Stereoselective total synthesis of tubulysin v

Article abstract of DOI:10.1002/cjoc.201200984

The total synthesis of tubulysin V was accomplished in 1.0% overall yield with linear 13 steps. Our synthetic strategy featured the following two reactions. One is zinc-mediated aza-Barbier reaction of (R)-N-tert- butanesulfinyl imine 8 with β-ester group functionalized allylic bromide 9 to afford the chiral homo-allylic amine (7); the other is to employ the methodology of aqueous indium-mediated aza-Barbier reaction previously developed by our group, giving the chiral homo-allylic amine 13 with high efficiency. The total synthesis of tubulysin V was accomplished in 1.0% overall yield with linear 13 steps. Our synthetic strategy featured the following two reactions. One is zinc-mediated aza-Barbier reaction of (R)-N-tert-butanesulfinyl imine 8 with β-ester group functionalized allylic bromide 9 to afford the chiral homo-allylic amine 7; the other is to employ the methodology of aqueous indium-mediated aza-Barbier reaction previously developed by our group, giving the chiral homo-allylic amine 13 with high efficiency. Copyright

Full text of DOI:10.1002/cjoc.201200984

FULL PAPER
DOI: 10.1002/cjoc.201200984
Stereoselective Total Synthesis of Tubulysin V†
Rui Wang, Ping Tian, and Guoqiang Lin*
CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
The total synthesis of tubulysin V was accomplished in 1.0% overall yield with linear 13 steps. Our synthetic
strategy featured the following two reactions. One is zinc-mediated aza-Barbier reaction of (R)-N-tert-butane-
sulfinyl imine 8 with β-ester group functionalized allylic bromide 9 to afford the chiral homo-allylic amine (7); the
other is to employ the methodology of aqueous indium-mediated aza-Barbier reaction previously developed by our
group, giving the chiral homo-allylic amine 13 with high efficiency.
Keywords tubulysin V, N-tert-butanesulfinyl imines, zinc, indium, aza-Barbier reaction
Introduction
R3
Tubulysins are a family of linear tetrapeptides first
isolated by Höfle and co-workers from myxobacterial
cultures in 2000.[1] These natural products display ex-
traordinary antimitotic activities[2] in disturbing the
microtubule construction and consequently inhibiting
tubulin polymerization with IC50 values ranging from
0.01 to 10 nmol/L. In fact, tubulysins have been demon-
strated to display better in vitro and in vivo anti-tumor
activities than epothilones.[2a] As shown in Figure 1,
tubulysins A, B, D, U, V are composed of the following
four units: non-proteinogenic N-methyl picolinic acid
(D-Mep), proteinogenic isoleucine (L-Ile), non-protein-
ogenic tubuvaline (Tuv), and terminal amino acid with
phenol (Tut, tubutyrosine) or phenyl (Tup, tubuphenyl-
alanine).
O
OR2
S
O
H
N
N
N
R1
N
N
H
O
CO2H
Tup
Mep
Ile
Tuv
Tubulysins
R1
R2 R3
A (1a)
B (1b)
D (1c)
U (1d)
V (1e)
CH2OCOCH2CH(CH3)2
Ac OH
Ac OH
CH2OCOCH2CH2CH3
CH2OCOCH2CH(CH3)2
Ac
Ac
H
H
H
H
H
H
Figure 1 Structures of tubulysins.
Due to its limitation from natural sources and high
demand in preclinical and clinical chemotherapeutics
studies, tubulysins have attracted considerable research
interests in chemical and biological community. In the
past decade, especially since the first asymmetric total
synthesis of tubulysin D accomplished by Ellman and
co-workers in 2006,[3] significant progress has been
achieved in the synthesis of such starry molecules.[4]
Recently, our group developed efficient approaches to
construct chiral homo-allylic amines from N-tert-bu-
tanesulfinyl imines through aza-Barbier-type reaction.[5]
Herein, we present our research results in the total syn-
thesis of tubulysin V by using these asymmetric meth-
odologies.
focus on the asymmetric syntheses of Tup and Tuv, and
the subsequent assembly of four units into tubulysin V.
Tup unit (2), a γ-amino acid derivative with two chiral
centers, can be achieved through the stereo-controlled
modifications of chiral γ-lactam 6, which is derived
from zinc-mediated aza-Barbier reaction of chiral imine
8 and β-ester group functionalized allylic bromide 9.
Tuv unit (3), a 2,4-disubstituted thiazole carboxylic acid,
was previously prepared through multi-component reac-
tion (MCR) of chiral aldehyde 10 derived from
β-D-homovaline, Schöllkopf isonitrile (methyl 3-dime-
thylamino-2-isocyano-acrylate, 11) and thioacetic acid
(12).[4a,6] In our synthesis, the chiral center in the key
aldehyde 10 can be established through indium-medi-
ated aza-Barbier reaction of chiral N-tert-butanesulfinyl
imine 14 and allylic bromide. Our practical and scalable
The retro-synthetic analysis of tubulysin V (1e), as
showed in Scheme 1, led to four amino acid derivatives,
Tup (2), Tuv (3), Ile (4) and Mep (5). In this paper, we
*
E-mail: lingq@sioc.ac.cn; Tel.: 0086-021-54925169; Fax: 0086-021-64166128
Received October 8, 2012; accepted November 4, 2012; published online December 11, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200984 or from the author.
Dedicated to the Memory of Professor Weishan Zhou.
†
40
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 40—48

Stereoselective Total Synthesis of Tubulysin V
Scheme 1 Retro-synthetic analysis of tubulysin V (1e)
Coupling Coupling
OH
Coupling
Ph
Ph
BocHN
CO2H
CO2H
BocHN
OH
O
O
H
N
CO2H
BocHN
N
N
N
N
N
N
H
H
Cbz
S
CO2Me
Tup (2)
S
O
CO2H
Mep (5)
Ile (4)
Tuv (3)
tubulysin V (1e)
O
S
O
S
O
HN
Boc
CO2Et
Br
Zn
N
Tup (2)
+
+
N
Ph
Ph
Ph
CO2Et
6
7
(R)-8
9
O
S
O
S
N
O
In
Br
HN
Tuv (3)
BocHN
H
10
13
(S)-14
forts, using 10% Pd/C under 20 atm of H2 or
[Rh(cod)2]BF4/monoPhos under 5 atm of H2, did not
provide any meaningful selectivities for such a
transformation.[4l,9] Therefore we turned our focus to the
reduction of the exo-carbon-carbon double bond at the
rigid five-membered lactam ring 15. Under Boc2O/
NEt3/DMAP conditions, N-Boc-protected γ-lactam 6
was obtained in 87% yield. The subsequent Pd-cataly-
zed hydrogenation afforded a mixture of two di-
astereomers in 1∶8 ratio, but in favour of the undesired
isomer 17. The chiral inversion of this α-methyl group
was realized through enolization using a strong base
t-BuOK at −60 ℃ and subsequent protonation, afford-
ing the desired isomer 18 with 6∶1 d.r. The absolute
configuration of α-methyl in compound 18 was assigned
as S by chemical correlation with the NOESY experi-
ments, i.e., the strong NOE effect between the α-H and
the benzylic hydrogen.
