Synthesis and biological evaluation of pretubulysin and derivatives

Article abstract of DOI:10.1002/ejoc.200900999

Pretubulysin, a biosynthetic precursor of the tubulysins, shows potent biological activity in the subnanomolar range towards various tumor cell lines. Its activity is only slightly less than those of the structurally more complex tubulysins. With a straig

Full text of DOI:10.1002/ejoc.200900999

FULL PAPER
DOI: 10.1002/ejoc.200900999
Synthesis and Biological Evaluation of Pretubulysin and Derivatives
Angelika Ullrich,[a] Jennifer Herrmann,[b] Rolf Müller,[b] and Uli Kazmaier*[a]
Dedicated to Dr. Dieter Häbich on the occasion of his 60th birthday
Keywords: Total synthesis / Antitumor agents / Myxobacteria / Natural products / Tubulysins / Pretubulysin
Pretubulysin, a biosynthetic precursor of the tubulysins,
shows potent biological activity in the subnanomolar range
towards various tumor cell lines. Its activity is only slightly
less than those of the structurally more complex tubulysins.
With a straightforward synthesis to hand, pretubulysin is an
ideal lead structure for the development of tubulysin-based
anticancer drugs
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
The tubulysins (1) are an interesting family of peptidic
secondary metabolites (Figure 1) of myxobacteria, first iso-
lated and characterized by Höfle and Reichenbach et al.[1]
The high cytotoxicities of these compounds are the result of
inhibition of tubulin polymerization, leading to apoptosis.[2]
This mode of action differs from those of paclitaxel and
the epothilones, and the tubulysins also exhibit significantly
higher bioactivities, with IC50 values in the low n range
towards various cancer cell lines.[3]
The structure of the tubulysins can be subdivided into
four amino acid building blocks, two of them represented
by an N-terminal N-methylated -pipecolic acid (-Mep),
connected to -Ile, the only proteinogenic amino acid. The
metabolites also contain two unusual amino acids: tubuva-
line (Tuv), a thiazole amino acid derived from valine, and
a C-terminal extended aromatic γ-amino acid. In some tub-
ulysin family members this γ-amino acid is tubuphenylalan-
ine (Tup, R2 = H), whereas others incorporate tubutyrosine
(Tut, R2 = OH). All tubulysins contain an acetoxy function
at the α-position of Tuv. The most unusual structural fea-
ture is the rare bis-acyl N,O-acetal moiety on the Tuv-
amide, which is found in all highly potent derivatives. Tubu-
lysins lacking this unique structure exhibit substantially re-
duced biological activity.[4] However, this unit poses the
Figure 1. Selected tubulysins (1) and pretubulysin (2).
greatest challenge to synthetic chemists. It is thus not sur-
prising that only one total synthesis of tubulysin D (1D), by
the Ellman group, has been reported to date.[5] Their studies
also indicated, however, that the N,O-acetal per se is not
necessary for high biological activity, a result supported by
work by Wipf et al.:[6] compounds incorporating a simple
N-methylamide at the same position can be equally po-
tent,[7] and only when the N-substituent is removed com-
pletely a significant drop in activity is observed.[4,8]
SAR studies have also focused on the N-terminal amino
acid Mep (which can be replaced by N-methylsarcosine)
and the C-terminal Tup, which can be substituted with a
wide range of alternative functionalities with retention of
biological activity.[7a]
[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,
P.O. Box 151150, 66041 Saarbrücken, Germany
Fax: +49-681-302-70202
During studies on the biosynthesis of the tubulysins, we
identified a range of biosynthetic intermediates in extracts
of the producing strain Angiococcus disciformis An d48, ap-
E-mail: rom@mx.uni-saarland.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900999.
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A. Ullrich, J. Herrmann, R. Müller, U. Kazmaier
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parently generated by alternative modes of operation of the
Despite the apparent simplicity of the Tup moiety, the
polyketide synthase (PKS)/nonribosomal peptide syn- stereoselective introduction of the α-methyl group is not a
thetase (NRPS) multienzyme assembly line.[10] One of the trivial issue and has represented a significant challenge in
characterized compounds was pretubulysin (2, Figure 1), a previous synthetic studies. Because our attempts to intro-
structure proposed earlier to be the first enzyme-free inter- duce the methyl group through an enolate alkylation of the
mediate in the pathway. Pretubulysin lacks the acetoxy ester modified with the Evans auxiliary[14] were unsuccess-
group (dTuv instead of Tuv) and the N,O-acetal function- ful (due to lactamization), we decided to use the more
ality, which is consistent with these groups being introduced straightforward approach of catalytic hydrogenation. The
by post-assembly line enzymes. The SAR studies on the tu- required α,β-unsaturated ester 8 was easily obtained from
bulysin analogues suggest that pretubulysin should also re- protected phenylalanine by DibalH reduction/Wittig ole-
tain significant biological activity. In addition, because it fination, as reported for dTuv (Scheme 2). No epimerization
lacks the configurationally labile acetoxy group, pretubuly- was observed in this one-pot reaction, whereas isolation of
sin is an ideal lead structure for the development of tubuly- the aldehyde intermediate resulted in nearly complete race-
sin-based anticancer drugs. We therefore pursued the total mization. Catalytic hydrogenation gave rise to the Tup de-
synthesis of pretubulysin[9] and adapted our route to afford rivative 9 as a 2:1 diastereomeric mixture. Hydrogenation
a range of derivatives.
of the free acid, as performed by Wipf,[12b] unfortunately
brought no improvement in selectivity. Interestingly, re-
duction of 8 to the corresponding allyl alcohol and catalytic
hydrogenation resulted in a reductive deoxygenation and
the formation of the protected amine 10.[15]
Results and Discussion
The synthesis of the central Ile-dTuv unit is shown in
Scheme 1. From the starting N-Boc-protected valine ester,
a DibalH reduction and in situ Wittig reaction of the re-
sulting aldehyde gave rise to the enantiomerically pure un-
saturated nitrile 3. Catalytic hydrogenation and subsequent
N-methylation to afford 4 proceeded without racemization.
The nitrile functionality was then converted into the
thioamide (compound 5),[11] which was subjected to a
Hantzsch thiazole synthesis. Trifluoroacetic acid anhydride
(TFA2O) was added to the hydroxythiazoline intermediate
to form the thiazole ring (compound 6).[12] Cleavage of the
Boc protecting group and coupling with Z-Ile gave rise to
the required dipeptide 7. BEP (2-bromo-1-ethylpyridinium
tetrafluoroborate) was used as a coupling reagent to avoid
the racemization frequently observed in couplings of N-
methyl amino acids and to achieve a high yield.[13]
Scheme 2. Synthetic attempts directed towards Tup.
This prompted us also to investigate homogeneous hy-
drogenations. Unfortunately, no hydrogenation of 8 was ob-
served in the presence of Wilkinson’s catalyst, and so we
focused on the reduction of substrate 12 with a methylene
side chain. Compound 12 was obtained from Boc-Phe by
coupling with Medrum’s acid[16] and subsequent reduction
of the keto group with NaBH4.[17] Treatment of the substi-
tuted Medrum’s acid 11 with Eschenmoser’s salt gave rise
Scheme 1. Synthesis of the Ile-dTuv fragment.
