Syntheses and evaluation of simplified pretubulysin analogues

Article abstract of DOI:10.1002/ejoc.201100155

The syntheses of new tubulysin analogues are described, in which the central amino acid, tubuvaline, is replaced by the rather simple building blocks phenyltubuvaline and phenoxytubuvaline. These analogues can be obtained in only two to three steps from e

Full text of DOI:10.1002/ejoc.201100155

FULL PAPER
DOI: 10.1002/ejoc.201100155
Syntheses and Evaluation of Simplified Pretubulysin Analogues
Jens L. Burkhart,[a] Rolf Müller,[b] and Uli Kazmaier*[a]
Keywords: Natural products / Antitumor agents / Synthesis design / Myxobacteria / Tubulysins
The syntheses of new tubulysin analogues are described, in
which the central amino acid, tubuvaline, is replaced by the
rather simple building blocks phenyltubuvaline and phen-
oxytubuvaline. These analogues can be obtained in only two
to three steps from easily accessible starting materials. Al-
though the new derivatives are less active than the tubulys-
ins, their activities towards U-2 OS tumor cells are still in the
nanomolar range.
tent drugs.[1] Aside from the maytansanoids[2] and epothi-
lones,[3] the peptides auristatine[4] and dolastatine 10[5] and
the family of the tubulysins[3,6] (Figure 1) are excellent can-
didates for this approach, based on their high biological
activity. The tubulysins, isolated by Höfle and co-workers
from myxobacteria,[7] especially Angiococcus disciformis An
d48 and Archangium gephyra Ar 315, belong to a nine-
Introduction
The development of selective anticancer drugs, able to dis-
criminate between normal and tumor cells, is an important
goal in modern cancer therapy. The highly promising ap-
proach to target tumor cells with monoclonal antibodies,
loaded with a suitable antitumor drug, requires highly po-
Figure 1. Peptidic tubulin polymerization inhibitors.
membered group of linear tetrapeptides (tubulysins A–I).[7]
Very recently, further derivatives were discovered in A. disci-
formis An d48 and Cystobacter SBCb004 by the Müller
group.[8] Common to all members of the tubulysin family
is their linear structure, beginning with an N-terminal N-
methylated d-pipecolinic acid (d-Mep), followed by the
only proteinogenic amino acid l-Ile. The next building
block is called tubuvaline (Tuv), an unusual thiazole amino
[a] Institute for Organic Chemistry, Saarland University,
P.O. Box 151150, 66041 Saarbrücken, Germany
Fax: +49-681-302-2409
E-mail: u.kazmaier@mx.uni-saarland.de
[b] Department of Pharmaceutical Biotechnology,
Saarland University und Helmholtz-Institut für Pharmazeutis-
che Forschung Saarland,
P.O. Box 151150, 66041 Saarbrücken, Germany
Fax: +49-681-302-70202
E-mail: rom@mx.uni-saarland.de
3050
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Syntheses and Evaluation of Simplified Pretubulysin Analogues
acid, bound through an exotic acylated N,O-acetal struc- N-methylamide at the same position can be equally po-
ture. The natural tubulysins differ mainly in the side chain tent,[21] a significant drop in activity is only observed when
of the N,O-acetal group. An α-methylated aromatic γ-amino the N substituent is removed completely .[22] SAR studies
acid is the C-terminal amino acid. In some tubulysins, this have also focused on the N-terminal amino acid, N-methyl
amino acid is derived from phenylalanine (tubulysins D–F, pipecolinic acid (Mep), which can probably be replaced by
and H), in others from tyrosin (tubulysins A–C, and G). N-methylsarcosine, and the C-terminal tubuphenylalanine
The newly explored derivatives mainly differ in the oxi- (Tup), which can be substituted with a wide range of alter-
dation and acylation state of the Tuv fragment and are native functionalities, with retention of biological ac-
probably biosynthetic intermediates.[9] Pretubulysin was tivity.[21a] These studies are consistent with the binding
found to be a central intermediate of the tubuphenlyalanine studies reported by Carlomagno et al.[12]
tubulysins D–F and H.[10]
The tubulysins belong to a group of natural products
that interact with the eucaryotic cytoskeleton by binding to
microtubuli. Inhibition of tubulin polymerization induces
Results and Discussion
As a result of biosynthetic studies, we postulated that
pretubulysin (Figure 1) was a common biosynthetic inter-
mediate.[10] When considering that the rather labile N,O-
acetal side chains in the Tuv fragment are missing, this
compound should be an ideal candidate to develop tubuly-
sin drugs. Especially because the biological activity of pre-
tubulysin is only slightly lower than tubulysin itself and still
in the low nanomolar range.[23] To simplify the structure
even more, we decided to replace the thiazole moiety with
a simple phenyl ring. In principle, this should allow the in-
troduction of a wide range of functionalities in this area to
modulate, for example, the pharmacokinetic properties of
this class of compound. With respect to the lack of a Tuv
acetoxy group in pretubulysin, the removal of the ste-
reogenic center α to the heterocycle should also allow us to
replace the remaining CH2 bond with other functionalities,
such as an ether bridge. Herein, we describe the synthesis
and biological activity of two new derivatives of pretubuly-
sin, which we call phenylpretubulysin (1) and phenoxypre-
tubulysin (2) (Figure 2).
apoptosis.[6,11] This mode of action differs from that of pa-
clitaxel and the epothilones. The tubulysins also show a
higher cytotoxicity towards a wide range of tumor cells in
the low nano- or even picomolar range.[7a] From a struc-
tural point of view, the tubulysins are in part very similar
to dolastatine 10, in particular, the left-hand side of the
molecule is conserved. A tertiary amine is found at the N
terminus of both types of peptides, followed by an apolar
amino acid (Val or Ile), and an N-alkylated apolar unusual
amino acid building block. On the C terminus both natural
products contain phenylalanine-derived unusual amino ac-
ids. The major difference is found in the area between the
sterically demanding side chain of the N-alkylated amino
acid and the C-terminal building block. Both natural prod-
ucts probably interact with the same binding side at the
vinca domain of the tubulin. Recent NMR spectroscopic
studies by the Carlomagno group indicated that the confor-
mations of these tubulin-bound peptides were very similar
and that the variable part in between the apolar left-hand
side and the aromatic substituent probably acted as a spacer
to bring the interacting parts of the natural products into
the correct orientation for binding.[12]
At first, the tubulysins seem to be relatively simple struc-
tures, especially with respect to other antitumor drugs, such
as paclitaxel. Nevertheless, to date, only two total syntheses
have been described in the literature. In 2006, the Ellman
group reported the first synthesis of tubulysin D,[13] and
recently Wessjohann et al. described the first synthesis of
tubulysin B.[14] In addition, the groups of Höfle[15] and
Dömling[16] patented syntheses of tubulysins. Very recently,
Figure 2. Pretubulysin derivatives 1 and 2.
The synthesis of the phenylpretubulysin intermediate 7
Tamura et al. reported the conversion of tubulysin D into (phenylpretubuvaline) is shown in Scheme 1. The starting
a cyclic derivative, bridging the N terminus of Tuv with an point was bromide 3, which was obtained by radical bromi-
OH group α to the thiazole ring.[17] Probably the major nation of methyl m-toluate.[24] Reaction with PPh3 under
problem during the total synthesis is the introduction of the standard conditions provided phosphonium salt 4,[25] which
N,O-acetal side chain found on the N terminus of the Tuv, was subjected to a Wittig reaction with tert-butyloxycarb-
which is found in all tubulysins. With respect to an applica- onyl (Boc-)protected valinal. The Wittig reaction was car-
tion as an anticancer drug, this rather labile structure ried out as a one-pot protocol by adding the Wittig salt to
should also be a critical candidate for enzymatic cleavage.
a freshly prepared solution of the aldehyde in CH2Cl2. The
Therefore, besides the total syntheses of the natural prod- required unsaturated derivative 5 was obtained in good
ucts, a range of syntheses towards tubulysin analogues[18] yield as a 4:1 mixture of E/Z isomers. In our case, the olefin
and building blocks[19] have also been reported, as well as geometry did not play any role because the double bond
detailed structure–activity (SAR) studies. These studies was removed in the next step by catalytic hydrogenation to
clearly indicate that the N,O-acetal is not necessary for high form 6. Subsequent N-methylation provided the protected
biological activity.[13,20] Compounds incorporating a simple phenylpretubuvaline 7. Attempts to introduce the N-methyl
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J. L. Burkhart, R. Müller, U. Kazmaier
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group at an earlier stage were less successful. If the one-pot lent optical purity (99% ee), accompanied by a small
sequence of diisobutylaluminum hydride (DIBALH) re- amount of aziridine 9.[28] Even increasing the reaction time
duction/Wittig olefination was carried out with the N-meth- to three days did not improve the yield. Subsequent N-
ylvaline derivative, the yield dropped to 44% and gave rise methylation occurred in nearly quantitative yield. Unsur-
to the unsaturated phenylpretubuvaline as a 1:1 isomeric prisingly, the attempt to obtain 10 directly by the Mitsu-
mixture (E/Z).
nobu reaction using protected N-methylvalinol failed com-
pletely. It is likely that nucleophilic attack of the phenolate
on the activated valinol derivative is also the critical step in
the former reaction.
Scheme 1. Synthesis of phenylpretubuvaline 7.
The synthesis of the corresponding phenoxypretubuv-
aline derivative 10 (Scheme 2) started with enantiomerically
pure Boc-valinol,[26] which was treated with methyl m-hy-
droxybenzoate under Mitsunobu conditions.[27] The cou-
pling product 8 was obtained in moderate yield, but excel- Scheme 2. Synthesis of phenoxypretubuvaline 10.
Scheme 3. Synthesis of phenylpretubulysin (1) and phenoxypretubulysin (2). TFA = trifluoroacetate.
