Total syntheses of tubulysins

Article abstract of DOI:10.1002/chem.201000963

The total syntheses of tetrapeptides tubulysins D (1b), U (1c), and V (1d), which are potent tubulin polymerization inhibitors, are described. The synthesis of Tuv (2), an unusual amino acid constituent of tubulysins, includes an 1,3-dipolar cycloaddition

Full text of DOI:10.1002/chem.201000963

DOI: 10.1002/chem.201000963
Total Syntheses of Tubulysins
Taku Shibue,[a] Toshihiro Hirai,[b] Iwao Okamoto,[b] Nobuyoshi Morita,[b] Hyuma Masu,[c]
Isao Azumaya,[c] and Osamu Tamura*[b]
Abstract: The total syntheses of tetra-
peptides tubulysins D (1b), U (1c),
and V (1d), which are potent tubulin
polymerization inhibitors, are de-
scribed. The synthesis of Tuv (2), an
unusual amino acid constituent of tu-
bulysins, includes an 1,3-dipolar cyclo-
addition reaction of chiral nitrone d-6
derived from d-gulose with N-acryloyl
camphor sultam (À)-9 employing the
double asymmetric induction, whereas
the synthesis of Tup (20), another un-
usual amino acid, involves a stereose-
lective Evans aldol reaction of (Z)-
boron enolate generated from (S)-4-
isopropyl-3-propionyl-2-oxazolidinone
with N-protected phenylalaninal and a
subsequent Barton deoxygenation pro-
tocol. We accomplished the total syn-
theses of tubulysins U (1c) and V (1d)
by using these methodologies, in which
the isoxazolidine ring was used as the
effective protective group for g-amido
alcohol functionality. Furthermore, to
understand the structure-activity rela-
tionship of tubulysins, we synthesized
tubulysin D (1b) and cyclo-tubulysin D
(1e) from 2-Me and 20, and ent-tubuly-
sin D (ent-1d) from ent-2-Me and ent-
20, respectively. The preliminary results
regarding their biological activities are
also reported.
Keywords:
amino
acids
·
antitumor agents · stereoselectivity ·
total synthesis · tubulysins
Introduction
Tubulysins A (1a) and D (1b), isolated from two species of
myxobacteria, Archangium gephyra and Angiococcus disci-
formis, respectively, are tetrapeptides that are each com-
posed of four amino acid fragments, N-methyl-d-pipecolic
acid (d-Mep), l-isoleucine (l-Ile), tubuvaline (Tuv), and tu-
buphenylalanine (Tup)/tubutyrosine (Tut) (Scheme 1).[1,2]
The linearly connected structures of tubulysins were deter-
[a] T. Shibue
Exploratory Research Laboratories
Kyorin Pharmaceutical Co. Ltd, 2399-1
Nogi, Nogi-Machi, Shimotsuga-Gun
Tochigi 329-0114 (Japan)
[b] T. Hirai, Dr. I. Okamoto, Dr. N. Morita, Prof. Dr. O. Tamura
Showa Pharmaceutical University
Higashi-tamagawagakuen, Machida
Tokyo 194-8543 (Japan)
Scheme 1. Structures of tubulysins and their components.
Fax : (+81)42-721-1579
E-mail: tamura@ac.shoyaku.ac.jp
mined by detailed two-dimensional NMR spectroscopic
analyses and chemical degradation of tubulysin D (1b) by
acidic hydrolysis.[3] Further X-ray diffraction of tubulysin A
(1a) established the absolute configurations of seven stereo-
centers.[2] The common structural characteristics of tubuly-
sins would include the existence of unusual amino acids, Tuv
and Tup/Tut, as well as the N,O-acetal side chain at the 14-
[c] Dr. H. Masu, Prof. Dr. I. Azumaya
Faculty of Pharmaceutical Sciences at Kagawa Campus
Tokushima Bunri University, 1314-1 Shido
Sanuki Kagawa 769-2193 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000963.
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FULL PAPER
N-positions of 1a and 1b.[4] The tubulysins display potent
antitumor activity by inhibiting tubulin polymerization and
by antiangiogenic factors.[5,6] It is worth noting that the in-
hibition activity of tubulysin D (1b), the most potent deriva-
tive, exceeds that of vinblastine, which is used as an antitu-
mor agent in clinical practice.[2,6] Due to their remarkable
biological activity and unique structural characteristics, in-
cluding unusual amino acids, such as Tuv and Tup, tubulysins
have attracted considerable attention as synthetic target
molecules as well as leads for the development of new anti-
cancer agents, and hence total syntheses and synthetic stud-
ies have already been reported.[4b,7–12] However, only a few
synthetic methods adaptable to analogue synthesis have
been explored, probably due to the lack of stereoselectivity
or efficiency for the syntheses of Tuv and Tup.[11] We have
recently developed an efficient method for the synthesis of
Tuv featuring the stereoselective 1,3-dipolar cycloaddition of
a nitrone derived from d-gulose with N-acryloyl camphor
sultam as well as a synthetic method for Tup by employing a
stereoselective aldol reaction followed by Barton deoxyge-
nation.[13] We describe herein efficient total syntheses of tu-
bulysins U (1c), V (1d), and D (1b) by using the above
methodologies. Furthermore, as a first step toward under-
standing the structure-activity relationship of tubulysins, we
synthesized ent-tubulysin D (ent-1b) and cyclo-tubulysin D
(1e), which was reported to be obtained from 1b by treat-
ment with dilute HCl.[3] Preliminary results regarding their
biological activities are also reported.
alcohol at the benzylic position in Tuv (2). Our choice for
the chiral nitrone II was gulose-derived nitrone
6
(Scheme 3). Both enantiomers of gulonic acid g-lactone (3)
are commercially available and the gulosyl group can be re-
moved under acidic conditions.
Scheme 3. Preparation of chiral nitrone d-6: a) CuSO4, conc. H2SO4, ace-
tone, 88%; b) DIBAL-H, THF, 98%; c) NH2OH·HCl, NaHCO3, EtOH/
H2O; d) isobutyraldehyde, MgSO4, CHCl3, 84% (2 steps). DIBAL-H=
diisobutylaluminium hydride.
Our synthetic studies began with the preparation of chiral
nitrone d-6 from commercially available d-gulonic acid g-
lactone (d-3) (Scheme 3). Four hydroxyl groups of lactone
d-3 were protected by diacetonide formation followed by
the reduction of carbonyl groups with DIBAL-H, leading to
lactol d-4.[15] The reaction of d-4 with hydroxylamine hydro-
chloride in the presence of sodium hydrogen carbonate
yielded the corresponding oxime d-5 as an 1:1 mixture of E
and Z isomers. Without separation of isomers, d-5 was con-
verted to nitrone d-6 by condensation with isobutylaldehyde
in a satisfactory yield. These successive transformations
from d-3 to d-6 were conducted on a multigram scale with-
out chromatographic purification. In a similar manner, ni-
trone l-6 was prepared from l-gulonic acid g-lactone (l-3).
We next examined the crucial 1,3-DC reaction of chiral
nitrone d-6 with vinyl thiazole 7[16] (Scheme 4). Nitrone d-6,
on treatment with 7 in refluxing toluene, underwent an 1,3-
DC reaction to give a mixture of cycloadducts 8a–d (8a/8b/
Results and Discussion
Synthesis of tubuvaline (Tuv): Our retrosynthetic analysis
(Scheme 2) for Tuv (2) pivoted on the use of an 1,3-dipolar
Scheme 2. Synthetic strategy for Tuv (2).
cycloaddition (1,3-DC) of nitrone.[14] Thus, isoxazoline I,
which may be available by means of an 1,3-DC of nitrone II
with 2-vinylthiazole III, would afford Tuv (2) by reductive
À
cleavage of the N O bond. To realize this plan, it would be
very important to choose an appropriate chiral auxiliary R*
in II, which would need 1) to be available in both enantio-
meric forms and 2) to be removed under conditions other
than hydrogenolysis due to the existence of the secondary
Scheme 4. 1,3-DC reaction of chiral nitrone d-6 with vinyl thiazole 7.
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O. Tamura et al.
1
8c/8d 38:6:28:28, as determined by H NMR spectroscopy).
xazoline V may provide a good foothold for the construc-
tion of the thiazole fragment of 2. The key compound I can
be obtained by condensation of V with a cysteine derivative,
followed by cyclization of the resulting IV. Taking into ac-
count both availability and crystallinity, we adopted Oppolz-
erꢁs camphor sultam as the chiral auxiliary X* of acrylate VI
(see (À)-N-acryloyl sultam 9 in Table 1).[19a]
After separation of the mixture, the stereochemistries of 8a
and 8d were determined by their X-ray crystallographic
data, and the stereochemistries of 8b and 8c were deducted
by their NOESY correlations and the structures 8a and
8d.[17] The stereochemistry of cycloadduct 8a corresponded
to that of Tuv.
To explain the stereoselection in the 1,3-DC reaction of
d-6 with 7, we proposed a plausible transition state from
these results (Scheme 5). Although nitrone d-6 exhibits
Table 1. 1,3-DC of chiral nitrone d-6 or l-6 with (À)-9.
Entry
6
Solvent T [8C] t [h] Yield [%] Diastereomeric ratio[a]
10 or 11/10’ or 11’
1
2
3
4
5
6
7
8
9
d
d
d
d
d
d
d
d
l
toluene 110
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
23:77[b]
EtOH
toluene
EtOH
THF
CH3CN
neat
78
40
40
40
40
40
40
40
24
48
24
48
48
48
48
Scheme 5. Plausible transition states for cycloaddition of d-6 and 7.
CH2Cl2
CH2Cl2
facial selectivity to some extent (8a+8c/8b+8d ca. 2:1),
endo/exo selectivity was not observed. Various reaction con-
ditions (solvents, temperature, and additives) were tested to
improve stereoselectivity, but they were all unsuccessful.[18]
Additionally, separation of cycloadduct 8a containing the
correct stereochemistry for 2 from stereoisomers 8b–d re-
quired several chromatographic purifications. It was, there-
fore, necessary to explore a more stereoselective 1,3-DC to
produce a cycloadduct containing the correct stereochemis-
try for 2.