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Synthesis and Biological Evaluation of Pretubulysin and Derivatives
to the required α-substituted acrylate 12. Unfortunately,
Because the yields in the coupling step could not be im-
attempts to obtain higher selectivity with this substrate were proved, we also investigated an alternative order of peptide
unsuccessful. bond formation. Cleavage of the Z protecting group and
Although the hydrogenation with this sterically less hin- subsequent peptide coupling with protected -Pip gave rise
dered substrate proceeded cleanly, in the presence of (R)- to the tripeptide 20, which could be saponified to afford the
Binap as chiral ligand the “wrong” diastereomer epi-9 was free acid 21 in quantitative yield.
formed preferentially. To make sure that this was not the
Coupling with dTup again proceeded without difficulty
result of a mismatch situation we also investigated the reac- to give the tetrapeptide 22. The sequence was completed by
tion in the presence of the enantiomeric ligand, but with cleavage of the Z protecting group and reductive methyl-
(S)-Binap the situation was even worse. The hydrogenation ation of the pipecolic acid. Finally, saponification and acidi-
was very slow and 9 was obtained as a more or less isomeric fication gave rise to demethylpretubulysin (23).
mixture (45% ds).
With this route to hand, the synthesis of pretubulysin (2)
Because Zanda et al. had reported the separation of the was accomplished in an analogous manner (Scheme 4). In
corresponding menthyl esters of 9 by chromatography,[8a] view of SAR studies by Wipf[6] and Ellman,[7] indicating a
we converted 8 into the menthyl ester 13 (Scheme 3). Its high degree of variability at the tubulysin C-terminus, we
hydrogenation provided 14 with a slightly better selectivity, also coupled 21 with Phe to determine whether or not the
with respect to the required diastereomer, than in the case shortened compound 25 might retain good biological ac-
of 8. After separation of the diastereomers, cleavage of the tivity.
protecting groups yielded 15 in enantiomerically pure
Pretubulysin (2) and its synthetic analogues 19, 23, and
form.[8a] To evaluate whether the α-methyl group has any 25 were evaluated for their cytotoxicities relative to tubulys-
influence on the biological activity, we also synthesized the ins A (1A) and D (1D), by measurement of their inhibition
demethyl derivative (dTup) 16 in an analogous manner.
of cell growth by an MTT assay (Table 1). Tubulysin D is
known to be the more toxic of the two metabolites, with a
potency approximately six times higher than that of tubuly-
sin A.[3] Human acute myeloid leukemia cells (HL-60) were
selected for initial activity screening, because in our hands
they are reproducibly sensitive relative to other cell lines.
As predicted from the earlier SAR studies, pretubulysin (2)
retained good activity relative to tubulysins A and D (cyto-
toxicity three and five times lower, respectively). However,
all of the other analogues were less active. Removal of the
C2-methyl group as in compound 23 led to a 13-fold rela-
tive reduction in cytotoxicity, whereas compound 19, in
which the N-terminal Mep had been replaced by a N-meth-
ylsarcosine unit, was essentially inactive (IC50:
620 ngmL–1). Derivative 25, in which the Tup had been re-
placed by Phe, was similarly affected (IC50: 790 ngmL–1).
In view of these results, tubulysins A and D and com-
pounds 2 and 23 were evaluated against a wide range of
other tumor cell lines. Both 2 and 23 exhibited activity
against all cell lines in the low- or subnanomolar range,
albeit reduced relative to tubulysins A and D, with 2 being
reproducibly the more potent. On average, the demethyl de-
rivative 23 is less active by a factor of 10, although a higher
activity was observed towards epithelial carcinoma cells
Scheme 3. Synthesis of Tup (15) and dTup (16).
This readily available analogue was also used to investi- (A431).
gate the final steps of our synthesis, firstly with a simplified
These results reveal several important SAR relationships,
analogue containing N-methylsarcosine instead of -Mep in addition to the data reported by other groups.[6–8] No-
(Scheme 4). The dipeptide 7 was saponified at the ester tably, the good potency of pretubulysin (2) confirms that
functionality, and coupling with 16 gave rise to the tripep- neither the N,O-acetal nor the acetoxy functionality of Tuv
tide 17 without significant lactam formation. The Z protect- are necessary for cytotoxicity, although there is a modest
ing group could not be removed by catalytic hydrogenolysis reduction in activity (ca. 10-fold) relative to a previously
(due to the thiazole moiety), so it was instead disconnected characterized analogue in which the acetoxy group was re-
under acidic conditions. Coupling with N-methylsarcosine, tained.[6b] Comparison of the data obtained for 2 and its
activated as its pentafluorophenyl ester (PFP), provided the C2-demethyl analogue 23 reveals that the methyl group in
corresponding tetrapeptide 18 in acceptable yield. Subse- Tup also provides an approximately 10-fold enhancement
quent saponification gave rise to the simplified tubulysin in biological activity. It has previously been demonstrated
derivative 19.
that the N-terminal Mep in tubulysin D can be replaced by
Eur. J. Org. Chem. 2009, 6367–6378
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A. Ullrich, J. Herrmann, R. Müller, U. Kazmaier
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Scheme 4. Synthesis of pretubulysin and simplified derivatives.
a sarcosine unit with essentially full retention of bioactiv- in activity. A similar drop was observed for the phenylala-
ity.[7] However, this modification in combination with the nine derivative 25, suggesting the importance of this por-
loss of the C2-methyl and both labile sites as in compound tion of the molecule in the absence of the acetoxy and N,O-
19 resulted in a drastic (five orders of magnitude) decrease acetal functionalities.
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Synthesis and Biological Evaluation of Pretubulysin and Derivatives
General Procedure for in-situ DibalH Reduction/Wittig Reaction: A
solution of DibalH in hexane (1 , 2.0 equiv.) was slowly added
dropwise at –78 °C to a solution of a Boc-protected amino acid
methyl ester (1.0 equiv.) in dry toluene or dichloromethane
(3 mLmmol–1). After the system had been stirred for 30 min at
–78 °C, the phosphonium salt (2.0 equiv.) and KOtBu (2.0 equiv.),
suspended in dry toluene or dichloromethane (2 mLmmol–1), were
added to the reaction mixture. The cooling bath was removed after
1 h, and the reaction mixture was stirred overnight at room tem-
perature, poured into saturated potassium sodium tartrate
(15 mLmmol–1), and vigorously stirred for 30 min. The aqueous
layer was extracted three times with EtOAc, and the combined or-
ganic layers were dried with Na2SO4. Purification by flash
chromatography provided the elongated unsaturated amino acid
derivative.
Table 1. Cytotoxicity of tubulysin, pretubulysin, and analogues
towards a wide range of tumor cell lines.
IC50 (ngmL–1)[a]
Cell line
Origin
1A
1D
2
23
HL-60
A431
A549
acute promyelocytic leukemia[b]
epithelial carcinoma[b]
lung carcinoma[b]
0.05 0.003 0.014 0.22
0.10 0.012 1.24 0.34
0.09 0.006 4.35 31.2
0.08 0.034 1.13 9.63
0.05 0.010 0.23 1.68
COS-7
kidney carcinoma[c]
HEK293T kidney[b]
HepG2
K562
hepatocell. carcinoma[b]
0.12 0.007 0.64 8.75
chronic myelogenous leukemia[b] 0.40 0.022 0.35 2.50
KB3.1
L929
cervical carcinoma[b]
0.16 0.018 2.32 26.3
subcutaneous connective tissue[d] 0.21 0.017 3.87 40.0
SH-SY5Y neuroblastoma[b]
0.21 0.011 0.61 2.90
0.02 0.004 0.21 3.16
0.19 0.034 0.56 3.23
0.05 0.003 0.04 0.38
SW480
U-2 OS
U937
colon adenocarcinoma[b]
bone osteosarcoma[b]
histiocytic lymphoma[b]
(4S,2E)-4-(tert-Butoxycarbonylamino)-5-methylhex-2-enenitrile (3):
The nitrile 3 was obtained from Boc--valine methyl ester (1.74 g,
7.52 mmol), DibalH (1  solution in hexane, 15 mL, 15 mmol), (cy-
anomethyl)triphenylphosphonium chloride (4.67 g, 13.8 mmol),
and KOtBu (1.55 g, 13.8 mmol) by the General Procedure for in
situ DibalH reduction/Wittig reaction as a white solid (1.20 g,
5.35 mmol, 71%, Ͼ99% ee) after flash chromatography (hexane/
EtOAc, 1. 9:1, 2. 8:2). Rf = 0.29 (Hex/EtOAc, 8:2). HPLC: Re-
prosil 100 Chiral-NR 8 µm, Hex/iPrOH, 95:5, 2 mLmin–1, tR[(R)-
[a] Values each represent the average of two measurements. Incu-
bation time: 5 d. [b] Human. [c] African green monkey. [d] Mouse.
