Synthesis of chondramide a analogues with modified β-tyrosine and their biological evaluation

Article abstract of DOI:10.1002/chem.201101978

Starting from cinnamates 9, obtained by Wittig reaction or Heck coupling, the diols 17 were prepared by asymmetric dihydroxylation. This was followed by a regioselective substitution of the 3-OH group with hydrazoic acid under Mitsunobu conditions. Methylation of the 2-OH group and reduction of the azide group led to the β-tyrosine derivatives 8. Condensation with the dipeptide acid 6 furnished the tripeptide part of the chondramides. The derived acids 21 were combined with the hydroxy ester 7 to the esters 22. Cleavage of the tert-butyl groups and intramolecular lactam formation gave rise to the chondramide A analogues 2 b-k. Growth inhibition assays showed most of the analogues to be biologically active. Some of them even reach the activity of jasplakinolide. It can be concluded that the 4-position of the aryl ring in the β-tyrosine of chondramide A tolerates structural modifications quite well.

Full text of DOI:10.1002/chem.201101978

FULL PAPER
DOI: 10.1002/chem.201101978
Synthesis of Chondramide A Analogues with Modified b-Tyrosine
and Their Biological Evaluation
Alexander Zhdanko,[a] Anke Schmauder,[a] Christopher I. Ma,[b] L. David Sibley,[b]
David Sept,[c] Florenz Sasse,[d] and Martin E. Maier*[a]
Abstract: Starting from cinnamates 9,
obtained by Wittig reaction or Heck
coupling, the diols 17 were prepared by
asymmetric dihydroxylation. This was
followed by a regioselective substitu-
tion of the 3-OH group with hydrazoic
acid under Mitsunobu conditions.
Methylation of the 2-OH group and re-
duction of the azide group led to the b-
tyrosine derivatives 8. Condensation
with the dipeptide acid 6 furnished the
tripeptide part of the chondramides.
The derived acids 21 were combined
with the hydroxy ester 7 to the esters
22. Cleavage of the tert-butyl groups
and intramolecular lactam formation
gave rise to the chondramide A ana-
logues 2b–k. Growth inhibition assays
showed most of the analogues to be
biologically active. Some of them even
reach the activity of jasplakinolide. It
can be concluded that the 4-position of
the aryl ring in the b-tyrosine of chon-
dramide A tolerates structural modifi-
cations quite well.
Keywords: chondramides
· cyclo-
peptides · Mitsunobu reaction · to-
tal synthesis · tyrosine analogues
Introduction
Very prominent examples of such cyclodepsipeptides, dis-
playing anti-tumor activity are the jasplakinolides and the
chondramides. Jasplakinolide (1) was isolated from the
marine sponge Jaspis splendans many years ago (Figure 1).[5]
Cyclodepsipeptides comprise a unique class of secondary
metabolides. They frequently contain unusual amino acids,
like d-amino acids or N-methylated amino acids and hy-
droxy acids that typically originate from the polyketide
pathway. Incorporation of the hydroxy acid results in an
ester bond, explaining the term “depsi”.[1,2] Many cyclodep-
sipeptides have been isolated from marine sponges and
found to display interesting biological activities.[3] Thus, cy-
clodepsipeptides with anti-HIV or anti-tumor activity are
known. With the peptide subunit cyclodepsipeptides are
clearly protein-like and therefore it is not surprising that
they very often modulate protein–protein interactions.[4]
[a] Dipl.-Chem. A. Zhdanko, Dr. A. Schmauder, Prof. Dr. M. E. Maier
Institut fꢀr Organische Chemie
Universitꢁt Tꢀbingen
Auf der Morgenstelle 18, 72076 Tꢀbingen (Germany)
Fax : (+49)7071-2975247
Figure 1. Structures of jasplakinolide (1) and the chondramides A–D (2–
5).
E-mail: martin.e.maier@uni-tuebingen.de
[b] Dr. C. I. Ma, Prof. Dr. L. D. Sibley
Department of Molecular Microbiology
Washington University School of Medicine
660 S. Euclid Ave., St. Louis, MO 63110-1093 (USA)
Independently, it was also found in a Jaspis sponge and
named jaspamide.[6] In the meantime jasplakinolide was iso-
lated from many other sponges. Furthermore, several addi-
tional jasplakinolides could be isolated by the groups of
Zampella[7] and Crews.[8] In contrast, so far only four natural
chondramides are known, namely chondramides A–D (2–5)
(Figure 1). They were isolated by the Hçfle/Reichenbach
group from myxobacteria.[9] The chondramides are quite
similar in structure to jasplakinolide, in that they contain a
tripeptide subunit consisting of an l-alanine, an N-methyl-d-
tryptophan, and an l-b-tyrosine. Jasplakinolide and the
[c] Prof. Dr. D. Sept
Department of Biomedical Engineering
Center for Computational Medicine and Bioinformatics
University of Michigan
1101 Beal Ave.
Ann Arbor, MI 48109-2110 (USA)
[d] Dr. F. Sasse
Abteilung Chemische Biologie
Helmholtz-Zentrum fꢀr Infektionsforschung
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101978.
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chondramides differ essentially in the hydroxy acid that
bridges the tripeptide part to form the cyclodepsipeptide.
Whereas jasplakinolide contains an 8-hydroxy acid, in the
chondramides the corresponding sector is a 7-hydroxy acid.
Accordingly, jasplakinolide features a 19-membered macro-
cycle, while the chondramides are 18-membered.
Over the years several total syntheses of jasplakinolide
have been achieved.[10] Most of them close the macrocyclic
ring by intramolecular amide or ester formation. The Wald-
mann/Arndt synthesis[10g] relies on a relay ring-closing meta-
thesis reaction. Furthermore, syntheses of simplified jaspla-
kinolide analogues have been reported.[11,12] Surprisingly,
syntheses of chondramides were achieved only recently. The
reason was that the configuration at the stereogenic centers
was not given in the original article. Even though it could be
assumed that the configurations in the peptide part and
even the overlapping hydroxy acid might be similar to the
ones in jasplakinolide. The Waldmann[13] and Kalesse[14]
groups independently secured the configuration of the hy-
droxy acid in the chondramides by synthesizing various ste-
reoisomers of chondramide C. Our group had developed a
concise synthesis of the hydroxy acid through an asymmetric
vinylogous aldol reaction.[15] Subsequently, we reported the
total synthesis of chondramide A (chon A).[16] Instead of a
b-tyrosine, this chondramide contains a 3-amino-2-methoxy-
3-arylpropanoic acid, whose configuration was determined
to be (2S,3S). The ester bond could be established either by
Yamaguchi esterification or via a Mitsunobu reaction[17] that
was then followed by macrolactam formation. While the b-
tyrosine derivative in chondramide A seems more compli-
cated on first sight, it is easily available by asymmetric dihy-
droxylation of a cinnamic ester precursor.
