Development of Novel Peptidomimetics Containing a Vinyl Sulfone Moiety as Proteasome Inhibitors

Article abstract of DOI:10.1002/cmdc.201100093

Proteasome inhibition is a topic of great interest in anticancer research. The proteolytic activity of this multicatalytic complex relies on three subunits, β1, β2 and β5, containing a caspase-like, a trypsin-like and a chymotrypsin-like active site, respectively. Several studies have demonstrated that, of the three activities, the chymotrypsin-like activity was the most necessary for cell viability and protein processing. Thus, most efforts towards the development of proteasome inhibitors have focused on the selective inhibition of the β5 subunit active site. Herein, we report the design and synthesis of a series of conformationally constrained tripeptidyl vinyl sulfones were determined to be good inhibitors of the chymotrypsin-like activity of proteasome, with K_I values in the sub-micromolar to micromolar range. These compounds were also tested against bovine pancreatic α-chymotrypsin and human cathepsinB and L, revealing a good selectivity for the target enzyme over these related enzymes. Molecular straitjackets! Conformationally constrained tripeptidyl vinyl sulfones were identified as good inhibitors of proteasome chymotrypsin-like activity, with K_I values in the sub-micromolar to micromolar range. Derivatives were also tested against bovine pancreatic α-chymotrypsin and human cathepsins B andL, revealing a good selectivity for the target enzyme.

Full text of DOI:10.1002/cmdc.201100093

MED
DOI: 10.1002/cmdc.201100093
Development of Novel Peptidomimetics Containing a Vinyl
Sulfone Moiety as Proteasome Inhibitors
Roberta Ettari,*[a] Cinzia Bonaccorso,[a] Nicola Micale,[a] Cornelia Heindl,[b]
Tanja Schirmeister,[b] Maria Luisa Calabrꢀ,[a] Silvana Grasso,[a] and Maria Zappalꢁ[a]
Proteasome inhibition is a topic of great interest in anticancer
research. The proteolytic activity of this multicatalytic complex
relies on three subunits, b1, b2 and b5, containing a caspase-
like, a trypsin-like and a chymotrypsin-like active site, respec-
tively. Several studies have demonstrated that, of the three ac-
tivities, the chymotrypsin-like activity was the most necessary
for cell viability and protein processing. Thus, most efforts to-
wards the development of proteasome inhibitors have focused
on the selective inhibition of the b5 subunit active site. Herein,
we report the design and synthesis of a series of conformation-
ally constrained tripeptidyl vinyl sulfones were determined to
be good inhibitors of the chymotrypsin-like activity of protea-
some, with KI values in the sub-micromolar to micromolar
range. These compounds were also tested against bovine pan-
creatic a-chymotrypsin and human cathepsin B and L, reveal-
ing a good selectivity for the target enzyme over these related
enzymes.
Introduction
The ubiquitin/proteasome system is the major nonlysosomal
proteolytic system for the degradation of abnormal or dam-
aged proteins, as well as proteins that are no longer required.[1]
Defects in the ubiquitin/proteasome pathway (UPP) can lead
to uncontrolled cell proliferation and tumor development. For
these reasons, UPP inhibition has become a new and signifi-
cant strategy for the drug development of cancer therapeutics.
In order to be recognized by the proteolytic system and tar-
geted for degradation, eukaryotic proteins are firstly marked
by addition of ubiquitin chains. Once tagged, these polyubi-
quitin proteins are degraded by 26S proteasome into ubiquitin
and short peptides, 3–25 amino acids in length, that are fur-
ther processed into recyclable amino acids.[2] The 26S protea-
some is composed of a 20S catalytic core capped by two 19S
regulatory complexes. The 19S caps contain a lid- and base-
like structure. The lid component is responsible for recognition
of polyubiquitinated substrates and deubiquitinating activity,
which allows recycling of ubiquitin moieties. The base compo-
nent consists of six ATPases required for the unfolding of sub-
strates and the opening of the narrow entry pore of the 20S
proteasome.[3]
tides with acidic residues in the P1 position; the b2 subunit
has a trypsin-like (T-L) activity and cleaves substrates with
basic residues at P1; the b5 subunit possesses a chymotrypsin-
like (ChT-L) activity and cleaves peptides with large hydropho-
bic residues at P1 (generally branched-chain amino acids).
Preferences of the various active subunits are determined
solely by the substrate binding pocket S1 formed by residue
45 of the catalytic subunit and by different residues of neigh-
boring b-subunits.[5] The S3 pocket is formed by residues of
the adjacent b-subunits and is thought to influence the bind-
ing efficiency of ligands and their substrate selectivity.[6] Thus,
manipulation of the corresponding P3 site of substrates opens
new perspectives for design and synthesis of specific inhibi-
tors. Crystal structure analysis also revealed that proteasome
does not possess S2 pocket specificity.[7] This feature provides
further advantages in the inhibitor design: bulky and space-de-
manding residues can be introduced in the P2 site, which en-
hance the specificity of the inhibitor and prevents nonspecific
inhibition of other proteases that have smaller binding pock-
ets.
Among the different enzymatic activities of proteasome, the
ChT-L activity has emerged as the biological function of great-
est interest and the focus of drug discovery efforts. Several
compounds, both natural and synthetic, have been found to
The 20S proteasome is a barrel-like structure with twofold
symmetry composed of four stacked rings of seven subunits
each, enclosing a central chamber. Seven different but related
a1–a7 subunits form the two outer rings, whereas the two
inner rings are composed of seven different b1–b7 subunits
that contain the proteolytic sites.[4] In eukaryotic proteasomes,
b1, b2 and b5 subunits contain proteolytically active sites,
whereas the other b-type subunits are inactive. Each subunit
uses the nucleophilic g-hydroxy group of the N-terminal Thr to
efficiently hydrolyze peptide bonds. They differ in substrate
specificity: the b1 subunit possesses a post-glutamyl peptide
hydrolyzing (PGPH) or caspase-like activity and cleaves pep-
[a] Dr. R. Ettari, Dr. C. Bonaccorso, Dr. N. Micale, Prof. M. L. Calabrꢀ,
Prof. S. Grasso, Prof. M. Zappalꢁ
Dipartimento Farmaco Chimico, University of Messina
Viale Annunziata, 98168, Messina (Italy)
Fax: (+39)090-355-613
E-mail: rettari@pharma.unime.it
[b] C. Heindl, Prof. Dr. T. Schirmeister
Institute of Pharmacy and Food Chemistry, University of Wuerzburg
Am Hubland, 97074 Wuerzburg (Germany)
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Peptidomimetic Proteasome Inhibitors
inhibit 20S proteasome and are quite selective for its ChT-L ac-
tivity.[5,8] Usually they are peptide-based compounds endowed
with an electrophilic warhead able to interact with the hydroxy
group of the active site Thr residue. Their classification is
based on the characteristics of their binding mode to the pro-
teolytic active sites, specificity, and reversibility of binding. Nat-
ural inhibitors include lactacystin and epoxomicin, both of
which bind to proteasome irreversibly. Synthetic inhibitors are
represented by peptide aldehydes, such as MG132, its vinyl sul-
fone (VS) analogue ZL3VS, and dipeptide boronate bortezomib
(velcade), the first proteasome inhibitor approved by US Food
and Drug Administration (FDA) for the treatment of hemato-
logical malignancies, such as multiple myeloma and mantle
cell lymphoma.[9]
confer optimal potency (e.g., the most potent vinyl sulfone,
AdaAhx3L3VS) and substrate selectivity (e.g., the most selective
vinyl sulfone, NL3VS).[10] So far, no remarkable subunit selectivi-
ty has been detected for this class of compounds.[11]
Beginning with this information, the main goal of the pres-
ent work was the design and synthesis of conformationally
constrained peptidomimetics with reduced peptidic character
compared with the lead vinyl sulfone, ZL3VS. This strategy has
been accomplished by bioisosteric replacement of the P3 Leu
residue with a pyridin-2-one core. This assembly should endow
the compounds with greater stability toward degradation by
enzymes, improved oral bioavailability, reduced conformational
freedom of peptides and thus subunit/substrate selectivity.
Further modifications to the new conformationally constrained
peptidomimetics entailed the P2 and P3 sites according to the
features mentioned above. Specifically, the Leu residue of the
P2 site was replaced by a Phe residue and the allyloxycarbonyl
(Alloc) group was used in place of the carbobenzyloxy (Cbz)
moiety. Finally, taking into account that a Leu residue at P1
site and the functional group downstream of the vinyl sulfone
are important structural requirements to direct the selectivity
towards ChT-L activity,[12] the pharmacophore portion of all
designed compounds (1–4) was based on these features.
Results and Discussion
Synthesis
The synthesis of carboxylic acids 10a–d with differently substi-
tuted N-terminal group, as mimics of the dipeptides Leu-Leu
and Leu-Phe, respectively, is outlined in Scheme 1. The re-
quired pyridone scaffold was synthesized from commercially
available 3-amino-2-pyridone 5, by protection of the amine
group as the carbamate (6a–b). The differently substituted P2
fragments were synthesized from the (S)-2-hydroxy-4-methyl-
pentanoic acid methyl ester 7a[13] or (S)-2-hydroxy-3-phenyl-
propionic acid methyl ester 7b,[13] obtained from the corre-
sponding commercially available acids, and activated into the
more reactive methanesulfonates 8a–b by reaction with mesyl
chloride in the presence of triethylamine. Intermediates 8a–b
were then condensed with the pyridone scaffolds 6a–b to
give the esters 9a–d, which were then hydrolized to afford the
desired carboxylic acids 10a–d.
Regarding vinyl sulfone series 1–2 bearing a Cbz group at
the N terminus (Scheme 2), the synthesis of tripeptides 12a–b
was realized by coupling the 3-methyl-1-vinyl-butylamine 11,
made according to the work by Albeck et al.,[14] and the car-
boxylic acids 10a–b in the presence of O-(7-azabenzotriazol-1-
yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU),
an efficient coupling reagent for sterically hindered substrates,
Peptide aldehydes were the first discovered inhibitors of 20S
proteasome and are still under active investigation. Generally,
aldehyde-containing inhibitors enter cells rapidly and reversibly
inhibit the enzyme by forming reversible covalent hemiacetal
bonds with the hydroxy group of the active site Thr. These
agents exhibit fast dissociation rates, are inactivated by oxida-
tion, and pumped out of the cells by the MDR carrier, affording
only short-lived inhibition. In addition, these compounds also
inhibit serine and cysteine proteases, and thus would not be
safe for use in patients. Vinyl sulfones are less reactive than al-
dehydes and bind to proteasome irreversibly by acting as Mi-
chael acceptors. However, they are not particularly specific for
proteasome as they also inhibit intracellular cysteine proteases,
such as cathepsins, and this reason may limit their application
in vivo.