Conclusions
In conclusion, we have shown that the complex struc-
tures of the tubulysins can be significantly reduced without
dramatic drops in their biological activities. Pretubulysin
(2), which was identified as direct biosynthetic precursor of
the tubulysins (1), has an activity similar to or slightly less
than that of tubulysin A (1A) (1–50-fold), but retains low-
or sub-nM activity. Taken together, these findings should
aid in future efforts to design simplified, yet highly potent
analogues of the tubulysins for evaluation as anticancer
agents.
3] = 5.91 min, tR[(S)-3] = 6.94 min; m.p. 62 °C. [α]2D0 = –5.7 (c =
3
1.0, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 0.90 (d, J6,5
=
3
6.7 Hz, 3 H, 6-H), 0.93 (d, J6Ј,5 = 6.7 Hz, 3 H, 6Ј-H), 1.43 (s, 9 H,
9-H), 1.84 (m, 1 H, 5-H), 4.11 (br. s, 1 H, 4-H), 4.50 (br. s, 1 H,
3
4
NH), 5.47 (dd, J2,3 = 16.5, J2,4 = 1.6 Hz, 1 H, 2-H), 6.62 (dd,
3J3,2 = 16.5, J3,4 = 5.5 Hz, 1 H, 3-H) ppm. 13C NMR (125 MHz,
3
CDCl3): δ = 18.0, 18.8, 28.3, 32.0, 57.4, 80.2, 100.4, 117.1, 154.0,
155.1 ppm. HRMS (CI): m/z calcd. for C12H21N2O2 [M + H]+
225.1603; found 225.1564. C12H20N2O2 (224.30): calcd. C 64.26, H
8.99, N 12.49; found C 64.04, H 9.25, N 12.86.
(4R)-4-(tert-Butoxycarbonyl-methylamino)-5-methylhexanenitrile (4):
A solution of 3 (2.77 g, 12.3 mmol) in MeOH (40 mL) was stirred
under hydrogen in the presence of Pd/C (10%, 138 mg) until re-
duction was complete. After filtration through celite, purification
by flash chromatography (hexane/EtOAc, 8:2) gave the saturated
nitrile as a white solid in 87% yield (2.41 g, 10.6 mmol). A solution
of this nitrile (2.39 g, 10.6 mmol) and methyl iodide (2.6 mL,
41.8 mmol) in dry DMF (40 mL) was treated at 0 °C with a suspen-
sion of NaH (60%, 964 mg, 24.1 mmol) in dry DMF (10 mL). The
reaction mixture was allowed to warm to room temperature over-
night. After dilution with water (200 mL) and saturated NH4Cl
(50 mL), the solution was extracted three times with EtOAc, and
the combined organic layers were dried with Na2SO4. The crude
product was purified by flash chromatography (hexane/EtOAc, 8:2)
to afford 4 (2.51 g, 10.4 mmol, 98%) as a colorless oil. Rf = 0.18
(Hex/EtOAc, 8:2). [α]2D0 = +7.0 (c = 1.0, CHCl3). 1H NMR analysis
Experimental Section
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
ethyl acetate (EtOAc) and hexane were generally used as eluents.
Analysis by TLC was carried out with commercially precoated
Polygram SIL-G/UV 254 plates (Machery–Nagel, Dueren). Visual-
ization was accomplished with the aid of UV light, KMnO4 solu-
1
tion, or iodine. H NMR and 13C NMR spectra were obtained at
room temperature with Bruker AV 400 and AV 500 spectrometers.
Chemical shifts are expressed in ppm relative to internal solvent.
Asterisked signals are estimated values from 13C- or HSQC-spectra.
Selected signals of minor isomers are extracted from the NMR
spectra of the isomeric mixtures. The enantiomeric and dia-
stereomeric ratios were determined by HPLC with a Shimadzu
10A VP with chiral columns (Reprosil 100 Chiral-NR 8 µm, Chi-
ralcel OD-H) or an achiral silica gel column (Lichrosorb 5 µ Si-
60 A). Optical rotation measurements were performed with a Per-
kin–Elmer 341 polarimeter, with concentrations given in g/100 mL.
Melting points were determined with a MEL-TEMP II apparatus
and are uncorrected. High-resolution mass spectra were recorded
with a Finnigan MAT 95Q instrument by the CI technique. Ele-
mental analyses were performed with a Leco CHN900 instrument.
1
at room temperature showed a 1:1 mixture of rotamers: H NMR
3
(400 MHz, CDCl3): δ = 0.83 (d, J6,5 = 7.0 Hz, 3 H, 6-H), 0.85 (d,
3
3J6,5 = 6.8 Hz, 3 H, 6-H), 0.93 (d, J6Ј,5 = 6.5 Hz, 3 H, 6Ј-H), 0.94
(d, 3J6Ј,5 = 6.5 Hz, 3 H, 6Ј-H), 1.43 (s, 9 H, 10-H), 1.45 (s, 9 H, 10-
H), 1.62–1.75 (m, 4 H, 3-Ha, 5-H), 1.91–2.03 (m, 2 H, 3-Hb), 2.12–
2.34 (m, 4 H, 2-Ha, 2-Hb), 2.64 (s, 6 H, 7-H), 3.62 (br. s, 1 H, 4-H),
3
3
3.68 (ddd, J4,3a/b ≈ 3J4,5 = 10.8, J4,3a/b = 3.3 Hz, 1 H, 4-H) ppm.
13C NMR (100 MHz, CDCl3): δ = 14.5, 19.6, 19.8, 20.0, 20.1, 25.9,
28.4, 28.8, 30.3, 30.5, 60.8, 61.1, 79.7, 80.2, 119.3, 119.7, 156.3,
156.6 ppm. HRMS (CI): m/z calcd. for C13H25N2O2 [M + H]+
241.1916; found 241.1922. C13H24N2O2 (240.35): calcd. C 64.97, H
10.06, N 11.66; found C 65.14, H 9.91, N 11.50.
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3
(4R)-4-[(tert-Butoxycarbonyl)methylamino]-5-methylhexanethio-
amide (5): Hydrogen sulfide was passed at –78 °C for 1 h through a
solution of nitrile 4 (1.85 g, 7.70 mmol) and triethylamine (5.4 mL,
38.4 mmol) in chloroform (15 mL).[11] The flask was sealed and
the reaction mixture was allowed to warm to room temperature
overnight. After stirring for 5 d at room temperature the solution
was washed with HCl (1 ), saturated NaHCO3, and water. After
drying of the organic phase over Na2SO4 and evaporation of the
solvent in vacuo, the thioamide 5 (1.94 g, 7.07 mmol, 92%) was
obtained after flash chromatography (hexane/EtOAc, 1:1) as a
white solid. Rf = 0.33 (Hex/EtOAc, 1:1). [α]2D0 = +20.7 (c = 1.0,
CHCl3). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.82 (d,
5-H), 3.63 (br. s, 1 H, 7-H), 4.39 (q, J14,15 = 7.0 Hz, 2 H, 14-H),
8.01 (s, 1 H, 3-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 20.1,
20.3, 30.1, 30.7, 30.8, 61.4, 79.2, 126.9, 146.8, 156.4, 161.5,
171.4 ppm. HRMS (CI): m/z calcd. for C18H31N2O4S [M + H]+
371.2004; found 371.1964.
