Proteasome inhibition by peptide-semicarbazones

Article abstract of DOI:10.1016/j.bmc.2008.02.042

Peptide-semicarbazones derived from Z-Trp-Trp-Phe-aldehyde inhibit the chymotryptic activity of the human proteasome at nanomolar concentrations, but are less active in a NFκB reporter gene assay. Cyclic semicarbazones, in contrast, combine a strong inhib

Full text of DOI:10.1016/j.bmc.2008.02.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4579–4588
Proteasome inhibition by peptide-semicarbazones
Johann Leban,* Marcus Blisse, Babett Krauss, Sandra Rath,
Roland Baumgartner and Markus H. J. Seifert
4SC AG, Am Klopferspitz 19a, 82152 Planegg-Martinsried, Germany
Received 1 October 2007; revised 7 February 2008; accepted 12 February 2008
Available online 15 February 2008
Abstract—Peptide-semicarbazones derived from Z-Trp-Trp-Phe-aldehyde inhibit the chymotryptic activity of the human protea-
some at nanomolar concentrations, but are less active in a NFjB reporter gene assay. Cyclic semicarbazones, in contrast, combine
a strong inhibitory effect on the enzyme with an inhibition of NFjB signaling in the nanomolar range. In addition, a practical syn-
thesis for scale-up of such compounds was developed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tide vinylsulfones, and peptide boronic acids. All these
compounds act as electrophilic traps and create covalent
intermediates, which can be in equilibrium with the
unmodified enzyme.
The controlled degradation of damaged or misfolded
proteins as well as the regulation of short lived proteins
plays a crucial role for proper cellular functions. This is
achieved by the ubiquitin/proteasome pathway, in which
the target protein is marked by a polyubiquitin chain
and, after recognition, is degraded in an ATP-dependent
manner by the 26S proteasome.1 The 26S proteasome by
itself is a multicatalytic threonine protease consisting of
two polyubiquitin recognizing 19S particles and the 20S
proteasome core, which houses at least three different
peptidase activities: post-glutamyl-, trypsin-, and chy-
motrypsin-like activities.2 The latter is thought to be
responsible for the rate limiting step in protein
degradation.
Tripeptide aldehydes based on the hydrophobic Trp-
Trp-Trp sequence were described in a patent as endothe-
lin converting enzyme inhibitors.4 In this patent, the
Trp-Trp-Trp-aldehyde sequence was shown to be a
superior inhibitor over the initial Ile-Ile-Trp-aldehyde
sequence. Ketoepoxide peptides of this series were later
published as potent proteasome inhibitors.5 Further, all
of the compounds of this patent4 were later revised to be
proteasome inhibitors.6 We confirm here that these com-
pounds are in fact inhibitors of the proteasome. In addi-
tion to the peptide semicarbazones, we synthesized
cyclic semicarbazone analogs and evaluated their inhib-
itory activity on the proteasome, determined the inhibi-
tory activity in a cell based NFjB reporter assay and
confirmed the biological activity in a PHA stimulated
T-cell proliferation assay.
Because of its central role in critical intracellular regula-
tory processes of cell division, growth activation, signal-
ing, and transcription, proteasome inhibitors have been
explored as agents to treat multiple myeloma. A peptide
boronate inhibitor (PS-341) was approved recently un-
der the name Velcade.3 Most of the inhibitors address
the chymotryptic activity via adduct formation with
the N-terminal threonine hydroxyl group as part of
the catalytically active center. These classes of covalent
binding inhibitors belong to a diverse group of com-
pounds such as epoxyketones, peptide aldehydes, pep-
2. Results and discussion
2.1. Synthesis
For the synthesis (Scheme 1), first the Boc-phenylalanine
aldehyde was used.7 The amino acid derived aldehyde
was converted to the Schiff base with the cyclic semi-
carbazide (aminohydantoin) in the presence of TMOB
and DIPEA. Deprotection with 3 N HCl in glacial acetic
acid yielded the deprotected Schiff base. This compound
Keywords: Proteasome; Semicarbazone; NFjB.
Corresponding author. Tel.: +49 89 700763 0; fax: +49 89 700763
28; e-mail: leban@4sc.com
*
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.042

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J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
Boc
N
CHO
a
H
Aa3
O
NH
O
H
b,c
O
Cbz
N
N
N
H
N
H
X
N
O
N
HN
Aa2
N
Boc
NH
O
Scheme 1. Reagents and conditions: (a) Ahyd, TMOB, DIPEA, rt, 24 h; (b) 3 N HCl in AcOH, rt; (c) HBTU, Cbz-Trp-Trp-OH, DMF,DIPEA, rt, 24 h.
was coupled with commercial Cbz protected dipeptides
in the presence of DIPEA with the coupling reagent,
HBTU.
The ability of the compounds to inhibit human 20S pro-
teasome was determined by fluorescence spectroscopy
using the substrate Suc-LLVV-AMC.8 As a secondary
assay, we used a NFjB gene reporter assay. These data
are shown in Table 1. A few compounds were also fur-
ther evaluated in their ability to inhibit PHA stimulated
T-cell proliferation.
This method resulted in a LC–MS pure product with the
expected mass. However, TLC indicated the presence of
isomers in a ratio of 3:1, where the minor isomer termed
Dia1 moved slightly further. The isomeric compounds
could be separated by pTLC in pure form and were
shown to have the same mass.
