Synthesis and biological characterization of argyrin F

Article abstract of DOI:10.1002/cmdc.201000080

Argyrin F unfolds its promising antitumor activity twice: First through stabilization of the tumor suppressor protein p27 and second by vascular damage.

Full text of DOI:10.1002/cmdc.201000080

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DOI: 10.1002/cmdc.201000080
Synthesis and Biological Characterization of Argyrin F
Leila Bꢀlow,[a] Irina Nickeleit,*[b] Anna-Katharina Girbig,[a] Tobias Brodmann,[a] Andreas Rentsch,[a] Ulrike Eggert,[a]
Florenz Sasse,[c] Heinrich Steinmetz,[c] Ronald Frank,[c] Teresa Carlomagno,[d] Nisar P. Malek,[b] and
Markus Kalesse*[a, c]
In the quest for new targets for antitumor therapy, the protea-
some and its inhibitors have been the focus of academic and
industrial research.[1] In this context, we recently reported the
p27 stabilization effect of argyrin A[2] through selective inhibi-
tion of the proteasome.[3] Consequently, increased levels of
p27, one of the pivotal tumor suppressor proteins, stop prolif-
eration of tumor cells. Additionally, argyrin A effects neovascu-
larization and therefore inhibits tumor progression through a
second mode-of-action. At the same time, argyrin A exhibits
biological activity at remarkably low concentration at which no
cytotoxic effects were observed. Even though these data al-
ready support the high potential of argyrin A as a potential
drug candidate, detailed structure–activity relationship studies,
and a molecular understanding of its mode-of-action are a pre-
requisite for the further optimization of its medicinal potential.
Since only limited structural modifications can be introduced
starting from the natural product, and the marginal amounts
of the minor metabolites derived from fermentation do not
allow for detailed biological investigations, we aimed at estab-
lishing a synthetic access to argyrin A and its analogues. Here,
we took advantage of the elegant construction of argyrin B
put forward by the Ley group, which dissected argyrin in three
equally complex segments (9, 10, 11) as delineated in
Figure 1.[4] Nevertheless, the modifications we sought to intro-
duce and the requirements imposed by practical considera-
tions necessitated modification in the overall synthesis and of
the three building blocks. At the outset of our synthetic
endeavors, we identified functional groups and structural ele-
ments as potential characteristics that are likely to be essential
for the biological activity or might be omitted in order to sim-
plify the synthesis of lead structures.
Figure 1. Natural occurring argyrins and their synthetic assembly based on
three segments 9, 10 and 11.
The unusual 4-methoxy tryptophan moiety attracted our at-
tention immediately since the site of the methoxy substitution
is biosynthetically the least accessed position.[5] Additionally, it
was reported that classic strategies used for amino acids syn-
theses failed for the construction of this unusual amino acid.[4]
As a solution to this problem, Ley et al. reported an enzymatic
kinetic resolution of the racemic precursor. However, efforts to
carry out the synthesis as described by Ley et al. were ham-
pered as the immobilized enzyme used in their synthesis was
no longer available, and other enzymes resulted in changing
yields in the range of 10–25%. In order to provide an efficient
access to this uncommon amino acid, we carefully analyzed
different chiral auxiliaries for their use in catalytic hydrogena-
tions and identified the DuanPhos ligand, in combination with
Rh(cod)2BF4, to provide the required amino acid in quantitative
yield (99%) and 99% ee (Scheme 1).[6]
[a] L. Bꢀlow, A.-K. Girbig, T. Brodmann, A. Rentsch, U. Eggert,
Prof. Dr. M. Kalesse
Biomolekulares Wirkstoffzentrum (BMWZ), Leibniz Universitꢁt Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
Fax: (+49)511-762-3011
E-mail: Markus.Kalesse@oci.uni-hannover.de
[b] Dr. I. Nickeleit, Prof. Dr. N. P. Malek
Dept. of Gastroenterology, Hepatology and Endocrinology
Institute for Molecular Biology, Hannover Medical School
30625 Hannover (Germany)
[c] Dr. F. Sasse, H. Steinmetz, Dr. R. Frank, Prof. Dr. M. Kalesse
Helmholtz Zentrum fꢀr Infektionsforschung
38124 Braunschweig (Germany)
[d] Prof. Dr. T. Carlomagno
Structural and Computational Biology, EMBL Heidelberg
69117 Heidelberg (Germany)
Next, we turned our attention to the synthesis of the dehy-
droalanine moiety. In order to circumvent the use of selenium
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000080.
