Stereoselective synthesis of tubuvaline methyl ester and tubuphenylalanine, components of tubulysins, tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.tetlet.2009.04.046

Synthetic studies of two components of tubulysins, tubulin polymerization inhibitors are described. The highly stereoselective synthesis of tubuvaline methyl ester (2) was accomplished by 1,3-dipolar cycloaddition of nitrone d-6 and acrylic acid derivativ

Full text of DOI:10.1016/j.tetlet.2009.04.046

Tetrahedron Letters 50 (2009) 3845–3848
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Stereoselective synthesis of tubuvaline methyl ester and tubuphenylalanine,
components of tubulysins, tubulin polymerization inhibitors
Taku Shibue a, Toshihiro Hirai b, Iwao Okamoto b, Nobuyoshi Morita b, Hyuma Masu c, Isao Azumaya c,
Osamu Tamura b,d,
*
a Discovery Research Laboratories, Kyorin Pharmaceutical Co. Ltd, 2399-1 Nogi, Nogi-Machi, Shimotsuga-Gun, Tochigi 329-0114, Japan
b Laboratory of Organic Chemistry, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan
c Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
d High Technology Research Center, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 February 2009
Revised 6 April 2009
Accepted 9 April 2009
Available online 14 April 2009
Synthetic studies of two components of tubulysins, tubulin polymerization inhibitors are described. The
highly stereoselective synthesis of tubuvaline methyl ester (2) was accomplished by 1,3-dipolar cycload-
dition of nitrone D-6 and acrylic acid derivatives 7 as a key step. The synthesis of tubuphenylalanine (3)
was conducted by an aldol reaction of a boron enolate of (S)-4-isopropyl-3-propionyl-2-oxazolidinone
(13) with aldehyde 14, readily prepared from phenylalanine, followed by Barton deoxygenation under
radical conditions.
Ó 2009 Elsevier Ltd. All rights reserved.
Tubulysins (1a–d), which are tetrapeptide derivatives, were
first isolated from the myxobacterial strains Archangium gephyra
and Angiococous disciformis, and they possess potent cell growth
inhibitory activity exceeding that of both vinblastine and dolasta-
tin10 (Fig. 1).1 Tubulysins inhibit tubulin polymerization2 and also
possess antiangiogenic properties.3 Since tubulysins are expected
to become excellent lead compounds for the development of new
anticancer agents that would inhibit tubulin polymerization, they
have attracted considerable attention as synthetic target mole-
cules. To date, the total syntheses of tubulysin D (1a), N14-deac-
etoxytubulysin H (1b), U (1c), V (1d), and synthetic approaches
have been reported.4–7 The chemical degradation of tubulysin D
(1a), the most active compound among tubulysins, and a compar-
ison of the resulting amino acids with the corresponding natural
and unnatural amino acids revealed that 1a is composed of four
alcohol structure in 2 can be made available by the reductive cleav-
age of the N–O bond of isoxazolidine I, and a thiazole ring can be
constructed by the cyclization of the cysteine moiety of I. Com-
pound I can be synthesized by the regio- and stereoselective 1,3-
dipolar cycloaddition of the appropriate nitrone III and acrylic acid
derivative IV, leading to II, followed by the removal of the chiral
auxiliary and condensation with the cysteine derivative. The ster-
eoselective synthesis of Tup (3) can be carried out by utilizing
the Evans aldol reaction9 of aldehyde VI, readily prepared from
phenylalanine, followed by the Barton deoxygenation of V under
radical conditions.10
O
OR2
O
H
N
amino acid fragments, N-methyl-D-pipecolic acid (D-Mep), L-isoleu-
N
N
R1
N
H
N
H
cine -Ile), tubuvaline (Tuv), and tubuphenylalanine (Tup)
(L
S
O
(Fig. 1).1,8 Among these four amino acids, the stereoselective syn-
theses of two unusual amino acids, Tuv and Tup, would be essen-
tial for an effective synthesis of tubulysins. We report herein an
efficient method for the stereoselective synthesis of Tuv featuring
the 1,3-dipolar cycloaddition of a nitrone, as well as a method for
stereoselective synthesis of Tup using an aldol reaction followed
by the Barton deoxygenation.