2.2. Structure–activity relationship
Compound 1 is a nanomolar proteasome inhibitor on
the human enzyme and exhibits a micromolar inhibi-
tory activity in the NFjB reporter assay. This activity
is at least 20-fold higher as the activity of the corre-
sponding aldehyde.4 The aminohydantoin analog (cpd
2) is at least 10-fold less active in inhibiting the enzyme
compared to cpd 1. When, for the sake of easier syn-
thesis, the P1 amino acid of cpd 1 is replaced with
phenylalanine, a minor reduction in enzyme inhibition
is accompanied by a large reduction of inhibitory activ-
ity in the NFjB reporter gene assay (cpd 3). The cyclic
semicarbazone with the same amino acid sequence,
however, retains the high activity in inhibiting the en-
zyme, but now in combination with a much better
activity in inhibiting NFjB (cpd 4). For the cyclic
semicarbazone compounds, Trp to Phe substitution
leads to an increase of both enzyme inhibition and cel-
lular activity by a factor of ꢀ10 (cpd 2 vs cpd 4). The
large difference in cellular activity between cpd 3 and
cpd 4 is likely due to a better cell penetration of the
cyclic semicarbazone compound. As expected, the D-
Phe isomer (cpd 5) is half as active on the enzyme as
the L-Phe isomer (cpd 4). A biotinyl group at the N-
terminal does not affect potency significantly, but the
activity in the NFjB reporter gene assay is lost (cpd
6). A replacement of the Cbz group with a pyridyl-
methyl-oxycarbonyl, for the sake of better solubility,
leads to lower activity (cpd 7). The introduction of a
p-hydroxy-phenyl-propionyl residue leads to slightly
higher activity as compared to the Cbz group (cpd
8); however, this advantage in enzyme inhibition is lost
on the cellular NFjB assay. This result indicates that
there exists a delicate balance between hydrophilicity
and cellular activity.
NMR experiments suggested that Dia1 (cpd 5) and Dia2
(cpd 4) differ only in the configuration of the a-carbon of
the phenylalanine derivative in position P1 (see Table 1).
For example, when pure isomer Dia2 was treated with
1 N HCl in dioxane for 1 h, again a mixture of isomers
in the ratio 1:1 was obtained, as shown by NMR
(Fig. 1). This experiment confirms that racemization oc-
curs when the semicarbazone is treated with acid.
To avoid the acid catalyzed racemization of the imine
we conceived an alternative synthesis route. This meth-
od proceeded through the coupling of deprotected
Weinreb amide with the Cbz protected dipeptides and
the reduction of the tripeptide Weinreb amide to the tri-
peptide aldehyde with LAH. The tripeptide aldehyde
was converted to the semicarbazone by the reaction with
aminohydantoin hydrochloride and DIPEA in the pres-
ence of TMOB for several days. By this method, single
isomer Dia2 (cpd 4) was obtained. Alternatively, exactly
the same synthesis procedure starting with Boc-D-phen-
ylalanine yielded a product, which was completely iden-
tical with the isomer Dia1 (cpd 5). These results confirm
the hypothesis about the nature of Dia1 and Dia2. Addi-
tionally, this synthesis was amenable to scale-up and
with minor modifications a batch of 170 g of compound
4 was prepared (Scheme 2).
Additional analogs with varying capping groups at the
N-terminus were prepared, by removing the Cbz group
by catalytic hydrogenation. This procedure leaves the
semicarbazone intact and acylation of the free amine
with activated esters or by the help of a coupling agent
resulted in additional analogs cpd 6–8 (Scheme 3).

J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
4581
Table 1. Inhibition of 20S proteasome (IC50/lM, SD 0.2 lM) and NFjB signaling (EC50/lM, SD 0.4 lM)
Compound
Structure
IC50
EC50
NH
O
NH
O
O
O
O
O
O
H
N
N
N
N
N
N
1
0.1
1.8
3.0
5.8
0.4
O
O
O
O
O
N
H
N
H
N
H
NH2
O
O
O
O
O
NH
NH
NH
NH
NH
NH
O
NH
N
O
H
N
2
3
4
5
1.1
0.2
0.2
0.4
N
H
N
H
NH
O
NH
O
O
H
N
N
H
N
H
N
H
NH2
NH
O
O
H
N
N
H
N
H
N
NH
O
NH
O
O
H
N
0.8
N
H
N
H
N
NH
O
(continued on next page)

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J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
Table 1 (continued)
Compound
Structure
IC50
EC50
NH
O
O
O
H
6
0.1
n.a.a
N
N
S
N
H
N
N
NH
H
O
NH
NH
O
HN
O
NH
O
O
O
H
N
N
7
1.6
4.9
O
N
N
N
NH
O
H
H
N
O
NH
NH
O
O
O
H
N
N
8
0.1
8.7
N
N
N
NH
O
H
H
O
HO
HN
a Not active.
son et al.8 have reported that semicarbazones of small
peptides are the inhibitors of Cathepsin K and that the
mechanism was most likely the formation of the peptide
aldehyde during the assay conditions.8 We, however,
have found that the compounds described here are ex-
tremely stable in acidic and basic conditions toward deg-
radation and need boiling to be degraded. We therefore
favor an alternative mode of action, where a covalent at-
tack of the Thr1 hydroxyl of the enzyme to the imine
carbon of the semicarbazone forms an aminal. Subse-
quent release of the aminohydantoin leads to an alde-
hyde adduct and blocks the Thr-1 nucleophile.
