Convergent synthesis and biological evaluation of syringolin a and derivatives as eukaryotic 20S proteasome inhibitors

Article abstract of DOI:10.1002/ejoc.201000317

A convergent synthesis of SylA was developed and consists of the synthesis of a fully functionalized macrocycle, which is subsequently coupled with a urea moiety. For cyclization, ring-closing metathesis of a conformationally preorganized precursor was employed. The established synthetic route was then applied to the synthesis of SylA derivatives by using various peptidic side chains for decoration of the SylA macrocycle. The resulting collection of SylA analogues was tested for proteasome inhibition, revealing PEGylated SylA derivatives as the most potent proteasome inhibitors.

Full text of DOI:10.1002/ejoc.201000317

FULL PAPER
DOI: 10.1002/ejoc.201000317
Convergent Synthesis and Biological Evaluation of Syringolin A and
Derivatives as Eukaryotic 20S Proteasome Inhibitors
Jérôme Clerc,[a] Barbara Schellenberg,[b] Michael Groll,[c] André S. Bachmann,[d]
Robert Huber,[e,f,g] Robert Dudler,[b] and Markus Kaiser*[a]
Keywords: Synthetic methods / Natural products / Biological activity / Metathesis / Inhibitors
A convergent synthesis of SylA was developed and consists
of the synthesis of a fully functionalized macrocycle, which
is subsequently coupled with a urea moiety. For cyclization,
ring-closing metathesis of a conformationally preorganized
precursor was employed. The established synthetic route
was then applied to the synthesis of SylA derivatives by
using various peptidic side chains for decoration of the SylA
macrocycle. The resulting collection of SylA analogues was
tested for proteasome inhibition, revealing PEGylated SylA
derivatives as the most potent proteasome inhibitors.
some inhibitors have lately entered advanced clinical trials[6]
or have been used as chemical tools for studying protea-
some function in live cells.[7]
Introduction
The ubiquitin proteasome system represents the central
degradation pathway in all eukaryotic cells.[1] Its core prote-
olysis system is the 20S proteasome, a chambered multicata-
lytic protease that features three distinct proteolytic activi-
ties.[2] The β5 subunit hosts chymotrypsin-like activity, the
β2 subunit hosts trypsin-like activity, and the β1 subunit
hosts caspase-like activity.[3] 20S proteasome inhibition rep-
resents a promising strategy for the chemotherapeutic treat-
ment of certain cancers, which was pointed out by the re-
cent FDA approval for the use of the proteasome inhibitor
Bortezomib (Velcade®) for treatment of relapsed and/or re-
fractory multiple myeloma. In addition, several clinical tri-
als are currently ongoing to assess its efficacy in other can-
cer types.[4] However, Bortezomib therapy is often ham-
pered by severe side effects and emerging drug resistances.[5]
Consequently, the discovery of alternative inhibitors is still
a persistent challenge and several small molecule protea-
In the last years, natural products (NPs) have been dem-
onstrated to be an invaluable source for the identification of
novel proteasome inhibitory lead structures. To date, several
NPs such as salinosporamide, epoxomicin, fetullamides,
TMC-95A, and argyrins have been reported as promising
proteasome inhibitors.[8] In 1998, the natural product syrin-
golin A (SylA, Figure 1) was isolated from strains of the
bacterial plant pathogen Pseudomonas syringae pv. syringae
(Pss),[9] in which it is biosynthesized under infection condi-
tions by a mixed nonribosomal peptide synthetase (NRPS)/
polyketide synthetase (PKS) cluster.[10] SylA exerts potent
biological activities such as inhibition of proliferation of
neuroblastoma and ovarian cancer cells,[11,12] resulting from
potent and, under physiological conditions, irreversible pro-
teasome inhibition.[12] Despite its irreversible binding mode,
SylA shows surprising selectivity for covalent proteasome
[a] Chemical Genomics Centre der Max-Planck-Gesellschaft,
Otto-Hahn-Str. 15, 44227 Dortmund, Germany
Fax: +49-231-9742-6479
E-mail: markus.kaiser@cgc.mpg.de
[b] Zurich-Basel Plant Science Center, Institute of Plant Biology,
University of Zürich,
Zollikerstr. 107, 8008 Zürich, Switzerland
[c] CIPSM, Department Chemie, TU München,
Lichtenbergstr. 4, 85747 Garching, Germany
[d] Cancer Research Center of Hawaii, University of Hawaii at
Manoa,
1236 Lauhala Street, Honolulu, HI 96813, USA
[e] Max-Planck-Institut für Biochemie,
Am Klopferspitz 18a, 82152 Martinsried, Germany
[f] Zentrum für Medizinische Biotechnologie, Universität
Duisburg-Essen,
45117 Essen, Germany
[g] Cardiff University,
Figure 1. Chemical structures of the natural product proteasome
inhibitors syringolin A (SylA), syringolin B (SylB), and synthetic
derivative SylA-LIP.
Cardiff CF10 3US, United Kingdom
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000317.
Eur. J. Org. Chem. 2010, 3991–4003
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Kaiser et al.
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inhibition, highlighting its potential as a promising lead
structure for drug discovery efforts.[13] However, even slight
structural variations of the natural product SylA have a sig-
nificant impact on proteasome inhibition and subsite selec-
tivity.[12,14] In fact, only the attachment of a lipophilic side
chain to the SylA core structure has resulted in the deriva-
tive SylA-LIP (Figure 1) that proved to be 100-times more
potent than the parent natural product SylA. Consequently,
derivatization of SylA holds promise to lead to proteasome
inhibitors with enhanced inhibitory and pharmacokinetic
properties. To this end, the establishment of a synthetic
route that allows the facile and rapid generation of SylA
derivatives would be highly desirable.
Here, we wish to report our findings on the implementa-
tion of such a synthesis route by employing a convergent
assembly strategy. In addition, we present its application to
the synthesis of SylA and SylA analogues and their subse-
quent biochemical evaluation.
Results and Discussion
In our previously published synthesis of SylA, we in-
stalled the fully functionalized ring system of SylA in the
penultimate step of the synthesis. This approach however
proved impractical for the synthesis of derivatives, as indi-
vidual optimization of reaction conditions was required for
Scheme 1. First retrosynthetic pathway to SylA by using macro-
lactamization as a key step for ring closure.
each different side-chain residue.[14] To overcome this 5.[18] First attempts to achieve formation of 7 in a one-pot
limitation, we envisaged a convergent assembly strategy, in approach by Jones oxidation failed, as only low yields of
which the molecule was divided into two major parts: The the desired product were obtained. Therefore, oxazolidine 5
synthesis of a fully functionalized SylA macrocycle and the was instead converted into 6 by a catalytic amount of p-
exocyclic residues (Scheme 1). The advantage of such an ap- toluenesulfonic acid in methanol under reflux conditions.
proach is that the SylA macrocycle can be easily decorated Then, a two-step oxidation of alcohol 6 was performed. In
with alternative side chains to yield SylA analogues. For the first step, Dess–Martin periodinane was used to provide
implementation of such a convergent strategy, an efficient the unstable aldehyde, which was then immediately oxidized
generation of the SylA macrocycle is however required.
to corresponding β,γ-dehydrolysine building block 7 by a
As a macrolactamization approach proved to be a suit- Pinnick oxidation procedure.[19]
able strategy for the synthesis of SylB (Figure 1),[14] we first
With 7 in hand, we continued the macrolactamization ap-
tested an analogous tactic for the synthesis of the SylA proach by attachment of an α,β-unsaturated valine residue
macrocycle. As depicted in Scheme 1, such an approach is (Scheme 3). To this end, literature-known 8[14] was depro-
straightforward, relying on simple, iterative peptide cou- tected at the N-terminus and coupled with 7 to yield dipep-
plings of previously prepared amino acid building blocks tide 9. Selective deprotection of the Troc protecting group
by standard peptide chemistry.
with zinc powder in ammonium acetate buffer pH 5.0, fol-
We therefore initiated our studies with the synthesis of lowed by methyl ester saponification then yielded macrolac-
the required β,γ-dehydrolysine derivative that could be ob- tamization precursor 10 in good yield.[20] For the realization
tained by a chiral pool approach by using Garner’s alde- of the macrolactamization, several different cyclization pro-
hyde 1 as the starting material.[15] The installation of the tocols such as O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetra-
critical trans-β,γ double bond was envisaged by a Johnson– methyluronium
hexafluorophosphate/1-hydroxy-7-aza-
Claisen rearrangement (Scheme 2). Accordingly, vinylmag- benzotriazole (HATU/HOAt) under dilution conditions
nesium bromide was added to ()-serine-derived Garner al- were tested, but disappointingly none of them delivered the
dehyde 1.[16] Resulting alcohol 2 was obtained in good desired product. LC–MS analysis of the corresponding reac-
yields as a mixture of diastereomers. Both isomers were tion mixtures revealed that desired product 11 was formed
treated with triethyl orthoacetate and propionic acid, yield- only in trace amounts, independently of our employed reac-
ing 3 with the desired trans double bond.[17] The ester was tion conditions. Instead, mostly dimers and trimers of 11
subsequently saponified to acid 4, which upon a modified were detected as side products, leading to the assumption
Curtius rearrangement with the use of diphenyl phosphoryl that high ring strain of the 12-membered macrocycle might
azide and an excess amount of trichloroethanol led directly be responsible for the unsuccessful cyclization. As ring-clos-
to orthogonally protected β,γ-dehydrolysinol derivative ing metathesis (RCM) has proven to be a versatile ap-
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Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors
Scheme 3. Attempted synthesis of macrocycle 11 using a macro-
lactamization approach. Reagents and conditions: (a) 1. 25% TFA/
DCM, 30 min; 2.
7 (1 equiv.), PyBop (1.5 equiv.), HOAt
(1.5 equiv.), DIEA (2 equiv.), DCM, 0 °C to room temp. overnight,
64% (two steps); (b) 1. Zn (150 equiv.), THF/NH4OAc buffer solu-
tion 1  pH 5.0 (5:1), 3 h; 2. 1  aq. LiOH (2 equiv.), MeOH/H2O
(3:1), 0 °C to room temp. over 30 min; 3. aq. HCl, 82% (three
steps).