Figure 2. Key structure-activity relationships for good biological activity
of jasplakinolides and chondramides illustrated with the jasplakinolide
structure.
kinolide analogues with this feature are still active even if
they lack some of the other methyl groups of the hydroxy
acid. For chondramide C analogues it could be shown that
the configuration at C6 and C7 of the hydroxy acid is less
critical.
Due to their interesting biology and structural similarity
analogues of the above-mentioned cyclodepsipeptides
should help to understand protein–protein interactions and
further advance the binding site model.[12] With an efficient
route to chondramide A we embarked on the synthesis of
chondramide A analogues with modified b-tyrosine deriva-
tives. In this paper we report on the synthesis of ten ana-
logues and their biological activity.
Results and Discussion
The potent antitumor activity of these cyclodepsipeptides
is due to their stabilizing effect on F-actin filaments.[18] Be-
cause of this property jasplakinolide became an important
tool in cell biology. Together with microtubules and the in-
termediate filaments, actin filaments (F-actin) make up the
cytoskeleton which has a key role in cell shape and division.
According to a binding model recently refined by the Wald-
mann group, chondramide C is located in a shallow binding
pocket made up by three independent actin subunits in the
filament.[19] The binding site is identical to the one of the bi-
cyclic heptapeptide phalloidin.[20] There seems to be a hy-
drophobic interaction of the indole moiety with a loop
region of one of the actin subunits (subunit X). The phenol-
ic group of the b-tyrosine occupies a larger cavity with the
hydroxyl group close to an Asp of G-actin monomer Y. It
seems that the hydroxy acid not only serves to provide a
scaffolding role, but rather parts (C1–C3) of it are close to
the protein surface of subunit Z and contribute substantially
to efficient binding. This model was supported by the activi-
ty of the available jasplakinolide and chondramide ana-
logues (Figure 2).[12] For example, omitting the phenolic ring
essentially results in an inactive jasplakinolide analogue.
With regard to the hydroxy acid, it turned out that the con-
figuration at C2 of the methyl-bearing carbon and the E-
configuration of the C4–C5 double bond are crucial. Jaspla-
Synthesis of the analogues: The synthetic route towards nat-
ural chondramide A (2a), recently disclosed from our labo-
ratory,[16] was put as a basis for synthesis of new chondra-
mide analogues, containing variations in the b-tyrosine
unit.[16] Accordingly, this implied the use of differently sub-
stituted b-amino esters 8 that had to be synthesized from
substituted trans-cinnamic esters 9. At this point, taking into
account the compatibility of the substituents at the aromatic
ring with all the synthetic steps, we have chosen eight cin-
namic esters 9b–e, 9g–i, and 9k as precursors for the de-
sired chondramide analogues (Scheme 1).
All the trans-cinnamic esters 9 were synthesized from
easily accessible commercial starting materials as outlined in
Scheme 2. Enoates 9b–e, 9k were prepared by Wittig reac-
tion of the corresponding aldehydes 10 with the stabilized
ylide [(carbomethoxy)methylene]triphenylphosphorane[21] in
dichloromethane. Enoate 9e could also be obtained by
Knoevenagel condensation with subsequent esterification in
high 93% yield. In case of enoate 9k, its low solubility in
methanol, allowed for efficient isolation of the product by
simple filtration. The 4-cyanocinnamate 9i was synthesized
by Heck reaction from 4-bromobenzonitrile under low cata-
lyst loading and ligand-free conditions, using PdACHTNUTRGNEUNG(OAc)2 as
catalyst.[22] This method is especially efficient for electron
deficient aromatic bromides and enoate 9i was obtained in
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Synthesis of Chondramide A Analogues
FULL PAPER
action to (E)-methyl 4-formylcinnamate (13) was followed
by NaBH4 reduction of the aldehyde group and silylation of
the primary alcohol. Cinnamate 9h with a CH2CH2OTBS
substituent in 4-position was prepared from the arylacetic
acid 14 through reduction with LiAlH4 and silylation of the
resulting primary alcohol. A subsequent halogen-metal ex-
change on aryl bromide 15 and quenching the anion with
DMF furnished aldehyde 16 which was extended via Wittig
reaction in good yield. The synthesis of the last derivative,
enoate 9h initially was also attempted using Heck reaction,
however, the corresponding substrate 15 did not react nei-
ther under ligand free conditions nor in the presence of
PPh3. Here, formation of palladium black was observed and
starting material was recovered unchanged. This is explained
by the low reactivity of the aromatic bromide, which is not
activated by an electron-withdrawing substituent. In all
cases the cinnamates 9 were obtained configurationally pure
and no cis-isomers could be detected by NMR in the prod-
ucts.
With several cinnamic esters in hands we next turned to
the synthesis of amino esters 8 (Scheme 3). Sharpless asym-
metric dihydroxylation (AD)[23] under standard conditions
Scheme 1. Synthetic strategy and key fragments for the synthesis of chon-
dramide A analogues 2b-k.
Scheme 2. Synthesis of cinnamic acid building blocks 9 by Wittig reaction
or Heck coupling; NMP=N-methyl-2-pyrrolidone.
essentially quantitative yield. This result stipulated the
choice of 4-bromobenzaldehyde as a precursor for prepara-
tion of the CH2OTBS-substituted enoate 9g, that was finally
done in three steps in high overall yield. Thus, the Heck re-
Scheme 3. Conversion of the cinnamates 9 to the b-tyrosine derivatives
19, respectively; DCE=dichloroethane, DEAD=diethyl azodicarboxy-
late.
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M. E. Maier et al.
(0.4 mol% Os catalyst, 2 mol% ligand) was used initially,
but this proved problematic providing the diols only in mod-
erate yield (30–68%). After some experimentation, possible
reasons for these problems became clear. Due to the elec-
tron-deficient nature of the alkenes, especially of the nitro
and cyano derivatives, dihydroxylation occurred with re-
duced rate and saponification of the methyl ester became a
significant side reaction. In the case of the nitro-substituted
diol 17e the corresponding acid was partially isolated and
characterized (see the supporting information). The dihy-
droxylation reactions with the NO2-, Ph-, and CN-substitut-
ed cinnamates were additionally hampered by very limited
solubility of the alkenes in the reaction medium. As soon as
the ester saponification seemed to be the only problem and
otherwise the reaction was clean, attempts were made to im-
prove the yield by addition of KHCO3 to the reaction mix-
ture, to make it less alkaline, but this was again not suffi-
ciently successful. Finally, the situation was significantly im-
proved when increased catalyst loading was used together
with buffering the reaction mixture with KHCO3. Indeed, in
the presence of 1 mol% K2OsO2(OH)4/1.5 mol% chiral
ligand, the reaction required somewhat shorter time and the
diol 17g was obtained in 93% yield. Although the synthesis
of other diols was not attempted under the optimized condi-
tions, we believe that increasing the catalyst loading (e.g.,
up to 2 mol%) should be the most efficient way to increase
the yield in the AD of these relatively unreactive and hardly
soluble alkenes. This is in line with the observations given in
the literature.[23] For three of the diols (17e, 17g, 17i) the
optical purity was assessed by means of HPLC on a chiral
column (see the Supporting Information), and in no case the
opposite enantiomer could be detected, which implies at
least 98% ee for these compounds.[16] For the other diols the
enantiomeric purity was tentatively assessed to be “high” by
contaminated with MeSO2NH2, resulting from the previous
step. We found MeSO2NH2 to have no influence on the re-
action.