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Eur. J. Org. Chem. 2011, 3050–3059

Syntheses and Evaluation of Simplified Pretubulysin Analogues
13C NMR spectra were obtained at room temperature with a
Bruker AV 400 spectrometer. Chemical shifts are expressed in ppm
relative to internal solvent. Selected signals of minor isomers were
extracted from the NMR spectra of the isomeric mixtures. The en-
antio- and diastereomeric ratios were determined by HPLC on a
Shimadzu 10A VP using a chiral column (Chiralcel OD-H) and an
achiral silica gel column (LiChrosorb 5 μ Si-60 A). Optical rotation
measurements were performed on a Perkin–Elmer 341 polarimeter,
with concentrations given in g/100 mL. Melting points were deter-
mined with a MEL-TEMP II apparatus and are uncorrected. High-
With these building blocks in hand, the synthesis of the
pretubulysin derivatives 1 and 2 was accomplished accord-
ing to Scheme 3. Cleavage of the Boc-protecting group and
coupling with Z-Ile gave rise to the required dipeptides 11
and 12. 2-Bromo-1-ethylpyridinium tetrafluoroborate
(BEP) was used as a coupling reagent to avoid racemiza-
tion, which has frequently been observed in couplings of N-
methyl amino acids, and to achieve high yields.[29] Subse-
quent removal of the protecting groups and coupling with
the N-terminal (R)-pipecolinic acid (Pip) and the C-ter- resolution mass spectra were recorded on a Finnigan MAT 95Q
instrument by using the CI technique. Elemental analyses were per-
formed on a Leco CHN900 instrument.
minal tubuphenylalanine (Tup) derivative gave rise to tetra-
peptides 15 and 16. Removal of the benzyloxycarbonyl (Z)-
protecting group and reductive methylation followed by sa-
Methyl (S)-3-[3-(tert-Butoxycarbonylamino)-4-methylpent-1-enyl]-
ponification of the ester moiety provided the required pre- benzoate (5): A solution of DIBALH (1 m) in hexane (9.00 mL,
9.00 mmol) was added dropwise at –78 °C to a solution of Boc-
protected methyl valinate (1.02 g, 4.41 mmol) in dry dichlorometh-
ane (25 mL). After stirring the reaction mixture for 2 h at –78 °C,
phosphonium salt 4[25] (2.38 g, 4.84 mmol) and KOtBu (544 mg,
4.85 mmol) were added and the reaction was warmed to room tem-
perature overnight. The reaction mixture was poured into 10%
aqueous tartaric acid (100 mL) and vigorously stirred for 30 min.
After separation of the layers, the aqueous layer was extracted
thrice with EtOAc. The combined organic layers were washed with
water and brine, dried with Na2SO4, and concentrated. The crude
tubulysin derivatives 1 and 2 in a straightforward manner.
The biological activity of our new pretubulysin deriva-
tives 1 and 2, as well as methyl ester 17, were tested, and
compared with tubulysins A and D as well as pretubulysin,
towards human bone osteosarcoma (U-2 OS) cells
(Table 1).[23] Whereas the tubulysins and pretubulysins
showed IC50 values in the sub-nanomolar range, phenylana-
logue 1 was two magnitudes of order less potent (ca.
100 nm). Methyl ester 17, which was probably a prodrug,
and the phenoxy derivative 2 showed a fourfold lower ac- product was purified by flash chromatography (hexane/EtOAc, 9:1)
to yield 5 (1.18 g, 3.54 mmol, 80%) as a colorless oil and a mixture
of E and Z isomers (E/Z = 4:1). Rf = 0.29 (hexane/EtOAc, 8:2).
tivity (Table 1).
1
3
Table 1. Cytotoxicity of tubulysins and analogues towards U-2 OS
cells.
(E)-5: H NMR (400 MHz, CDCl3): δ = 0.96 (d, J13,12 = 6.8 Hz,
3 H, 13-H), 0.97 (d, 3J13Ј,12 = 6.8 Hz, 3 H, 13Ј-H), 1.47 (s, 9 H, 16-
H), 1.87 (m, 1 H, 12-H), 3.93 (s, 3 H, 1-H), 4.15 (br. s, 1 H, 4-H),
IC50 [ngmL–1]
3
3
4.63 (br. s, 1 H, NH), 6.17 (dd, J10,9 = 15.9, J10,11 = 6.5 Hz, 1 H,
Tubulysin A
Tubulysin D
Pretubulysin D
Phenylpretubulysin (1)
Phenoxypretubulysin (2)
Methyl ester 17
0.19
0.034
0.56
78.6
318.2
284.6
3
3
3
10-H), 6.53 (d, J9,10 = 15.9 Hz, 1 H, 9-H), 7.38 (dd, J5,6 = J5,4
3
= 7.7 Hz, 1 H, 5-H), 7.53 (d, J4,5 = 7.8 Hz, 1 H, 4-H), 7.89 (d,
3J6,5 = 7.8 Hz, 1 H, 6-H), 8.05 (s, 1 H, 8-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 18.3 (q, C-13), 18.8 (q, C-13Ј), 28.4 (q, C-
16), 32.7 (d, C-12), 52.1 (q, C-1), 57.7, (d, C-11), 79.4 (s, C-15),
127.2 (d, C-8), 128.4 (d, C-6), 128.6 (d, C-5), 129.7 (d, C-9), 130.4
(s, C-3), 130.6 (d, C-10), 130.8 (s, C-4), 137.3 (s, C-7), 155.5 (s, C-
1
14), 167.0 (s, C-2) ppm. (Z)-5: H NMR (400 MHz, CDCl3): δ =
3
0.88 (d, J13,12 = 6.6 Hz, 6 H, 13-H, 13Ј-H), 1.42 (s, 9 H, 16-H),
Conclusions
1.78 (m, 1 H, 12-H), 3.91 (s, 3 H, 1-H), 4.40 (br. s, 1 H, 4-H), 4.53
3
3
We have shown that the complex structure of tubulysins
can be significantly reduced by replacing the Tuv subunit
with a relatively simple spacer unit. Although the activity
is significantly lower than that of the natural products and
pretubulysin, the activity is still in the nanomolar range.
Therefore, these compounds might be interesting for further
developments, especially based on their easy synthetic avail-
ability.
(br. s, 1 H, NH), 5.55 (dd, J10,9 = 11.8, J10,11 = 6.5 Hz, 1 H, 10-
3
3
H), 6.54 (d, J9,10 = 9.6 Hz, 1 H, 9-H), 7.41 (dd, J5,6
= =
3J5,4
3
7.7 Hz, 1 H, 5-H), 7.64 (br. s, 1 H, 4-H), 7.91 (d, J6,5 = 7.8 Hz, 1
H, 6-H), 7.94 (s, 1 H, 8-H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 18.2 (q, C-13), 18.4 (q, C-13Ј), 28.3 (q, C-16), 33.4 (d, C-12),
52.1 (q, C-1), 53.4, (d, C-11), 79.1 (s, C-15), 128.1 (d, C-6), 128.5
(d, C-5), 129.9 (d, C-4), 130.1 (d, C-9), 130.2 (s, C-3), 132.1 (d, C-
10), 133.0 (s, C-8), 137.0 (s, C-7), 155.2 (s, C-14), 167.0 (s, C-2)
ppm. C19H27NO4 (333.42): calcd. C 68.44, H 8.16, N 4.20; found
C 68.43, H 8.54, N 4.20. HRMS (CI): calcd. for C15H20NO4 [M –
C4H7]+ 278.1392; found 278.1380.
Experimental Section
Methyl
(R)-3-[3-(tert-Butoxycarbonylamino)-4-methylpentyl]ben-
General Remarks: Reactions with dry solvents were carried out in
oven-dried glassware (100 °C) under nitrogen. Solvents were dried
as follows: THF was distilled from LiAlH4, CH2Cl2 from CaH2,
MeOH from Mg, and toluene from Na. The products were purified
by flash chromatography on silica gel (0.063–0.2 mm). Mixtures of
EtOAc and hexanes were generally used as eluents. Analysis by
TLC was carried out on commercially precoated Polygram SIL-G/
zoate (6): A solution of 5 (513 mg, 1.53 mmol, E/Z mixture) in
MeOH (7 mL) was stirred under hydrogen in the presence of 5%
Pd/C (50 mg) until the reduction was complete (3 h). After fil-
tration through Celite, the solvent was removed in vacuo and the
crude product was purified by flash chromatography (hexane/
EtOAc, 8:2) to yield 6 (470 mg, 1.40 mmol, 92%) as a white solid;
m.p. 72–73 °C. Rf = 0.31 (hexane/EtOAc, 8:2). HPLC: Chiracel
UV 254 plates (Machery–Nagel, Düren). Visualization was ac- OD-H (250ϫ4.6), hexane/2-propanol = 95:5, 1 mLmin–1, tR[(R)-
1
complished with UV light, KMnO4 solution, or ninhydrin. H and
6] = 6.61 min, tR[(S)-6] = 8.19 min. [α]2D1 = –7.6 (c = 1.0, ee = 99%,
Eur. J. Org. Chem. 2011, 3050–3059
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J. L. Burkhart, R. Müller, U. Kazmaier
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CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.87 (d, 3J13,12 = 6.8 Hz,
3 H, 13-H), 0.90 (d, 3J13Ј,12 = 6.8 Hz, 3 H, 13Ј-H), 1.45 (s, 9 H, 16-
H), 1.56 (m, 1 H, 10-Ha), 1.69–1.83 (m, 2 H, 10-Hb, 12-H), 2.67
4
4
8.0 Hz, 1 H, 5-H), 7.54 (dd, J8,6 = 2.2, J8,4 = 1.5 Hz, 1 H, 8-H),
7.63 (ddd, J4,5 = 7.8, J4,6 = J4,8 = 1.1 Hz, 1 H, 4-H) ppm. 13C
3
4
4
NMR (100 MHz, CDCl3): δ = 18.7 (q, C-12), 19.6 (q, C-12Ј), 28.4
(m, 1 H, 9-Ha), 2.75 (m, 1 H, 9-Hb), 3.50 (m, 1 H, 11-H), 3.90 (s, (q, C-15), 29.5 (d, C-11), 52.2 (q, C-1), 55.2 (d, C-10), 68.4 (t, C-
3 H, 1-H), 4.35 (d, 3JNH,11 = 9.4 Hz, 1 H, NH), 7.32–7.39 (m, 2 H,
9), 79.3 (s, C-14), 114.8 (d, C-8), 119.7 (d, C-6), 122.2 (d, C-4),
5-H, 6-H), 7.84–7.86 (m, 2 H, 4-H, 8-H) ppm. 13C NMR 129.4 (d, C-5), 131.5 (s, C-3), 155.7 (s, C-13), 158.7 (s, C-7), 166.9
(100 MHz, CDCl3): δ = 17.6 (q, C-13), 19.1 (q, C-13Ј), 28.4 (q, C- (s, C-2) ppm. C18H27NO5 (337.41): calcd. C 64.07, H 8.07, N 4.15;
16), 32.4 (d, C-12), 32.6 (t, C-9), 34.7 (t, C-10), 52.0 (q, C-1), 55.4,
(d, C-11), 79.0 (s, C-15), 127.1 (d, C-4), 128.4 (d, C-5), 129.4 (d,
C-8), 130.2 (s, C-3), 133.1 (d, C-6), 142.5 (s, C-7), 156.0 (s, C-14),
167.2 (s, C-2) ppm. C19H29NO4 (335.44): calcd. C 68.03, H 8.71,
N 4.18; found C 68.02, H 8.63, N 4.20. HRMS (CI): calcd. for
C19H30NO4 [M + H]+ 336.2175; found 336.2169.
found C 64.01, H 7.81, N 3.75. HRMS (CI): calcd. for C18H28NO5
[M + H]+ 338.1967; found 338.1962.
