A Straightforward Approach towards Cyclic Photoactivatable Tubulysin Derivatives

Article abstract of DOI:10.1002/anie.201405650

The development of a new photolabile protecting group containing an additional allyl functionality allows the synthesis of cyclic photoactivatable natural products. Cyclization occurs between the allyl moiety in the protecting group and a second double bond in the target molecule by means of ring-closing metathesis. Cyclization should increase the metabolic stability towards proteases. On the other hand, the conformational change should cause diminished biological activity. As illustrated for tubulysin derivatives, cyclic and photoactivatable drug candidates can easily be obtained in only two steps from simple building blocks through Ugi reaction and ring-closing metathesis. The photolabile protecting group is introduced by means of the isocyanide component during the Ugi reaction.

Full text of DOI:10.1002/anie.201405650

.
Angewandte
Communications
DOI: 10.1002/anie.201405650
Photoactivation
A Straightforward Approach towards Cyclic Photoactivatable
Tubulysin Derivatives**
Judith Hoffmann and Uli Kazmaier*
[
14]
Abstract: The development of a new photolabile protecting
group containing an additional allyl functionality allows the
synthesis of cyclic photoactivatable natural products. Cycliza-
tion occurs between the allyl moiety in the protecting group and
a second double bond in the target molecule by means of ring-
closing metathesis. Cyclization should increase the metabolic
stability towards proteases. On the other hand, the conforma-
tional change should cause diminished biological activity. As
illustrated for tubulysin derivatives, cyclic and photoactivatable
drug candidates can easily be obtained in only two steps from
simple building blocks through Ugi reaction and ring-closing
metathesis. The photolabile protecting group is introduced by
means of the isocyanide component during the Ugi reaction.
mones.
photochemical release of ATP.
Several different photocleavable groups are suitable
One of the first described examples was the
[15]
[
10a,11]
candidates for these types of applications.
benzyl group (NB) plays a dominant role (Figure 1), although
the o-nitrobenzaldehyde formed in the cleavage step might
The o-nitro-
Figure 1. Photocleavable protecting groups.
S
ince ancient times light has been used for the treatment of
[
1]
[16]
skin diseases. In ancient Egypt, psoralen already found
application in UV therapy, a protocol known today as PUVA
cause damage in cellular systems. The introduction of two
additional methoxy groups to NB (DMNB) lowers the
quantum yield, but also shifts the absorption maximum to
longer wavelengths (365 nm), making DMNB an ideal
candidate for application in biological systems.
Our research group has been involved in the syntheses of
peptidic natural products the past few years. An especially
interesting class of compounds are the tubulysins and their
derivatives. We are particularly interested in pretubulysin,
a biosynthetic precursor of the tubulysins (Figure 2), which
[
2]
(
psoralen + UVA) therapy . Psoralen and its more
frequently used 8-methoxy derivative boost the sensitivity
of the skin towards UV irradiation, increasing the efficiency
of the light therapy. Another approach, photodynamic
therapy, combines UV irradiation with the administration of
porphyrin derivatives as photosensitizers, and is used in tumor
therapy.
A rather modern concept uses photoswitches, molecules
which change their conformation upon irradiation.
Although azobenzenes are by far the most commonly used
photoswitches, other substance classes such as 1,2-dithieny-
lalkenes are also widespread. The light-induced conforma-
tional change can be used to open or close ion channels and
to modulate the affinity of drugs towards their receptor.
Besides these medical applications, photoactivatable com-
[
17]
[
3]
[
18]
[
4]
[5]
[
6]
[
7]
[
8]
[9]
[
10]
pounds find also application in cell biology. Here, biolog-
ically active molecules can be deactivated by modification
with a photolabile linker, and upon irradiation, the active
compound is released into the cell. This approach can be
applied for the release of neurotransmitters
[
11]
[
12]
[
13]
and hor-
Figure 2. Tubulysins and pretubulysin.
[*] J. Hoffmann, Prof. Dr. U. Kazmaier
Institute for Organic Chemistry, Saarland University
P.O. Box 151150, 66041 Saarbrꢀcken (Germany)
E-mail: u.kazmaier@mx.uni-saarland.de
inhibits the growth of a wide range of tumor cells in the low
[19]
nanomolar range. Like the tubulysins, pretubulysin inter-
Homepage: http://www.uni-saarland.de/fak8/kazmaier
**] This work was supported by the Deutsche Forschungsgemeinschaft
FOR 1406, Ka880/10-1/2). J.H. gratefully acknowledges a fellow-
[20]
acts with the microtubuli skeleton, suppressing angiogen-
[
[21]
esis,
and is therefore an excellent candidate for the
(
[22]
development of antitumor drugs. Structure–activity rela-
tionship (SAR) studies indicate that pretubulysin-derived
esters are 3–6 times less active than pretubulysin, while
amides are even less active (10–20fold) depending on the
derivative. Therefore one might assume that these derivatives
might act as prodrugs.
ship from the Studienstiftung des Deutschen Volkes. We thank J.