CO2H
N-methyl-
D
-pipecolic acid
L
-isoleucine
-Ile)
tubuphenylalanine
(Tup)
tubuvaline
(Tuv)
(
D-Mep)
(L
R1
R2
Our synthetic plan for the synthesis of tubuvaline methyl ester
(Tuv-Me, 2) and Tup (3) is depicted in Scheme 1. The 1,3-amino
tubulysin D (1a)
H (1b)
CH2OCOCH2CH(CH3)2 COCH3
CH2OCOCH3
COCH3
COCH3
H
U (1c)
V (1d)
H
H
* Corresponding author.
E-mail address: tamura@ac.shoyaku.ac.jp (O. Tamura).
Figure 1.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.046

3846
T. Shibue et al. / Tetrahedron Letters 50 (2009) 3845–3848
the stereoselectivity of the 1,3-dipolar cycloaddition.11 Taking this
*
into account, we chose a gulose-derived auxiliary as R for nitrone
OH
P1
-6 in Scheme 2)12,13 and Oppolzer’s camphor sul-
tam as the X of acrylate IV (see N-acryloyl sultam 7 in Table 1).
N
O
III (see nitrone
D
N
H2N
CO2Me
H
N
*
H
S
CO2Me
Our synthesis commenced with the preparation of nitrone
from 2,3:5,6-O-diisopropylidene- -gulofuranose (
-4)12,13 (Scheme
2). Treatment of -4 with hydroxylamine hydrochloride in the
presence of sodium hydrogen carbonate gave oxime -5 as a 1:1
mixture of E and Z isomers, which, without separation, was reacted
with isobutylaldehyde to afford nitrone -6 in 83% yield from -4.
In a similar manner, nitrone -6 was prepared from 2,3:5,6-O-diiso-
propylidene- -gulofuranose ( -4) in 90% yield (2 steps).
Next, we attempted the crucial cycloaddition of nitrone
with (2R)-N-(acryloyl)bornane-10,2-sultams (7)14 (Table 1). The
exposure of nitrone -6 to 7 in refluxing toluene led to cycloaddi-
D-6
O
Tuv-Me (2)
I
D
D
SP2
D
nitrone
cycloaddition
D
R*
N
O
N+
R*
O-
COX*
D
D
COX
L
II
IV
III
L
L
R* = Chiral Auxiliary X* = Chiral Auxiliary
D-6
D
P3HN
Evans
H2N
tion, to give (3R,5R)-isoxazolidine 815 as the major product (76%)
along with other isomers 9 (24%, mixture of isomers)16 (run 1).
Cycloadduct 8 was readily separated from the other isomers by
column chromatography. The stereochemistry of 8 was precisely
determined by X-ray crystallography (Fig. 2), which revealed that
8 had the correct stereochemistry for the synthesis of Tuv-Me
(2).17 Use of a protic polar solvent (EtOH) slightly improved stere-
oselectivity (run 2). At a rather low temperature (40 °C), this cyclo-
addition proceeded to exhibit higher selectivity (runs 3 and 4).
Among reactions in several solvents (runs 5–8), the reaction in
refluxing CH2Cl2 gave the best results, and cycloadduct 8 (85%)
aldol reaction
OH
COY
P3HN
CHO
CO2H
Tup (3)
V
VI
Scheme 1.
O
H
O
H
O
OH
OH
O
NOH
NH2OH·HCl
O
was obtained (run 8). On the other hand, the reaction of L-6 derived
NaHCO3, EtOH-H2O
O
O
O
O
from L-4 with 7 in refluxing CH2Cl2 yielded a mixture of four iso-
mers of cycloadduct. The yield of cycloadduct 8015 which exhibited
the correct stereochemistry for 2 (Fig. 2)17 was diminished to 19%
yield.18 These results clearly demonstrated that the combination of
D-4
D
-5
H
H
O
H
O
nitrone
-6 and 7 was a mismatched pair.