Methyl (S)-3-{2-[(tert-Butoxycarbonyl)methylamino]-3-methylbut-
oxy}benzoate (10): To a solution of 8 (551 mg, 1.63 mmol) in anhy-
drous DMF (10 mL) methyl iodide (410 μL, 6.59 mmol) was
added. After cooling to 0 °C NaH (60% in oil) (170 mg,
Methyl (R)-3-{3-[(tert-Butoxycarbonyl)methylamino]-4-methylpent- 4.25 mmol) was added. The reaction mixture was warmed to room
yl}benzoate (7): Methyl iodide (280 μL, 4.50 mmol) was added to a
solution of 6 (373 mg, 1.11 mmol) in anhydrous DMF (8 mL). Af-
ter cooling to 0 °C, NaH (60% in oil, 102 mg, 2.50 mmol) was
added. The reaction mixture was warmed to room temperature and
stirred for 20 h before it was quenched with water, acidified to pH
6 with saturated NH4Cl, and extracted with EtOAc. The combined
organic layers were washed twice with water, 5% Na2S2O3, water,
and brine. The organic phase was dried with Na2SO4 and concen-
trated. The crude material was purified by flash chromatography
(hexane/EtOAc, 8:2) to yield 7 (380 mg, 1.09 mmol, 98%) as a col-
orless oil and a mixture of rotamers. Rf = 0.33 (hexane/EtOAc,
temperature and stirred for 21 h, before it was quenched with water,
acidified to pH 6 with saturated NH4Cl, and extracted with EtOAc.
The combined organic layers were washed twice with water, 5%
Na2S2O3, water, and brine; dried with Na2SO4; and concentrated
in vacuo. The crude material was purified by flash chromatography
(hexane/EtOAc, 8:2) to yield 10 (542 mg, 1.54 mmol, 95%) as a
colorless oil and a mixture of rotamers. Rf = 0.35 (hexane/EtOAc,
8:2). [α]2D0 = –49.0 (c = 1.2, CHCl3). Major rotamer: 1H NMR
3
(400 MHz, CDCl3): δ = 0.93 (d, J12,11 = 6.6 Hz, 6 H, 12-H), 1.41
(s, 9 H, 15-H), 1.98 (m, 1 H, 11-H), 2.80 (s, 3 H, 16-H), 3.90 (s, 3
3
H, 1-H), 4.06–4.15 (m, 3 H, 9-H, 10-H), 7.06 (d, J6,5 = 8.2 Hz, 1
8:2). [α]2D1 = –7.5 (c = 0.9, CHCl3). Major rotamer: 1H NMR
H, 6-H), 7.32 (dd, J5,6 = J5,4 = 8.0 Hz, 1 H, 5-H), 7.53 (s, 1 H,
3
3
3
(400 MHz, CDCl3): δ = 0.83 (d, J13,12 = 6.6 Hz, 3 H, 13-H), 0.90 8-H), 7.62 (d, 3J4,5 = 7.6 Hz, 1 H, 4-H) ppm. 13C NMR (100 MHz,
(d, 3J13Ј,12 = 6.6 Hz, 3 H, 13Ј-H), 1.43 (s, 9 H, 16-H), 1.57–1.67 (m,
2 H, 10-Ha, 12-H), 1.91 (m, 1 H, 10-Hb), 2.55 (m, 2 H, 9-H), 2.65 C-16), 52.1 (q, C-1), 60.3 (d, C-10), 67.5 (t, C-9), 79.3 (s, C-14),
CDCl3): δ = 19.9 (q, C-12), 27.0 (d, C-11), 28.4 (q, C-15), 30.4 (q,
(s, 3 H, 17-H), 3.82 (m, 1 H, 11-H), 3.90 (s, 3 H, 1-H), 7.31–7.38
114.6 (d, C-8), 119.9 (d, C-6), 122.1 (d, C-4), 129.4 (d, C-5), 131.4
(s, C-3), 156.3 (s, C-13), 158.6 (s, C-7), 166.9 (s, C-2) ppm. Minor
(m, 2 H, 5-H, 6-H), 7.83–7.87 (m, 2 H, 4-H, 8-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 20.1 (q, C-13), 20.3 (q, C-13Ј), 28.1 (q, C- rotamer: 1H NMR (400 MHz, CDCl3): δ = 1.01 (d, 3J12,11 = 6.4 Hz,
17), 28.4 (q, C-1), 30.6 (d, C-12), 31.7 (t, C-10), 32.7 (t, C-9), 52.0
(q, C-1), 60.5 (d, C-11), 79.3 (s, C-15), 127.1 (d, C-4), 128.4 (d, C-
5), 129.0 (d, C-8), 130.2 (s, C-3), 133.1 (d, C-6), 142.7 (s, C-7), 156.6
(s, C-14), 167.2 (s, C-2) ppm. Minor rotamer: 1H NMR (400 MHz,
3 H, 12-H), 1.02 (d, 3J12Ј,11 = 6.4 Hz, 3 H, 12Ј-H), 1.46 (s, 9 H, 15-
H), 2.09 (m, 1 H, 11-H), 2.81 (s, 3 H, 16-H), 3.90 (m, 1 H, 10-H)
ppm. 13C NMR (100 MHz, CDCl3): δ = 19.8 (q, C-12), 20.1 (q, C-
12Ј), 27.8 (d, C-11), 61.6 (d, C-10), 68.3 (t, C-9), 79.5 (s, C-14),
114.9 (d, C-8), 120.1 (d, C-6), 122.2 (d, C-4), 129.4 (d, C-5), 156.4
3
3
CDCl3): δ = 0.84 (d, J13,12 = 6.7 Hz, 3 H, 13-H), 0.92 (d, J13Ј,12
= 6.6 Hz, 3 H, 13Ј-H), 1.47 (s, 9 H, 16-H), 2.72 (s, 3 H, 17-H), 3.59 (s, C-13), 158.8 (s, C-7) ppm. C19H29NO5 (351.44): calcd. C 64.93,
(br. s, 1 H, 11-H), 3.90 (s, 3 H, 1-H) ppm. 13C NMR (100 MHz,
H 8.32, N 3.99; found C 64.90, H 8.09, N 4.22. HRMS (CI): calcd.
CDCl3): δ = 19.6 (q, C-13), 19.9 (q, C-13Ј), 28.5 (q, C-1), 30.9 (d, for C19H30NO5 [M + H]+ 352.2124; found 352.2135.
C-12), 31.5 (t, C-10), 32.5 (t, C-9), 52.0 (q, C-1), 79.0 (s, C-15),
127.1 (d, C-4), 128.3 (d, C-5), 130.1 (s, C-3), 133.0 (d, C-6), 142.4
(s, C-7), 167.2 (s, C-2) ppm. C20H31NO4 (349.46): calcd. C 68.74,
Methyl 3-{(R)-3-[(2S,3S)-2-(Benzyloxycarbonylamino)-N,3-dimeth-
ylpentanamido]-4-methylpentyl}benzoate (11): At 0 °C, a solution of
HCl in dioxane (4 m, 2.50 mL, 10.0 mmol) was added to compound
7 (357 mg, 1.02 mmol). The solvent was evaporated in vacuo after
H 8.94, N 4.01; found C 68.46, H 8.97, N 4.32. HRMS (CI): calcd.
for C20H32NO4 [M + H]+ 350.2331; found 350.2378.
complete deprotection (2 h) and the hydrochloride salt was dried
Methyl (S)-3-[2-(tert-Butoxycarbonylamino)-3-methylbutoxy]ben- in vacuo. This salt was dissolved in dry dichloromethane (10 mL)
zoate (8): Diisopropyl azodicarboxylate (DIAD) (112 μL,
0.53 mmol, 94% purity) was added to a solution of Boc-protected
valinol (116 mg, 57 mmol), methyl 3-hydroxybenzoate (81 mg,
0.53 mmol), and triphenylphosphane (140 mg, 0.53 mmol) in dry
THF (2 mL) at 0 °C. The reaction mixture was stirred for 24 h and
then concentrated in vacuo. The crude product was purified by
flash chromatography (hexane/EtOAc, 9:1, 8:2) to yield of 8
(90 mg, 0.27 mmol, 51%) as a colorless oil. Rf = 0.32 (hexane/
together with Z-protected (S)-isoleucine (320 mg, 1.21 mmol) and
2-bromo-1-ethylpyridinium tetrafluoroborate (BEP) (310 mg,
1.13 mmol) before diisopropylethylamine (0.52 mL, 3.06 mmol)
was added dropwise at –10 °C. The cooling bath was removed after
20 min and the reaction mixture was stirred at room temperature
for 20 h. The reaction mixture was diluted with dichloromethane
and washed with 1 m aqueous HCl, saturated aqueous NaHCO3,
water, and brine; dried with Na2SO4; and concentrated in vacuo.