To gain the desired stereoselectivity, the dipolarophile has
to react from the re face. The low stereoselectivity of the cy-
cloaddition prompted us to consider the use of a dipolaro-
phile with independent facial selectivity. We anticipated ob-
taining the desired stereochemistry by double asymmetric
induction of an 1,3-DC reaction of chiral nitrone II (R*=
gulosyl) with acrylate VI containing the appropriate chiral
auxiliary (X*) (Scheme 6). The carbonyl functionality in iso-
[a] Unless otherwise noted, diastereomeric ratios of 10 to 10’ were deter-
mined by the H NMR spectra of the mixtures. [b] This sample contained
three isomers detected by HPLC.
1
Heating d-6 with (À)-9[19] in refluxing toluene gave the
desired endo-cycloadduct 10 in 76% yield along with anoth-
er stereoisomer 10’ (24%, mixture of isomers) (Table 1,
entry 1). The stereochemistry of 10 was precisely deter-
mined by X-ray crystallography, which revealed that 10 had
the correct stereochemistry for the synthesis of Tuv (2).[13,17]
To improve the stereoselectivity, various reaction solvents
and temperatures were examined next (Table 1, entries 2–8),
and the use of refluxing dichloromethane was found to opti-
mize conditions for providing adduct 10 (entry 8). Thus, the
reaction of d-6 with 9 in refluxing dichloromethane for 48 h
afforded adduct 10 as pure crystals in 85% isolated yield. It
should be noted that the cycloaddition can be conducted on
a 60 g-scale without any chromatographic separation (see
the Experimental Section).
In contrast, the 1,3-DC reaction of l-6, instead of d-6,
with (À)-9 gave a mixture of four isomers of cycloadducts
(Table 1, entry 9). The separation of mixtures by HPLC re-
vealed that the yield of adduct 11 bearing the correct stereo-
chemistry for Tuv was only 19%.[17] These results clearly
showed that the combination of nitrone d-6 and (À)-9 repre-
sents a matched pair, whereas that of l-6 and (À)-9 is a mis-
matched pair.
The high stereoselectivity of the 1,3-DC reaction in ni-
trone d-6 and (À)-9 can be explained by considering their
transition states (Scheme 7). It is generally accepted that di-
poles, such as nitrile oxide, silyl nitronates, and cyclopropa-
nediene, attack the re face of (À)-N-acryloyl sultam 9, which
Scheme 6. Revised retrosynthetic plan for Tuv.
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Total Syntheses of Tubulysins
FULL PAPER
tivated manganese dioxide to provide thiazole 14, the so-
called N,O-dehydroTuv derivative, in 75% yield (two steps).
À
Finally, reductive cleavage of the N O bond of 14 by heat-
ing with molybdenum hexacarbonyl in acetonitrile/water
(10:1),[23] and the subsequent removal of the Fmoc group by
exposure to diethylamine afforded Tuv-Me (2-Me) in 91%
yield (two steps).
Scheme 7. Facial selectivity of 9 and plausible transition state providing
adduct 10.
Synthesis of Tup: The stereoselective synthesis of Tup (20)
was initiated by the Evans aldol reaction of the (Z)-boron
enolate of (S)-4-isopropyl-3-propionyl-2-oxazolidinone (15)
with N-protected l-phenylalaninal 16 affording aldol 17 with
high selectivity and good yield (Scheme 9).[24–26] In these re-
has predominantly the (s)-cis conformation.[19b] Chiral ni-
trone d-6 also tends to react with a re face. Thus, the combi-
nation of d-6 and 9 caused double asymmetric induction to
provide cycloadduct 10 in high yield.
Since cycloadduct 10 already has the correct stereocenters
regarded as those in Tuv, we turned our attention to the syn-
thesis of 2 from adduct 10 (Scheme 8). Stepwise removal of
Scheme 9. Synthesis of Tup (20): a) Bu2BOTf, DIPEA, 16, CH2Cl2, then
30% H2O2 aq., MeOH, 92% (for 17a), 77% (for 17b); b) TCDI, THF,
95% (for 18a), quant. (for 18b); c) Bu3SnH, AIBN, toluene; d) LiOH,
30% H2O2 aq., THF/H2O, 70% (from 18a), 74% (from 18b); e) H2,
10% Pd/C, 4m HCl/dioxane, THF, 95% (from 19a), f) 4m HCl/dioxane,
CH2Cl2 (from 19b). AIBN=azobisisobutyronitrile; Boc=tert-butoxycar-
bonyl; Cbz=carbobenzyloxy; TCDI=1,1’-thiocarbonyldiimidazole.
Scheme 8. Synthesis of Tuv-Me (2-Me): a) LiOH, THF/H2O, 89%;
b) HClO4 aq., CH3CN; c) Fmoc-Cl, NaHCO3, dioxane/H2O, 99%
(2 steps); d) l-(S)-Tr-cysteine methyl ester, HATU, DIPEA, CH2Cl2,
82%; e) Ph3P=O, Tf2O, CH2Cl2; f) MnO2, CH2Cl2, 75% (2 steps);
g) Mo(CO)6, CH3CN/H2O, 89%, h) Et2NH, CH3CN, quant. DIPEA=
actions, the diastereomer of 17 was not detected. Leitheis-
erꢁs group reported that, when N-(tert-butoxycarbonyl)-d-
phenylalaninal was used instead of 16 (P=Boc), the yield of
the aldol adduct was poor.[25] So it can be seen that this
aldol reaction is again controlled by double asymmetric in-
duction. Next, the secondary hydroxyl group of aldol 17 was
removed by the Barton–McCombie procedure.[27] Thus, 17
was converted to thiocarbamate 18 by treatment with TCDI.
Subsequent treatment with Bu3SnH in the presence of a cat-
alytic amount of AIBN and removal of the chiral auxiliary
with lithium peroxide provided the N-protected Tup (19). It
should be noted that in the homolytic deoxygenation step,
tin residues were effectively removed by using KF-silica.[28]
Finally, deprotection of the N-protecting group of 19 fur-
nished Tup·HCl (20·HCl).[7] With regard to total yield in this
synthetic sequence, the Cbz group was slightly better than
the Boc group for protecting the nitrogen atom. This synthe-
sis of Tup (20) is more stereoselective than reported synthe-
ses to date; hence, Tup (20) was readily available in multi-
gram quantities.
N,N-Diisopropylethylamine;
Fmoc=9-fluorenylmethoxycarbonyl;
HATU=(7-azabenzotriazol-1-yl)tetramethyluronium
hexafluorophos-
phate.
the two chiral auxiliaries in 10 was conducted by employing
lithium hydroxide followed by perchloric acid to give
(3R,5R)-3-isopropylisoxazolidine-5-carboxylic acid, of which
the nitrogen atom was protected by a Fmoc group to afford
carboxylic acid 12 in 88% yield from 10. The acid 12 was
coupled with l-(S)-tritylcysteine methyl ester[21] in the pres-
ence of HATU and DIPEA, yielding the fully protected cys-
teine-containing dipeptide 13. When dipeptide 13 was ex-
posed to bis(triphenyl)oxodiphosphonium trifluoromethane-
sulfonate,[22] prepared from triphenylphosphin oxide and
triflic anhydride, cyclodehydration proceeded smoothly to
construct a thiazoline ring, which was then oxidized with ac-
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O. Tamura et al.
Syntheses of tubulysins
ester in 22 and the subsequent condensation with Tup (20)
afforded tetrapeptide 23. Synthesis of tubulysin V (1d) was
À
Syntheses of tubulysins U and V: With efficient approaches
to the two unusual amino acids in tubulysins in hand, we
accomplished by reductive cleavage of the N O bond to
generate a g-amido alcohol functionality with molybdenum
hexacarbonyl in refluxing acetonitrile/water.[29] Finally, ace-
tylation of the secondary alcohol in the Tuv moiety fur-
nished tubulysin U (1c) in 91%
aimed to synthesize tubulysins U (1c) and
V (1d)
(Scheme 10). In this synthesis, the isoxazoline ring played an
yield.
Syntheses of tubulysin D, cyclo-
tubulysin D, and ent-tubuly-
sin D: To synthesize tubuly-
sin D (1b) from Tuv-Me (2-Me)
and Tup (20), we adopted a re-
ported synthetic sequence (see
Scheme 11 and the Supporting
information).[7] Thus, deacetyl
tubulysin D (24) was converted
to tubulysin D (1b) by acetyla-
tion of the secondary alcohol
on Tuv. Furthermore, treatment
of 24 with 1m HCl aq. at 608C
afforded cyclo-tubulysin D (1e)
with excellent yield via an inter-
mediate N-acyl methylene-imi-
nium ion.[3]
Scheme 10. Synthesis of tubulysins U (1c) and V (1d): a) Et2NH, CH3CN, 84%; b) Boc-l-Ile, HATU, DIPEA,
CH2Cl2, 96%; c) TFA, CH2Cl2, quant.; d) d-Mep, DECP, DIPEA, DMF, 85%; e) LiOH, THF/H2O, 96%;
f) PFP, DIC, CH2Cl2 then 20 HCl, DIPEA, DMF, 87%; g) [Mo(CO)6], CH3CN/H2O, 71%; h) Ac2O, pyridine
then H2O/dioxane, 91%. DECP=diethyl cyanophosphonate; TFA=trifluoroacetic acid.
We next synthesized the ent-
important role as a protecting group for the amine and the
hydroxyl groups. Deprotection of the Fmoc group in thia-
zole 14, which possessed the correct stereochemistry for tu-
bulysins, by treatment with diethylamine provided N,O-de-
hydroTuv 21 in 84% yield. The condensation reaction of 21
with Boc-l-Ile proceeded smoothly under the usual condi-
tions to afford the dipeptide in 96% yield. Removal of the
Boc group in the l-Ile moiety under acidic conditions, fol-
lowed by coupling with d-Mep,[7] by using DECP and
DIPEA,[28] gave tripeptide 22. Saponification of the methyl
tubulysin D (ent-1b) to compare its biological activity with
that of 1b. All of the synthetic steps depicted in Schemes 3,
9, and 10 were repeated by starting from the enantiomers.
Thus, ent-Tuv-Me (ent-2-Me) was synthesized from l-6 and
ent-9, whereas ent-Tup·HCl (ent-20·HCl) was prepared from
ent-15 and ent-16. Total synthesis of ent-tublysin D (ent-1b)
from ent-2-Me and ent-20 was achieved.
Biological activities: Biological studies were carried out with
synthesized samples of tubulysins U (1c), V (1d), and D
Scheme 11. Synthesis of tubulysin D (1b), cyclo-tubulysin D (1e), and ent-tubulysin D (ent-1b): a) Ac2O, pyridine then H2O/dioxane, 93%; b) 1m HCl
aq., THF, 97%.
