Synthesis and biological evaluation of tubulysin D analogs related to stereoisomers of tubuvaline

Article abstract of DOI:10.1016/j.bmcl.2010.10.118

The synthesis and biological evaluation of stereoisomers in tubulysin D are described. The stereoselective synthesis of all possible stereoisomers of C-11 and C-13 positions in tubulysin D was achieved by employing 1′-epi-Tuv-Me, 3′-epi-Tuv-Me, and ent-Tu

Full text of DOI:10.1016/j.bmcl.2010.10.118

Bioorganic & Medicinal Chemistry Letters 21 (2011) 431–434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of tubulysin D analogs related
to stereoisomers of tubuvaline
Taku Shibue a, Iwao Okamoto b, Nobuyoshi Morita b, Hiroshi Morita c, Yusuke Hirasawa c, Takahiro Hosoya c,
Osamu Tamura b,
⇑
a Exploratory Research Laboratories, Kyorin Pharmaceutical Co. Ltd, 2399-1 Nogi, Nogi-Machi, Shimotsuga-Gun, Tochigi 329-0114, Japan
b Showa Pharmaceutical University, Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan
c Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of stereoisomers in tubulysin D are described. The stereoselective
synthesis of all possible stereoisomers of C-11 and C-13 positions in tubulysin D was achieved by employ-
ing 10-epi-Tuv-Me, 30-epi-Tuv-Me, and ent-Tuv-Me and their biological properties were evaluated. It is
clear that the stereochemistries of the C-11 and C-13 positions in tubulysin D have no practical impact
on the inhibition of tubulin polymerization but play a role in the potent antiproliferative activities.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 August 2010
Revised 9 October 2010
Accepted 25 October 2010
Available online 30 October 2010
Keywords:
Tubulysin D
Inhibition of tubulin polymerization
Antiproliferative activity
Microtubules are composed of tubulin molecules, which are
heterodimers of two closely related globular polypeptides, -tubu-
development of new anticancer agents due to their remarkable
biological activities and unique structural characteristics.12
To date, much effort has been devoted to the synthesis and bio-
logical evaluation of natural tubulysins and their analogs.13–23
Among them, WZY-III-69A (2),17 3,18 and pretubulysin (4)23 dis-
played nanomolar cell growth inhibition (Fig. 2). For potent cell
growth inhibition, it appears that (i) the labile N,O-acetal at the
a
lin and b-tubulin. The microtubule is certified as one of the targets
for effective chemotherapy of cancers.1 The anticancer agents that
act on microtubules can be classified into two major categories
according to their mechanism of action.2 One category consists of
microtubule-stabilizing agents such as paclitaxel,3 epothilones,4
and discodermolide.5 The other category includes microtubule
destabilizing agents such as vinblastine and vincristine, which in-
hibit microtubule polymerization.6
R3
The tubulysin A (1a) and D (1b), isolated from the two different
species of myxobacteria, Archangium gephyra and Angiococcus disc-
iformis, respectively, are novel tetrapeptides that display potent
antitumor activity by inhibiting tubulin polymerization.7–10 In par-
ticular, the antiproliferative activities of tubulysin D (1b), the most
potent derivative among the tubulysins shown in Figure 1, have
been reported to exceed those of vinblastine.8,11
O
OR2
11
O
H
N
13
N
N
N
Me
3
14
N
H
7
O
R1
S
CO2H
1
Tubulysins 1a and 1b are composed of four amino acid frag-
ments, N-methyl-D-pipecolic acid (D-Mep), L-isoleucine (L-Ile),
N-methyl-
tubuphenylalanine
/ tubutyrosine
(Tup) / (Tut)
D
-pipecolic acid
L
-isoleucine tubuvaline
(D-Mep)
(L
-Ile) (Tuv)
tubuvaline (Tuv), and tubuphenylalanine (Tup)/tubutyrosine
(Tut). The structural features of 1a and 1b would indicate the pres-
ence of unusual amino acids Tup (Tut) and Tuv, which has a labile
N,O-acetal side chain at the N-14 position.8,11 The tubulysins have
received much attention in recent years as lead compounds for the
R1
R2
R3
tubulysins
(1a)
(
(1c)
(1d)
CH2OCOCH2CH(CH3)2
CH2OCOCH2CH(CH3)2
A
D
U
V
Ac
Ac
Ac
H
OH
H
H
1b
)
H
H
H
⇑
Corresponding author. Tel.: +81 (42) 721 1578; fax: +81 (42) 721 1579.
E-mail address: tamura@ac.shoyaku.ac.jp (O. Tamura).
Figure 1. Structures of tubulysins and their components.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.118