EtOAc, 8:2). HPLC: Chiracel OD-H (250ϫ4.6), hexane/EtOH = The crude product was purified by flash chromatography (hexane/
99:1, 1 mLmin–1, tR[(R)-8] = 11.55 min, tR[(S)-8] = 12.64 min. EtOAc, 7:3) to provide dipeptide 11 (472 mg, 0.95 mmol, 93% over
[α]2D0 = –60.7 (c = 0.7, ee = 99%, CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 0.98 (d, J12,11 = 6.8 Hz, 3 H, 12-H), 0.99 (d, J12Ј,11
2 steps) as a colorless oil and a mixture of rotamers. Rf = 0.25
(hexane/EtOAc, 7:3). [α]2D1 = –15.6 (c = 1.4, CHCl3). Major rotamer:
1H NMR (400 MHz, CDCl3): δ = 0.77 (d, 3J13,12 = 6.6 Hz, 3 H, 13-
3
3
= 6.8 Hz, 3 H, 12Ј-H), 1.45 (s, 9 H, 15-H), 2.02 (m, 1 H, 11-H),
2
3.74 (br. s, 1 H, 10-H), 3.99 (s, 3 H, 1-H), 4.06 (dd, J9a,9b = 9.3, H), 0.89 (t, 3J19,18 = 7.4 Hz, 3 H, 19-H), 0.94 (d, 3J13Ј,12 = 6.5 Hz, 3
3J9a,10 = 3.2 Hz, 1 H, 9-Ha), 4.10 (dd, 2J9b,9a = 9.3, 3J9b,10 = 4.0 Hz,
H, 13Ј-H), 1.01 (d, J20,17 = 6.7 Hz, 3 H, 20-H), 1.15 (m, 1 H, 18-
Ha), 1.52–1.72 (m, 3 H, 10-Ha, 12-H, 18-Hb), 1.80 (m, 1 H, 17-H),
1.94 (m, 1 H, 10-Hb), 2.50 (m, 2 H, 9-H), 2.98 (s, 3 H, 14-H), 3.90
3
3
1 H, 9-Hb), 4.79 (d, 3JNH,10 = 9.0 Hz, 1 H, NH), 7.09 (ddd, J6,5
=
=
4
4
3
3
8.2, J6,8 = 2.6, J6,4 = 0.8 Hz, 1 H, 6-H), 7.34 (dd, J5,6 = J5,4
3054
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3050–3059

Syntheses and Evaluation of Simplified Pretubulysin Analogues
(s, 3 H, 1-H), 4.34 (m, 1 H, 11-H), 4.58 (dd, 3J16,NH = 9.3, 3J16,17
=
MeOH (7 mL) was stirred under hydrogen in the presence of 10%
Pd/C (24 mg) until the deprotection was complete (1 h). After fil-
6.8 Hz, 1 H, 16-H), 5.09 (m, 2 H, 22-H), 5.51 (d, 3JNH,16 = 9.4 Hz, 1
H, NH), 7.25–7.34 (m, 7 H, 5-H, 6-H, 24-H, 25-H, 26-H), 7.83 (s, tration through Celite, the solvent was removed in vacuo and the
1 H, 8-H), 7.86 (m, 1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3): free amine was dried in vacuo. Isobutyl chloroformate (63 μL,
δ = 11.2 (q, C-19), 16.0 (q, C-20), 19.5 (q, C-13), 20.0 (q, C-13Ј),
0.47 mmol) was added to a solution of Z-protected (R)-pipecolic
23.8 (t, C-18), 29.2 (q, C-14), 30.2 (d, C-12), 31.4 (t, C-10), 32.7 (t, acid (125 mg, 0.47 mmol) and N-methylmorpholine (155 μL,
C-9), 37.5 (d, C-17), 52.0 (q, C-1), 55.8 (d, C-16), 59.3 (d, C-11), 1.41 mmol) in dry THF (7 mL) at –20 °C. After 20 min, the free
66.7 (t, C-22), 127.2 (d, C-4), 127.8, 128.0, 128.4, 128.5 (4d, C-5,
C-24, C-25, C-26), 129.1 (d, C-8), 130.3 (s, C-3), 132.9 (d, C-6),
amine dissolved in dry THF (6 mL) was added and the mixture
was warmed to room temperature overnight. Water was added and
the mixture was extracted thrice with EtOAc. The combined or-
136.4 (s, C-23), 142.2 (s, C-7), 156.4 (s, C-21), 167.1 (s, C-2), 173.1
1
(s, C-15) ppm. Minor rotamer: H NMR (400 MHz, CDCl3): δ = ganic layers were washed with 1 m aqueous HCl, water, saturated
1.04 (d, 3J20,17 = 6.6 Hz, 3 H, 20-H), 1.87 (m, 1 H, 17-H), 2.01 (m, aqueous NaHCO3, water, and brine; dried with Na2SO4; and con-
2
3
1 H, 10-H), 2.35 (dt, J9a,9b = 13.2, J9,10 = 4.3 Hz, 1 H, 9-Ha),
centrated in vacuo. The crude product was purified by flash
2.91 (s, 3 H, 14-H), 3.60 (m, 1 H, 11-H), 3.89 (s, 3 H, 1 H), 4.67 chromatography (hexane/EtOAc, 6:4) to yield tripeptide 13
3
3
(dd, J16,NH = 9.7, J16,17 = 6.4 Hz, 1 H, 16-H), 7.80 (s, 1 H, 8-H)
ppm. 13C NMR (100 MHz, CDCl3): δ = 11.3 (q, C-19), 16.1 (q, C-
20), 20.3 (q, C-13), 20.5 (q, C-13Ј), 23.6 (t, C-18), 27.2 (q, C-14),
38.0 (d, C-17), 55.2 (d, C-16), 63.1 (d, C-11), 66.8 (t, C-22), 132.9
(d, C-6), 136.3 (s, C-23), 141.9 (s, C-7), 156.1 (s, C-21), 167.1 (s, C-
(680 mg, 1.12 mmol, 89% over 2 steps) as a white solid and a mix-
ture of rotamers; m.p. 38–40 °C. Rf = 0.22 (hexane/EtOAc, 6:4).
[α]2D1 = –7.2 (c = 1.0, CHCl3). Major rotamer: 1H NMR (400 MHz,
3
CDCl3): δ = 0.76 (d, J13,12 = 6.6 Hz, 3 H, 13-H), 0.85 (br. s, 3 H,
3
19-H), 0.94 (d, J13Ј,12 = 6.5 Hz, 3 H, 13Ј-H), 0.97 (br. s, 3 H, 20-
2), 172.8 (s, C-15) ppm. HRMS (CI): calcd. for C29H41N2O5 [M + H), 1.09 (m, 1 H, 18-Ha), 1.25–1.71 (m, 8 H, 10-Ha, 12-H, 18-Hb,
H]+ 497.3015; found 497.3005.
23-Ha, 24-H, 25-H), 1.81 (m, 1 H, 17-H), 1.95 (m, 1 H, 10-Hb),
2.31 (m, 2 H, 23-Hb), 2.49 (m, 2 H, 9-H), 2.89 (br. s, 1 H, 26-Ha),
3.01 (s, 3 H, 14-H), 3.90 (s, 3 H, 1-H), 4.08 (br. s, 1 H, 26-Hb), 3.34
Methyl 3-{(S)-2-[(2S,3S)-2-(Benzyloxycarbonylamino)-N,3-dimeth-
ylpentanamido]-3-methylbutoxy}benzoate (12): According to the
preparation of dipeptide 11, compound 12 was obtained by depro-
tection of 10 (512 mg, 1.48 mmol) in HCl/dioxane (4 m, 3.80 mL,
15.0 mmol). Subsequent peptide coupling with Z-protected l-iso-
leucine (429 mg, 1.62 mmol), BEP (448 mg, 1.64 mmol), and di-
isopropyl ethylamine (0.75 mL, 4.41 mmol) in dry dichloromethane
(15 mL) gave rise to 12 (693 mg, 1.39 mmol, over 2 steps) as a col-
orless oil and a mixture of rotamers. Rf = 0.21 (hexane/EtOAc,
7:3). [α]2D0 = –38.0 (c = 1.3, CHCl3). Major rotamer: 1H NMR
(400 MHz, CDCl3): δ = 0.84–0.90 (m, 6 H, 12-H, 18-H), 0.98 (d,
3J19,16 = 6.7 Hz, 3 H, 19-H), 1.01 (d, 3J12Ј,11 = 6.9 Hz, 3 H, 12Ј-H),
1.15 (m, 1 H, 17-Ha), 1.60 (m, 1 H, 17-Hb), 1.77 (m, 1 H, 16-H),
2.03 (m, 1 H, 11-H), 3.06 (s, 3 H, 13-H), 3.90 (s, 3 H, 1-H), 4.10
3
3
(m, 1 H, 11-H), 4.84 (dd, J16,NH = 8.7, J16,17 = 7.9 Hz, 1 H, 16-
H), 4.91 (br. s, 1 H, 22-H), 5.17 (m, 2 H, 28-H), 6.70 (br. s, 1 H,
NH), 7.27–7.40 (m, 7 H, 5-H, 6-H, 30-H, 31-H, 32-H), 7.82 (s, 1
H, 8-H), 7.87 (m, 1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 11.1 (q, C-19), 15.9 (q, C-20), 19.6 (q, C-13), 20.0 (q, CЈ-13),
20.4 (t, C-25), 24.1 (t, C-18), 24.8 (t, C-24), 25.6 (t, C-23), 29.2 (q,
C-14), 30.2 (d, C-12), 31.4 (t, C-10), 32.8 (t, C-9), 37.2 (d, C-17),
42.3 (t, C-26), 52.1 (q, C-1), 53.7 (d, C-16), 55.0 (d, C-22), 59.3 (d,
C-11), 67.6 (t, C-28), 127.3 (d, C-4), 127.8, 128.0, 128.5 (4d, C-5,
C-30, C-31, C-32), 129.1 (d, C-8), 130.3 (s, C-3), 132.9 (d, C-6),
136.4 (s, C-29), 142.2 (s, C-7), 156.4 (s, C-21), 167.2 (s, C-2), 170.5
(s, C-21), 173.1 (s, C-15) ppm. Minor rotamer: 1H NMR (400 MHz,
3
CDCl3): δ = 1.04 (d, J20,17 = 6.5 Hz, 3 H, 20-H), 2.01 (m, 1 H,
3
3
(m, 2 H, 9-H), 4.56 (dd, J15,NH = 9.0, J15,16 = 7.5 Hz, 1 H, 15-
10-Hb), 2.26 (m, 1 H, 23-Hb), 2.64 (m, 2 H, 9-H), 2.81 (s, 3 H, 14-
H), 2.63 (m, 1 H, 11-H), 3.88 (s, 3 H, 1-H), 4.20 (br. s, 1 H, 26-
Hb), 4.34 (m, 1 H, 11-H), 5.05–5.07 (m, 2 H, 16-H, 28-Ha), 6.58
(br. s, 1 H, NH) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.3 (q,
C-19), 16.2 (q, C-20), 20.3 (q, C-13), 20.6 (t, C-25), 23.6 (t, C-18),
24.6 (t, C-24), 26.1 (t, C-23), 27.3 (q, C-14), 31.3 (d, C-12), 32.2 (t,
C-10), 33.2 (t, C-9), 38.2 (d, C-17), 52.0 (q, C-1), 53.0 (d, C-16),
55.4 (d, C-22), 63.3 (d, C-11), 67.5 (t, C-28), 155.5 (s, C-21) ppm.
HRMS (CI): calcd. for C35H50N3O6 [M + H]+ 608.3699; found
608.3729.