In regard to vinyl sulfones, one of the most suitable recogni-
tion motif seems to be the tripeptide sequence Leu-Leu-Leu
bearing a long side chain at the N-terminal residue that could
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R. Ettari et al.
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16a–c was first attempted on
amine 11, which was found to
be unreactive under CMR condi-
tions, presumably because the
amine polar functionality is able
to bind the catalyst, removing it
from the catalytic cycle.[16] There-
fore, the vinyl sulfone moiety
was introduced by CMR on N-
trityl-3-methyl-1-vinyl-butylamine
(14) and the intermediates 15a–
c deprotected to give the de-
sired intermediates 16a–c.
Scheme 1. Reagents and conditions: a) benzyl or allyl chloroformate, NaHCO3/dioxane (7:3), 08C!RT, 12 h;
b) MsCl, Et3N, CH2Cl2, RT, 2 h, N2; c) NaH, DMF, 08C!RT, 1 h; then, 6a or 6b, RT, 12 h, N2; d) LiOH, MeOH/H2O (1:1),
RT, 5 h.
Biological activity
All synthesized compounds 1–4
were tested for their inhibitory capacity on purified 20S protea-
some isolated from human erythrocytes, using the appropriate
Scheme 2. Reagents and conditions: a) HATU, DIPEA, DMF, 1008C, MW, 2 h;
b) Hoveyda catalyst, CH2Cl2, 1008C, MW, 2 h.
and employing diisopropylethylamine (DIPEA) as a base. This
reaction, first attempted by stirring the mixture at room tem-
perature overnight, was optimized performing the reaction
under microwave irradiation. This strategy provided the de-
sired coupling products 12a–b in shorter reaction times, with
higher yields, and with minimal by-product formation, when
compared to standard conditions. The introduction of the re-
quired vinyl sulfone warhead on the terminal olefins 13a–b
was achieved by cross-metathesis reaction (CMR), an easy and
inexpensive catalytic carbon-bond-forming reactions, using
Hoveyda–Grubbs second generation catalyst,[15] a phosphine-
free nitrogen-containing heterocyclic carbene ruthenium com-
plex that is easy to handle and able to tolerate a wide range of
functional groups, with minimal substrate protection neces-
sary.
Scheme 3. Reagents and conditions: a) Hoveyda catalyst, CH2Cl2, 1008C, MW,
2 h; b) 6n HCl/acetone, D, 5 h; c) HATU, DIPEA, DMF, 1008C, MW, 2 h.
fluorogenic substrate for each of the proteolytic activities (Suc-
Leu-Leu-Val-Tyr-AMC for ChT-L; Boc-Leu-Arg-Arg-AMC for T-L;
Z-Leu-Leu-Glu-AMC for PGPH). First, compounds underwent a
preliminary screening for ChT-L activity at 20 mm using an
equivalent volume of dimethyl sulfoxide (DMSO) as a negative
control. The screening showed that all compounds, with the
exception of 4a, inhibited more than 40% of the enzyme ac-
tivity. Thus, continuous assays (progress curve method, from 0
to 100 mm) were performed to determine the first-order rate
constants of inhibition kinac (minꢀ1), the dissociation constants
KI (mm), and the second-order rate constants of inhibition k2nd
(Mꢀ1 minꢀ1), as k2nd =kinac/KI (Table 1).
Regarding the vinyl sulfone series 3–4, bearing an Alloc
group at the N terminus, the introduction of the pharma-
cophore portion was realized at a different step in the synthet-
ic route, by coupling intermediates 10c–d with the amines
16a–c, already functionalized by olefin metathesis, under mi-
crowaves irradiation (Scheme 3). This strategy was selected to
avoid a predictable two-directional CMR at both of the termi-
nal olefins of the final tripeptides. The synthesis of amines
All compounds exhibit good affinity for the proteasome b5
subunit with KI values in the sub-micromolar to micromolar
range. Generally, compounds with an Alloc group as the side
chain at the P3 site exhibit better affinity and inhibitory poten-
cy with respect to their Cbz group-containing derivatives, how-
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Peptidomimetic Proteasome Inhibitors
Table 1. Inhibition of chymotrypsin-like proteasome activity by vinyl sul-
fones 1–4.
Table 2. Inhibition of the post-glutamyl peptide hydrolyzing proteasome
activity by selected vinyl sulfones.
[a]
[a]
[a]
[a]
Compd
K2nd
kinac
KI[a]
[mm]
Compd
K2nd
kinac
KI[a]
[mm]
[mꢀ1 minꢀ1
]
[minꢀ1
]
[mꢀ1 minꢀ1
]
[minꢀ1
]
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
47000
61000
13000
177000
154000
66000
53000
102000
77000
0.044
0.056
0.058
0.99
0.051
0.079
0.049
0.062
0.073
0.94
0.91
4.5
5.6
0.33
1.2
0.93
0.61
0.95
1a
1b
1c
2a
2c
31000
16000
1900
6600
5000
3200
300[c]
0.095
0.083
0.044
[b]
3.1
5.2
23
[b]
0.032
0.047
6.9
15
3c
ZL3VS
[a] Values represent the mean of three independent determinations; vari-
ability is less than 10%. [b] Plot of kobs versus [I] is restricted to the linear
range, thus the individual constants kinac and KI could not be determined.
[c] Data reported in Reference [17] as Kobs/[I] and converted into
mꢀ1 minꢀ1 for comparison purposes only.
30% inhibition at 20 mM
105000
47000
1740[b]
0.039
0.090
0.37
1.93
ZL3VS
[a] Values represent the mean of three independent determinations; vari-
ability is less than 10%. [b] Data reported in Reference [17] as Kobs/[I] and
converted into mꢀ1 minꢀ1 for comparison purposes only.
(k2nd =6,600mꢀ1 minꢀ1). None of these peptide-like vinyl sul-
fones exhibited any inhibition in the T-L activity assay at
20 mm.
The selectivity of these compounds towards the target
enzyme was verified by testing them against bovine pancreatic
a-chymotrypsin and human cathepsins B and L at 20 mm. The
preliminary screening against a-chymotrypsin showed no sig-
nificant inhibition with values ranging from 4.2% (3a) to 35%
(3c). Regarding the cathepsins, since the vinyl sulfone warhead
is expected to be the most reactive toward cysteine thiolates,
the degree of inhibition is slightly higher, ranging from 27%
(2a) to 38% (1b) and from 21% (1c) to 37% (1b) for cathep-
sins B and L, respectively.
ever, this is only true of compounds with a Leu residue at the
P2 site, for example, 3b (KI =0.61 mm; k2nd =102000mꢀ1 minꢀ1
)
and 3c (KI =0.95 mm; k2nd =77000mꢀ1 minꢀ1) versus 1b (KI =
0.91 mm; k2nd =61000mꢀ1 minꢀ1) and 1c (KI =4.5 mm; k2nd
=
13000mꢀ1 minꢀ1). Conversely, compounds with a Phe residue
at the P2 site show higher inhibitory potency compared to
compounds with a Cbz group at the P3 site, that is, 2a (k2nd
177000mꢀ1 minꢀ1), 2b (k2nd =154000mꢀ1 minꢀ1) and 2c (k2nd
66000mꢀ1 minꢀ1) versus 1a (k2nd =47000mꢀ1 minꢀ1), 1b (k2nd
=
=
=
61000mꢀ1 minꢀ1) and 1c (k2nd =13000mꢀ1 minꢀ1). Modifications
to the pharmacophore fragment follow a common trend with
higher affinity and inhibitory potency for ethyl-vinyl sulfones
with respect to the methyl and phenyl analogues, for example,
Conclusions
We designed and synthesized novel constrained peptido-
mimetics bearing a vinyl sulfone (VS) warhead (compounds 1–
4), which proved to be potent irreversible inhibitors of protea-
some chymotrypsin-like activity. Peptidomimetic derivatives
2b, 3b and 4b, which possess high inhibitory potencies cou-
pled with a good target selectivities, could be considered as
lead compounds for further development in the design of
novel antitumor agents.
3b (KI =0.61 mm,
k ) versus 3a (KI =
2nd =102000mꢀ1 minꢀ1
0.93 mm, k2nd =53000mꢀ1 minꢀ1). Ethyl derivative 2b represents
an exception to this trend as it is less potent than its methyl
analogue 2a but possesses a higher affinity, that is, 2b (k2nd
154000mꢀ1 minꢀ1
KI =0.33 mm) versus 2a (k2nd =177000
mꢀ1 minꢀ1, KI =5.6).
=
,
Noteworthy, the most potent compound of this series (2a,
2nd =177000mꢀ1 minꢀ1) also has the weakest affinity towards
k
the enzyme (KI =5.6 mm), but the highest first-order rate con-
stant of inhibition (ki =0.99 minꢀ1). Interestingly, all the synthe-
sized vinyl sulfones 1–4, with the exception of compound 4a,
turned out to be more potent than the elected lead ZL3VS,[17]
validating our rational drug design strategy.
Experimental Section
Chemistry
All reagents and solvents were obtained from commercial suppliers
and were used without further purification. HPLC grade acetonitrile
was obtained from Merck. Elemental analyses were carried out on
a C. Erba 1106 elemental analyzer for C, H and N, and the results
were within ꢁ0.4% of the theoretical values. Reactions under mi-
crowave (MW) irradiation were performed on a CEM Discover ap-
paratus. Merck silica gel 60 F254 plates were used for analytical TLC;
flash column chromatography was performed on Merck silica gel
(200–400 mesh) or conducted using prepacked cartridges on a MP-
LC Buchi system. Semipreparative HPLC was performed on a
Waters 1525 binary HPLC pump system equipped with a Rheodyne
model 3725i injector (2 mL sample loop). Reverse-phase chroma-
The main issue of these newly synthesized vinyl sulfones is
their subunit selectivity. As shown in Table 2, six out of twelve
compounds did not pass the preliminary screening for protea-
some PGPH inhibitory activity, using the same selection criteria
as for the ChT-L activity. Compounds considered for continu-
ous assays (1a–c, 2a, 2c, 3c) displayed moderate inhibition of
proteasome b1 subunit, with KI values in the micromolar range
and second-order rate constants one or two order of magni-
tude less than those detected for the b5 subunit, that is, 2c
and 3c (k2nd =5000 and 3200mꢀ1 minꢀ1, respectively) or 2a
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R. Ettari et al.