432
T. Shibue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 431–434
OAc
O
H
O
O
O
OAc
O
O
N
N
H
N
Me
N
Me
N
Me
N
Me
N
H
N
Me
N
N
H
S
O
S
O
O
Me
CO2H
2
3
WZY-III-69A ( )
OH
O
O
H
N
H
N
N
N
N
Me
N
H
N
Me
N
Me
N
H
N
H
S
S
O
CO2H
CO2H
4
5
pretubulysin ( )
11-epi-tubulysin V ( )
Figure 2.
OH
N
a
O
OAc
FmocHN
O
CO2Me
H
N
11
13
N
S
N
N
3
14
N
H
7
9
S
O
Me
O
CO2H
O
1
NO2
O
b
O
1b
N
FmocHN
CO2Me
S
10
O
OAc
O
OAc
O
OAc
OH
11
13
13
11
c
N
N
O
N
O
N
O
FmocHN
CO2Me
S
O
O
O
11
OH
6
7
8
1'
N
HOTs·H2N
CO2Me
Scheme 1.
S
12 ·TsOH
N-14 position would not be essential,17 (ii) the
D-Mep of the N-ter-
OH
3'
minal amino acid could be replace with another amino acid such as
N-methylsarcosine,17 (iii) the Tup of the C-terminal amino acid
would be able to be converted into a simple structure, (iv) the acet-
oxy group in Tuv is perhaps not necessary. Although it has been re-
ported that the antiproliferative activities of 11-epi-tubulysin V (5)
shows less activity than tubulysin V (1d), there is no remarkable
difference between 5 and 1d on regarding the activity of tubulin
inhibition.21 On the other hand, we have also reported a synthesis
of tubulysin D (1b), ent-D (ent-1b), U (1c), and V (1d), and their
preliminary biological activities against HEp-2 cell (human epider-
moid carcinoma of the larynx) in vitro.24 However, the details of
the relationship between the two stereochemistries of Tuv and
their biological properties remain unclear. We have therefore envi-
sioned the stereoselective synthesis of all possible stereoisomers of
Tuv in tubulysin D (1b), and have strived to synthesize 11-epi-
tubulysin D (6), 13-epi-tubulysin D (7), and 11,13-di-epi-tubulysin
D (8) (Scheme 1). We report herein the synthesis and biological
evaluation, including inhibition of tubulin polymerization and
antiproliferative activity, of the tubulysin D (1b) and its analogs.
The syntheses of 11-epi-tubulysin D (6) and 13-epi-tubulysin D
(7) require 10-epi-Tuv-Me (12) and 30-epi-Tuv-Me (ent-12), respec-
tively (Scheme 2). Mitsunobu reactions25,26 of N-Fmoc-Tuv-Me (9)
and ent-N-Fmoc-Tuv-Me (ent-9)24 were employed for the prepara-
tion of 12 and ent-12, respectively. Thus, secondary alcohol 9, on
treatment with p-nitrobenzoic acid in the presence of diethyl
N
a-c
HOTs·H2N
CO2Me
9
ent-
S
12
ent- ·TsOH
Scheme 2. Reagents and conditions: (a) p-NO2-C6H4-CO2H, DEAD, PPh3, THF, 93%
(for 10), 91% (for ent-10); (b) NaN3, MeOH, quant (for 11), quant (for ent-11); (c)
Et2NH, MeCN then TsOH, MeCN, 83% (for 12ꢀTsOH), 93% (for ent-12ꢀTsOH).
azodicarboxylate (DEAD) and triphenylphosphine, underwent a
Mitsunobu inversion to give 10 in 93% yield. Mild and selective
cleavage of the p-nitrobenzoic ester in 10 was accomplished by
using excess sodium azide in methanol to give 10-epi-N-Fmoc-
Tuv-Me (11) in excellent yield.27 Removal of the 9-fluorenylmeth-
yloxycarbonyl (Fmoc) group of 11 by treatment with diethylamine
and subsequent treatment with p-toluenesulphonic acid afforded
10-epi-Tuv-Meꢀtosylate (12ꢀTsOH), which is the corresponding
fragment in 6. The 30-epi-Tuv-Me (ent-12), which contains amino
acids requisite for the synthesis of 7, was prepared from ent-924
in the same synthetic sequence described above for the prepara-
tion of 12.
With the desired 10-epi-Tuv-Me (12) and 30-epi-Tuv-Me (ent-12)
in hand, the stage was set for the synthesis of epi-tubulysin D. To
synthesized epi-tubulysin Ds from 12 and ent-12, we adopted a re-

