
various other fields of biological and biomedical
research.
Methylation of the cysteinyl-glycine amide (II) is very
well accepted by cytosolic GSTs, indicated by a com-
parable inhibition pattern to compound I. The cys-gly
amide nitrogen is presumably not directly involved in
H-bonding. Furthermore, the increased steric bulk can
be accommodated within the active site of the GST iso-
enzymes. Loss of water-bridged interactions may
explain the slight decrease in inhibition. The importance
of the g-glutamyl-cysteine amide for enzyme–substrate
interactions is clearly seen in compound III. When this
peptide bond is methylated, a drastic decrease in inhi-
bition was seen. This may be a result of the loss of a
crucial H-bond, or may be a consequence of steric cla-
shes with the enzyme. Compound IV, completely lack-
ing the cysteinyl–glycine peptide bond is a poor
inhibitor of cytosolic GSTs. The drop in inhibition is a
result of omission of the carbonyl oxygen, which is an
important H-bond acceptor.28 The increased flexibility
of the alkyl moiety is probably also an important factor,
as enthropic effects may prevent this compound from
obtaining its optimal bioactive conformation. Among
the gGT stable inhibitors (III, V and VI), the reduced
peptide V isostere seems to have the most favorable
inhibition characteristics.
Experimental
Materials and methods
All solvents used were of analytical grade unless stated
otherwise in the text. THF was freshly distilled prior to
use. Other solvents were stored on molecular sieves.
Protected amino acids and derivatives were purchased
from Bachem AG (Bubendorf, Switzerland). Glu-
tathione was purchased from Boehringer (Mannheim,
Germany). Ethacrynic acid and bovine g-glutamyl-
transpeptidase were obtained from Sigma-Aldrich (St.
Louis, MO, USA).
For structure verification, NMR peaks were assigned by
1
HH-, and CH-COSY experiments. H and 13C NMR
spectra were recorded on Bruker AC-200, Bruker WM-
300 or Bruker DMX-600 spectrometers, operating at
200/50.1, 300/75 and 600/150 MHz, respectively. Che-
mical shifts are given in ppm (d) relative to the internal
standard tetramethylsilane. Mass spectra were recorded
with Finnigan MAT TSQ70 triple quadrupole, or with
Perkin Elmer Sciex API 165 mass spectrometers. HPLC
purification was performed on a Kratos spectroflow 400
system equipped with a Supelcosil SPLC-18-DB semi-
preparative column. A linear gradient of 5-50% aceto-
nitrile in H2O/0.1%TFA was used to purify end pro-
ducts. For biological evaluation, all compounds were at
least 90% pure, as determined by RP HPLC.
Conclusions
A series of pseudopeptide glutathione analogues was
prepared by solution-phase peptide synthesis. Initial
stability studies indicate that the g-glutamyl modified
compounds are stabilized against degradation by g-glu-
tamyltranspeptidase. Because this enzyme is mainly
responsible for the degradation of GSH conjugates in
the bloodstream, metabolic stabilization is a requisite
for in vivo use of GSH conjugate analogues.
Chemical synthesis
Fmoc-Cys(Acm)-Src-OtBu (2). Fmoc-Cys(Acm)-OH
(830 mg, 2 mmol) was added to a solution of HOBt (270
mg, 2 mmol) in 4 mL dry THF at 0 ꢁC. A solution of
sarcosine–OtBuꢂ HCl (363 mg, 2 mmol) and N-methyl-
morpholine (NMM, 220 mL, 2 mmol) in 4 mL di-
chloromethane (DCM) was added in one portion.
Diisopropylcarbodiimide (DIC, 310 mL, 2 mmol) was
added dropwise to the reaction mixture. After 1 h at
0 ꢁC, the suspension was stirred for 20 h at room tem-
perature. DCM (50 mL) was added to the reaction
mixture, which was then washed repeatedly with satu-
rated NaHCO3, 0.1 M HCl and water. Pure 2 was col-
lected after silica gel column chromatography as white
foam. Yield 1.05 g (97%). Rf 0.2 (DCM/MeOH, 95:5).
1H NMR (CDCl3): d 1.45 (9H, s, tBu), 1.98 (3H,
CH3Acm), 2.78–3.0 (2H, m, CbH2Cys), 3.16 (3H, s,
CH3Src), 3.75–4.1 (2H, m, CH2Acm), 4.1–4.45 (3H, m,
CH2Src, CaHCys), 4.2–4.45 (3H, m, CH–CH2 Fmoc),
6.4 (1H, d, J=9.1 Hz, NHCys), 7.24–7.75 (8H, m, aro-
matic), 7.4 (1H, m, NHAcm). 13C NMR (CDCl3): d
23.06 (CH3Acm), 27.67 (tBu), 33.28 (CH2S), 36.31
(CH3Acm), 41.37 (CH2Acm), 50.44 (CH2Src), 50.59
All ethacrynic acid conjugates inhibit rat liver cytosolic
GSTs. Results indicate that alterations of the glu-
tathione backbone are not equally well accepted by the
different isoenzymes, and thus already provides iso-
enzyme selectivity. More elaborate inhibition studies on
purified GST isoenzymes are needed to make firm con-
clusions regarding selectivity. The reduced peptide iso-
stere may be considered as the most interesting lead for
the development of in vivo active GST inhibitors, as is
completely stable towards g-GT and can be accom-
modated in the GST active site. In this study ethacrynic
acid was used as model electrophile to obtain potent,
but non-selective GSH conjugate analogues. Isoenzyme
selectivity can be achieved by replacing the ethacrynic
acid moiety by a more suitable hydrophobic substrate,
thereby also avoiding the inherent instability of EA.
Apart from GST, several other enzymes such as glyox-
alase I, DNA-dependent protein kinase, leukotriene-C4-

200
D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
(CaCys), 81.98 (CqtBu), 119.7–128.07 (Fmoc), 140.96,
143.51 (Fmoc), 156.19 (CO Fmoc), 167.86 (CO ester),
170.89 (CO Cys), 171.59 (CO Acm).
with ether and used without further purification. Yield
276 mg (100%). 1H NMR (MeOD): d 2.2 (4H, m,
CbH2Glu), 2.55 (4H, m, CgH2Glu), 2.85 and 3.2 (4H, m,
CbH2Cys), 2.9 (2H, s, CaHGlu) 3.25 (3H, s, CH3Src), 4.05
(4H, m, CH2Gly), 4.1–4.6 (2H, m, CaHCys), 8.55 (2H, d,
NHamide). 13C NMR (MeOD): d 32.1 (CbCys), 36.9
(CH3Src), 40.6 (CbGlu), 50.0 (CaCys), 50.5 (CH2Src),
51.3 (CgGlu), 53.4 (CaGlu), 171.5–173.8 (4ÂC¼O).