As cycloadduct 8 showed the correct stereochemistry, an elab-
D-6 and alkene 7 represented a matched pair, and that of
O
N+
O-
isobutylaldehyde
L
O
N+
O-
MgSO4, CHCl3
2 steps, 83%
O
D
-gulosyl
oration of adduct 8 was carried out to obtain Tuv-Me (2) (Scheme
3). Treatment of 8 with lithium hydroxide at room temperature fol-
lowed by treatment with perchloric acid subsequently gave [(3R)-
3-isopropylisoxazoline-5-yl]carboxylic acid, the nitrogen of which
was protected by an Fmoc group to afford acid 10 in 86% yield from
D-6
Scheme 2.
8. Next, 10 was condensed with L
-S-tritylcysteine methyl ester19
under usual conditions, yielding the fully protected cysteine-con-
taining dipeptide 11. When dipeptide 11 was exposed to bis(tri-
phenyl)oxodiphosphonium trifluoromethanesulfonate,20 which
In the synthesis of Tuv-Me (2), the mild and selective removal of
each chiral auxiliary (R or X ) in cycloadduct II is essential. It was
also expected that double asymmetric induction would improve
*
*
Table 1
1,3-Dipolar cycloaddition of the chiral nitrone D-6 or L-6 with 7
N
O
7
S
R*
R*
N
O
O
+
N
O
N+
O-
R*
O
COX*
COX*
D
L
-6: R* = -gulosyl
8: R* =
8': R* =
D
L
-gulosyl 9: R* =
D
L
-gulosyl
-gulosyl
-6: R* =D
L-gulosyl
-gulosyl 9': R* =
Run
6
Solvents
Temp (°C)
Time (h)
Yield (%)
Diastereomer ratioa 8 or 80:9 or 90
1
2
3
4
5
6
7
8
9
D
D
D
D
D
D
D
D
-6
Toluene
EtOH
Toluene
EtOH
THF
CH3CN
Neat
110
78
40
40
40
40
40
40
40
1
6
Quant
Quant
Quant
Quant
Quant
Quant
Quant
Quant
83
76:24
78:22
83:17
81:19
80:20
79:21
82:18
85:15
23:77b
-6
-6
-6
-6
-6
-6
-6
24
48
24
48
48
48
48
CH2Cl2
CH2Cl2
L
-6
Diastereomer ratios of 8 to 9 were determined by the 1H NMR spectra of the mixtures.
This sample 90 contained three isomers detected by HPLC.
a
b

T. Shibue et al. / Tetrahedron Letters 50 (2009) 3845–3848
3847
Figure 2. ORTEP drawings for 8 and 80.
1) LiOH,
THF-H2O
Bu2BOTf, DIPEA
CH2Cl2
BocHN
CHO
BocHN
D-gulosyl
Fmoc
N
O
N
O
OH
O
2) HClO4, CH3CN
3) FmocCl, NaHCO3
dioxane-H2O
O
14
O
COX*
CO2H
N
then 30% H2O2 aq.,
MeOH, 77%
O
O
O
3 steps, 86%
N
10
8
13
15
Fmoc
BocHN
1) LiOH,
N
O
1) Ph3P=O, Tf2O
CH2Cl2
L
-S-Tr-cysteine
30% H2O2aq.,
methylester
H
N
1) TCDI, THF
THF-H2O, 77%
H
3·HCl
CO2Me
2) MnO2,CH2Cl2
2 steps, 75%
HATU, DIPEA
CH2Cl2, 82%
O
2) n-Bu3SnH, AIBN
toluene,
2) 4 M HCl-dioxane,
89%
O
O
N
2 steps, 86%
STr
O
11
16
Fmoc
N
1) Mo(CO)6,
CH3CN-H2O
Scheme 4.
