Total synthesis of tubulysin U and V

Article abstract of DOI:10.1002/anie.200601259

Multicomponent method: Tubulysins are among the most potent cytotoxic agents known. Now the first total synthesis of some members has been achieved by utilizing a rapid three-component reaction for the synthesis of the unusual central thiazole amino acid

Full text of DOI:10.1002/anie.200601259

Angewandte
Chemie
Total Synthesis
DOI: 10.1002/anie.200601259
Total Synthesis of Tubulysin U and V**
Alexander Dömling,* Barbara Beck, Uwe Eichelberger,
Sukumar Sakamuri, Sanjay Menon, Quin-Zene Chen,
Yingchun Lu, and Ludger A. Wessjohann*
In memory of Ivar Ugi
Tubulysins (1) are compounds of extraordinary potency,
rapidly degrading the tubulin cytoskeleton, with tubulysin D
being the most active tubulin-modifier known so far.[1,2] The
tubulysins were first described by Höfle, Reichenbach, and
co-workers in 2000.[3,4] Several representatives are active with
GI50 values (growth inhibition of 50%) in the low picomolar
range against the NCI-60 cancer cell-line panel, and some are
highly antiangiogenic.[5] Semisynthetic tubulysins, derived
from isolated material, show promising in vivo anticancer
properties and are candidates for antibody conjugates. Thus,
tubulysins are attractive leads as novel anticancer agents.[5]
However, so far, tubulysins can only be produced by a
fermentation process that yields less than 10 mgLÀ1 by several
rather tedious chromatographic purification steps. Thus, and
[*] Priv.-Doz. Dr. A. Dömling, Dr. B. Beck
R&D Biopharmaceuticals GmbH
Am Klopferspitz 19a, 82152 Martinsried (Germany)
Fax: (+49)89-7007-6499
E-mail: alexander.doemling@rdbiopharma.de
Dr. U. Eichelberger, Prof. L. A. Wessjohann
Leibniz-Institut für Pflanzenbiochemie
Abteilung für Natur- und Wirkstoffchemie
Weinberg 3, 06120 Halle (Germany)
Fax: (+49)345-5582-1309
E-mail: wessjohann@ipb-halle.de
Dr. B. Beck, Dr. S. Sakamuri, Dr. S. Menon, Dr. Q.-Z. Chen, Dr. Y. Lu
Morphochem Inc.
11 Deer Park Drive, Suite 116, Monmouth Junction, NJ 08852 (USA)
[**] U.E. and L.A.W. acknowledge support from the State of Saxony-
Anhalt as part of a HWP grant. We thank Dr. A. Porzel for special
NMR analysis and Prof. G. Höfle for providing reference spectra and
preliminary activity and isolation information.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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in order to reveal the structure–activity relationship for these
compounds, a total synthesis is highly desirable.
properly connect either the Ile or Mep residues to the rest of
the molecule.
Tubulysins are structurally related to the marine natural
product class of dolastatins and the anticancer drug
LU 103793. Similar to dolastatins 10[6] and 15,[7] tubulysins
are unusual peptides, mainly composed of the uncommon or
hydrophobic amino acids N-methylpipecolic acid (Mep),
isoleucine (Ile), tubuvaline (Tuv), and tubuphenylalanine
(Tup) or tubutyrosine (Tut), with an N- to C-terminal distance
of ꢀ 18 Š, an aromatic amino acid at the C terminus, and a
tertiary amino terminus. Two major series of tubulysins can be
distinguished (Table 1). Those with labels from the beginning
Herein we report the first stereoselective total synthesis of
two members of the tubulysin family. A retrosynthetic
analysis of the “pentapeptidic” tubulysin U (1u, Scheme 1)
Table 1: Selected members of the tubulysin family.[a] [8]
Tubulysin
R1
R2
R3
CH2O (CO) CH2CH(CH3)2
À
À
A
…
D
E
OH
Ac
À
À
H
H
H
H
Ac
Ac
Ac
Ac
CH2O (CO) CH2CH(CH3)2
À
À
Scheme 1. Retrosynthesis of tubulysin U (1u). LG=leaving group,
EWG=electron-withdrawing group, Aux*=chiral auxiliary, MCR=mul-
ticomponent reaction.