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tography was carried out on a Merck Chromolith SemiPrep RP-18e
column (100ꢃ10 mm i.d.), at a temperature of 208C, with a mobile
phase of water/acetonitrile (90:10) and a flow rate of 5 mLminꢀ1. A
Waters 2489 UV/Vis dual-wavelength absorbance detector was
used, and chromatograms were processed using Waters HPLC
Breeze 2 software. The detector wavelength was set at 266 and
310 nm. All solutions employed in the analysis were filtered
through 25 mmꢃ0.45 mm polypropylene (PP) membrane mem-
3H, OCH3), 5.31 (m, 1H, CHOSO2), 7.38–7.49 ppm (m, 5H, ArH);
Anal. calcd (%) for C11H14O5S: C 51.15, H 5.46, found: C 51.33, H
5.28.
(S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-4-
methyl-pentanoic acid methyl ester (9a): A suspension of NaH
(118 mg, 2.95 mmol) in DMF at 08C under N2 was treated with 6a
(690 mg, 2.83 mmol) and stirred for 1 h. Methanesulfonate 8a was
then added (552 mg, 2.46 mmol) and the reaction mixture was
stirred for a further 12 h at RT. The reaction was quenched with sa-
turated aq NaHCO3 and extracted with EtOAc. The organic phase
was dried (Na2SO4), filtered and concentrated in vacuo. The crude
was purified by flash column chromatography (light petroleum/
EtOAc, 6:4) to give the title compound 9a as a pale yellow oil
(675 mg, 64%): Rf =0.80 (light petroleum/EtOAc, 6:4); 1H NMR
(300 MHz, CDCl3): d=0.92 (d, J=6.6 Hz, 6H, CH(CH3)2), 0.98 (d, J=
6.6 Hz, 6H, CH(CH3)2), 1.77–2.04 (m, 3H, CH2CH(CH3)2), 3.74 (s, 3H,
OCH3), 5.19 (s, 2H, CH2O), 5.64 (m, 1H, NCHCO), 6.30 (t, J=7.3 Hz,
ArH), 6.99 (dd, J=1.6, 7.3 Hz, 1H, ArH), 7.34–7.39 (m, 5H, ArH), 7.85
(s, 1H, NH), 8.02 ppm (d, J=7.3 Hz, ArH); 13C NMR (75 MHz, CDCl3):
d=21.7, 23.2, 24.9, 39.7, 52.9, 67.2, 76.7, 106.89, 120.2, 127.3, 127.4,
128.5, 128.6, 129.5, 136.5, 153.6; 157.5, 171.0 ppm; Anal. calcd (%)
for C20H24N2O5: C 64.50, H 6.50, N 7.52, found: C 64.38, H 6.41, N
7.76.
1
brane. H and 13C NMR spectra were recorded on a Varian Gemini
1
300 spectrometer at 300 K and 300 and 75 MHz, respectively. H
chemical shifts (d) are reported in parts per million (ppm) relative
to the internal tetramethylsilane (TMS) and coupling constants (J)
are reported in hertz (Hz). 13C NMR chemical shifts (d) are reported
relative to the residual solvent peak (CHCl3, central peak, d=
77.0 ppm).
(2-Oxo-1,2-dihydropyridin-3-yl)-carbamic acid benzyl ester (6a):
A solution of 5 (900 mg, 8.17 mmol) in saturated aq NaHCO3/diox-
ane (7:3, 50 mL) was treated dropwise with benzyl chloroformate
(1.84 mL, 12.26 mmol) at 08C. The reaction mixture was then
stirred at RT for 12 h, diluted with EtOAc, and washed with HCl
(3n). The organic phase was dried (Na2SO4), filtered and concen-
trated in vacuo. The crude was by flash column chromatography
(light petroleum/EtOAc, 2:8) to afford 6a as a white solid (1.65 g,
90%): Rf =0.39 (light petroleum/EtOAc, 2:8); mp: 175–1788C;
1H NMR (300 MHz, CDCl3): d=5.21 (s, 2H, CH2Bz), 6.32 (t, J=
7.0 Hz, 1H, ArH), 6.98 (dd, J=7.0, 1.6 Hz, 1H, ArH), 7.26–7.40 (m,
5H, ArH), 7.79 (s, 1H, NH), 8.11 ppm (d, J=7.0 Hz, 1H, ArH);
13C NMR (75 MHz, CDCl3): d=67.5, 108.1, 122.7, 126.3, 128.6, 129.0,
129.7, 136.3, 153.7, 159.2 ppm; Anal. calcd (%) for C13H12N2O3: C
63.93, H 4.95, N 11.47, found: C 63.85, H 4.83, N 11.64.
(S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-3-
phenyl-propionic acid methyl ester (9b): Compound 6a (730 mg,
3.0 mmol) was reacted with methanesulfonate 8b (646 mg,
2.5 mmol) according to the same procedure described for 9a to
give the title compound 9b as a pale yellow oil (670 mg, 55%,):
Rf =0.70 (light petroleum/EtOAc, 6:4); 1H NMR (300 MHz, CDCl3):
d=3.25–3.53 (m, 2H, CH2Ph), 3.93 (s, 3H, OCH3), 5.36 (s, 2H, CH2O),
5.51 (m, 1H, NCHCO), 6.25 (t, J=7.0 Hz, ArH), 6.87 (d, J=7.0 Hz,
ArH), 7.22–7.52 (m, 10H, ArH), 8.01 (s, 1H, NH), 8.12 ppm (d, J=
7.0 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=39.7, 52.8, 78.9, 67.2,
76.8, 106.8, 120.2, 127.3, 127.4, 128.5, 128.6, 129.3, 129.6, 129.9,
130.5, 136.4, 153.6, 157.4, 171.0 ppm; Anal. calcd (%) for
C23H22N2O5: C 67.97, H 5.46, N 6.89, found: C 68.29, H 5.31, N 6.75.
(2-Oxo-1,2-dihydropyridin-3-yl)-carbamic acid allyl ester (6b):
Compound 5 (600 mg, 5.45 mmol) was reacted with allyl chlorofor-
mate (0.90 mL, 8.17 mmol) according to the same procedure de-
scribed for 6a. The title compound was obtained after purification
by flash column chromatography (light petroleum/EtOAc, 2:8) as
pale pink solid (845 mg, 80%): Rf =0.35 (light petroleum/EtOAc,
2:8); mp: 140–1438C; 1H NMR (300 MHz, CDCl3): d=4.84 (d, J=
5.5 Hz, 2H, CH2O), 5.43 (d, J=10.2 Hz, 2H, CH2 =), 5.54 (d, J=
10.2 Hz, 2H, CH2 =), 6.12 (m, 1H, CH), 6.51 (t, J=7.0 Hz, 1H, ArH),
7.18 (d, J=7.0 Hz, 1H, ArH), 7.92 (s, 1H, NH), 8.28 ppm (d, J=
7.0 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=66.3, 108.1, 118.6, 122.9,
126.4, 129.6, 132.6, 153.6, 159.2 ppm; Anal. calcd (%) for C9H10N2O3:
C 55.67, H 5.19, N 14.43, found: C 55.81, H 5.03, N 14.31.
(S)-2-(3-Allyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-4-methyl-
pentanoic acid methyl ester (9c): Compound 6b (600 mg,
3.0 mmol) was reacted with methanesulfonate 8a (560 mg,
2.5 mmol) according to the same synthetic procedure described
for 9a to give the title compound 9c as a pale yellow oil (550 mg,
60%): Rf =0.76 (light petroleum/EtOAc, 6:4); 1H NMR (300 MHz,
CDCl3): d=0.92 (d, J=6.6 Hz, CH(CH3)2), 0.98 (d, J=6.6 Hz,
CH(CH3)2), 1.88–2.04 (m, 3H, CH2CH), 3.74 (s, 3H, OCH3), 4.66 (d, J=
5.5 Hz, 2H, CH2O), 5.25 (d, J=10.4 Hz, 1H, CH2 =), 5.36 (d, J=
17.3 Hz, 1H, CH2 =), 5.69 (m, 1H, NCHCO), 5.95 (m, 1H, =CH), 6.30
(t, J=7.1 Hz, ArH), 6.99 (d, J=7.1 Hz, 1H, ArH), 7.85 (bs, 1H, NH),
8.02 ppm (d, J=7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=21.8,
23.3, 25.0, 40.0, 53.4, 56.8, 66.2, 107.1, 118.4, 120.2, 126.9, 129.6,
133.5, 153.4, 157.3, 171.1 ppm; Anal. calcd (%) for C16H22N2O5: C
59.62, H 6.88, N 8.69, found: C 59.39, H 6.97, N 8.81.
(S)-2-Methanesulfonyloxy-4-methyl-pentanoic acid methyl ester
(8a): A solution of 7a (360 mg, 2.46 mmol) in dry CH2Cl2 (40 mL)
was treated with Et3N (622 mg, 6.15 mmol) and methanesulfonyl
chloride (561 mg, 4.9 mmol). The resulting solution was stirred,
under N2, at RT for 2 h. The reaction mixture was washed with
water, dried (Na2SO4), filtered and concentrated in vacuo to give
8a as a pale yellow oil (546 mg, 99%): Rf =0.68 (light petroleum/
EtOAc, 4:6, detected by KMnO4); 1H NMR (300 MHz, CDCl3): d=
0.99–1.00 (m, 6H, CH(CH3)2), 1.64–1.80 (m, 2H, CH2), 1.85 (m, 1H,
CH(CH3)2), 3.15 (s, 3H, CH3SO2), 3.80 (s, 3H, OCH3), 5.07 ppm (m,
1H, CHOSO2); Anal. calcd (%) for C8H16O5S: C 42.84, H 7.19, found:
C 42.99, H 7.06.