Boc-Glu[Cys(Acm)-Src-OtBu]-OtBu (8). DBU (300 mL,
2 mmol) was added to a solution of the protected pep-
tide 2 (1.05 g, 1.94 mmol) in DCM (10mL). After 15
min at room temperature, TLC analysis confirmed
complete Fmoc removal. The solvent was evaporated
under reduced pressure and the residue was co-evapo-
rated with dry toluene. The crude H-Cys(Acm)-Src-
OtBu (3) was used without further purification.
H-Glu[Cys(EA)-Src-OH]-OH, II. Compound 18 was
dissolved in 6 mL argon flushed nPrOH/H2O (2:1). The
pH was adjusted to 8.5 with 25% aq NH3 solution. Tri-
n-butylphosphine (106 mg, 0.43 mmol) was added, after
which the solution was stirred under argon for 60 min.
Ethacrynic acid (303 mg, 1 mmol), dissolved in 2 mL
EtOH was then added. The pH was adjusted to 8 by
addition of satd. NaHCO3 solution. After at least 4 h,
the solvent was evaporated. The residue was further
purified by LH20 gelfiltration using MeOH/H2O (7:3)
as eluent. LC–MS confirmed the presence and identity
of the desired compound II. Residual traces of tri-n-
butylphosphine–EA complex were removed by RP-
A solution of HOBt (270 mg, 2 mmol) and Boc-Glu-
OtBu (30, 607 mg, 2 mmol) in 2 mL DCM was chilled
and then added to an ice-cold solution of the amine 5 in
DCM. DIC (310 mL, 2 mmol) was then added dropwise.
The reaction mixture was stirred for two h at 0 ꢁC and
16 h at 4 ꢁC. The residue obtained after evaporation of
the solvent was taken up in 100 mL EtOAc and washed
with satd. aqueous NaHCO3, 1 M NaHSO4 and water.
Silica gel column chromatography with 1% MeOH/
DCM as eluent gave the pure tripeptide 8 as a foam,
which solidified after further drying. Yield 995 mg
(85%). Rf 0.45 (DCM/MeOH, 95:5). 1H NMR (CDCl3):
d 1.4 (27H, Boc, tBu), 1.95 (3H, s, CH3Acm), 1.65–2.3
(2H, m, CbH2Glu), 1.97 (3H, s, CH3Acm), 2.29 (2H, m,
CgH2Glu), 2.6–3.05 (2H, m, CbH2Cys), 3.12 (3H, s,
CH3Src), 3.68–4.1 (2H, m, CH2Acm), 4.2 (2H, m,
CH2Src), 4.35 (1H, m, CaHGlu), 5.08 (1H, m, CaHCys),
5.38 (1H, d, NHBoc), 7.05 (1H, d, NHCys), 7.45 (1H, d,
1
HPLC. Rf 0.3 (nBuOH/H2O/AcOH, 15:3:2). H NMR
(D2O): d 0.85 (3H, m, CH3EA), 1.55 and 1.71 (2H, m,
CH2EA), 2.11 (2H, m, CbH2Glu), 2.45 (2H, m,
CgH2Glu), 2.67–2.8 and 2.85–3.0 (2H, m, CbH2Cys), 2.9
(2H, d, J=6.3 Hz, CH2EA), 3.1 (3H, s, CH3Src), 3.53
(1H, m, CH-EA), 3.74 (1H, m, CaHGlu), 3.8–4.06 (2H,
m, CH2Src), 4.63 (2H, s, CH2EA), 4.74–4.95 (1H,
CaHCys), 6.95 and 7.60 (2Â1H, m, aromatic). 13C
NMR: 11.45 (CH3EA), 25.4 (4ÂCH2, CbGlu, CbCys,
2ÂCH2EA), 37.4 (CH3Src), 50.3 (CH EA), 52.5
(CaCys), 53.1 (CH2Src), 54.5 (CgGlu), 56.5 (CaGlu),
68.6 (OCH2EA), 111.9 and 129.7 (CH, aromatic), 123.9
and 131.95 (C–Cl, aromatic), 158.1–175.65 (5ÂCO).
Mass spectrometry (ES–MS): m/e 624, [M+H]+.
NHAcm). 13C NMR (CDCl3/MeOD):
d
22.57
(CH3Acm), 27.6, 27.97 (Boc, tBu), 31.7 (CbGlu), 33.36
(CbCys), 36.3 (CH3Src), 41.49 (CH2Acm), 48.7 (CaCys),
50.4 (CH2Src), 52.04 (CgGlu) 53.38 (CaGlu), 81.6–82.6
(Cq, Boc, tBu), 155.68 (CO, Boc), 167.8 (CO, Src-tBu
ester), 170.8–172.24 (CO, Acm, Cys, Glu, Glu-tBu
ester). Mass spectrometry (ES-MS): m/e: 60, [M+H]+;
627, [M+Na]+.
Fmoc-Cys(Acm)-Gly-OtBu (1). To a solution of Fmoc-
Cys(Acm)-OH (829 mg, 2 mmol) in 4 mL dry THF at
0 ꢁC, HOBt (270 mg, l2 mmol) was added. Then an ice-
cold solution of H-Gly-OtBu.HCl (335 mg, 2 mmol)
and NMM (220 mg, 2 mmol) in 4 mL of dry DCM was
added. Subsequently, DIC (310 mg, 2 mmol) was intro-
duced dropwise. After 1 h at 0 ꢁC and 16 h at 4 ꢁC, the
solution was evaporated under vacuum. The residue
was dissolved in 50 mL EtOAc and washed with satd.
aq NaHCO3, 0.5 M HCl and water. After drying and
evaporation of the organic phase, the residue was chro-
matographed on silica gel to give the protected dipep-
tide 1 as a foam. Yield 1.03 g (98%), Rf 0.42 (DCM/
{Boc-Glu[Cys-Src-OtBu]-OtBu}2 (17). Peptide 8 (603
mg, 1 mmol) was dissolved in 10 mL MeOH. A solution
of I2 (508 mg, 2 mmol) in 5 mL MeOH was added
dropwise over 30 min. After 10 min TLC analysis indi-
cated complete disulfide formation. The solution was
decolorized by addition of 1 N sodium thiosulfate solu-
tion. EtOAc (100 mL) was added and the organic phase
was washed with thiosulfate solution and water. After
evaporation of the solvent, product 17 was purified by
gelfiltration chromatography on a Sephadex LH20 col-
umn, using DCM/MeOH (1:1) as solvent. Yield 458 mg
(86%). Rf 0.6 (DCM/MeOH, 9:1). 13C NMR (CDCl3): d
27.22, 27.55 (Boc, tBu), 31.46 (CbCys), 35.92 (CH3Src),
39.37 (CbGlu), 48.05 (CaCys), 50.17 (CH2Src), 51.62
(CgGlu), 53.45 (CaGlu), 79.13-82.26 (Cq, Boc, tBu),
155.73 (CO, Boc), 167.5 (CO, Src-OtBu ester), 170.6–
172.14 (CO, Cys, Glu, Glu-OtBu ester).