Scheme 2. Synthesis of protected β,γ-dehydrolysine building block
7. Reagents and conditions: (a) vinylmagnesium bromide (3 equiv.),
THF, 30 min at –78 °C and then 2 h at room temp., 75%, syn/anti,
1:3; (b) triethyl orthoacetate (9 equiv.), propionic acid (0.2 equiv.),
xylenes, reflux, 24 h, 90%; (c) 1  aq. LiOH (3 equiv.), MeOH/H2O
(3:1), 0 °C to room temp. over 30 min, Ͼ98%; (d) 1. diphenyl phos-
phoryl azide (1 equiv.), Et3N (1.2 equiv.), toluene, 30 min at room
temp. and then 4 h at reflux; 2. trichloroethanol (3 equiv.) at 50 °C
and then 20 h at reflux, 93% (two steps); (e) pyridinium p-toluene-
sulfonate (PPTS, 0.2 equiv.), MeOH, overnight, reflux, Ͼ98%;
(f) 1. Dess–Martin periodinane (2 equiv.), DCM, 2 h, 0 °C; 2. aq.
NaClO2 (7 equiv.), aq. NaH2PO4 (4 equiv.), tBuOH/2-methyl-2-
butene (3:1), 0 °C to room temp. over 30 min, 69% (two steps).
proachalso for the synthesis of strained macrocycles, we
therefore revised our synthetic approach to include RCM as
a key reaction for ring closure (Scheme 4).[21]
The corresponding retrosynthesis however leads to a
RCM tripeptide precursor that features a vinylglycine resi-
due at its N-terminus. As vinylglycines are known to easily
undergo unwanted isomerization to the α,β-conjugated sys-
tem under peptide coupling conditions, the use of a pre-
viously reported phenyl selenyl derivative that can be trans-
formed into a vinylglycine just prior to RCM was envisaged
Scheme 4. Retrosynthesis for the generation of the core macrocycle
by using ring-closing metathesis (RCM) as a key step.
However, as observed during the macrolactamization ap-
instead.[22] Further disconnections at the peptide bonds re- proach, all our attempts to cyclize 15 to macrocycle 11
sult in three major building blocks that are all rapidly ac- failed again. Although different Grubbs catalysts and reac-
cessible by known methods.
tion conditions were screened, desired product 11 could not
Accordingly, ()-homoserine was converted into pro- be isolated; instead, only dimers and trimers both in the
tected vinylglycine derivative 12 by following the protocol ring-opened and ring-closed forms and various other side
of Berkowitz (Scheme 5). Saponification of 8, coupling with products were detected by LC–MS analysis. Thus, to
commercially available 3-butenylamine, and Boc deprotec- achieve ring closure, an alteration of the conformation prior
tion yielded amine building block 13. Coupling of 12 and to ring closure was necessary. We imagined that this could
13 by using PyBop/HOAt activation led to intermediate 14, be achieved through simple modification of the RCM pre-
which upon one-pot oxidation/elimination of the phenyl se- cursor by replacing the central double bond by a spatially
lenyl group yielded RCM precursor 15 in good yields.[23]
different five-membered ring system, arising from dihydrox-
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M. Kaiser et al.
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ylation and subsequent acetonide formation. Reinstallment
of the double bond could then be achieved by a Corey–
Winter elimination (Scheme 6).
To put this into practice, α,β-unsaturated methyl ester 8
was dihydroxylated to 16 with a high diastereomeric ratio
(96%dr) by using osmium tetroxide and 4-methyl morph-
oline N-oxide (NMO, Scheme 7).[24] Protection of diol 16
as an acetonide led to conformationally preorganized inter-
mediate 17. Methyl ester saponification and derivatization
by peptide coupling with 3-butenylamine yielded 18. Its
conversion into 19 was achieved by chemoselective Boc de-
protection with TMSOTf and 2,6-lutidine,[25] followed by
coupling with phenylselenyl building block 12. One-pot oxi-
dation and elimination yielded the vinylglycinyl intermedi-
Scheme 5. Attempted synthesis of core macrocycle 11 by using a
RCM approach. Reagents and conditions: (a) 1. 1  aq. LiOH
(6 equiv.), MeOH/H2O (3:1), 0 °C to room temp. over 30 min; 2. 3-
butenylamine hydrochloride (1.1 equiv.), PyBop (1.1 equiv.), DIEA
(3 equiv.), DCM, 0 °C to room temp. overnight, 82% (two steps),
3. 20% TFA/DCM, 30 min, Ͼ98%; (b) 12 (1 equiv.), 13 (1 equiv.),
PyBop (1.01 equiv.), HOAt (1.01 equiv.), DIEA (2.7 equiv.), DCM,
from 0 °C to room temp. overnight, 93%; (c) 30% aq. H2O2/DIEA
(1:1), DCM, 50 °C, 3 h, 67 %.
Scheme 7. Synthesis of the SylA macrocycle core structure by using
a RCM approach with structural preorganization. Reagents and
conditions: (a) OsO4 (0.05 equiv.), NMO (1.5 equiv.), Ac2O/H2O
(2:1), 48 h at room temp., 85%; (b) 2,2-DMP (30 equiv.), PPTS
(0.05 equiv.), DCM, reflux, Ͼ98%; (c) 1. 1  aq. LiOH (3 equiv.),
MeOH/H2O (1:1), 0 °C to room temp. over 30 min; 2. 3-buten-
ylamine hydrochloride (1.2 equiv.), PyBop (1.5 equiv.), HOAt
(1.5 equiv.), DIEA (2 equiv.), DCM, 0 °C to room temp. overnight,
80% (two steps); (d) 1. 2,6-lutidine (2 equiv.), TMSOTf (1.5 equiv.),
DCM, room temp., 15 min; 2. 12 (1.3 equiv.), PyBop (1.5 equiv.),
HOAt (1.5 equiv.), DIEA (2 equiv.), DCM, from 0 °C to room
temp. overnight, 87% (two steps); (e) 30% aq. H2O2/DIEA (1:1),
DCM, 3 h at 50 °C, 93%; (f) Grubbs II catalyst (0.15 equiv.), tolu-
ene, 18 h, 90 °C, 49%; (g) 1. 2,6-lutidine (2 equiv.), TMSOTf
(1.5 equiv.), DCM, room temp., 15 min; 2. 2,2,2-trichloroethyl
chloroformate (1.1 equiv.), NaHCO3 (2 equiv.), THF, 0 °C to room
temp. over 90 min, 81% (two steps); (h) 1. MW, 150 W, 140 °C,
30 min, pTsOH·H2O, MeOH/H2O/THF (2:2:1); 2. (Im)2CS,
DMAP, THF, 80 °C, overnight, 86% (two steps); (i) P(OMe)3,
130 °C, 2.5 h, 88%.
Scheme 6. Retrosynthesis for the generation of the core macrocycle
by using ring-closing metathesis (RCM) on a conformationally pre-
oriented precursor.
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Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors
Table 1. Catalyst and reaction conditions screen for optimizing the RCM conversion of 20 into 21.
Entry
Catalyst
E/Z selectivity
Time
[h]
Temperature
Solvent
Yield 21
Conversion
[%][b]
[°C]
[%][a]
1
2
3
4
5
6
Grubbs I
Grubbs II
Grubbs II
10:1
24
24
20
20
24
48
r.t.
r.t.
110
90
r.t.
r.t.
DCM
DCM
toluene
toluene
DCM
DCM
n.i.[c]
42
39
25
95
95
95
5
1.1:1.0
3.1:1.0
2.6:1.0
n.d.
Grubbs II
49
Hoveyda–Grubbs I
Hoveyda–Grubbs II
n.i.[c]
22
1.1:1.0
90
[a] Yield of (E) isomer only. [b] Determined by LC–MS (ESI). [c] n.i.: not isolated.
ate and RCM precursor 20. For the subsequent key RCM ture of SylA, we planned to synthesize and couple all four
reaction, several reaction conditions and RCM catalysts stereoisomers of the urea dipeptide unit to the macrocycle.
were screened (Table 1). We started our investigations with Thus, commercial isocyanate 25 was treated with either the
Grubbs I catalyst (Table 1, Entry 1). Whereas the Grubbs I ()- or ()-configured tert-butyl valine hydrochloride salt
catalyst provided 21 with the desired stereoselectivity (E/Z (30 or ent-30, respectively) to generate bis-protected ureas
= 10:1), only low conversion and thus yield was observed. 26 and 27, which upon treatment with wet formic acid
We then tested the Grubbs II catalyst, performing the RCM yielded acids 28 and 29 in excellent yields (Scheme 8). For
at room temperature for 24 h. To our delight, ring closure the synthesis of the two other isomers, ()- or ()-config-
was achieved in high yield but with a poor stereoselectivity. ured salt 30 or ent-30, respectively, was employed again by
In fact, 42% of the desired (E) isomer and 39% of the (Z) using triphosgene followed by addition of ()-valine methyl
isomer could be isolated. An improvement in selectivity ester to form corresponding ureas 31 and 33. Acidic cleav-
could be obtained when the reaction was performed at age of the tert-butyl ester group with wet formic acid then
110 °C in toluene (E/Z = 3.1:1; Table 1, Entry 3). However, yielded the two alternative ureas 32 and 34.
under these reaction conditions, side products arose that led
to serious purification problems. Adjacent optimization of
reaction conditions finally allowed the generation of 21
with 2.6:1 E/Z diastereoselectivity and 49% yield of the de-
sired (E) isomer if the reaction was performed for 20 h in
toluene at 90 °C (Table 1, Entry 4).
The Grubbs–Hoveyda I catalyst however was even less
reactive than the Grubbs I catalyst, resulting in almost no
conversion to the cyclized products (Table 1, Entry 5). The
Grubbs-Hoveyda II catalyst (Table 1, Entry 6) showed com-
parable selectivity to that of the Grubbs II catalyst (Table 1,
Entry 2) under similar reaction conditions but required a
much longer reaction time to achieve the same level of con-
version. We therefore decided to apply the Grubbs II-cata-
lyzed RCM at 90 °C in toluene (Table 1, Entry 4) for the
conversion of 20 into 21, performing the reaction with a
similar yield on a gram scale. As the next step, selective
deprotection of the acetonide group to install the required
precursor for the Corey–Winter elimination was envisaged.