After the two chiral centers were established in
compound 18, the key Tup unit (2) was successfully
obtained through base-promoted ring-opening and the
subsequent methylation with diazomethane. The overall
yield from compound 8 to the Tup unit (2) was 51%
yield in six linear steps. Because the two diastereomers
17 and 18 were inseparable by silica gel chromatogra-
phy, the Tup unit (2) was obtained as a mixture of two
diastereomers in 6∶1 ratio, which was directly used in
the final assembly (Scheme 2).
Barbier-type reactions of both imines 14 and 8 with the
corresponding allylic bromides allow for facile prepara-
tion of both chiral homo-allylic amines 13 and 7, thus
providing a convenient approach to synthesize tubulysin
V.
Results and Discussion
The detailed synthesis of Tup unit (2) is shown in
Scheme 2. The chiral amino group in compound 7 was
introduced through zinc-mediated aza-Barbier reaction
between N-tert-butanesulfinyl imine 8 derived from
phenylacetaldehyde and allylic bromide 9. While opti-
mizing the reaction conditions for the coupling, we
found that excess anhydrous lithium chloride (up to six
equiv.) not only significantly accelerated the reaction,
but also improved the diastereo-selectivity.[7] Other at-
tempts, using either HMPA as reaction solvent along
with H2O as additive or THF as solvent along with
In(OTf)3 as additive,[5a,5b] led to low yields and/or low
diastereo-selectivities. When the enantiopure (R)-N-tert-
butanesulfinyl imine 8 was applied as the reaction sub-
strate, chiral homo-allylic amine 7 was obtained in
quantitative yield and 90% de. The newly formed chiral
center was assigned as S-configuration based on the
nucleophilic attack of the allyl group from less-hindered
direction opposite to tert-butyl group.[5] To further
confirm the absolute configuration of this new chiral
center, we prepared the N-Bn-protected γ-lactam
derivates 16, whose optical rotation was consistent with
the known compound.[8] The optical purity of compound
7 could be enriched to 99% de after single recrystalli-
zation using CH2Cl2/Et2O as co-solvent. Orginally we
wanted to prepare the desired chiral α-methyl carbox-
ylate through diastereo-selective hydrogenation of the
double bond in compound 7. Unfortunately, all the ef-
The synthesis of Tuv unit (3) is shown in Scheme 3.
The chiral amino group in compound 13 was introduced
using aqueous aza-Barbier reaction previously devel-
oped in our group.[5b] This allylation of chiral (S)-N-tert-
butanesulfinyl imine 14 was mediated by indium in
saturated aqueous NaBr solution, affording the desired
R-isomer 13 in 76% yield and 92% de. The newly
formed chiral center was assigned as R configuration
Chin. J. Chem. 2013, 31, 40—48
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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41

Wang, Tian & Lin
FULL PAPER
Scheme 2 Synthesis of Tup unit (2)
Scheme 3 Synthesis of Tuv unit (3)
O
S
O
S
O
O
b
a
CO2Et
S
HN
Br
S
b
a
+
N
HN
N
Br
+
Ph
Ph
9
13
(S)-14
(R)-8
CO2Et
7
O
c
d
quant. yield, 90% de
99% de (after single recrystallization)
BocHN
BocHN
H
19
10
O
O
Bn
c
[α]26 -55.2 (c 1.0, CHCl3)
Found:
D
N
HN
[α]25 -61.0 (c 1.2, CHCl3) [10]
Literature:
D
Ph
Ph
16
OAc
S
OH
N
15
[α]20 -91.6 (c 1.9, CHCl3)
Found:
N
CO2Me
CO2H
D
e
BocHN
BocHN
[α]20 -71.0 (c 1.6, CHCl3) [8]
d
Literature:
D
S
20
3
O
O
Boc
Boc
f
e
N
N
Chemical structure of compounds 11 and 12:
Ph
Ph
O
6
17
S
N
O
O
N+
C-
OH
12
Ph
Boc
N
g
Ph
11
BocHN
H
H H
CO2Me
Reagents and conditions: a) In (power), NaBr (sat.), 76%, 92% de;
b) HCl (g)/MeOH (2 mol/L); then Boc2O, NaHCO3, 80%; c) OsO4,
NMO; then NaIO4, 78% for 2 steps; d) 11, 12, BF3•OEt2, THF, −78
℃, 54%, d.r.＝1∶2.4; e) NaOH, THF/H2O, quant
NOE
18
2
Reagents and conditions: a) Zn/LiCl, DMF, r.t., quant., 90% de.; b)
HCl (g)/dioxane (4 mol/L); then NaOH, H2O/THF, 83%, 99% ee; c)
NaH, BnBr, THF, 0 ℃ to r.t., 91%; d) NEt3, Boc2O, DMAP, CH2Cl2,
87%; e) H2 (1 atm), 10% Pd/C, EtOAc, r.t., 74%, d.r. (17∶18)＝8∶
1; f) t-BuOK, THF, −60 ℃, quant., d.r. (18∶17)＝6∶1; g) LiOH,
THF/ H2O, r.t., quant.; then CH2N2/Et2O, 95%
pound 21 in 72% yield. After protection of the hydroxyl
group in compound 21 as its acetate and subsequent
N-Boc deprotection, the Ile unit was introduced by am-
ide coupling with N-Boc isoleucine 4. Partial racemiza-
tion of the α-carbon chiral center in Ile unit was ob-
served in this coupling to give compound 23 as a mix-
ture of two diastereomers in 5∶1 ratio in 76%,[4a]
which was directly used in the next transformation
without purification. Another N-Boc deprotection of 23
in this assembling sequence allowed the introduction of
Mep unit by coupling with N-Cbz picolinic acid 5 under
HATU/NMM conditions, producing the skeleton 24 in
66% yield. Further modifications of 24 into the natural
product tubulysin V involved the conversion of N-Cbz
to N-Me, which was achieved by the deprotection of
Cbz group with TMSI[11] and subsequent reductive
amination[12] with paraformaldehyde to give compound
25 in 68% yield. The resulting methyl ester 25 was hy-
drolyzed with aqueous 1.0 mol/L LiOH solution to give
tubulysin V (1e) in 73% yield. The resulting crude tu-
bulysin V was still a mixture of two diastereomers,
which was successfully separated by preparative HPLC.
All the spectrum features of the pure tubulysin V are
consistent with the literature data.[4b]
according to our previous report,[5b] as well as the com-
parison of optical rotation with the known compound
10.[10] Removal of N-tert-butyl sulfinamide in 13 and
subsequent protection with Boc2O gave the homoallylic
amine 19 in 80% yield. The oxidative cleavage of the
carbon-carbon double bond in 19 was conducted
through dihydroxylation (OsO4/NMO) and subsequent
treatment with NaIO4, providing the desired aldehyde
10 in 78% yield. Following the procedure reported by
Wessjohann and co-workers,[4a] the thiazole ring was
constructed from aldehyde 10, Schöllkopf isonitrile
[methyl 3-dimethylamino-2-isocyano-acrylate, (11)] and
thioacetic acid 12 in a one-pot fashion, affording com-
pound 20 in 54% yield. This MCR generated a chiral
hydroxyl group in 1∶2.4 diastereoselectivity. Fortu-
nately, both isomers could be separated by silica gel
chromatography. Basic treatment of compound 20 re-
sulted in the hydrolysis of methyl ester and the removal
of acetyl group, giving the desired acid 3 in quantitative
yield. Overall, the Tuv unit (3) was prepared from imine
14 in six linear steps with 8% total yield and 92% ee.
