Total synthesis and biological evaluation of tubulysin U, tubulysin V, and their analogues

Article abstract of DOI:10.1021/jm8013579

A stereoselective total synthesis of the cytotoxic natural products tubulysin U, tubulysin V, and its unnatural epimer epitubulysin V, is reported. Simplified analogues containing N,N-dimethyl- D-alanine as a replacement for the N-terminal N-Me-pipecolinic acid residue of the tubulysins are also disclosed. Biological evaluation of these natural products and analogues provided key information with regard to structural and stereochemical requirements for antiproliferative activity and tubulin polymerization inhibition.

Full text of DOI:10.1021/jm8013579

238
J. Med. Chem. 2009, 52, 238–240
Chart 1
Total Synthesis and Biological Evaluation of
Tubulysin U, Tubulysin V, and Their
Analogues
Ranganathan Balasubramanian,† Bhooma Raghavan,†
Adrian Begaye,‡ Dan L. Sackett,‡ and Robert A. Fecik*,†
Department of Medicinal Chemistry, 308 HarVard Street SE, 8-101
WeaVer-Densford Hall, UniVersity of Minnesota, Minneapolis,
Minnesota 55455-0353, and Laboratory of IntegratiVe and Medical
Biophysics, National Institute of Child Health and Human
DeVelopment, National Institutes of Health, 9 Memorial DriVe,
Building 9, Room 1E129, Bethesda, Maryland 20892-0924
ReceiVed October 27, 2008
Tuv, and (3) the assembly of the tetrapeptide, as a consequence
of the sterically congested Tuv region.
Abstract: A stereoselective total synthesis of the cytotoxic natural
products tubulysin U, tubulysin V, and its unnatural epimer epi-
tubulysin V, is reported. Simplified analogues containing N,N-dimethyl-
D-alanine as a replacement for the N-terminal N-Me-pipecolinic acid
residue of the tubulysins are also disclosed. Biological evaluation of
these natural products and analogues provided key information with
regard to structural and stereochemical requirements for antiproliferative
activity and tubulin polymerization inhibition.
The total synthesis of tubulysins D,4 U, and V5 and numerous
analogues6 has allowed for determination of structure-activity
relationship (SAR) studies for these natural products. In a report
by Wipf and co-workers, it was established that the Mep residue
at the N-terminus of the tubulysins could be replaced with the
acyclic amino acid N-methylsarcosine.6b This observation was
corroborated by Ellman and co-workers who reported that
incorporation of N-methylsarcosine in the place of Mep provided
a tubulysin analogue with equal antiproliferative activity to
tubulysin D.6c The latter group extended the SAR of this family
of peptides by stating that a basic tertiary amine was required
at the Mep residue for activity, a finding that was confirmed by
our research group.6d,e
The tubulysins (1-7, Chart 1) are a group of extremely potent
tubulin polymerization inhibitors that rapidly disintegrate the
cytoskeleton of dividing cells and induce apoptosis.1 Tubulysins
are produced in very limited quantities by fermentation of the
myxobacteriaAngiococcusdisciformisandArchangiumgephyra.1a,b
Biosynthesis of tubulysins is accomplished by an unusual mixed
nonribosomal peptide synthetase-polyketide synthase
(NRPS-PKS)a system.2 This has led to extensive investigations
into their total synthesis and the synthesis of novel analogues.3-6
The most active natural product known so far in this family is
tubulysin D (2), which retains its anticancer activity against the
multidrug resistant P-glycoprotein-expressing human KB-V1