T. Shibue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 431–434
433
O
OH
O
OTES
H
N
N
N3
N
N
Me
N
O
CO2Me
a,b
d,e
f
N
R
CO2Me
12
·TsOH
S
O
S
O
13 : R = H
c
15
14 : R = CH2OCOCH2CH(CH3)2
O
OH
O
OAc
11
H
O
H
h,i
N
N
N
N
N
O
N
Me
CO2R
N
O
N
Me
N
H
3
7
S
O
S
O
O
O
CO2H
1
6
11-epi-tubulysin D ( )
16 : R = H
17 : R = C6F5
g
O
OAc
O
H
13
a-i
N
N
N
O
N
Me
3
ent- 12·TsOH
N
7
H
S
O
O
CO2H
1
7
13-epi-tubulysin D ( )
OH
N
OAc
11
O
O
a-i
H
N
13
H2N
CO2Me
N
N
O
N
Me
N
H
S
3
7
S
O
O
ent-Tuv-Me (18)
CO2H
1
11,13-di-epi-tubulysin D (8)
Scheme 3. Reagents and conditions: (a) N3-Ile-Cl, DIPEA, CH2Cl2, 97% (for 6), 91% (for 7), 88% (for 8); (b) TESOTf, 2,6-lutidine, CH2Cl2, quant (for 6), quant (for 7), quant (for 8);
(c) KHMDS, ClCH2OCOCH2CH(CH3)2, THF, 71% (for 6), 84% (for 7), 88% (for 8); (d) -Mep, PFP, EtOAc, then 10% Pd-C, H2, 60% (for 6), 40% (for 7), 76% (for 8); (e) AcOH-THF-H2O,
D
89% (for 6), 78% (for 7), 83% (for 8); (f) Me3SnOH, Cl(CH2)2Cl, 47% (for 6), 57% (for 7), 41% (for 8); (g) PFP, DIC, CH2Cl2; (h) DIPEA, TupꢀHCl, DMF, 64% (for 6), 68% (for 7), 45% (for
8); (i) Ac2O, pyridine then 1,4-dioxane-H2O, 71% (for 6), 90% (for 7), 94% (for 8).
ported synthetic sequence.14 The condensation reaction of 12 with
N3-Ile-Cl followed by protection of the secondary alcohol with a
triethylsilyl group, provided 13 (Scheme 3).
sin D (7) and 11,13-di-epi-tubulysin D (8) was accomplished in the
same manner described above starting from ent-12 and ent-Tuv-
Me (18), respectively.
N-Alkylation of the amide nitrogen in 13 was executed by treat-
ment with KHMDS and chloromethyl isobutyl carbonate to afford
14. The coupling reaction of azide dipeptide 14 with D-Mep under
The synthesized tubulysin D analogs (6–8) were evaluated for
the inhibition of tubulin polymerization using purified porcine
brain tubulin, and assayed for in vitro cytotoxicity against four hu-
man cancer cell lines, including HL60 (human blood premyelocytic
leukemia), HCT116 (human colon cancer), MCF7 (human breast
adenocarcinoma), and A549 (human lung adenocarcinoma epithe-
lial). The biological data are presented in Table 1. The inhibitory
activity values for tubulin polymerization of 11-epi-tubulysin D
(6) and 13-epi-tubulysin D (7) were comparable to those of tubuly-
sin D (1b) and vinblastine, which are known to be capable of inhib-
hydrogenation conditions followed by the deprotection of triethyl-
silyl ether gave tripeptide 15. Selective cleavage of the methyl ester
in 15 was conducted by treatment with Me3SnOH in 1,2-dichloro-
ethane at 60 °C to provide the desired acid 16 in 47% yield along
with undesired cleavage of the N-O-acetal function (48%). The acti-
vated ester 17 prepared from 16 with pentafluorophenol (PEP)
with N,N0-diisopropylcarbodiimide (DIC) was condensed with Tup
to afford deacetyl-11-epi-tubulysin D. Finally, the synthesis of
11-epi-tubulysin D (6) was achieved by acetylation of the second-
ary hydroxyl group. The stereoselective synthesis of 13-epi-tubuly-
iting tubulin polymerization [IC50
(lM): 1.4 for 6, 1.7 for 7, 1.7 for
1b, and 1.6 for vinblastine], whereas IC50 (6.3
lM) of 11,13-di-epi-
tubulysin D (8) showed rather weaker activity than the other ana-
Table 1
Biological activity of tublysin D and its analogs
Compound
Inhibition of Tubulin
Antiproliferative activity (IC50, nM)
polymerization (IC50, lM)
HL60
HCT116
MCF7
A549
6
7
8
1b
1.4
1.7
6.3
1.7
1.6
3.1
98
3600
0.0047
1.0
13
920
>10,000
0.0031
27
280
240
>10,000
0.67
0.79
43
1200
>10,000
0.013
17
Vinblastine