With both Tup unit (2) and Tuv unit (3) in hand, we
turned to the final assembly of the four units to produce
tubulysin V (1e) (Scheme 4). After the removal of
N-Boc protection of Tup unit (2) using trifluoroacetic
acid, the resulting amino acid was coupled with Tuv unit
(3) under HATU/HOAt/NEt3 condition, affording com-
Conclusions
In summary, we have developed new asymmetric
syntheses of Tup and Tuv units, and assembled them to
produce tubulysin V. The preparation of Tup and Tuv
units both featured the aza-Barbier reaction, from chiral
42
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 40—48

Stereoselective Total Synthesis of Tubulysin V
Scheme 4 Total synthesis of tubulysin V (1e)
analyses were performed on Jasco 2089 or Water 510
liquid chromatography. Each of the target compounds
was purified by silica gel (300－400 mesh) column
chromatography.
Ph
Ph
OR2
O
a, b
N
(S)-Ethyl-4-((R)-1,1-dimethylethylsulfinamido)-2-
methylene-5-phenylpentanoate (7) To a stirred solu-
tion of (R,E)-2-methyl-N-(2-phenyl-ethylidene)propane-
2-sulfinamide (8) (223 mg, 1.0 mmol) and ethyl
2-(bromomethyl)acrylate (9) (772 mg, 4.0 mmol) in
anhydrous DMF (5 mL) were added active zinc powder
(262 mg, 4.0 mmol) and anhydrous lithium chloride
(254 mg, 6.0 mmol) under N2 atmosphere. The resulting
mixture was stirred at room temperature for 40 h, TLC
showed the imine was completely consumed. The mix-
ture was diluted with ethyl acetate (50 mL), then care-
fully quenched with 1.0 mol/L HCl (aq). The organic
layer was washed with saturated aqueous NaHCO3,
brine, and dried over Na2SO4. The crude product was
purified by flash silica gel chromatography (hexanes∶
EtOAc＝4∶1) to provide a colorless sticky liquid 7
(338 mg, 100%). [α]D22 ＋1.2 (c 1.0, CHCl3) 92% de;
1H NMR (400 MHz, CDCl3) δ: 7.32－7.21 (m, 5H),
6.23 (s, 1H), 5.56 (s, 1H), 4.17 (dd, J＝11.6, 5.6 Hz,
1H), 3.79－3.72 (m, 1H), 3.21 (d, J＝6.4 Hz, 1H), 3.08
(dd, J＝11.2, 4.0 Hz, 1H), 2.96 (dd, J＝11.2, 5.6 Hz,
1H), 2.52 (dd, J＝11.2, 4.0 Hz, 1H), 2.40 (dd, J＝11.2,
7.6 Hz, 1H), 1.27 (t, J＝5.6 Hz, 3H), 1.13 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ: 166.9, 137.7, 136.9, 129.8,
128.4, 127.5, 126.5, 60.7, 56.9, 56.1, 42.8, 37.4, 22.5,
14.0; FT-IR (film) ν: 2984, 1715, 1185, 1068, 703, 424
cm−1; ESI-MS: [M＋H]＋ 338; ESI-HRMS: [M＋H]＋
calcd for C18H28NO3S 338.1783, found 338.1784.
BocHN
c
N
BocHN
H
S
CO2Me
CO2Me
21: R2 = H
22: R2 = Ac
2
Ph
O
OAc
O
d, e
BocHN
N
N
N
H
H
S
CO2Me
Ph
23
O
OAc
S
O
H
N
f, g
N
N
R
N
N
H
H
O
CO2Me
24: R = Cbz
25: R = Me
h, i
Ph
O
OH
N
O
j
H
N
N
N
N
H
H
O
S
CO2H
tubulysin V (1e)
Reagents and conditions: a) TFA/CH2Cl2, quant.; b) 3, HATU,
HOAt, NEt3, DMF, 72%; c) Ac2O, Pyr., 77%; d) HCl-dioxane, quant.;
e) 4, HATU, DIPEA, CH2Cl2, 76%; f) HCl-dioxane, quant.; g) 5,
HATU, NMM, CH2Cl2, 66%; h) TMSI, CH2Cl2, quant.; i) (HCHO)n,
NaBH(OAc)3, 68%; j) LiOH (1.0 mol/L), 73%
(S)-5-Benzyl-3-methylenepyrrolidin-2-one
(15)
To a solution of 7 (338 mg, 1.0 mmol) in anhydrous
MeOH (5 mL) was added hydrochloric acid (0.2 mL,
8.4 equiv. in dioxane) at 0 ℃. The resulting mixture
was stirred at 0 ℃ for 2 h, TLC showed the reaction
was complete. The reaction mixture was concentrated in
vacuo and the crude product was used in next step
without further purification. To a solution of the above
crude compound in THF (10 mL) and water (2 mL) was
added catalytic NaOH (2 mg, 0.050 mmol). The result-
ing mixture was stirred at ambient temperature over-
night. The mixture was extracted with ethyl acetate (30
mL×3). The combined organic layers were washed
with brine, dried over Na2SO4. The solvents were re-
moved in vacuo, and the residue was purified by flash
silica gel chromatography (hexanes∶EtOAc＝4∶1) to
give a white solid 15 (155 mg, 83%). The optical purity
of this material was enriched from 88% ee to 99% ee by
single recrystallization in CH2Cl2-Et2O. [α]2D4 −94.5 (c
(R)-N-tert-butanesulfinyl imine 8 and (S)-N-tert-butane-
sulfinyl imine 14 to the chiral homoallylic amines 7 and
13, respectively. The assembly of the four units (Mep,
Ile, Tuv and Tup) was realized through conventional
amide couplings to give skeleton 24, which was further
modified to give tubulysin V (1e). The biological testing
of tubulysin V and other synthetic analogs around this
natural product will be reported in future communica-
tion.
Experimental
General information All reagents were purchased
from commercial sources and used without further puri-
fication unless otherwise noted. Optical rotations were
recorded on a Jasco P-1030 polarimeter. IR spectra were
recorded on a Perkin-Elmer 983 or Digibal FT-IR spec-
trometer using KBr pellet or film. The 1H NMR spectra
were collected on Varian Mercury 300 Bruker Av300,
Bruker DPX300, BrukerAv400, BrukerAv500 instru-
ments using deuterated solvents with tetramethylsilane
(TMS) as internal standard. EI-MS was recorded on
HP-5989A apparatus. ESI-HRMS was recorded on
Bruker ApeXIII 7.0 TESLA FTMS apparatus. HPLC
1
1.0, CHCl3) 88% ee; H NMR (400 MHz, CDCl3) δ:
7.33－7.17 (m, 5H), 6.98 (br s, 1H), 5.97 (s, 1H), 5.33
(s, 1H), 3.93－3.86 (m, 1H), 2.92 (dd, J＝17.2, 7.6 Hz,
1H), 2.80 (d, J＝6.8 Hz, 2H), 2.55 (dd, J＝16.8, 3.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ: 170.4, 139.1,
137.1, 129.1, 128.8, 126.9, 116.2, 52.5, 43.4, 32.6;
FT-IR (film) ν: 3226, 1702, 1644, 1454, 1280, 936, 699
Chin. J. Chem. 2013, 31, 40—48
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43

Wang, Tian & Lin
FULL PAPER
cm−1; ESI-MS: [M＋Na]＋ 188; ESI-HRMS: [M＋Na]＋
calcd for C12H13NONa 210.0885, found 210.0889. Anal.
calcd for C12H13NO: C 76.98, H 7.00, N 7.48; found C
76.90, H 7.03, N 7.37.