O
2
N
2) Et2NH, CH3CN
2 steps, 91%
S
CO2Me
Finally, removal of the chiral auxiliary with lithium peroxide in
THF–H2O and the subsequent deprotection of the Boc group suc-
cessfully produced Tupꢀhydrochloride (3ꢀHCl).23
12
Scheme 3.
In summary, we explored a novel synthetic route to yield
tubuvaline methyl ester (2) and tubuphenylalanine hydrochloride
(3ꢀHCl). These two synthetic approaches are now available for pro-
ducing tubulysin analogues required for the investigation of struc-
ture–activity relationships. The syntheses of natural tubulysins and
novel tubulysins analogues that cannot be derived from natural re-
sources are under investigation.
had been prepared from triphenylphosphinoxide and triflic anhy-
dride, cyclodehydration proceeded smoothly to the construction
of a thiazoline moiety, which was oxidized by activated manganese
dioxide to provide thiazole 12 in 75% yield (2 steps). Finally, reduc-
tive cleavage of the N–O bond of 12 by heating the sample with
molybdenum hexacarbonyl in acetonitrile-water (10:1),21 and the
subsequent removal of the Fmoc group by treatment with diethyl-
amine afforded Tuv-Me (2)15 in 91% yield (2 steps).
Acknowledgments
We are grateful to Dr. T. Ishizaki of Kyorin Pharmaceutical Co.
Ltd for his extensive support, as well as to Dr. Y. Fukuda of Kyorin
Pharmaceutical Co. Ltd for his valuable suggestions and discussions
of this study.
The synthesis of Tup (3) was initiated by the stereoselective al-
dol reaction of aldehyde 14 with the (Z)-boron enolate of (S)-4-iso-
propyl-3-propionyl-2-oxazolidinone (13), yielding adduct 15 in
77% yield (Scheme 4).9,22,23 Removal of the secondary hydroxyl
group of 15 was then carried out according to the Barton–McCom-
bie procedure.10 Thus, exposure of 15 to 1,10-thiocarbonyldiimi-
dazole (TCDI) followed by treatment with Bu3SnH in the
presence of a catalytic amount of 2,20-azobis(2-methylpropionitri-
le) (AIBN) gave the deoxygenated product 16 in 86% yield (2 steps).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.04.046.

3848
T. Shibue et al. / Tetrahedron Letters 50 (2009) 3845–3848
17. The crystallographic data were collected on
a
CCD detector. The crystal
References and notes
structure was solved by direct methods SHELXS-97 and refined by full-matrix
least-squares SHELXL-97 (Sheldrick, G.M. Acta Crystallogr., Sect. A 64, 2008, 112–
122). All non-hydrogen atoms were refined anisotropically. All hydrogen atoms
were included as their calculated positions. Crystallographic data for 8:
C29H46N2O9S, M = 598.74, monoclinic, a = 10.853(1), b = 11.583(1), c =
12.862(1) Å, b = 109.902(1)°, V = 1520.4(3) Å3, T = 120 K, space group P21,
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a = 10.468(1), b = 21.676(3), c = 13.561(2) Å, b = 93.987(1)°, V = 3069.4(7) Å3,
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F
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0.05(6), CCDC-719635. Two independent molecules are included an
asymmetric unit of the crystal.
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18. The separation of the four isomers was performed by HPLC with a chiral
column to give pure samples of 80. Separation conditions were as follows:
Daicel Chiralpak IA, / 2.0 cm ꢂ 25 cm; hexane/2-propanol/EtOH = 70:15:15;
flow rate 10 mL/min: HPLC analysis—Daicel Chiralpak IA, / 0.46 cm ꢂ 25 cm;
hexane/2-propanol/EtOH = 70:15:15, flow rate 0.5 mL/min; tR 11.3 min (80),
13.0, 15.5, and 19.7 min (other isomer 90). The stereochemistry of 80 was
determined by X-ray crystallography (Fig. 2).
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Hecht, S. M. J. Am. Chem. Soc. 2003, 125, 8218.
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15. For experimental procedure of 8 and spectral data of tubuvaline methyl ester
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16. The stereochemistries of two isomers were not determined.