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Total Syntheses of Tubulysins
FULL PAPER
pump, a Hitachi D-7500 chromato integrator, a GL Sciences UV702 UV/
Vis detector, a GL Sciences PU716 HPLC pump, and a CO705 column
oven.
(1b), cyclo-tubulysin D (1e), and ent-tubulysin D (ent-1b).
The antiproliferative activity against HEp-2 cells (human
epidermoid carcinoma of the larynx) in vitro was evaluated
by using vinblastine as a positive control. The antiprolifera-
tive activity of 1b in HEp-2 cells was shown to be more
potent than that of vinblastine; thus the IC50 [ngmLÀ1]
values were 0.21 and 7.0 for vinblastine. The activities of tu-
bulysin U (1c) and cyclo-tubulysin D (1e) showed the same
strength of activity as that of vinblastine (IC50 [ngmLÀ1]: 1.4
for 1c and 13.0 for 1e). On the other hand, 1d and ent-1b
exhibited much weaker activities (IC50 [ngmLÀ1]: 730 for 1d
and 520 for ent-1b). It is noteworthy that ent-1b is about
2000-fold less active than 1b, which is the most active com-
pound in tubulysins, in this antiproliferative assay. Here we
clarified the antiproliferative activity of 1e and ent-1b for
the first time.
1,3-DC reaction of chiral nitrone d-6 with (2R)-N-(acryloyl)bornane-
10,2-sultams (9): A mixture of d-6 (45.5 g 138 mmol) and (2R)-N-(acryl-
oyl)bornane-10,2-sultams (9) (33.8 g, 126 mmol) in CH2Cl2 (250 mL) was
heated under reflux for 48 h. After concentration, the residue was recrys-
tallized from EtOH (300 mL) to give 10 (61.2 g 82% from 9) as a color-
less solid. Flash chromatography (silica gel, hexane/AcOEt 7:3) of the
mother liquor gave further 10 (1.13 g, 2%) as a colorless solid. An ana-
lytical sample of 10 (colorless crystal) was obtained by further recrystalli-
zation from hexane/AcOEt. M.p. 190–1928C; [a]2D6 =À111 (c=1.00 in
CHCl3); 1H NMR (400 MHz, CDCl3): d=0.93 (d, J=6.7 Hz, 3H), 0.95
(d, J=6.7 Hz, 3H), 0.98 (s, 3H), 1.19 (s, 3H), 1.26 (s, 3H), 1.32–1.46 (m,
2H), 1.38 (s, 3H), 1.41 (s, 3H), 1.43 (s, 3H), 1.60–1.70 (m, 1H), 1.82–1.98
(m, 3H), 2.08 (dd, J=8.0, 14.0 Hz, 1H), 2.22–2.32 (m, 1H), 2.45 (ddd, J=
1.8, 7.9, 12.8 Hz, 1H), 2.65 (td, J=7.4, 13.5 Hz, 1H), 3.44 (d, J=14.1 Hz,
1H), 3.47–3.52 (m, 1H), 3.53 (d, J=14.1 Hz, 1H), 3.73 (dd, J=6.7,
8.6 Hz, 1H), 3.91 (dd, J=4.9, 8.0 Hz, 1H), 4.08 (dd, J=3.6, 8,7 Hz, 1H),
4.19 (dd, J=6.7, 8.6 Hz, 1H), 4.34 (td, J=6.7, 8.6 Hz, 1H), 4.64–4.68 (m,
2H), 4.99 (d, J=6.1 Hz, 1H), 5.00 ppm (brt, J=7.9 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=19.7, 19.9, 20.1, 20.9, 24.9, 25.3, 26.1, 26.5, 26.6,
30.5, 32.5, 32.9, 38.0, 44.7, 47.8, 48.7, 53.1, 65.6, 66.0, 68.3, 75.7, 77.9, 80.3,
Conclusion
83.97, 84.0, 96.6, 109.6, 112.4, 170.6 ppm; IR (ATR): n˜ =1702, 2958 cmÀ1
;
We established efficient and concise routes to two unusual
amino acids, Tuv (2) and Tup (20), in tubulysins and synthe-
sized tubulysins U (1c), V (1d), and D (1b), cyclo-tubuly-
sin D (1e), and ent-tubulysin D (ent-1d). The synthesis of 2-
Me features a stereoselective 1,3-DC reaction of d-6 with
(À)-9, whereas synthesis of 20 was conducted by employing
a stereoselective aldol reaction and Barton deoxygenation.
These stereoselective and scalable methods enabled us to
synthesize sufficient quantities of tubulysins for a biological
assay. Thus, the total syntheses of 1c and 1d were accom-
plished, in which the isoxazoline ring played an important
role as a protecting group for the g-amido alcohol function-
ality. For the biological assay, 1b was also synthesized from
Tuv-Me (2-Me) and Tup (20). The assay clarified the anti-
proliferative activities of the synthesized tubulysins, espe-
cially 1e and ent-1b, for the first time. Further biological
evaluation, including that of the synthetic tubulysins and
their analogues, is currently underway, and the results will
be reported in due course.
MS (ESI+): m/z: 599 [M+H]+; HRMS (ESI+): m/z: calcd for
C29H47N2O9S: 599.3002 [M+H]+; found: 599.3007; elemental analysis
calcd (%) for C29H46N2O9S: C 58.17, H 7.74, N 4.68; found: C 58.06, H
7.59, N 4.57; the stereochemistry of 10 was determined by X-ray crystal-
lography.[13, 17]
Compound ent-10 (54.3 g, 84%) was prepared from l-6 (39.5 g,
120 mmol) with (2S)-N-(acryloyl)bornane-10,2-sultams (ent-9) (29.3 g,
109 mmol) in the same manner as described for 10. Colorless crystal;
m.p. 190–1918C; [a]2D3 =+117 (c=1.02 in CHCl3); MS (ESI+): m/z: 599
[M+H]+; HRMS (ESI+): m/z: calcd for C29H47N2O9S: 599.3002 [M+H]+;
found: 599.3005; elemental analysis calcd (%) for C29H46N2O9S: C 58.17,
1
H 7.74, N 4.68; found: C 58.01, H 7.79, N 4.64; H and 13C NMR and IR
spectra of this sample were identical to those described for 10.
AHCTUNGTERG(NNUN 3R,5R)-2-[(9H-Fluoren-9-yl)methoxycarbonyl]-3-isopropylisoxazolidine-
5-carboxylic acid (12) and its enantiomer (ent-12): A solution of LiOH
(12.6 g, 526 mmol) in H2O (130 mL) at 48C was added to a solution of 10
(63.0 g, 105 mmol) in THF (260 mL) and MeOH (130 mL), and the mix-
ture was stirred at the same temperature for 1 h. After concentration, the
mixture was poured into H2O (300 mL) and the pH of the mixture was
adjusted to pH 9 with 10% H2SO4 aq. The mixture was extracted with
AcOEt, then the aqueous layer was adjusted to pH 3 with 10% H2SO4
aq. The mixture was extracted with AcOEt, the organic extract was
washed with brine, dried over Na2SO4, filtered, and then concentrated in
vacuo. The residue was triturated with hexane to give (3R,5R)-2-
{(3aR,4R,6S,6aR)-6-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethylte-
trahydrofuro[3,4-d,1,3]dioxol-4-yl}-3-isopropylisoxazolidine-5-carboxylic
acid (40.2 g 89%) as a colorless solid. This material was used for the next
step without further purification. To a solution of this material (5.17 g,
12.9 mmol) in MeCN (64 mL) was added 60% HClO4 aq. (4.31 mL,
25.7 mmol) at 48C, and the mixture was stirred at room temperature for
1 h. After concentration, the residue was dissolved in 1,4-dioxane
(32 mL), and then a suspension of NaHCO3 (10.8 g, 129 mmol) in H2O
(32 mL) and Fmoc-Cl (4.00 g, 15.4 mmol) was added at 48C. The mixture
was stirred at room temperature for 21 h. After concentration, the resi-
due was diluted with H2O and Et2O and the whole was washed with
Et2O. The aqueous layer was adjusted to pH 3 with 10% H2SO4 aq. The
mixture was extracted with AcOEt, the organic extract was washed with
brine, dried over Na2SO4, filtered, and then concentrated in vacuo. The
residue was triturated with hexane to give 12 (4.85 g, 99%) as a colorless
amorphous solid. [a]2D5 =+57.3 (c=0.50 in MeOH); 1H NMR (400 MHz,
CDCl3): d=0.69 (d, J=6.1 Hz, 3H), 0.71 (d, J=6.7 Hz, 3H), 1.53 (oct,
J=6.1 Hz, 1H), 1.92–2.06 (m, 1H), 2.34 (ddd, J=3.1, 9.2, 12.8 Hz, 1H),
3.24–3.40 (m, 1H), 4.17 (brt, J=4.9 Hz, 1H), 4.50–4.62 (m, 2H), 4.77
(dd, J=4.9, 10.4 Hz, 1H), 7.26–7.34 (m, 2H), 7.39 (dt, J=2.4, 7.3 Hz,
Experimental Section
General information: All melting points are uncorrected. 1H NMR spec-
tra were measured with a 300 or 400 MHz spectrometer. Measurements
of 13C NMR spectra were measured with a 75 or 100 MHz spectrometer.
The chemical shifts are expressed in parts per million (d value) downfield
from tetramethylsilane, by using tetramethylsilane (d =0) and/or residual
solvents, such as chloroform (d =7.26), as an internal standard. Splitting
patterns are indicated as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad peak). Unless otherwise noted, all the experiments
were carried out by using anhydrous solvents under an atmosphere of
argon. Throughout this study, Merck precoated TLC plates (Silica gel 60
F254, 0.25 mm) were used for TLC analysis, and all the spots were visual-
ized by using UV light followed by coloring with phosphomolybdic acid
or anisaldehyde. Silica gel 60N (40–50 mm, neutral; Kanto Chemical Co.,
Inc., Tokyo, Japan) and Wakogel 100C18 (63–212 mm, Wako Chemical
Co., Inc., Tokyo, Japan) were used for the flash column chromatography.