(S)-2-(3-Allyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-3-phenyl-
propionic acid methyl ester (9d): Compound 6b (442 mg,
2.28 mmol) was reacted with methanesulfonate 8b (491 mg,
1.9 mmol) according to the same synthetic procedure described
for 9a to give the title compound 9d as a pale yellow oil (552 mg,
68%): Rf =0.66 (light petroleum/EtOAc, 6:4); 1H NMR (300 MHz,
CDCl3): d=3.19 (dd, J=6.9, 13.7 Hz, 1H, CH2Ar), 3.38 (dd, J=4.4,
13.7 Hz, 1H, CH2Ar), 3.88 (s, 3H, OCH3), 4.80 (d, J=5.5 Hz, 2H,
CH2O), 5.40 (d, J=10.2 Hz, 1H, CH2 =), 5.51 (d, J=17.0 Hz, 1H,
CH2 =), 6.12 (m, 1H, =CH), 6.35 (t, J=7.4 Hz, 1H, ArH), 6.91 (d, J=
(S)-2-Methanesulfonyloxy-3-phenyl-propionic acid methyl ester
(8b): Starting from 7b (350 mg, 1.9 mmol) and following the same
procedure described for 8a gave the title compound 8b as a pale
yellow oil (486 mg, 99%): Rf =0.73 (light petroleum/EtOAc, 4:6);
1H NMR (300 MHz, CDCl3): d=2.87 (s, 3H, CH3SO2), 3.26 (dd, J=
8.8, 14.3 Hz, 1H, CH2) 3.43 (dd, J=4.1, 14.3 Hz, 1H, CH2), 3.90 (s,
1232
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ChemMedChem 2011, 6, 1228 – 1237

Peptidomimetic Proteasome Inhibitors
7.4 Hz, 1H, ArH), 7.22–7.41 (m, 5H, ArH), 7.95 (s, 1H, NH), 8.18 ppm
(d, J=7.4 Hz, 1H, ArH); 13C NMR (75 MHz, CDCl3): d=36.3, 53.2,
62.6, 66.2, 106.7, 118.4, 120.4, 127.5, 127.9, 128.5, 129.3, 129.6,
129.9, 132.7, 135.3, 136.3, 153.6, 157.2, 169.8 ppm; Anal. calcd (%)
for C19H20N2O5: C 64.04, H 5.66, N 7.86, found: C 64.25, H 5.71, N
7.62.
mixture was stirred under the same conditions for an additional
1 h. The solution was diluted with CH2Cl2 and washed with H2O.
The organic phase was dried (Na2SO4), filtered and evaporated in
vacuo. The crude was purified by flash column chromatography
(light petroleum/EtOAc, 8:2) to give compound 12a as a pale
yellow oil (150 mg, 60%): Rf =0.78 (light petroleum/EtOAc, 8:2);
1H NMR (300 MHz, CDCl3): d=0.91–0.97 (m, 12H, CH(CH3)2), 1.40–
1.64 (m, 4H, CH2CH), 1.79–2.12 (m, 2H, CH2CH), 4.24 (m, 1H,
NHCH), 4.81–4.99 (m, 2H, CH=CH2), 5.21 (s, 2H, CH2O), 5.65 (m, 1H,
NCHCO), 5.83 (m, 1H, CH=CH2), 6.30 (t, 1H, J=7.1 Hz, ArH), 6.99 (d,
1H, J=7.1 Hz, ArH), 7.34–7.39 (m, 5H), 7.85 (s, 1H, NH), 8.02 ppm
(d, 1H, J=7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=22.5, 23.3,
24.8, 25.5, 40.9, 45.2, 52.4, 64.2, 67.0, 109.5, 111.7, 117.3, 127.1,
127.7, 129.0, 131.8, 134.2, 136.2, 137.7, 153.3, 158.4, 171.0 ppm;
Anal. calcd (%) for C26H35N3O4: C 68.85, H 7.78, N 9.26, found: C
68.99, H 7.91, N 9.02.
(S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-4-
methyl-pentanoic acid (10a): A solution of ester 9a (675 mg,
1.8 mmol) in MeOH/H2O (1:1, 50 mL) at 08C was treated with LiOH
(86 mg, 3.6 mmol) and stirred at 208C for 5 h. The reaction was
concentrated in vacuo, and the residue was treated with 10% citric
acid (until pH 4) and extracted with EtOAc. The organic phase was
dried (Na2SO4), filtered and concentrated to give acid 10a as a
brown oil (612 mg, 95%): 1H NMR (300 MHz, CDCl3): d=0.91 (d,
J=6.6 Hz, CH(CH3)2), 0.98 (d, J=6.6 Hz, CH(CH3)2), 1.87–2.03 (m, 3H,
CH2CH), 5.20 (s, 2H, CH2O), 5.63 (m, 1H, NCHCO), 6.29 (t, J=7.2 Hz,
ArH), 6.98 (d, J=7.2 Hz, 1H, ArH), 7.35–7.39 (m, 5H, ArH), 7.84 (s,
1H, NH), 8.01 (d, J=7.2 Hz, ArH), 9.25 ppm (bs, 1H, COOH); Anal.
calcd (%) for C19H22N2O5: C 63.68, H 6.19, N 7.82, found: C 63.79, H
6.31, N 7.62.
Benzyl-1-((S)-1-((S)-5-methylhex-1-en-3-ylamino)-1-oxo-3-phenyl-
propan-2-yl)-2-oxo-1,2-dihydropyridin-3-ylcarbamate (12b): Acid
10b (278 mg, 0.71 mmol) was reacted with amine 11 (120 mg,
1.06 mmol) according to the same synthetic procedure described
for compound 12a to give the title compound 12b as a pale
yellow oil (225 mg, 65%): Rf =0.73 (light petroleum/EtOAc, 8:2);
1H NMR (300 MHz, CDCl3): d=0.93 (d, 6H, J=6.5 Hz, CH(CH3)2),
1.22–1.50 (m, 3H, CH2CH), 3.24 (m, 1H, CH2Ph), 3.67 (m, 1H,
CH2Ph), 4.51 (m, 1H, NHCH), 4.99–5.17 (m, 3H, CH=CH2 and
NCHCO), 5.35 (s, 2H, CH2O), 5.73 (m, 1H, CH=CH2), 6.35 (s, 1H, NH),
6.48 (t, J=7.1 Hz, ArH), 7.30–7.68 (m, 11H, ArH), 7.92 (bs, 1H, NH),
8.21 ppm (d, 1H, J=7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=
23.3, 24.8, 35.3, 45.2, 52.4, 66.9, 69.1, 109.4, 111.7, 117.3, 125.9,
127.1, 127.6, 127.7, 128.6, 129.0, 131.8, 134.3, 134.1, 136.2, 137.7,
153.3, 158.3, 171.1 ppm; Anal. calcd (%) for C29H33N3O4: C 71.44, H
6.82, N 8.62, found: C 71.71, H 6.67, N 8.56.
(S)-2-(3-Benzyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-3-
phenyl-propionic acid (10b): Ester 9b (500 mg, 1.23 mmol) was
reacted with LiOH (59 mg, 2.46 mmol) according to the same syn-
thetic procedure described for compound 10a to give the title
1
compound 10b as a brown oil (470 mg, 97%): H NMR (300 MHz,
CDCl3): d=3.23–3.50 (m, 2H, CH2Ph), 5.34 (s, 2H, CH2O), 5.50 (m,
1H, NCHCO), 6.23 (t, J=7.3 Hz, ArH), 6.85 (d, J=7.3 Hz, ArH), 7.20–
7.51 (m, 10H), 8.00 (s, 1H, NH), 8.11 (d, J=7.3 Hz, ArH), 9.29 ppm
(bs, 1H, COOH); Anal. calcd (%) for C22H20N2O5: C 67.34, H 5.14, N
7.14, found: C 67.53, H 5.05, N 7.01.
(S)-2-(3-Allyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-4-methyl-
pentanoic acid (10c): Ester 9c (550 mg, 1.55 mmol) was reacted
with LiOH (74 mg, 3.10 mmol) according to the same synthetic pro-
cedure described for compound 10a to give the title compound
Benzyl-1-((S)-4-methyl-1-((S,E)-5-methyl-1-(methylsulfonyl)hex-1-
en-3-ylamino)-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (1a): A solution of 12a (60 mg, 0.132 mmol) in dry
CH2Cl2 (5 mL) was treated with methyl vinyl sulfone 13a (140 mg,
1.32 mmol) and Hoveyda–Grubbs second generation catalyst [(1,3-
bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-dichloro-(o-isopro-
poxyphenylmethylene)ruthenium] (16 mg, 0.026 mmol). The result-
ing mixture was heated under microwave irradiation at 1008C for
2 h, the concentrated in vacuo. Purification first by flash column
chromatography (light petroleum/EtOAc, 7:3) and subsequently by
semipreparative RP-HPLC gave the title compound 1a as a white
1
10c as a brown oil (468 mg, 90%): H NMR (300 MHz, CDCl3): d=
0.84 (d, J=4.9 Hz, CH(CH3)2), 0.86 (d, J=4.9 Hz, CH(CH3)2), 1.84–2.07
(m, 3H, CH2CH), 4.59 (d, J=5.2 Hz, 2H, CH2O), 5.16 (d, J=10.4 Hz,
1H, CH2 =), 5.28 (d, J=17.0 Hz, 1H, CH2 =), 5.56 (m, 1H, NCHCO),
5.86 (m, 1H, =CH), 6.28 (t, J=7.3 Hz, ArH), 6.94 (dd, J=1.6, 7.3 Hz,
1H, ArH), 7.84 (bs, 1H, NH), 8.00 (d, J=7.3 Hz, ArH) 9.29 ppm (bs,
1H, COOH); Anal. calcd (%) for C15H20N2O5: C 58.43, H 6.54, N 9.09,
found: C 58.31, H 6.66, N 9.16.