Ethyl (R)-2-{3-[(N-Benzyloxycarbonyl-(S)-isoleucyl)methylamino]-4-
methylpentyl}thiazole-4-carboxylate (7): A solution of HCl in diox-
ane (4 , 7.5 mL, 30 mmol) was added at 0 °C to thiazole 6 (1.11 g,
3.00 mmol). The solvent was evaporated in vacuo after complete
deprotection (TLC monitoring) and the hydrochloride salt was
dried in high vacuum. N-Deprotected 6, Z-protected -isoleucine
(876 mg, 3.30 mmol), and 2-bromo-1-ethylpyridinium tetrafluo-
roborate (BEP, 905 mg, 3.30 mmol) were dissolved in dry dichloro-
methane (30 mL) and diisopropyl ethylamine (2.04 mL, 12 mmol)
was added dropwise to this solution at –10 °C.[13] The cooling bath
was removed after 20 min and the reaction mixture was stirred at
room temperature overnight. The dipeptide 7 (1.42 g, 2.64 mmol,
88%) was obtained after flash chromatography (hexane/EtOAc,
3J6,5 = 6.5 Hz, 3 H, 6-H), 0.93 (d, J6Ј,5 = 6.8 Hz, 3 H, 6Ј-H), 1.44
3
2
3
(s, 9 H, 10-H), 1.60 (m, 1 H, 5-H), 1.70 (dddd, J3a,3b = 14.6, J3a,4
= 12.3, 3J3a,2b = 6.8, 3J3a,2a = 3.5 Hz, 1 H, 3-Ha), 2.04 (dddd, 2J3b,3a
3
3
= 14.6, J3b,2a = 11.0, J3b,2b ≈ 3J3b,4 = 3.0 Hz, 1 H, 3-Hb), 2.39
(ddd, 2J2a,2b = 12.6, 3J2a,3b = 11.0, 3J2a,3a = 3.5 Hz, 1 H, 2-Ha), 2.61
2
3
3
(s, 3 H, 7-H), 2.73 (dddd, J2b,2a = 12.6, J2b,3a = 6.8, J2b,3b = 3.5,
4J2b,4 = 1.5 Hz, 1 H, 2-Hb), 3.68 (ddd, J4,3a = 12.3, J4,5 = 10.3,
3J4,3b = 2.5 Hz, 1 H, 4-H), 7.48 (br. s, 1 H, NH), 8.71 (br. s, 1 H,
NHЈ) ppm. 13C NMR (100 MHz, CDCl3): δ = 19.9, 20.2, 27.9,
28.4, 28.7, 29.8, 41.9, 60.0, 80.2, 158.0, 210.4 ppm. Selected signals
of the minor rotamer: 1H NMR (400 MHz, CDCl3): δ = 1.42 (s,
9 H, 10-H), 2.24 (m, 1 H, 3-Hb), 2.43–2.57 (m, 2 H, 2-Ha, 2-Hb),
2.65 (s, 3 H, 7-H), 3.61 (br. s, 1 H, 4-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 19.9, 28.5, 41.5, 79.8, 153.3, 211.6 ppm. HRMS (CI):
m/z calcd. for C13H27N2O2S [M + H]+ 275.1793; found 275.1769.
C13H26N2O2S (274.42): calcd. C 56.90, H 9.55, N 10.21; found C
57.04, H 9.25, N 10.10.
3
3
1. 7:3, 2. 1:1) as a yellow oil. Rf = 0.34 (Hex/EtOAc, 1:1). [α]2D0
=
–22.9 (c = 1.0, CHCl3). Major rotamer: 1H NMR (400 MHz,
3
3
CDCl3): δ = 0.75 (d, J9,8 = 6.5 Hz, 3 H, 9-H), 0.86 (t, J15,14
=
3
7.4 Hz, 3 H, 15-H), 0.95 (d, J16,13 = 6.5 Hz, 3 H, 16-H), 0.96 (d,
3J9Ј,8 = 6.5 Hz, 3 H, 9Ј-H), 1.11 (ddq, J14a,14b = 13.6, J14a,13
=
2
3
3
3
9.7, J14a,15 = 7.3 Hz, 1 H, 14-Ha), 1.37 (t, J24,23 = 7.0 Hz, 3 H,
24-H), 1.57 (dqd, J14b,14a = 13.6, J14b,15 = 7.6, J14b,13 = 3.0 Hz,
1 H, 14-Hb), 1.63–1.79 (m, 2 H, 8-H, 13-H), 1.88 (m, 1 H, 6-Ha),
2.14 (m, 1 H, 6-Hb), 2.83 (dt, J5a,5b = 15.2, J5a,6 = 6.3 Hz, 1 H,
5-Ha), 2.89 (dt, J5b,5a = 15.2, J5b,6 = 6.3 Hz, 1 H, 5-Hb), 2.94 (s,
2
3
3
2
3
2
3
3
3 H, 10-H), 4.32 (m, 1 H, 7-H), 4.39 (q, J23,24 = 7.0 Hz, 1 H, 23-
3
3
H), 4.52 (dd, J12,NH = 9.5, J12,13 = 6.8 Hz, 1 H, 12-H), 5.05 (d,
Ethyl (R)-2-{3-[(tert-Butoxycarbonyl)methylamino]-4-methylpentyl}-
thiazole-4-carboxylate (6): Ethyl bromopyruvate (90%, 770 µL,
5.52 mmol) was added dropwise at –10 °C to a solution of thioam-
ide 5 (1.37 g, 4.99 mmol) in dry acetone (7.5 mL).[12] After 1.5 h
the reaction mixture was poured into a vigorously stirred mixture
of dichloromethane (25 mL) and saturated KHCO3 (25 mL). The
aqueous layer was extracted with dichloromethane, and the solvent
was evaporated in vacuo after drying of the combined organic lay-
ers over Na2SO4. Pyridine (890 µL, 11.0 mmol) was added at
–20 °C to a solution of the hydroxythiazoline intermediate in dry
dichloromethane (6 mL), and trifluoroacetic anhydride (1.05 mL,
5.49 mmol) was added dropwise. The reaction mixture was allowed
to warm to 0 °C over 2 h and was stirred overnight at room tem-
perature. The solution was diluted with dichloromethane (10 mL)
and washed twice with saturated NaHCO3. The aqueous layers
were extracted with dichloromethane, and the combined organic
layers were washed with KHSO4 (1 ) and dried with Na2SO4. The
crude product was purified by flash chromatography (hexane/
EtOAc, 8:2, 7:3, 1:1) to afford the thiazole 6 (1.56 g, 4.22 mmol,
84%, Ͼ99% ee) as a pale yellow oil. Rf = 0.23 (Hex/EtOAc, 7:3).