Molecular modeling of the educt complex of cpd 4
and the 20S proteasome (PDB ID 1IRU) revealed that
the 2-carbonyl of the hydantoin is likely to be coordi-
nated by the N-terminal nitrogen of Thr1 with the semi-
carbazone coming close to the c-oxygen of Thr1 (Fig. 2,
ppm
11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
Figure 1. 1H NMR spectra of Dia2 (bottom) after treatment with HCl
in dioxane (middle), for comparison the spectra of pure Dia1 is shown
above (top).
˚
distance 3.4 A). The model therefore showed that the
semicarbazone can—in principle—be cleaved by the
chymotryptic site of the proteasome. Such a mechanism
could be described as an enzyme activated prodrug ap-
proach, since the peptide aldehyde inhibitor is released
from the semicarbazone (prodrug) only in the presence
of the enzyme.
2.3. Mode of action
It is interesting to propose a mode of action for the inhi-
bition of threonine proteases by semicarbazones. Adki-

J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
4583
O
H
N
N
O
Boc
a,b,c
Aa3
O
d
H
Cbz
N
Aa3
N
N
O
O
H
H
H
O
N
Cbz
N
Aa2
N
NH
N
N
H
H
Aa2
O
O
O
Scheme 2. Synthesis method B. Reagents and conditions: (a) 3 HCl dioxane, 1 h; (b) HBTU, DIPEA, Cbz-Trp-Trp3-OH, DMF, rt, 24 h; (c) LAH,
THF, ꢁ55 ꢁC; (d) AhydHCl, DIPEA, TMOB, rt, 48 h.
Aa3
Aa3
O
a
H
O
H
Cbz
N
N
N
N
O
H2N
N
O
H
H
O
N
H
O
N
Aa2
N
NH
Aa2
N
NH
O
O
O
O
N
Aa3
O
O
O
H
NO2
N
O
N
N
O
H
H
b
N
O
N
Aa2
N
NH
O
Scheme 3. Synthesis method C. Reagents and conditions: (a) H2, Pd/C 4 h, rt; (b) DIPEA, DMF, rt, 24 h.
a-carbon adjacent to the aldehyde group. Further, they
can form adducts with proteins. Compounds with pro-
tected aldehydes such as semicarbazones represent a sta-
ble alternative to peptide aldehydes as proteasome
inhibitors.
2.4. Specificity of enzyme inhibition and immunosuppres-
sive effect
In order to examine the subunit specificity of these com-
pounds, proteasome inhibition of compounds 1 and 4
was measured at the peptidylglutamyl peptide hydrolyz-
ing (PGPH) site. Inhibition values were compared to the
values obtained at the chymotrypsin-like (CT) site.
Compound 1 showed a 60-fold selectivity for the chymo-
tryptic site (IC50-CT = 0.06 lM, IC50-PGPH = 3.7 lM)
whereas compound 4 is only six times more selective to-
ward the chymotryptic site (IC50-CT = 0.2 lM, IC50-
PGPH = 1.2 lM). These results are in good agreement
with the prevailing substrate specificity of the chymo-
trypsin-like (hydrophobic) and the peptidylglutamyl
peptide hydrolyzing (acidic) site. Due to the incompati-
Figure 2. Model of the non-covalent complex of cpd 4 and the
chymotryptic site of the 20S proteasome. (A) amino acids surrounding
the binding site. (B) Surface representation of the binding site.
Peptide aldehydes as proteasome inhibitors are well
known, but have not obtained a practical use for stabil-
ity reasons. They tend to polymerize and racemize at the

4584
J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
Table 2. Inhibition of human PBMC proliferation
eral blood mononuclear cell; Pd/C, palladium on
charcoal; PGPH, peptidylglutamyl peptide hydrolyzing
site; PHA, phytohemagglutinin; Phe, phenylalanine;
PDB, protein data bank; pHPLC, preparative HPLC;
ps, picoseconds; pTLC, preparative thin-layer chroma-
tography; quint., quintet; Rt, retention time; rt, room
temperature; s, singlet; SD, standard deviation; SDS, so-
dium dodecyl sulfate; SEAP, secreted alkaline phospha-
tase; t, triplet; THF, tetrahydrofurane; TLC, thin-layer
chromatography; TMOB, trimethyl-ortho-formate;
TNF, tumor necrosis factor; Trp, tryphtophane; W,
pseudo; ll, microliter; lm, micrometer; lM,
micromolar.
Compound
IC50 (lM)
4
1
6
0.7
1.4
29
bility of the tryptic substrate with SDS used for protea-
some activation, the tryptic site could not be
characterized.
Proteasome inhibitors block the T-cell signaling path-
way through their inhibition of NFjB and exert an
immunosuppressive effect.9 To solidify the assumption
that these compounds have immunosuppressive activity,
we tested a subset of compounds in a PBMC prolifera-
tion assay. Due to the phytohemagglutinin stimulus
(PHA) used in the assay, the measured effect can be
attributed mainly to T-cells. A significant suppressive ef-
fect was detected (Table 2).9 As can be seen with the
NFjB cell assay, the biotinylated analog has little activ-
ity despite its activity on the proteasome. The other
compounds have potent activity in this assay, which is
indicative of their immunosuppressive activity.
4.2. General methods
4.2.1. Analytical TLC. Merck aluminum sheets, silica gel
60 F254.
4.2.2. Preparative TLC. Merck TLC plates, silica gel 60
254, 0.5, 1.0, or 2.0 mm.