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tide and condensation with TBTU and HOBt. Finally,
the tert-butyl protecting group was selectively re-
moved without affecting the exo-methylene group or
peptidic linkages through treatment with trifluoro-
acetic acid (TFA) at 08C. Based on the building blocks
and protecting group strategies described above,
several argyrin analogues were synthesized.
Scheme 1. Enantioselective hydrogenation of olefin 13. Reagents and conditions: a) Rh-
(cod)2BF4/DuanPhos, H2 (10 bar), MeOH, RT, 24 h, 99% (99% ee).
In order to provide derivatives for structure–activi-
ty relationship (SAR) studies, argyrins A (1), F (6) and
G (7), and derivatives lacking the methoxy group at
compounds for the introduction of the exo-methylene moiety,
we reasoned that the dehydroalanine moiety would be chemi-
cally stable as long as the amino group was substituted by an
electron-withdrawing group.[7] Consequently, we performed a
copper(I)-catalyzed elimination of the serine hydroxy group
from dipeptide 14, which was generated under standard pep-
tide-coupling conditions (Scheme 2). The next peptide cou-
the tryptophan moiety (24–26) were synthesized.[8] Additional-
ly, analogues lacking the exo-methylene group (29) and con-
taining an extra methyl group provided by replacing the gly-
cine residue to alanine (27, 28) were generated.
Initially, the proteasome inhibition efficacy of the different
argyrins and related analogues were investigated, in addition
to measurements of their antiproliferative activity against SW-
480 colon carcinoma cells (Table 1). It became apparent that
Scheme 3. Synthesis of thiazole moiety 19. Reagents and conditions:
a) 1. DCC, HOBT, NH3; 2. Belleau’s reagent, 70%; b) Ethyl bromo pyruvate,
78%.
Scheme 2. Synthesis of dehydroalanine segment 16. Reagents
and conditions: a) CuCl, EDC, CH2Cl2, 96%; b) 1. LiOH,THF/MeOH/
H2O; 2. HCl-Sar-OEt, PyBroP, DIPEA, CH2Cl2, 08C!RT, 74%.
pling was performed after hydrolysis of the ester
group with the ammonium salt of the sarcosin ester
and with PyBroP as the coupling reagent to provide
building block 16.
Finally, we had to provide a route that would allow
for the incorporation of a hydroxymethyl group in
the thiazole dipeptide. Based on our analysis of po-
tential protecting groups, we used tert-butyl protect-
ed serine (17), which was converted into the corre-
sponding thioamide (18) using DCC, NH3 and Bel-
leau’s reagent. Treatment with ethyl bromo pyruvate
provided building block 19 necessary for introducing
a
hydroxy moiety as observed for argyrin F
(Scheme 3).
Representative for all argyrin derivatives that are
described in the context of their biological activity,
the synthesis of argyrin F (6) is outlined in Scheme 4.
The synthesis starts with the coupling of thiazole 19
to the N terminus of the tryptophan-containing
building block 20, which was obtained by standard
peptide-coupling conditions.[3] Subsequent hydrolysis
of the ester moiety and coupling with dehydroala-
nine segment 16 provides the open-chain precursor
of argyrin F. Ring closure was achieved by liberation
of both the C and the N terminus of the linear pep-
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the methoxy group of tryptophan Trp2 and the exo-
methylene group are essential for biological activity.
An exchange of the glycine by alanine residues
reduced the observed activity. This was especially
severe in the case of d-alanine.
The additional hydroxy group in the thiazole seg-
ment in argyrin F (6) on the other side does not
seem to influence the pharmacodynamics. However,
this moiety might be a beneficial derivatization in
order to increase the water solubility of the argyrin
analogues and to improve their pharmacokinetic
parameters. Remarkably, the presence of several hy-
droxy groups only partially restored the proteasome
inhibition activity in analogues lacking the methoxy
indole moiety. This supports the hypothesis that the
methoxy group at tryptophan Trp2 contributes to
specific interactions with the target rather than by
changing the hydrophobic character of the molecule.
Surprisingly, the presence of an ethyl group at posi-
tion R3 (argyrin E and G) decreases the activity signifi-
cantly, which can be seen by comparing the activities
of argyrin F and argyrins E or G. These data demon-
strate that argyrin F has approximately the same
activity as argyrin A but with a higher solubility, and
therefore it exhibits possible advantages in its phar-
macokinetic behavior (water solubility: argyrin A,
5 mgmLꢀ1; argyrin F, 24 mgmLꢀ1).