HPLC: Chiralcel OD-H, Hex/iPrOH, 9:1, 1 mLmin–1, tR[(S)-6] =
2J18a,18b = 12.4 Hz, 1 H, 18-Ha), 5.08 (d, J18b,18a = 12.4 Hz, 1 H,
2
3
18-Hb), 5.40 (d, JNH,12 = 9.5 Hz, 1 H, NHIle), 7.18–7.36 (m, 5-H,
arom. H), 8.01 (s, 1 H, 3-H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 11.2, 14.3, 16.0, 19.5, 20.0, 23.8, 29.4, 29.7, 30.1, 30.6, 37.4, 55.7,
59.1, 61.3, 66.7, 126.9, 127.8, 128.0, 128.4, 136.4, 146.9, 156.4,
161.3, 170.9, 173.3 ppm. Selected signals of the minor rotamer: 1H
3
NMR (400 MHz, CDCl3): δ = 1.04 (d, J9Ј,8 = 6.8 Hz, 3 H, 9Ј-H),
2.23 (m, 1 H, 6-Ha), 2.74 (s, 3 H, 10-H), 3.58 (ddd, 3J7,6a/b ≈ 3J7,8
10.2, J7,6a/b = 3.3 Hz, 1 H, 7-H), 4.57 (dd, J12,NH = 9.6, J12,13
=
=
3
3
3
6.3 Hz, 1 H, 12-H), 7.98 (s, 1 H, 3-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 11.3, 16.1, 20.3, 20.4, 23.5, 27.3, 29.8, 31.3, 37.8, 55.2,
62.6, 66.9, 128.4, 136.3, 156.2, 170.5, 172.5 ppm. HRMS (CI): m/z
calcd. for C27H40N3O5S [M + H]+ 518.2689; found 518.2721.
C27H39N3O5S (517.68): calcd. C 62.64, H 7.59, N 8.12; found C
62.30, H 7.61, N 8.24.
Ethyl
(4S,2E)-4-[(tert-Butoxycarbonyl)amino]-2-methyl-5-phenyl-
pent-2-enoate (8):[12b,18] The unsaturated ester 8 was obtained as a
white solid (4.78 g, 14.3 mmol, 82%, Ͼ99% ee) from Boc--phenyl-
alanine methyl ester (4.90 g, 17.5 mmol), DibalH (1  in hexane,
35 mL, 35 mmol), (1-ethoxycarbonyl-ethyl)-triphenylphosphonium
bromide (15.5 g, 35.0 mmol), and KOtBu (4.03 g, 35.9 mmol) by
the General Procedure for in situ DibalH reduction/Wittig reaction
and flash chromatography (hexane/EtOAc, 95:5, 9:1). Rf = 0.31
(Hex/EtOAc, 8:2). HPLC: Reprosil 100 Chiral-NR 8 µm, Hex/iP-
rOH, 8:2, 2 mLmin–1, tR[(R)-8] = 5.96 min, tR[(S)-8] = 6.86 min;
1
6.91 min, tR[(R)-6] = 9.12 min. [α]2D0 = –13.3 (c = 1.0, CHCl3). H
NMR analysis at room temperature showed a 54:46 mixture of
rotamers; major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.83
3
3
(d, J9,8 = 6.8 Hz, 3 H, 9-H), 0.93 (d, J9Ј,8 = 6.5 Hz, 3 H, 9Ј-H),
3
1.38 (t, J15,14 = 7.2 Hz, 3 H, 15-H), 1.44 (s, 9 H, 13-H), 1.65 (m,
1 H, 8-H), 1.82 (m, 1 H, 6-Ha), 2.12 (m, 1 H, 6-Hb), 2.63 (s, 3 H,
m.p. 68 °C. [α]2D0 = +33.0 (c = 1.0, CHCl3). 1H NMR (500 MHz,
3
3
10-H), 2.95 (t, J5,6 = 8.1 Hz, 2 H, 5-H), 3.82 (m, 1 H, 7-H), 4.40 CDCl3): δ = 1.26 (t, J15,14 = 7.3 Hz, 3 H, 15-H), 1.38 (s, 9 H, 13-
3
4
2
(q, J14,15 = 7.2 Hz, 2 H, 14-H), 8.03 (s, 1 H, 3-H) ppm. 13C NMR H), 1.68 (d, J10,3 = 1.5 Hz, 3 H, 10-H), 2.76 (dd, J5a,5b = 13.3,
3
(100 MHz, CDCl3): δ = 14.4, 19.6, 19.9, 28.2, 28.5, 30.0, 30.5, 30.7,
3J5a,4 = 7.0 Hz, 1 H, 5-Ha), 2.90 (m, 1 H, 5-Hb), 4.16 (q, J14,15
=
60.3, 61.4, 79.6, 128.6, 147.0, 156.6, 161.4, 171.7 ppm. Selected sig-
nals of the minor rotamer: H NMR (400 MHz, CDCl3): δ = 0.94
7.3 Hz, 2 H, 14-H), 4.55 (br. s, 1 H, NH), 4.64 (br. s, 1 H, 4-H),
1
3
4
3
6.49 (dd, J3,4 = 9.1, J3,10 = 1.5 Hz, 1 H, 3-H), 7.15 (d, J7,8 =
3
3
3
(d, J9Ј,8 = 6.8 Hz, 3 H, 9Ј-H), 1.38 (t, J15,14 = 7.0 Hz, 3 H, 15-H), 7.0 Hz, 2 H, 7-H), 7.20 (t, J9,8 = 7.3 Hz, 1 H, 9-H), 7.26 (dd,
1.41 (s, 9 H, 13-H), 2.68 (s, 3 H, 10-H), 2.94 (t, J5,6 = 7.9 Hz, 2 H, 3J8,7 ≈ 3J8,9
=
7.2 Hz, 2 H, 8-H) ppm. 13C NMR (100 MHz,
3
6372
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6367–6378

Synthesis and Biological Evaluation of Pretubulysin and Derivatives
3
CDCl3): δ = 12.5, 14.2, 28.3, 41.1, 50.1, 60.6, 79.6, 126.6, 128.4,
(m, 1 H, 5-Ha), 2.26 (m, 1 H, 5-Hb), 2.84 (d, J3,4 = 6.0 Hz, 2 H,
129.2, 129.5, 136.7, 140.2, 154.9, 167.7 ppm.
3-H), 3.89 (br. s, 1 H, 2-H), 4.21 (m, 1 H, 4-H), 4.44 (br. s, 1 H,
NH), 7.15–7.23 (m, 3 H, 7-H, 9-H), 7.28 (dd, 3J8,7 = 3J8,9 = 7.3 Hz,
2 H, 8-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 26.0, 28.2, 28.6,
31.4, 41.8, 44.3, 49.9, 79.6, 105.0, 126.7, 128.6, 129.3, 137.1, 156.7,
165.5, 165.7 ppm.