Subsequently, azides 18 were smoothly methylated by tri-
methyloxonium tetrafluoroborate in the presence of proton
sponge to give the a-methoxy propanoates 19. The cyano
derivative 19i was hydrolyzed with H2O2/K2CO3 to provide
amide 19j, the precursor for chondramide analogue 2j. The
hydrolysis was performed in DMSO, MeOH and DMF
media and was found to be the fastest in DMSO (complete
conversion was observed already in 1–2 min), but in this
case the product was isolated only as inseparable mixture
with dimethyl sulfone as by-product. Pure amide 19j was
obtained in MeOH, but in this case the reaction took much
longer time and gave the product in reduced yield most
likely due to hydrolysis of the ester.
Azides 19 were conveniently hydrogenated in presence of
Pd/C to afford the amino esters 8. Nitro derivative 19i was
selectively reduced by treatment with PPh3 followed by hy-
drolysis of the iminophosphorane intermediate to afford
amine 8e, that was used without further purification.
In several steps amino esters were transformed into chon-
dramide analogs 2b–k (Scheme 4). First, peptide coupling
with the known dipeptide acid,[11c,25] 6 afforded tripeptides
20 in good yields. Subsequent saponification of the methyl
ester, either with Me3SnOH (20b–d) or simply with NaOH
[20e (NO2), 20i (CN), 20j (CONH2), 20k (Ph)] occurred in
quantitative yields and without epimerization. But saponifi-
cation of the CH2OTBS- and (CH2)2OTBS-substituted tri-
peptide esters 20g and 20h was accompanied by partial de-
protection of the alcohol functions. Still, pure acids 21g and
21h were successfully isolated by chromatography in re-
duced, but good yields (~80%). Thus, there was no need to
call upon the milder Me3SnOH procedure. On the next
stage, acids 21b–d were initially esterified with hydroxy
ester 7-epi-7 under Yamaguchi conditions to give the seco-
compounds 22b–d in reasonable yields. However, later we
found it more convenient to perform the esterification reac-
tion under Mitsunobu conditions since the synthesis of hy-
droxy ester 7 is shorter than the one for 7-epi-7.[15] There-
fore, the acids 21 were esterified with alcohol[15] 7 under
Mitsunobu conditions in quite good yields. Here, the correct
use of either DEAD or DIAD reagents in each case is im-
portant for successful separation of the esterification prod-
uct. Otherwise, the corresponding hydrazodicarboxylate by-
product would be inseparable (or hardly separable) from
the product. Unfortunately, the acid 21j (CONH2) could not
be esterified under Mitsunobu conditions. Other methods
(Yamaguchi esterification, DCC/DMAP conditions) were
unsuccessful either. Eventually, the corresponding chon A
derivative was prepared by nitrile hydrolysis on the macro-
cycle 2j (see below). For macrolactam formation, the NBoc
protected tert-butyl esters 22 were subjected to double de-
protection under acidic conditions. Initially, the reaction was
carried out in a mixture of TFA/CH2Cl2 (1:30) as previously
described.[16] The progress of the deprotection was easily
monitored by TLC and NMR and under these conditions
1
the observation of no significant side peaks in the H and
13C NMR spectra on later steps when diastereomers would
appear.
Diols 17 were next transformed to b-azides 18 by means
of a Mitsunobu reaction with hydrazoic acid.[24] Initial ex-
periments revealed only moderate conversion of the diols,
accompanied by significant nitrogen evolution. This side re-
action was caused by hydrazoic acid decomposition of the
ternary mixture in the presence of triphenylphosphine and
DEAD. Mixing all three components together caused rapid
nitrogen evolution, while the binary mixture of HN3 and
PPh3 was stable at room temperature. This undesired side
reaction was diminished when the DEAD solution was in-
troduced into the reaction as the last component at ꢀ258C
rather than at 08C. Thereafter, the mixture was allowed to
stand at room temperature until the reaction stopped.
Under these conditions, the desired azides were obtained in
better yields as pure stereoisomers in accordance with litera-
ture data.[24] The structure and purity were additionally con-
firmed by careful comparison of NMR spectra with previ-
ously described compounds (see supporting information). In
these Mitsunobu reactions, diols 17e (NO2), 17i (CN), and
17k (Ph) were used as hardly separable mixtures, slightly
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Synthesis of Chondramide A Analogues
FULL PAPER
(CH2OH) and 2h [(CH2)2OH] the crude cyclization prod-
ucts were stirred in aqueous K2CO3/MeOH for 5–10 min
before purification. This resulted in complete hydrolysis of
the corresponding TFA esters. Unfortunately, the presence
of the benzylic trifluoroacetate negatively affected cycliza-
tion of the amino acid generated from 22g (CH2OTBS),
yielding after short hydrolysis with aqueous K2CO3/MeOH
two products as judged by TLC. The more polar substance
turned out to be the desired chondramide A analogue 2g.
The second substance was shown to be the corresponding
1
benzotriazolyl ether that was characterized by H NMR and
HRMS analysis (see Supporting Information for details).
Thus, for this analogue it can be concluded that the TBS
group is not ideal and revision of the synthetic scheme
would be required in this case for an optimized synthesis.
But for the other cases the described synthetic Scheme is
considered reliable. With the NO2-substituted analogue 2e
we attempted reduction of the nitro group to obtain the
NH2 derivative 2 f. This was tried with catalytic hydrogena-
tion and sodium dithionite. Both methods afforded the de-
sired amino chondramide 2 f in moderate yield. Finally, the
CONH2-substituted analogue 2j was prepared by hydrolysis
of the cyano group of 2i with H2O2/K2CO3/DMSO under
mild conditions in good unoptimized yield.
Biology: The biological activity of the chondramide A (chon
A) analogues 2 was evaluated in a cytotoxicity assay on
human primary foreskin fibroblast (HFF) cells. These data
are included in Table 1. We also included the natural prod-
Table 1. Cytotoxicity of jasplakinolide (1) and chondramide A deriva-
tives 2.[a]
Entry Compound
EC50 (HFF) [mm] IC50 (L-929) [mm]
0.028ꢁ0.004
1
jasplakinolide_np (1)
2
3
chon A_np (2a)
chon B_np (3)
0.118ꢁ0.023
0.044ꢁ0.005
0.036ꢁ0.007
0.109ꢁ0.007
0.027ꢁ0.002
0.066ꢁ0.008
0.029ꢁ0.002
0.040ꢁ0.006
0.057ꢁ0.001
0.052ꢁ0.010
0.056ꢁ0.013
4
chon C_np (4)
5
6
7
8
chon A_synth (2a)
chon A_Me (2b)
chon A_OMe (2c)
chon A_F (2d)
0.0079ꢁ0.0013
0.0083ꢁ0.0021
0.0080ꢁ0.0013
0.017ꢁ0.005
0.031ꢁ0.002
0.035ꢁ0.002
0.033ꢁ0.004
0.011ꢁ0.001
0.223ꢁ0.042
0.025ꢁ0.002
Scheme 4. Synthesis of chondramide A analogues 2b–j. Esters 22b–d
were also prepared by Yamaguchi esterification using hydroxy ester 7-
epi-7; TBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tet-
rafluoroborate, HOBt=1-hydroxy-benzotriazole, DIAD=diisopropyl
azodicarboxylate. [a] For compound 2, R=CH2OH and (CH2)2OH, re-
spectively.