CH2O (CO) CH2CH2CH3
À
À
F
CH2O (CO) CH2CH3
À
À
G
…
U
V
CH2O (CO) CH3
H
H
Ac
H
H
H
leads to the four amino acids Mep, Ile, Tuv, and Tup, of which
Tuv is biosynthetically derived from valine, an acetate
equivalent, and cysteine as a hidden fifth amino acid. For
the chemical synthesis, we introduced two disconnections to
yield three equally complex and large substructures. For the
C-terminal moiety, the stereocontrolled alkylation of azir-
idine 7 by using a chiral auxiliary (such as 8) appeared most
useful. Tubuvaline, the complex-looking centerpiece of all
tubulysins, resolves unexpectedly into a set of three easily
available building blocks (4–6) if one considers a recently
discovered thiazole synthesis,[12] an isonitrile-based multi-
component reaction (MCR) based on the multiple reactivity
of Schöllkopf type isonitriles, 6.[13] The N terminus contains
the commercially available amino acids pipecolinic acid and
Ile. When earlier attempts toward the synthesis of tubulysins
are considered, the formation of the Ile–Tuv amide bond is
expected to constitute the most severe problem. According to
the established C- to N-terminus peptide-assembly routine,
the successive coupling of Ile and Mep should be preferred,
due to the lower propensity for racemization. However,
coupling of a preformed Mep–Ile dipeptide is more con-
vergent and may be more attractive if racemization is not an
issue or if the central Ile–Tuv amide bond formation reaction
gives only a low yield.
…
Z
OH
H
H
[a] Mep=N-methylpipecolinic acid, Ile=isoleucine, Tuv=tubuvaline,
Tup =tubuphenylalanine, Tut=tubutyrosine.
of the alphabet (for example, the tubulysin A or D series, with
Tut or Tup, respectively) have both the Tuv hydroxy group
(R2) and an N,O-acetal involving the amide nitrogen atom of
tubuvaline (R3) acylated. The series with labels from later in
the alphabet (for example, tubulysins U–Z) has these two
functions partially or completely missing (Table 1).[9] Usually,
this latter series is less active, but the members studied so far
(tubulysins W–Z) reach the activity range of, for example,
taxol or the epothilones.
Despite several attempts, no complete synthesis of a
tubulysin has been described so far.[10,11] The total synthesis is
challenging for several reasons. Only one amino acid of the
tetrapeptide (Ile) occurs naturally as a separate compound.
The central fragment, Tuv, constitutes a complex, thiazole-
containing amino acid with two stereocenters. In the series
labeled with earlier alphabet letters, the bis-acyl N,O-acetal at
Tuv makes a total synthesis more difficult, since this func-
tional group is not compatible with most protecting-group
strategies. The C-terminal amino acid, Tup, constitutes a g-
amino acid comprising two stereocenters as well. Most
importantly, the middle part of all tubulysins is sterically
highly crowded, and peptide-bond formation often fails to
According to the retrosynthesis, tubuphenylalanine (3) is
available from commercial (S)-N-Ts-2-benzylaziridine (7a;
Ts = toluene-4-sulfonyl), by ring opening with a propanoyl
anion under the control of pseudoephedrine as a chiral
auxiliary connected to propionic acid, as in amide 10
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(Scheme 2).[14,15] The desired syn diastereomer is formed in
70% relative yield and is separated from the minor anti
isomer by chromatography. Amide hydrolysis and removal of
the tosylate paves the way to Tup and its various protected
Scheme 3. One-pot synthesis of protected l-tubuvaline by a new MCR
to form 2-hydroxymethyl thiazoles. a) 1. BF3·Et2O, Boc-l-homovaline
aldehyde, THF, À788C; 2. simultaneous addition of thioacetic acid and
Schöllkopf isonitrile in THF by syringe pump over 30 min; then room
temperature overnight; 40%, 3:1, major isomer separated; b) NaOH,
THF/H2O (3:1), 62%.
catalysis (see below), as has been demonstrated with tubuly-
sin A.[8]
d-Mep-l-Ile is the third major building block (Scheme 4).
The commercial isoleucine 4-nitrophenyl (pNP) ester, Boc-
Scheme 2. Synthesis of differently protected tubuphenylalanine (Tup)
derivatives by the pseudoephedrine route. a) Propionic anhydride,
Et3N, CH2Cl2, 93%; b) 1. LDA, LiCl, THF, À788C; 2. (S)-(+)-2-benzyl-1-
(toluene-4-sulfonyl)-aziridine, THF, À208C, 86% (81:19 d.r. mixture of
C-2 diastereomers, major isomer shown separated and processed
further); c) 4m H2SO4/dioxane, reflux, 91%; d) MeOH, conc. HCl,
reflux, 85%; e) Boc2O, DMAP, CH3CN, 100%; f) Mg (powder), MeOH,
ultrasound, 70%; g) 4n HCl in dioxane, 100%. Boc=tert-butoxycar-
bonyl, DMAP=4-dimethylaminopyridine, LDA=lithium diisopropyla-
mide, THF=tetrahydrofuran.