Analytical and preparative HPLC was carried out by using an apparatus
equipped with a Hitachi L-7405 UV-detector, a Hitachi L-7100 HPLC
Chem. Eur. J. 2010, 16, 11678 – 11688
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11683

O. Tamura et al.
2H), 7.50 (d, J=7.4 Hz, 1H), 7.53 (d, J=7.4 Hz, 1H), 7.71 (d, J=7.4 Hz,
1H); 7.73 ppm (d, J=7.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=18.2,
18.8, 30.8, 34.9, 47.3, 66.1, 67.9, 78.6, 119.85, 119.90, 124.6, 124.8, 127.3,
127.5, 127.8, 128.0, 141.3, 141.4, 142.8, 143.4, 160.2, 173.0 ppm; IR (ATR):
n˜ =1708, 2960 cmÀ1; MS (CI+): m/z: 382 [M+H]+; HRMS (CI+): m/z:
calcd for C22H24NO5: 382.1654 [M+H]+; found: 382.1665. Compound ent-
12 (31.9 g, 95%) was prepared from ent-10 (54.2 g, 90.5 mmol) in the
141.24, 141.26, 143.4, 143.6, 146.6, 157.2, 161.4, 170.1 ppm; IR (ATR): n˜ =
1717, 2957 cmÀ1; MS (ESI+): m/z: 479 [M+H]+; HRMS (ESI+): m/z:
calcd for C26H27N2O5S: 479.1641 [M+H]+; found: 479.1647.
Compound ent-14 (2.61 g, 64%) was prepared from ent-13 (6.33 g,
8.54 mmol) in the same manner as described for 14. Colorless amorphous
solid; [a]2D5 =À13.0 (c=0.51 in MeOH); MS (ESI+): m/z: 479 [M+H]+;
HRMS (ESI+): m/z: calcd for C26H27N2O5S: 479.1641 [M+H]+; found:
479.1638. 1H and 13C NMR and IR spectra of this sample were identical
with those described for 14.
same manner as described for 12. Colorless amorphous solid; [a]D23
=
À58.0 (c=0.50 in MeOH); MS (CI+): m/z: 382 [M+H]+; HRMS (CI+):
m/z: calcd for C22H24NO5: 382.1654 [M+H]+; found: 382.1680; 1H and
13C NMR and IR spectra of this sample were identical to those described
for 12.
N-Fmoc-tubuvaline methyl ester (N-Fmoc-Tuv-Me) and its enantiomer
(ent-N-Fmoc-Tuv-Me): [Mo(CO)6] (82.8 mg, 0.31 mmol) was added to a
solution of 14 (100 mg, 0.21 mmol) in MeCN (2 mL) and H2O (0.2 mL)
and the mixture was stirred at 708C for 16 h. After concentration, the
residue was diluted with AcOEt (5 mL) and a 10% aq. solution of citric
acid (5 mL). NaIO4 was added to the mixture until the aqueous layer
became clear, and the whole was extracted with AcOEt. The organic ex-
tract was washed with a 10% aq. solution of NaS2O3 and brine, dried
over Na2SO4, filtered, and then concentrated in vacuo. Flash chromatog-
raphy (silica gel, hexane/AcOEt 3:2) of the residue gave N-Fmoc-Tuv-
Me (91.1 mg, 91%) as a colorless solid. An analytical sample of this ma-
terial (colorless crystal) was obtained by recrystallization from hexane/
AcOEt. M.p. 127–1288C; [a]2D5 =À19.8 (c=0.50 in MeOH); 1H NMR
(400 MHz, CDCl3): d=0.93 (d, J=6.7 Hz, 3H), 0.94 (d, J=6.7 Hz, 3H),
1.67–1.83 (m, 2H), 2.09 (ddd, J=2.4, 11.7, 14.1 Hz, 1H), 3.69–3.79 (m,
1H), 3.95 (s, 3H), 4.19 (t, J=6.1 Hz, 1H), 4.46 (dd, J=6.1, 10.4 Hz, 1H),
4.60–4.72 (m, 3H), 4.78 (d, J=4.9 Hz, 1H), 7.24–7.30 (m, 2H), 7.34 (dd,
J=1.2, 7.3 Hz, 1H), 7.40 (brt, J=7.3 Hz, 1H), 7.55–7.61 (m, 1H), 7.75
(d, J=7.3 Hz, 1H), 8.15 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=
18.3, 19.4, 32.2, 41.2, 47.5, 52.4, 53.1, 66.6, 68.8, 120.0, 120.1, 124.7, 124.9,
127.0, 127.1, 127.6, 127.69, 127.74, 141.37, 141.41, 143.3, 143.7, 146.5,
158.2, 162.0, 176.4 ppm; IR (ATR): n˜ =1523, 1715, 2953, 3342 cmÀ1; MS
(ESI+): m/z: 481 [M+H]+; HRMS (ESI+): m/z: calcd for C26H29N2O5S:
481.1797 [M+H]+; found: 481.1793; elemental analysis calcd (%) for
C26H28N2O5S: C 64.98, H 5.87, N 5.83; found: C 64.66, H 5.86, N 5.77.
Methyl (R)-2-[(3R,5R)-2-[(9H-fluoren-9-yl)methoxycarbonyl]-3-isopro-
pylisoxazolidine-5-carboxamido]-3-(triphenylmethylthio)propionate (13)
and its enantiomer (ent-13): iPr2NEt (0.27 mL, 1.56 mmol) and HATU
(324 mg, 0.85 mmol) were added to a solution of 12 (270 mg, 0.71 mmol)
and l-(S)-Tr-cysteine methyl ester hydrochloride[21] (441 mg, 1.07 mmol)
in CH2Cl2 (7 mL) at 48C and the mixture was stirred at the same temper-
ature for 30 min. The mixture was allowed to warm to room temperature
and stirring was continued for 24 h. The mixture was poured into saturat-
ed aqueous NaHCO3 solution and extracted with CH2Cl2, dried over
Na2SO4, filtered, and then concentrated in vacuo. Flash chromatography
(silica gel, hexane/AcOEt 1:2) of the residue gave 13 (431 mg, 82%) as
colorless amorphous solid. [a]2D5 =+21.4 (c=0.50 in MeOH); 1H NMR
(400 MHz, CDCl3): d=0.87 (d, J=6.7 Hz, 6H), 1.82 (oct, J=6.7 Hz,
1H), 2.40–2.53 (m, 3H), 2.73 (dd, J=7.3, 12.2 Hz, 1H), 3.61 (s, 3H), 3.82
(brq, J=6.7 Hz, 1H), 4.27 (t, J=6.7 Hz, 1H), 4.32–4.42 (m, 1H), 4.40–
4.52 (m, 2H), 4.62 (dd, J=6.1, 7.3 Hz, 1H), 7.10–7.15 (m, 3H), 7.15–7.24
(m, 6H), 7.26–7.35 (m, 8H), 7.39 (t, J=7.6 Hz, 2H), 7.56 (d, J=7.3 Hz,
1H), 7.60 (d, J=7.3 Hz, 1H), 7.76 (d, J=7.3 Hz, 2H), 8.14 ppm (d, J=
7.9 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=17.6, 19.0, 30.9, 33.5, 34.7,
46.9, 51.2, 52.5, 64.3, 67.2, 68.4, 79.7, 119.89, 119.93, 124.9, 125.1, 126.8,
127.09, 127.12, 127.7, 127.8, 127.9, 129.5, 141.3, 141.4, 143.4, 143.9, 144.2,
157.8, 170.3, 170.7 ppm; IR (ATR): n˜ =1682, 1714, 1736 cmÀ1; MS (ESI+):
m/z: 741 [M+H]+; HRMS (ESI+): m/z: calcd for C45H45N2O6S: 741.2998
[M+H]+; found: 741.3002.
Compound ent-N-Fmoc-Tuv-Me (13.3 g, 86%) was prepared from ent-14
(15.5 g, 32.4 mmol) in the same manner as described for N-Fmoc-Tuv-
Me. Colorless crystal; m.p. 128–1308C; [a]2D5 =+17.5 (c=0.50 in MeOH);
MS (ESI+): m/z: 481 [M+H]+; HRMS (ESI+): m/z: calcd for
C26H29N2O5S: 481.1797 [M+H]+; found: 481.1793; elemental analysis
calcd (%) for C26H28N2O5S: C 64.98, H 5.87, N 5.83; found: C 64.69, H
Compound ent-13 (48.0 g, 89%) was prepared from ent-12 (27.9 g,
73.1 mmol) and d-(S)-Tr-cysteine methyl ester hydrochloride (45.4 g,
110 mmol) in the same manner as described for 13. Colorless amorphous
solid; [a]2D5 =À27.1 (c=0.50 in MeOH); MS (ESI+): m/z: 758
[M+NH4]+; HRMS (ESI+): m/z: calcd for C45H48N3O6S: 758.3264
[M+NH4]+; found: 758.3269; 1H and 13C NMR and IR spectra of this
sample were identical with those described for 13.
1
5.82, N 5.75; H and 13C NMR and IR spectra of this sample were identi-
cal with those described for N-Fmoc-Tuv-Me.
Tubuvalin methyl ester (2-Me) and its enantiomer (ent-2-Me): Et2NH
(0.65 mL, 6.24 mmol) was added to
a solution of N-Fmoc-Tuv-Me
N-Fmoc-N,O-dehydrotubuvaline methyl ester (14) and its enantiomer
(ent-14): Tf2O (20.3 mL, 121 mmol) was added to a solution of Ph3P=O
(42.6 g, 57.5 mmol) in CH2Cl2 (400 mL) at À108C and the mixture was
(100 mg, 0.21 mmol) in MeCN (0.7 mL) and the mixture was stirred at
room temperature for 2 h. After concentration, flash chromatography
(silica gel, CH2Cl2/MeOH/NH4OH 50:5:1) of the residue gave 2-Me
(54.3 mg, quant) as a colorless solid. An analytical sample of 2-Me (color-
less solid) was obtained by recrystallization from EtOH. M.p. 158–
1608C; [a]2D6 =+73.2 (c=0.50 in DMSO); 1H NMR (400 MHz,
[D6]DMSO): d=0.78 (d, J=6.1 Hz, 3H), 0.81 (d, J=6.1 Hz, 3H), 1.42–
1.54 (m, 1H), 1.64–1.72 (m, 2H), 2.54–2.62 (m, 1H), 3.80 (s, 3H), 5.07
(brt, J=6.1 Hz, 1H), 8.42 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=17.6, 18.8, 34.0, 40.9, 51.9, 52.5, 69.0, 128.6, 145.7, 161.4,
180.0 ppm. IR (ATR): n˜ =1704, 2950, 3346 cmÀ1; MS (ESI+): m/z: 259
[M+H]+; HRMS (ESI+): m/z: calcd for C11H19N2O3S: 259.1116 [M+H]+;
found: 259.1120; elemental analysis calcd (%) for C11H18N2O3S·0.1H2O:
C 50.79, H 7.05, N 10.77; found: C 50.66, H 6.81, N 10.67.
stirred at the same temperature for 1 h.