9
chon A_NO2 (2e)
chon A_NH2 (2 f)
chon A_CH2OH (2g)
10
11
12
13
14
15
chon A_ (CH2)2OH (2h) 0.059ꢁ0.010
chon A_CN (2i)
0.027ꢁ0.001
1.424ꢁ0.017
0.213ꢁ0.016
chon A_CONH2 (2j)
chon A_Ph (2k)
the conversion was not complete even after 2 d. In particu-
lar, the carboxylic ester cleavage is quite slow. However
with more concentrated acid (1:10) complete and clean de-
protection was observed for each compound after about
20 h. Interestingly, under these reaction conditions the TBS
groups of 22g and 22h were quantitatively transformed to
the corresponding TFA esters, as suggested by NMR of the
crude products. In each case the crude amino acids were
subjected to macrolactamization in the presence of TBTU,
which afforded chondramide A analogs 2e (NO2), 2i (CN),
and 2k (Ph) directly. In order to obtain the analogues 2g
[a]np=natural product (isolated), synth=synthetic compound.
ucts jasplakinolide and other chondramides in this assay. As
can be seen, several of the chon A analogues are slightly
more active than the natural product itself. Three of the an-
alogues [2b (Me), 2d (F), 2i (CN)] are even comparable to
jasplakinolide (1) itself (EC50 in the range of 30 nm). These
are characterized by small substituents. Other analogues
show intermediate activity, being about half as active as jas-
plakinolide (EC50 in the range of 60 nm). These analogues
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M. E. Maier et al.
water and dried in vacuo to afford crude but pure (2E)-3-(4-nitropheny-
l)acrylic acid in quantitative yield (Note: the free acid is soluble in ace-
tone, DMSO, but hardly soluble in CH2Cl2, EtOAc). It was taken up in
methanol (20 mL), thionyl chloride (1.3 equiv) was carefully added and
the resulting suspension was heated at reflux for 1 h. At this time TLC
(EtOAc/petroleum ether, 1:2) indicated complete consumption of the
acid and the volume of precipitates visually increased. The product was
obtained by recrystallization directly from the reaction mixture as fol-
lows. The reaction mixture was concentrated in vacuo till a thick suspen-
sion resulted, that was filtered on a glass frit and the precipitate was
washed with a small amount of cold methanol to afford 1.52 g (93%) of
pure methyl (2E)-3-(4-nitrophenyl)acrylate (9e) as a slightly yellow solid.
Analytical data were in agreement with literature data.[26]
include 2c (OMe), 2e (NO2), 2 f (NH2), 2g (CH2OH), and
2h ((CH2)2OH). Somewhat surprising is the activity of ana-
logue 2k (Ph) (EC50 =213 nm). With this large substituent
one would not expect that the analogue would fit in the
binding pocket.[12] Although the amide substituent in 2j is
not very large, this compound is much less active. A similar
trend in activity can be seen on the mouse fibroblast cell
line L-929, which is more sensitive to these cyclodepsipepti-
des.
In general, it can be concluded that the aryl ring on the
tyrosine derivative accepts a range of substituents. One ex-
planation could be that the 4-position of the aryl ring pro-
trudes out of the binding pocket. Thus, some of the pre-
pared derivatives might be used for the preparation of la-
beled derivatives. The recent finding that jasplakinolide V
with a catechol in place of the phenol substituent is also
quite active shows this region to be less sensitive to structur-
al modifications than the other parts.[8b]
AHCTUNGRETG(NNUN 2S,3R)-Methyl 3-(4-cyanophenyl)-2,3-dihydroxypropanoate (17e): A
mixture of K3Fe(CN)6 (7.22 g, 22.0 mmol, 3 equiv), K2CO3 (3.04 g,
22.0 mmol, 3 equiv), MeSO2NH2 (0.70 g, 7.37 mmol, 1 equiv),
K2OsO2(OH)4 (10 mg, 0.027 mmol, 0.0037 equiv), and the ligand
(DHQD)2PHAL (57 mg, 0.073 mmol, 0.01 equiv) was stirred in a mixture
of water (35 mL) and tBuOH (35 mL) until dissolved and then the solu-
tion was cooled to 08C in an ice bath. At this point cinnamate 9e (1.52 g,
7.34 mmol) was added and the reaction mixture was allowed to reach
room temperature slowly while being stirred overnight, at which time a
yellow suspension was formed and complete or almost complete conver-
sion was observed according to TLC (petroleum ether/EtOAc, 1:1). Then
solid Na2SO3 (9.2 g, 73.4 mmol, 10 equiv) was added and the mixture was
stirred for several min. The suspension was filtered, and the filter cake
was washed with EtOAc. The filtrate was transferred to a separatory
funnel, the organic phase was separated, and the water phase was ex-
tracted twice with EtOAc. The combined organic extracts were washed
with saturated NaCl solution, dried with Na2SO4, filtered, and evaporat-
ed. The residue was purified by flash chromatography (CH2Cl2/MeOH,
95:5) to afford pure diol 17e (0.698 g, 39%) as a colorless solid. Some-
times it was difficult to purify the diol from CH3SO3NH2 (as a contami-
nant in different compounds comes at d=3.08–3.10 (s, 3H), 4.67–
4.79 ppm (brs, 2H)), but the sulfone amide impurity did not influence
the next Mitsunobu azidation as was established later. Also, a better pro-
cedure but for another substrate (17g) utilizing 1% of the catalyst is de-
scribed in the Supporting information. 1H NMR (400 MHz, CDCl3): d=
2.81 (brs, 1H, OH), 3.13 (brs, 1H, OH), 3.86 (s, 3H, OCH3), 4.39 (brs,
1H, 2-H), 5.14 (brs, 1H, 3-H), 7.59 (d, J=8.4 Hz, 2H, Ar), 8.23 ppm (d,
J=8.4 Hz, 2H, Ar).
Conclusion
Based on our previous synthesis of chondramide A, a range
of analogues was prepared that feature modifications in the
4-position of the b-tyrosine derivative. The required building
blocks, 3-aryl-3-amino-2-methoxypropanoates 8 were pre-
pared from cinnamates 9 by asymmetric dihydroxylation, re-
gioselective Mitsunobu substitution with hydrazoic acid, O-
methylation and reduction of the azide group. Subsequently,
amino esters 8 were condensed with dipeptide acid 6. After
hydrolysis of the methyl ester function, esterification of the
tripeptide acids 21 with the 7-hydroxy ester 7 or 7-epi-7, re-
spectively furnished the seco-compounds 22. Deprotection
of both the amino and the ester group was followed by mac-
rolactam formation to give analogues 2.