Unfortunately, all our trials to chemoselectively deprotect
the acetonide failed. As a consequence, a protecting group
exchange was performed, yielding Troc-protected amine 22
(Scheme 7). With 22 in hand, microwave-assisted acidic
cleavage of the acetonide to the dihydroxy intermediate and
further conversion into thiocarbonate 23 with 1,1Ј-thio-
carbonyldiimidazole and DMAP provided the necessary
precursor for the Corey–Winter elimination. Elimination
due to the desired (E)-configured double bond of SylA core
Scheme 8. Synthesis of all four stereoisomers of the urea building
macrocycle 24 was then achieved by heating 23 in trimethyl
blocks. Reagents and conditions: (a) 30 (1 equiv.), DIEA (2 equiv.),
DCM, room temp., overnight, 90%; (b) ent-30 (1 equiv.), DIEA
(2.5 equiv.), DCM, room temp., 1 h, 91%; (c) wet HCO2H, 28:
91%, 29: 91%, 32: Ͼ98%, 34: 92%; (d) triphosgene (1.1 equiv.),
DIEA (2.2 equiv.), DCM, 5 min at room temp. then ()-valine
methyl ester hydrochloride (1 equiv.), DIEA (2.2 equiv.), DCM,
phosphite for 2.5 h at 130 °C.[26]
To complete the synthesis of SylA, the urea building
block for attachment to the macrocyclic core was also re-
quired. For further biological assays and to study the influ-
ence of the side-chain stereochemistry on the overall struc- 10 min at room temp., 31: 30%, 33: 76%.
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Zn-mediated cleavage of the Troc-protecting group of 24 Table 2. Yields for the last two steps of the synthesis of SylA deriva-
tives.
to free amine 35, followed by peptide coupling of the pre-
viously synthesized urea building blocks then led to the
SylA derivatives 36a–d (Scheme 9 and Table 2). Finally,
methyl ester cleavage under mild conditions with aluminum
tribromide and tetrahydrothiophene yielded the desired nat-
ural product SylA (37a) and three isomers thereof (i.e., 37b–
d).[27]
Scheme 9. Synthesis of four SylA stereoisomers. Reagents and con-
ditions: (a) Zn (150 equiv.), THF/AcOH (1:1), 3 h, Ͼ98%; (b) urea
28, 29, 32, or 34 (1.1 equiv.), PyBop (1.2 equiv.), HOAt (1.2 equiv.),
DIEA (2 equiv.), DMF, 0 °C to room temp. over 40 min, for yields
see Table 2; (c) AlBr3 (8 equiv.), tetrahydrothiophene, room temp.,
1 h, for yields see Table 2.
To gain some insight into the impact of the stereochemis-
try of the side chain on the structure of SylA, we investi-
gated the NMR spectra of 37a–d (Figure 2). It turned out
that as expected the α-protons of the exocyclic valine resi-
dues but interestingly also the signals of the β-olefinic pro-
ton of the β,γ-unsaturated lysine were most sensitive to the
stereochemical environment. All signals of the NMR spec-
trum of the “all ()” isomer 37a matched those of natural
SylA, as reported previously by us. In contrast, derivatives
37c and 37d, which feature a ()-valine residue adjacent to
the macrocycle displayed a shift of ≈0.48 ppm at the β-ole- Figure 2. Overlay of the characteristic part of the 1H NMR spectra
of 37a (SylA--), 37b (SylA--), 37c (SylA--), and 37d (SylA-
finic proton of the β,γ-unsaturated lysine, indicating that
-).
this modification has a considerable effect on the overall
arrangement of the molecule. The signals of the α-protons
of the exocyclic valine residues were also unique for each more active than SylA, the attachment of this lipid chain
isomer, resulting in an individual pattern for isomers 37a–d however reduced its overall water solubility and conse-
in the NMR spectra.
quently limited its pharmacokinetic suitability.
With our modified convergent synthesis in hand, we then
We therefore envisaged the synthesis of SylA derivatives
turned our attention to rationally designed derivatives of that featured a PEG moiety instead of the lipophilic chain
SylA. Previous studies revealed that the attachment of a of SylA-LIP, thereby hopefully retaining its potent bio-
lipophilic chain to the SylA core structure resulted in a de- logical activity but enhancing its water solubility. Conse-
rivative SylA-LIP with a significantly higher inhibition po- quently, an analogous PEG derivative of SylA-LIP was gen-
tency (Figure 1).[14] Although SylA-LIP was Ͼ100-fold erated, starting from triethylene glycol monomethyl ether
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Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors
38 (Scheme 10). Transformation of 38 into tosylate 39, fol-
lowed by nucleophilic displacement with sodium azide and
subsequent triphenylphosphane-mediated reduction led to
amine 40.[28] Reaction with disuccinimidyl carbonate then
resulted in the PEG succinimidyl carbamate 41 as a precur-
sor for urea formation.[29] To complete the synthesis of the
PEG derivative, macrocycle 35 was then coupled with Boc-
()-valine to yield 42. Subsequent Boc deprotection and
coupling with 41 then led to desired SylA-PEG1 derivative
43.
Scheme 11. Synthesis of SylA-PEG2 derivative 47. Reagents and
conditions: (a) TosCl (1 equiv.), Et3N (1.5 equiv.), DCM, 2 h at
0 °C then 30 min at room temp., 53%; (b) 1. NaN3 (2.5 equiv.),
DMF, 10 h at 67 °C; 2. PPh3 (1.1 equiv.), Et2O, 1 h at 0 °C then
90 min at room temp., 59%; (c) 37a (0.66 equiv.), PyBop
(0.8 equiv.), HOAt (0.8 equiv.), DIEA (2 equiv.), DMF, 0 °C to
room temp. over 40 min, 79%.
Table 3. Inhibition of the chymotryptic activity of human 20S pro-
teasome by SylA derivatives.
Entry
Urea
KiЈ C-L activity [nM]
1
2
3
4
5
6
7
37b (SylA--)
37c (SylA--)
37d (SylA--)
43 (SylA-PEG1)
44 (SylA-PEG2)
11117Ϯ2222
Ͼ100000
33962Ϯ11317
586Ϯ69
401Ϯ37
natural SylA (37a, SylA--) 843Ϯ8.4[a]
lipophilic SylA (SylA-LIP)
8.65Ϯ1.33[b]
[a] Literature values from ref.[12] [b] Literature values from ref.[14]
which the terminal valine residue was converted into a ()-
isomer displayed significantly reduced activity, being
roughly 10-times less potent than natural SylA. Structural
analysis of the SylA/yeast 20S proteasome complex has re-
vealed a pattern of hydrogen bonds between aspartate-144
of the proteasomal β6 subunit and the carboxyl moiety of
the terminal valine residue. It is therefore tempting to
speculate that the observed reduced activity of 37b versus
that of natural SylA (37a) is a consequence of a lack of
hydrogen bonds between these two residues. Nevertheless,
37b is still a potent proteasome inhibitor and could there-
fore still be used as a small molecule probe in plant biology
studies. Interestingly, PEG derivatives 43 and 47 proved to
be the most potent inhibitors of the small SylA collection,
and there were found to be slightly more potent than the
parent compound SylA but less potent than the lipophilic
SylA derivative SylA-LIP.
Scheme 10. Synthesis of SylA PEG derivative 43. Reagents and
conditions: (a) TosCl (1 equiv.), Et3N (1.5 equiv.), DCM, 2 h at
0 °C then 30 min at room temp., 76%; (b) 1. NaN3 (2.5 equiv.),
DMF, 10 h at 67 °C; 2. PPh3 (1.1 equiv.), Et2O, 1 h at 0 °C then
90 min at room temp., 82%; (c) DSC (2 equiv.), Et3N (2 equiv.),
DMF, room temp., 90 min, Ͼ98%; (d) Boc-()-valine (1.2 equiv.),
PyBop (1.5 equiv.), HOAt (1.5 equiv.), DIEA (2 equiv.), DMF, 0 °C
to room temp. over 1 h, 95%; (e) 1. 25% TFA/DCM, 30 min; 2. 41
(8 equiv.), DIEA (3 equiv.), DMF, 0 °C to room temp. overnight,
68%.
In addition, PEG derivative 47 featuring a shorter PEG
chain than 43 but with the dipeptide urea moiety of SylA
was synthesized (Scheme 11). To this end, first amine 46
was synthesized in an analogous manner as 40. Amine 46
was then coupled to SylA-- (37a) with PyBop/HOAt to
yield desired SylA-PEG2 derivative 47 in good yields.
To evaluate the biological activity of the newly synthe-
sized derivatives, biochemical activity assays with the use of
human 20S proteasome were performed and compared with
previously prepared SylA derivatives (Table 3).
Conclusions
In summary, a convergent synthesis of syringolin A and
As expected, natural SylA (37a) proved to be the most derivatives thereof has been developed that should enable
potent proteasome inhibitor of all four SylA isomers. Intro- rapid synthesis of SylA derivatives for further optimization
duction of a ()-configured valine, as in 37c or 37d, adja- of the proteasomal inhibition potency of SylA. To this end,
cent to the macrocycle however reduced significantly the a synthetic route has been built up that consists of the gen-
proteasome inhibition potency. Also, SylA derivative 37b in eration of the SylA macrocycle by RCM of a conformation-
Eur. J. Org. Chem. 2010, 3991–4003
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Kaiser et al.
FULL PAPER
Methyl (4S)-tert-Butoxycarbonylamino-(2S,3R)-dihydroxy-5-meth-
ylhexanoate (16): To a solution of 8 (643 mg, 2.50 mmol, 1 equiv.)
dissolved in acetone/water (2:1, 22.5 mL) in a 100-mL flask was
consecutively added 4-methylmorpholine N-oxide (440 mg,
3.75 mmol, 1.5 equiv.) and osmium tetroxide solution (4 wt.-%/
H2O, 764 µL, 125 µmol, 0.05 equiv.). The flask was flushed with
argon, and the reaction mixture was stirred for 2 d. The reaction
was quenched by the addition of a saturated aqueous NaHSO3
solution, and the acetone was evaporated in vacuo. Ethyl acetate
and further water were added, the layers were separated in a funnel,
ally preorganized precursor. The resulting fully function-
alized macrocycle can then be decorated with various pep-
tide chains for derivatization. Using this approach, four
SylA isomers were prepared and subsequently tested for in-
hibition, thereby verifying the previous stereochemical as-
signment of SylA and probing the impact of stereochemis-
try on its biological activity. In addition, two PEGylated
derivatives were generated that proved to be more potent
than natural SylA. Subsequent plant biology studies with
these derivatives are currently being pursued and will be and the organic layer was dried with Na2SO4, filtered through Ce-
lite, and concentrated to dryness to give a crude mixture of dia-
stereoisomers. Product 16 (583 mg, 2.00 mmol, 80%) was obtained
by recrystallization (cyclohexane) as a pure single diastereoisomer
as colorless crystals. The residual mixture was then purified by
flash column chromatography (70% diethyl ether in petroleum
ether) to afford another portion of 16 (38 mg, 0.13 mmol, 5%) as
colorless crystals (overall yield of 85%). TLC (70% diethyl ether in
reported in due course.