136.9, 129.5, 128.6, 126.7, 82.9, 57.3, 41.3, 37.3, 30.4,
28.1, 16.6; FT-IR (film) ν: 2978, 2938, 1785, 1749,
1714, 1456, 1369, 1351, 1307, 1257, 1151, 988, 781,
703 cm−1; ESI-MS: [M＋Na]＋ 312; ESI-HRMS: [M＋
＋
(S)-1,5-Dibenzyl-3-methylenepyrrolidin-2-one (16)
To a stirred solution of 15 (29 mg, 0.16 mmol) in THF
(2 mL) at 0 ℃ was added NaH (7 mg, 0.18 mmol, 60%
in mineral) in one portion. After the reaction mixture
was stirred for 30 min at this temperature, BnBr (24 μL,
0.2 mmol) was added and the mixture was allowed to
warm to ambient temperature overnight. Sat. aqueous
NH4Cl was added to quench the reaction, the crude
product was extracted with Et2O, and dried over Na2SO4.
Organic solvents were evaporated and the residue was
purified by flash silica gel chromatography to provide
16 (39 mg, 91%). [α]2D3 −91.6 (c 1.9, CHCl3); (literature:
Na]
312.1570.
(3S,5R)-tert-Butyl-5-benzyl-3-methyl-2-oxopyrroli-
calcd for C17H23NO3Na 312.1577, found
dine-1-carboxylate (18) To a stirred solution of com-
pound 17 (47 mg, 0.16 mmol) in anhydrous THF (1 mL)
at −60 ℃ was added catalytic potassium t-BuOK (2
mg, 0.018 mmol), and the resulting mixture was stirred
at this temperature for 2 h. 1.0 mol/L sodium bisulfate
(1 mL) was added to quench the reaction, the crude
product was extracted with ether (10 mL×3), washed
with brine, and dried over Na2SO4. Solvents were con-
centrated in vacuo, the crude product was then purified
by silica gel flash chromatography (hexanes∶EtOAc＝
4∶1) to provide a colorless liquid 18 (47 mg, 100%).
1
[α]2D0 −71.0 (c 1.6, CHCl3))[8]; H NMR (400 MHz,
CDCl3) δ: 7.36－7.21 (m, 8H), 7.03 (d, J＝8.0 Hz, 2H),
6.01 (d, J＝4.0 Hz, 1H), 5.28 (s, 1H), 5.18 (d, J＝16.0
Hz, 1H), 4.12 (d, J＝16.0 Hz, 1H), 3.69－3.63 (m, 1H),
3.11 (dd, J＝12.0, 4.0 Hz, 1H), 2.63－2.57 (m, 1H),
2.48－2.43 (m, 2H); 13C NMR (100 MHz, CDCl3) δ:
168.4, 139.1, 137.00, 136.6, 129.4, 129.0, 128.9, 128.5,
127.9, 127.0, 116.3, 55.4, 45.2, 40.0, 30.7.
1
[α]2D7 ＋115.2 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3) (major isomer) δ: 7.29－7.14 (m, 5H), 4.24 (td,
J＝8.8, 3.2 Hz, 1H), 3.07 (dd, J＝13.2, 3.6 Hz, 1H),
2.69 (dd, J＝13.2, 9.2 Hz, 1H), 2.39－2.29 (m, 1H),
2.00 (dd, J＝12.0, 8.0 Hz, 1H), 1.54 (s, 9H), 1.57－1.50
(m, 1H), 1.08 (d, J＝7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) (major isomer) δ: 176.5, 150.0, 137.3, 129.3,
128.7, 126.8, 82.7, 56.9, 39.1, 36.1, 30.7, 28.1, 15.3;
FT-IR (film) ν: 3399, 3029, 2977, 2935, 2877, 1786,
1750, 1712, 1456, 1369, 1309, 1255, 1150, 975, 779,
701 cm−1; ESI-MS: [2M＋Na]＋ 601; ESI-HRMS: [M
＋ Na] ＋ calcd for C17H23NO3Na 312.1574, found
312.1570.
(S)-tert-Butyl-5-benzyl-3-methylene-2-oxopyrro-
lidine-1-carboxylate (6) To a stirred solution of 15
(94 mg, 0.50 mmol) in CH2Cl2 (2.5 mL) were added
DMAP (62 mg, 0.50 mmol), Et3N (71 μL, 0.51 mmol)
and Boc2O (220 mg, 1.0 mmol). The reaction mixture
was kept at room temperature for 24 h. Solvent was re-
moved and the residue was purified by flash silica gel
chromatography (hexanes/EtOAc＝10/1) to afford a
colorless liquid 6 (126 mg, 87%). [α]2D6 ＋32.3 (c 1.9,
CHCl3); 1H NMR (400 MHz, CDCl3) δ: 7.33－7.17 (m,
5H), 6.15 (s, 1H), 5.43 (s, 1H), 4.38 (t, J＝8.0 Hz, 1H),
3.23 (dd, J＝13.2, 3.2 Hz, 1H), 2.69－2.61 (m, 1H),
2.57－2.49 (m, 2H), 1.60 (s, 9H); 13C NMR (100 MHz,
CDCl3) δ: 166.3, 150.4, 138.4, 136.8, 129.4, 128.7,
126.8, 120.4, 83.2, 55.9, 40.7, 28.9, 28.1; FT-IR (film) ν:
3500, 1705, 1369, 1232, 533 cm−1; ESI-MS: [M＋Na]＋
310; ESI-HRMS: [M＋Na]＋ calcd for C17H21NO3Na
310.1410, found 310.1414.