3
H), 4.65 (br. s, 1 H, 10-H), 5.09 (s, 2 H, 21-H), 5.51 (d, JNH,15
=
3
4
9.3 Hz, 1 H, NH), 7.03 (dd, J6,5 = 8.3, J6,4 = 2.0 Hz, 1 H, 6-H),
7.27–7.34 (m, 6 H, 5-H, 23-H, 24-H, 25-H), 7.51 (s, 1 H, 8-H), 7.63
3
(d, J4,5 = 7.8 Hz, 1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 11.2 (q, C-18), 15.5 (q, C-19), 19.5 (q, C-12), 19.8 (q, C-12Ј),
24.0 (t, C-17), 27.0 (d, C-11), 30.5 (q, C-13), 37.7 (d, C-16), 52.1
(q, C-1), 55.5 (d, C-15), 58.3 (d, C-10), 66.7 (d, C-21), 67.0 (t, C-
9), 114.5 (d, C-8), 119.7 (d, C-6), 122.3 (d, C-4), 127.8, 127.9, 128.4
(3d, C-23, C-24, C-25), 129.4 (d, C-5), 131.5 (s, C-3), 136.4 (s, C-
22), 156.4 (s, C-20), 158.5 (s, C-7), 166.7 (s, C-2), 173.2 (s, C-14)
ppm. Minor rotamer: 1H NMR (400 MHz, CDCl3): δ = 1.01 (d,
3J12,11 = 6.9 Hz, 3 H, 12-H), 1.12 (d, 3J12Ј,11 = 6.6 Hz, 3 H, 12Ј-H),
1.86 (m, 1 H, 16-H), 2.82 (s, 3 H, 13-H), 3.87 (s, 3 H, 1-H), 3.94–
Methyl 3-((S)-2-{(2S,3S)-N,3-Dimethyl-2-[(R)-1-(3-phenylpro-
panoyl)piperidine-2-carboxamido]pentanamido}-3-methylbutoxy)ben-
zoate (14): According to the preparation of tripeptide 13, com-
pound 14 was obtained by deprotection of 12 (629 mg, 1.26 mmol)
in MeOH (7 mL). Subsequent peptide coupling with Z-protected
(R)-pipecolic acid (366 mg, 1.39 mmol), isobutyl chloroformate
(185 μL, 1.39 mmol), and N-methylmorpholine (155 μL,
1.41 mmol) in dry THF (7 mL) gave rise to 14 (680 mg, 1.12 mmol,
89% over 2 steps) as a white solid and a mixture of rotamers; m.p.
38–40 °C. Rf = 0.13 (hexane/EtOAc, 7:3). [α]2D0 = –7.2 (c = 1.0,
CHCl3). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.79–
3
3
4.01 (m, 3 H, 9-H, 10-H), 4.78 (dd, J15,NH = 9.4, J15,16 = 6.3 Hz,
2
1 H, 15-H), 5.00 (d, J21a,21b = 12.3 Hz, 1 H, 21-Ha), 5.60 (d,
3JNH,15 = 9.5 Hz, 1 H, NH), 7.00 (m, 1 H, 6-H), 7.20 (dd, J5,6
=
3
3J5,4 = 8.0 Hz, 1 H, 5-H), 7.48 (s, 1 H, 8-H), 7.61 (m, 1 H, 4-H)
ppm. 13C NMR (100 MHz, CDCl3): δ = 11.4 (q, C-18), 16.1 (q, C-
19), 20.2 (q, C-12), 23.6 (t, C-17), 37.9 (d, C-16), 37.9 (d, C-16),
52.1 (q, C-1), 62.1 (d, C-10), 66.6 (d, C-21), 114.0 (d, C-8), 120.3
(d, C-6), 122.3 (d, C-4), 127.9, 128.0, 128.4 (3d, C-23, C-24, C-25),
129.4 (d, C-5), 131.2 (s, C-3), 156.2 (s, C-20), 158.3 (s, C-7), 173.4
(s, C-14) ppm. HRMS (CI): calcd. for C28H39N2O6 [M + H]+
499.2808; found 499.2804.
3
0.88 (m, 6 H, 12-H, 18-H), 0.95 (d, J19,16 = 5.5 Hz, 3 H, 19-H),
3
1.04 (d, J12,11 = 6.5 Hz, 3 H, 12-H), 1.09 (m, 1 H, 17-Ha), 1.26–
1.44 (m, 2 H, 23-Ha, 24-Ha), 1.45–1.66 (m, 4 H, 17-Hb, 22-Ha, 23-
Methyl
3-((R)-3-{(2S,3S)-N,3-Dimethyl-2-[(R)-1-(3-phenylpro- Hb, 24-Hb), 1.80 (m, 1 H, 16-H), 2.00 (m, 1 H, 11-H), 2.30 (m, 1
panoyl)piperidine-2-carboxamido]pentanamido}-4-methylpentyl)ben-
zoate (13): A solution of dipeptide 11 (230 mg, 0.38 mmol) in
H, 22-Hb), 2.88 (m, 1 H, 25-Ha), 3.09 (s, 3 H, 13-H), 3.91 (s, 3 H,
1-H), 4.02–4.12 (m, 3 H, 9-H, 25-Hb), 4.67 (br. s, 1 H, 10-H), 4.80
Eur. J. Org. Chem. 2011, 3050–3059
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3055

J. L. Burkhart, R. Müller, U. Kazmaier
FULL PAPER
(dd, J15,NH = J15,16 = 8.5 Hz, 1 H, 15-H), 4.91 (br. s, 1 H, 21-H),
3
3
(t, C-35), 49.0 (d, C-5), 51.8 (q, C-42), 53.8 (d, C-25), 54.9 (d, C-
31), 58.9 (d, C-20), 67.7 (t, C-37), 124.1 (d, C-13), 126.5, 127.8,
3
5.17 (m, 2 H, 27-H), 6.59 (br. s, 1 H, NH), 7.02 (dd, J6,5 = 8.1,
4J6,4 = 1.6 Hz, 1 H, 6-H), 7.25–7.40 (m, 6 H, 5-H, 29-H, 30-H, 31- 127.7, 128.0, 128.3, 128.4, 128.6, 129.4 (s, 8d, C-8, C-9, C-10, C-
H), 7.50 (s, 1 H, 8-H), 7.63 (d, J4,6 = 7.8 Hz, 1 H, 4-H) ppm. 13C
NMR (100 MHz, CDCl3): δ = 11.0 (q, C-18), 15.4 (q, C-19), 19.5
12, C-14, C-17, C-39, C-40, C-41), 131.4 (s, C-15), 136.2 (s, C-38),
137.7 (s, C-7), 142.1 (s, C-16), 155.6 (s, C-36), 167.0 (s, C-11), 170.4
3
(q, C-12), 19.8 (q, C-12Ј), 20.4 (t, C-23), 24.4 (t, C-17), 24.8 (t, C- (s, C-30), 173.5 (s, C-24), 177.0 (s, C-1) ppm. Selected minor rot-
1
3
24), 25.7 (t, C-22), 26.7 (d, C-11), 27.3 (q, C-13), 37.2 (d, C-16), amer: H NMR (400 MHz, CDCl3): δ = 1.02 (d, J22,11 = 6.5 Hz,
42.3 (t, C-25), 52.1 (q, C-1), 53.5 (d, C-15), 55.0 (C-21), 58.3, (C- 3 H, 22-H), 2.78 (s, 3 H, 23-H), 3.57 (m, 1 H, 20-H), 4.20 (m, 1 H,
10), 66.9 (t, C-9), 67.6 (t, C-27), 114.4 (d, C-8), 119.7 (d, C-6), 122.3
(d, C-4), 127.8, 128.0, 128.5 (3d, C-29, C-30, C-31), 129.4 (d, C-5),
131.5 (s, C-3), 136.4 (s, C-28), 156.4 (s, C-26), 158.5 (s, C-7), 166.8
35-Hb), 4.97–5.05 (m, 3 H, 25-H, 37-H), 7.50–7.52 (m, 2 H, 13-H,
17-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.2 (q, C-28), 16.2
(q, C-29), 17.5 (q, C-3), 20.3 (q, C-22), 20.5 (q, C-22Ј), 27.4 (q, C-
23), 31.3 (d, C-21), 32.0 (t, C-19), 32.9 (t, C-18), 63.2 (d, C-20),
(s, C-2), 170.6 (s, C-20), 173.3 (s, C-14) ppm. Minor rotamer: 1H
3
NMR (400 MHz, CDCl3): δ = 1.01 (d, J12,11 = 6.7 Hz, 3 H, 12- 67.5 (t, C-37), 137.9 (s, C-7), 141.7 (s, C-16), 155.5 (s, C-36), 176.9
3
H), 1.11 (d, J12Ј,11 = 6.5 Hz, 3 H, 12Ј-H), 2.11 (m, 1 H, 11-H),
(s, C-1) ppm. HRMS (CI): calcd. for C47H66N4O7 [M + 2H]2+
2.36 (m, 1 H, 22-Hb), 2.85 (s, 3 H, 13-H), 2.98 (m, 1 H, 25-Ha), 798.4931; found 798.4973.
3.89 (s, 3 H, 1-H), 4.20 (br. s, 1 H, 25-Hb), 6.77 (d, 3JNH,13 = 9.2 Hz,
Benzyl (R)-2-{(2S,3S)-1-[((S)-1-{3-[(2R,4S)-5-Methoxy-4-methyl-5-
1 H, NH), 7.10 (m, 1 H, 6-H), 7.48 (s, 1 H, 8-H), 7.61 (m, 1 H, 4-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.4 (q, C-18), 16.1 (q,
C-19), 20.1 (q, C-12), 20.3 (q, C-12Ј), 26.1 (t, C-22), 30.5 (q, C-13),
37.7 (d, C-16), 52.1 (q, C-1), 55.4 (C-21), 62.1 (d, C-10), 67.2 (t, C-
9), 67.5 (t, C-27), 113.0 (d, C-8), 120.7 (d, C-6), 122.6 (d, C-4),
131.3 (s, C-3), 158.4 (s, C-7), 170.1 (s, C-20) ppm. HRMS (CI):
calcd. for C34H48N3O7 [M + H]+ 610.3492; found 610.3454.