F
4.2.3. Flash chromatography. Acros silica gel 60 A,
0.035–0.070 mm. Flash Master Personal or Flash Mas-
ter II, Jones Chromatography, UK.
1
4.2.4. NMR spectra. Bruker Avance 300 MHz. The H
3. Conclusions
NMR spectra were recorded at 300 MHz; concentra-
tion, 1–5 mg/ml; temperature, 305 K. The 13C NMR
spectra at 75.5 MHz; concentration, 5–20 mg/ml; tem-
perature, 305 K. The residual solvent peaks were used
as the internal standards (DMSO-d6: dH 2.49, dC 39.5;
CDCl3: dH 7.24, dC 77.0; CD3OD: dH 3.30, dC 49.0).
Alternatively, TMS was used as a standard (indicated
with TMS).
In conclusion, we have prepared a novel class of protea-
some inhibitors derived from peptide aldehydes with
good cellular activity in inhibiting NFjB signaling.
The compounds have good stability in acidic and basic
solutions and show low toxicity in vivo. Such com-
pounds have great potential to be useful for the treat-
ment of autoimmune diseases and cancer.
4.2.5. Analytical LC–MS. Waters 2700 Autosampler. 2·
Waters 600 Multisolvent Delivery System, Waters 600
Controller. Fifty micloliters of sample loop. Column,
Chromolith Speed ROD RP18e (Merck, Darmstadt),
50 · 4.6 mm, with 2 lm prefilter (Merck). Eluent A,
H2O + 0.1% HCO2H; eluent B, MeCN. Gradient, 2%
B to 100% B within 4 min, then isocratic for 0.90 min,
then back to 2% B within 0.15 min, then isocratic for
0.50 min; flow, 3 ml/min. Waters LCZ single quadrupole
mass spectrometer with electrospray source. MS meth-
od, MS8minPM-80-800-20 V; positive/negative ion
mode scanning, m/z 80–800 or 80–900 in 1 s; capillary,
3.5 kV; cone voltage, 20 V; multiplier voltage, 400 V;
probe and desolvation gas temperature, 120 ꢁC and
350 ꢁC, respectively. Waters 2487 Dual k Absorbance
Detector, set to 254 nm. Software, Waters Masslynx V
4.0.
4. Experimental
4.1. Abbreviations
Ahyd, aminodydantoin; ATP, adenosine triphosphate;
Boc, tert-butyloxycarbonyl; br, broad; BrdU, 5-brom-
2-desoxyuridin; Cbz, benzyloxycarbonyl; cpd, com-
pound; CT, chymotrypsin-like site; d, doublet; DCM,
dichloromethane; Dia, diastereomer; DIPEA, diisopro-
pylethylamine;
DMAP,
4-dimethylaminopyridine;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
DNA, deoxyribonucleic acid; EC50, half maximal effec-
tive concentration, EDTA, ethylenediaminetetraacetic
acid; ELISA, enzyme linked immunoassay; g, gram,
H2, hydrogen; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluoro-phosphate; HCl, hydro-
chloric acid; Hepes, 2-(4-2-hydroxyethyl)-1-piperazinyl
sulfate; h(s) hour(s); HV, high vacuum; IC50, half max-
imal inhibitory concentration; J, coupling constant; kV,
kilovolt; LAH, lithium aluminum hydride; LC–MS, li-
quid chromatography mass spectroscopy; M, molar;
m, multiplet; MD, molecular dynamics; MeCN, acetoni-
trile; MeOH, methanol; min(s), minute(s); ml, milliliter;
mmol, millimol; NFjB, nuclear factor jB; nm, nanome-
ter; NMR, nuclear magnetic resonance; PBMC, periph-
4.2.6. Preparative LC–MS. Waters 2700 Autosampler,
Waters 600 Multisolvent Delivery System with prepara-
tive pump heads, Waters 600 Controller, 5000 ll Sample
loop. At-column dilution: Waters 600 Multisolvent
Delivery System with analytical pump heads; Waters
600 Controller; solvent, MeCN/MeOH 80: 20 (v/v); flow
rate, 0.20 or 1 ml/min. Column, Waters X-Terra RP18,
7 lm, 19 · 150 mm with X-Terra RP18 guard cartridge
7 lm, 19 · 10 mm, used at flow rate 20 ml/min. Eluent

J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
4585
A, H2O containing 0.1% (v/v) HCO2H or H2O contain-
MS for C41H42N6O6: 715.2 MH+.
ing 0.1% (v/v) NEt3; eluent B, MeCN. Different linear
gradients, individually adapted to sample. Injection vol-
ume, 0.5–5 ml, depending on sample. Make-up solvent,
MeOH/MeCN/H2O/HCO2H 80:15:4.95:0.05 (v/v/v/v).
Make-up pump, Waters Reagent Manager, flow rate
0.5 ml/min. Waters ZQ single quadrupole mass spec-
trometer with electrospray source. Positive or negative
ion mode scanning m/z 105–950 in 1 s; capillary, 4 kV;
cone voltage, 20 V; multiplier voltage, 600 V; probe
and desolvation gas temperature, 120 ꢁC and 250 ꢁC,
respectively. Waters Fraction Collector II with mass-
triggered fraction collection. Waters 2487 Dual k Absor-
bance Detector, set to 254 nm. Software, Waters Mass-
lynx V 4.0.