Preparation of hydrazoic acid solution: CAUTION: Hydrazoic acid is a
highly volatile, toxic and explosive liquid in individual state. However, in
solution it is stable and safe. In this study solutions up to 5m were used.
Sodium azide (3.0 g, 46 mmol) was mixed with water (1.5 mL) and tolu-
ene (10 mL). The suspension was cooled to near 08C and concentrated
H2SO4 (~2 g, ~1.09 mL, 20 mmol) was carefully added while cooling and
shaking the round bottom flask (stirring with magnetic stirring bar is not
sufficient). Crystals were kneaded with a spatula shortly after the addi-
tion of the acid. Then the mixture was filtered under positive pressure
and dried with Na2SO4 (there was no water phase remained, and no need
to separate it from toluene solution). To rapidly estimate the resulting
concentration of hydrazoic acid, a known amount of NaOH was dissolved
in a small amount of water, phenolphthalein was added and the pink so-
lution was titrated with the hydrazoic acid solution from an analytical
pipette (the concentration was found to be 3.3m against theoretical
4.0m).
In cell growth assays several of the derivatives surpassed
natural chondramide A in its biological activity. Among the
derivatives only the amide derivative 2j was not very active.
Otherwise, it appears that the aryl ring of the b-tyrosine tol-
erates a broad range of substituents in the 4-position. These
derivatives should be useful for probing a number of cellular
questions in different systems that rely on actin filaments
for important aspects of biology.
Experimental Section
Only part of the experimental material is given here. For further details
and for compound characterization see Supporting Information. Proce-
dures are given for the sequence leading to chondramide A analogue 2e
(NO2) and the reduction of 2e to analogue 2 f (NH2).
AHCTUNGTERG(NNUN 2S,3S)-Methyl 3-azido-2-hydroxy-3-(4-nitrophenyl)propanoate (18e): To
a stirred solution of diol 17e (0.228 g, 0.946 mmol), triphenylphosphine
(0.297 g, 1.14 mmol, 1.2 equiv), hydrazoic acid (0.86 mL, 3.3m in toluene,
3 equiv) in THF (2.0 mL) at ꢀ258C was added DEAD (0.56 mL, 0.535 g,
1.23 mmol, 40% wt. solution in toluene, 1.3 equiv), then the cooling bath
was removed (slight evolution of N2 was observed) and the resulting mix-
ture was stirred overnight at ambient temperature (TLC control: petrole-
um ether/EtOAc, 1:1; NMR control: a sample portion was taken from
the reaction mixture, evaporated and directly analyzed by NMR). Then
the reaction mixture was concentrated in vacuo. The residue was purified
by flash chromatography (petroleum ether/EtOAc, 4:1 to 2:1) to yield
(E)-Methyl 4-nitrocinnamate (9e) (by Knoevenagel reaction): A solution
of nitrobenzaldehyde (1.51 g, 0.01 mol), malonic acid (1.14 g, 1.1 equiv),
and pyridine (0.25 mL, 0.31 equiv) in ethanol (2 mL) was heated at
reflux. Already after 10–15 min a white solid began to precipitate. After
1.5 h, when TLC (EtOAc/petroleum ether, 1:2) indicated complete con-
sumption of the aldehyde, the reaction mixture was cooled and acidified
with diluted aqueous HCl. The precipitate was filtered, washed with
13354
www.chemeurj.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 13349 – 13357

Synthesis of Chondramide A Analogues
FULL PAPER
18e (0.145 g, 58%) as a slightly orange oil which solidified into a waxy
solid upon standing. Rf (petroleum ether/EtOAc, 1:1) = 0.55; ½aꢂ2D0 = +
7.9, 7.1, 0.8 Hz, 1H, Trp HAr), 7.21 (d, J=8.1 Hz, 1H, b-Tyr NH), 7.31 (d,
J=7.9 Hz, 1H, Trp HAr), 7.34 (d, J=8.3 Hz, 2H, Ar), 7.57 (d, J=7.9 Hz,
1H, Trp HAr), 8.06 (d, J=8.3 Hz, 2H, Ar), 8.21 ppm (s, 1H, Trp NH);
13C NMR (100 MHz, CDCl3): d=17.8 (Ala CH3), 23.5 (CH2), 28.3 (C-
1
101.1 (c = 1.00, CH2Cl2); H NMR (400 MHz, CDCl3): d=3.11 (brs, 1H,
OH), 3.73 (s, 3H, OCH3), 4.58 (d, 1H, 2-H), 5.08 (d, J=3.8 Hz, 1H, 3-
H), 7.53 (d, J=8.7 Hz, 2H, Ar), 8.23 ppm (d, J=8.7 Hz, 2H, Ar);
13C NMR (100 MHz, CDCl3): d=53.1 (OCH3), 66.4 (C-3), 73.7 (C-2),
123.7 (Car), 128.7 (Car), 141.8 (Car), 148.1 (Car), 171.3 ppm (CO2CH3);
HMRS (ESI): m/z calcd for C10H10N4O5 [M+Na]+: 289.05434; found:
289.05431. Note. The product 18e was slightly contaminated with diethyl
hydrazodicarboxylate (~3–5 mol%), having signals d=1.28ꢁ0.01 (t,
6H), 4.22ꢁ0.01 ppm (q, 4H). This impurity did not cause problems in
the subsequent steps.
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Trp), 111.2, 118.5, 119.5, 122.2 (2C, Trp), 123.4 (2C, Ar), 127.1 (quat.
Trp), 128.7 (2C, Ar), 136.1 (quat. Trp), 144.3 (quat. Ar), 147.5 (quat. Ar),
155.2 (Boc), 169.5, 169.6, 174.5 ppm; HMRS (ESI): m/z calcd for
C31H39N5O9 [M+Na]+: 648.26400; found: 648.26502.