Experimental Section
General Methods: Unless otherwise noted, all reagents and solvents
were purchased from Acros, Fluka, Sigma–Aldrich, or Merck and
used without further purification. Dry solvents were purchased as
anhydrous reagents from commercial suppliers. LC–MS analyses
were performed with an HPLC system from Agilent (1200 series)
with an Eclipse XDB-C18, 5 µm (column dimensions:
150ϫ4.60 mm) column from Agilent and a Thermo Finnigan LCQ
Advantage Max ESI-Spectrometer. Two gradients were used for the
analyses using H2O with 0.1% formic acid (solvent A) and acetoni-
trile with 0.1% formic acid (solvent B) at a flow rate of 1 mLmin–1.
Gradient 1: from 0 to 1 min: 75% solvent A/25% solvent B; from
1 to 10 min: from 75% solvent A/25% solvent B to 0% solvent A/
100% solvent B; from 10 to 12 min: 0% solvent A/100% solvent B;
from 12 to 15 min: from 0% solvent A/100% solvent B to 90% sol-
vent A/10% solvent B. Gradient 2: from 0 to 1 min: 90% solvent A/
10% solvent B; from 1 to 10 min: from 90% solvent A/10% sol-
vent B to 0% solvent A/100% solvent B; from 10 to 12 min: 0%
solvent A/100% solvent B; from 12 to 15 min: from 0% solvent A/
100% solvent B to 90% solvent A/10% solvent B. Preparative
HPLC was conducted with a Varian HPLC system (Pro Star 215)
with a VP 250/21 Nucleosil C18PPN-column from Macherey-Na-
gel. The corresponding gradients are described in the synthesis sec-
tion. NMR spectra were recorded with a Varian Mercury 400 sys-
tem (400 and 100 MHz for 1H and 13C NMR, respectively), a
Bruker Avance DRX 500 system (500 and 125 MHz for 1H and
13C NMR, respectively), or a Varian Unity Inova 600 system (600
1
petroleum ether): Rf = 0.23. HPLC (gradient 2): tR = 7.55 min. H
NMR (400 MHz, CDCl3): δ = 4.78 (d, J = 10.0 Hz, 1 H), 4.32 (br.
s, 1 H), 4.01 (br. s, 1 H), 3.81–3.86 (m, 1 H), 3.81 (s, 3 H), 3.52–
3.59 (m, 1 H), 2.59 (br. s, 1 H), 2.08–2.18 (m, 1 H), 1.43 (s, 9 H),
0.98 (d, J = 6.8 Hz, 3 H), 0.91 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 173.04, 157.49, 80.36, 72.16, 71.17, 57.35,
52.73, 28.38, 27.99, 20.11, 16.67 ppm. HRMS (ESI): calcd. for
C13H25O6NH+ [M + H]+ 292.1755; found 292.1757.
Methyl (5R)-[(1S)-tert-Butoxycarbonylamino-2-methylpropyl]-2,2-
dimethyl-[1,3]dioxolane-(4S)-carboxylate (17): To a solution of 16
(3.53 g, 12.12 mmol, 1 equiv.) dissolved in dichloromethane
(45 mL) in a 250-mL, flame-dried flask was added 2,2-dimeth-
oxypropane (45 mL, 364.00 mmol, 30 equiv.) and pyridinium p-tol-
uenesulfonate (153 mg, 0.61 mmol, 0.05 equiv.). The flask was
flushed with argon, and the solution was heated to reflux for 5 h.
After evaporation to dryness, desired product 17 (3.93 g,
11.88 mmol, Ͼ98%) was obtained as a colorless solid. TLC (15%
ethyl acetate in cyclohexane): Rf = 0.26. HPLC (gradient 1): tR
=
1
9.78 min. H NMR (400 MHz, CDCl3): δ = 4.46 (d, J = 6.0 Hz, 1
H), 4.41–4.45 (m, 1 H), 4.11 (dd, J = 9.2, 6.4 Hz, 1 H), 3.76 (s, 3
H), 3.69–3.76 (m, 1 H), 2.09 (sept.d, J = 6.8, 3.6 Hz, 1 H), 1.44 (s,
3 H), 1.42 (s, 9 H), 1.41 (s, 3 H), 0.94 (d, J = 6.8 Hz, 3 H), 0.86 (d,
J = 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.87,
156.07, 111.88, 79.50, 77.97, 57.50, 52.47, 28.52, 28.39, 27.23,
26.02, 19.83, 15.70 ppm. HRMS (ESI): calcd. for C16H29O6NH+
[M + H]+ 332.2068; found 332.2069.
1
1
and 150 MHz for H and 13C NMR, respectively). H NMR spec-
tra are reported in the following manner: chemical shifts calculated
with reference to solvent standards based on tetramethylsilane,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept., sep-
tuplet; m, multiplet), coupling constant(s), and number of protons.
TLC analyses were performed with TLC aluminum sheets
20ϫ20 cm silica gel 60 F254 from Merck. HRMS measurements
were performed with a LC–HRMS (ESI-FT) machine from
Thermo Electron Corporation. Microwave-assisted reactions were
conducted by using a focused microwave unit (Discover® Reactor
from CEM Corporation). The instrument consists of a continuous
tert-Butyl [(1S)-{(5R)-But-3-enylcarbamoyl-2,2-dimethyl-[1,3]diox-
olan-4S-yl}-2-methylpropyl]carbamate (18): To a solution of 17
(1.40 g, 4.23 mmol, 1 equiv.) dissolved in methanol/water (1:1,
20 mL) in a 50-mL flask was added a 1  aq. lithium hydroxide
solution (13 mL, 533 mg, 12.69 mmol, 3 equiv.) at 0 °C. The mix-
ture was stirred for 30 min at room temperature. After evaporation
of methanol, a 20% aq. citric acid solution was added to acidify the
reaction mixture. The mixture was extracted with dichloromethane
(3ϫ50 mL), and the organic layer was dried with Na2SO4, filtered,
focused microwave power delivery system with operator-selectable and concentrated to yield the cleaved intermediate (1.31 g,
power output from 0–300 W. In all experiments, the microwave 4.15 mmol, Ͼ98%) as a white powder. To this intermediate (1.33 g,
power was held constant to ensure reproducibility. Reactions were
performed in 10-mL glass vessels, which were sealed with a septum
and locked into a pressure device, which controlled the pressure in
the reaction vessel (maximum 10 bar). The specified reaction time
corresponds to the total irradiation time. The temperature was
monitored by an infrared temperature sensor positioned below the
reaction vessel. The indicated temperature correlates with the maxi-
mum temperature reached during each experiment.
4.20 mmol, 1 equiv.) was then added a solution of 3-butenylamine
hydrochloride (0.54 g, 5.10 mmol, 1.2 equiv.), HOAt (858 mg,
6.30 mmol, 1.5 equiv.), PyBop (3.28 g, 6.30 mmol, 1.5 equiv.) dis-
solved in dichloromethane (5 mL) in a 10-mL flask. N,N-Diiso-
propylethylamine (1.46 mL, 8.40 mmol, 2 equiv.) was added at 0 °C,
and the resulting mixture was stirred overnight at room tempera-
ture. The reaction was stopped by quenching with a 20% aq. citric
acid solution, and 18 was extracted from the mixture with chloro-
3998
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 3991–4003

Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors
form (3 ϫ50 mL). The combined organic layer was dried with
Na2SO4, filtered, and concentrated. The crude product was purified
by flash column chromatography (20% ethyl acetate in cyclohex-
ane) to afford 18 (1.27 g, 3.43 mmol, 82%) as a colorless solid. TLC
(30% ethyl acetate in cyclohexane): Rf = 0.49. HPLC (gradient 2):
dition of ethyl acetate (50 mL) and a 10% aqueous KHSO4 solu-
tion (50 mL) generated a biphasic mixture, which was separated in
a funnel. The organic phase was washed with a 5% aqueous
NaHCO3 solution (50 mL) and brine (50 mL), dried with Na2SO4,
filtered, and concentrated. The crude product was purified by flash
tR = 10.79 min. 1H NMR (400 MHz, CDCl3): δ = 6.63 (br. s, 1 H), column chromatography (20% ethyl acetate in cyclohexane) to af-
5.75 (ddt, J = 17.2, 10.4, 6.8 Hz, 1 H), 5.19 (d, J = 9.2 Hz, 1 H), ford 20 (861 mg, 1.90 mmol, 93%) as a colorless solid. TLC (25%
5.06–5.13 (m, 2 H), 4.30 (d, J = 6.0 Hz, 1 H), 4.06 (dd, J = 9.2,
6.0 Hz, 1 H), 3.64–3.73 (m, 1 H), 3.37–3.46 (m, 1 H), 3.24–3.33 (m, 10.27 min. H NMR (400 MHz, CDCl3): δ = 6.95 (d, J = 8.8 Hz,
1 H), 2.24–2.31 (m, 2 H), 2.01–2.11 (m, 1 H), 1.45 (s, 3 H), 1.43 (s, 1 H), 6.67 (t, J = 5.6 Hz, 1 H), 5.93 (ddd, J = 17.2, 10.4, 6.4 Hz, 1
ethyl acetate in cyclohexane): Rf = 0.16. HPLC (gradient 2): tR =
1
9 H), 1.36 (s, 3 H), 0.96 (d, J = 7.2 Hz, 3 H), 0.87 (d, J = 7.2 Hz, 3
H), 5.76 (ddt, J = 17.6, 9.6, 6.8 Hz, 1 H), 5.49 (br. s, 1 H), 5.38
(ddd, J = 17.2, 1.2, 1.2 Hz, 1 H), 5.23 (ddd, J = 10.4, 1.2, 1.2 Hz,
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.40, 156.60, 135.05,
117.49, 111.36, 79.17, 78.36, 58.11, 37.99, 33.77, 29.59, 28.47, 1 H), 5.08–5.14 (m, 2 H), 4.67 (br. s, 1 H), 4.15 (d, J = 6.4 Hz, 1
27.28, 26.32, 19.81, 16.25 ppm. HRMS (ESI): calcd. for
H), 4.06 (dd, J = 9.2, 6.8 Hz, 1 H), 3.98 (ddd, J = 9.2, 3.6, 3.6 Hz,
1 H), 3.36–3.45 (m, 1 H), 3.25–3.34 (m, 1 H), 2.25–2.31 (m, 2 H),
2.09 (sept.d, J = 6.8, 3.6 Hz, 1 H), 1.45 (s, 3 H), 1.44 (s, 9 H), 1.36
(s, 3 H), 0.94 (d, J = 7.2 Hz, 3 H), 0.90 (d, J = 7.2 Hz, 3 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 171.31, 170.21, 155.08, 134.78,
134.20, 117.41, 117.22, 111.33, 79.61, 78.45, 78.02, 57.51, 56.89,
37.90, 33.59, 30.03, 28.29, 26.90, 25.82, 19.50, 16.49 ppm. HRMS
(ESI): calcd. for C23H39O6N3H+ [M + H]+ 454.2912; found
454.2906.