To a solution of 1.4 g (2 mmol) of Cbz-Trp-Trp-Phe-N-
methyl O-methylhydroxylamine in 30 ml dry THF, 3 ml
(3 mmol) of a 1 M solution of LAH was added dropwise
in an ice bath. The reaction was stirred for another 5 h at
rt and then quenched with 5% citric acid. The mixture
was extracted with ethyl acetate and washed with 10%
Na2CO3 and saturated NaCI. The organics were dried
over MgSO4, filtered, and concentrated in the vacuum.
The completion of the reaction was shown by TLC and
MS and the tripeptide aldehyde further reacted without
purification. (1 g 100% crude yield).
MS for C39H37N5O5 656.2 (MH+).
4.3. Compound 1
To a solution of 1.0 g (1.5 mmol) of the above-made tri-
peptide aldehyde, 0.34 g (3 mmol) semicarbazideÆHCl
and 0.53 ml (3 mmol) DIPEA were added and the mix-
ture was stirred for 48 h at rt. The solvent was removed
in the vacuum. The product became crystalline after
adding water and was collected by filtration. The prod-
uct was purified by preparative HPLC. By this method,
0.57 g LC–MS pure product was obtained (53%).
Was prepared as described in.4
4.4. Compound 2
[1-[1-[1-Formyl-2-(1H-indol-3-yl)-ethylcarbamoyl] -2-
(1H-indol-3-yl)-ethylcarbamoyl]-2-(1H-indol-3-yl)-ethyl]-
carbamic acid benzyl ester was prepared as described
in.4 To
a
solution of 1.66 g of this product
MS for C40H40N8 713.16 (MH+).
(2.39 mmol) in 100 ml TMOB, 0.6 g (2.39 mmol) amin-
ohydantoinÆHCl and 0.7 ml (2.629 mmol) DIPEA were
added and the mixture was stirred for 24 h at rt. The
solvent was removed in the vacuum and the residue
became crystalline with water. After filtration and dry-
ing in the vacuum, 1.8 g crude product was obtained.
The product was purified by flash chromatography on
silica gel using DCM/MeOH 95:5 and 0.9 g (47%)
pure product was obtained.
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm, TMS)
d 2.67–3.11 (6H, m); 4.30 (1H, m); 4.61 (2H, m); 4.93
(2H, m); 6.27 (2H, s); 6.93–7.33 (19H, m); 7.53–7.60
(2H, m); 8.00 (1H, d, J = 8.0 Hz); 8.17 (1H, d,
J = 7.7 Hz); 9.88 (1H, s); 10.76 (2H, m).
4.6. Compound 4
4.6.1. Method A. To a mixture of 5.68 g (22.8 mmol) (1-
benzyl-2-oxo-ethyl)-carbamicacid tert-butyl ester9 and
3.45 g (22.8 mmol) 1-aminohydantoin hydrochloride
(Aldrich) in 150 ml trimethylorthoformate, 3.4 ml DIE-
PA was added slowly under ice cooling. Ten microliters
of dry DMF was added and the reaction mixture was stir-
red for 72 h at rt. The solvent was removed in the vacuum
and the residue purified by flash chromatography on sil-
ica gel using DCM/MeOH 95:5. This procedure yielded
6.2 g (79.2% yield) pure [1-benzyl-2-(2,4-dioxo-imidazo-
lin-1-ylimino)-ethyl]-carbaminic acid tert-butyl ester.
MS for C44H41N9O6 792.00 (MH+).
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm, TMS)
d 2.84–3.20 (6H, m); 3.94 (2H, m); 4.32 (1H, m); 4.60
(1H, m); 4.67 (1H, m); 4.94 (2H, s); 6.76 (1H, d,
J = 5.5 Hz); 6.90–7.35 (19H, m); 7.60–7.52 (3H, m);
8.05 (1H, d, J = 8.1 Hz); 8.20 (1H, d, J = 7.8 Hz);
10.76 (3H, m); 11.11 (1H, s).
4.5. Compound 3
Boc-L-Phenylalanine-n-methyl-o-methylhydroxylamine
7.6 g was dissolved in 100 ml 2 M HCl in glacial acetic
acid and stirred at rt for 2 h. The solvent was removed
in the vacuum and the residue washed with ether sev-
eral times. The residue was dried in the HV to yield
7.0 g of an oily residue. An aliquot of 3.0 g
(12.25 mmol) was dissolved in 50 ml dry DMF. To this
solution, 6.4 g (12.25 mmol) Cbz-Trp-Trp-OH, 4.6 g
(12.25 mmol) HBTU, and 6.5 ml DIPEA (36.77 mmol)
were added and the reaction mixture was stirred for
12 h at rt. The reaction mixture was diluted with
200 ml ethyl acetate and successively washed with
20% bicarbonate and citric acid. The ethyl acetate
phase was dried with magnesium sulfate, the solvent re-
moved in the vacuum and dried in the HV to yield
7.5 g white foam (86%).
MS: MH+-Boc 246.87 95% (346.39 Mass).
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm) d 1.3
(9H, s); 2.6–3.0 (4H, m); 4.2 (2H, s), 4.3–4.4 (1H, m);
6.9–7.0 (H, m); 7.1–7.3 (5H, m); 11.0 (1H, s), 12.7 (1H, s).
1.0 g (3 mmol) of [1-benzyl-2-(2,4-dioxo-imidazolin-1-
ylimino)-ethyl]-carbaminic acid tert-butyl ester was dis-
solved in 50 ml of 2 M HCl in glacial acetic acid and stir-
red for 1 h at rt. The solvent was removed on the
rotavapor and the residue made crystalline with ether,
collected by filtration and dried in the HV to get 0.8 g
deprotected material.