A solution of 13 (42.6 g,
57.5 mmol) in CH2Cl2 (174 mL) was added to the reaction mixture at
À108C and the mixture was stirred at room temperature for 6 h. The
mixture was added to saturated aqueous NaHCO3 solution at 48C and
was extracted with CH2Cl2. The organic extract was washed with brine,
dried over Na2SO4, filtered, and then concentrated in vacuo. Flash chro-
matography (silica gel, hexane/AcOEt 3:2) of the residue gave the corre-
sponding thiazolidine derivative as a yellow amorphous solid. MnO2
(58.0 g, 667 mmol) was added to a solution of this material in CH2Cl2
(450 mL) and the mixture was stirred at room temperature for 24 h. The
mixture was filtrated through a pad of Celite and the filtrate was concen-
trated in vacuo. Flash chromatography (silica gel, hexane/AcOEt 3:2) of
the residue gave 14 (20.5 g, 75%) as a colorless amorphous solid. [a]D24
=
Compound ent-Tuv-Me (ent-2-Me, 2.45 g, 95%) was prepared from ent-
N-Fmoc-Tuv-Me (4.81 g, 10.0 mmol) in the same manner as described for
2-Me. Colorless solid; m.p. 168–1698C; [a]2D4 =À67.1 (c=0.50 in DMSO);
MS (ESI+): m/z: 259 [M+H]+; HRMS (ESI+): m/z: calcd for
C11H19N2O3S: 259.1116 [M+H]+; found: 259.1114; elemental analysis
calcd (%) for C11H18N2O3S: C 51.14, H 7.02, N 10.84; found: C 51.06, H
+12.5 (c=0.50 in MeOH); 1H NMR (400 MHz, CDCl3): d=0.92 (d, J=
7.3 Hz, 6H), 1.89 (oct, J=7.3 Hz, 1H), 2.60 (ddd, J=5.5, 7.9, 13.3 Hz,
1H), 2.79 (ddd, J=4.2, 8.5, 13.3 Hz, 1H), 3.88 (s, 3H), 4.07 (dt, J=5.5,
7.3 Hz, 1H), 4.16 (t, J=6.1 Hz, 1H), 4.34 (dd, J=6.7, 10.9 Hz, 1H), 4.50
(dd, J=6.7, 10.3 Hz, 1H), 5.51 (dd, J=4.2, 7.9 Hz, 1H), 7.23–7.33 (m,
2H), 7.37 (t, J=8.6 Hz, 1H), 7.39 (t, J=7.9 Hz, 1H), 7.56 (d, J=7.9 Hz,
1H), 7.59 (d, J=7.3 Hz, 1H), 7.73 (d, J=7.4 Hz, 1H), 7.75 (d, J=7.9 Hz,
1H), 8.09 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=17.6, 19.1, 31.4,
36.6, 47.0, 52.3, 64.5, 67.6, 79.1, 119.83, 119.85, 125.0, 127.1, 127.7, 128.9,
1
6.95, N 10.80; H and 13C NMR and IR spectra of this sample were iden-
tical with those described for 2-Me.
Benzyl (2S,3S,4S)-3-hydroxy-5-[(S)-4-isopropyl-2-oxooxazolidin-3-yl]-4-
methyl-5-oxo-1-phenylpentan-2-ylcarbamate (17a) and its enantiomer
11684
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11678 – 11688

Total Syntheses of Tubulysins
FULL PAPER
(ent-17a): Bu2BOTf (1m in CH2Cl2, 46.2 mL, 46.2 mmol) and iPr2NEt
(8.78 mL, 50.4 mmol) were added to a solution of (S)-4-isopropyl-3-pro-
pionyloxazolidin-2-one (15) (7.78 g, 42.0 mmol) in CH2Cl2 (44 mL) at
48C and the mixture was stirred at the same temperature for 45 min.
After cooling to À788C, a solution of N-(benzyloxycarbonyl)-l-phenyla-
laninal 16a[26a] (13.1 g, 46.2 mmol) in CH2Cl2 (40 mL) was added at the
same temperature and the solution was allowed to slowly warm to room
temperature for 17 h. The mixture was poured into 80 mL of potassium
phosphate buffer (pH 7.0) and the mixture was extracted with CH2Cl2.
The organic extract was washed with brine, dried over Na2SO4, filtered,
and then concentrated in vacuo. The residue was dissolved in MeOH
(330 mL) and cooled to 48C, then 30% H2O2 aqueous solution (99 mL)
was added slowly and the mixture was stirred at the same temperature
for 4 h. After addition of H2O (100 mL), the mixture was concentrated in
vacuo to remove MeOH. The resulting aqueous solution was extracted
with AcOEt and the organic extract was washed successively with 5%
NaHCO3 solution and brine, dried over Na2SO4, filtered, and then con-
centrated in vacuo. Flash chromatography (silica gel, hexane/AcOEt 2:1)
of the residue gave 17a as a colorless amorphous solid (18.2 g, 92%).
[a]2D3 =+24.9 (c=0.50 in MeOH); 1H NMR (400 MHz, CDCl3): d=0.87
(d, J=7.3 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H), 1.31 (d, J=6.7 Hz, 3H),
2.06–2.14 (m, 1H), 2.20–2.30 (m, 1H), 2.88 (d, J=7.3 Hz, 2H), 3.84–3.96
(m, 2H), 3.95 (brq, J=7.6 Hz, 1H), 4.15 (dd, J=1.8, 8.6 Hz, 1H), 4.36 (t,
J=8.6 Hz, 1H), 4.46 (ddd, J=2.4, 6.7, 7.9 Hz, 1H), 4.94 (d, J=12.2 Hz,
1H), 4.99 (brs, 1H), 5.02 (d, J=11.6 Hz, 1H), 7.16–7.24 (m, 3H), 7.24–
7.36 ppm (m, 7H); 13C NMR (100 MHz, CDCl3): d=15.0, 15.2, 17.9, 29.1,
38.8, 40.8, 53.7, 58.5, 63.9, 66.9, 73.4, 126.5, 127.97, 128.0, 128.46, 128.53,
129.1, 136.4, 137.8, 154.0, 156.3, 175.8 ppm; IR (ATR): n˜ =1514, 1689,
1774 cmÀ1; MS (ESI+): m/z: 469 [M+H]+; HRMS (ESI+): m/z: calcd
for C26H33N2O6: 469.2339 [M+H]+; found: 469.2343.
gave benzyl (2R,4S)-5-[(S)-4-isopropyl-2-oxooxazolidin-3-yl]-4-methyl-5-
oxo-1-phenylpentan-2-ylcarbamate (1.08 g, 80%) as
a colorless oil.
[a]2D6 =+64.9 (c=0.50 in MeOH); 1H NMR (400 MHz, CDCl3): d=0.88
(d, J=6.7 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H), 1.20 (d, J=6.7 Hz, 3H), 1.27
(ddd, J=3.1, 11.6, 14.1 Hz, 1H), 2.12 (ddd, J=3.1, 11.6, 14.1 Hz, 1H),
2.21–2.33 (m, 1H), 2.68 (dd, J=7.3, 14.1 Hz, 1H), 2.83 (dd, J=6.3,
14.1 Hz, 1H), 3.84–4.02 (m, 2H), 4.15 (dd, J=2.4, 8.6 Hz, 1H), 4.36 (t,
J=8.6 Hz, 1H), 4.44 (brd, J=10.4 Hz, 1H), 4.45–4.53 (m, 1H), 4.94 (d,
J=12.2 Hz, 1H), 5.01 (d, J=12.2 Hz, 1H), 7.12–7.18 (m, 2H), 7.18–7.24
(m, 1H), 7.24–7.36 ppm (m, 7H); 13C NMR (100 MHz, CDCl3): d=15.3,
17.9, 19.0, 29.2, 34.1, 39.3, 41.9, 49.6, 58.6, 63.9, 66.8, 126.5, 128.0, 128.1,
128.5, 129.3, 136.5, 137.4, 154.2, 156.4, 176.8 ppm; IR (ATR): n˜ =1202,
1264, 1385, 1686, 1770 cmÀ1; MS (ESI+): m/z: 453 [M+H]+; HRMS
(ESI+): m/z: calcd for C26H33N2O5: 453.2390 [M+H]+; found: 453.2384.
An aqueous solution of LiOH (1.11 g, 46.4 mol) and 30% H2O2
(4.26 mL, 139 mmol) were added to a solution of the deoxygenated com-
pound obtained above (10.5 g, 23.2 mmol) in THF (180 mL) and H2O
(60 mL) at 48C and the mixture was stirred at the same temperature for
3 h. The reaction mixture was poured into a 1.5m aqueous solution of
Na2SO3 and was adjusted to pH 3 with 2m HCl aq. at 48C. The resulting
aqueous solution was extracted with CH2Cl2 and the organic extract was
washed brine, dried over Na2SO4, filtered, and then concentrated in
vacuo. Flash chromatography (silica gel, hexane/AcOEt 2:3) of the resi-
due gave 19a (6.90 g, 87%) as a colorless solid. An analytical sample of
19a (colorless solid) was obtained by recrystallization from hexane/
AcOEt. M.p. 88–898C; [a]2D6 =+21.5 (c=0.50 in CHCl3); 1H NMR
(400 MHz, [D6]DMSO): d=1.04 (d, J=6.7 Hz, 3H), 1.35 (ddd, J=5.5,
9.2, 13.5 Hz, 1H), 1.74 (ddd, J=4.3, 9.8, 14.1 Hz, 1H), 2.35–2.47 (m, 1H),
2.59–2.70 (m, 1H), 2.63 (d, J=6.7 Hz, 1H), 3.62–3.76 (m, 1H), 4.92 (d,
J=12.8 Hz, 1H), 4.98 (d, J=12.2 Hz, 1H), 7.10–7.20 (m, 4H), 7.20–7.36
(m, 6H), 12.0 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO): d=18.0,
35.9, 38.0, 41.0, 50.7, 64.8, 125.9, 127.4, 127.6, 128.0, 128.3, 129.1, 137.4,
138.7, 155.6, 177.0 ppm; IR (ATR): n˜ =1263, 1536, 1687, 3322 cmÀ1; MS
(ESI+): m/z: 342 [M+H]+; HRMS (ESI+): m/z: calcd for C20H24NO4:
342.1705 [M+H]+; found: 342.1705; elemental analysis calcd (%) for
C20H23NO4: C 70.36, H 6.79, N 4.10; found: C 70.29, H 6.74, N 4.08.