1
MeOH, 95:5). H NMR (CDCL3): d 1.45 (9H, s, tBu),
1.92 (3H, s, CH3 Acm), 2.97 (2H, m, Hb Cys), 3.96 (2H,
m, Acm), 4.2–4.5 (3H, m, CH–CH2 Fmoc), 4.25–4.65
(3H, Ha Cys, CH2 Gly), 6.47 (1H, d, J=8.4 Hz), 7.3
(2H, 2ÂNH), 7.24–7.74 (8H, m, aromatic). 13C NMR
(CDCl3): d 23.15 (CH3, Acm), 27.7 (3ÂCH3, tBu), 33.4
(Cb, Cys), 40.6 (CH2, Acm), 41.9 (CH2, Fmoc), 81.8
(Cq, tBu), 119.7–127.5 (aromatic, Fmoc), 141.0 and
143.6 (4C, Fmoc), 156.6 (CO, Fmoc), 168.6 (CO, Cys),
171.6 (2Â CO, Acm, tBu ester).
{H-Glu[Cys-Src-OH]-OH}2 (18). Trifluoroacetic acid (5
mL) and water (100 mL) was added to 17 (458 mg, 0.43
mmol). After 4 h at room temperature, the deprotected
disulfide 18 was precipitated by addition of ice-cold
diethylether. The product was repeatedly coevaporated
H-Cys(Acm)-Gly-OtBu (4). Compound 1 (791 mg, 1.5
mmol) was dissolved in 5 mL dry DCM. DBU (224 mg,

D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
201
1.5 mmol) was added at room temperature. After 15
min at room temperature, the solution was evaporated
under reduced pressure. The crude product was used
without further purification. Rf 0.41 (DCM/MeOH, 9:1).
oil. Yield 181mg (60%). Rf 0.4 (DCM/MeOH, 95:5). 1H
NMR (CDCl3): d 1.45 (27H, s, Boc, tBu), 1.89 (2H, m,
CbH2Glu), 2.05 (3H, s, CH3Acm), 2.45 (2H, m,
CgH2Glu), 2.97 (3H, s, N–CH3), 2.22–3.27 (2H, m,
CbH2Cys), 3.85 (2H, d, J=5.8 Hz, CH2Gly), 4.24 (1H,
m, CaHGlu), 4.31–4.65 (2H, m, CH2Acm), 5.32 (1H, m,
CaHCys), 5.4 (1H, d, J=8.0 Hz, NHBoc), 7.11 (1H, m,
NHGly), 7.33 (1H, m, NHAcm).
oNs-Cys(Acm)-Gly-OtBu (5). The crude Fmoc depro-
tected dipeptide 4 (456 mg, 1.5 mmol) was dissolved in
10 mL dry DCM. The solution was cooled on an ice/
water bath, after which DiPEA (311 mg, 1.8 mmol) and
2-nitrobenzenesulfonylchloride (399 mg, 1.8 mmol) were
added. After 16 h at ambient temperature, the solvent
was evaporated and product 5 was purified by silica gel
column chromatography. Yield 525 mg (74% over two
steps). Rf 0.38 (DCM/MeOH, 95:5). 1H NMR (CDCl3):
d 1.45 (9H, s, tBu), 2.04 (3H, s, CH3Acm), 2.86 (2H, m,
CbH2Cys), 3.75 (2H, m, CH2Acm), 4.12 (1H, CaHCys),
4.5 (2H, m, CH2Gly), 6.95 (1H, m, NHGly), 7.45 (1H,
m, NHAcm), 7.6 (1H, m, NH-oNS), 7.72–8.18 (4H, m,
aromatic).
{Boc-Glu[MeCys-GlyOtBu]-OtBu}2 (19). Product 19
was prepared as described for compound 17. Yield 143
mg (89%). Rf 0.45 (DCM/MeOH, 95:5). 1H NMR
(CDCl3): d 1.45 (54H, s, Boc, tBu), 1.9 (4H, m,
CbH2Glu), 2.45 (4H, m, CgH2Glu), 2.19–3.36 (4H, m,
CbCys), 3.02 (6H, s, N–CH3), 3.88 (4H, d, J=5.8 Hz,
CH2Gly), 4.19 (2H, m, CaHGlu), 5.27 (2H, d, J=8.05
Hz, NHBoc), 5.3 (2H, m, CaHCys), 7.51 (2H, m,
NHGly).
{H-Glu[MeCys-Gly-OH]-OH}2 (20). Acid-labile pro-
tecting groups were removed as described for compound
18. Product 20 was obtained after triturating with die-
thyl ether and used without further purification.
oNs-MeCys(Acm)-Gly-OtBu (6). DIAD (223 mL, 1.15
mmol) was added to a stirred solution of triphenylpho-
sphine (302 mg, 1.15 mmol) in dry 3 mL THF at 0 ꢁC.
After 30 min, a white precipitate had formed, indicating
the formation of the phosphonium intermediate. Pep-
tide 5 (283 mg, 0.6 mmol) and 1.2 mmol dry MeOH (50
mL), dissolved in 1 mL dry THF, were added dropwise
to the ice-cold reaction mixture. The solution was stir-
red for 2 h at 0 ꢁC, and for 24 h at room temperature.
The solvent was evaporated, and pure 6 was obtained
after silica gel column chromatography. Yield> 95%.
H-Glu[MeCys(EA)-Gly-OH]-OH, III. Disulfide reduc-
tion and ethacrynic acid conjugation was performed
essentially the same as for compound (I). Compound II
was purified after LH20 gelfiltration (70% MeOH/
1
water) and RP-HPLC. H NMR (D2O): d 0.85 (3H, m,
CH3EA), 1.5–1.7 (2H, m, CH2EA), 2.2 (2H, m,
CbH2Glu), 2.45 (2H, m, CgH2Glu), 2.2–3.2 (2H, m,
CbH2Cys), 2.9 (2H, d, J=6.3 Hz, CH2EA), 2.95 (3H, s,
N–CH3), 3.5 (1H, m, CH–EA), 3.7 (1H, m, CaHGlu),
3.9 (2H, d, CH2Gly), 4.63 (2H, s, CH2EA), 5.3 (1H,
CaHCys), 6.95 and 7.60 (2Â1H, m, aromatic). Mass-
spectrometry (ES-MS): m/e 624, [M+H]+.
1
Rf 0.42 (DCM/MeOH, 95:5). H NMR (CDCl3): d 1.47
(9H, s, tBu), 2.04 (3H, s, CH3Acm), 2.68–3.27 (2H, m,
CbH2Cys), 3.03 (3H, s, CH3N), 3.69–4.04 (2H, m,
CH2Acm), 4.17 (1H, m, CaHCys), 4.5 (2H, m, CH2Gly),
6.79 (1H, m, NHGly), 7.25 (1H, m, NHAcm), 7.4–8.15
(4H, m, aromatic).