oxo-1-phenylpentan-2-ylcarbamoyl]phenoxy}-3-methylbutan-2-yl)-
methylamino]-3-methyl-1-oxopentan-2-ylcarbamoyl}piperidine-1-carb-
oxylate (16): According to the preparation of tetrapeptide 15, com-
pound 14 (610 mg, 1.00 mmol) was subjected to saponification with
1 m NaOH (1.25 mL, 1.5 mmol) in MeOH (10 mL). Subsequent
peptide coupling of the crude acid (395 mg, 0.66 mmol) with Tup-
OMe·HCl (190 mg, 0.74 mmol), isobutyl chloroformate (0.92 mL,
0.69 mmol), and N-methylmorpholine (175 μL, 1.59 mmol) in dry
Benzyl (R)-2-{(2S,3S)-1-[((R)-1-{3-[(2R,4S)-5-Methoxy-4-methyl-5-
oxo-1-phenylpentan-2-ylcarbamoyl]phenyl}-4-methylpentan-3-yl)meth- THF (6 mL) gave rise to 16 (366 mg, 0.46 mmol, 70%) as a white
ylamino]-3-methyl-1-oxopentan-2-ylcarbamoyl}piperidine-1-carb- solid and a mixture of rotamers; m.p. 58–60 °C. Rf = 0.20 (hexane/
oxylate (15): A mixture of methyl ester 13 (180 mg, 0.30 mmol) and
1 m aqueous NaOH (0.45 mL, 0.45 mmol) in MeOH (3 mL) was
stirred at 60 °C until complete saponification occurred (3 h). The
solvent was evaporated in vacuo and the residue was dissolved in
water, acidified to pH 1 with 1 m aqueous KHSO4, and extracted
thrice with EtOAc. The combined organic layers were dried with
Na2SO4 and the solvent was evaporated in vacuo to give the crude
acid as a white foam. Isobutyl chloroformate (39 μL, 0.30 mmol)
was added to a solution of the crude acid and N-methylmorpholine
(78 μL, 0.71 mmol) in dry THF (6 mL) at –20 °C. After 20 min
Tup-OMe·HCl (78 mg, 0.30 mmol) was added and the mixture was
warmed to room temperature overnight. Water was added and the
mixture was extracted thrice with EtOAc. The combined organic
layers were washed with 1 m aqueous HCl, water, saturated aqueous
NaHCO3, water, and brine; dried with Na2SO4; and concentrated
in vacuo. The crude product was purified by flash chromatography
(hexane/EtOAc, 1:1) to yield tetrapeptide 15 (165 mg, 0.21 mmol,
73% over 2 steps) as a white solid and a mixture of rotamers; m.p.
59–61 °C. Rf = 0.23 (hexane/EtOAc, 1:1). [α]2D0 = +23.1 (c = 0.9,
CHCl3). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.74 (d,
EtOAc, 1:1). [α]2D0 = +1.2 (c = 1.4, CHCl3). Major rotamer: 1H
NMR (400 MHz, CDCl3): δ = 0.78–0.88 (m, 6 H, 21-H, 27-H),
3
0.94 (d, 3J28,25 = 6.5 Hz, 3 H, 28-H), 1.04 (d, J21,20 = 6.5 Hz, 3 H,
3
21-H), 1.10 (m, 1 H, 26-Ha), 1.18 (d, J3,2 = 7.15 Hz, 3 H, 3-H),
1.32–1.45 (m, 2 H, 32-Ha, 32-Hb), 1.45–1.65 (m, 4 H, 26-Ha, 31-
2
3
3
Ha, 32-Hb, 33-Hb), 1.69 (ddd, J4a,4b = 14.2, J4,2 = 9.7, J4,5
=
4.2 Hz, 1 H, 4-Ha), 1.81 (m, 1 H, 25-Ha), 1.93–2.05 (m, 2 H, 4-Hb,
2
20-H), 2.28 (m, 1 H, 31-Hb), 2.66 (m, 1 H, 2-H), 2.89 (dd, J6a,6b
3
= 13.8, J6a,5 = 6.8 Hz, 1 H, 6-Ha), 2.93 (m, 1 H, 34-Ha), 2.98 (dd,
3
2J6b,6a = 13.6, J6b,5 = 5.9 Hz, 1 H, 6-Hb), 3.08 (s, 3 H, 22-H), 3.63
(s, 3 H, 41-H), 4.00–4.25 (m, 3 H, 18-H, 34-Hb), 4.41 (m, 1 H, 5-
H), 4.69 (m, 1 H, 19-H), 4.78 (m, 1 H, 24-H), 4.87 (m, 1 H, 30-H),
2
2
5.15 (d, J36a,36b = 12.6 Hz, 1 H, 36-Ha), 5.20 (d, J36b,36a
=
12.5 Hz, 1 H, 36-Hb), 6.23 (br. s, 1 H, NH), 6.79 (br. s, 1 H, NH),
3
6.94 (d, 3J15,14 = 7.8 Hz, 1 H, 15-H), 7.16 (d, J13,14 = 7.7 Hz, 1 H,
13-H), 7.19–7.26 (m, 3 H, 8-H, 10-H), 7.27–7.38 (m, 9 H, 9-H, 14-
H, 17-H, 38-H, 39-H, 40-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 11.0 (q, C-27), 15.4 (q, C-28), 17.3 (q, C-3), 19.4 (q, C-21), 19.7
(q, C-21Ј), 20.3 (t, C-32), 24.4 (t, C-26), 24.6 (t, C-33), 25.8 (t, C-
31), 26.8 (d, C-20), 30.4 (d, C-20), 30.4 (d, C-2), 37.0 (t, C-4), 37.1
(d, C-25), 40.7 (t, C-6), 42.3 (t, C-34), 49.0 (d, C-5), 51.8 (q, C-41),
3J22,21 = 6.6 Hz, 3 H, 22-H), 0.84 (m, 3 H, 28-H), 0.89 (d, 3J22,21
=
6.5 Hz, 3 H, 22Ј-H), 0.93–1.03 (m, 4 H, 27-Ha, 29-H), 1.16 (d, 3J3,2 53.7 (d, C-24), 54.9 (d, C-30), 58.2 (d, C-19), 66.5 (t, C-18), 67.7
= 7.1 Hz, 3 H, 3-H), 1.28–1.48 (m, 2 H, 33-Ha, 34-Ha), 1.48–1.76 (t, C-36), 112.6 (d, C-17), 118.4 (d, C-15), 118.7 (d, C-13), 126.5,
(m, 7 H, 4-Ha, 19-Ha, 21-H, 27-Hb, 32-Ha, 33-Hb, 34-Hb), 1.76– 127.8, 128.0, 128.4, 128.5, 129.4, 129.5 (7d, C-8, C-9, C-10, C-14,
1.92 (m, 2 H, 19-Hb, 26-H), 1.98 (m, 1 H, 4-Hb), 2.27 (m, 1 H, 32- C-38, C-39, C-40), 136.0 (s, C-12), 136.3 (s, C-37), 137.6 (s, C-7),
2
Hb), 2.50 (m, 1 H, 18-H), 2.64 (m, 1 H, 2-H), 2.88 (dd, J6a,6b
=
156.6 (s, C-35), 158.7 (s, C-16), 166.5 (s, C-11), 170.2 (s, C-29),
3
13.6, J6a,5 = 6.8 Hz, 1 H, 6-Ha), 2.82–3.08 (m, 3 H, 6-Hb, 23-H,
35-Ha), 3.60 (s, 3 H, 42-H), 4.06 (m, 1 H, 35-Hb), 4.31 (m, 1 H, (400 MHz, CDCl3): δ = 1.01 (d, J21,20 = 6.7 Hz, 3 H, 21-H), 1.11
20-H), 4.41 (m, 1 H, 5-H), 4.76–4.91 (m, 2 H, 25-H, 31-H), 5.11 (d, J21,20 = 6.4 Hz, 3 H, 21-H), 2.09 (m, 1 H, 20-H), 2.84 (s, 3 H,
(d, J37a,37b = 12.5 Hz, 1 H, 37-Ha), 5.18 (d, J37b,37a = 12.5 Hz, 1
173.5 (s, C-23), 177.0 (s, C-1) ppm. Minor rotamer: 1H NMR
3
3
2
3
22-H), 3.61 (s, 3 H, 41-H), 3.89 (m, 1 H, 19-H), 6.55 (br. s, 1 H,
H, 37-Hb), 6.41 (br. s, 1 H, NH), 6.78 (br. s, 1 H, NH), 7.18–7.36
NH), 7.01 (m, 1 H, 15-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
(m, 12 H, 8-H, 9-H, 10-H, 14-H, 15-H, 39-H, 40-H, 41-H), 7.46 11.4 (q, C-27), 16.1 (q, C-28), 17.4 (q, C-3), 20.0 (q, C-21), 26.1 (t,
(m, 1 H, 13-H), 7.58 (m, 1 H, 17-H) ppm. 13C NMR (100 MHz, C-31), 36.4 (d, C-20), 40.8 (t, C-6), 42.0 (t, C-34), 49.1 (d, C-5),
CDCl3): δ = 11.0 (q, C-28), 15.8 (q, C-29), 17.3 (q, C-3), 19.5 (q,
C-22), 19.9 (q, C-22Ј), 20.2 (t, C-33), 24.2 (t, C-27), 24.6 (t, C-34),
51.7 (q, C-41), 55.4 (d, C-30), 62.3 (d, C-19), 67.5 (t, C-36), 137.8
(s, C-7), 155.4 (s, C-35), 158.6 (s, C-16), 166.5 (s, C-11), 173.0 (s,
25.9 (t, C-32), 29.4 (q, C-23), 30.2 (d, C-21), 31.3 (t, C-19), 32.6 (t, C-23), 176.9 (s, C-1) ppm. HRMS (CI): calcd. for C39H55N4O7
C-18), 36.4 (d, C-2), 37.0, 37.1 (d, t, C-4, C-26), 40.7 (t, C-6), 42.2
[M – C7H7O]+ 691.4071; found 691.4100.