C19H34O5N2H+ [M + H]+ 371.2541; found 371.2540.
tert-Butyl {(1S)-[(1S)-{(5R)-But-3-enylcarbamoyl-2,2-dimethyl-[1,3]-
dioxolan-(4S)-yl}-2-methylpropylcarbamoyl]-3-phenylselanylpropyl}-
carbamate (19): To a solution of 18 (710 mg, 1.92 mmol, 1 equiv.)
dissolved in dichloromethane (2 mL) under an atmosphere of argon
in a 10-mL flame-dried flask was added 2,6-lutidine (446 µL,
3.84 mmol, 2 equiv.) and trimethylsilyl trifluoromethanesulfonate
(522 µL, 2.88 mmol, 1.5 equiv.), and the resulting mixture was
stirred for 15 min. The reaction was quenched upon addition of a
saturated aqueous NH4Cl solution. The pH of the water phase was
adjusted to 9 by addition of a 2  aqueous NaOH solution and
was extracted with dichloromethane. The combined organic layer
was washed with brine, dried with Na2SO4, filtered, and concen-
trated to yield the deprotected intermediate (508 mg, 1.88 mmol,
Ͼ98%) as a white powder. This intermediate (512 mg, 1.90 mmol,
1 equiv.), 12 (878 mg, 2.45 mmol, 1.3 equiv.), PyBop (1.48 g,
2.85 mmol, 1.5 equiv.), and HOAt (388 mg, 2.85 mmol, 1.5 equiv.)
were then dissolved in dichloromethane (10 mL) in a 25-mL flask.
The solution was cooled to 0 °C and N,N-diisopropylethylamine
(662 µL, 3.80 mmol, 2 equiv.) was added. The reaction was stirred
overnight at room temperature, quenched by addition of a 20%
aqueous citric acid solution, and extracted with chloroform
(3ϫ50 mL). The combined organic layer was dried with Na2SO4,
filtered, and concentrated. The crude product was purified by flash
column chromatography (20% ethyl acetate in cyclohexane) to af-
ford 19 (1.03 g, 1.69 mmol, 89%) as a colorless solid. TLC (25%
21: A solution of 20 (737 mg, 1.620 mmol, 1 equiv.) dissolved in
toluene (800 mL) under an atmosphere of argon in a 1-L flame-
dried flask was heated to 90 °C. A solution of Grubbs 2nd genera-
tion catalyst (207 mg, 0.243 mmol, 0.15 equiv.) in toluene (25 mL)
was added over 8 h with a syringe pump to the preheated mixture.
The resulting solution was stirred for 10 h at 90 °C. After concen-
tration to dryness, the crude product was purified by flash column
chromatography (50% ethyl acetate in cyclohexane) to afford 21
(335 mg, 0.787 mmol, 49%) as a light-brown solid. The product
was pure enough to be used in the next step without further purifi-
cation. Nevertheless, a second flash column chromatography was
performed to completely eliminate the remaining trace amounts of
ruthenium residues. TLC (60% ethyl acetate in cyclohexane): Rf =
0.29. HPLC (gradient 2): tR = 8.56 min. 1H NMR (400 MHz,
CDCl3): δ = 5.63–5.77 (m, 2 H), 5.47–5.55 (m, 2 H), 5.12 (ddd, J
= 15.2, 10.0, 0.8 Hz, 1 H), 4.46 (dd, J = 10.0, 8.4 Hz, 1 H), 4.34 (t,
J = 8.0 Hz, 1 H), 3.83–3.95 (m, 2 H), 3.68 (d, J = 8.0 Hz, 1 H),
2.82–2.90 (m, 1 H), 2.36–2.44 (m, 1 H), 1.98–2.04 (m, 1 H), 1.83–
1.94 (m, 1 H), 1.36 (s, 9 H), 1.34 (s, 3 H), 1.33 (s, 3 H), 0.88 (d, J
= 6.8 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 170.86, 169.67, 154.82, 131.54, 130.49,
ethyl acetate in cyclohexane): Rf = 0.27. HPLC (gradient 2): tR
11.68 min. H NMR (400 MHz, CDCl3): δ = 7.42–7.46 (m, 2 H),
7.17–7.23 (m, 3 H), 6.97 (d, J = 8.4 Hz, 1 H), 6.64 (t, J = 5.6 Hz,
=
1
1 H), 5.72 (ddt, J = 17.6, 9.6, 6.8 Hz, 1 H), 5.19 (d, J = 8.0 Hz, 1 111.65, 79.93, 79.81, 76.14, 58.04, 56.71, 37.07, 34.62, 30.09, 28.43,
H), 5.04–5.10 (m, 2 H), 4.16–4.25 (m, 1 H), 4.10 (d, J = 6.8 Hz, 1 26.90, 26.27, 19.75, 15.98 ppm. HRMS (ESI): calcd. for
H), 4.03 (dd, J = 8.8, 6.8 Hz, 1 H), 3.96 (ddd, J = 8.8, 3.6, 3.6 Hz,
1 H), 3.33–3.42 (m, 1 H), 3.17–3.26 (m, 1 H), 2.83–2.90 (m, 2 H),
2.16–2.28 (m, 3 H), 2.07 (sept.d, J = 6.8, 3.6 Hz, 1 H), 1.91–2.00
(m, 1 H), 1.42 (s, 3 H), 1.40 (s, 9 H), 1.33 (s, 3 H), 0.90 (d, J =
6.8 Hz, 3 H), 0.87 (d, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 171.58, 171.15, 155.38, 134.87, 132.51, 130.08, 129.01,
126.79, 117.49, 111.29, 79.76, 78.41, 78.24, 56.85, 54.96, 37.90,
33.64, 32.98, 29.90, 28.33, 26.95, 25.88, 23.22, 19.57, 16.40 ppm.
HRMS (ESI): calcd. for C29H45O6N380SeH+ [M + H]+ 612.2546;
found 612.2543. HRMS (ESI): calcd. for C29H45O6N378SeH+ [M +
H]+ 610.2554; found 610.2558.
C21H35O6N3H+ [M + H]+ 426.2599; found 426.2596.
22: To a solution of 21 (295 mg, 0.69 mmol, 1 equiv.) dissolved in
dichloromethane (4 mL) under an atmosphere of argon in a 10-
mL flame-dried flask was added 2,6-lutidine (161 µL, 1.38 mmol,
2 equiv.) and trimethylsilyl trifluoromethanesulfonate (188 µL,
1.04 mmol, 1.5 equiv.) at room temperature, and the resulting mix-
ture was stirred for 15 min. Addition of a saturated aqueous NH4Cl
solution quenched the reaction. The pH was adjusted to 9 by ad-
dition of a 2  NaOH solution, and the desired product was ex-
tracted from the water phase with dichloromethane. The combined
organic layer was washed with brine, dried with Na2SO4, filtered,
tert-Butyl {(1S)-[(1S)-{(5R)-But-3-enylcarbamoyl-2,2-dimethyl- and concentrated to yield the deprotected intermediate (221 mg,
[1,3]dioxolan-4S-yl}-2-methylpropylcarbamoyl]allyl}carbamate (20): 0.68 mmol, 98%) as a white solid. This intermediate (166 mg,
To a solution of 19 (925 mg, 2.04 mmol) dissolved in dichlorometh-
ane (85 mL) in a 250-mL flask. was added hydrogen peroxide (30%
in water, 10 mL) and N,N-diisopropylethylamine (10 mL), and the
resulting mixture was heated to 50 °C for 3 h. The reaction was
quenched by addition of a saturated aqueous CuSO4 solution. Ad-
510 µmol, 1 equiv.) and sodium hydrogen carbonate (86 mg,
1.02 mmol, 2 equiv.) were then dissolved under an atmosphere of
argon in tetrahydrofuran (15 mL) in a 25-mL flame-dried flask.
2,2,2-Trichloroethyl chloroformate (76 µL, 560 µmol, 1.1 equiv.)
was added dropwise at 0 °C, and the resulting mixture was stirred
Eur. J. Org. Chem. 2010, 3991–4003
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3999

M. Kaiser et al.
for 90 min at room temperature. The solvents were removed in 24: A solution of 23 (70 mg, 139 µmol, 1 equiv.) dissolved in tri-
FULL PAPER
vacuo, and the remaining residue was partitioned between a satu-
rated ammonium chloride solution (50 mL) and dichloromethane
(50 mL). The aqueous layer was extracted three more times with
dichloromethane, and the combined organic layer was finally dried
with Na2SO4. After concentration to dryness, the crude product
was purified by flash column chromatography (50% ethyl acetate
in cyclohexane) to afford 22 (212 mg, 423 µmol, 83%) as a colorless
solid. TLC (50% ethyl acetate in cyclohexane): Rf = 0.40. HPLC
methyl phosphite (1.0 mL) under an atmosphere of argon in a 10-
mL flame-dried flask was heated at reflux in a prewarmed oil bath
at 130 °C for 2.5 h. After concentration to dryness, the crude prod-
uct was purified by flash column chromatography (6% methanol
in dichloromethane) to yield 24 (52 mg, 122 µmol, 88%) as a color-
less solid. Importantly, to assure high yields during this reaction, a
minimal amount of trimethyl phosphite should be employed in the
reaction, thereby facilitating precipitation of desired product 24.