D-2-Aminoadipicacid-OEt (21). D-2-aminoadipic acid
29 (1.61g, 10mmol) was suspended in 30 mL dry ethanol
in an N2 atmosphere. TMS-Cl (2.78 mL, 22 mmol) was
added dropwise to the suspension. The resulting clear
solution was stirred for 24 h at room temperature. The
solvent was evaporated under reduced pressure and the
crude ester was redissolved in 60 mL EtOH. Pyridine
(4.5 mL) was added, after which a white precipitate
formed immediately. After 6 h at 4 ꢁC, the crystals were
collected and washed with ether. Recrystallization from
water containing a trace of pyridine yielded pure
H-MeCys(Acm)-Gly-OtBu (7). Thiophenol (256 mL, 2.5
mmol) and DiPEA (345 mL, 2 mmol) were added to a
solution of 6 (243 mg, 0.5 mmol) in 5 mL DMF. After
16 h at room temperature, TLC analysis indicated
complete oNs-cleavage. Toluene (5 mL) was added to
the reaction mixture. The DMF/toluene mixture was
added on a silica gel column, which was equilibrated with
pure toluene. The column was flushed until the bright
yellow apolar o-Ns cleavage products and excess thio-
phenol had eluted. Elution with a gradient of EtOAc in
toluene gave the methylated dipeptide 7 as an oil. Yield
156 mg (98%). Rf 0.14 (DCM/MeOH, 95:5). NMR
analysis confirmed complete removal of oNs group.
1
monoester 21 as a white solid. Yield 1.569 g (83%). H
NMR (D2O): d 1.24 (3H, t, J=7.2 Hz, CH3ethyl), 1.72
(2H, m, CgH2), 1.9 (2H, m, CbH2), 2.45 (2H, m, CdH2),
3.92 (1H, t, J=5.9 Hz, CaH), 4.15 (2H, q, CH2ethyl).
Boc-Glu[MeCys(Acm)-GlyOtBu]-OtBu
(9).
Boc-
Glu(OH)-OtBu(30, 152 mg, 0.5 mmol) and 7 (156 mg,
0.5 mmol) were added to a stirred solution of HOBt (68
mg, 0.5 mmol) in 5 mL dry DCM at 0 ꢁC. DIC (77 mL,
0.5 mmol) was then slowly added to the solution. After
24 h at room temperature, the solvent was evaporated
and the residue was dissolved in 100 mL EtOAc. The
organic layer was then washed repeatedly with satd.aq
NaHCO3 solution, 0.1 M HCl solution and water.
Removal of the solvent and purification over a silica gel
column gave the pure N-methylated tripeptide 9 as an
Boc-Glu[Aminoadipicacid-OEt]-OtBu (10). Compound
21 (945mg, 5mmol) was suspended in 50 mL DMF,
containing DiPEA (950 mL, 5.5 mmol) and Boc-
Glu(OSu)-OtBu (5 mmol). The mixture was stirred
overnight, after which the clear solution was con-
centrated under reduced pressure and acidified to
pH=3 with 1M HCl. EtOAc (200 mL) was added and
the organic phase was washed with water. The organic
layer was dried over MgSO4 and evaporated. Pure
dipeptide 10 was obtained as colorless oil after silica gel

202
D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
column chromatography. Yield 85%. Rf 0.63 (DCM/
1
m/e (EI-MS): 475, 443, 417, 389, 361, 329, 317, 300, 273
and smaller fragments. Fragmentation is in accordance
with the expected structure.
MeOH, 9:1). H NMR (CDCl3): d 1.25 (3H, t, J=7.2
Hz, CH3Ethyl), 1.4 (18H, s, Boc, tBu), 1.7 (4H, m,
CbH2Aad, CgH2Aad), 1.9 (2H, m, CbH2Glu), 2.35 (4H,
m CgH2Glu, CdH2Aad), 3.95 (1H, m, CaHAad), 4.1
(2H, q, CH2ethyl), 5.55 (1H, d, NHBoc), 7.25 (1H, d,
NHamide).
Boc-Glu[2-amino-1-mercapto(S-EA)-6-hexanoicacid]-OtBu
(22). The thioacetate moiety and the ethyl ester of com-
pound 12 were simultaneously cleaved using 5 equiva-
lents of Tesser’s base (Dioxane/MeOH/2 M aq NaOH,
15:4:1) in an argon atmosphere. After 3 h at room tem-
perature, ethacrynic acid (303 mg, 1 mmol) in 5 mL etha-
nol was added to the mixture. The mixture was allowed to
stir for at least 4 h. The pH was then brought to 3 with 1 M
HCl solution. Solvents were evaporated under reduced
pressure. The residue was dissolved in ethyl acetate and
washed several times with 0.1M HCl solution. The
ethacrynic acid conjugate 22 was collected by evapora-
tion of the organic layer and was used without further
purification. Rf 0.45 (DCM/MeOH/AcOH, 95:5:1).
Boc-Glu[2-amino-1-hydroxy-6-hexanoicacid-OEt]-OtBu
(11). Compound 10 (1 mmol) was dissolved in 5 mL
THF at ꢀ10 ꢁC. NMM (110 mL, 1 mmol) and ethyl-
chloroformate (96 mL, 1 mmol) were added and the
resulting suspension was stirred for 15 min at 0 ꢁC.
NaBH4 (121 mg, 3.2 mmol) was added in one portion.
Methanol was added dropwise, carefully controlling the
rate of reduction. After addition of 10 mL methanol,
the mixture was allowed to stir for 30 min at room
temperature. The pH of the solution was acidified (pH
5) by addition of 1M aq H2SO4. EtOAc (50 mL) was
added and the resulting solution was washed with water.
The organic phase was dried over MgSO4 and evapo-
rated under reduced pressure. The residue was purified
by silica gel column chromatography, using EtOAc as
eluent. The pure dipeptide alcohol 11 was obtained as
colorless oil. Yield 84%. Rf 0.2 (EtOAc/hexanes/AcOH,
ꢀ-Glu[2-amino-1-mercapto(S-EA)-6-hexanoicacid-OEt]-
OH, IV. Trifluoroacetic acid/1%H2O (10 mL) was
added to 22. After 3 h at room temperature, ice-cold
diethylether was added. The precipitated product IV
was washed with diethylether and further purified by
Sephadex LH20 gelfiltration, using MeOH/water (7:3)
as eluent. Rf 0.30 (nBuOH/H2O/AcOH, 15:3:2). 1H
NMR (D2O): d 0.86 (3H, t, CH3EA), 1.35–1.9 (6H, m,
CH2EA, CgH2Glu, CbH2Hex), 2.1–2.35 (4H, m,
CbH2Glu, CdH2Hex), 2.37–2.4 (2H, CgH2Glu), 2.52–
2.95 (4H, m, CH2EA, CH2–S), 3.56 (1H, CHEA), 3.74–
3.86 (2H, CaHGlu, CaHHex), 4.65 (2H, s, OCH2 EA),
6.95 (1H, d, Arom), 7.60 (1H, d, Arom.). Mass spec-
trometry (ES–MS) m/e: 595, [M+H]+.