This material was dissolved in 30 ml dry DMF and 1.48 g
(2.8 mmol) Cbz-Trp-Trp-OH and 1.0 g (2.8 mmol)

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J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
HBTU were added. Finally, 1.5 ml (8.4 mmol) DIPEA
was added and the solution was stirred at rt for 12 h.
The solution was diluted with 200 ml ethyl acetate and
washed with an 20% aqueous solution of sodium bicar-
bonate and citric acid. The ethyl acetate phase was dried
with magnesium sulfate and the solvent removed in the
vacuum. Purification by flash chromatography on silica
gel using DCM/MeOH as eluent yielded 1.8 g (85%) of
LC–MS pure material. The material showed two closely
moving spots on TLC eluting with DCM/MeOH (95:5)
and an estimated ratio of 3 to 1.
Phe as a starting material. The product obtained was
identical (NMR, LC–MS, and TLC) with Dia1 obtained
in the synthesis method A of compound 4.
4.8. Compound 6
One gram of compound 4 prepared by method B was
dissolved in 100 ml MeOH and 3 ml acetic acid. Pd/C
(10%) was added and the mixture was stirred under
hydrogen for 3 h. The suspension was filtered over a
short pad of Celite and washed with MeOH. The prod-
uct was concentrated in vacuum. After diethyl ether was
added, a precipitate was formed, filtered off and dried in
vacuum. Yield: 0.8 g. This material was used for the syn-
theses of cpd 6–8.
A batch of 100 mg of this material was submitted to pre-
parative TLC and 20 mg of the major spot (lower mov-
ing) was obtained and this material was termed Dia2.
Another 10 mg of the material moving faster was ob-
tained and the material was termed Dia1.
MS for C34H34N8O4 619.2 (MH+).
Dia 1: MS (C40H40N8O5) 753.2 (MH+).
The product from above Trp-Trp-Phe-Ahyd 0.33 g
(0.49 mmol) and D(+)-biotine 118 mg (0.49 mmol) was
dissolved in 3 ml, DIPEA 169 ll (0.97 mmol) and
HBTU 184 mg (0.49 mmol) were added and the reaction
was stirred for 16 h at rt. After water was added, a pre-
cipitate was formed and filtered off, washed with water
and dried. The product was further purified by prepara-
tive HPLC.
Dia 1: 1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm) d
0
3.03–2.72 (6H, m, Hb;b Phe, Trp1, Trp2); 4.10 (2H, s,
CH2 Hyd); 4.26 (1H, m, HaTrp2); 4.54 (1H, m, HaTrp1);
4.69 (1H, dddd, J = 9.1, 8.4, 5.9, 4.5 Hz, HaPhe); 4.92
(2H, m, CH2 Z); 6.95 (1H, d, J = 4.5 Hz, HHyd); 7.33–
6.92 (19H, m, Harom.(Trp1,
Z,Phe); Hd(Trp1,
;
Trp2)
Trp2,
NH Trp2); 7.53 (1H, d, J = 7.6 Hz, Hn2 Trp1); 7.56 (1H, d,
J = 8.0 Hz, Hn2 Trp2); 7.91 (1H, d, J = 8.3 Hz, NHTrp1);
8.34 (1H, d, J = 8.4 Hz, NHPhe); 10.73 (1H, d,
J = 4.5 Hz, He1Trp2); 10.74 (1H, d, J = 4.2 Hz, He1Trp1);
MS for C44H48N10O6S 845.3 (MH+).
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm, TMS)
d 1.16 (2H, m); 1.37 (4H, m); 2.00 (2H, m); 2.75–3.12
(6H, m); 3.99 (2H, s); 4.02 (1H, m); 4.24 (1H, m); 4.56
(3H, m); 6.32 (2H, m); 6.72 (1H, d, J = 5.0 Hz); 6.92–
7.32 (14H, m); 7.55 (2H, m); 7.89 (2H, m); 8.11 (1H,
d, J = 8.0 Hz); 10.73 (1H, d, J = 2.0 Hz); 10.80 (1H, d,
J = 1.8 Hz); 11.15 (1H, s).
11.73 (1H, s, NHHyd
)
Dia 2: MS (C40H40N8O5) 753.2 (MH+).
Dia 2: 1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm) d
0
3.14–2.78 (6H, m, Hb;b Phe, Trp1, Trp2); 3.98 (2H, s,
CH2 Hyd); 4.31 (1H, m, HaTrp2); 4.57 (1H, m, HaTrp1);
4.63 (1H, dddd, J = 8.2, 7.0, 6.7, 4.9 Hz, HaPhe); 4.93
(2H, m, CH2 Z); 6.70 (1H, d, J = 4.9 Hz, HHyd); 7.35–
6.93 (19H, m, Harom.(Trp1, Trp2, Z, Phe); Hd(Trp1, Trp2); NH
Trp2); 7.57 (1H, d, J = 7.9 Hz, Hn2 Trp1); 7.60 (1H, d,
J = 7.4 Hz, Hn2 Trp2); 8.03 (1H, d, J = 8.4 Hz, NHTrp1);
8.18 (1H, d, J = 8.2 Hz, NHPhe); 10.75 (1H, d,
J = 2.1 Hz, He1 Trp2); 10.79 (1H, d, J = 1.9 Hz, He1 Trp1);
11.14 (1H, s, NHHyd).