Scheme 4. Synthesis of d-Mep–l-Ile as the activated pNP ester. a) HBr
in acetic acid (33%), 08C, 91%; b) 1. N-methyl d-pipecolic acid, Et3N,
CH2Cl2, À158C; 2. ethyl chloroformate, À108C; 3. l-Ile-pNP, À108C,
43%. pNP=4-nitrophenyl.
forms 12–16, of which the Tup methyl ester hydrochloride,
14·HCl, was used for the further synthesis. Racemization was
not observed. Several alternative routes to this building block,
for example, the use of Evans methodology, were tried but did
not lead to the product at all or gave lower yields and
significantly lower de values (see also reference [3]).
19, can be N-deprotected. Activated as a mixed anhydride, d-
Mep[17] reacts chemoselectively and without detectable race-
mization with 19 to give the Mep–Ile dipeptide already
activated at the C terminus. The coordinated levels of
carbonyl activation and careful control of the reaction
temperature save several steps of protection–deprotection–
activation, by utilizing pNP as a carboxyl-protecting moiety in
the first coupling and as an activation moiety in the second,
upcoming amide coupling.
Finally, suitably monoprotected Tup 14 and Tuv 18 were
coupled without racemization with 3-(diethoxyphosphoryl-
oxy)-1,2,3-benzotriazin-4-(3H)-one (DEPBT, Scheme 5).[18]
After Boc deprotection of 22, the second coupling step
(Scheme 5) with Cbz-Ile activated as a pNP-ester, Cbz-19,
also proceeds in very good yield and without racemization.
The subsequent deprotection of the benzyloxycarbonyl group
is clean. However, the final coupling step with d-Mep (20),
activated by various methods (for example, TBTU, 2(-7-aza-
Tubuvaline is available as its Boc-protected methyl ester
17 in a very straightforward one-pot process following a newly
discovered three-component synthesis of 2-hydroxymethyl-
thiazoles (Scheme 3).[12] Thus, Schöllkopf isonitrile (6a,
available in one step from glycine isonitrile),[13] Boc-protected
l-homovaline aldehyde (Boc-4),[16] and thioacetic acid (5)
gave 17 in 40% yield, with a 3:1ratio of diastereomers. These
values constitute considerable improvements over both the
first thiazole MCRs[12] and the alternative routes.[11] The major
diastereomer shows the right configuration at C-11, as
determined by comparison with data for the Cbz-Tuv ethyl
ester (Cbz = benzyloxycarbonyl) published by Wipf et al.[11]
and the original NMR data for tubulysin A.[2] Basic hydrolysis
allows cleavage of the ester to give building block 18, which is
also suitable for the synthesis of tubulysins V and U (see
below). The selective hydrolysis of the methyl ester (R1),
while leaving the acetate (R2) intact, is possible by enzymatic
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Communications
Scheme 5. Assembly of tubulysin U (1u) by prolongation of the N terminus. a) 1. 18, DIPEA, DEPBT, DMF, 08C; 2. 14, DMF, 78%; b) 3-step 1-pot
procedure: 1. TFA, CH2Cl2; 2. d-Mep–l-Ile-pNP (21), Et3N, DMF; 3. Ac2O, pyridine, 83%, 1:1 mixture of diastereomers; c) porcine liver esterase,
phosphate buffer (pH 7.2), 368C, 61%. Alternative route: d) 3-step procedure (b) with Cbz-Ile-pNP (step 2), only one diastereomer detected, 85%;
e) 1. Pd/C/H2, MeOH, 93%; 2. TBTU, DIPEA, DMF, d-Mep, only one diastereomer detected, 10%. DIPEA=diisopropylethylamine, DMF=N,N-
dimethylformamide, TBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, TFA=trifluoroacetic acid
(5, 0.33 mL, 4.64 mmol) in THF (1.5 mL) and a solution of 6a[13]
1H-benzotriazole)-1,1,3,3-tetramethyluronium tetrafluorobo-
(0.72 g, 4.64 mmol) in THF (1.5 mL) were added simultaneously by
using a double-syringe pump over a period of 30 min. The reaction
rate (TATU), or 1-hydroxy-1H-benzotriazole (HOBt)), gave
protected tubulysins as complex mixtures with a maximum
mixture was stirred for 1h at À788C, allowed to come to room
10% yield of isolated product. These were separable by GC
temperature, and stirred overnight. The reaction was quenched with
but hardly by preparative LC. This was not satisfactory, and
water (15 mL) and, after addition of Et2O (15 mL), the resulting
thus the more convergent direct coupling of Boc-Tuv–Tup-
Me, 22, with Mep–Ile-pNP, 21, was tried. A one-pot proce-
dure, including amino deprotection of the Tuv–Tup part,
coupling, and reacetylation of the resulting methyl ester of
tubulysin V, 23v, gave the corresponding tubulysin U ester
23u in an excellent 83% yield. Unfortunately, but not
unexpectedly, quantitative epimerization occurs at the a-
carbon atom of Ile during dipeptide transfer (indicated by an
asterisk in Scheme 5). Nevertheless, separation of the 1:1
diasteromeric mixture gives over 40% of the desired tubu-
lysin U ester 23u with the correct stereochemistry; this more
convergent process is thus much more efficient than the less-
racemization-prone stepwise route.