Ethyl (4S)-4-[(tert-Butoxycarbonyl)amino]-2-methyl-5-phenylpenta-
noate (9): A solution of 8 (219 mg, 0.657 mmol) in MeOH (8 mL)
was stirred under hydrogen with Pd/C (10%, 20 mg) until no
double bond could be detected (TLC), after which the catalyst was
filtered off through celite. Purification by flash chromatography
Methyl (S)-4-[(tert-Butoxycarbonyl)amino]-2-methylene-5-phenyl-
pentanoate (12):[20] As described in the literature, 12 was obtained
from 11 (250 mg, 0.662 mmol) and N,N-dimethylmethyleneammo-
nium iodide (308 mg, 1.66 mmol) in dry MeOH (8 mL) as white
solid (169 mg, 0.529 mmol, 80%) after column chromatography
(hexane/EtOAc, 9:1) afforded
9 as a colorless oil (195 mg,
0.581 mmol, 88%, dr 2:1). Rf = 0.29 (Hex/EtOAc, 8:2). Major dia-
stereomer: 1H NMR (500 MHz, CDCl3): δ = 1.12 (d, J10,2
=
3
3
7.3 Hz, 3 H, 10-H), 1.21 (t, J15,14 = 7.2 Hz, 3 H, 15-H), 1.37 (s,
2
3
9 H, 13-H), 1.40 (m, 1 H, 3-Ha), 1.86 (ddd, J3b,3a = 13.3, J3b,2/4
1
(hexane/EtOAc, 8:2). Rf = 0.29 (Hex/EtOAc, 8:2); m.p. 102 °C. H
3
= 9.7, J3b,4/2 = 3.7 Hz, 1 H, 3-Hb), 2.55 (m, 1 H, 2-H), 2.75 (d,
NMR (400 MHz, CDCl3): δ = 1.36 (s, 9 H, 13-H), 2.30 (m, 1 H,
3-Ha), 2.53 (dd, 2J3b,3a = 14.1, 3J3b,4 = 4.2 Hz, 1 H, 3-Hb), 2.75 (dd,
2J5a,5b = 13.6, 3J5a,4 = 6.8 Hz, 1 H, 5-Ha), 2.86 (m, 1 H, 5-Hb), 3.73
(s, 3 H, 14-H), 3.99 (m, 1 H, 4-H), 4.44 (br. s, 1 H, NH), 5.56 (d,
2J10a,10b = 1.0 Hz, 1 H, 10-Ha), 6.19 (d, 2J10b,10a = 1.0 Hz, 1 H, 10-
3J5,4 = 5.7 Hz, 1 H, 5-H), 3.85 (m, 1 H, 4-H), 4.09 (q, J14,15
=
3
3
7.0 Hz, 2 H, 14-H), 4.30 (d, JNH,4 = 8.8 Hz, 1 H, NH), 7.15 (d,
3
3J7,8 = 7.3 Hz, 2 H, 7-H), 7.18 (t, J9,8 = 7.3 Hz, 1 H, 9-H), 7.26
3
(dd, J8,7
=
3J8,9 = 7.3 Hz, 2 H, 8-H) ppm. 13C NMR (125 MHz,
CDCl3): δ = 14.2, 17.6, 28.4, 36.4, 37.9, 41.4, 49.8, 60.4, 79.0, 126.3,
3
3
Hb), 7.15–7.23 (m, 3 H, 7-H, 9-H), 7.28 (dddd, J8,7 = J8,9 = 7.2,
128.3, 129.5, 137.9, 155.1, 176.2 ppm. Selected signals of the minor
4J8,8’ 5J8,7Ј = 1.4 Hz, 2 H, 8-H) ppm. 13C NMR (100 MHz,
=
diastereomer: 1H NMR (500 MHz, CDCl3): δ = 1.10 (d, J10,2
=
3
CDCl3): δ = 28.3, 36.5, 41.3, 51.3, 51.9, 79.0, 126.4, 127.4, 128.4,
129.4, 137.3, 138.0, 155.3, 167.7 ppm.
7.3 Hz, 3 H, 10-H), 1.22 (t, 3J15,14 = 7.2 Hz, 3 H, 15-H), 1.49 (ddd,
3
3
2J3a,3b = 14.1, J3a,2/4 = 7.3, J3a,4/2 = 3.7 Hz, 1 H, 3-Ha), 1.71 (m,
2
3
1 H, 3-Hb), 2.44 (m, 1 H, 2-H), 2.69 (dd, J5a,5b = 13.6, J5a,4
=
(1R,2S,5R)-(–)-Menthyl (4S,2E)-4-[(tert-Butoxycarbonyl)amino]-2-
methyl-5-phenylpent-2-enoate (13): A mixture of ethyl ester 8
(668 mg, 2.00 mmol) and NaOH (1 , 2.4 mL, 2.4 mmol) in diox-
ane (20 mL) was heated to 80 °C for 2 h. The solvent was evapo-
rated in vacuo, and the residue was dissolved in water. After extrac-
tion with EtOAc, and discarding of the organic layer the aqueous
layer was acidified to pH 2 with HCl (1 ) and extracted twice with
EtOAc. The combined organic layers were dried with Na2SO4 and
the solvent was evaporated in vacuo to give the corresponding acid
(582 mg, 1.91 mmol, 95%) as a white solid. A solution of DCC
(483 mg, 2.34 mmol) in diethyl ether (3 mL) was added at 0 °C to
a solution of this acid (657 mg, 2.15 mmol), (–)-menthol (840 mg,
5.38 mmol), and DMAP (26 mg, 0.213 mmol) in diethyl ether
(21.5 mmol). The reaction mixture was allowed to warm to room
temperature overnight. After filtration of the precipitated urea the
crude product was purified by flash chromatography (hexane/
EtOAc, 95:5) to give the unsaturated menthyl ester 13 (690 mg,
1.56 mmol, 72%) as a white solid. Rf = 0.43 (Hex/EtOAc, 8:2); m.p.
89–90 °C. [α]2D0 = –27.8 (c = 1.0, CHCl3). 1H NMR (400 MHz,
7.0 Hz, 1 H, 5-Ha), 2.79 (dd, 2J5b,5a = 13.6, J5b,4 = 5.7 Hz, 1 H, 5-
3
3
Hb), 3.75 (m, 1 H, 4-H), 4.10 (q, J14,15 = 7.0 Hz, 2 H, 14-H) ppm.
13C NMR (125 MHz, CDCl3): δ = 14.2, 17.3, 37.0, 37.6, 42.4, 50.3,
60.4, 155.3 ppm. HRMS (CI): m/z calcd. for C19H30NO4 [M +
H]+ 336.2175; found 336.2187.
(R)-N-(tert-Butoxycarbonyl)-4-methyl-1-phenylpentan-2-amine (10):[19]
A solution of DibalH in hexane (1 , 1.3 mL, 1.3 mmol) was added
dropwise at 0 °C to a solution of ester 8 (136 mg, 0.41 mmol) in
dichloromethane (2.5 mL). After 2.5 h the reaction mixture was
poured into saturated potassium sodium tartrate and stirred vigor-
ously. The aqueous layer was extracted three times with dichloro-
methane and the combined organic layers were dried with Na2SO4.
The allyl alcohol was obtained by flash chromatography (hexane/
EtOAc, 7:3, 6:4) as a white solid (83 mg, 0.285 mmol, 70%). A
solution of this allyl alcohol (69 mg, 0.237 mmol) in MeOH
(2.8 mL) was stirred under hydrogen with Pd/C (10%, 7 mg) until
complete hydrogenation (TLC) and the catalyst was filtered off
through a pad of celite. Purification by flash chromatography (hex-
ane/EtOAc, 8:2) afforded the amide 10 as a white solid (58 mg,
0.209 mmol, 88%). Rf = 0.60 (Hex/EtOAc, 1:1); m.p. 115 °C. [α]2D0
3
CDCl3): δ = 0.74 (d, J21,20 = 6.8 Hz, 3 H, 21-H), 0.85 [dddd,
2J17ax,17eq ≈ 3J17ax,16ax ≈ 3J17ax,18(ax) = 11.8, 3J17ax,16eq = 2.8 Hz, 1 H,
3
3
17-Hax], 0.88 (d, J22,18 = 6.3 Hz, 3 H, 22-H), 0.89 (d, J21Ј,20
=
1
= +36.1 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3): δ = 0.85
6.8 Hz, 3 H, 21Ј-H), 0.93 [ddd, J19ax,19eq ≈ 3J19ax,18ax ≈ J19ax,14(ax)
2
3
3
3
(d, J1,2 = 6.6 Hz, 3 H, 1-H), 0.87 (d, J1Ј,2 = 7.0 Hz, 3 H, 1Ј-H),
2
=
11.6 Hz, 1 H, 19-Hax], 1.05 [dddd, J16ax,16eq ≈ 3J16ax,15(ax)
3
3
1.22 (dd, J3,2/4 = 7.3, J3,4/2 = 7.0 Hz, 2 H, 3-H), 1.38 (s, 9 H, 12-
≈ 3J16ax,17ax = 12.9, J16ax,17eq = 3.0 Hz, 1 H, 16-Hax], 1.39 [dddd,
3
H), 1.66 (m, 1 H, 2-H), 2.73 (m, 2 H, 5-H), 3.77 (m, 1 H, 4-H),
3J15(ax),14(ax) ≈ 3J15(ax),16ax = 10.0, J15(ax),16eq
≈
3J15(ax),20 = 3.0 Hz,
3
3
4.22 (br. s, 1 H, NH), 7.15 (d, J7,8 = 7.3 Hz, 2 H, 7-H), 7.18 (t,
1 H, 15-H(ax)], 1.39 (s, 9 H, 13-H), 1.48 [m, 1 H, 18-H(ax)], 1.63–
3
3
3J9,8 = 7.3 Hz, 1 H, 9-H), 7.26 (dd, J8,7 = J8,9 = 7.3 Hz, 2 H, 8-
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 23.2, 24.8, 28.4, 41.8,
43.6, 49.6, 78.9, 126.2, 128.2, 129.6, 138.3, 155.4 ppm.