1
10:10:1). H NMR (CDCl3): d 1.25 (3H, t, J=7.2 Hz,
CH3ethyl), 1.44–1.47 (18H, 2Âs, Boc, tBu), 1.52–1.77
(6H, m, CbH2, CgH2 Hex+CbH2Glu), 2.07–2.36 (4H,
CgH2 Glu+CdH2 Hex), 3.53–3.78 (2H, CH2O), 3.9 (1H,
m, CaH Hex), 4.08–4.17 (3H, m, CH2ethyl+CaH Glu),
5.4 (1H, d, J=8.2 Hz, NH Boc), 6.5 (1H, d, J=8.2 Hz,
NH amide). 13C NMR (CDCl3): d 13.91 (CH3Et), 21.1
(CgHex), 28.0 (Boc, tBu), 28.67 (CbGlu), 30.13 (CgGlu),
32.28 (CbHex), 33.59 (CdHex), 50.99 (CaHex), 53.21
(CaGlu), 60.03 (CH2ethyl), 64.21 (CH2–OH), 79.62,
81.77 (Cq, Boc, tBu), 155.80 (CO Boc), 171.32, 172.32,
173.29 (CO).
Boc-Glutaminol-OtBu (13). Boc-Glu-OtBu (30, 304 mg,
1 mmol) was dissolved in 3 mL freshly distilled THF
and cooled to ꢀ40 ꢁC under a stream of N2 gas. Trie-
thylamine (167 mL, 1.2 mmol) was added to the cold
solution, followed by dropwise addition of isobutyl-
chloroformate (157 mL, 1.2 mmol) in 1 mL THF. The
resulting suspension was stirred at ꢀ20 ꢁC for 45 min.
The suspension was then filtered into a rigorously stir-
red solution of NaBH4 in 2mL THF/H2O (8:1) at 0 ꢁC.
The reduction was then allowed to stir for 3 h at room
temperature. The solvent was then removed under
reduced pressure and compound 13 was purified by
silica gel column chromatography, using EtOAc/hex-
anes (1:1) as eluent. Yield 257mg (89%). Rf 0.4 (DCM/
MeOH, 95:5). 1H NMR (CDCl3): d 1.45 (18H, 2Âs,
Boc, tBu), 1.54–1.95 (4H, m, CbH2, CgH2), 3.65 (2H, t,
J=5.9 Hz, CdH2), 4.16 (1H, m, CaH), 5.34 (1H, d,
J=8.4 Hz, NH).
Boc-Glu[2-amino-1-mercaptoacetyl-6-hexanoicacid-OEt]-
OtBu (12). DIAD (291 mL, 1.5 mmol) was added to a
stirred solution of triphenylphosphine (394 mg, 1.5
mmol) in 3.5 mL dry THF at 0 ꢁC. A white precipitate
formed immediately. After 30 min at 0 ꢁC, a solution of
peptide alcohol 11 (0.75 mmol) and thioacetic acid (107
mL, 1.5 mmol) in 1.5 mL THF was added dropwise to
the suspension. After 1 h at 0 ꢁC, the solution was stir-
red for 16 h at room temperature. The solvent was eva-
porated under reduced pressure. The residue was
subjected to silica gel column chromatography, using
EtOAc/hexanes (3:7) as eluent. Product 12 was collected
as a colorless oil. Yield 89%. Rf 0.52 (EtOAc/hexanes,
1:1). 1H NMR (CDCl3): d 1.25 (3H, t, J=7.2 Hz,
CH3ethyl), 1.45 (18H, 2Âs, Boc, tBu), 1.5–1.95 (5H, m,
CbH2Hex, CgH2Hex, CbH2Glu), 2.05–2.37 (7H, m,
CH3SAc, CdH2Hex, CgH2Glu), 3.05 (2H, m, CH2–S),
4.1 (3H, m, CH2ethyl, CaHHex), 5.29 (1H, d, J=7.2
Hz, NHBoc), 6.45 (1H, d, J=7.2 Hz, NHamide). 13C
NMR: d 13.9 (CH3 ethyl ester), 20.97 (CgHex), 27.6,
28.0 (Boc, tBu), 30.15 (CH3 SAc), 32.25–33.37
(CbCgGlu, CH2-S, CbCdHex), 48.56 (CaHex), 53.57
(CaGlu), 59.9 (CH2ethyl ester), 79.13 (CqBoc), 81.34 (Cq
tBu), 155.61 (CO, Boc), 171.23, 171.8 (2ÂCO, ester),
172.89 (CO Glu), 195.15 (CO, SAc). Mass spectrometry
Boc-Glu[W (CH2N-oNS)-Cys(Acm)-Gly-OtBu]-OtBu
(14). To a stirred solution of triphenylphosphine (394
mg, 1.5 mmol) in 15 mL freshly distilled THF, DIAD
(291 mL, 1.5 mmol) was added at ꢀ50 ꢁC in an argon
atmosphere. After 15 min, a white suspension had
formed, to which a solution of alcohol 13 (257 mg, 0.89
mmol) and oNs-protected dipeptide 5 (425 mg, 0.9
mmol) in 7 mL THF was added dropwise. The tem-
perature was carefully maintained at ꢀ50 ꢁC during the
peptide/alcohol addition. Afterwards, the reaction

D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
203
mixture was slowly allowed to reach room temperature
and was stirred overnight. The solvent was removed and
product 14 was purified over a silica gel column. The
product was collected as an oil. Yield 630 mg (96%). Rf
0.65 (DCM/MeOH, 9:1). 1H NMR (CDCl3): d 1.45
(27H, s, Boc, tBu), 1.5–1.85 (4H, m, CbH2Glu,
CgH2Glu), 1.97 (3H, s, CH3Acm), 2.69–3.25 (2H, m,
CbH2Cys), 3.63–3.97 (2H, m, CH2Acm), 4.03 (2H, m,
CH2Gly), 4.45 (1H, m, CaHGlu), 4.76 (1H, m, CaH-
Cys), 5.2 (1H, d, J=7.2 HZ), 7.39–8.15 (5H, NHGly,
CH–aromatic). 13C NMR (CDCl3): d 22.82 (CH3Acm),
26.3 (CgGlu), 27.82, 28.15 (Boc, tBu), 29.82 (CbGlu),
31.3 (CbCys), 40.56 (CH2Acm), 42.0 (CH2Gly), 45.59
(CdGlu), 53.32 (CaGlu), 59.18 (CaCys), 79.37, 81.77 (Cq
Boc, tBu), 124.0–133.56 (4C, aromatic), 147.85 (C-
NO2), 155.34 (CO Boc), 168.2-171.59 (4ÂCO).
compound 17. Yield 93%. Rf 0.45 (DCM/MeOH, 95:5).