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T. Shibue et al. / Bioorg. Med. Chem. Lett. 21 (2011) 431–434
logs. It should be noted that changing one of the stereochemistries
at the C-11 and C-13 positions in 1b does not influence inhibition
activity of tubulin polymerization. However, inversion of both
configurations at the C-11 and C-13 positions in 1b resulted in a
decrease in the inhibitory activity.
References and notes
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The antiproliferative activities of 6 were dramatically decreased
compared with 1b in a tested human cancer cell line. Compound 7,
which was a stereoisomer at the C-13 position in 1b, also showed a
pronounced decline in antiproliferative activities. When compared
to 7, compound 6 showed similar activity in MCF7 cells, whereas it
exhibited potent activities in other cells. Compound 8, bearing an
inverted configuration at the two stereocenters in 1b, exhibited
much less activity than the other compounds in this assay. Our re-
sults indicate that the two stereochemistries of Tuv in tubulysin D
(1b) play a major role in the potent antiproliferative activities. The
inverted configuration at either the C-11 or C-13 position impairs
the antiproliferative activities. Interestingly, these results regard-
ing the antiproliferative activity do not correlate well with the
inhibition of tubulin polymerization. In particular, the epi-tubuly-
sin Ds (6 and 7) were found to possess tubulin polymerization
inhibitory activity similar to that of tubulysin D (1b), whereas
the di-epi-tubulysin D (8) exhibited much less activity than 1b.
One possibility is that an inverted configuration at either the C-
11 or C-13 position in 1b might induce the change in the cell mem-
brane permeability.19
In summary, we have succeeded in carrying out stereoselective
synthesis of all possible stereoisomers of Tuv in tubulysin D (1b),
and have clarified the relationship between two stereochemistries
of Tuv in 1b as well as the biological properties. It has been dem-
onstrated that an inverted configuration at either the C-11 or C-
13 position in 1b does not have a practical impact on the inhibition
of tubulin polymerization but dose play a role in the potent anti-
proliferative activities. Further investigation related to biological
studies of these and other tubulysin analogs is currently under
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Chem. Eur. J. 2010, 16, 11678. In this report, the antiproliferative activities of 1b
in HEp-2 cells have been shown to be more potent than those of vinblastine. In
contrast, ent-1b has exhibited much weaker activities [IC50
: 0.21 ng/mL
(0.25 nM) for 1b, 520 ng/mL (630 nM) for ent-1b and 7.0 ng/mL (8.6 nM) for
vinblastine]. The activity of 1c, which lacks the N,O-acetal side chain at the N-
14 position in 1b, exceeds that of vinblastine, but is slightly less active than 1b.
Acknowledgment
Meanwhile, tubulysin
V (1d), which is a deacetylated form of 1c, is
approximately 100-fold less active than 1c and vinblastine [IC50: 1.4 ng/mL
(2.0 nM) for 1c and 730 ng/mL (1090 nM) for 1d].
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We are grateful to Dr. Y. Fukuda of Kyorin Pharmaceutical Co.
Ltd for his valuable suggestions for and discussions of this study.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.118.