4
1.71 (m, 2 H, 16-Heq, 17-Heq), 1.69 (d, J10,3 = 1.5 Hz, 3 H, 10-H),
3
3
1.83 [qqd, J20,21
=
3J20,21Ј = 6.8, J20,15(ax) = 2.8 Hz, 1 H, 20-H],
1.98 (m, 1 H, 19-Heq), 2.77 (dd, 2J5a,5b = 13.3, 3J5a,4 = 7.0 Hz, 1 H,
(R)-5-{2-[(tert-Butoxycarbonyl)amino]-3-phenylpropyl}-2,2-dimeth- 5-Ha), 2.91 (dd, 2J5b,5a = 13.3, 3J5b,4 = 5.5 Hz, 1 H, 5-Hb), 4.54 (br.
yl-1,3-dioxane-4,6-dione (11):[17] As described in the literature, Boc-
-phenylalanine (5.32 g, 20.1 mmol) and Meldrum’s acid[16] (3.21 g,
22.3 mmol) were coupled with the aid of dicyclohexyl carbodiimide
(4.76 g, 23.1 mmol) and DMAP (3.95 g, 32.3 mmol) in dichloro-
methane (150 mL). The intermediate was reduced with sodium
borohydride (1.90 g, 50.2 mmol) and acetic acid (13.5 mL,
236 mmol) to give 11 as a white solid (4.35 g, 9.66 mmol, 48%)
after column chromatography (hexane/EtOAc, 8:2). Rf = 0.49 (Hex/
EtOAc, 1:1); m.p. 111–113 °C. 1H NMR (400 MHz, CDCl3): δ =
s, 1 H, NH), 4.64 (br. s, 1 H, 4-H), 4.68 [ddd, J14(ax),15(ax)
3
3
≈ 3J14(ax),19ax = 10.8, J14(ax),19eq = 4.5 Hz, 1 H, 14-H(ax)], 6.44 (dd,
4
3J3,4 = 9.2, J3,10 = 1.5 Hz, 1 H, 3-H), 7.12 (m, 2 H, 7-H), 7.19 (tt,
4
3
3J9,8 = 7.3, J9,7 = 1.9 Hz, 1 H, 9-H), 7.25 (dddd, J8,7 ≈ 3J8,9
7.1, J8,8Ј
=
≈
5J8,7Ј = 1.8 Hz, 2 H, 8-H) ppm. 13C NMR (100 MHz,
4
CDCl3): δ = 12.7, 16.4, 20.8, 22.0, 23.5, 26.4, 28.3, 31.4, 34.3, 40.9,
41.2, 47.2, 50.0, 74.5, 79.7, 126.7, 128.4, 129.6, 139.6, 154.9,
167.1 ppm. C27H41NO4 (443.63): calcd. C 73.10, H 9.32, N 3.16;
found C 73.15, H 9.08, N 3.21. HRMS (CI): m/z calcd. for
1.33 (s, 9 H, 12-H), 1.71 (s, 3 H, 14-H), 1.75 (s, 3 H, 14Ј-H), 2.13 C27H42NO4 [M + H]+ 444.3114; found 444.3077.
Eur. J. Org. Chem. 2009, 6367–6378
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6373

A. Ullrich, J. Herrmann, R. Müller, U. Kazmaier
FULL PAPER
1
(–)-Menthyl (2SR,4R)-4-[(tert-Butoxycarbonyl)amino]-2-methyl-5- MeOH); m.p. 134–137 °C. H NMR (400 MHz, CDCl3): δ = 1.13
3
2
3
phenylpentanoate (14/epi-14):[8a] A solution of menthyl ester 13
(599 mg, 1.35 mmol) in methanol (13.5 mL) was stirred under hy-
drogen with Pd/C (5%, 60 mg) until complete hydrogenation was
observed (TLC) and the catalyst was filtered off through a pad of
celite. NMR of the crude product showed a 1:3 mixture of epi-14/
14. The diastereomers were separated by column chromatography
(1. hexane/diethyl ether, 9:1, 2. hexane/EtOAc, 8:2) to give epi-14
(d, J10,2 = 7.0 Hz, 3 H, 10-H), 1.78 (ddd, J3a,3b = 13.8, J3a,2/4 =
3
2
3
8.9, J3a,2/4 = 3.8 Hz, 1 H, 3-Ha), 1.97 (ddd, J3b,3a = 13.8, J3b,2/4
= 10.7, J3b,2/4 = 3.0 Hz, 1 H, 3-Hb), 2.90 (dd, J5a,5b = 13.6, J5a,4
3
2
3
2
= 8.9 Hz, 1 H, 5-Ha), 2.92 (m, 1 H, 2-H), 3.27 (dd, J5b,5a = 13.6,
3J5b,4 = 5.3 Hz, 1 H, 5-Hb), 3.58 (s, 3 H, 11-H), 3.60 (br. s, 1 H, 4-
H), 7.20–7.26 (m, 3 H, 7-H, 9-H), 7.30 (dd, J8,7 ≈ 3J8,9 = 7.2 Hz,
3
2 H, 8-H), 8.52 (br. s, 3 H, NH3+) ppm. 13C NMR (100 MHz,
(89 mg, 0.200 mmol, 15%, Ͼ99% ds), 14 (361 mg, 0.810 mmol, CDCl3): δ = 17.7, 35.9, 36.0, 39.7, 51.9, 52.2, 127.3, 128.9, 129.3,
60%, 98% ds), and a mixed fraction (121 mg, 0.272 mmol, 20%,
135.4, 175.7 ppm.
epi-14:14 2.9:1).
Methyl (4R)-4-Amino-5-phenylpentanoate, Hydrochloride (16): Boc-
-phenylalanine methyl ester (4.27 g, 15.3 mmol) was reduced to
the aldehyde with DibalH (1  solution in hexane, 33 mL,
33 mmol) by the General Procedure for in situ DibalH reduction/
Wittig reaction and elongated with methyl (triphenyl-λ5-phosphan-
yliden)acetate (10.4 g, 31.1 mmol). The Wittig product was ob-
tained after flash chromatography (hexane/EtOAc, 8:2) as white
solid (2.59 g, 8.47 mmol, 55%). A solution of the Wittig product
(1.37 g, 4.49 mmol) was stirred under hydrogen with Pd/C (10%,
68 mg) until complete hydrogenation was observed (TLC) and the
catalyst was filtered off through a pad of celite. Purification by
flash chromatography (hexane/EtOAc, 8:2) afforded the saturated
intermediate as a white solid (1.33 g, 4.34 mmol, 96%). A solution
of HCl in dioxane (4 , 6.25 mL, 25 mmol) was added at 0 °C to
the hydrogenation product (768 mg, 2.5 mmol). The solvent was
evaporated in vacuo after complete deprotection (TLC monitoring)
and the hydrochloride salt was dried in high vacuum to give the salt
16 (599 mg, 2.46 mmol, 98%) as a white solid; m.p. 117–119 °C.