1H NMR (CDCL3): d 1.45 (72H, s, Boc, tBu), 1.5–1.82
(8H, m, CbH2Glu, CgH2Glu), 2.8–3.6 (8H, m, CdH2Glu,
CbH2Cys), 3.9 (4H, d, CH2Gly), 4.12 (2H, m, CaHGlu),
4.9 (2H, m, CaHCys), 5.15 (2H, d, NHBoc), 7.45 (2H,
m, NHGly).
Boc-Glu[W (CH2N-Boc)-Cys(EA)-Gly-OtBu]-OtBu (24).
The protected disulfide 23 (0.37 mmol) was dissolved in
5 mL nPrOH/H2O (4:1). The pH was adjusted to 8.5
with 25% aq NH4OH solution and the mixture was
flushed with argon. After addition of tri-n-butylpho-
sphine (92 mL, 0.37 mmol), the resulting mixture was
stirred for 1 h at room temperature. Ethacrynic acid
(303 mg, 1 mmol), dissolved in 1 mL EtOH was added.
After at least 4 h at rt, the solvent was evaporated. The
residue was purified by silica gel column chromato-
graphy, using DCM/MeOH (95:5) as eluent. The pro-
tected EA conjugate was collected as an oil. Yield 389
mg (65%). Rf 0.35 (DCM/MeOH, 95:5). 1H NMR
(CDCl3): d 0.9 (3H, t, CH3EA), 1.45 (36H, s, Boc, tBu),
1.5–1.89 (6H, m, CbH2Glu, CgH2Glu, CH2EA), 2.15–
2.7 (2H, m, CbH2Cys), 2.97–3.4 (2H, m, CdH2Glu), 3.57
(1H, m, CHEA), 3.9 (2H, d, CH2Gly), 4.2 (1H, m,
CaHGlu), 4.78 (2H, s, CH2EA), 5.15 (1H, d, NHBoc),
6.78, 7.15 (2Â1H, dd, EA aromatic).
Boc-Glu[W (CH2NH)-Cys(Acm)-Gly-OtBu]-OtBu
(15).
Compound 14 (584 mg, 0.8 mmol) was dissolved in 15
mL dry DMF in an N2 atmosphere. Thiophenol (409
mL, 4 mmol) and DiPEA (552 mL, 3.2 mmol) were
added, after which the mixture was stirred overnight.
Toluene was added to the reaction mixture and applied
to a silica gel column. Flushing with toluene was con-
tinued until excess thiophenol and brightly colored
deprotection by-products had eluted. The eluent was
then replaced by EtOAc/hexanes, after which product
15 could be isolated as an oil. Yield 436 mg (100%). Rf
H-Glu[W (CH2NH)-Cys(EA)-Gly-OtBu]-OH,
V.
1
0.26 (DCM/MeOH, 95:5). H NMR (CDCL3): d 1.41
Deprotection of 24 was performed by addition of 5 mL
TFA/H2O (99:1). After 4 h at rt, the product was pre-
cipitated by addition of ice-cold diethylether. Further
purification of the precipitate by Sephadex LH20 col-
umn chromatography, eluent: MeOH/H2O (7:3), yiel-
ded compound IV as a fluffy white powder after
lyophilization. LC–MS analysis indicated the presence
of the desired product and a tri-i-butylphosphine–EA
complex. The pure reduced tripeptide–EA conjugate
was obtained after HPLC purification. Rf 0.3 (nBuOH/
H2O/AcOH, 15:3:2). 1H NMR (D2O): d 0.86 (3H, t,
CH3EA), 1.55 (2H, m, CH2EA), 1.74–1.92 (4H, m,
CbH2, CgH2) 2.4 (2H, m, CdH2) 2.85 (2H, dd,
CbH2Cys), 3.0 (2H, m, CH2EA), 3.74 (1H, d, CaHEA)
3.78 (2H, d, CH2Gly), 4.1 (1H, m, CaHGlu), 4.65 (2H,
s, CH2EA), 6.9, 7.6 (2Â1H, dd, EA aromatic). Mass
spectrometry (ES-MS): m/e 596, [M+H]+.
(27H, ss, Boc, tBu), 1.54 (2H, m, CgH2Glu), 1.74 (2H,
m, CbH2Glu), 1.98 (3H, s, CH3Acm), 2.52–3.05 (2H,
CbH2Cys), 2.85 (2H, s, CbH2Cys), 2.93 (2H, m,
CdH2Glu), 3.6 and 3.69 (1H, m, CaHCys), 3.89 (2H, d,
J=5.9 Hz, CH2Gly), 4.18 (1H, m, CaHGlu), 4.3 (2H, m,
CH2Acm), 5.2 (1H, d, J=8.4 Hz, NHBoc), 7.19 (1H, m,
NHAcm), 7.92 (1H, m, NHGly).
Boc-Glu[W (CH2N-Boc)-Cys(Acm)-Gly-OtBu]-OtBu (16).
Reduced peptide isostere 15 (436 mg, 0.8 mmol) was
dissolved in 20 mL dry acetonitrile. DiPEA (0.155 mL,
0.9 mmol) and BOC2O (350 mg, 1.6 mmol) were added.
The mixture was stirred at 60 ꢁC for 24–48 h, until no
starting material could be detected by TLC. EtOAc (100
mL) was added and the organic phase was washed with
satd aq NaHCO3, 0.1 M HCl and water. The organic
solvent was evaporated under reduced pressure. Com-
pound 16 was obtained as an oil after silica gel column
chromatography, using EtOAc/hexanes as eluent. Yield
Boc-Glo(ONp)-OtBu (25). Boc-Ser(OtBu) (261.2 mg, 1.5
mmol) was dissolved in 20 mL DMF. Bis(4-nitrophe-
nyl)carbonate (456 mg, 1.5 mmol) and DiPEA (259 mL,
1.5 mmol) were added, after which the resulting solution
was stirred for 16 h at room temperature. EtOAc (100
mL) was added to the mixture, which was then repeatedly
washed with saturated NaHCO3 until the aq layer was no
longer yellow. After drying and evaporation of the
organic layer, the product was purified by silica gel col-
umn chromatography. The carbonate 25 was collected
as a foam. Yield 582 mg (91%). Rf 0.85 (DCM/MeOH,
1
516 mg (100%). Rf 0.3 (DCM/MeOH, 95:5). H NMR
(CDCl3): d 1.45 (27H, ss, Boc, tBu), 1.5–1.8 (4H, m,
CbH2Glu, CaH2Glu), 2.01 (3H, s, CH3Acm), 2.85 and
3.2 (2H, m, CbH2Cys), 3.17 (2H, m, CdH2Glu), 3.9 (2H,
m, CH2Gly), 4.23 (1H, m, CaHGlu), 4.54 (2H, m,
CH2Acm), 4.67 (1H, m, CaHCys), 5.18 (1H, d, J=8.0
Hz) 6.9 (2H, m, NHAcm, NHGly). 13C NMR (CDCl3/
MeOD): d 21.94 (CH3Acm), 25.03 (CgGlu), 27.27–27.61
(Boc, tBu), 28.94 (CbGlu), 29.97 (CbCys), 40.2
(CH2Acm), 41.32 (CH2Gly), 44.32 (CdGlu), 53.66
(CaGlu), 57.84 (CaCys), 78.0–81.2 (4Â Cq, Boc, tBu),
155.55, 156.03 (2ÂCOBoc), 168.2–171.53 (4ÂCO).