1
solid (14 mg, 20%): Rf =0.65 (light petroleum/EtOAc, 7:3); H NMR
(S)-2-(3-Allyloxycarbonylamino-2-oxo-2H-pyridin-1-yl)-3-phenyl-
propionic acid (10d): Ester 9d (552 mg, 1.87 mmol) was reacted
with LiOH (90 mg, 3.74 mmol) according to the same synthetic pro-
cedure described for compound 10a to give the title compound
(300 MHz, CDCl3): d=0.86–0.90 (m, 12H, CH(CH3)2), 1.68–2.08 (m,
6H, CH2CH), 3.01 (s, 3H, CH3), 4.32 (m, 1H, NCH), 4.63 (m, 1H,
NCHCO), 5.21 (s, 2H, CH2O), 6.30 (t, 1H, J=7.3 Hz, ArH), 6.94 (d,
1H, J=15.2 Hz, =CHSO2), 6.96 (d, 1H, J=7.3 Hz, ArH), 7.12 (dd, 1H,
J=4.2, 15.2 Hz, CH=), 7.34–7.39 (m, 5H), 7.83 (s, 1H, NH),
8.04 ppm (d, 1H, J=7.3 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=
22.4, 22.5, 23.2, 23.4, 39.1, 43.2, 45.3, 47.1, 55.8, 66.1, 109.5, 111.8,
127.3, 129.0, 129.4, 131.8, 134.5, 141.1, 141.2, 148.5, 158.4,
171.2 ppm; Anal. calcd (%) for C27H37N3O6S: C 61.00, H 7.01, N 7.90,
found: C 61.17, H 7.12, N 7.79.
1
10d as a brown oil (499 mg, 94%): H NMR (300 MHz, CDCl3): d=
3.20 (dd, J=6.9, 13.7 Hz, 1H, CH2Ph), 3.35 (dd, J=4.4, 13.7 Hz, 1H,
CH2Ph), 4.81 (d, J=5.5 Hz, 2H, CH2O), 5.38 (d, J=10.2 Hz, 1H,
CH2 =), 5.53 (d, J=17.0 Hz, 1H, CH2 =), 6.09 (m, 1H, =CH), 6.30 (t,
J=7.4 Hz, 1H, ArH), 6.92 (d, J=7.4 Hz, 1H, ArH), 7.20–7.38 (m, 5H,
ArH), 7.93 (s, 1H, NH), 8.15 (d, J=7.4 Hz, 1H, ArH), 9.23 ppm (bs,
1H, COOH); Anal. calcd (%) for C18H18N2O5: C 63.15, H 5.30, N 8.18,
found: C 63.31, H 5.15, N 8.07.
Benzyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-yl-
amino)-4-methyl-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-
ylcarbamate (1b): Starting from olefin 12a (100 mg, 0.22 mmol)
and ethyl vinyl sulfone 13b (264 mg, 2.2 mmol) and following the
same procedure described for 1a gave the title compound 1b as a
white solid (26 mg, 22%): Rf =0.70 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.89–0.94 (m, 12H, CH(CH3)2), 1.41 (t,
3H, SO2CH2CH3), 1.61–2.01 (m, 6H, CH2CH), 3.07–3.11 (m, 2H,
Benzyl-1-((S)-4-methyl-1-((S)-5-methylhex-1-en-3-ylamino)-1-oxo-
pentan-2-yl)-2-oxo-1,2-dihydropyridin-3-ylcarbamate (12a): A so-
lution of acid 10a (201 mg, 0.56 mmol) in CH2Cl2 was treated with
HATU (300 mg, 0.84 mmol), amine 11 (126 mg, 0.84 mmol) and
DIPEA (0.10 mL, 0.56 mmol) and stirred at 1008C for 1 h under mi-
crowave irradiation. After that time, additional HATU (100 mg,
0.28 mmol) and DIPEA (0.048 mL, 0.28 mmol) were added, and the
ChemMedChem 2011, 6, 1228 – 1237
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R. Ettari et al.
MED
SO2CH2CH3), 4.22 (m, 1H, NCH), 4.67 (m, 1H, NCHCO), 5.20 (s, 2H,
CH2O), 6.29 (t, 1H, J=7.0 Hz, ArH), 6.95 (d, 1H, J=15.2 Hz, =
CHSO2), 6.99 (d, 1H, J=7.0 Hz, ArH), 7.05 (dd, 1H, J=4.2, 15.2 Hz,
CH=), 7.36–7.41 (m, 5H), 7.74 (s, 1H, NH), 7.99 ppm (d, 1H, J=
7.0 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=5.1, 22.4, 22.5, 23.2,
23.4, 39.1, 45.4, 45.3, 47.1, 55.7, 66.1, 109.5, 111.8, 127.3, 129.0,
129.4, 131.8, 134.5, 141.0, 141.3, 148.4, 158.4, 171.2 ppm; Anal.
calcd (%) for C28H39N3O6S: C 61.63, H 7.20, N 7.70, found: C 61.47, H
7.52, N 7.59.
141.0, 141.2, 148.5, 158.4, 171.2 ppm; Anal. calcd (%) for
C31H37N3O6S: C 64.23, H 6.43, N 7.25, found: C 64.51, H 6.29, N 7.11.
Benzyl-1-((S)-1-((S,E)-5-methyl-1-(phenylsulfonyl)hex-1-en-3-yl-
amino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-
ylcarbamate (2c): Starting from olefin 12b (150 mg, 0.30 mmol)
and phenyl vinyl sulfone 13c (504 mg, 3.0 mmol) and following
the same procedure described for 1a gave the title compound 2c
as a white solid (60 mg, 32%): Rf =0.65 (light petroleum/EtOAc,
7:3); 1H NMR (300 MHz, CDCl3): d=0.73 (m, 1H, CH2CH), 0.94 (d,
6H, J=6.2 Hz, CH(CH3)2), 1.17 (t, 2H, J=7.4 Hz, CH2CH), 1.59 (t, 2H,
J=7.4 Hz, CH2CH), 3.04 (dd, 1H, J=4.4 Hz, 12.4 Hz, CH2Ph), 3.28
(dd, 1H, J=7.0 Hz, 12.4 Hz, CH2Ph), 4.17 (m, 1H, NHCH), 5.27 (s,
2H, CH2O), 5.66 (t, 1H, J=6.4 Hz, NCHCO), 5.90 (d, 1H, J=7.3 Hz,
ArH), 6.56 (d, 1H, J=15.2 Hz, =CHSO2), 6.75 (dd, 1H, J=6.1 Hz,
15.2 Hz, CH=), 6.81 (d, 1H, J=7.3 Hz, ArH), 7.17–7.38 (m, 15H),
7.79 (bs, 1H, NH), 8.11 ppm (d, 1H, J=7.3 Hz, ArH); 13C NMR
(75 MHz, CDCl3): d=23.2, 23.3, 35.5, 45.2, 47.1, 60.6, 66.1, 109.5,
111.7, 126.0, 127.3, 127.8, 128.3, 128.7, 129.0, 129.4, 129.8, 131.8,
133.8, 134.6, 139.5, 139.5, 141.0, 141.2, 148.4, 158.4, 171.2 ppm;
Anal. calcd (%) for C35H37N3O6S: C 66.97, H 5.94, N 6.69, found: C
66.61, H 6.19, N 6.81.
Benzyl-1-((S)-4-methyl-1-((S,E)-5-methyl-1-(phenylsulfonyl)hex-1-
en-3-ylamino)-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (1c): Starting from olefin 15a (100 mg, 0.22 mmol) and
phenyl vinyl sulfone 13c (370 mg, 2.2 mmol) and following the
same procedure described for 1a gave the title compound 1c as a
white solid (26 mg 20%): Rf =0.79 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.88–0.93 (m, 12H, CH(CH3)2), 1.78–
2.06 (m, 6H, CH2CH), 4.23 (m, 1H, NCH), 4.69 (m, 1H, NCHCO), 5.20
(s, 2H, CH2O), 6.29 (t, 1H, J=7.1 Hz, ArH), 6.91 (d, 1H, J=15.2 Hz, =
CHSO2), 6.93 (d, 1H, J=7.1 Hz, ArH), 7.06 (dd, 1H, J=4.2, 15.2 Hz,
CH=), 7.33–7.55 (m, 10H), 7.86 (s, 1H, NH), 8.02 ppm (d, 1H, J=
7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=22.4, 22.5, 23.2, 23.4,
39.1, 45.2, 47.1, 55.7, 66.1, 109.5, 111.8, 127.2, 128.3, 129.0, 129.4,
129.8, 131.8, 133.8, 134.5, 139.4, 141.0, 141.2, 148.4, 158.4,
171.2 ppm; Anal. calcd (%) for C32H39N3O6S: C 64.73, H 6.62, N 7.08,
found: C 64.57, H 6.74, N 7.17.
(S)-[1-(2-Methanesulfonyl-vinyl)-3-methyl-butyl]-tritylamine
(15a): Starting from olefin 14 (125 mg, 0.35 mmol) and methyl
vinyl sulfone 13a (371 mg, 3.5 mmol) and using the same proce-
dure described for 1a gave the title compound 15a, following pu-
rification by flash column chromatography (light petroleum/EtOAc,
9:1), as a brown oil (68 mg, 45%): Rf =0.30 (light petroleum/EtOAc,
98:2); 1H NMR (300 MHz, CDCl3): d=0.69 (d, 6H, J=6.4 Hz,
CH(CH3)2), 0.74 (d, 6H, J=6.4 Hz, CH(CH3)2), 1.38–1.54 (m, 3H,
CH2CH), 2.92 (s, 3H, SO2CH3), 3.10 (m, 1H, NHCH), 6.59 (d, J=
15.2 Hz, 1H, =CHSO2), 6.81 (dd, J=15.2 Hz, 4.2 Hz, 1H, CH=), 7.12–
7.78 ppm (m, 15H, ArH); Anal. calcd (%) for C27H31NO2S: C 74.79, H
7.21, N 3.23, found: C 74.58, H 7.39, N 3.31.