Herrmann and V. Schmitt (R. Mꢀller research group, Helmholtz
Institute for Pharmaceutical Reasearch Saarland) for the determi-
nation of the biological activities.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201405650.
1
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We recently became interested in applying the concept of
drug photoactivation for the directed treatment of easily
accessible tumors, such as melanoma and colon cancer. Our
aim was to convert pretubulysin into an “inactive” prodrug,
which could be activated by light (Figure 3). The major
excess of phosgene for 2 h. The excess phosgene was removed
to provide the chloroformate AMNBOC-Cl (5) in pure form
and in almost quantitative yield. This acid chloride was found
to be highly reactive and sensitive towards hydrolysis, but it
can be stored at 48C for weeks without significant decom-
position.
While the synthesis of tubulysin derivatives with a central
N-methylamide bond, such as in the case of pretubulysin, is
[
25]
straightforward, the synthesis of other N-substituted deriv-
[26]
atives is far from trivial for steric reasons. An interesting
approach towards the synthesis of N-substituted tubulysins
[
27]
was reported recently by Wessjohann et al.,
which took
[
28]
advantage of Ugi reactions. The products obtained, called
tubugis, showed excellent biological activities comparable to
those of the natural products. The structure of the tubulysin
side chain was defined by the structure of the isocyanide used.
We used this concept for the synthesis of photoactivatable
tubulysin derivatives, introducing the photolabile group via
the isocyanide. The natural tubulysins vary primarily in the
structure of the aliphatic acylal side chain. To introduce
a photolabile protecting group at this position, a heterofunc-
tionality is required in the side chain. Since it was not known
what type of functionalities would be accepted, we decided to
introduce not only a protected hydroxy group but also an
amine and a carboxyl functionality. The latter ones should
generate ionic side chains on cleavage, what should have
a rather strong effect on the biological activity. The required
isocyanides were obtained according to Scheme 2. The
Figure 3. Photocleavable pretubulysin derivatives.
difference between pretubulysin and the tubulysins is the
missing N-acylal side chain. Therefore, this position should be
suitable for the introduction of a photolabile side chain as
well, allowing cyclization with the C-terminus of the peptide.
One might expect that the conformation of the cyclic
pretubulysin derivative should differ significantly from the
open-chain form, resulting in a (significantly) diminished
binding affinity towards tubulin. Upon irradiation, the ring is
cleaved and the molecule should take up its natural con-
formation for binding. In this case, the photolabile protecting
group is still bound to prebutulysin through the ester linkage.
Proteolytic cleavage of this bond might liberate the active
drug.
Based on our good experience with peptide cyclizations
[
23]
through ring-closing metathesis (RCM),
we explored
a short synthetic sequence for the 4-allyloxy-5-methoxy-3-
nitrobenzyl (AMNB) protecting group, starting from vanillin
(1, Scheme 1). While the O-allylation of 1 proceeded almost
quantitatively, subsequent nitration was not a trivial issue
owing to several side reactions. However, when exactly
Scheme 2. Syntheses of photolabile isocyanides: a) 4, TBTU [O-(benzo-
triazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate]
1
equiv KNO was used in trifluoroacetic acid, the desired
3
product 3 could be obtained in good yield and purity.
Reduction of the aldehyde provided the photolabile benzyl-
alcohol AMNBO (4), which can be used directly for the
protection of carboxylic acids. For the synthesis of the
corresponding carbonates/carbamates, 4 was stirred with an
(
1.3 equiv), DMAP (0.3 equiv), DIPEA (2.0 equiv), CH Cl , RT, 16 h;
2 2
b) 4m HCl/dioxane, CH Cl , RT, 2 h; c) HCOOEt, NEt , TsOH (cat.),
528C, 20 h; d) POCl
e) 5, pyridine (1.0 equiv), THF, RT, 20 h.
2
2
3
(1.0 equiv), NEt (3.0 equiv), CH Cl , 08C, 1 h;
3 2 2
3
photolabile, protected carboxylic acid derivative 7 could be
obtained directly from Boc-Gly-OH and 4 using standard
transformations. Generation of the isocyanide turned out to
be the only critical step. Even when the reaction mixture had
been stirred at 08C for 1 h, conversion was still not complete
but the overall yield of 65% (for three steps) was acceptable.
Prolonging the reaction time or running the reaction at higher
temperature resulted in lower yield, caused by side reactions.
The isocyanides containing a photolabile carbamate and
carbonate group (9a and 9b) were prepared in an analogous
fashion, while completely anhydrous conditions were essen-
tial for good results. In addition, for the synthesis of reference
samples, the analogous non-photolabile isocyanides were
synthesized (see the Supporting Information).