1
95:5). H NMR (CDCl3): d 1.4 (9H, s, Boc), 1.5 (9H, s,
tBu), 4.61 (3H, m, CaH, CbH2), 5.62 (1H, d, J=6.5 Hz,
NH), 7.4 (2H, aromatic) and 8.25 (2H, aromatic).
{Boc-Glu[W (CH2N-Boc)-Cys-Gly-OtBu]-OtBu}2 (23).
Acm-deprotection was performed as described for
Boc-Glo[Cys(Acm)-Gly-OtBu]-OtBu (26). A solution of
4 (456 mg, 1.5 mmol) in 2.5 mL dioxane was added to a

204
D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
solution of 25 (582 mg, 1.35 mmol) in 2.5 mL dioxane.
The mixture was stirred at 80 ꢁC overnight. After eva-
poration of the solvent, the residue was dissolved in 50
mL EtOAc. Washing with satd aq Na2CO3 was repe-
ated until the aq layer was colorless. Silica gel column
chromatography gave the pure tripeptide 26 as a foam.
m, CH2EA), 3.75 (1H, d, CaHEA) 3.9 (2H, d, CH2Gly),
4.3–4.5 (3H, m, CaHGlo, CbH2Glo), 4.65 (2H, s,
CH2EA), 4.9 (2H, m, CaHCys), 6.9 and 7.6 (2Â1H, dd,
EA aromatic). Mass spectrometry (ES–MS) m/e 610,
[MꢀH]ꢀ (Negative mode)
1
Yield 622 mg (78%). Rf 0.48 (DCM/MeOH, 9:1). H
Stability towards ꢀ-Glutamyl transpeptidase
NMR (CDCl3): d 1.45 (27H, s, Boc+tBu), 2.0 (3H, s,
CH3 Acm), 2.8–3.0 (2H, m, CbH2Cys), 3.8–4.1 (2H, m,
CH2 Acm), 4.1–4.6 (6H, m, CH2 Gly, CaHCys CaH,
CbH2 Glo), 5.6 (1H, d, NH Boc), 6.2 (1H, d, NH Glo),
7.4 (2H, 2ÂNH, Acm and Gly amide). 13C NMR
(CDCl3): d 22.6 (CH3 ACM), 27.6–28.0 (9ÂCH3, Boc,
tBu), 33.6 (Cb Cys), 40.4 (CH2 Acm), 41.7 (CH2 Gly),
The rate of degradation was determined by incubating
250 mM of the GS-EA analogues at 37 ꢁC with 0.25 mg/
mL bovine kidney g-glutamyltranspeptidase in 0.1 M
Tris–HCl buffer, pH=7.4, supplemented with 0.1 mM
EDTA. At selected time-intervals, a 100 mL sample was
taken, heat-inactivated, and stored on ice until HPLC-
analysis. Analysis was performed by RP-HPLC on an
Alltech Platinum C18 column, using 50 mM ammonium
acetate buffer pH=3.8, supplemented with 10–20%
acetonitrile (optimized for the various peptide analo-
gues) as eluent. Disappearance of the parent peak and
emergence of the corresponding dipeptide-EA conjugate
was detected by UV absorbency at 270 nm. Remaining
percentage of the parent compounds was determined as
ratio of peak areas relative to the initial (t=0 min)
value. Results were corrected for non-peptidase medi-
ated degradation of the ethacrynic acid conjugates.
53.7 (Ca Cys, Ca Glo), 65 (Cb Glo), 79.5–82.2 (3ÂCquart
,
Boc, tBu), 155.1 (CO, Boc), 155.8 (CO, Glo), 168.4 (CO,
Gly-tBu ester), 168.7 (CO, Acm), 170.7 (CO, Glo-tBu
ester), 171.3 (CO, Cys)
(Boc-Glo[Cys-Gly-OtBu]-OtBu)2 (27). Compound 26
(591 mg, 1 mmol) was dissolved in 7 mL of dry MeOH.
A solution of I2 (508 mg, 2 mmol) in 3 mL MeOH was
added dropwise during 30 min. After 10 min at room
temperature, the mixture was cooled to 0 ꢁC and deco-
lorized with 1 M aq Na2S2O3 solution. The resulting
mixture was diluted with 100 mL EtOAc and washed
with 1M aq Na2S2O3 solution and water. After drying
and evaporation of the organic phase, the residue was
dissolved in DCM/MeOH (1:1) and purified by Sepha-
dex LH20 gelfiltration chromatography, using DCM/
MeOH as eluent. The pure disulfide 27 was obtained as
a white foam. Yield 494 ml (95%). Rf 0.51 (DCM/
Inhibition of rat liver cytosolic GSTs
Freshly isolated rat livers were emulsified in 0.1 M KCl
using a Potter–Elvehjem homogeniser. After initial cen-
trifugation for 30 min at 9000g, the resulting super-
natant was subjected to a second centrifugation for 75
min at 105g. The cytosolic protein containing super-
natant (S100) was used for the GST inhibition experi-
ments. GST inhibition experiments were performed
according to Habig et al., modified for a 96-well assay.
All solutions were kept on ice until use. Various con-
centrations of the inhibitors were incubated at 37 ꢁC
with rat liver cytosol (1 mL, containing 12 mg of total
protein) in sodium-phosphate buffer, pH 6.5, supple-
mented with 0.1 mM EDTA and 1 mM GSH. Reaction
was started by adding 4 mL CDNB in ethanol (final
concentration=1mM, maximal ethanol content=2% v/
v). Formation of the GS-CDNB conjugate was followed
at 340 nm for 5 min. Initial reaction velocities were
corrected for the spontaneous (not GST catalyzed)
reaction rates. Inhibition was determined as percentage
of the non- inhibited reaction rate. Experiments were
performed three times with duplicate measurements.