4.9. Compound 7
Carbonic acid 4-nitrophenyesterpyridin-4-methylester:
to a vigorously stirred mixture of 0.6 g (5.5 mmol) of
3-pyridinylmethanol and 1 g of potassium carbonate in
20 ml of DCM under cooling (ice bath) was added drop-
wise 1.4 g (7 mmol) of p-nitrophenyl chloroformate in
10 ml of DCM, and a suspension was stirred for 24 h
at rt. The precipitate was filtered off and DCM was
evaporated at reduced pressure. The residue was washed
with 100–150 ml of cold water and dried. Recrystalliza-
tion of the residual solid from benzene afforded 1 g
(73%) of desired compound. 1H NMR (d, ppm, CDCl3):
5.33 (s, 2H, CH2), 7.36–7.41 (m, 3H, Ar, HetAr), 7.79–
7.82 (m, 1H, HetAr), 8.27–8.31 (m, 2H, Ar), 8.65–8.67,
8.72–8.73 (m, 2H, HetAr).
4.6.2. Method B. To a solution of 0.5 g (0.76 mmol) of
the Cbz-tripeptide aldehyde as prepared in the synthesis
of compound 2, 0.115 g (0.76 mmol) AhydHCl and
0.133 ml (0.74 mmol) DIPEA were added and the mix-
ture was stirred for 48 h at rt. The solvent was removed
in the vacuum. The product became crystalline after
adding water and was collected by filtration. By this
method, 0.5 g product was obtained. Purification by
flash chromatography yielded 0.3 g (50%) pure mate-
rial. The product was identical (LC–MS, TLC, and
NMR) with the product Dia2 obtained in compound
4, method A.
The intermediate product from cpd 6 Trp-Trp-Phe-
Ahyd 300 mg (0.49 mmol) and carbonic acid 4-nitro-
phenyesterpyridin-4-methylester 133 mg (0.49 mmol)
was dissolved in 3 ml dry DMF, DIPEA 169 ll
(0.97 mmol) was added and the reaction stirred for
16 h at rt. After water was added, a precipitate was
formed and filtered off, washed with water and ether
and dried. The product was further purified by prepara-
tive HPLC.
4.7. Compound 5
Synthesis was completely identical to the synthesis of
compound 4 with the exception of the use of Boc-D-

J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
4587
MS for C41H39N9O6 754.23 (MH+).
4.12. NFjB reporter gene assay
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm, TMS) d
2.80–3.18 (6H, m); 3.99 (2H, s); 4.31 (1H, m); 4.56 (1H,
m); 4.63 (1H, m); 4.98 (2H, s); 6.71 (1H, d, J = 4.7 Hz);
6.92–7.40 (15H, m); 7.58 (2H, m); 8.05 (1H, d,
J = 8.0 Hz); 8.18 (1H, d, J = 8.1 Hz); 8.50 (2H, m);
10.75 (1H, s); 10.80 (1H, s); 11.15 (1H, s).
The NF-jB reporter gene assay was prepared with
A549-NF-jB-SEAP cell line (CCS cell culture service,
Hamburg, Germany) according to manufacturer’s
instructions. In short, A549 cells stable transfected with
pNF-jB-SEAP reporter gene plasmid were plated at
2 · 104/well and allowed to attach overnight. The cells
were subsequently incubated for 5 h with described com-
pounds at 100, 30, 10, 3, 1, 0.3, 0.1, and 0 lM and then
stimulated with 10 ng/ml TNF-a for 22 h. The superna-
tant of the cell was analyzed for SEAP activity using a
chemiluminescent SEAP reporter gene assay (Roche,
Mannheim, Germany), and a cell viability assay was
prepared using a CellTiter-BluTM Cell Viability Assay
(Promega, Mannheim, Germany). For each concentra-
tion of the compound, four replicates were measured.
4.10. Compound 8
The intermediate product from cpd 6 Trp-Trp-Phe-
Ahyd 300 mg (0.49 mmol) and 3-(4-hydroxyphenyl)-
propionic acid 81 mg (0.49 mmol) was dissolved in 3
ml dry DMF, DIPEA 169 ll (0.97 mmol) and HBTU
184 mg (0.49 mmol) were added and the reaction was
stirred for 16 h at rt. After water was added, a precipi-
tate was formed and filtered off, washed with water
and dried. The product was further purified by pHPLC
and 60 mg (30%) pure product was obtained.
4.13. T-cell proliferation assay
Cells (Mononuclear cells) were isolated from human
peripheral blood by AccuspinTM System-Histopaqueꢂ-
1077 (Sigma, Germany). After washing, the cells were di-
luted to approximately 100,000–200,000 cells/well in a
sterile 96-well flat bottomed MP (Corning, Netherlands).
T-lymphocytes were stimulated by the addition of 20 lg/
ml phytohemagglutinin-L (Roche, Germany). The incu-
bation at 37 ꢁC, 5% CO2, 90% humidity was made in the
presence of different concentrations of the compounds.
All cells were incubated for 48 h at 37 ꢁC, 5% CO2,
90% relative humidity over a concentration range of
0.4–50 lM compound solutions with a final volume per
well of 100 ll. After the initial incubation period,
10 lM BrdU (Roche, Germany) was added for an addi-
tional 4 h incubation. The culture medium employed was
RPMI 1640, which contained 10% heat inactivated fetal
bovine serum, 2mM L-glutamine, 100 U/ml penicillin G,
and 100 lg/ml streptamycin sulfate. After 48 h incuba-
tion, the cells were labeled by adding 10 ll BrdU-Solu-
tion (Roche, Germany) and reinsulated for further 4 h.