(2S,4R)-Methyl-4-((tert-butoxycarbonyl)amino)-
2-methyl-5-phenylpentanoate (2) To a solution of
compound 18 (47 mg, 0.16 mmol) in THF (1.5 mL) was
added LiOH (1.0 mol/L, 0.5 mL) at ambient tempera-
ture. The resulting mixture was stirred at room tem-
perature for 3 h. Organic solvent was removed, water
(10 mL) was added before 5% potassium bisulfate solu-
tion was added to adjust pH＝3, the mixture was ex-
tracted with ethyl acetate (10 mL×3), and dried over
Na2SO4. Organic solvent was removed, and the residue
was purified by silica gel flash chromatography (hex-
anes∶EtOAc＝1∶1) to afford crude acid compound
(50 mg, 100%). To a stirred solution of above crude acid
(326 mg, 1.1 mmol) in ether (8 mL) was added diazo-
methane solution at 0 ℃ until the light yellow color
persisted. The reaction mixture was stirred at room
temperature overnight. Solvent was removed, and the
residue was purified by silica gel flash chromatography
(hexanes∶EtOAc＝4∶1) to afford compound 2 (322
(3R,5R)-tert-Butyl-5-benzyl-3-methyl-2-oxopyrroli-
dine-1-carboxylate (17) To a stirred solution of com-
pound 6 (96 mg, 0.33 mmol) in ethyl acetate (2 mL)
was added 10% Pd/C (25 mg). The mixture was evacu-
ated and refilled with H2 for 3 times, then kept under 1
atm H2 (ballon) for 24 h. The mixture was filtered
through a short silica-gel column. Organic solvent was
removed, the residue was purified by flash silica gel
chromatography (hexanes∶EtOAc＝10∶1) to afford a
colorless product 17 (72 mg, 74%). [α]2D8 ＋81.9 (c 1.1,
1
mg, 95%). [α]2D4 ＋14.4 (c 1.0, CHCl3); H NMR (400
MHz, CDCl3) δ: 7.35－7.21 (m, 5H), 4.45－4.43 (m,
1H), 3.93－3.73 (m, 1H), 3.71 (s, 3H), 2.82 (d, J＝6.0
Hz, 1H), 2.66－2.42 (m, 1H), 1.94－1.90 (m, 1H), 1.41
(s, 9H), 1.41－1.38 (m, 1H), 1.20 (d, J＝7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ: 176.8, 155.2, 137.9,
129.5, 128.3, 126.4, 79.0, 51.7, 49.7, 41.5, 38.0, 36.3,
28.4, 17.6; FT-IR (film) ν: 3371, 2977, 1737, 1713,
1
CHCl3); H NMR (400 MHz, CDCl3) (major isomer) δ:
7.31－7.16 (m, 5H), 4.22－4.16 (m, 1H), 3.46 (dd, J＝
12.8, 3.2 Hz, 1H), 2.59 (dd, J＝12.8, 9.6 Hz, 1H),
2.49－2.43 (m, 1H), 2.15－2.07 (m, 1H), 1.59 (s, 9H),
1.40－1.33 (m, 1H), 1.15 (d, J＝7.2 Hz, 3H); 13C NMR
(100 MHz, CDCl3) (major isomer) δ: 177.1, 150.4,
44
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 40—48

Stereoselective Total Synthesis of Tubulysin V
1518, 1498, 1456, 1366, 1254, 1172, 701 cm−1; ESI-MS:
[M＋Na]＋ 344; ESI-HRMS: [M＋Na]＋ calcd for
C18H27NO4Na 344.1824, found 344.1832.
Solvent was concentrated in vacuo and the residue was
purified by silica gel flash chromatography (hexanes∶
EtOAc＝2∶1) to afford diol (326 mg). To a stirred so-
lution of the above diol (326 mg, 1.3 mmol) in THF (7
mL) and water (7 mL) was added sodium periodate (430
mg, 2.0 mmol) at 0 ℃, the reaction mixture was al-
lowed to warm to ambient temperature and stirred over-
night. The mixture was extracted with ethyl acetate,
neutralized with sodium bisulfate, washed with brine
and dried over Na2SO4. Solvents were concentrated in
vacuo and the residue was purified by silica gel flash
chromatography (hexanes∶EtOAc＝10∶1) to give
compound 10 (250 mg, 78% for 2 steps). [α]2D6 −55.2 (c
1.0, CHCl3); (literatre: [α]2D5 −61.0 (c 1.2, CHCl3));[10]
1H NMR (400 MHz, CDCl3) δ: 9.67 (s, 1H), 4.81 (d,
J＝8.8 Hz, 1H), 3.86－3.85 (m, 1H), 2.54－2.37 (m,
2H), 1.80－1.72 (m, 1H), 1.35 (s, 9H), 0.86 (d, J＝6.8
Hz, 6H); 13C NMR (100 MHz, CDCl3) δ: 201.5, 155.5,
79.2, 51.4, 46.6, 32.1, 28.3, 18.9, 18.2.
Methyl-2-((1R,3R)-1-acetoxy-3-((tert-butoxycar-
bonyl)amino)-4-methylpentyl)thiazole-4-carboxylate
(20) To a stirred solution of BF3•Et2O (0.20 mL, 1.2
mmol) in THF (2.5 mL) in dry-ice bath was added
compound 10 (131 mg, 0.61 mmol) in THF (0.2 mL).
The mixture was stirred for 10 min at −78 ℃. Simulta-
neously, thioacetic acid 12 (47 μL, 0.61 mmol) in THF
(0.2 mL) and 3-(dimethylamino)-2-isocyano-acrylate
(11) (94 mg, 0.61 mmol) in THF (0.2 mL) were added
using a double syringe pump over a period of 30 min.
The reaction mixture was stirred at −78 ℃ for 2 h, then
warmed to ambient temperature, and stirred overnight.
The reaction was quenched with water (10 mL) and
Et2O (10 mL) was added, the resulting mixture was
stirred for 30 min. Aqueous layer was extracted with
ether. The combined organic layers were washed with
5% aqueous citric acid, saturated NaHCO3 solution,
brine, and dried over Na2SO4. The solvents were con-
centrated and the crude product was purified carefully
by silica gel flash chromatography (hexanes∶EtOAc＝
4∶1) to afford desired product 20 (37 mg) along with
diastereomeric epi-20 (90 mg) in 54% overall yield.
[α]2D5 ＋11.5 (c 1.7, CHCl3); 1H NMR (400 MHz,
CDCl3) δ: 8.14 (s, 1H), 6.13 (dd, J＝11.2, 2.8 Hz, 1H),
4.41 (d, J＝10.4 Hz, 1H), 3.95 (s, 3H), 3.80－3.73 (m,
1H), 2.18 (s, 3H), 2.05－2.01 (m, 1H), 1.89 (br s, 1H),
1.76－1.68 (m, 1H), 1.42 (s, 9H), 0.93 (d, J＝6.8 Hz,
3H), 0.91 (d, J＝6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 171.9, 170.0, 161.6, 155.7, 146.8, 127.5, 79.3,
70.1, 52.5, 51.3, 38.1, 32.8, 28.3, 20.8, 19.1, 17.8.
epi-20: [α]2D5 −27.7 (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3) δ: 8.16 (s, 1H), 6.10 (t, J＝6.4 Hz, 1H), 4.37 (d,
J＝9.2 Hz, 1H), 3.88 (s, 3H), 3.54－3.49 (m, 1H),
2.34－2.33 (m , 1H), 2.09 (s, 3H), 2.14－2.05 (m, 1H),
1.76－1.69 (m, 1H), 1.39 (s, 9H), 0.83 (d, J＝6.8 Hz,
3H), 0.80 (d, J＝6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 170.2, 169.7, 161.6, 155.3, 146.9, 128.0, 79.0,
71.0, 59.5, 52.4, 36.7, 32.3, 28.3, 20.9, 18.7, 17.4.
(S)-2-Methyl-N-((R)-2-methylhex-5-en-3-yl)pro-
pane-2-sulfinamide (13) To a stirred solution of 14
(175 mg, 1.0 mmol) in saturated aqueous sodium bro-
mide solution (10 mL) were added indium powder (460
mg, 4.0 mmol) and allylic bromide (0.33 mL, 4.0 mmol).
The reaction mixture was stirred overnight. Saturated
aqueous sodium bisulfate solution was added to quench
the reaction, the mixture was extracted with ethyl ace-
tate, and dried over anhydrous sodium sulfate. Solvent
was concentrated in vacuo. The crude product was puri-
fied by silica gel flash chromatography (hexanes∶
EtOAc＝4∶1) to afford compound 13 (165 mg, 76%).