Compound ent-17a (14.8 g, 89%) was prepared from (R)-4-isopropyl-3-
propionyloxazolidin-2-one (ent-15) (6.53 g, 35.3 mmol) and N-(benzyloxy-
carbonyl)-d-phenylalaninal (11.0 g, 38.8 mmol) in the same manner as de-
scribed for 17a. Colorless amorphous solid; [a]2D4 =À21.8 (c=0.50 in
MeOH); MS (ESI+): m/z: 469 [M+H]+; HRMS (ESI+): m/z: calcd for
C26H33N2O6: 469.2339 [M+H]+; found: 469.2336; 1H and 13C NMR and
IR spectra of this sample were identical with those described for 17a.
Compound ent-19a (6.96 g, 74% from ent-18a) was prepared from ent-
18a (15.9 g, 27.5 mmol) in the same manner as described for 19a. Color-
less solid; m.p. 88–898C; [a]D28 =À16.8 (c=0.50 in CHCl3); MS (ESI+):
m/z: 342 [M+H]+; HRMS (ESI+): m/z: calcd for C20H24NO4: 342.1705
[M+H]+; found: 342.1698; elemental analysis calcd (%) for C20H23NO4:
O-(2S,3S,4S)-1-[(S)-4-Isopropyl-2-oxooxazolidin-3-yl]-2-methyl-1-oxo-5-
phenyl-4-(phosphinylideneamino)pentan-3-yl
1H-imidazole-1-carbo-
thioate (18a) and its enantiomer (ent-18a): TCDI (1.71 g, 9.58 mmol)
was added to a solution of 17a (1.50 g, 3.19 mmol) in THF (30 mL) and
the mixture was heated under reflux for 18 h. After consumption of 17a
by monitoring on TLC, the mixture was concentrated in vacuo. Flash
chromatography (silica gel, hexane/AcOEt 3:1) of the residue gave 18a
(1.75 g, 95%.) as a yellow amorphous solid. [a]2D5 =À37.6 (c=0.50 in
MeOH); 1H NMR (400 MHz, CDCl3): d=0.90 (d, J=6.7 Hz, 3H), 0.93
(d, J=6.7 Hz, 3H), 1.31 (d, J=7.3 Hz, 3H), 2.22–2.34 (m, 1H), 2.56 (dd,
J=10.4, 14.7 Hz, 1H), 3.06 (dd, J=4.3, 14.7 Hz, 1H), 4.19 (dd, J=3.0,
9.2 Hz, 1H), 4.32–4.44 (m, 2H), 4.44–4.56 (m, 2H), 4.80 (br d, J=9.8 Hz,
1H), 4.87 (d, J=12.2 Hz, 1H), 4.99 (d, J=12.2 Hz, 1H), 6.38 (dd, J=2.4,
9.8 Hz, 1H), 7.06–7.34 (m, 10H), 7.64 (brs, 1H), 8.39 ppm (s, 1H);
13C NMR (100 MHz, CDCl3): d=14.5, 15.4, 17.9, 29.3, 38.3, 39.6, 53.2,
58.6, 64.3, 67.2, 85.5, 117.8, 126.8, 127.9, 128.2, 128.5, 128.6, 128.8, 131.2,
135.9, 136.4, 137.2, 154.1, 156.6, 173.8, 184.3 ppm; IR (ATR): n˜ =1220,
1385, 1693, 1777 cmÀ1; MS (ESI+): m/z: 579 [M+H]+; HRMS (ESI+):
m/z: calcd for C30H35N4O6S: 579.2277 [M+H]+; found: 579.2270.
1
C 70.36, H 6.79, N 4.10; found: C 70.24, H 6.82, N 4.08; H and 13C NMR
and IR spectra of this sample were identical with those described for
19a.
Tubuphenylalanine hydrochloride (20·HCl) and its enantiomer (ent-
20·HCl): 10% Pd/C (200 mg) and 4m HCl/dioxane solution (14.6 mL,
58.6 mmol) were added to a solution of 19a (2.00 g, 5.86 mmol) in THF
(15 mL) and the mixture was stirred at room temperature under a hydro-
gen atmosphere for 5 h. The mixture was filtrated through a pad of
Celite and then concentrated in vacuo. The residue was triturated with
Et2O to give 20·HCl salt (1.35 g, 95%) as a colorless solid. M.p. 140–
1418C; [a]2D4 =+6.4 (c=1.00 in MeOH) (lit.:[7] [a]2D5 =+3.2 (c=1.00 in
MeOH)); 1H NMR (400 MHz, D2O, internal standard [D4]3-(trimethylsi-
lyl)propionic acid sodium salt, [D4]TSP): d=1.19 (d, J=6.7 Hz, 3H), 1.74
(td, J=6.7, 14.7 Hz, 1H), 2.04 (ddd, J=6.1, 8.6, 14.7 Hz, 1H), 2.70 (td,
J=6.7, 8.6 Hz, 1H), 2.92 (dd, J=7.9, 14.1 Hz, 1H), 3.04 (dd, J=6.7,
14.1 Hz, 1H), 3.61 (brquin, J=6.7 Hz, 1H), 7.29–7.46 ppm (m, 5H);
13C NMR (100 MHz, D2O, internal standard [D4]TSP): d=19.7, 38.4,
38.8, 41.3, 54.2, 130.5, 132.1, 132.4, 138.4, 183.1 ppm; IR (ATR): n˜ =1580,
1711, 2965 cmÀ1; MS (CI+): m/z: 208 [M+H]+; HRMS (CI+): m/z:
calcd for C12H18NO2: 208.1338 [M+H]+; found: 208.1361; elemental anal-
ysis calcd (%) for C12H17NO2·HCl·0.25H2O: C 58.06, H 7.51, N 5.64;
found: C 57.92, H 7.30, N 5.52.
Compound ent-18a (16.3 g, 90%) was prepared from ent-17a (14.7 g,
31.4 mmol) in the same manner as described for 18a. Yellow amorphous
solid; [a]2D5 =+44.0 (c=0.50 in MeOH); MS (ESI+): m/z: 579 [M+H]+;
HRMS (ESI+): m/z: calcd for C30H35N4O6S: 579.2277 [M+H]+; found:
579.2279; 1H and 13C NMR and IR spectra of this sample were identical
with those described for 18a.
N-Cbz-tubuphenylalanine (19a) and its enatiomer (ent-19a): nBu3SnH
(1.61 mL, 5.98 mmol) and AIBN (3.2 mg, 29.9 mm) were added to a solu-
tion of 18a (1.73 g, 2.99 mmol) in toluene (30 mL) and the mixture was
heated under reflux for 30 min. After consumption of 18a by monitoring
on TLC, the mixture was concentrated in vacuo. Flash chromatography
(silica gel containing 10% w/w KF,[30] hexane/AcOEt 4:1) of the residue
ent-Tubuphenylalanine hydrochloride (ent-20·HCl, 1.27 g, 89%) was pre-
pared from ent-19a (2.0 g, 5.86 mmol) in the same manner as described
for 20·HCl. Colorless solid; m.p. 143–1448C; [a]2D5 =À4.8 (c=1.00 in
MeOH); MS (ESIÀ): m/z: 206 [MÀH]À; HRMS (ESIÀ): m/z: calcd for
C12H16NO2: 206.1181 [MÀH]À; found: 206.1189; elemental analysis calcd
Chem. Eur. J. 2010, 16, 11678 – 11688
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11685

O. Tamura et al.
(%) for C12H17NO2·HCl·0.25H2O: C 58.06, H 7.51, N 5.64; found: C
57.96, H 7.23, N 5.60; 1H and 13C NMR and IR spectra of this sample
were identical with those described for 20·HCl.
to warm to room temperature and stirring was continued for 18 h. After
concentration, the residue was poured into CH2Cl2 and saturated aqueous
NaHCO3 solution and was extracted with CH2Cl2, dried over Na2SO4, fil-
tered, and then concentrated in vacuo. Flash chromatography (silica gel,
AcOEt/MeOH 7:1) of the residue gave 22 (4.54 g, 85%) as a colorless
solid. An analytical sample of 22 was obtained by trituration with iPr2O.
M.p. 114–1158C; [a]2D5 =+77.4 (c=0.50 in MeOH); 1H NMR (400 MHz,
[D6]DMSO, 808C): d=0.58 (brd, J=6.7 Hz, 3H), 0.68 (brt, J=7.3 Hz,
3H), 0.90 (brd, J=6.7 Hz, 3H), 0.91 (brd, J=6.7 Hz, 3H), 1.08–1.24 (m,
1H), 1.28–1.66 (m, 7H), 1.97 (brtd, J=3.1, 11.6 Hz, 1H), 2.09 (brs, 3H),
2.08–2.18 (m, 1H), 2.45 (dd, J=3.1, 10.4 Hz, 1H), 2.60–2.70 (m, 1H),
2.76–2.86 (m, 2H), 3.83 (s, 3H), 4.40–4.56 (m, 2H), 5.59 (dd, J=4.3,
7.4 Hz, 1H), 7.24 (brd, J=9.8 Hz, 1H), 8.55 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO, 248C; mixture of rotamers): d=10.6, 15.3, 16.6,
18.6, 22.8, 23.3, 24.8, 29.6, 29.9, 33.5, 34.9, 43.8, 52.1, 52.4, 54.7, 60.8, 68.4,
77.1, 131.3, 145.6, 161.0, 167.9, 168.1, 172.3 ppm; IR (ATR): n˜ =1212,
1449, 1536, 1623, 1682, 1734, 2937, 3293 cmÀ1; MS (ESI+): m/z: 495
[M+H]+; HRMS (ESI+): m/z: calcd for C24H39N4O5S: 495.2641 [M+H]+;
found: 495.2648; elemental analysis calcd (%) for C24H38N4O5S: C 58.28,
H 7.74, N 11.33; found: C 58.07, H 7.68, N 11.22.