3056
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3050–3059

Syntheses and Evaluation of Simplified Pretubulysin Analogues
3
3
Methyl (2S,4R)-4-[3-((R)-3-{(2S,3S)-N,3-Dimethyl-2-[(R)-1-methyl-
piperidine-2-carboxamido]pentanamido}-4-methylpentyl)benzamido]-
2-methyl-5-phenylpentanoate (17): A solution of tetrapeptide 15
(253 mg, 0.32 mmol) in MeOH (4 mL) was stirred under hydrogen
in the presence of 10% Pd/C (27 mg) until deprotection was com-
plete (3 h). After filtration through Celite, the solvent was removed
in vacuo and the free amine was dried in vacuo. This crude amine
was dissolved in dry MeOH (4 mL) and paraformaldehyde (29 mg,
0.32 mmol) was added. After the solution had been stirred for 3 h
3 H, 21-H), 0.88 (t, J27,26 = 7.4 Hz, 3 H, 27-H), 0.96 (d, J28,25 =
3
6.7 Hz, 3 H, 28-H), 1.02 (d, J21.20 = 6.3 Hz, 3 H, 21-H), 1.17 (d,
3J3,2 = 7.2 Hz, 3 H, 3-H), 1.12–1.25 (m, 2 H, 26-Ha, 32-Ha), 1.36
(m, 1 H, 31-Ha), 1.42–1.72 (m, 5 H, 4-Ha, 26-Hb, 32-Hb, 33-H),
1.77 (m, 1 H, 31-Hb), 1.87 (m, 1 H, 25-H), 1.93–2.04 (m, 3 H, 4-
3
Hb, 20-H, 34-Ha), 2.21 (s, 3 H, 35-H), 2.45 (dd, J30,31a = 10.7,
3J30,31b = 2.1 Hz, 1 H, 30-H), 2.65 (m, 1 H, 2-H), 2.86–2.91 (m, 2
2
3
H, 6-Ha, 34-Hb), 2.97 (dd, J6b,6a = 13.7, J6b,5 = 5.9 Hz, 1 H, 6-
Hb), 3.08 (s, 3 H, 22-H), 3.62 (s, 3 H, 36-H), 4.08 (m, 2 H, 18-H),
3
at room temperature, sodium cyanoborohydride (22 mg, 4.40 (m, 1 H, 5-H), 4.65 (br. s, 1 H, 19-H), 4.74 (dd, J24,NH
=
3
0.36 mmol) was added and the reaction mixture was stirred for fur-
ther 16 h. The methanol was removed, the residue was diluted with
saturated aqueous NaHCO3, and the solution was extracted thrice
with dichloromethane. The combined organic layers were washed
with brine, dried with Na2SO4, and concentrated in vacuo. The
crude product was purified by flash chromatography (dichloro-
methane/MeOH, 97:3, 95:5) to yield the N-methylated tetrapeptide
3J24,25 = 9.0 Hz, 1 H, 24-H), 6.21 (d, JNH,5 = 7.3 Hz, 1 H, NH),
6.94 (dd, 3J15,14 = 8.2, 4J15,13 = 2.2 Hz, 1 H, 15-H), 7.01 (d, 3JNH,24
= 9.3 Hz, 1 H, NH), 7.15–7.35 (m, 8 H, 8-H, 9-H, 10-H, 13-H, 14-
H, 17-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 10.9 (q, C-27),
15.5 (q, C-28), 17.3 (q, C-3), 19.5 (q, C-21), 19.8 (q, C-21Ј), 23.2
(t, C-34), 24.8 (t, C-26), 25.1 (t, C-33), 26.9 (d, C-20), 30.3 (t, C-
31), 30.7 (q, C-22), 36.4 (d, C-2), 37.0 (t, C-4), 37.2 (d, C-25), 40.7
17 (168 mg, 0.25 mmol, 78% over 2 steps) as a white solid and a (t, C-6), 44.8 (q, C-35), 48.9 (d, C-59), 51.8 (q, C-36), 52.8 (d, C-
mixture of rotamers; m.p. 45–47 °C. Rf = 0.26 (dichloromethane/
24), 55.4 (t, C-34), 58.1 (d, C-19), 67.1 (t, C-18), 69.7 (d, C-30),
112.5 (d, C-17), 118.3 (d, C-15), 118.7 (d, C-13), 126.5 (d, C-10),
1
MeOH, 95:5). [α]2D1 = +35.2 (c = 1.2, CHCl3). Major rotamer: H
3
NMR (400 MHz, CDCl3): δ = 0.73 (d, J22,21 = 6.6 Hz, 3 H, 22- 128.4, 129.4 (2d, C-8, C-9), 129.5 (d, C-14), 136.1 (s, C-12), 137.6
3
3
H), 0.86 (d, J22Ј,21 = 6.4 Hz, 3 H, 22Ј-H), 0.89 (t, J28,27 = 7.4 Hz,
(s, C-7), 158.8 (s, C-16), 166.5 (s, C-11), 173.2, 174.4 (2s, C-7, C-13),
3
3
3 H, 28-H), 0.99 (d, J29,26 = 6.8 Hz, 3 H, 29-H), 1.16 (d, J3,2
=
177.0 (s, C-1) ppm. Minor rotamer: 1H NMR (400 MHz, CDCl3): δ
3
3
7.2 Hz, 3 H, 3-H), 1.15–1.25 (m, 2 H, 27-Ha, 33-Ha), 1.36 (m, 1 H,
32-Ha), 1.45–1.74 (m, 7 H, 4-Ha, 19-Ha, 21-H, 27-Hb, 33-Hb, 34-
H), 1.79–1.92 (m, 3 H, 19-Hb, 26-H, 32-Hb), 1.97–2.04 (m, 2 H, 4-
Hb, 35-Ha), 2.24 (s, 3 H, 36-H), 2.40–2.50 (m, 2 H, 18-Ha, 31-H),
= 1.10 (d, J21,20 = 6.5 Hz, 3 H, 21-H), 1.14 (d, J3,2 = 7.3 Hz, 3
H, 3-H), 2.05 (m, 1 H, 20-H), 2.25 (s, 3 H, 35-H), 2.45 (m, 1 H,
2
3
30-H), 2.78 (dd, J6a,6b = 13.5, J6a,5 = 7.7 Hz, 1 H, 6-Ha), 2.84 (s,
2
3
3 H, 22-H), 3.03 (dd, J6b,6a = 13.8, J6b,5 = 5.7 Hz, 1 H, 6-Hb),
3
2.55 (m, 1 H, 18-Hb), 2.65 (m, 1 H, 2-H), 2.84–2.93 (m, 2 H, 6-Ha, 3.58 (s, 3 H, 36-H), 3.85 (m, 1 H, 19-H), 5.22 (dd, J24,NH = 9.5,
35-Hb), 2.95–3.04 (m, 4 H, 6-Hb, 23-H), 3.60 (s, 3 H, 37-H), 4.28 3J24,25 = 4.4 Hz, 1 H, 24-H), 6.75 (d, JNH,5 = 8.4 Hz, 1 H, NH),
3
3
3
(br. s, 1 H, 20-H), 4.41 (m, 1 H, 5-H), 4.81 (dd, J25,26 = J25,NH
=
7.48 (s, 1 H, 17-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.6
(q, C-27), 16.5 (q, C-28), 17.6 (q, C-3), 20.0 (q, C-21), 20.4 (q, C-
21Ј), 23.5 (t, C-34), 27.4 (d, C-20), 27.8 (q, C-22), 36.4 (d, C-2),
3
8.9 Hz, 1 H, 25-H), 6.60 (d, JNH,5 = 8.2 Hz, 1 H, NH), 7.06 (d,
3JNH,25 = 9.5 Hz, 1 H, NH), 7.18–7.33 (m, 7 H, 8-H, 9-H, 10-H, 14-
H, 15-H), 7.51–7.56 (m, 13-H, 17-H) ppm. 13C NMR (100 MHz, 41.4 (t, C-6), 45.1 (q, C-35), 49.4 (d, C-59), 51.7 (q, C-36), 53.3 (d,
CDCl3): δ = 10.9 (q, C-28), 15.9 (q, C-29), 17.6 (q, C-3), 19.6 (q,
C-22), 20.0 (q, C-22Ј), 23.2 (t, C-33), 24.7 (t, C-27), 25.1 (t, C-34),
C-24), 55.5 (t, C-34), 62.4 (d, C-19), 66.9 (t, C-18), 70.2 (d, C-30),
113.8 (d, C-17), 117.3 (d, C-15), 119.8 (d, C-13), 126.4 (d, C-10),
29.5 (q, C-23), 30.2 (d, C-21), 30.4 (t, C-32), 31.4 (t, C-19), 32.6 (t, 135.6 (s, C-12), 138.0 (s, C-7), 158.5 (s, C-16), 166.2 (s, C-11), 173.0,
C-18), 36.4 (d, C-2), 37.2, 37.3 (d, t, C-4, C-26), 41.2 (t, C-6), 44.9
(q, C-36), 49.1 (d, C-5), 51.7 (q, C-37), 53.0 (d, C-25), 55.4 (t, C-
35), 68.4 (d, C-20), 69.6 (d, C-31), 124.6 (d, C-13), 126.4 (d, C-10),
127.1 (d, C-17), 128.6 (d, C-14), 128.4, 129.4 (2d, C-8, C-9), 131.2
(d, C-15), 134.5 (s, C-12), 137.9 (s, C-7), 142.0 (s, C-16), 166.9 (s,
C-11), 173.3 (s, C-30), 174.2 (s, C-24), 176.9 (s, C-1) ppm. Minor
174.2 (2s, C-7, C-13), 176.8 (s, C-1) ppm. HRMS (CI): calcd. for
C39H59N4O6 [M + H]+ 679.4434; found 679.4454.
Phenylpretubulysin Trifluoroacetate (1·Tfa): A mixture of N-meth-
ylated tetrapeptide 17 (66 mg, 97 μmol) and 1 m NaOH (0.20 mL,
0.20 mmol) in dioxane (1 mL) was stirred at 80 °C until complete
saponification occurred (4 h). The solvent was removed in vacuo
and the residue was dissolved in water, acidified to pH 1 with tri-
fluoroacetic acid, and extracted thrice with EtOAc. The combined
organic layers were dried with Na2SO4 and the solvent was evapo-
rated in vacuo. Purification by flash chromatography (dichloro-
methane/MeOH, 9:1) provided the TFA salt of 1 (68 mg, 88 μmol,
91%) as a white solid and a mixture of rotamers; m.p. 110–112 °C.
Rf = 0.30 (dichloromethane/MeOH, 9:1). [α]2D0 = –21.3 (c = 1.1,
MeOH). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.80 (d,
3J22,21 = 6.4 Hz, 3 H, 22-H), 0.89–1.00 (m, 6 H, 28-H, 29-H), 1.05
1
rotamer: H NMR (400 MHz, CDCl3): δ = 0.91–0.95 (m, 6 H, 22-
H, 29-H), 2.22 (s, 3 H, 36-H), 2.79 (s, 3 H, 23-H), 3.56 (m, 1 H,
3
3
20-H), 3.61 (s, 3 H, 37-H), 5.01 (dd, J25,NH = 9.7 Hz, J25,26
=
5.6 Hz, 1 H, 25-H), 6.53 (d, JNH,5 = 8.4 Hz, 1 H, NH) ppm. 13C
NMR (100 MHz, CDCl3): δ = 11.4 (q, C-28), 16.5 (q, C-29), 20.3
(q, C-22), 20.6 (q, C-22Ј), 23.9 (t, C-33), 31.9 (t, C-19), 40.9 (t, C-
6), 49.1 (q, C-36), 49.0 (d, C-5), 69.8 (d, C-31) ppm. C40H60N4O5
(676.93): calcd. C 70.97, H 8.93, N 8.28; found C 70.10, H 8.67, N
7.92. HRMS (CI): calcd. for C40H61N4O5 [M + H]+ 677.4643;
found 677.4634.