Finally, selective methyl ester hydrolysis in the presence of
the acetate and, most importantly, without epimerization or
other acid- or base-induced deterioration of tubulysin U was
achieved by selective enzymatic hydrolysis with porcine liver
esterase to give the title compound 1u in 61% yield.
Tubulysin V (1v), the deacetylated derivative, is likewise
obtainable by either full ester hydrolysis, and indeed it forms
as a minor byproduct even under controlled enzyme hydrol-
ysis conditions, or by omitting the reacetylation step after the
first coupling.
mixture was stirred for 30 min. After separation of the phases, the
aqueous layer was washed with Et2O (3 ” 10 mL). The combined
organic layers were washed with a solution of citric acid (5%, 2 ”
10 mL), sat. NaHCO3 solution (2 ” 10 mL), and brine (2 ” 10 mL),
dried over Na2SO4, filtered, and concentrated in vacuo. The obtained
oil was purified by flash chromatography on silica (hexanes/EtOAc
7:3) to yield two diastereomers: 17a (0.60 g) and 17b (0.21g) in 43%
combined yield (diastereomeric ratio 3:1). For further data, see the
Supporting Information.
Synthesis of 1u/v by coupling of Tup–Tuv-Me with Mep–Ile-pNP
and selective enzymatic methyl ester hydrolysis:
Synthesis of 23: TFA (300 mL, 1.77 mmol) was added to 22
(66 mg, 0.12 mmol) in CH2Cl2 (3.5 mL). The mixture was stirred for
1h at room temperature and all volatile matter was removed in vacuo.
The residue was dissolved in DMF (3.5 mL) and this was followed by
subsequent addition of Et3N (150 mL, 1mmol) and 21 (60 mg,
0.16 mmol). After stirring for 38 h at room temperature, the solvent
was evaporated under high vacuum (p < 1mbar, 25 8C). The crude
residue was dissolved in pyridine (3.5 mL) and, after addition of
acetic acid (0.7 mL), the mixture was stirred for 3 h at room
temperature. Evaporation of all volatile matter in vacuo yielded the
crude product, which was purified by reversed-phase HPLC (RP-18,
MeOH/H2O/TFA 65:35:0.1). Yield = 71mg (83%).
Synthesis of 1u: A phosphate buffer (2 mL, pH 7.3, 0.02m) was
added to 23 (6 mg, 8.2 mmol) in dimethylsulfoxide (0.15 mL) and the
mixture was vigorously stirred for 5 min. The temperature was kept
constant at 368C and porcine liver esterase (0.15 mL, 250 unitsmgÀ1
)
Overall, we have developed a short, stereoselective, and
convergent route to selected tubulysin family members. The
rapid access will form the basis for further structure–activity
relationship and biological studies of this promising class of
anticancer natural products.
was added. After stirring for 4 h (detection of only one product;
careful TLC control), the mixture was extracted with EtOAc (2 ”
15 mL) and the combined organic extracts were freed of solvent in
vacuo without heating. The crude product was purified by reversed-
phase HPLC (RP-18, MeOH/H2O/TFA 65:35:0.1). Yield = 3.6 mg
(61%) of 1u. Longer treatment with porcine liver esterase yielded
increasing amounts of 1v.
Experimental Section
MCR synthesis of 17: Boc-4 (1g, 4.64 mmol) in THF (1.5 mL) was
added to BF3·Et2O (1.16 mL, 9.29 mmol) in THF (2.5 mL) at À788C.
The resulting mixture was stirred for 5 min at À788C. Thioacetic acid
Received: March 31, 2006
Revised: July 7, 2006
Published online: October 2, 2006
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Keywords: antitumor agents · multicomponent reactions ·
.
natural products · peptide coupling · thiazoles
ˇ
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