N,O-Dehydrotubuvaline methyl ester (21): Et2NH (10.5 mL, 101 mmol)
was added to a solution of 14 (1.62 g, 3.38 mmol) in MeCN (11 mL) at
48C and the mixture was stirred at room temperature for 2 h. After con-
centration, Et2O (20 mL) and 1m HCl aq. (20 mL) were added to the res-
idue and the mixture was stirred at room temperature for 15 min. After
separation, the aqueous layer was washed with benzene and the pH ad-
justed to 9 with NaHCO3. The resulting aqueous solution was extracted
with AcOEt, and the organic extract was washed brine, dried over
Na2SO4, filtered, and then concentrated in vacuo. Flash chromatography
(silica gel, hexane/AcOEt 2:3) of the residue gave 21 (729 mg, 84%) as a
colorless solid. An analytical sample of 21 (colorless solid) was obtained
by recrystallization from iPr2O. M.p. 117–1188C; [a]2D4 =+9.6 (c=1.05 in
MeOH); 1H NMR (400 MHz, CDCl3): d=0.98 (d, J=6.7 Hz, 3H), 1.04
(d, J=6.7 Hz, 3H), 1.68 (broct, J=6.8 Hz, 1H), 2.28–2.51 (brs, 1H),
2.58–2.76 (brs, 1H), 3.18 (brq, J=7.6 Hz, 1H), 3.95 (s, 3H), 5.44 (dd, J=
3.1, 9.2 Hz, 1H), 5.45–5.80 (brs, 1H), 8.17 ppm (s, 1H); 13C NMR
(100 MHz, CDCl3): d=20.0, 20.6, 31.1, 52.5, 66.5, 80.3, 128.1, 147.1, 161.8,
173.9 ppm; IR (ATR): n˜ =1238, 1710 cmÀ1; MS (EI+): m/z: 256 [M]+;
HRMS (EI+): m/z: calcd for C11H16N2O3S: 256.0882 [M]+; found:
256.0911; elemental analysis calcd for C11H16N2O3S: C 51.54, H 6.29, N
10.93; found: C 51.19, H 6.12, N 10.87.
N-Methyl-d-pipecorinyl-l-isoleucinyl-N,O-dehydrotubuvalinyl-tubuphe-
nylalanine (23): LiOH (26.3 mg, 1.10 mmol) was added to a solution of
22 (495 mg, 1.00 mmol) in THF (3.3 mL) and H2O (1.7 mL) at 48C and
the mixture was stirred at room temperature for 16 h. After concentra-
tion, the residue was poured into H2O, and the pH of the mixture was ad-
justed to pH 4 with HCl aq. (1.00 molLÀ1) and then concentrated in
vacuo. Flash chromatography (silica gel, CH2Cl2/MeOH/NH4OH 20:5:1)
of the residue gave N-methyl-d-pipecorinyl-l-isoleucinyl-N,O-dehydrotu-
buvaline (462 mg, 96%) as a colorless solid. M.p. 122–1248C; [a]2D6 =+
26.0 (c=0.51 in MeOH); 1H NMR (400 MHz, [D6]DMSO, 808C): d=
0.59 (brd, J=6.7 Hz, 3H), 0.69 (brt, J=7.3 Hz, 3H), 0.90 (brd, J=
6.1 Hz, 3H), 0.94 (brd, J=6.7 Hz, 3H), 0.96–1.00 (m, 1H), 1.10–1.60 (m,
7H), 1.63 (brd, J=11.0 Hz, 2H), 1.99 (td, J=2.4, 11.0 Hz, 1H), 2.11 (brs,
3H), 2.10–2.18 (m, 1H), 2.45–2.55 (m, 1H), 2.60–2.70 (m, 1H), 2.74–2.90
(m, 2H), 4.40–4.50 (m, 1H), 4.50–4.60 (m, 1H), 5.72 (dd, J=4.9, 7.9 Hz,
1H), 7.20–7.40 ppm (m, 1H), 8.34 (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=10.7, 15.3, 16.6, 18.6, 22.7, 23.3, 24.7, 29.5, 29.9, 33.5,
34.9, 43.7, 52.4, 54.7, 60.8, 68.2, 77.3, 128.9, 149.6, 162.4, 166.7, 168.0,
N-Methyl-d-pipecorinyl-l-isoleucinyl-N,O-dehydrotubuvaline
methyl
ester (22): HATU (7.79 g, 20.5 mmol) and iPr2NEt (3.57 mL, 20.5 mmol)
were added to a solution of 21 (3.50 g, 13.6 mmol) and Boc-l-Ile (4.74 g,
20.5 mmol) in CH2Cl2 (68 mL) at 48C and the mixture was stirred at the
same temperature for 30 min. The resulting mixture was allowed to warm
to room temperature and stirring was continued for 74 h. The reaction
mixture was poured into saturated aqueous NH4Cl solution. After sepa-
ration, the aqueous layer was extracted with AcOEt. The combined or-
ganic extracts were washed with brine, dried over Na2SO4, filtered, and
then concentrated in vacuo. Flash chromatography (silica gel, hexane/
AcOEt 3:2) of the residue gave N-Boc-l-isoleucinyl-N,O-dehydrotubuva-
line methyl ester (6.16 g, 96%) as a colorless oil. [a]2D4 =+39.5 (c=0.50 in
1
MeOH); H NMR (400 MHz, CDCl3): d=0.74–0.88 (m, 6H), 0.96 (d, J=
7.3 Hz, 3H), 0.98 (d, J=7.3 Hz, 3H), 1.00–1.12 (m, 1H), 1.41 (brs, 9H),
1.40–1.62 (m, 2H), 2.28–2.46 (m, 1H), 2.66–2.80 (m, 2H), 3.95 (s, 3H),
4.38–4.62 (m, 2H), 4.96–5.08 (m, 1H), 5.60 (t, J=6.7 Hz, 1H), 8.21 ppm
(brs, 1H); 13C NMR (100 MHz, CDCl3): d=11.2, 15.7, 16.6, 19.1, 23.8,
28.2, 29.7, 35.2, 36.9, 52.6, 55.7, 62.2, 78.3, 79.5, 128.7, 146.9, 155.6, 161.4,
168.1, 168.7 ppm; IR (ATR): n˜ =1164, 1215, 1242, 1455, 1498, 1641, 1711,
2964 cmÀ1; MS (ESI+): m/z: 470 [M+H]+; HRMS (ESI+): m/z: calcd
for C22H36N3O6S: 470.2325 [M+H]+; found: 470.2327.
172.0 ppm; IR (ATR): n˜ =1275, 1361, 1459, 1591, 1638, 2961, 3221 cmÀ1
;
MS (ESI+): m/z: 481 [M+H]+; HRMS (ESI+): m/z: calcd for
C23H37N4O5S: 481.2485 [M+H]+; found: 481.2483; elemental analysis
calcd (%) for C23H36N4O5S·1.75H2O: C 53.94, H 7.77, N 10.94; found: C
53.90, H 7.50, N 11.20.
1,3-Diisopropylcarbodiimide (42.0 mL, 0.27 mmol) was added to a solu-
tion of carboxylic acid (118 mg, 0.25 mmol) prepared above and penta-
fluorophenol (67.8 mg, 0.37 mmol) in CH2Cl2 (1.3 mL) at 48C and the
mixture was stirred at room temperature for 24 h. After concentration,
AcOEt (2 mL) was added and the resulting mixture was filtered and the
filtrate was concentrated in vacuo to give the crude pentafluorophenyl
ester, which was used without further purification. iPr2NEt (0.21 mL,
1.23 mmol) and 20·HCl (120 mg, 0.49 mmol) were added to a solution of
the crude pentafluorophenyl ester in DMF (1 mL) at 48C and the mix-
ture was stirred at the same temperature for 30 min. The resulting mix-
ture was allowed to warm to room temperature and stirring was contin-
ued for 24 h. After concentration, the residue was poured into H2O and
was extracted with CH2Cl2, dried over Na2SO4, filtered, and then concen-
trated in vacuo. Preparative TLC of the residue on silica gel (CH2Cl2/
MeOH 12:1) gave 23 (143 mg, 87%) as a colorless amorphous solid. An
analytical sample of 23 (colorless powder) was obtained by freeze-drying
a solution of 23 in MeCN/H2O. M.p. 82–848C; [a]2D6 =+51.2 (c=0.50 in
MeOH); 1H NMR (400 MHz, CD3OD): d=0.45 (brs, 3H), 0.66 (brs,
3H), 0.90–1.10 (m, 8H), 1.15 (brd, J=7.3 Hz, 3H), 1.24–1.50 (m, 3H),
1.50–1.75 (m, 4H), 1.75–1.90 (m, 2H), 1.96–2.04 (m, 1H), 2.22–2.46 (m,
5H), 2.46–2.58 (m, 1H), 2.60–2.68 (m, 1H), 2.82–3.00 (m, 3H), 3.00–3.15
(m, 2H), 4.30–4.45 (m, 1H), 4.55–4.65 (m, 1H), 4.60–4.75 (m, 1H), 5.72
(dd, J=3.1, 7.9 Hz, 1H), 7.15–7.20 (m, 1H), 7.20–7.30 (m, 4H), 8.21 ppm
(brs, 1H); 13C NMR (100 MHz, CD3OD): d=11.4, 16.0, 17.0, 18.7, 19.1,
23.7, 25.0, 25.5, 31.1, 31.6, 34.5, 36.5, 38.8, 39.2, 41.9, 44.1, 50.9, 55.4, 56.5,
63.0, 69.9, 79.1, 127.0, 127.3, 129.4, 130.5, 139.6, 151.2, 162.4, 168.6, 169.8,
TFA (11 mL) was added to a solution of this material (6.04 g, 12.9 mmol)
in CH2Cl2 (53 mL) at 48C and the mixture was stirred at the same tem-
perature for 1 h. After this time, the mixture was allowed to warm to
room temperature. After further stirring for 1.5 h, the mixture was con-
centrated and the residue was partitioned between CH2Cl2 and saturated
aqueous NaHCO3 solution. The organic extract was dried over Na2SO4,
filtered, and then concentrated in vacuo. Flash chromatography (silica
gel, AcOEt/MeOH 15:1) of the residue gave l-isoleucinyl-N,O-dehydro-
tubuvaline methyl ester (4.74 g, quant.) as a colorless oil. [a]2D5 =+57.3
(c=0.51 in MeOH); 1H NMR (400 MHz, CDCl3): d=0.80 (d, J=6.7 Hz,
3H), 0.81 (t, J=6.7 Hz, 3H), 0.97 (d, J=7.4 Hz, 3H), 0.99 (d, J=7.3 Hz,
3H), 1.02–1.16 (m, 1H), 1.48–1.60 (m, 2H), 1.60–1.66 (m, 2H), 2.26–2.42
(m, 1H), 2.71 (brt, J=6.7 Hz, 2H), 3.40–3.62 (brs, 1H), 3.96 (s, 3H),
4.47 (q, J=6.1 Hz, 1H), 5.59 (t, J=6.7 Hz, 1H), 8.23 ppm (s, 1H);
13C NMR (100 MHz, CDCl3): d=11.2, 16.2, 16.8, 19.1, 23.5, 30.1, 35.3,
37.0, 52.6, 56.9, 62.0, 78.3, 128.6, 147.0, 161.4, 168.5, 173.0 ppm; IR
(ATR): m/z: 1214, 1242, 1467, 1638, 1729, 2961 cmÀ1; MS (ESI+): m/z:
370 [M+H]+; HRMS (ESI+): m/z: calcd for C17H28N3O4S: 370.1801
[M+H]+; found: 370.1801.