1
(gradient 2): tR = 9.32 min. H NMR (400 MHz, [D6]DMSO): δ =
7.99 (d, J = 9.7 Hz, 1 H), 7.90 (d, J = 6.3 Hz, 1 H), 7.54 (d, J = ent 2): tR = 7.79 min. H NMR (400 MHz, [D6]DMSO): δ = 7.96–
TLC (6% methanol in dichloromethane): Rf = 0.30. HPLC (gradi-
1
10.2 Hz, 1 H), 5.61 (ddd, J = 14.8, 11.2, 3.8 Hz, 1 H), 5.40 (dd, J
= 15.1, 9.6 Hz, 1 H), 4.82 (d, J = 12.4 Hz, 1 H), 4.69 (d, J =
12.4 Hz, 1 H), 4.54 (dd, J = 9.6, 6.3 Hz, 1 H), 4.35 (dd, J = 10.5,
8.05 (m, 2 H), 7.45 (t, J = 7.1 Hz, 1 H), 6.69 (dd, J = 15.5, 5.5 Hz,
1 H), 6.08 (d, J = 15.6 Hz, 1 H), 5.66 (dt, J = 15.4, 7.7 Hz, 1 H),
5.38 (dd, J = 15.9, 7.9 Hz, 1 H), 4.82 (d, J = 12.4 Hz, 1 H), 4.75
7.7 Hz 1 H), 3.82 (ddd, J = 10.4, 10.2, 3.4 Hz, 1 H), 3.73 (d, J = (d, J = 12.3 Hz, 1 H), 4.67 (dd, J = 7.9, 7.7 Hz, 1 H), 4.03–4.11
7.6 Hz, 1 H), 3.66–3.77 (m, 1 H), 2.70–2.78 (m, 1 H), 2.22–2.30 (m, (m, 1 H), 3.08–3.22 (m, 2 H), 2.26–2.35 (m, 1 H), 1.90–2.01 (m, 1
1 H), 1.88–2.00 (m, 2 H), 1.33 (s, 3 H), 1.32 (s, 3 H), 0.86 (d, J = H), 1.69–1.79 (m, 1 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.91 (d, J =
6.9 Hz, 3 H), 0.79 (d, J = 6.9 Hz, 3 H) ppm. 13C NMR (100 MHz,
[D6]DMSO): δ = 171.17, 167.69, 153.46, 130.41, 130.23, 109.63,
96.14, 79.82, 76.71, 73.32, 57.56, 55.11, 35.57, 32.32, 29.31, 26.73,
6.6 Hz, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 168.97,
166.21, 153.91, 143.29, 133.53, 124.90, 121.56, 96.03, 73.48, 56.07,
55.50, 42.29, 35.07, 31.43, 19.70, 19.20 ppm. HRMS (ESI): calcd.
for C16H22O4N3Cl3H+ [M + H]+ 426.0749; found 426.0748. HRMS
(ESI): calcd. for C16H22O4N3Cl237ClH+ [M + H]+ 428.0719; found
25.94,
19.44,
15.79 ppm.
HRMS
(ESI):
calcd.
for
C19H28O6N3Cl3H+ [M + H]+ 500.1117; found 500.1113. HRMS
(ESI): calcd. for C19H28O6N3Cl237ClH+ [M + H]+ 502.1087; found 428.0717.
502.1082.
(8S)-Amino-(5S)-isopropyl-1,6-diazacyclododeca-(3E,9E)-diene-2,7-
23: In a 10-mL vessel was placed 22 (20 mg, 40 µmol, 1 equiv.), p-
toluenesulfonic acid monohydrate (7.6 mg, 40 µmol, 1 equiv.),
methanol/water/tetrahydrofuran (2:2:1, 5 mL), and a magnetic stir-
ring bar. The vessel was sealed with a septum, placed into the mi-
crowave cavity and locked with the pressure device. Constant mi-
crowave irradiation of 150 W as well as simultaneous air-cooling
(300 kPa, 45 Psi) were used during the entire reaction time (30 min,
140 °C, resulting reaction pressure 12 bar). After cooling to room
temperature, the solvent was removed under reduced pressure to
afford the dihydroxy intermediate (18 mg, 39 µmol, Ͼ98%) as a
colorless solid. The product was pure enough to be used in the next
step without further purification. After performing this reaction
several times, all product fractions were pooled, and the resulting
residue of the dihydroxy derivative (124 mg, 269 µmol, 1 equiv.) was
dissolved in tetrahydrofuran (80 mL) under an atmosphere of ar-
gon in a 250-mL flame-dried flask. To this solution was added 1,1Ј-
thiocarbonyl diimidazole (480 mg, 2.69 mmol, 10 equiv.) and 4-(di-
methylamino)pyridine (329 mg, 2.69 mmol, 10 equiv.). The re-
sulting reaction mixture was heated to 80 °C and stirred at this
temperature overnight. After cooling to room temperature, a small
portion of silica gel was added, and the solvent was removed under
reduced pressure. The adsorbed crude product was purified by flash
column chromatography (50% ethyl acetate in cyclohexane) to
yield 23 (119 mg, 237 µmol, 88%) as a colorless solid. TLC (50%
dione (35): Compound 24 (34 mg, 81 µmol, 1 equiv.) was dissolved
in tetrahydrofuran (1 mL) in a 10-mL flask. Acetic acid was added
(1 mL), followed by zinc powder (798 mg, 12.2 mmol, 150 equiv.),
which was added in portions over 30 min. After 3 h of vigorous
stirring, the mixture was filtered through a small plug of Celite and
washed with ethyl acetate. After evaporation to dryness, 35 (20 mg,
79 µmol, 98%) was obtained as a colorless solid. TLC (1% triethyl-
amine + 5% methanol in dichloromethane): Rf = 0.31. HPLC (gra-
dient 2): tR = 2.06 min; NMR (400 MHz, [D6]DMSO): δ = 8.42 (d,
J = 8.3 Hz, 1 H), 7.48 (t, J = 6.9 Hz, 1 H), 6.73 (dd, J = 15.5,
5.2 Hz, 1 H), 6.05 (d, J = 15.6 Hz, 1 H), 5.68 (dt, J = 15.8, 7.8 Hz,
1 H), 5.50 (d, J = 15.8, 7.5 Hz, 1 H), 4.30 (d, J = 7.4 Hz, 1 H),
4.10–4.17 (m, 1 H), 3.10–3.31 (m, 2 H), 2.27–2.37 (m, 1 H), 1.94–
2.05 (m, 1 H), 1.70–1.80 (m, 1 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.92
(d, J = 6.8 Hz, 3 H) ppm. HRMS (ESI): calcd. for C13H21O2N3H+
[M + H]+ 252.1707; found 252.1708.
SylA-L-L Methyl Ester (36a): Compound 28 (14 mg, 50 µmol,
1.1 equiv.), 35 (11.6 mg, 46 µmol, 1 equiv.), PyBop (30 mg,
56 µmol, 1.2 equiv.), and HOAt (8 mg, 56 µmol, 1.2 equiv.) were
dissolved in N,N-dimethylformamide (1.0 mL) in a 10-mL flask.
The solution was cooled to 0 °C and N,N-diisopropylethylamine
(16 µL, 92 µmol, 2 equiv.) was added. The reaction was stirred for
40 min at room temperature. After concentration to dryness, the
crude product was purified by flash column chromatography (10%
ethyl acetate in cyclohexane): Rf = 0.30. HPLC (gradient 2): tR
=
9.29 min. 1H NMR (400 MHz, [D6]DMSO): δ = 8.52 (d, J = methanol in dichloromethane) to yield 36a (22.2 mg, 44 µmol,
9.8 Hz, 1 H), 8.07 (d, J = 6.1 Hz, 1 H), 7.82 (d, J = 10.4 Hz, 1 H), 95%) as a colorless solid. TLC (8% methanol in dichloromethane):
1
5.62 (ddd, J = 14.8, 11.1, 3.5 Hz, 1 H), 5.48 (dd, J = 15.2, 9.5 Hz, Rf = 0.19. HPLC (gradient 2): tR = 6.84 min. H NMR (500 MHz,
1 H), 4.98 (dd, J = 10.5, 9.9 Hz, 1 H), 4.84 (d, J = 12.4 Hz, 1 H),
4.74 (d, J = 9.6 Hz, 1 H), 4.70 (d, J = 12.4 Hz, 1 H), 4.51 (dd, J =
9.4, 6.2 Hz, 1 H), 4.23 (ddd, J = 10.5, 10.4, 3.8 Hz, 1 H), 3.63–3.76
[D6]DMSO): δ = 8.04 (d, J = 7.2 Hz, 1 H), 8.00 (d, J = 8.7 Hz, 1
H), 7.44 (t, J = 7.1 Hz, 1 H), 6.68 (dd, J = 15.5, 5.5 Hz, 1 H), 6.42
(d, J = 8.8 Hz, 1 H), 6.22 (d, J = 9.1 Hz, 1 H), 6.09 (d, J = 15.5 Hz,
(m, 1 H), 2.90 (dd, J = 12.8, 4.6 Hz, 1 H), 2.29–2.36 (m, 1 H), 1 H), 5.60 (dt, J = 15.6, 7.7 Hz, 1 H), 5.41 (dd, J = 15.9, 7.7 Hz, 1
1.92–2.06 (m, 2 H), 0.90 (d, J = 6.9 Hz, 3 H), 0.84 (d, J = 6.8 Hz, H), 4.86 (t, J = 7.4 Hz, 1 H), 4.00–4.11 (m, 3 H), 3.61 (s, 3 H),
3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 190.08, 171.56,
3.10–3.24 (m, 2 H), 2.24–2.32 (m, 1 H), 1.86–2.02 (m, 3 H), 1.69–
163.17, 153.59, 130.49, 130.36, 96.08, 83.69, 82.58, 73.37, 57.73, 1.78 (m, 1 H), 0.95 (d, J = 6.7 Hz, 3 H), 0.90 (d, J = 6.7 Hz, 3 H),
53.56, 36.27, 31.84, 29.07, 19.06, 15.65 ppm. HRMS (ESI): calcd.
0.82–0.87 (m, 9 H), 0.78 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR
(125 MHz, [D6]DMSO): δ = 190.46, 176.03, 174.32, 173.30, 172.93,
164.17, 131.91, 130.03, 83.95, 82.19, 58.59, 58.21, 56.74, 54.93,
52.26, 37.01, 32.75, 31.29, 29.21, 26.78, 19.24, 19.11, 18.95, 18.10,
for C17H22O6N3Cl3SH+ [M + H]+ 502.0368; found 502.0365.
HRMS (ESI): calcd. for C17H22O6N3Cl237ClSH+ [M
504.0338; found 504.0331.
+
H]+
4000
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 3991–4003

Syringolin A and Derivatives as Eukaryotic 20S Proteasome Inhibitors
17.64, 15.41 ppm. HRMS (ESI): calcd. for C25H41O6N5H+ [M +
H]+ 508.3130; found 508.3134.