cervix carcinoma cell line (IC50 ) 0.31 nM).1b This activity
profile makes the tubulysins exciting leads for the treatment of
multidrug resistant cancers.
Structurally, tubulysins are tetrapeptides comprising of N-
methylpipecolinic acid (Mep) at the N-terminus, isoleucine (Ile,
the only proteinogenic amino acid) as the second residue, the
unique thiazole-containing tubuvaline (Tuv) as the third residue,
and two possible γ-amino acids at the C-terminus (tubutyrosine
(Tut) or tubuphenylalanine (Tup)). An acetylated alcohol and
an N,O-acetal in tubuvaline are found in most members of this
natural product family. Tubulysins U-X (3-6) and Z (7) are
the only natural tubulysins in which one or both of these
functional groups are not present. Although these are relatively
simple molecules to envision a total synthesis of, there are a
number of challenging synthetic issues as summarized in a
recent review:3 (1) the installation of the extremely sensitive
N,O-acetal, (2) the synthesis of the complex thiazole-containing
We have recently reported the synthesis and initial biological
activity of a series of simplified tubulysin analogues lacking
the N,O-acetal and having the acetate group of Tuv replaced
with a ketone.6d,e Systematic modifications of the Mep residue
(N-desmethyl, stereochemistry, and ring size) and Tup residue
(desmethyl and dimethyl substituents in place of the stereogenic
R-methyl group) have enabled us to establish the minimal
structural requirements for cytotoxicity. Our simplified ana-
logues having a ketone in the Tuv residue are perfect precursors
to tubulysins U and V (3 and 4). Usually, this latter series of
tubulysins is less active, but tubulysins U and V were reported
to have bioactivity similar to paclitaxel and the epothilones.1b
Herein, we report the total synthesis and biological activity of
tubulysins U and V and a series of simplified analogues in which
the Mep residue has been replaced with N,N-dimethyl-D-alanine.
These data extend our SAR studies of these important natural
products.
Before attempting the reduction of a full-length tetrapeptide
precursor to tubulysin V, we performed a nonstereoselective
NaBH4 reduction on dipeptide 86d (Scheme 1). This reaction
was used as a model to assess the selectivity of reduction and
to establish the stereochemistry of the two alcohol products. A
mixture of alcohols 9 and 10 were obtained with 1:3 selectivity
and were readily separable by chromatography. The stereo-
chemistry of the major product 10 was determined by modified
Mosher analysis7 of the 1H NMR spectrum of MTPA esters 11
and 12, formed by reaction of alcohol 10 with (R)- and (S)-
MTPA-Cl, respectively.
* To whom correspondence should be address. Phone: 612-626-6523.
Fax: 612-624-0139. E-mail: fecik001@umn.edu.
†
University of Minnesota.
National Institute of Child Health and Human Development.
‡
a Abbreviations: Ala, alanine; CBS, Corey-Bakshi-Shibata; Ile, iso-
leucine; Mep, N-methylpipecolinic acid; MTPA, R-methoxy-R-trifluorom-
ethylphenylacetic acid; NRPS, nonribosomal peptide synthetase; PKS,
polyketide synthase; Pip, pipecolinic acid; SAR, structure-activity relation-
ship; Tup, tubuphenylalanine; Tut, tubutyrosine; Tuv, tubuvaline.
We next sought to examine the reduction of tetrapeptide 13 to
give a protected precursor of tubulysin V. Reduction of tetrapeptide
13 with NaBH4 also afforded a mixture of the two diastereomeric
10.1021/jm8013579 CCC: $40.75
2009 American Chemical Society
Published on Web 12/22/2008