Following incubation, the media plus BrdU and drug
was removed and the cells were fixed and the DNA
was denatured in a single step using FixDenat (Roche,
Germany) The anti-BrdU-POD (Roche, Germany)
binds to the BrdU incorporated in newly synthesized, cel-
lular DNA. The immune complexes were detected by the
subsequent substrate reaction. The reaction product was
quantified by measuring the absorbance at the respective
wavelength using an ELISA reader. The EC50 values
were determined using a fitting function.
MS for C43H42N8O6 767.24 (MH+).
1H NMR (300.13 MHz, 305 K, DMSO-d6, ppm, TMS)
d 2.24 (2H, t, J = 7.4 Hz); 2.54 (2H, t, J = 7.4 Hz);
2.78–3.10 (6H, m); 4.00 (2H, s), 4.47–4.66 (3H, m);
6.61 (2H, m); 6.72 (1H, d, J = 4.9 Hz); 6.95–7.33 (16H,
m); 7.55 (2H, m); 7.92 (2H, m); 8.08 (1H, d,
J = 8.1 Hz); 9.06 (1H, s); 10.74 (1H, d, J = 2.0 Hz);
10.79 (1H, d, J = 2.0 Hz); 11.09 (1H, s).
4.11. Proteasome assay
Compounds were characterized by monitoring the
inhibitory effect on the chymotryptic activity of the hu-
man 20S proteasome (Biomol). The assay was per-
formed using a Tecan Ultra plate reader and the
fluorogenic substrates Suc-LLVY-AMC (Bachem) for
the chemotryptic and Z-LLE-ßBachem) for the pepti-
dyglutamyl peptide hydrolyzing and Bz-VGR-AMC
(Bachem) for the tryptic activity, respectively. In a
black 96-well polypropylene plate, 2 ll of compound,
dissolved in 100% DMSO, was mixed with 50 ll sub-
strate solution (25 mM Hepes, pH 7.5, 0.5 mM EDTA
and 75 lM Suc-LLVY-AMC or Z-LLE-ßNA or Bz-
VGR-AMC). Reaction was initiated by adding 150 ll
proteasome solution (1.3 lg 20S proteasome in
25 mM Hepes, pH 7.5, 0.5 mM EDTA, 0.033% (w/v)
SDS, pre-incubated for 10 min at room temperature).
Substrate hydrolysis was followed by fluorescence spec-
troscopy (excitation wavelength: 360 nm; emission
wavelength: 465 nm) for 20 min at 30 ꢁC and signals
were calculated as relative fluorescence units (RFU)
per minute. However, no signal could be obtained
when using Bz-VGR-AMC as a substrate. This sub-
strate might be incompatible with SDS used for protea-
some activation. For the determination of the IC50
values (concentration of inhibitor required for 50%
inhibition), eight inhibitor concentrations were applied.
Data points were recorded in triplicates on single mea-
surement day. To obtain dose–response curves and
IC50 values, data were fitted to a four parameter logis-
tic function using SigmaPlot.
4.14. Molecular modeling
The potential binding modes of the compounds were ex-
plored by refinement of docking poses generated by Pro-
Pose.10,11 The active site definition for docking and
˚
refinement contained subunits b5 and b6 of the 2.7 A
structure of the mammalian 20S proteasome (PDB ID
1IRU). Promising binding modes were selected
manually and refined by molecular dynamics simula-
tions (MD) utilizing Moloc.12 The refinement protocol
comprised an initial energy minimization, a short MD
run (ꢀ10 ps), and a final energy minimization step. Fig-

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J. Leban et al. / Bioorg. Med. Chem. 16 (2008) 4579–4588
6. Spaltenstein, A.; Leban, J.; Sherman, D.; Miller, J.;
Huang, J.; Viveros, U.; Sigafoos, J. Keystone Symposium
on Proteolytic Enzymes and Inhibitors in Biology, Key-
stone, CO, March 25–31, 1998.
ure 2 was prepared with the help of pymol 0.99 (open
source).13
7. Sharma, R. P.; Gore, M. G.; Akhtar, M. J. Chem. Soc.,
Chem. Commun. 1979, 20, 875–877.
Acknowledgments
8. Adkison, K. K.; Barrett, D. G.; Deaton, D. N.; Gampe, R.
T.; Hassel, A. M.; Long, S. T.; McFadyen, R. B.; Miller,
A. B.; Miller, L. R.; Payne, J. A.; Shewchuk, L. M.; Wells-
Knecht, K. J.; Willard, D. H.; Wright, L. L. Bioorg. Med.
Chem. Lett. 2006, 16, 978–983.
We thank Stefan Strobl and Daniel Vitt for helpful dis-
cussions and for support; Viola Trentinaglia, Marcel
Baierl, and Martin Kralik are acknowledged for techni-
cal assistance.
9. Schulze-Luehrmann, J.; Ghoshi, S. Immunity 2006, 25,
701–715.
References and notes
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ProPose: a docking engine based on a fully configurable
protein–ligand interaction model. J. Mol. Model. 2004, 10,
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1. Lee, D. H.; Goldberg, A. L. Trends Cell Biol. 1998, 8, 397–
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2. Groll, M.; Ditzel, l.; Lowe, J.; Stock, D.; Bochter, M.;
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A.; Viveros, O. H.; Wan, D. C. EP 1029 869 B1.
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R.; Viveros, O. H.; Sigafoos, J.; Crouch, R. Tetrahedron
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