Tripeptide acid 21e (NO2): To a solution of methyl ester 20e (55.3 mg,
0.0884 mmol) in THF (0.4 mL) were added water (0.6 mL), methanol
(0.3 mL) and NaOH (7.5 mg, 0.188 mmol, 2.1 equiv). The initial biphasic
mixture became homogeneous with progressing saponification. After
being stirred for 1 h at room temperature until complete conversion (con-
trolled by TLC), the mixture was diluted with water (5 mL) and ethyl
acetate (8 mL). It was carefully acidified with 1m NaHSO4 to pH ~2
before the layers were separated and the aqueous phase extracted once
with ethyl acetate (8 mL). The combined organic layers were washed
with water, saturated NaCl solution, dried with Na2SO4, filtered, and con-
centrated in vacuo to afford the crude acid 21e as a colorless foam. Rf
(EtOAc/AcOH 100:1) = 0.4; 1H NMR (400 MHz, CDCl3): d=0.83 (d,
J=6.9 Hz, 3H, Ala CH3), 1.39 (s, 9H, tBu), 2.93 (s, 3H, NCH3), 3.18 (dd,
J=15.3, 9.9 Hz, 1H, CH2), 3.28–3.34 (m, 1H, CH2), 3.34 (s, 3H, OCH3),
3.98 (d, J=6.3 Hz, 1H, CHOCH3), 4.42–4.49 (m, 1H, Ala CH), 5.40 (dd,
J=7.6, 7.1 Hz, 1H, b-Tyr CH), 5.46 (d, J=7.4 Hz, 1H, Ala NH), 5.59
(dd, J=9.9, 7.4 Hz, 1H, Trp CH), 6.88 (s, 1H, Trp HAr), 7.05 (app t, J=
7.0 Hz, 1H, Trp HAr), 7.13 (app t, J=7.0 Hz, 1H, Trp HAr), 7.21 (d, J=
8.6 Hz, 1H, b-Tyr NH), 7.28 (d, J=7.9 Hz, 1H, Trp HAr), 7.41 (d, J=
8.4 Hz, 2H, Ar), 7.53 (d, J=7.9 Hz, 1H, Trp HAr), 8.04 (d, J=8.4 Hz, 2H,
Ar), 8.36 ppm (s, 1H, Trp NH); 13C NMR (100 MHz, CDCl3): d=17.0
ACHTUNGTRENNUNG(2S,3S)-Methyl 3-azido-2-methoxy-3-(4-nitrophenyl)propanoate (19e): To
a solution of a-hydroxy ester 18e (0.495 g, 1.86 mmol) in dry 1,2-di-
chloroethane (1.9 mL) was added trimethyloxonium tetrafluoroborate
(0.495 g, 3.35 mmol, 1.8 equiv) and proton sponge (0.876 g, 4.09 mmol,
2.2 equiv). The flask was covered with alumina foil. After stirring the sus-
pension at 408C overnight, a small probe was taken from the reaction
mixture and quenched with EtOAc/HClaq for TLC (petroleum ether/
EtOAc, 1:1), that indicated full conversion. The reaction mixture was
cooled, treated with EtOAc/H2O, and acidified with 1n HCl to pH 2–3.
The precipitate was filtered off and the filtrate was separated. The aque-
ous phase was extracted once with EtOAc and the combined organic ex-
tracts were washed with water, and saturated NaCl solution, dried with
Na2SO4, filtered, and concentrated in vacuo. The residue was chromato-
graphed (petroleum ether/EtOAc, 3:1 to 2:1) to yield 19e (0.48 g, 92%)
as a slightly orange oil. Rf (petroleum ether/EtOAc, 2:1) = 0.54; ½aꢂ2D0 = +
49.5 (c = 1.00, CH2Cl2); 1H NMR (400 MHz, CDCl3): d=3.38 (s, 3H,
OCH3), 3.75 (s, 3H, CO2CH3), 3.99 (d, J=6.1 Hz, 1H, 2-H), 4.89 (d, J=
6.1 Hz, 1H, 3-H), 7.55 (d, J=8.7 Hz, 2H, Ar), 8.22 ppm (d, J=8.7 Hz,
2H, Ar); 13C NMR (100 MHz, CDCl3): d=52.5 (OCH3), 59.3 (OCH3),
65.0 (C-3), 83.0 (C-2), 123.8 (Car), 129.0 (Car), 142.4 (Car), 148.1 (Car),
169.6 ppm (CO2CH3); HMRS (ESI): m/z calcd for C11H12N4O5 [M+Na]+
: 303.06999; found: 303.07002.
(Ala CH3), 23.2 (CH2), 28.3 (C
ACHTUNGTRENNUNG
(b-Tyr CH), 56.7 (Trp CH), 58.8 (OCH3), 80.8 (CAHCUTNGTRENNUNG
(CHOCH3), 110.2 (quat. Trp), 111.2, 118.4, 119.5, 122.1, 122.3, 123.5 (2C,
Ar), 127.1 (quat. Trp), 128.4 (2C, Ar), 136.1 (quat. Trp), 145.2 (quat. Ar),
147.4 (quat. Ar), 156.3 (Boc), 169.5, 170.7, 174.8 ppm; HMRS (ESI): m/z
calcd for C30H37N5O9 [M+Na]+: 634.24835; found: 634.24819.
Reduction of azide 19e and coupling of amine 8e with acid 6 to tripep-
tide 20e (NO2): A solution of azide 19e (89.6 mg, 0.32 mmol) and PPh3
(92.2 mg, 0.352 mmol, 1.1 equiv) in of THF (1 mL) was stirred at 40–
508C for 1 h for clean and complete conversion to the corresponding imi-
nophosphorane (TLC control: CH2Cl2/MeOH/NH3, 10:1:0.1, Rf 0.5 for
the iminophosphorane). Selected 1H NMR (400 MHz, CDCl3) data for
the iminophosphorane: d=7.91 (d, 2H), 3.67 (s, 3H), 3.13 ppm (s, 3H).