Benzyl-1-((S)-1-((S,E)-5-methyl-1-(methylsulfonyl)hex-1-en-3-yl-
amino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-
ylcarbamate (2a): Starting from olefin 12b (150 mg, 0.30 mmol)
and methyl vinyl sulfone 13a (318 mg, 3.0 mmol) and following
the same procedure described for 1a gave the title compound 2a
as a white solid (46 mg, 27%): Rf =0.46 (light petroleum/EtOAc,
7:3); 1H NMR (300 MHz, CDCl3): d=0.72 (m, 1H, CH2CH), 0.92 (d,
6H, J=6.2 Hz, CH(CH3)2), 1.16 (t, 2H, J=7.4 Hz, CH2CH), 1.58 (t, 2H,
J=7.4 Hz, CH2CH), 2.86 (s, 3H, SO2CH3), 3.02 (dd, 1H, J=4.4 Hz,
12.4 Hz, CH2Ph), 3.26 (dd, 1H, J=7.0 Hz, 12.4 Hz, CH2Ph), 4.16 (m,
1H, NHCH), 5.25 (s, 2H, CH2O), 5.64 (t, 1H, J=6.4 Hz, NCHCO), 5.89
(d, 1H, J=7.3 Hz, ArH), 6.55 (d, 1H, J=15.2 Hz, =CHSO2), 6.72 (dd,
1H, J=6.1 Hz, 15.2 Hz, CH=), 6.79 (d, 1H, J=7.3 Hz, ArH), 7.15–
7.29 (m, 10H), 7.79 (bs, 1H, NH), ppm 8.12 (d, 1H, J=7.3 Hz, ArH);
13C NMR (75 MHz, CDCl3): d=23.2, 23.4, 35.5, 43.2, 45.3, 47.1, 60.7,
66.1, 109.5, 111.8, 126.0, 127.3, 127.8, 128.7, 129.0, 129.4, 131.8,
134.5, 139.5, 141.1, 141.2, 148.5, 158.4, 171.2 ppm; Anal. calcd (%)
for C30H35N3O6S: C 63.70, H 6.24, N 7.43, found: C 63.53, H 6.35, N
7.59.
(S)-[1-(2-Ethanesulfonyl-vinyl)-3-methyl-butyl]-tritylamine (15b):
Starting from olefin 14 (125 mg, 0.35 mmol) and ethyl vinyl sulfone
13b (420 mg, 3.5 mmol) and using the same procedure described
for 1a gave the title compound 15b, following flash column chro-
matography (light petroleum/EtOAc, 9:1), as a brown oil (78 mg,
50%): Rf =0.34 (light petroleum/EtOAc, 98:2); 1H NMR (300 MHz,
CDCl3): d=0.70 (d, 6H, J=6.4 Hz, CH(CH3)2), 0.78 (d, 6H, J=6.4 Hz,
CH(CH3)2), 1.30 (t, J=7.5 Hz, SO2CH2CH3) 1.40–1.56 (m, 3H, CH2CH),
2.97 (q, J=7.5 Hz, SO2CH2CH3), 3.11 (m, 1H, NHCH), 6.58 (d, J=
15.2 Hz, 1H, =CHSO2), 6.80 (dd, J=15.2 Hz, 4.2 Hz, 1H, CH=), 7.12–
7.78 ppm (m, 15H, ArH); Anal. calcd (%) for C28H33NO2S: C 75.13, H
7.43, N 3.13, found: C 75.28, H 7.22, N 3.24.
Benzyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-yl-
amino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-
ylcarbamate (2b): Starting from olefin 12b (150 mg, 0.30 mmol)
and ethyl vinyl sulfone 13b (360 mg, 3.0 mmol) and following the
same procedure described for 1a gave the title compound 2b as a
white solid (52 mg, 30%): Rf =0.51 (light petroleum/EtOAc, 7:3);
1H NMR (300 MHz, CDCl3): d=0.73 (m, 1H, CH2CH), 0.94 (d, 6H, J=
6.2 Hz, CH(CH3)2), 1.19 (t, 1H, J=7.4 Hz, CH2CH), 1.37 (t, 3H, J=
6.8 Hz, SO2CH2CH3), 1.60 (t, 1H, J=7.4 Hz, CH2CH), 3.04 (dd, 1H, J=
4.4 Hz, 12.4 Hz, CH2Ph), 3.13 (q, 2H, J=6.8 Hz, SO2CH2CH3), 3.28
(dd, 1H, J=7.0 Hz, 12.4 Hz, CH2Ar), 4.18 (m, 1H, NHCH), 5.27 (s, 2H,
CH2O), 5.66 (t, 1H, J=6.4 Hz, NCHCO), 5.90 (d, 1H, J=7.3 Hz, ArH),
6.57 (d, 1H, J=15.6 Hz, =CHSO2), 6.74 (dd, 1H, J=6.1 Hz, 15.6 Hz,
CH=), 6.81 (d, 1H, J=7.3 Hz, ArH), 7.19–7.32 (m, 10H), 7.80 (bs,
1H, NH), 8.15 ppm (d, 1H, J=7.3 Hz, ArH); 13C NMR (75 MHz,
CDCl3): d=5.2, 23.2, 23.3, 35.5, 45.4, 45.3, 47.1, 60.6, 66.1, 109.5,
111.8, 126.0, 127.3, 127.8, 128.7, 129.0, 129.4, 131.8, 134.5, 139.5,
(S)-[1-(2-Benzenesulfonyl-vinyl)-3-methyl-butyl]-tritylamine
(15c): Starting from olefin 14 (125 mg, 0.35 mmol) and phenyl
vinyl sulfone 13c (589 mg, 3.5 mmol) and using the same proce-
dure described for 1a gave the title compound 15c, following
flash column chromatography (light petroleum/EtOAc, 9:1), as a
brown oil (69 mg, 40%): Rf =0.57 (light petroleum/EtOAc, 98:2);
1H NMR (300 MHz, CDCl3): d=0.70 (d, 6H, J=6.4 Hz, CH(CH3)2),
0.78 (d, 6H, J=6.4 Hz, CH(CH3)2), 1.40–1.56 (m, 3H, CH2CH), 3.11
(m, 1H, NHCH), 6.58 (d, J=15.2 Hz, 1H, =CHSO2), 6.80 (dd, J=
15.2 Hz, 4.2 Hz, 1H, CH=), 7.12–7.80 ppm (m, 20H, ArH); Anal.
calcd (%) for C32H33NO2S: C 77.54, H 6.71, N 2.83, found: C 77.32, H
6.85, N 2.98.
(S)-1-(2-Methanesulfonyl-vinyl)-3-methyl-butylamine (16a): A so-
lution of olefin 15a (68 mg, 0.15 mmol) in acetone was treated
dropwise over 5 min with 6n HCl (40 mL, 0.22 mmol) and then re-
fluxed for 5 h. The solvent was evaporated in vacuo, the residue
1234
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1228 – 1237

Peptidomimetic Proteasome Inhibitors
was taken up in CH2Cl2, and the solution was extracted with 1n
HCl. The aqueous phase was brought to pH 10 by addition of
Na2CO3(s) and extracted with CH2Cl2. The organic phase was dried
(Na2SO4), filtered and concentrated in vacuo to give compound
(q, 2H, J=6.8 Hz, SO2CH2CH3), 4.18 (m, 1H, NHCH), 4.78 (d, 2H, J=
6.2 Hz, CH2O), 5.10 (d, 2H, J=10.2, CH2 =), 5.15 (t, 1H, J=7.5 Hz,
NCHCO), 5.84 (m, 1H, CH2 =CH), 5.92 (d, 1H, J=7.1 Hz, ArH), 6.70
(d, 1H, J=7.1 Hz, ArH), 6.74 (dd, 1H, J=6.0 Hz, 15.1 Hz, CH=), 6.85
(d, 1H, J=15.1 Hz, =CHSO2), 7.85 (bs, 1H, NH), 7.99 ppm (d, 1H,
J=7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=5.2, 22.4, 22.5, 23.2,
23.3, 39.2, 45.2, 45.4, 47.2, 55.8, 65.2, 109.5, 111.8, 116.4, 129.4,
131.8, 133.6, 134.5, 141.0, 148.4, 158.5, 171.1 ppm; Anal. calcd (%)
for C24H37N3O6S: C 58.16, H 7.52, N 8.48, found: C 58.33, H 7.74, N
8.19.
1
16a as a brown oil (12 mg, 41%); H NMR (300 MHz, CDCl3): d=
0.68 (d, 6H, J=6.4 Hz, CH(CH3)2), 0.73 (d, 6H, J=6.4 Hz, CH(CH3)2),
1.36–1.53 (m, 3H, CH2CH), 2.90 (s, 3H, SO2CH3), 3.09 (m, 1H,
NHCH), 6.57 (d, J=15.2 Hz, 1H, =CHSO2), 6.80 ppm (dd, J=15.2 Hz,
4.2 Hz, 1H, CH=); Anal. calcd (%) for C8H17NO2S: C 50.23, H, 8.96,
N 7.32, found: C 50.01, H, 8.82, N 7.59.
Allyl-1-((S)-4-methyl-1-((S,E)-5-methyl-1-(phenylsulfonyl)hex-1-
en-3-ylamino)-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (3c): Starting from acid 10c (56 mg, 0.18 mmol) and
amine 18c (50 mg, 0.20 mmol) and using the same procedure de-
scribed for 12a gave the title compound 3c, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
(S)-1-(2-Ethanesulfonyl-vinyl)-3-methyl-butylamine (16b): Start-
ing from olefin 15b (68 mg, 0.15 mmol) and following the same
procedure described for 16a gave the title compound 16b as a
1
brown oil (14 mg, 46%): H NMR (300 MHz, CDCl3): d=0.69 (d, 6H,
J=6.4 Hz, CH(CH3)2), 0.75 (d, 6H, J=6.4 Hz, CH(CH3)2), 1.29 (t, J=
7.5 Hz, SO2CH2CH3) 1.38–1.54 (m, 3H, CH2CH), 2.95 (q, J=7.5 Hz,
SO2CH2CH3), 3.09 (m, 1H, NHCH), 6.56 (d, J=15.2 Hz, 1H, =CHSO2),
6.81 ppm (dd, J=15.2 Hz, 4.2 Hz, 1H, CH=); Anal. calcd (%) for
C9H19NO2S: C 52.65, H 9.33, N 6.82, found: C 52.92, H 9.18, N 6.67.