In order to further characterize the biochemical in-
teractions of argyrin F with the proteasome, Michae-
lis–Menten kinetics were generated (see Supporting
Information). The results indicated that argyrin F acts
as a competitive, reversible inhibitor (trypsin-like, Ki =
112 nm; chymotrypsin-like, Ki =76 nm; caspase-like,
Ki =81 nm).
Taking all the biochemical data together, it became
apparent that argyrin A and F are the most potent
derivatives among the analogues tested so far. Given
the comparable activities of argyrins A and F as inhib-
itors of the 20S proteasome, we next asked whether
both compounds would also exhibit similar in vitro
Scheme 4. Synthesis of argyrin F. Reagents and conditions: a) LiOH, THF/MeOH/H2O; b) H2,
Pd/C; c) EDC, HOBt, DIPEA, 82% (two steps); d) LiOH, THF, MeOH/H2O; e) TFA, CH2Cl2;
f) EDC, HOBt, DIPEA, 85% (two steps); g) 1. LiOH, THF/MeOH/H2O; 2. TFA, CH2Cl2; 3. TBTU,
HOBt, DIPEA, 88% (three steps); h) TFA, CH2Cl2, 83%.
and in vivo activities. We had previously shown that the bio-
logical activities of argyrin A depend on the stabilization of the
tumor suppressor protein p27kip1. Therefore, we investigated
whether argyrin F would show a similar dependency on p27
stabilization. First, we determined the kinetics of p27 induction
in cells treated with argyrin A or F. As shown in Figure 2, treat-
ment with argyrin F resulted in a dose-dependent expression
of p27 in SW-480 colon carcinoma cells. Next, we suppressed
p27 expression using specific siRNAs in HeLa cells and mea-
sured the number of apoptotic cells after incubation with ar-
gyrin F. As shown in Figure 2, argyrin F induced apoptotic cell
death in HeLa cells after 48 h incubation. Conversely, cells in
which p27 expression was suppressed due to siRNA treatment
did not respond to argyrin F, a result that is in agreement with
our previous observations using argyrin A. Together, these
experiments suggested that argyrin F, like argyrin A, requires
p27 expression for the induction of its biological phenotype.
Table 1. Inhibition of proteasome activity.
Compound
Remaining proteasome activity
Trypsin-like Caspase-like Chymotrypsin-like
IC50 [nm]
SW-480
Argyrin A (1) 45ꢁ4.5
29ꢁ5.2
52ꢁ3.0
38ꢁ8.3
50ꢁ6.0
65ꢁ4.0
35ꢁ7.0
60ꢁ6.5
52ꢁ5.5
65ꢁ7.0
68ꢁ4.6
72ꢁ13.3
54ꢁ2.5
65ꢁ3.0
100ꢁ6.5
30ꢁ6.0
58ꢁ6.5
43ꢁ5.5
64ꢁ5.2
70ꢁ5.5
28ꢁ2.5
65ꢁ4.5
51ꢁ8.0
55ꢁ3.5
62ꢁ19
92ꢁ3.0
50ꢁ3.0
87ꢁ2.5
87ꢁ6.5
3.8ꢁ0.3
Argyrin B (2)
Argyrin C (3)
55ꢁ8.5
40ꢁ7.0
4.6ꢁ0.6
1.5ꢁ1.1
3.6ꢁ2.0
520ꢁ270
4.2ꢁ0.4
63ꢁ55
Argyrin D (4) 62ꢁ8.0
Argyrin E (5)
Argyrin F (6)
70ꢁ3.0
38ꢁ6.5
Argyrin G (7) 75ꢁ3.0
Argyrin H (8) 60ꢁ7.0
30ꢁ2
Argyrin 24
Argyrin 25
Argyrin 26
Argyrin 27
Argyrin 28
Argyrin 29
70ꢁ7.5
75ꢁ2.7
74ꢁ2.9
72ꢁ4.5
89ꢁ2.5
85ꢁ6.5
1050ꢁ180
3800ꢁ43
>4000
90ꢁ0.2
2300ꢁ180
3600ꢁ400
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Next, we tested the ability of argyrin A, argyrin F,
and argyrin 29, a derivative without the exo-methyl-
ene group, to suppress the growth of colon carcino-
ma xenotransplant tumors derived from SW-480 cells
in nude mice. We found that argyrin 29 (no exo-
methylene group) was unable to inhibit the protea-
some in vitro and shows no detectable antitumor ac-
tivity in vivo. These data provide direct evidence that
the proteasome inhibitory activity of the argyrins is
required for their antitumor activities. Next, we com-
pared the activities of argyrin A and F against colon
carcinoma xenotransplants. We found that both
compounds lead to a clear reduction in tumor
volume when compared with untreated mice. Strik-
ingly, however, argyrin F-treated tumors remained a
very small size even after treatment was stopped at
day 21. This remarkable activity was detected in
more than 50% of all tumors in mice treated with ar-
gyrin F. These tumors did not regrow after treatment
was stopped. Conversely, most of the argyrin A-treat-
ed tumors reappeared after discontinuation of the
treatment (Figure 3).