Then water (0.1 mL) was added and the mixture was further stirred at
40–508C for ~8 h. Because the Rf values of the iminophosphorane, Ph3P=
O and the resulting amine were all the same, the reaction progress was
conveniently monitored by analyzing small evaporated probes taken
from the reaction mixture by NMR. Selected 1H NMR (400 MHz,
CDCl3) data for the amine: d=8.15 (d, 2H), 3.65 (s, 3H), 3.39 ppm (s,
3H). When appropriately clean and high (~86%) conversion was ach-
ieved, the mixture was evaporated to yield 0.166 g of a sticky oil, contain-
ing ~36% w/w amine 8e (assuming the conversion was 80% as the
lowest). Then, to a solution of this crude mixture (108 mg), containing
amine 8e (approx. 38.9 mg, 0.153 mmol, 1.24 equiv of the amine) in
DMF (2.3 mL) were added acid 6 (47.7 mg, 0.123 mmol), HOBt (24.9 mg,
0.184 mmol, 1.5 equiv), iPr2NEt (0.064 mL, 0.369 mmol, 3 equiv). At
ꢀ108C TBTU (59 mg, 0.184 mmol, 1.5 equiv) was added and the reaction
was stirred for 5–6 h at room temperature. The mixture was diluted with
water (5 mL) and extracted with ethyl acetate (3ꢃ8 mL). The combined
organic layers were washed with 1n NaHSO4 solution (5 mL), saturated
NaHCO3 solution (5 mL), saturated NaCl solution (5 mL), dried with
Na2SO4, filtered, and concentrated in vacuo. Purification of the residue
by flash chromatography (petroleum ether/EtOAc, 2:1) gave tripeptide
20e (58.7 mg, 76%) as a white foam. Rf (petroleum ether/EtOAc, 2:1) =
0.48; ½aꢂ2D0 = +4.0 (c = 1.00, CH2Cl2); 1H NMR (400 MHz, CDCl3): d=
0.97 (d, J=6.9 Hz, 3H, Ala CH3), 1.40 (s, 9H, tBu), 2.98 (s, 3H, NCH3),
3.17 (dd, J=15.5, 9.4 Hz, 1H, CH2), 3.38 (s, 3H, OCH3), 3.38 (m, 1H,
CH2), 3.61 (s, 3H, CO2CH3), 4.07 (d, J=5.1 Hz, 1H, CHOCH3), 4.48–
4.54 (m, 1H, Ala CH), 5.33 (d, J=7.4 Hz, 1H, Ala NH), 5.44 (dd, J=8.1,
5.1 Hz, 1H, b-Tyr CH), 5.53 (dd, J=8.9, 7.4 Hz, 1H, Trp CH), 6.89 (s,
1H, Trp HAr), 7.08 (ddd, J=7.9, 7.1, 0.8 Hz, 1H, Trp HAr), 7.16 (ddd, J=
Depsipeptide 22e (NO2): The crude acid 21e (52 mg, 0.0851 mmol) and
alcohol 7 (32.8 mg, 0.128 mmol, 1.5 equiv) were dissolved in THF (1 mL)
and Ph3P (40 mg, 0.152 mmol, 1.8 equiv) was added at 08C. This was fol-
lowed by the dropwise addition of DIAD (0.030 mL, 0.152 mmol,
1.8 equiv). The cooling bath was removed and the mixture stirred over-
night at room temperature. The reaction mixture was concentrated in
vacuo and the residue purified by flash chromatography (petroleum
ether/EtOAc 2:1 to 1:1) to give ester 22e (62 mg, 86%) as a colorless
foam. Rf (petroleum ether/EtOAc 1:1) = 0.30; ½aꢂ2D0 =ꢀ4.0 (c = 1.00,
CH2Cl2); 1H NMR (400 MHz, CDCl3): d=0.77 (d, J=6.6 Hz, 3H, CH3),
0.91 (d, J=6.1 Hz, 3H, CH3), 0.96 (d, J=6.8 Hz, 3H, Ala CH3), 1.01 (d,
J=6.8 Hz, 3H, CH3), 1.40 (s, 9H, tBu), 1.41 (s, 9H, tBu), 1.56 (s, 3H,
CH3), 1.92 (dd, J=13.9, 7.6 Hz, 1H, CH2), 2.33 (dd, J=13.9, 6.8 Hz, 1H,
CH2), 2.40–2.51 (m, 2H, 2 CH), 2.97 (s, 3H, NCH3), 3.17 (dd, J=15.4,
9.6 Hz, 1H, CH2), 3.37 (dd, J=15.4, 7.1 Hz, 1H, CH2), 3.40 (s, 3H,
OCH3), 4.06 (d, J=4.5 Hz, 1H, CHOCH3), 4.46–4.61 (m, 2H, Ala CH,
CO2CH), 4.84 (d, J=9.9 Hz, 1H, =CH), 5.34 (d, J=7.3 Hz, 1H, Ala
NH), 5.43 (dd, J=8.3, 4.5 Hz, 1H, b-Tyr CH), 5.52 (dd, J=9.6, 7.1 Hz,
1H, Trp CH), 6.88 (s, 1H, Trp HAr), 7.07 (ddd, J=7.8, 7.1, 0.8 Hz, 1H,
Trp HAr), 7.15 (ddd, J=8.1, 7.1, 0.8 Hz, 1H, Trp HAr), 7.21 (d, J=8.3 Hz,
1H, b-Tyr NH), 7.30 (d, J=8.1 Hz, 1H, Trp HAr), 7.39 (d, J=8.6 Hz, 2H,
Ar), 7.56 (d, J=7.8 Hz, 1H, Trp HAr), 8.04 (d, J=8.6 Hz, 2H, Ar),
8.25 ppm (s, 1H, Trp NH); 13C NMR (100 MHz, CDCl3): d=16.4, 16.6,
17.1, 17.6, 17.9, 23.5 (CH2), 28.0 (C
37.5, 38.6, 43.4 (CH2), 46.7 (Ala CH), 53.8 (b-Tyr CH), 56.8 (Trp CH),
59.3 (OCH3), 76.4 (CO2CH), 79.6 (C(CH3)3), 79.9 (C(CH3)3), 81.4
ACHTUNTGRENNG(U CH3)3), 28.3 (CCAHTUNGTREN(NUNG CH3)3), 30.9 (NMe),
A
ACHTUNGTRENNUNG
(CHOCH3), 110.5 (quat. Trp), 111.1, 118.4, 119.5, 122.1, 122.2, 123.3 (2C,
Ar), 127.1 (quat. Trp), 127.5 (=CH), 129.1 (2C, Ar), 134.0 (=C<), 136.1
(quat. Trp), 144.3, 147.4, 155.2 (Boc), 168.6, 169.5, 174.5, 175.7 ppm;
HMRS (ESI): m/z calcd for C45H63N5O11 [M+Na]+: 872.44163; found:
872.44212.
Chem. Eur. J. 2011, 17, 13349 – 13357
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13355