3
3
3
(d, J22,21 = 6.7 Hz, 3 H, 22-H), 1.18 (d, J3,2 = 7.0 Hz, 3 H, 3-H),
Methyl (2S,4R)-4-[3-((S)-2-{(2S,3S)-N,3-Dimethyl-2-[(R)-1-methyl- 1.26 (m, 1 H, 27-Ha), 1.51–1.73 (m, 3 H, 4-Ha, 27-Hb, 33-Ha), 1.73–
piperidine-2-carboxamido]pentanamido}-3-methylbutoxy)benzamido]- 1.88 (m, 4 H, 4-Hb, 19-Ha, 32-Ha, 34-Ha), 1.88–2.03 (m, 5 H, 19-
2-methyl-5-phenylpentanoate (18): According to the preparation of
N-methylated tetrapeptide 17, compound 18 was obtained by de-
protection of 16 (282 mg, 0.35 mmol) in MeOH (4 mL). Subse-
quent reductive methylation with paraformaldehyde (31 mg,
0.34 mmol) and sodium cyanoborohydride (24 mg, 0.38 mmol) in
MeOH (4 mL) gave rise to 18 (177 mg, 0.26 mmol, 76%) as a white
solid and a mixture of rotamers; m.p. 56–58 °C. Rf = 0.29 (dichlo-
romethane/MeOH, 9:1). [α]2D0 = +6.8 (c = 1.4, CHCl3). Major rot-
Hb, 21-H, 26-H, 33-Hb, 34-Hb), 2.15 (m, 1 H, 32-Hb), 2.48 (m, 1
H, 18-Ha), 2.51–2.62 (m, 2 H, 2-H, 18-Hb), 2.73 (s, 3 H, 36-H),
3
2.89 (d, J6,5 = 6.9 Hz, 2 H, 6-H), 3.06 (m, 1 H, 35-Ha), 3.13 (s, 3
3
3
H, 23-H), 3.49 (m, 1 H, 35-Hb), 3.78 (dd, J31,32a = 11.8, J31,32b
=
2.4 Hz, 1 H, 31-H), 4.18 (br. s, 1 H, 20-H), 4.39 (m, 1 H, 5-H),
3
4.71 (d, J25,26 = 7.9 Hz, 1 H, 25-H), 7.16 (m, 1 H, 10-H), 7.20–
7.28 (m, 4 H, 8-H, 9-H), 7.31–7.36 (m, 2 H, 14-H, 15-H), 7.50–
7.58 (m, 2 H, 13-H, 17-H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 11.3 (q, C-28), 16.2 (q, C-29), 18.6 (q, C-3), 20.3 (q, C-22), 20.6
1
3
amer: H NMR (400 MHz, CDCl3): δ = 0.84 (d, J21,20 = 6.6 Hz,
Eur. J. Org. Chem. 2011, 3050–3059
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3057

J. L. Burkhart, R. Müller, U. Kazmaier
(q, C-22Ј), 22.3 (t, C-33), 24.0 (t, C-34), 25.5 (t, C-27), 30.2 (t, C- also thank Jennifer Herrmann at the Department of Pharmaceuti-
FULL PAPER
32), 30.5 (q, C-23), 31.3 (d, C-21), 32.6 (t, C-19), 34.1 (t, C-18),
37.5 (d, C-26), 38.0 (d, C-2), 39.2 (t, C-4), 42.4 (q, C-36), 42.9 (t,
C-6), 51.1 (d, C-5), 56.2 (t, C-35), 56.3 (d, C-25), 61.7 (d, C-20),
cal Biotechnology for investigation of the biological activities.
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2
68.1 (d, C-31), 118.3 (q, JC,F = 1170 Hz, CF3COOH), 125.9 (d,
C-13), 127.4 (d, C-10), 128.3 (d, C-17), 129.3 (d, C-8), 129.6 (d, C-
14), 130.5 (d, C-9), 132.5 (d, C-15), 136.2 (s, C-12), 139.9 (s, C-7),
3
143.6 (s, C-16), 163.0 (q, JC,F = 132 Hz, CF3COOH), 169.3 (s, C-
11), 170.3 (s, C-30), 174.5 (s, C-24), 180.2 (s, C-1) ppm. Minor
rotamer: 1H NMR (400 MHz, CDCl3): δ = 1.09 (d, 3J22,21 = 6.7 Hz,
3
3 H, 22-H), 1.36 (d, J3,2 = 6.4 Hz, 3 H, 3-H), 2.21 (m, 1 H, 32-
Hb), 2.73 (s, 3 H, 36-H), 3.13 (s, 3 H, 23-H), 3.49 (m, 1 H, 35-Hb),
2
2
3.85 (dd, J31,32a = 11.8, J31,32b = 2.4 Hz, 1 H, 31-H), 5.05 (d,
3J25,26 = 5.5 Hz, 1 H, 25-H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 11.7 (q, C-28), 16.5 (q, C-29), 21.1 (q, C-22), 28.4 (q, C-23), 43.0
(t, C-6), 51.2 (d, C-2), 55.4 (d, C-25), 65.1 (d, C-20), 125.7 (d, C-
13), 128.6 (d, C-17), 132.3 (d, C-15), 143.4 (s, C-16), 173.7 (s, C-
24) ppm. HRMS (CI): calcd. for C39H59N4O5 [M + H]+ 663.4485;
found 663.4474.
Phenoxypretubulysin Trifluoroacetate (2·Tfa): According to the
preparation of 1·Tfa, compound 2·Tfa was obtained by saponifica-
tion of 18 (103 mg, 0.15 mmol) with 1 m NaOH (0.30 mL,
0.30 mmol) in dioxane (1.5 mL) to yield 2·Tfa (112 mg, 0.14 mmol,
94%) as a white solid and a mixture of rotamers; m.p. 116–118 °C.
Rf = 0.19 (dichloromethane/MeOH, 9:1). [α]2D0 = –32.5 (c = 1.0,
MeOH). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.88 (d,
3
3J21,20 = 6.5 Hz, 3 H, 21-H), 0.92 (t, J27,26 = 7.6 Hz, 3 H, 27-H),
1.02 (d, 3J28,25 = 6.5 Hz, 3 H, 28-H), 1.07 (d, 3J21,20 = 6.3 Hz, 3 H,
3
21-H), 1.18 (d, J3,2 = 7.0 Hz, 3 H, 3-H), 1.22 (m, 1 H, 26-Ha),
1.56–1.71 (m, 3 H, 4-Ha, 26-Ha, 32-Ha), 1.76–1.82 (m, 2 H, 31-Ha,
33-Ha), 1.84–1.94 (m, 3 H, 25-H, 32-Hb, 33-Hb), 1.96–2.08 (m, 2
H, 4-Hb, 20-H), 2.13 (m, 1 H, 31-H), 2.57 (m, 1 H, 2-H), 2.72 (s,
3
2
3 H, 35-H), 2.88 (d, J6,5 = 6.8 Hz, 2 H, 6-H), 3.07 (ddd, J34a34b
3
3
= J34a,33a = 12.8, J34a,33b = 2.6 Hz, 1 H, 34-Ha), 3.15 (s, 3 H, 22-
H), 3.48 (m, 1 H, 34-Hb), 3.75 (dd, 3J30,31a = 12.0, 3J30,31b = 2.8 Hz,
1 H, 30-H), 4.17 (m, 1 H, 5-H), 4.61–4.63 (m, 2 H, 19-H, 24-H),
3
7.03 (d, J15,14 = 7.2 Hz, 1 H, 15-H), 7.16 (m, 1 H, 10-H), 7.20–
7.35 (m, 7 H, 9-H, 13-H, 14-H, 17-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 11.2 (q, C-27), 15.6 (q, C-28), 18.5 (q, C-3), 20.1 (q,
C-21), 20.4 (q, C-21Ј), 22.3 (t, C-32), 24.1 (t, C-33), 26.0 (t, C-26),
28.1 (d, C-20), 30.1 (t, C-31), 31.2 (q, C-22, C-H, COSY), 37.7 (d,
C-25), 37.9 (d, C-2), 39.2 (t, C-4), 42.3 (t, C-6), 42.8 (q, C-35), 51.1
(d, C-5), 56.0 (d, C-24), 56.2 (t, C-34), 60.5 (d, C-19), 67.6 (t, C-
18), 68.1 (d, C-30), 114.0 (d, C-17), 119.0 (d, C-15), 120.8 (d, C-
13), 127.4 (d, C-10), 129.3, 130.5 (2d, C-8, C-9), 130.6 (d, C-14),
137.7 (s, C-12), 139.9 (s, C-7), 160.0 (s, C-16), 169.2, 169.8 (2s, C-
1
11, C-29), 175.0 (s, C-23), 180.0 (s, C-1) ppm. Minor rotamer: H
3
NMR (400 MHz, CDCl3): δ = 1.14 (d, J21,20 = 6.9 Hz, 3 H, 21-
H), 2.54 (s, 3 H, 35-H), 2.78 (s, 3 H, 22-H), 2.97 (2J34a34b = 3J34a,33a
3
= 12.7, J34a,33b = 2.9 Hz, 1 H, 34-Ha), 3.42 (m, 1 H, 34-Hb), 3.81
3
3
(dd, J30,31a = 11.9, J30,31b = 3.1 Hz, 1 H, 30-H), 4.06–4.08 (m, 2
3
H, 18-Ha, 19-H), 4.26 (m, 1 H, 18-Hb), 7.10 (d, J15,14 = 7.2 Hz, 1
H, 15-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.5 (q, C-27),
16.4 (q, C-28), 20.3 (q, C-21), 20.8 (q, C-21Ј), 39.3 (t, C-4), 42.4 (t,
C-6), 43.0 (q, C-35), 55.3 (d, C-5), 64.0 (d, C-19), 120.7 (d, C-13),
137.5 (s, C-12), 139.9 (s, C-7), 168.8, 169.5 (2s, C-11, C-29), 174.5
(s, C-23) ppm. HRMS (CI): calcd. for C38H57N4O6 [M + H]+
665.4278; found 665.4251.
Acknowledgments
Financial support by the Deutsche Forschungsgemeinschaft
(DFG) (FOR 1406, Ka 880/10-1) is gratefully acknowledged. We
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Received: February 2, 2011
Published Online: April 18, 2011
Eur. J. Org. Chem. 2011, 3050–3059
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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