1
solid (35 mg, 36%): Rf =0.73 (light petroleum/EtOAc, 7:3); H NMR
(300 MHz, CDCl3): d=0.73 (m, 1H, CH2CH), 0.93 (d, 12H, J=6.2 Hz,
CH(CH3)2), 1.05 (d, 12H, J=6.2 Hz, CH(CH3)2), 1.21 (t, 2H, J=7.2 Hz,
CH2CH), 1.50 (t, 2H, J=7.2 Hz, CH2CH), 1.55 (m, 1H, CH2CH), 1.64 (t,
2H, J=7.2 Hz, CH2CH), 1.78 (t, 2H, J=7.2 Hz, CH2CH), 4.19 (m, 1H,
NHCH), 4.81 (d, 2H, J=6.2 Hz, CH2O), 5.20 (d, 2H, J=10.2, CH2 =),
5.69 (t, 1H, J=7.5 Hz, NCHCO), 5.86 (m, 1H, CH2 =CH), 5.89 (d, 1H,
J=7.3 Hz, ArH), 6.13 (d, 1H, J=7.3 Hz, ArH), 6.67 (dd, 1H, J=
6.1 Hz, 15.2 Hz, CH=), 6.79 (d, 1H, J=15.2 Hz, =CHSO2), 7.36–7.74
(m, 5H, Ar), 7.84 (bs, 1H, NH), 8.01 ppm (d, 1H, J=7.3 Hz, ArH);
13C NMR (75 MHz, CDCl3): d=22.4, 22.5, 23.2, 23.3, 39.2, 45.3, 47.2,
55.7, 65.2, 109.5, 111.7, 116.4, 128.3, 129.4, 129.8, 131.9, 133.7,
133.8, 134.6, 139.4, 141.1, 148.4, 158.5, 171.1 ppm; Anal. calcd (%)
for C28H37N3O6S: C 61.86, H 6.86, N 7.73, found: C 61.67, H 6.71, N
7.99.
(S)-1-(2-Benzenesulfonyl-vinyl)-3-methyl-butylamine (16c): Start-
ing from olefin 15c (69 mg, 0.14 mmol) and following the same
procedure described for 16a gave the title compound 16c as a
1
brown oil (16 mg, 45%): H NMR (300 MHz, CDCl3): d=0.69 (d, 6H,
J=6.4 Hz, CH(CH3)2), 0.75 (d, 6H, J=6.4 Hz, CH(CH3)2), 1.40–1.55 (m,
3H, CH2CH), 3.13 (m, 1H, NHCH), 6.50 (d, J=15.2 Hz, 1H, =CHSO2),
6.75 (dd, J=15.2 Hz, 4.2 Hz, 1H, CH=), 7.36–7.74 ppm (m, 5H, Ar);
Anal. calcd (%) for C13H19NO2S: C 61.63, H 7.56, N 5.53, found: C
61.82, H 7.25, N 5.78.
Allyl-1-((S)-4-methyl-1-((S,E)-5-methyl-1-(methylsulfonyl)hex-1-
en-3-ylamino)-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (3a): Starting from acid 10c (60 mg, 0.20 mmol) and
amine 16a (44 mg, 0.22 mmol) and using the same procedure de-
scribed for 12a gave the title compound 3a, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
Allyl-1-((S)-1-((S,E)-5-methyl-1-(methylsulfonyl)hex-1-en-3-ylami-
no)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (4a): Starting from acid 10d (62 mg, 0.18 mmol) and
amine 16a (40 mg, 0.20 mmol) and using the same procedure de-
scribed for 12a gave the title compound 4a, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
1
solid (32 mg, 34%): Rf =0.50 (light petroleum/EtOAc, 7:3); H NMR
(300 MHz, CDCl3): d=0.72 (m, 1H, CH2CH), 0.91 (d, 12H, J=6.2 Hz,
CH(CH3)2), 1.04 (d, 12H, J=6.2 Hz, CH(CH3)2), 1.19 (t, 2H, J=7.2 Hz,
CH2CH), 1.51 (t, 2H, J=7.2 Hz, CH2CH), 1.56 (m, 1H, CH2CH), 1.64 (t,
2H, J=7.2 Hz, CH2CH), 1.79 (t, 2H, J=7.2 Hz, CH2CH), 2.85 (s, 3H,
SO2CH3), 4.18 (m, 1H, NHCH), 4.80 (d, 2H, J=6.2 Hz, CH2O), 5.21 (d,
2H, J=10.2, CH2 =), 5.67 (t, 1H, J=7.5 Hz, NCHCO), 5.85 (m, 1H,
CH2 =CH), 5.91 (d, 1H, J=7.1 Hz, ArH), 6.11 (d, 1H, J=7.1 Hz, ArH),
6.65 (dd, 1H, J=6.1 Hz, 15.2 Hz, CH=), 6.78 (d, 1H, J=15.2 Hz, =
CHSO2) 7.88 (bs, 1H, NH), 7.96 ppm (d, 1H, J=7.1 Hz, ArH);
13C NMR (75 MHz, CDCl3): d=22.4, 22.5, 23.2, 23.3, 39.2, 43.2, 45.3,
47.1, 55.7, 65.1, 109.5, 111.8, 116.4, 129.4, 131.9, 133.6, 134.5, 141.1,
148.4, 158.5, 171.1 ppm; Anal. calcd (%) for C23H35N3O6S: C 57.36, H
7.33, N 8.72, found: C 57.12, H 7.54, N 8.61.
1
solid (35 mg, 38%): Rf =0.31 (light petroleum/EtOAc, 7:3); H NMR
(300 MHz, CDCl3): d=0.84 (d, 6H, J=6.4 Hz, CH(CH3)2), 0.90 (d, 6H,
J=6.4 Hz, CH(CH3)2), 1.52–1.94 (m, 6H, CH2CH), 3.00 (s, 3H,
SO2CH3), 3.15 (dd, 1H, J=6.9 Hz, 13.7 Hz, CH2Ph), 3.38 (dd, J=
4.4 Hz, 13.7 Hz, 1H, CH2Ph), 4.75 (d, J=5.5 Hz, 2H, CH2O), 4.15–4.19
(m, 1H, NHCH), 4.40 (m, 1H, NCHCO), 5.33 (d, 1H, J=10.2 Hz,
CH2 =), 5.45 (d, 1H, J=17.0 Hz, CH2 =), 6.00 (m, 1H, CH2 =CH),
6.30 (t, 1H, J=7.1 Hz, ArH), 6.81 (d, 1H, J=15.2 Hz, =CHSO2), 6.94
(d, 1H, J=7.1 Hz, ArH), 6.99 (dd, 1H, J=15.2 Hz, 4.2 Hz, CH=),
7.21–7.33 (m, 5H), 7.79 (s, 1H, NH), 8.01 ppm (d, 1H, J=7.1 Hz,
ArH); 13C NMR (75 MHz, CDCl3): d=23.2, 23.4, 35.5, 43.2, 45.3, 47.1,
60.6, 65.1, 109.5, 111.8, 116.5, 126.1, 127.8, 128.7, 129.5, 131.8,
133.6, 134.5, 139.5, 141.1, 148.4, 158.5, 171.1 ppm; Anal. calcd (%)
for C26H33N3O6S: C 60.56, H 6.45, N 8.15, found: C 60.78, H 6.23, N
8.02.
Allyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-ylami-
no)-4-methyl-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (3b): Starting from acid 10c (74 mg, 0.24 mmol) and
amine 16b (52 mg, 0.26 mmol) and using the same procedure de-
scribed for 12a gave the title compound 3b, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
Allyl-1-((S)-1-((S,E)-1-(ethylsulfonyl)-5-methylhex-1-en-3-ylami-
no)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (4b): Starting from acid 10d (62 mg, 0.18 mmol) and
amine 16b (50 mg, 0.20 mmol) and using the same procedure de-
scribed for 12a gave the title compound 4b, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
1
solid (42 mg, 35%): Rf =0.55 (light petroleum/EtOAc, 7:3); H NMR
(300 MHz, CDCl3): d=0.97–0.99 (m, 12H, CH(CH3)2), 1.22 (m, 1H,
CH2CH), 1.32 (t, 1H, J=7.2 Hz, CH2CH), 1.38 (t, 3H, J=6.8 Hz,
SO2CH2CH3), 1.40 (m, 1H, CH2CH), 1.47 (t, 3H, J=7.2 Hz, CH2CH),
1.54 (t, 3H, J=7.2 Hz, CH2CH), 2.01 (t, 3H, J=7.2 Hz, CH2CH), 3.43
1
solid (38 mg, 40%): Rf =0.34 (light petroleum/EtOAc, 7/3); H NMR
ChemMedChem 2011, 6, 1228 – 1237
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1235

R. Ettari et al.
MED
(300 MHz, CDCl3): d=0.84 (d, 6H, J=6.4 Hz, CH(CH3)2), 0.90 (d, 6H,
J=6.4 Hz, CH(CH3)2), 1.41 (t, 3H, SO2CH2CH3), 1.52–1.94 (m, 6H,
CH2CH), 3.07 (q, 2H, SO2CH2), 3.15 (dd, 1H, J=6.9 Hz, 13.7 Hz,
CH2Ph), 3.38 (dd, 1H, J=4.40 Hz, 13.7 Hz, CH2Ph), 4.76 (d, 2H, J=
5.5 Hz, CH2O), 4.19 (m, 1H, NHCH), 4.38 (m, 1H, NCHCO), 5.35 (d,
1H, J=10.2 Hz, CH2 =), 5.46 (d, 1H, J=17.0 Hz, CH2 =), 5.99 (m,
1H, CH2 =CH), 6.33 (t, 1H, J=7.1 Hz, ArH), 6.82 (d, 1H, J=15.2 Hz,
=CHSO2), 6.96 (d, 1H, J=7.1 Hz, ArH), 6.97 (dd, 1H, J=15.2 Hz,
4.2 Hz, CH=), 7.19–7.28 (m, 5H), 7.81 (s, 1H, NH), 8.00 ppm (d, 1H,
J=7.1 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=5.1, 23.2, 23.3, 35.5,
45.3, 45.4, 47.1, 60.7, 65.1, 109.6, 111.8, 116.5, 126.1, 127.8, 128.7,
129.5, 131.8, 133.6, 134.5, 139.5, 141.1, 148.3, 158.4, 171.1 ppm;
Anal. calcd (%) for C27H35N3O6S: C 61.23, H 6.66, N 7.93, found: C
61.58, H 6.38, N 7.72.
Dissociation constants KI were obtained by correction to zero sub-
strate concentration using Equation (3), the Cheng–Prusoff equa-
tion. Second-order rate constants were calculated using Equa-
tion (4). In cases where the plots of kobs versus inhibitor concentra-
tion [I] were restricted to the linear range only, k2nd values were cal-
culated using Equation (5). The Km values were determined in sepa-
rate experiments: ChT-L activity with Suc-Leu-Leu-Val-Tyr-AMC
(13 mm), PGPH activity with Cbz-Leu-Leu-Glu-AMC (53 mm).