1
[α]2D3 ＋75.2 (c 1.3, CHCl3) 92% de; H NMR (400
MHz, CDCl3) δ: 5.69－5.61 (m, 1H), 5.01 (d, J＝6.0 Hz,
1H), 4.99 (s, 1H), 3.06 (d, J＝4.8 Hz, 1H), 3.01－2.96
(m, 1H), 2.23－2.22 (m, 1H), 2.17－2.13 (m, 1H),
1.75－1.69 (m, 1H), 1.07 (s, 9H), 0.76 (d, J＝1.2 Hz,
3H), 0.75 (d, J＝1.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 134.4, 118.3, 59.7, 55.6, 36.7, 30.8, 22.4,
18.2, 17.6.
(R)-tert-Butyl(2-methylhex-5-en-3-yl)carbamate
(19) To a stirred solution of compound 13 (55 mg,
0.25 mmol) in anhydrous MeOH (2 mL) in an ice-water
bath was added hydrochloric acid (0.2 mL, 8.4 equiv. in
dioxane), the resulting mixture was stirred for 2 h. Sol-
vent was concentrated in vacuo, the crude product was
used in next step without further purification. To a
stirred solution of the above crude product in CH2Cl2 (2
mL) was added saturated aqueous sodium bisulfate so-
lution (1 mL) and Boc2O (80 mg, 0.36 mmol). The reac-
tion mixture was stirred at ambient temperature over-
night. The crude product was extracted with CH2Cl2,
washed with brine and dried over Na2SO4. Solvent was
concentrated in vacuo and the crude product was puri-
fied by silica gel flash chromatography (hexanes∶
EtOAc ＝ 10 ∶ 1) to provide 19 (43 mg, 80%).
[α]2D4 −34.4 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3)
δ: 5.78－5.67 (m, 1H), 5.03－4.98 (m, 2H), 4.35－4.33
(m, 1H), 3.45－3.44 (m, 1H), 2.22－2.17 (m, 1H), 2.09
－2.02 (m, 1H), 1.71－1.68 (m, 1H), 1.38 (s, 9H), 0.87
(d, J＝6.8 Hz, 3H), 0.83 (d, J＝6.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ: 155.8, 135.0, 117.0, 78.7, 55.0,
36.9, 31.3, 28.30, 27.3, 19.2, 17.6; FT-IR (film) ν: 3319,
2979, 1678, 1539, 1367, 1178, 910, 676 cm−1; ESI-MS:
[M＋Na]＋ 236; ESI-HRMS: [M＋Na]＋ calcd for
C12H23NO2Na 236.1623, found 236.1621.
(R)-tert-Butyl(4-methyl-1-oxopentan-3-yl)carba-
mate (10) To a solution of compound 19 (318 mg, 1.5
mmol) in acetone (13.5 mL) and water (1.5 mL) was
added osmium tetraoxide (0.031 mmol, 5.0 mg/mL in
water, 1.6 mL) and NMO (443 mg, 3.8 mmol) at 40 ℃.
The resulting mixture was stirred at room temperature
for 34 h. Sodium thiosulfate was added to quench the
reaction. The crude product was extracted with ethyl
acetate, washed with brine, and dried over Na2SO4.
Chin. J. Chem. 2013, 31, 40—48
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
45

Wang, Tian & Lin
FULL PAPER
1
2-((1R,3R)-3-(tert-Butoxycarbonylamino)-1-hy-
droxy-4-methylpentyl)thiazole-4-carboxylic acid (3)
To a stirred solution of 20 (90 mg, 0.23 mmol) in
tetrahydrofuran (2 mL) and water (1 mL) was added
NaOH (18 mg, 0.45 mmol) at ambient temperature. The
reaction mixture was stirred for 24 h. 5% potassium
bisulfate was added to adjust pH＝3, the mixture was
extracted with CH2Cl2, washed with brine, and dried
over Na2SO4. Solvents were concentrated in vacuo to
afford a white solid 3 (80 mg, 100%), which was used in
next step without further purification.
(147 mg, 77%). [α]D25 ＋11.2 (c 0.6, CHCl3); H NMR
(400 MHz, CDCl3) δ: 8.01 (s, 1H), 7.22－7.20 (m, 5H),
7.30 (d, J＝4.4 Hz, 1H), 6.05 (d, J＝10.8 Hz, 1H),
4.39－4.37 (m, 2H), 3.83－3.75 (m, 1H), 3.63 (s, 3H),
2.96－2.87 (m, 2H), 2.65－2.55 (m, 1H), 2.17 (s, 3H),
2.05－1.62 (m, 5H), 1.43 (s, 9H), 1.16 (d, J＝7.2 Hz,
3H), 0.97－0.93 (m, 6H); 13C NMR (100 MHz, CDCl3)
δ: 176.8, 170.5, 170.1, 160.4, 155.6, 150.0, 137.6, 129.5,
128.4, 123.3, 79.4, 69.7, 51.7, 48.4, 41.2, 37.7, 36.5,
32.7, 29.5, 28.4, 26.7, 20.6, 19.0, 17.5; FT-IR (film) ν:
3338, 3027, 2924, 1710, 1663, 1541, 1367, 1220, 1171,
1085, 1042, 741, 605 cm−1; ESI-MS: [M＋H]＋ 590;
ESI-HRMS: [M ＋ H] ＋ calcd for C30H43N3O7SNa
612.2715, found 612.2714.
(2S,4R)-Methyl-4-(2-((1R,3R)-3-(tert-butoxycarbonyl-
amino)-1-hydroxy-4-methylpentyl)thiazole-4-carbox-
amido)-2-methyl-5-phenylpentanoate (21)
To a
stirred solution of compound 2 (2.0 g, 6.2 mmol) in
CH2Cl2 (32 mL) in an ice-water bath was added
trifluoroacetic acid (8 mL). The reaction mixture was
warmed to ambient temperature and stirred at this tem-
perature for 4 h. Solvents were removed and the crude
amino acid salt was used in next step without further
purification. To a stirred solution of compound 3 (91 mg,
0.24 mmol) in DMF (2 mL) were added HATU (114 mg,
0.30 mmol), HOAt (40 mg, 0.29 mmol) and NEt3 (74 μL,
0.53 mmol) at 0 ℃. The resulting mixture was stirred
for additional 5 min. The above amino acid salt pre-
pared from compound 2 (100 mg, 0.39 mmol) in DMF
(2 mL) was added and the mixture was warmed to am-
bient temperature and stirred at this temperature for 24 h.
Upon completion of the reaction, the mixture was ex-
tracted with ether. The organic layer was washed with
1.0 mol/L hydrochloric acid, saturated NaHCO3, brine,
and dried over Na2SO4. Organic solvent was concen-
trated in vacuo and the crude product was purified by
silica gel flash chromatography (hexanes∶EtOAc＝2∶
1) to afford 21 (95 mg, 72%). [α]2D4 ＋10.4 (c 1.5,
(2S,4R)-Methyl-4-(2-((6S,9R,11R)-6-sec-butyl-9-
isopropyl-2,2-dimethyl-4,7,13-trioxo-3,12-dioxa-5,8-
diazatetradecan-11-yl)thiazole-4-carboxamido)-2-
methyl-5-phenylpentanoate (23) A solution of com-
pound 22 (22 mg, 0.037 mmol) in hydrochloric acid (2
mL, 8.0 equiv. in dioxane) was stirred at 0 ℃ for 2 h.