Scheme 1. Synthesis of photolabile protecting groups suitable for
subsequent metatheses: a) allyl bromide, K CO , EtOH, 788C, 16 h;
2
3
b) KNO (1.0 equiv), CF COOH, RT, 16 h; c) NaBH , EtOH, RT, 4 h;
3
3
4
d) COCl (20% in toluene, 10 equiv), THF, RT, 2 h.
2
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Scheme 4. Expected product from the photocleavage of 11c.
a o-nitrosobenzaldehyde derivative (Scheme 4) should be
[
16]
formed upon photocleavage.
This linear product should
show the same molecular mass as 11c. Indeed, besides traces
of unreacted starting material 11c, a new product with the
2
+
same mass (m/z 503, [M+2H] ) could be identified. In
addition, a peak at m/z 496 was observed, correlating with the
product resulting from the reduction of the nitroso group to
2
+
the corresponding aniline ([M+2À14] ). This is in agreement
with the typical ionization pattern of aromatic nitroso
compounds.
Scheme 3. Syntheses of photocleavable tubulysin derivatives:
[36]
a) CH Cl , RT, 5 d; b) benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-
2
2
To determine whether the biological activity of the cyclic
compounds 11 is indeed lower than that of the potential
cleavage products 12, we measured their cytotoxicity (IC )
imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium
(
Grubbs II catalyst, 15 mol%), HCl (5.0 equiv), CH Cl , 1. 408C, 1 h;
2 2
2
. RT, 1 h.
5
0
towards a panel of tumor cell lines and compared the results
with those obtained for the linear peptides 12 (Figure 4). The
results obtained with the HCT-116 cell line are summarized in
Table 1. Interestingly, the cyclic carbonate 11a (IC :
The Ugi reactions were carried out in CH Cl (instead of
2
2
the commonly used MeOH or trifluoroethanol) to avoid side
reactions caused by nucleophilic attack of the solvent at the
5
0
À1
À1
120 ngmL ) and the ester 11c (IC : 63 ngmL ) both
5
0
[
28]
reaction intermediates (Scheme 3). Paraformaldehyde was
found to be more suitable than formaldehyde solution,
because of its lower reactivity (fewer side reactions). Usage
of the neutral form of the dipeptide serving as the acid
component was crucial, since the yields dropped significantly
when the corresponding amine hydrochloride was used.
Under these conditions, the reaction time was comparatively
long (5 days), but the reactions proceeded very cleanly and
the desired products could be obtained in good yields.
Figure 4. Potential cleavage products after irradiation and ester
cleavage.
The subsequent ring-closing metathesis also required
[
29]
some optimization studies.
0c as the substrate, only traces of the cyclization product
could be obtained, even when 0.6 equiv of Grubbs catalyst
In initial experiments using
1
Table 1: Biological activity of derivatives 11a–c and 12a–c determined by
[
30]
was used. We assumed that the reaction failed because of
coordination of the N-terminal tertiary amine on the Ru
catalyst. Possible changes to suppress this catalyst deacti-
À1
MTT assay (HCT-116/CHO-K1 cell line, IC50 [mgmL ]).
[
31]
Cyclic, photoactivatable tubulysin
derivatives
Open-chain, deprotected refer-
ence samples
[
32]
+ [33]
vation included the addition of Ti(OiPr)4 and H . After
11a
11b
11c
0.12/0.35
0.57/4.54
0.063/n.d .
12a
12b
12c
0.02/0.10
2.12/6.34
0.58/0.64
investigating several options we found that the addition of
5
.0 equiv of HCl and the use of 15 mol% Grubbs II catalyst
[
a]
gave the best results. Under these conditions the desired
[a] n.d.: not determined.
product 11c could be obtained in 72% yield (purity > 95%)
[35]
after reversed-phase flash chromatography. Similar results
could be obtained for the other Ugi products under these
conditions as well. This protocol provided cyclic tubulysin
derivatives in only two steps from rather simple building
blocks (see the Supporting Information).
To verify the photolabile characteristics of our products,
cyclopeptide 11c was irradiated for two hours with a UV-
LED lamp (365 nm). The reaction was monitored and
analyzed by HPLC/MS. According to the known mechanism,
showed comparatively high activity, while the carbamate
À1
11b was significantly less active (IC : 570 ngmL ).
5
0
When we examined the linear derivatives 12 closely it
became apparent that charged side chains are probably not
tolerated, since the activity of the amine 12b as well as the
carboxylic acid 12c was significantly lower than that of the
cyclic compounds (by a factor of 4–10). On the other hand, an
1
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OH functionality seems to be accepted. The activity of the
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magno et al.
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In conclusion we demonstrated that the photoactivation
of cyclic drug candidates could be realized with tubulysin
derivatives. The synthesis of photoactivatable peptides pro-
ceeded in only two steps from easily available building blocks.
The biological data obtained provided important information
about the influence of functionalized side chains on the
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activations in tumor cells and tumor tissues.
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Keywords: antitumor agents · natural products ·
photoactivation · pretubulysin · tubulysin
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