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2 239
Scheme 3. Total Synthesis of Tubulysins U and V
Scheme 1. Nonselective Reduction of Dipeptide 8
Scheme 2. Nonstereoselective Reduction of Tetrapeptide 13
Scheme 4. Synthesis of Epi-Tubulysin V
alcohol products 14 with little selectivity (2.4:1, Scheme 2). Each
diastereomer was isolated and individually characterized. The
stereochemistry of each diastereomer was later determined by
spectral comparison to the immediate precursor to tubulysin V.
CBS reduction8 of tetrapeptide 13 gave intractable mixtures, so
we then attempted the reduction of a tripeptide intermediate.
Tripeptide intermediate 15 was found to be optimal for
achieving stereoselective reduction of the Tuv ketone. Reduction
of tripeptide 15 with NaBH4 and the Luche reagent9 failed to
produce any significant diastereoselectivity. We were gratified
to find that treatment of tripeptide 15 with the (S)-CBS or (R)-
CBS reagent in the presence of BH3 ·DMS almost exclusively
afforded the reduced tripeptides 16 and 17, respectively
(Schemes 3 and 4). The stereochemical outcome of these
isolation of the methyl ester to a significant extent (38%). Using
acetonitrile as the mobile phase smoothly afforded the natural
product in an analytically pure form. Acetylation of the
secondary hydroxyl group of Tuv contained in tubulysin V (4)
under standard conditions afforded a second natural product,
tubulysin U (3, Scheme 3). Epi-tubulysin V (18) was synthesized
and isolated in an identical fashion starting from reduced
tripeptide 17 (Scheme 4).
A small series of simplified analogues in which the N-terminal
residue of the tubulysins (Mep) was replaced with the acyclic
N,N-dimethyl-D-alanine was synthesized to extend our previous
findings and those of Wipf and Ellman. Synthesis of these
analogues follows our published route for the synthesis of other
series of simplified analogues.6d,e Tripeptides 15, 19, and 20
were deprotected, coupled with N,N-dimethyl-D-Ala, and sa-
ponified to afford tetrapeptides 21-23 (Scheme 5).
1
reactions was supported by the following: (1) the H NMR
spectrum of 4 matched that of tubulysin V as previously
reported5a and (2) the findings of Zanda and co-workers who
reported that the (S)-CBS reagent with BH3 ·DMS reacted nearly
exclusively from the si face of the ketone of a racemic Tuv
intermediate to give the alcohol products possessing the natural
configuration as established by X-ray crystallography.5a The 1H
NMR spectrum of the immediate tetrapeptide benzyl ester
precursor of tubulysin V was identical to the minor diastereomer
14, enabling us to assign the stereochemistry of the purified
diastereomers 14.
Tubulysins U and V and analogues 18 and 21-23 were
evaluated for antiproliferative activity in 1A9 ovarian cancer cells
and MCF-7 breast cancer cells and for in vitro inhibition of tubulin
polymerization (Table 1).6d,e,8 The hemiasterlin analogue HTI-286
(SPA110) was used as a positive control in all assays.
The total synthesis of tubulysin V (4) was completed by
amino group deprotection of reduced tripeptide 16, followed
by coupling with N-Me-D-Pip and saponification of the benzyl
ester (Scheme 3). Interestingly, purification of tubulysin V by
reverse-phase HPLC under the conditions reported by Do¨mling
and co-workers5b using MeOH as the mobile phase resulted in
Tubulysin V is about 30- to 45-fold more active than epi-
tubulysin V in the antiproliferative assays, suggesting that the
absolute configuration of the secondary alcohol in tubuvaline is
important for activity. Acetylation of this alcohol to produce
tubulysin U caused a dramatic improvement in potency by 3 orders
of magnitude, possibly because of its increased lipophilicity and

240 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 2
Letters
Scheme 5. Synthesis of Simplified Tubulysin Analogues 21-23
Acknowledgment. This work was generously supported by
the American Cancer Society, Grant RSG-05-105-01-CDD (to
R.A.F.) and was supported in part by intramural funds from
the National Institute of Child Health and Human Development,
NIH. Funding for NMR instrumentation in the Department of
Biochemistry, Molecular Biology, and Biophysics was provided
by the University of Minnesota Medical School, NSF (Grant
BIR-961577), and the Minnesota Medical Foundation.
Supporting Information Available: Experimental procedures,
spectroscopic data, and NMR spectra for all new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Table 1. Biological Activity of Tubulysins U and V and Analogues
inhibition of proliferation
(IC50, µM)b
tubulin
inhibition
compda
1A9
MCF-7
0.0004
0.24
(IC50, µM)c
3 (tubulysin U)
4 (tubulysin V)
18 (epi-tubulysin V)
21 (FT-040)Tc
22 (FT-039)
0.00065
0.12
1.9
1.1
1.3
3.6
12.5
8.5
5.1
8.1
>50
NDd
0.17
0.3
23 (FT-038)
14.2
12.2
HTI-286 (SPA110)
0.0002
0.00015
0.7
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JM8013579