1
MeOH, 9:1). H NMR (CDCl3): d 1.45 (54H, s, Boc,
tBu), 2.95 (4H, m, CbH2), 3.9 (4H, m, CH2Gly), 4.3–4.5
(6H, CaHGlo, CbH2Glo), 4.98 (2H, m, CaHCys), 5.4
(2H, d, J=8 Hz, NHBoc), 5.9 (2H, d, J=8.2 Hz,
NHGlo), 7.8 (2H, m, NHGly)
(H-Glo[Cys-Gly-OH]-OH)2 (28). Disulfide 27 (494 mg,
0.48 mmol) was dissolved in 5 mL TFA/100mL H2O.
After 3 h at room temperature, the deprotected peptide
was precipitated by addition of 15 mL ice-cold diethy-
lether. The disulfide (TFA salt) 28 was used without
further purification.
H-Glo[Cys(EA)-Gly-OH]-OH, VI. The deprotected dis-
ulfide 28 (296 mg, 0.48 mmol) was dissolved in 15 mL
nPrOH/H2O (2:1). Aqueous ammonia solution (25%)
was added until pH=8.5. After flushing with N2, tri-n-
butylphosphine (123 mL, 0.5 mmol) was added and the
solution was stirred for 60 min at room temperature. A
solution of ethacrynic acid (455 mg, 1.5 mmol) in 5 mL
nPrOH was added. After adjusting the solution to pH 8
with 1 M NaOH, stirring was maintained for 4 h. The
solvent was evaporated under reduced pressure, after
which the residue was dissolved in water. The aqueous
solution was extracted with EtOAc and purified by
Sephadex LH20 column chromatography, using
MeOH/H2O (7:3) as eluent. Product VI was further
purified by reversed phase HPLC to yield a fluffy white
powder after lyophilization. Rf 0.35 (nBuOH/H2O/
Acknowledgements
This work was financially supported by the Dutch
Cancer Society, grant RUL 97-1407.
References and Notes
1. Anderson, M. E. Chem. Biol. Interact. 1998, 11–112, 1.
2. Hayes, J. D.; Pulford, D. J. Crit. Rev. Biochem. Mol. Biol.
1995, 30, 445.
1
AcOH, 15:3:2). H NMR (D2O): d 0.9 (3H, t, CH3EA),
1.55 (2H, m, CH2EA), 2.9 (2H, dd, CbH2Cys), 3.0 (2H,

D. Burg et al. / Bioorg. Med. Chem. 10 (2002) 195–205
205
3. Minhas, H. S.; Thornalley, P. J. Biochem. Pharmacol. 1995,
49, 1475.
4. Lyttle, M. H.; Hocker, M. D.; Hui, H. C.; Caldwell, C. G.;
Aaron, D. T.; Engqvist Goldstein, A.; Flatgaard, J. E.; Bauer,
K. E. J. Med. Chem. 1994, 37, 189.
5. Klotz, P.; Slaoui, H. A.; Baneres, J. L.; Duckert, J. F.;
Rossi, J. C.; Kerbal, A. J. Med. Chem. 1998, 41, 2278.
6. Kunze, T. Arch. Pharm. Weinheim. 1996, 329, 503.
7. Kunze, T.; Heps, S. Biochem. Pharmacol. 2000, 59, 973.
8. Luisi, G.; Calcagni, A.; Pinnen, F. Tetrahedron Lett. 1993,
34, 2391.
9. Chen, W. J.; Lee, D. Y.; Armstrong, R. N. J. Org. Chem.
1986, 51, 2848.
10. Lucente, G.; Luisi, G.; Pinnen, F. Farmaco. 1998, 53, 721.
11. Adang, A. E.; Brussee, J.; van der Gen, A.; Mulder, G. J.
Biochem. J. 1990, 269, 47.
12. Adang, A. E.; Brussee, J.; Meyer, D. J.; Coles, B.; Ket-
terer, B.; van der Gen, A.; Mulder, G. J. Biochem. J. 1988,
255, 721.
18. Calcagni, A.; Dupre, S.; Lucente, G.; Luisi, G.; Pinnen,
F.; Rossi, D.; Spirito, A. Arch. Pharm. Weinheim. 1996, 329,
498.
19. Mitsunobu, D. Synthesis 1981, 1.
20. Turner, J. J.; Wilschut, N.; Overkleeft, H. S.; Klaffke, W.;
van der Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 1999,
40, 7039.
21. Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107,
4342.
22. Coste, J.; Frerot, E.; Jouin, P.; Castro, B. Tetrahedron
Lett. 1991, 32, 1967.
23. Kokotos, G. Synthesis 1990, 299.
24. Marraud, M.; DuPont, V.; Grand, V.; Zerkout, S.; Lecoq,
A.; Boussard, G.; Vidal, J.; Collet, A.; Aubry, A. Biopolymers
1993, 33, 1135.
25. Paris, M.; Pothion, C.; Heitz, A.; Martinez, J.; Fehrentz,
J . A.Tetrahedron Lett. 1998, 39, 1341.
26. Habig, W. H.; Pabst, M. J.; Jacoby, W. B. J. Biol. Chem.
1974, 249, 7130.
13. Ouwerkerk-Mahadevan, S.; van Boom, J. H.; Dreef,
T. M.; Ploemen, J. H.; Meyer, D. J.; Mulder, G. J. Biochem. J.
1995, 308, 283.
14. Ouwerkerk-Mahadevan, S.; van Boom, J. H.; Mulder,
G. J. J. Pharmacol. Exp. Ther. 1996, 276, 923.
15. Ouwerkerk-Mahadevan, S.; Mulder, G. J. Chem. Biol.
Interact. 1998, 111–112, 163.
27. Satoh, K.; Sato, R.; Takahata, T.; Hayakari, M.; Tsuchida,
S.; Hatayama, I. Arch. Biochem. Biophys. 1999, 361, 271.
28. Oakley, A. J.; LoBello, M.; Battistoni, A.; Ricci, G.;
Rossjohn, J.; Villar, H. O.; Parker, M. W. J. Mol. Biol. 1997,
274, 84.
29. Shen, H.; Schultz, M. P.; Tew, K. D. J. Pharmacol. Exp.
Ther. 1999, 290, 1101.
16. Awasthi, S.; Srivastava, S. K.; Ahmad, F.; Ahmad, H.;
Ansari, G. A. Biochim. Biophys. Acta 1993, 1164, 173.
17. Ploemen, J.; van Ommen, B.; van Bladeren, P. J. Biochem.
Pharmacol. 1990, 40, 1631.
30. Keppler, D.; Leier, I.; Jedlitschky, G.; Konig, J. Chem.
Biol. Interact. 1998, 111–112, 153.
31. Vince, R.; Brownell, J.; Akella, L. B. Bioorg. Med. Chem.
Lett. 1999, 9, 853.