(dd, J = 15.4, 5.3 Hz, 1 H), 6.42 (d, J = 8.7 Hz, 1 H), 6.26 (d, J =
9.1 Hz, 1 H), 6.10 (d, J = 15.5 Hz, 1 H), 5.55–5.64 (m, 1 H), 5.34–
5.42 (m, 1 H), 4.87–4.93 (m, 1 H), 4.12 (dd, J = 9.1, 5.6 Hz, 1 H),
4.05–4.11 (m, 1 H), 4.03 (dd, J = 8.7, 5.3 Hz, 1 H), 3.61 (s, 3 H),
3.09–3.24 (m, 2 H), 2.23–2.31 (m, 1 H), 1.84–2.01 (m, 3 H), 1.69–
1.77 (m, 1 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.91 (d, J = 6.6 Hz, 3 H),
0.86 (d, J = 6.8 Hz, 3 H), 0.84 (d, J = 6.9 Hz, 3 H), 0.80 (d, J =
6.7 Hz, 3 H), 0.77 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR (125 MHz,
[D6]DMSO): δ = 172.98, 171.24, 169.03, 166.25, 157.41, 143.20,
133.11, 126.03, 121.46, 57.82, 57.27, 55.44, 53.52, 51.37, 42.52,
34.98, 31.36, 30.18, 19.66, 19.19, 19.08, 19.02, 17.79, 17.44 ppm.
HRMS (ESI): calcd. for C25H41O6N5H+ [M + H]+ 508.3130; found
508.3127.
SylA-L-D Methyl Ester (36b): Compound 32 (7 mg, 25 µmol,
1.1 equiv.), 35 (5.8 mg, 23 µmol, 1 equiv.), PyBop (15 mg, 28 µmol,
1.2 equiv.), and HOAt (4 mg, 28 µmol, 1.2 equiv.) were dissolved in
N,N-dimethylformamide (1.0 mL) in a 10-mL flask. The solution
was cooled to 0 °C and N,N-diisopropylethylamine (8 µL, 46 µmol,
2 equiv.) was added. The reaction was stirred for 40 min at room
temperature. After concentration to dryness, the crude product was
purified by flash column chromatography (10% methanol in
dichloromethane) to yield 36b (10.1 mg, 20 µmol, 87%) as a color-
less solid. TLC (8% methanol in dichloromethane): Rf = 0.30.
HPLC (gradient 2): tR = 6.93 min. 1H NMR (500 MHz, [D6]-
DMSO): δ = 8.10 (d, J = 7.0 Hz, 1 H), 8.01 (d, J = 8.2 Hz, 1 H),
7.43 (t, J = 6.9 Hz, 1 H), 6.68 (dd, J = 15.5, 5.4 Hz, 1 H), 6.51 (d,
J = 8.9 Hz, 1 H), 6.27 (d, J = 9.2 Hz, 1 H), 6.10 (d, J = 15.5 Hz,
1 H), 5.55–5.64 (m, 1 H), 5.42 (dd, J = 16.0, 7.7 Hz, 1 H), 4.86–
4.92 (m, 1 H), 4.05–4.12 (m, 3 H), 3.62 (s, 3 H), 3.09–3.24 (m, 2
H), 2.24–2.32 (m, 1 H), 1.85–2.02 (m, 3 H), 1.70–1.78 (m, 1 H),
0.95 (d, J = 6.6 Hz, 3 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.85 (d, J =
6.9 Hz, 3 H), 0.83 (d, J = 6.5 Hz, 3 H), 0.82 (d, J = 6.5 Hz, 3 H),
0.77 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR (125 MHz, [D6]DMSO):
δ = 173.04, 171.33, 168.88, 166.20, 157.24, 143.12, 133.08, 125.81,
121.45, 57.45, 57.10, 55.42, 53.55, 51.43, 42.51, 34.98, 31.41, 31.36,
30.58, 19.70, 19.14, 19.09, 18.87, 17.64, 17.47 ppm. HRMS (ESI):
calcd. for C25H41O6N5H+ [M + H]+ 508.3130; found 508.3126.
SylA-L-L (37a): Compound 36a (20.0 mg, 39.4 µmol, 1 equiv.) and
aluminum bromide (84 mg, 316 µmol, 8 equiv.) were dissolved in
tetrahydrothiophene (2 mL) under an atmosphere of argon in a 10-
mL flame-dried flask. The resulting mixture was stirred for 1 h at
room temperature. After concentration to dryness, the remaining
residue was purified by preparative HPLC (using H2O with 0.1%
TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B) at a
flow rate of 25 mLmin–1. Gradient: from 0 to 10 min: 90% sol-
vent A/10% solvent B; from 10 to 30 min: from 90% solvent A/
10% solvent B to 70% solvent A/30% solvent B; from 30 to 50 min:
from 70% solvent A/30% solvent B to 40% solvent A/60% sol-
vent B; from 50 to 60 min: from 40% solvent A/60% solvent B to
0% solvent A/100% solvent B; from 60 to 80 min: 0% solvent A/
100% solvent B) to yield 37a (SylA--; 16.3 mg, 33.1 µmol, 84%)
as a colorless solid. TLC (2% acetic acid + 15% methanol in
dichloromethane): Rf = 0.32. HPLC (gradient 2): tR = 6.13 min. 1H
NMR (400 MHz, [D6]DMSO): δ = 12.35 (br. s, 1 H), 8.03 (d, J =
8.4 Hz, 1 H), 7.99 (d, J = 6.7 Hz, 1 H), 7.40–7.48 (m, 1 H), 6.68
(dd, J = 15.2, 4.3 Hz, 1 H), 6.32 (d, J = 8.9 Hz, 1 H), 6.25 (d, J =
9.0 Hz, 1 H), 6.10 (d, J = 15.4 Hz, 1 H), 5.59 (dt, J = 15.5, 7.1 Hz,
1 H), 5.40 (dd, J = 15.5, 7.5 Hz, 1 H), 4.82–4.88 (m, 1 H), 4.01–
4.10 (m, 2 H), 3.97 (dd, J = 8.7, 4.7 Hz, 1 H), 3.07–3.25 (m, 2 H),
2.23–2.32 (m, 1 H), 1.86–2.03 (m, 3 H), 1.69–1.78 (m, 1 H), 0.94
(d, J = 6.2 Hz, 3 H), 0.90 (d, J = 6.3 Hz, 3 H), 0.80–0.88 (m, 9 H),
0.77 (d, J = 6.5 Hz, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO):
δ = 173.95, 171.37, 168.83, 166.18, 157.60, 143.19, 132.97, 125.90,
121.51, 57.50, 57.35, 55.42, 53.53, 42.50, 34.96, 31.36, 31.02, 30.12,
19.71, 19.21, 19.17, 19.13, 17.58, 17.53 ppm. HRMS (ESI): calcd.
for C24H39O6N5H+ [M + H]+ 494.2973; found 494.2978.
SylA-D-L Methyl Ester (36c): Compound 29 (7 mg, 25 µmol,
1.1 equiv.), 35 (5.8 mg, 23 µmol, 1 equiv.), PyBop (15 mg, 28 µmol,
1.2 equiv.), and HOAt (4 mg, 28 µmol, 1.2 equiv.) were dissolved in
N,N-dimethylformamide (1.0 mL) in a 10-mL flask. The solution
was cooled to 0 °C and N,N-diisopropylethylamine (8 µL, 46 µmol,
2 equiv.) was added. The reaction was stirred for 40 min at room
temperature. After concentration to dryness, the crude product was
purified by flash column chromatography (10% methanol in
dichloromethane) to yield 36c (9.6 mg, 19 µmol, 76%) as a colorless
solid. TLC (8% methanol in dichloromethane): Rf = 0.30. HPLC
1
(gradient 2): tR = 6.95 min. H NMR (500 MHz, [D6]DMSO): δ =
8.16 (d, J = 7.5 Hz, 1 H), 8.00–8.06 (m, 1 H), 7.43 (t, J = 6.4 Hz,
1 H), 6.68 (dd, J = 15.5, 3.2 Hz, 1 H), 6.53 (d, J = 8.9 Hz, 1 H),
6.31 (d, J = 9.1 Hz, 1 H), 6.10 (d, J = 15.4 Hz, 1 H), 5.55–5.66 (m,
1 H), 5.34–5.42 (m, 1 H), 4.87–4.94 (m, 1 H), 4.16 (dd, J = 8.9,
5.6 Hz, 1 H), 4.05–4.12 (m, 2 H), 3.62 (s, 3 H), 3.09–3.22 (m, 2 H),
2.23–2.32 (m, 1 H), 1.84–2.02 (m, 3 H), 1.69–1.77 (m, 1 H), 0.89–
0.97 (m, 6 H), 0.85 (d, J = 6.8 Hz, 3 H), 0.82 (d, J = 6.8 Hz, 3 H),
0.80 (d, J = 6.7 Hz, 3 H), 0.76 (d, J = 6.7 Hz, 3 H) ppm. 13C NMR
(125 MHz, [D6]DMSO): δ = 173.02, 171.26, 169.02, 166.23, 157.26,
143.18, 132.97, 126.03, 121.49, 57.48, 57.06, 55.43, 53.64, 51.44,
42.61, 34.99, 31.58, 31.34, 30.58, 19.65, 19.19, 19.06, 18.89, 17.63,
17.42 ppm. HRMS (ESI): calcd. for C25H41O6N5H+ [M + H]+
508.3130; found 508.3127.
SylA-L-D (37b): Compound 36b (10.0 mg, 19.7 µmol, 1 equiv.) and
aluminum bromide (42 mg, 158 µmol, 8 equiv.) were dissolved in
tetrahydrothiophene (1 mL) under an atmosphere of argon in a 10-
mL flame-dried flask. The resulting mixture was stirred for 1 h at
room temperature. After concentration to dryness, the remaining
residue was purified by preparative HPLC (using H2O with 0.1%
TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B) at a
flow of 25 mL/min. Gradient: from 0 to 10 min: 90% solvent A/
10% solvent B; from 10 to 30 min: from 90% solvent A/10% sol-
vent B to 70% solvent A/30% solvent B; from 30 to 50 min: from
70% solvent A/30% solvent B to 40% solvent A/60% solvent B;
from 50 to 60 min: from 40% solvent A/60% solvent B to 0% sol-
vent A/100% solvent B; from 60 to 80 min: 0% solvent A/100%
solvent B) to yield 37b (SylA--; 9.0 mg, 18.3 µmol, 93%) as a
colorless solid. TLC (2% acetic acid + 15% methanol in dichloro-
SylA-D-D Methyl Ester (36d): Compound 34 (7 mg, 25 µmol,
1.1 equiv.), 35 (5.8 mg, 23 µmol, 1 equiv.), PyBop (15 mg, 28 µmol,
1.2 equiv.), and HOAt (4 mg, 28 µmol, 1.2 equiv.) were dissolved in
N,N-dimethylformamide (1.0 mL) in a 10-mL flask. The solution
was cooled to 0 °C and N,N-diisopropylethylamine (8 µL, 46 µmol,
2 equiv.) was added. The reaction was stirred for 40 min at room
temperature. After concentration to dryness, the crude product was
purified by flash column chromatography (10% methanol in
dichloromethane) to yield 36d (12.0 mg, 24 µmol, 95%) as a color-
less solid. TLC (8% methanol in dichloromethane): Rf = 0.21.