M. E. Maier et al.
Chondramide 2e (NO2): To a stirred solution of compound 22e (62.7 mg,
0.0738 mmol) in CH2Cl2 (2.2 mL) was added TFA (0.22 mL, 0.34 g,
2.98 mmol) at 08C. The reaction mixture was allowed to warm to room
temperature and after stirring for 22 h, the solvent was removed in vacuo
(TLC control: CH2Cl2/MeOH/NH3, 10:1:0.1). For azeotropic removal of
TFA the residue was taken up in toluene (3ꢃ0.5 mL) and concentrated
in vacuo each time. The crude product was dissolved in DMF (30 mL)
and iPr2NEt (64 mL, 47.7 mg, 0.369 mmol, 5 equiv), HOBt (19.9 mg,
0.147 mmol, 2 equiv) and TBTU (47.4 mg, 0.147 mmol, 2 equiv) were
added. The solution was stirred at room temperature for 20 h and then
diluted with water (20 mL) and EtOAc (20 mL). The aqueous layer was
extracted with EtOAc (3ꢃ20 mL) and the combined organic layers were
washed with 5% aqueous KHSO4 solution (20 mL), water (20 mL), satu-
rated NaHCO3 solution (20 mL), water (2ꢃ20 mL) and saturated NaCl
solution (20 mL). The combined organic extracts were dried over
Na2SO4, filtered, and concentrated in vacuo. The crude product was puri-
fied by flash chromatography (petroleum ether/EtOAc, 1:3 to 0:1) to
give depsipeptide 2e (27.3 mg, 48%) as a colorless foam. Rf (EtOAc) =
0.41; ½aꢂ2D0 = +18.5 (c = 1.00, CH2Cl2); 1H NMR (400 MHz, CDCl3): d=
0.84 (d, J=6.6 Hz, 3H, CH3), 0.85 (d, J=6.1 Hz, 3H, CH3), 1.07 (d, J=
6.8 Hz, 3H, CH3), 1.17 (d, J=6.8 Hz, 3H, CH3), 1.64 (s, 3H, CH3), 1.88
(d, J=13.1 Hz, 1H, CH2), 2.36–2.51 (m, 3H, 2 CH, CH2), 2.95 (s, 3H,
NCH3), 3.17 (dd, J=15.2, 8.6 Hz, 1H, CH2), 3.22 (s, 3H, OCH3), 3.31
(dd, J=15.2, 7.8 Hz, 1H, CH2), 3.75 (d, J=8.1 Hz, 1H, CHOCH3), 4.77–
4.83 (m, 2H, Ala CH, CO2CH), 4.90 (d, J=9.1 Hz, 1H, =CH), 5.30 (t,
J=8.3 Hz, 1H, b-Tyr CH), 5.60 (t, J=8.1 Hz, 1H, Trp CH), 6.50 (d, J=
7.3 Hz, 1H, Ala NH), 6.82 (s, 1H, Trp HAr), 7.06 (d, J=8.8 Hz, 1H, b-Tyr
NH), 7.10 (ddd, J=8.1, 7.1, 0.8 Hz, 1H, Trp HAr), 7.17 (ddd, J=8.1, 7.1,
0.8 Hz, 1H, Trp HAr), 7.24 (d, J=8.6 Hz, 2H, Ar), 7.32 (d, J=8.1 Hz, 1H,
Trp HAr), 7.59 (d, J=8.1 Hz, 1H, Trp HAr), 8.06 (d, J=8.6 Hz, 2H, Ar),
8.11 ppm (s, 1H, Trp NH); 13C NMR (100 MHz, CDCl3): d=15.8, 16.6,
17.3, 18.6, 20.0, 23.6 (CH2), 30.2 (NCH3), 37.0, 40.2, 44.1 (CH2), 45.2 (Ala
CH), 54.4 (b-Tyr CH), 56.0 (Trp CH), 58.2 (OCH3), 76.8 (CO2CH), 81.9
(CHOCH3), 110.2 (quat. Trp), 111.2, 118.5, 119.6, 122.2, 122.3, 123.4,
127.1 (quat. Trp), 127.8 (=CH), 127.9, 134.3 (=C<), 136.1 (quat. Trp),
144.5, 147.3, 169.77, 169.82, 174.1, 174.5 ppm; HMRS (ESI): m/z calcd for
C36H45N5O8 [M+Na]+: 698.31603; found: 698.31672.
6.96 (m, 3H, b-Tyr NH, Ar), 7.11 (ddd, J=8.1, 7.8, 1.0 Hz, 1H, Trp HAr),
7.17 (ddd, J=8.1, 7.8, 1.0 Hz, 1H, Trp HAr), 7.30 (d, J=7.8 Hz, 1H, Trp
HAr), 7.90 (d, J=7.8 Hz, 1H, Trp HAr), 7.87 ppm (s, 1H, Trp NH);
13C NMR (100 MHz, CDCl3): d=15.3, 16.6, 17.6, 18.6, 20.5, 23.3 (CH2),
30.1 (NCH3), 37.1, 40.3, 43.8 (CH2), 45.3 (Ala CH), 53.9 (b-Tyr CH), 55.7
(Trp CH), 58.0 (OCH3), 77.2 (CO2CH), 82.6 (CHOCH3), 110.7 (quat.
Trp), 111.0, 115.0 (2C, Ar), 118.6, 119.4, 122.05, 122.11, 127.2 (quat. Trp),
127.7 (quat. Ar), 127.9 (2C, Ar), 128.4 (=CH), 134.1 (=C<), 136.1 (quat.
Trp), 145.9, 169.6, 170.6, 174.0, 174.5 ppm; HMRS (ESI): m/z calcd for
C36H47N5O6 [M+Na]+: 668.34186; found: 668.34224.
Cytotoxicity assay and EC50 determination: Human foreskin fibroblast
(HFF) cells grown in Dulbeccoꢄs modified Eagleꢄs medium with 10%
fetal bovine serum and 10 mgmLꢀ1 gentamicin were seeded at 2000 cells
per well in 96-well microtiter plates. After 2 h of incubation at 378C in a
humidified atmosphere with 5% CO2, jasplakinolide and chon A ana-
logues were added to reach the final concentrations of 0.0049–5 mm (in
two-fold increments). The number of viable cells was determined 72 h
later using the CellTiter 96 AQueous One Solution Cell Proliferation
Assay (Promega). Absorbance at 570 l (A570) measured was normalized
to percentage of control (untreated) and plotted against logarithmic con-
versions of compound concentrations using Prism 5.0 (GraphPad Soft-
ware). The log (inhibitor) vs. response curve with variable slope was gen-
erated from triplicates of data to calculate EC50 values with Prism 5.0.
MTT test with the L-929 mouse fibroblasts: Growth inhibitory activity
on L-929 mouse fibroblasts were determined after incubation with serial
dilutions of the compounds for 5 d using an MTT assay.[27,28] The IC50 was
estimated from the concentration dependent activity curves.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft, an NIH
(grant AI073155) and the Fonds der Chemischen Industrie is gratefully
acknowledged. We thank Dr. Dorothee Wistuba (Institute of Organic
Chemistry, Tꢀbingen) for measuring the HRMS spectra. In addition, we
acknowledge help with chiral separations from Dr. Silvia Marten, Head
of Applications and Columns Department, Knauer GmbH, Berlin, Ger-
many. Furthermore, we would like to thank Wera Collisi (HZI Braun-
schweig) for excellent technical assistance with cytotoxicity assays.
Chondramide 2 f (NH2): By catalytic hydrogenation: A solution of chon-
dramide 2e (6.1 mg, 9.03 mmol) in methanol (0.5 mL) was hydrogenated
overnight in a round bottom flask connected to a hydrogen filled balloon,
using 10% Pd on carbon (TLC control: CH2Cl2/MeOH, 10:1). Upon
complete conversion, the solvent was evaporated and the residue purified
by flash chromatography (CH2Cl2/MeOH, 30:1 to 20:1) to afford chon-
dramide 2 f (2.7 mg, 47%) as white foam. A mixed fraction, containing
2 f and another unknown compound as major component was also isolat-
ed. Complete conversion was observed, but the reaction was not very
clean, likely because of formation of RNO and/or RNHOH, as suggested
by LC/MS examination of the mixed fraction. No evidence for competi-
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1
tive hydrogenation of the double bond was found (by H NMR).
By reduction with sodium dithionite: Alternatively, reduction of chondra-
mide 2e (5.8 mg, 8.58 mmol) was carried out in THF (0.5 mL), H2O
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6.8 Hz, 3H, Ala CH3), 1.15 (d, J=7.1 Hz, 3H, CH3), 1.63 (s, 3H, CH3),
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Synthesis of Chondramide A Analogues
FULL PAPER
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Received: June 27, 2011
Published online: October 20, 2011
Chem. Eur. J. 2011, 17, 13349 – 13357
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13357