iPr2NEt (3.77 mL, 21.7 mmol) and DECP (2.74 mL, 16.2 mmol) were
added to a solution of this material (4.0 g, 10.8 mmol) and d-Mep[7]
(1.86 g 13.0 mmol) in DMF (54 mL) at 48C and the mixture was stirred
at the same temperature for 30 min. The resulting mixture was allowed
11686
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11678 – 11688

Total Syntheses of Tubulysins
FULL PAPER
173.6, 181.4 ppm; IR (ATR): n˜ =1453, 1492, 1539, 1647, 2961 cmÀ1; MS
(ESI+): m/z: 670 [M+H]+; HRMS (ESI+): m/z: calcd for C35H52N5O6S:
670.3638 [M+H]+; found: 670.3642; elemental analysis calcd (%) for
C35H51N5O6S·H2O: C 61.11, H 7.77, N 10.18; found: C 60.94, H 7.59, N
10.10.
13C NMR (100 MHz, CD3OD): d=11.4, 16.4, 18.7, 19.5, 20.4, 23.8, 25.6,
25.8, 27.3, 31.1, 34.5, 37.8, 38.7, 39.3, 42.1, 44.1, 50.9, 54.7, 55.8, 56.5, 69.8,
74.0, 74.1, 124.7, 127.3, 129.3, 130.5, 139.6, 150.8, 162.88, 162.91, 172.9,
173.1, 174.1, 181.3 ppm; IR (ATR): n˜ =1542, 1649, 2963 cmÀ1; MS (ESI+):
m/z: 684 [M+H]+; HRMS (ESI+): m/z: calcd for C36H54N5O6S: 684.3795
[M+H]+; found: 684.3793; elemental analysis calcd (%) for
C36H53N5O6S·H2O: C 61.60, H 7.90, N 9.98; found: C 61.62, H 7.83, N
9.59.
Tubulysin V (1d): [Mo(CO)6] (31.7 mg, 0.12 mmol) was added to a solu-
tion of 23 (67.0 mg, 0.10 mmol) in MeCN (1 mL) and H2O (0.1 mL) and
the mixture was stirred at 908C for 1 h. After concentration, preparative
TLC (CH2Cl2/MeOH 12:1) and subsequent reverse-phase chromatogra-
phy (H2O/CH3CN 2:1) of the residue gave the tubulysin V (1k) (48.2 mg,
72%) as a colorless solid. M.p. 118–1208C; [a]2D7 =À40.4 (c=0.50 in
H2O); 1H NMR (400 MHz, CD3OD): d=0.91(t, J=7.3 Hz, 3H), 0.95 (d,
J=6.7 Hz, 3H), 0.96 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 1.15
(brd, J=6.7 Hz, 3H), 1.17–1.26 (m, 1H), 1.32–1.42 (m, 1H), 1.52–1.92
(m, 10H), 1.96–2.06 (m, 1H), 2.08–2.18 (m, 1H), 2.33 (s, 3H), 2.34–2.42
(m, 1H), 2.46–2.60 (m, 1H), 2.93 (d, J=6.7 Hz, 2H), 2.98 (dd, J=3.1,
11.6 Hz, 2H), 3.04–3.12 (m, 1H), 4.04–4.12 (m, 1H), 4.20 (d, J=8.6 Hz,
1H), 4.28–4.40 (m, 1H), 4.79 (dd, J=2.4, 10.4 Hz, 1H), 7.10–7.20 (m,
1H), 7.20–7.35 (m, 4H), 8.02 ppm (brs, 1H); 13C NMR (100 MHz,
CD3OD): d=11.0, 16.2, 18.8, 19.0, 19.5, 23.6, 25.5, 26.1, 31.1, 33.8., 37.5,
39.1, 39.2, 40.8, 41.7, 44.1, 51.0, 52.7, 56.4, 59.8, 69.71, 69.79, 124.2, 127.4,
129.3, 130.5, 139.6, 151.0, 163.1, 173.6, 173.9, 179.3, 182.3 ppm; IR (ATR):
n˜ =1495, 1539, 1646, 2960, cmÀ1; MS (ESI+): m/z: 672 [M+H]+; HRMS
(ESI+): m/z: calcd for C35H54N5O6S: 672.3795 [M+H]+; found: 672.3791;
elemental analysis calcd (%) for C35H53N5O6S·0.75H2O: C 61.33, H 8.01,
N 10.22; found: C 61.35, H 7.84, N 10.18.
Acknowledgements
We are grateful to Dr. Y. Fukuda of Kyorin Pharmaceutical Co. Ltd. for
his valuable suggestions and discussions. We also thank Ms. M. Yamazaki
and Mr. H. Ebisu (Discovery Research Laboratories, Kyorin Pharmaceut-
ical Co. Ltd.) for performing the biological assays.
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Tubulysin U (1c): Ac2O (0.17 mL, 1.75 mmol) was added to a solution of
1k (147 mg, 0.22 mmol) in pyridine (2.1 mL) at 48C and the mixture was
stirred at room temperature for 10 h. To the reaction mixture was added
1,4-dioxane (3.8 mL) and H2O (3.8 mL) at 48C and the mixture was fur-
ther stirred at room temperature for 11 h and then concentrated in
vacuo. Flash chromatography (silica gel, CH2Cl2/MeOH 9:1) of the resi-
due gave tubulysin U (1c) (143 mg, 92%) as a colorless powder. An ana-
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iPr2O. M.p. 102–1038C; [a]2D3 =À11.7 (c=0.21 in MeOH) (lit.:[8a] [a]2D3
=
1
À1.42 (c=1.50 in MeOH)); H NMR (400 MHz, CD3OD): d=0.92 (t, J=
7.3 Hz, 3H), 0.95–0.96 (m, 6H), 0.99 (d, J=6.7 Hz, 3H), 1.15 (d, J=
6.7 Hz, 3H), 1.16–1.26 (m, 1H), 1.34–1.48 (m, 1H), 1.52–2.04 (m, 10H),
2.06–2.14 (m, 1H), 2.15 (s, 3H), 2.18–2.28 (m, 1H), 2.41, (s, 3H), 2.40–
2.50 (m, 1H), 2.50–2.60 (m, 1H), 2.92 (dd, J=1.8, 6.7 Hz, 2H), 3.04–3.16
(m, 2H), 3.92–4.02 (m, 1H), 4.21 (d, J=8.6 Hz, 1H), 4.30–4.40 (m, 1H),
5.90 (dd, J=2.4, 10.4 Hz, 1H), 7.10–7.20 (m, 1H), 7.20–7.26 (m, 4H),
8.07 ppm (brs, 1H); 13C NMR (100 MHz, CD3OD): d=11.1, 16.2, 18.5,
18.9, 19.5, 20.7, 23.5, 25.4, 25.9, 31.0, 33.8, 37.6, 38.0, 38.9, 39.0, 41.7, 44.0,
51.1, 52.0, 56.3, 59.7, 69.6, 71.3, 124.9, 127.4, 129.3, 130.5, 139.7, 151.0,
162.7, 171.73, 171.76, 173.3, 173.8, 182.0 ppm; IR (ATR): n˜ =1220, 1495,
1540, 1650, 1744, 2961 cmÀ1; MS (ESI+): m/z: 714 [M+H]+; HRMS
(ESI+): m/z: calcd for C37H56N5O7S: 714.3900 [M+H]+; found:
714.3896; elemental analysis calcd (%) for C37H55N5O7S·H2O: C 60.71, H
7.69, N 9.44; found: C 60.56, H 7.69, N 9.44.
ˇ
´
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Cyclo-tubulysin D (1e): A mixture of deacetyl-tubulysin D (24) (23.4 mg,
29.8 mm) and 1m HCl aq. (2 mL) in THF (2 mL) was stirred at 608C for
24 h. After concentration, the residue was poured into CH2Cl2/MeOH
(10:1, 10 mL) and phosphate buffer (pH 7.4, 10 mL). The mixture was ex-
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5
(19.8 mg, 97%) as a colorless amorphous solid. This material appeared to
exist as an 84:16 mixture of rotamers in CD3OD. [a]2D9 =+49.1 (c=0.34
in MeOH); 1H NMR (400 MHz, CD3OD): d=0.85 (d, J=6.7 Hz, 3H),
0.94 (t, J=7.3 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 1.12 (d, J=6.7 Hz, 3H),
1.15 (d, J=6.7 Hz, 3H), 1.15–1.42 (m, 3H), 1.50–1.76 (m, 5H), 1.76–1.90
(m, 3H), 1.94–2.10 (m, 2H), 2.24–2.60 (m, 4H), 2.33 (s, 3H), 2.90 (brd,
J=7.3 Hz, 3H), 3.08 (brd, J=11.6 Hz, 1H), 4.25 (m, 1Hꢂ14/100), 4.30–
4.40 (m, 1H), 4.45 (m, 1Hꢂ86/100), 4.57 (d, J=10.4 Hz, 1Hꢂ14/100),
4.75 (d, J=7.9 Hz, 1H), 5.02 (d, J=11.6 Hz, 1Hꢂ86/100), 5.33 (dd, J=
1.8, 11.6 Hz, 1H), 5.76 (d, J=11.6 Hz, 1Hꢂ86/100), 5.76 (d, J=9.8 Hz,
1Hꢂ14/100), 7.10–7.18 (m, 1H), 7.20–7.26 (m, 4H), 8.06 ppm (s, 1H);
Chem. Eur. J. 2010, 16, 11678 – 11688
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11687

O. Tamura et al.
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[16] Prepared as described in the Supporting Information.
[17] Crystallographic data: CCDC-760558 (8a), -760559 (8d), -719636
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Received: April 15, 2010
Published online: August 23, 2010
11688
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 11678 – 11688