HPLC (gradient 2): tR = 6.75 min. 1H NMR (500 MHz, [D6]-
1
methane): Rf = 0.40. HPLC (gradient 2): tR = 6.51 min. H NMR
(500 MHz, [D6]DMSO): δ = 12.43 (br. s, 1 H), 8.07 (d, J = 6.6 Hz,
1 H), 8.00 (d, J = 8.5 Hz, 1 H), 7.43 (t, J = 6.7 Hz, 1 H), 6.68 (dd,
J = 15.4, 4.9 Hz, 1 H), 6.40 (d, J = 9.1 Hz, 1 H), 6.28 (d, J =
8.9 Hz, 1 H), 6.10 (d, J = 15.6 Hz, 1 H), 5.60 (dt, J = 15.8, 7.2 Hz,
DMSO): δ = 8.00–8.12 (m, 2 H), 7.43 (t, J = 6.9 Hz, 1 H), 6.69 1 H), 5.42 (dd, J = 15.8, 8.1 Hz, 1 H), 4.88 (t, J = 6.6 Hz, 1 H),
Eur. J. Org. Chem. 2010, 3991–4003
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4001

M. Kaiser et al.
FULL PAPER
4.05–4.12 (m, 2 H), 4.04 (dd, J = 8.2, 5.2 Hz, 1 H), 3.10–3.24 (m,
2 H), 2.24–2.31 (m, 1 H), 1.93–2.03 (m, 2 H), 1.85–1.93 (m, 1 H),
2.02 (m, 2 H), 1.84–1.90 (m, 1 H), 1.70–1.77 (m, 1 H), 0.95 (d, J
= 6.4 Hz, 3 H), 0.91 (d, J = 6.4 Hz, 3 H), 0.86 (d, J = 6.6 Hz, 3
1.69–1.78 (m, 1 H), 0.95 (d, J = 6.4 Hz, 3 H), 0.90 (d, J = 6.4 Hz, H), 0.83 (d, J = 6.7 Hz, 3 H), 0.80 (d, J = 6.8 Hz, 3 H), 0.77 (d, J
3 H), 0.85 (d, J = 6.8 Hz, 3 H), 0.83 (d, J = 6.4 Hz, 3 H), 0.82 (d, = 6.7 Hz, 3 H) ppm. 13C NMR (150 MHz, [D6]DMSO): δ = 174.09,
J = 6.4 Hz, 3 H), 0.77 (d, J = 6.5 Hz, 3 H) ppm. 13C NMR
(125 MHz, [D6]DMSO): δ = 174.06, 171.52, 169.00, 166.26, 157.38,
171.41, 169.08, 166.42, 157.64, 143.42, 133.15, 125.65, 121.34,
57.38, 57.21, 55.30, 53.47, 42.36, 34.80, 31.29, 31.20, 29.88, 19.93,
143.26, 133.17, 125.73, 121.52, 57.15, 57.07, 55.41, 53.57, 42.52, 19.56, 18.97, 18.84, 17.78, 17.36 ppm. HRMS (ESI): calcd. for
35.03, 31.52, 31.45, 30.57, 19.81, 19.21, 19.15, 19.11, 17.56, C24H39O6N5H+ [M + H]+ 494.2973; found 494.2966.
17.44 ppm. HRMS (ESI): calcd. for C24H39O6N5H+ [M + H]+
494.2973; found 494.2969.
Human 20S Proteasome Assays: Proteasome assays were performed
and evaluated as previously reported in ref.[12] by using commer-
SylA-
D
-
L
(37c): Compound 36c (9.0 mg, 17.7 µmol, 1 equiv.) and
cially available human erythrocyte 20S proteasome from Biomol.
Compounds 37b–d, 43, and 47 were dissolved as a stock solution
in DMSO, and a dilution series in DMSO was prepared for de-
termining the corresponding KiЈ values. For each data point, three
independent measurements were performed (n = 3).
aluminum bromide (38 mg, 142 µmol, 8 equiv.) were dissolved in
tetrahydrothiophene (1 mL) under an atmosphere of argon in a 10-
mL flame-dried flask. The resulting mixture was stirred for 1 h at
room temperature. After concentration to dryness, the remaining
residue was purified by preparative HPLC (using H2O with 0.1%
TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B) at a
flow of 25 mL/min. Gradient: from 0 to 10 min: 90% solvent A/
10% solvent B; from 10 to 30 min: from 90% solvent A/10% sol-
vent B to 70% solvent A/30% solvent B; from 30 to 50 min: from
70% solvent A/30% solvent B to 40% solvent A/60% solvent B;
from 50 to 60 min: from 40% solvent A/60% solvent B to 0% sol-
vent A/100% solvent B; from 60 to 80 min: 0% solvent A/100%
solvent B) to yield 37c (SylA--; 4.6 mg, 9.3 µmol, 53%) as a col-
orless solid. TLC (2% acetic acid + 15% methanol in dichloro-
Supporting Information (see footnote on the first page of this arti-
cle): Synthesis of compounds 2–10, 12–15, 26–29, 31–34, and 39–
47.
Acknowledgments
Financial support from the Swiss National Science Foundation
(Grant 3100A0-115970, to R. D.) is gratefully acknowledged.
1
methane): Rf = 0.40. HPLC (gradient 2): tR = 6.56 min. H NMR
[1] A. Hershko, A. Ciechanover, Annu. Rev. Biochem. 1998, 67,
425–479.
[2] M. Groll, L. Ditzel, J. Löwe, D. Stock, M. Bochtler, H. D. Bar-
tunik, R. Huber, Nature 1997, 386, 463–471.
[3] A. K. Nussbaum, T. P. Dick, W. Keilholz, M. Schirle, S. Stev-
anovic, K. Dietz, W. Heinemeyer, M. Groll, D. H. Wolf, R.
Huber, H. G. Rammensee, H. Schild, Proc. Natl. Acad. Sci.
USA 1998, 95, 12504–12509.
(500 MHz, [D6]DMSO): δ = 12.39 (br. s, 1 H), 8.12 (d, J = 7.8 Hz,
1 H), 8.05 (d, J = 8.7 Hz, 1 H), 7.43 (t, J = 6.9 Hz, 1 H), 6.69 (dd,
J = 15.6, 5.3 Hz, 1 H), 6.42 (d, J = 9.0 Hz, 1 H), 6.32 (d, J =
9.0 Hz, 1 H), 6.10 (d, J = 15.5 Hz, 1 H), 5.62 (dt, J = 15.4, 7.6 Hz,
1 H), 5.37 (dd, J = 16.1, 7.7 Hz, 1 H), 4.92 (t, J = 7.6 Hz, 1 H),
4.16 (dd, J = 8.9, 5.5 Hz, 1 H), 4.06–4.12 (m, 1 H), 4.05 (dd, J =
8.9, 4.7 Hz, 1 H), 3.10–3.23 (m, 2 H), 2.24–2.31 (m, 1 H), 1.93–
2.03 (m, 2 H), 1.84–1.92 (m, 1 H), 1.70–1.78 (m, 1 H), 0.95 (d, J
= 6.6 Hz, 3 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.86 (d, J = 6.7 Hz, 3
H), 0.82 (d, J = 6.8 Hz, 3 H), 0.80 (d, J = 6.8 Hz, 3 H), 0.76 (d, J
= 6.8 Hz, 3 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 173.86,
171.34, 169.07, 166.19, 157.36, 143.21, 133.10, 125.83, 121.46,
57.20, 57.10, 55.44, 53.62, 42.50, 34.98, 31.56, 31.35, 30.53, 19.64,
19.19, 19.07, 19.03, 17.40 ppm. HRMS (ESI): calcd. for
C24H39O6N5H+ [M + H]+ 494.2973; found 494.2967.
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SylA-D-D (37d): Compound 36d (11.8 mg, 23.2 µmol, 1 equiv.) and
aluminum bromide (50 mg, 186 µmol, 8 equiv.) were dissolved in
tetrahydrothiophene (1 mL) under an atmosphere of argon in a 10-
mL flame-dried flask. The resulting mixture was stirred for 1 h at
room temperature. After concentration to dryness, the remaining
residue was purified by preparative HPLC (using H2O with 0.1%
TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B) at a
flow of 25 mL/min. Gradient: from 0 to 10 min: 90% solvent A/
10% solvent B; from 10 to 30 min: from 90% solvent A/10% sol-
vent B to 70% solvent A/30% solvent B; from 30 to 50 min: from
70% solvent A/30% solvent B to 40% solvent A/60% solvent B;
from 50 to 60 min: from 40% solvent A/60% solvent B to 0% sol-
vent A/100% solvent B; from 60 to 80 min: 0% solvent A/100%
solvent B) to yield 37d (SylA--; 7.8 mg, 15.8 µmol, 68%) as a
colorless solid. TLC (2% acetic acid + 15% methanol in dichloro-
1
methane): Rf = 0.35. HPLC (gradient 2): tR = 6.21 min. H NMR
(600 MHz, [D6]DMSO): δ = 8.03–8.10 (m, 2 H), 7.44 (br. s, 1 H),
6.69 (dd, J = 15.2, 4.3 Hz, 1 H), 6.31 (d, J = 8.9 Hz, 1 H), 6.27 (d,
J = 9.0 Hz, 1 H), 6.10 (d, J = 15.4 Hz, 1 H), 5.60 (dt, J = 15.7,
5.6 Hz, 1 H), 5.37 (dd, J = 15.5, 7.6 Hz, 1 H), 4.90 (t, J = 6.3 Hz,
1 H), 4.12 (dd, J = 8.1, 5.9 Hz, 1 H), 4.05–4.11 (m, 1 H), 3.98 (dd,
J = 8.3, 4.8 Hz, 1 H), 3.10–3.24 (m, 2 H), 2.24–2.32 (m, 1 H), 1.92–
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Received: March 7, 2010
Published Online: May 19, 2010
Eur. J. Org. Chem. 2010, 3991–4003
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4003