Figure 2. Argyrin F activity depends on the induction of p27 expression. SW-480 cells
were treated with argyrin F, p27 siRNA or both for either 24 hr (left) or 48 hr (right). The
sub-G1 fraction of apoptotic cells was determined by flow cytometric analysis and p27
expression by immunoblotting using a p27 specific antibody.
To understand this phenotype in more detail, we
studied the effect of argyrin F on endothelial cells.
Our previous studies suggested that the antitumor
activity of argyrin A in vivo was in large part caused
by antiangiogenic and direct vessel-damaging activi-
ties of the compound. We therefore tested whether
argyrin F would have similar activities against endo-
thelial cells. As shown in Figure 4a, we found that at
identical concentrations argyrin F had an even more
pronounced effect on human umbilical vein endo-
thelial cells (HUVECs) in that it induced apoptosis in
the vast majority of cells. We also observed an
almost complete inhibition of tube formation in cells
treated with argyrin F as compared with argyrin A.
Next we assayed the effects of argyrin F on xeno-
transplanted tumors. For this we stained the tumor
tissue with CD31 antibody to detect endothelial cells
and determined the microvessel density after treat-
ment. As shown in Figure 4, we found that argyrin F
leads to an even faster destruction of blood vessels
in vivo compared with argyrin A.
Figure 3. The proteasome inhibitory activity of argyrin F is required for its antitumor ac-
tivities. SW-480 colon car/jcinoma cells were mixed with Matrigel and injected under the
skin of nu/nu-mice to establish xenotransplant tumors. Treatment with argyrin A, or ar-
gyrin F was started when the tumors reached a volume of 150 mm3. The graphs show
quantification of tumor volumes at the indicated time points compared with the starting
&
*
^
In conclusion, we have identified argyrin F (6) as
the most promising antitumor compound, exerting
the same biological effects as argyrin A. Besides its
potential to stabilize p27, due to a competitive, re-
size, which was set as 100, after treatment with argyrin A ( ) and F ( ) or a control ( ).
n=3 (0.066 mgkgꢀ1 argyrin A and F); n=3 (control: PBS/DMSO).
versible inhibition of the proteasome, argyrin F shows vascular-
damaging effects, which increases the ability of argyrin F to
reduce the size of solid tumors in mice models. These two bio-
logical effects exerted by argyrin F make this natural product a
promising compound that has the potential to become a
unique and unprecedented drug in antitumor therapy. Addi-
tionally, we were able to identify the pivotal structural require-
ments, such as the methoxy group in the tryptophan Trp2, the
exo-methylene group and the alanine building block, necessary
for proteasome inhibition. The SAR studies presented here will
not only help to understand the molecular interaction of argyr-
in F with the proteasome, but they open the door for further
optimization of its biological profile and pharmacological pa-
rameters.
Experimental Section
Experimental details and full analytic characterization of all argyrins
reported here are provided in the Supporting Information.
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Figure 4. Argyrin A and argyrin F damage existing tumor blood vessels. a) Human umbilical vein endothelial cells (HUVECs) were incubated with argyrin A or
argyrin F for 24 h. The enclosed network of tubular structures were photographed and quantified. Scale bars=100 mm; b) After a single injection of argyrin A
and argyrin F (0.066 mgkgꢀ1) tumors were explanted at the indicated time points and blood vessels in tumor tissue were stained for CD31 (red). Scale
bars=25 mm; c) Quantification of data in panel b.
All experiments were performed after review by and in accordance
with the animal rights and protection agencies of Lower Saxony,
Germany.
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Keywords: antitumor agents · drug design · inhibitors ·
natural products · structure–activity relationships
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Received: February 24, 2010
Published online on March 31, 2010
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