The solvent was concentrated in vacuo to provide the
corresponding amino acid salt as a white solid, which
was used in next step without further purification. To a
stirred solution of Boc-isoleucine 4 (22 mg, 0.095 mmol)
and HATU (16 mg, 0.042 mmol) in CH2Cl2 (1 mL) at 0
℃ was added DIPEA (11 μL, 0.067 mmol), the result-
ing mixture was stirred for 10 min at the same tempera-
ture. The above crude amino acid salt prepared from
compound 22 (20 mg, 0.038 mmol) in CH2Cl2 (1 mL)
was injected into the mixture, and the resulting mixture
was stirred overnight. The reaction was quenched by
adding 5% sodium bisulfate in an ice-water bath, the
residue was extracted with CH2Cl2, washed with water,
saturated bisulfate solution and brine, dried over anhy-
drous Na2SO4. Solvent was removed and the residue
was purified by silica gel flash chromatography to pro-
vide a colorless oil 23 (20 mg, 76%), which was a mix-
ture of two diastereomers in 5∶1 ratio (by LC-MS).
1
CHCl3); H NMR (400 MHz, CDCl3) δ: 8.04 (s, 1H),
7.29－7.24 (m, 5H), 7.12 (d, J＝9.2 Hz, 1H), 5.27 (s,
1H), 4.90 (d, J＝10.8 Hz, 1H), 4.65 (d, J＝8.8 Hz, 1H),
4.43－4.42 (m, 1H), 3.77－3.70 (m, 1H), 3.65 (s, 3H),
2.98－2.88 (m, 2H), 2.62 (m, 1H), 2.07－1.96 (m, 2H),
1.87－1.81 (m, 2H), 1.62－1.55 (m, 1H), 1.49 (s, 9H),
1.18 (d, J＝6.8 Hz, 3H), 1.03 (d, J＝6.0 Hz, 6H); 13C
NMR (100 MHz, CDCl3) δ: 176.7, 175.5, 160.9, 157.9,
149.7, 137.6, 129.7, 128.4, 126.5, 123.0, 80.5, 68.8,
52.3, 51.7, 48.2, 41.6, 41.1, 38.6 (37.8), 36.5, 32.3, 29.7
(28.4), 19.4, 18.4 (17.8).
1
[α]2D6 −2.9 (c 0.9, CHCl3); H NMR (400 MHz, CDCl3)
(major) δ: 8.04 (s, 1H), 7.30－7.19 (m, 5H), 6.20－6.12
(m, 1H), 6.00－5.92 (m, 1H), 5.05－4.95 (m, 1H), 4.45
－4.41 (m, 1H), 4.20－4.10 (m, 1H), 3.90－3.80 (m,
1H), 3.65 (s, 3H), 3.48－3.40 (m, 1H), 3.04－2.88 (m,
2H), 2.70－2.60 (m, 1H), 2.20 (s, 3H), 2.10－1.96 (m,
6H), 1.68－1.58 (m, 2H), 1.46 (s, 9H), 1.20－1.18 (d,
J＝7.6 Hz, 3H), 0.98－0.96 (m, 12H); 13C NMR (100
MHz, CDCl3) (major) δ: 176.6, 171.8, 170.0, 169.9,
160.4, 156.1, 150.1, 137.7, 129.5, 128.4, 126.5, 123.4,
80.1, 69.7, 59.8, 51.6, 50.0, 48.6, 42.0, 41.3, 38.6, 37.8,
36.5, 32.2, 28.3, 26.5, 24.7, 20.8, 19.1, 17.7, 15.8, 11.0.
(R)-Benzyl-2-((2S,3S)-1-((1R,3R)-1-acetoxy-1-(4-
((2R,4S)-5-methoxy-4-methyl-5-oxo-1-phenylpentan-
2-ylcarbamoyl)thiazol-2-yl)-4-methylpentan-3-yl-
amino)-3-methyl-1-oxopentan-2-ylcarbamoyl)piperi-
dine-1-carboxylate (24) A solution of compound 23
(1.3 g, 1.8 mmol) in hydrochloric acid (4 mL, 8.0 equiv.
in dioxane) in an ice-water bath was stirred for 2 h. Or-
ganic solvent was concentrated in vacuo to give the
(2S,4R)-Methyl-4-(2-((1R,3R)-1-acetoxy-3-(tert-
butoxycarbonylamino)-4-methylpentyl)thiazole-4-
carboxamido)-2-methyl-5-phenylpentanoate
(22)
To a stirred solution of compound 21 (177 mg, 0.32
mmol) in anhydrous pyridine (2 mL) was added acetic
anhydride (1.0 mL) and the resulting mixture was
stirred for 12 h. Water was added to quench the reaction,
organic solvent was concentrated in vacuo, the crude
mixture was extracted with ethyl acetate, washed with
brine, and dried over Na2SO4. Solvent was concentrated
in vacuo and the residue was purified by silica gel flash
chromatography (hexanes∶EtOAc＝4∶1) to give 22
46
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 40—48

Stereoselective Total Synthesis of Tubulysin V
crude amino acid salt as a white solid (930 mg), which
was used in next step without further purification. To a
stirred solution of N-Cbz picolinic acid 5 (620 mg, 2.4
mmol) in CH2Cl2 (2 mL) were added HATU (900 mg,
2.4 mmol) and NMM (350 μL, 3.1 mmol) in an ice-
water bath. The mixture was stirred for additional 30
min, then the above crude amino acid salt prepared from
compound 23 (930 mg, 1.5 mmol) in CH2Cl2 (2 mL)
was injected. The reaction mixture was warmed to am-
bient temperature overnight. Water was added to quench
the reaction, the organic layer was subsequently washed
with 5% KHSO4, H2O, sat. NaHCO3, H2O, brine, and
dried over Na2SO4. The crude product was purified by
silica gel flash chromatography (hexanes∶EtOAc＝2∶
1) to afford a white solid 24 (817 mg, 66%). [α]D25
4.4 Hz, 3H), 3.54 (s, 1H), 2.97－2.87 (m, 3H), 2.70－
2.50 (m, 2H), 2.20 (s, 6H), 2.00－1.40 (m, 7H), 1.40－
1.00 (m, 10H), 1.00－0.88 (m, 15H); 13C NMR (125
MHz, CDCl3) δ: 176.6, 175.2, 171.1, 170.1, 169.9,
160.5, 160.4, 150.0, 137.7, 129.5, 128.4, 128.4, 126.5,
123.3, 69.8, 69.6, 57.9, 55.4, 51.8, 50.0, 48.6, 48.5, 44.9,
41.9, 41.3, 37.8, 37.6, 37.5, 36.9, 36.4, 34.8, 32.2, 31.9,
30.8, 29.7, 29.3, 25.1, 24.9, 23.3, 22.7, 20.8, 18.9;
FT-IR (film) ν: 3292, 2925, 2854, 1737, 1652, 1541,
1260, 1224, 737, 702 cm−1; ESI-MS: [M＋H]＋ 728;
ESI-HRMS: [M ＋ H] ＋ calcd for C38H57N5O7SNa
728.4039, found 728.4052.