KI ¼KIapp=ð1 þ ½Sꢂ=KmÞ
k2nd ¼kinac=KI
ð3Þ
ð4Þ
ð5Þ
k2nd ꢃkobs½Iꢂꢀ1ð1 þ ½SꢂKm
Þ
ꢀ1
Assaying the chymotryptic activity of 20S proteasome: Human 20S
proteasome was incubated at 308C at a final concentration of
0.004 mgmLꢀ1 with test compound present at varying concentra-
tions. The reaction buffer consisted of 50 mm Tris pH 7.5, 10 mm
NaCl, 25 mm KCl, 1 mm MgCl2, 0.03% sodium dodecyl sulfate
(SDS), and 5% DMSO. Product release from substrate hydrolysis
(75 mm) was monitored continuously over a period of 30 min.
Allyl-1-((S)-1-((S,E)-5-methyl-1-(phenylsulfonyl)hex-1-en-3-ylami-
no)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl-
carbamate (4c): Starting from acid 10d (48 mg, 0.14 mmol) and
amine 16c (48 mg, 0.16 mmol) and using the same procedure de-
scribed for 12a gave the title compound 4c, following preliminary
purification by flash column chromatography (light petroleum/
EtOAc, 7:3) and subsequent semipreparative RP-HPLC, a white
1
Assaying the tryptic activity of the 20S proteasome: Human 20S pro-
teasome was incubated at 308C at a final concentration of
0.0025 mgmLꢀ1 with test compound at varying concentrations.
The reaction buffer consisted of 50 mm Tris buffer pH 7.4, 50 mm
NaCl, 0.5 mm EDTA, 0.03% SDS, and 7.5% DMSO. Product release
from substrate hydrolysis (85 mm) was monitored continuously
over a period of 30 min.
solid (27 mg, 34%); Rf =0.59 (light petroleum/EtOAc, 7:3); H NMR
(300 MHz, CDCl3): d=0.83 (d, 6H, J=6.4 Hz, CH(CH3)2), 0.91 (d, 6H,
J=6.4 Hz, CH(CH3)2), 1.51–1.93 (m, 6H, CH2CH), 3.12 (dd, 1H, J=
6.9 Hz, 13.7 Hz, CH2Ph), 3.36 (dd, 1H, J=4.40 Hz, 13.7 Hz, CH2Ph),
4.74 (d, 2H, J=5.5 Hz, CH2O), 4.18 (m, 1H, NHCH), 4.39 (m, 1H,
NCHCO), 5.34 (d, 1H, J=10.2 Hz, CH2 =), 5.45 (d, 1H, J=17.0 Hz,
CH2 =), 5.99 (m, 1H, CH2 =CH), 6.29 (t, 1H, J=7.0 Hz, ArH), 6.79 (d,
1H, J=15.2 Hz, =CHSO2), 6.96 (d, 1H, J=7.0 Hz, ArH), 7.01 (dd, 1H,
J=15.2 Hz, 4.2 Hz, CH=), 7.26–7.53 (m, 10H), 7.77 (s, 1H, NH),
8.03 ppm (d, 1H, J=7.0 Hz, ArH); 13C NMR (75 MHz, CDCl3): d=
23.2, 23.3, 35.5, 45.3, 47.1, 60.7, 65.1, 109.6, 111.8, 116.4, 126.1,
127.8, 128.3, 128.7, 129.5, 129.8, 131.8, 133.6, 133.8, 134.6, 139.4,
139.6, 141.1, 148.5, 158.5, 171.2 ppm; Anal. calcd (%) for
C31H35N3O6S: C 64.45, H 6.11, N 7.27, found: C 64.69, H 6.28, N 7.11.
Assaying the post-glutamyl-peptide hydrolyzing activity of the 20S
proteasome: Human 20S proteasome was incubated at 308C at a
final concentration of 0.004 mgmLꢀ1 with the test compound at
varying concentrations. The reaction buffer consisted of 50 mm Tris
buffer pH 7.5 containing 25 mm KCl, 10 mm NaCl, 1 mm MgCl2,
0.03% SDS, and 5% DMSO. Product release from substrate hydro-
lysis (80 mm) was monitored continuously over a period of 30 min.
Assays for bovine pancreatic a-chymotrypsin inhibition: The enzyme
(250 mgmLꢀ1) was incubated at 258C with test compound. The re-
action buffer consisted of 50 mm Tris buffer pH 8.0 containing
100 mm NaCl, 5 mm EDTA and 7.5% DMSO. Product release from
substrate hydrolysis (75 mm final concentration, Suc-Leu-Tyr-AMC)
was determined over a period of 10 min.
Biology
In vitro 20S proteasome inhibition assays: The three distinct proteo-
lytic activities of human 20S proteaseome (Biomol GmbH, Ham-
burg, Germany) were measured by monitoring the hydrolysis of
peptidyl 7-amino-4-methyl coumarin substrates (all obtained from
Bachem), Suc-Leu-Leu-Val-Tyr-AMC, Boc-Leu-Arg-Arg-AMC, and
Cbz-Leu-Leu-Glu-AMC, for ChT-L, T-L or PGPH enzyme activity, re-
spectively. Hydrolysis of the given substrates was measured using
a Varian Cary Eclipse fluorescence spectrophotometer (Varian,
Darmstadt, Germany) at 308C with a 370 nm excitation filter and a
465 nm emission filter. Preliminary screening for inhibition of the
three proteolytic activities of 20S proteaseome was performed at
20 mm inhibitor concentrations using an equivalent amount of
DMSO as a negative control. Compounds showing at least 40% in-
hibition at 20 mm were subjected to detailed assays. The inhibition
constants were obtained from progress curves (30 min) at various
concentrations of inhibitor by fitting the progress curves to Equa-
tion (1), yielding the pseudo-first order rate constants of inhibition
kobs, which were then fitted to the inhibitor concentrations using
Equation (2), yielding the first-order rate constant kinac and the ap-
Assays for cathepsin B and L inhibition: Inhibition of cathepsin B
and L was determined using the methods previously described.[18]
Cbz-Phe-Arg-AMC was used as the substrate (80 mm for cathe-
psin B, 5 mm for cathepsin L).
Acknowledgements
This work was financially supported by the Ministero dell’Istru-
zione, dell’Universita‘ e della Ricerca Scientifica e Tecnologica
(MIUR-PRIN2008). We would like to thank the Deutscher Akade-
mischer Austausch Dienst (DAAD) (Vigoni project 2009/10) for
partial support of this work. TS thanks the Deutsche Forschungs-
gemeinschaft (DFG) (SFB630) for financial support.
parent dissociation constant KIapp
F ¼Að1ꢀexpðꢀkobstÞÞ þ B
kobs ¼kinac½Iꢂ=ðKIapp þ ½IꢂÞ
.
Keywords: chymotrypsin-like
peptidomimetics · proteasome · vinyl sulfones
activity
·
inhibitors
·
ð1Þ
ð2Þ
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ChemMedChem 2011, 6, 1228 – 1237

Peptidomimetic Proteasome Inhibitors
[1] G. DeMartino, T. Gillette, Cell. 2007, 129, 659–662.
[2] a) A. Ciechanover, Angew. Chem. 2005, 117, 6095–6119; Angew. Chem.
Int. Ed. 2005, 44, 5944–5967; b) A. L. Goldberg, Biochem. Soc. Trans.
2007, 35, 12–17.
[3] a) M. Groll, M. Bajorek, A. Kohler, L. Moroder, D. M. Rubin, R. Huber,
M. H. Glickman, D. Finley, Nat. Struct. Biol. 2000, 7, 1062–1067; b) B. C.
Braun, M. Glickman, R. Kraft, B. Dahlmann, P. M. Kloetzel, D. Finley, M.
Schmidt, Nat. Cell Biol. 1999, 1, 221–226; c) E. Strickland, K. Hakala, P. J.
Thomas, G. N. DeMartino, J. Biol. Chem. 2000, 275, 5565–5572.
[4] M. Groll, L. Ditzel, J. Lowe, M. Bochtler, H. Bartunik, R. Huber, Nature
1997, 386, 463–471.
[5] L. Borissenko, M. Groll, Chem. Rev. 2007, 107, 687–717.
[6] M. Groll, T. Nazif, R. Huber, M. Bogyo, Chem. Biol. 2002, 9, 655–662.
[7] M. Groll, M. Bochtler, H. Brandstetter, T. Clausen, R. Huber, ChemBioChem
2005, 6, 222–256.
[8] S. Meiners, A. Ludwig, V. Stangl, K. Stangl, Med. Res. Rev. 2008, 28, 309–
327.
[11] a) B. M. Kessler, D. Tortorella, M. Altun, A. F. Kisselev, E. Fiebiger, B. G.
Hekking, H. L. Ploegh, H. S. Overkleeft, Chem. Biol. 2001, 8, 913–929;
b) T. Nazif, M. Bogyo, Proc. Natl. Acad. Sci. USA 2001, 98, 2967–2972.
[12] M. Bogyo, J. S. McMaster, M. Gaczynska, D. Tortorella, A. L. Goldberg, H.
Ploegh, Proc. Natl. Acad. Sci. USA 1997, 94, 6629–6634.
[13] K. N. Fanning, A. G. Jamieson, A. Sutherland, Org. Biomol. Chem. 2005,
3, 3749–3756.
[14] A. Albeck, R. Persky, J. Org. Chem. 1994, 59, 653–657.
[15] R. Ettari, N. Micale, J. Organomet. Chem. 2007, 692, 3574–3576.
[16] A. F. Newton , S. J. Roe , J. C. Legeay, P. Aggarwal, C. Gignoux, N. J.
Birch, R. Nixon, M. L. Alcaraz, R. A. Stockman, Org. Biomol. Chem. 2009,
7, 2274–2277.
[17] G. de Bettignies, O. Coux, Biochimie 2010, 92, 1530–1545.
[18] R. Vicik, M. Busemann, C. Gelhaus, N. Stiefl, J. Scheiber, W. Schmitz, F.
Schulz, M. Mladenovic, B. Engels, M. Leippe, K. Baumann, T. Schirmeister,
ChemMedChem 2006, 1, 1126–1141.
[9] A. Field-Smith, G. J. Morgan, F. E. Davies, Ther. Clin. Risk Manage. 2006,
2, 271–279.
Received: February 15, 2011
[10] M. Bogyo, S. Shin, J. S. McMaster, H. L. Ploegh, Chem. Biol. 1998, 5, 307–
320.
Published online on April 19, 2011
ChemMedChem 2011, 6, 1228 – 1237
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