(2S,4R)-4-(2-((1R,3R)-1-Hydroxy-4-methyl-3-((2S,
3S)-3-methyl-2-((R)-1-methylpiperidine-2-carbox-
amido)pentanamido)pentyl)thiazole-4-carboxamido)-
2-methyl-5-phenylpentanoic acid (1e) To a stirred
solution of compound 25 (20 mg, 0.027 mmol) in THF
(1 mL) in an ice-water bath was added LiOH (1.0 mol/L,
0.5 mL). The mixture was warmed to ambient tempera-
ture and stirred for 3 d. Organic solvent was removed
and 20% aqueous trifluoroacetic acid was slowly added
to acidify the mixture at 0 ℃. The mixture was diluted
with ethyl acetate, then washed with brine and dried
over Na2SO4. Solvent was removed and the residue was
purified by preparative TLC to afford a mixture of two
diastereomers (13.5 mg), which was further purified by
prepative HPLC to provide pure tubulysin V (1e) as a
white solid (10 mg, 54%). [α]2D6 −11.9 (c 0.73, MeOH);
(literature: [α]2D3 −11.4 (c 0.70, MeOH));[4b] 1H NMR
(500 MHz, CD3OD) δ: 8.02 (s, 1H), 7.231－7.15 (m,
5H), 4.38－4.33 (m, 1H), 4.21 (d, J＝8.5 Hz, 1H), 4.08
－4.05 (m, 1H), 3.75 (d, J＝9.5 Hz, 1H), 3.47 (d, J＝
12.0 Hz, 1H), 3.07 (t, J＝13.0 Hz, 1H), 2.88 (d, J＝7.0
Hz, 2H), 2.76 (s, 3H), 2.57－2.50 (m, 1H), 2.31 (s, 1H),
2.16－2.07 (m, 2H), 2.03－1.98 (m, 1H), 1.94－1.74
(m, 7H), 1.64－1.54 (m, 3H), 1.32－1.19 (m, 6H), 1.16
(d, J＝7.0 Hz, 3H), 0.99 (d, J＝7.0 Hz, 3H), 0.96 (d,
J＝1.5 Hz, 3H), 0.95 (d, J＝1.5 Hz, 3H), 0.91 (t, J＝7.5
Hz, 3H); 13C NMR (125 MHz, CD3OD) δ: 180.1, 179.5,
173.8, 169.5, 163.5, 151.1, 139.7, 130.8, 130.2, 129.6
(129.5), 127.7, 126.6, 124.6, 70.1, 68.6, 60.4, 56.5, 53.1,
50.9, 43.3, 42.5, 41.1, 39.5, 38.1, 37.9, 34.0, 31.0, 30.5,
26.2, 24.3, 22.7, 19.8, 18.8, 16.4, 11.5; FT-IR (film) ν:
3381, 2963, 1742, 1687, 1515, 1257, 1223, 1174, 767
cm−1; ESI-MS: [M＋H]＋ 672; ESI-HRMS: [M＋H]＋
calcd for C35H54N5O6S 672.3791, found 672.3789.
1
＋12.1 (c 0.6, CHCl3); H NMR (500 MHz, CDCl3) δ:
8.02 (s, 1H), 7.36－7.15 (m, 13H), 6.45－6.43 (m, 1H),
5.93 (d, J＝8.8 Hz, 1H), 5.18－5.14 (m, 3H), 4.82 (d,
J＝2.4 Hz, 1H), 4.50－4.40 (m, 1H), 2.89 (s, 1H), 2.99
－2.80 (m, 4H), 2.70－2.58 (m, 1H), 2.19 (s, 3H), 2.18
－1.80 (m, 5H), 1.80－1.59 (m, 10H), 0.95－0.87 (m,
16H); 13C NMR (125 MHz, CDCl3) δ: 177.2, 176.6,
171.5, 171.0, 169.9, 160.5, 160.3, 156.6, 150.1, 149.9,
136.7, 136.2, 129.5, 128.6, 128.4, 128.4, 128.2, 127.9,
126.5, 123.4, 123.4, 69.7, 67.8, 60.4, 58.5, 6.11, 51.7,
51.6, 50.2, 48.5, 42.5, 42.0, 41.2, 37.8, 37.6, 37.4, 36.8,
36.4, 35.5, 35.4, 32.30, 29.7, 25.7, 24.6, 21.0, 20.8, 20.3,
19.1; FT-IR (film) ν: 3324, 2961, 2927, 1652, 1541,
1227, 700 cm−1; ESI-MS: [M＋H]＋ 848; ESI-HRMS:
[M＋Na]＋ calcd for C45H61N5O9SNa 870.4078, found
870.4082.
(2S,4R)-Methyl-4-(2-((1R,3R)-1-acetoxy-4-methyl-
3-((2S,3S)-3-methyl-2-((R)-1-methylpiperidine-2-
carboamido)pentanamido)pentyl)thiazole-4-carbox-
amido)-2-methyl-5-phenylpentanoate (25)
To a
stirred solution of compound 24 (11 mg, 0.013 mmol) in
anhydrous CH2Cl2 (1 mL) at 0 ℃ was added TMSI (3
μL, 0.029 mmol). The resulting mixture was stirred at
this temperature for 6 h. 5% KHSO4 was added care-
fully to quench the reaction, the organic phase was sub-
sequently washed with water, sat. NaHCO3, water, brine,
and dried over Na2SO4. Solvent was removed and the
crude product was purified by silica gel flash chroma-
tography (hexanes∶EtOAc＝1∶1) to afford a white
solid (10 mg) which was directly used in next step. To a
stirred solution of the above product (10 mg, 14 μmol)
in ethyl acetate (1 mL) was added acetic acid (125 μL,
2.2 mmol), paraformaldehyde (2 mg) and sodium tri-
acetoxyborohydride (30 mg, 0.20 mmol) at ambient
temperature. The mixture was stirred for 3 d, then care-
fully quenched with sat. NaHCO3. The organic layer
was washed with brine and dried over Na2SO4. Solvent
was removed and the residue was purified by silica gel
flash chromatography (hexanes∶EtOAc＝1∶1) to af-
ford a white solid 25 (6.8 mg, 68% for 2 steps).
Acknowledgement
Financial support from the Major State Basic Re-
search Development Program (No. 2010CB833206), the
Chinese Academy of Sciences (No. 2008DP173071)
and the State Key Laboratory of Bio-organic and Natu-
ral Products Chemistry (Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences) is gratefully
acknowledged. We also thank Dr. Hanqing Dong (OSI
Pharmaceuticals, USA) for helpful discussion in prepa-
ration of this manuscript.
1
[α]2D4 −0.3 (c 0.2, MeOH); H NMR (500 MHz, CDCl3)
δ: 8.02 (s, 1H), 7.29－7.19 (m, 5H), 5.94－5.91 (m, 1H),
4.50－4.40 (m, 1H), 4.12－4.09 (m, 2H), 3.62 (d, J＝
Chin. J. Chem. 2013, 31, 40—48
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
47

Wang, Tian & Lin
FULL PAPER
Morita, N.; Masu, H.; Azumaya, I.; Tamura, O. Chem. Eur. J. 2010,
16, 11678; (o) Shibue, T.; Hirai, T.; Okamoto, I.; Morita, N.; Masu,
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