[α]2D0 = –7.4 (c = 1.0, MeOH). 1H NMR (400 MHz, CDCl3): δ =
Rf(epi-14) = 0.42 (hexane/EtOAc, 8:2); m.p. 110–111 °C (epi-14).
[α]2D0 = –36.0 (c = 1.0, CHCl3, epi-14). 1H NMR (400 MHz,
3
CDCl3): δ = 0.72 (d, J21,20 = 6.8 Hz, 3 H, 21-H), 0.84 [dddd,
2J17ax,17eq ≈ 3J17ax,16ax ≈ 3J17ax,18(ax) = 12.9, 3J17ax,16eq = 3.6 Hz, 1 H,
3
3
17-Hax], 0.86 (d, J21Ј,20 = 7.0 Hz, 3 H, 21Ј-H), 0.87 (d, J22,18
=
6.5 Hz, 3 H, 22-H), 0.90 [ddd, J19ax,19eq ≈ 3J19ax,18ax
≈
3J19ax,14(ax)
2
2
=
11.8 Hz, 1 H, 19-Hax], 1.02 [dddd, J16ax,16eq ≈ 3J16ax,15(ax)
≈ 3J16ax,17ax = 13.0, 3J16ax,17eq = 3.3 Hz, 1 H, 16-Hax], 1.11 (d, 3J10,2
= 7.3 Hz, 3 H, 10-H), 1.29–1.47 [m, 2 H, 15-H(ax), 18-H(ax)], 1.38
2
3
3
(s, 9 H, 13-H), 1.49 (ddd, J3a,3b = 14.2, J3a,2/4 = 8.5, J3a,2/4
=
4.0 Hz, 1 H, 3-Ha), 1.61–1.69 (m, 2 H, 16-Heq, 17-Heq), 1.73 (ddd,
3
3
2J3b,3a = 14.2, J3b,2/4 = 11.0, J3b,2/4 = 5.5 Hz, 1 H, 3-Hb), 1.80
3
3
[qqd, J20,21
=
3J20,21Ј = 6.9, J20,15(ax) = 2.8 Hz, 1 H, 20-H], 1.93
(m, 1 H, 19-Heq), 2.44 (m, 1 H, 2-H), 2.73 (dd, J5a,5b = 13.5, 3J5a,4
2
2
3
= 6.5 Hz, 1 H, 5-Ha), 2.79 (dd, J5b,5a = 13.5, J5b,4 = 5.0 Hz, 1 H,
3
5-Hb), 3.87 (br. s, 1 H, 4-H), 4.33 (d, JNH,4 = 7.8 Hz, 1 H, NH),
3
3
4.62 [ddd, J14(ax),15(ax)
≈
3J14(ax),19ax = 10.9, J14(ax),19eq = 4.3 Hz,
3
3
1 H, 14-H(ax)], 7.14 (d, J7,8 = 7.0 Hz, 2 H, 7-H), 7.19 (tt, J9,8
=
4
3
3J8,9 = 7.2,
3
2
2.02 (td, J3,2 ≈ 3J3,4 = 7.0 Hz, 2 H, 3-H), 2.54 (dt, J2a,2b = 16.8,
7.4, J9,7 = 1.3 Hz, 1 H, 9-H), 7.26 (dddd, J8,7
≈
4J8,8Ј ≈ 5J8,7Ј = 1.4 Hz, 2 H, 8-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 16.2, 16.8, 20.8, 22.0, 23.3, 26.2, 28.4, 31.4, 34.3, 36.9,
37.2, 40.8, 41.2, 47.0, 49.8, 74.1, 79.2, 126.3, 128.3, 129.5, 137.9,
155.4, 176.1 ppm.
2
3
3J2a,3 = 7.5 Hz, 1 H, 2-Ha), 2.62 (dt, J2b,2a = 16.8, J2b,3 = 7.5 Hz,
1 H, 2-Hb), 2.93 (dd, 2J5a,5b = 13.7, 3J5a,4 = 8.9 Hz, 1 H, 5-Ha), 3.27
2
3
(dd, J5b,5a = 13.7, J5b,4 = 5.2 Hz, 1 H, 5-Hb), 3.55 (s, 3 H, 10-
H), 3.60 (br. s, 1 H, 4-H), 7.20–7.26 (m, 3 H, 7-H, 9-H), 7.29 (dd,
3J8,7 ≈ 3J8,9 = 7.3 Hz, 2 H, 8-H), 8.51 (br. s, 3 H, NH3+) ppm. 13C
NMR (100 MHz, CDCl3): δ = 27.0, 30.1, 39.3, 51.8, 53.2, 127.3,
128.9, 129.4, 135.5, 127.9 ppm. HRMS (EI): m/z calcd. for
C11H14NO [M – CH3O]+ 176.1075; found 176.1059.
Rf(14) = 0.38 (hexane/EtOAc, 8:2); m.p. 91–92 °C (14). [α]2D0
=
1
–17.2 (c = 1.0, CHCl3, 14). H NMR (400 MHz, CDCl3): δ = 0.72
(d, 3J21,20 = 7.0 Hz, 3 H, 21-H), 0.84 [dddd, 2J17ax,17eq ≈ 3J17ax,16ax
≈
3J17ax,18(ax) = 12.9, 3J17ax,16eq = 3.5 Hz, 1 H, 17-Hax], 0.87 (d, 3J21Ј,20
3
= 7.0 Hz, 3 H, 21Ј-H), 0.88 (d, J22,18 = 6.5 Hz, 3 H, 22-H), 0.91
General Procedure for Mixed-Anhydride Peptide Coupling: Isobutyl
chloroformate (1.1 equiv.) was added dropwise at –20 °C to a solu-
tion of carboxylic acid (1.0 equiv.) and N-methylmorpholine
(1.1 equiv.) in dry THF (10 mLmmol–1). After 10 min, either a
solution of free amine (1.0 equiv.) in dry THF (2 mLmmol–1) was
added dropwise or the amine was added as the solid hydrochloride
(1.0 equiv.) after addition of further N-methylmorpholine
(1.1 equiv.). The mixture was allowed to warm to room temperature
overnight. The white precipitate (hydrochloride salt of N-methyl-
morpholine) was filtered off and the filtrate was concentrated in
vacuo. The residue was dissolved in EtOAc, and washed with water,
which was extracted afterwards twice with EtOAc. The combined
organic layers were washed with saturated NaHCO3 and dried with
Na2SO4.
2
[ddd, J19ax,19eq ≈ 3J19ax,18ax
≈
3J19ax,14(ax) = 11.7 Hz, 1 H, 19-Hax],
2
3
3
1.03 [dddd, J16ax,16eq ≈ 3J16ax,15(ax) ≈ J16ax,17ax = 13.0, J16ax,17eq
=
3.4 Hz, 1 H, 16-Hax], 1.13 (d, 3J10,2 = 7.3 Hz, 3 H, 10-H), 1.30–1.54
[m, 3 H, 3-Ha, 15-H(ax), 18-H(ax)], 1.37 (s, 9 H, 13-H), 1.61–1.69
(m, 2 H, 16-Heq, 17-Heq), 1.78–1.91 (m, 2 H, 3-Hb, 20-H), 1.97 (d,
2J19eq,19ax = 11.5 Hz, 1 H, 19-Heq), 2.54 (m, 1 H, 2-H), 2.75 (dd,
3
2J5a,5b = 13.2, J5a,4 = 6.1 Hz, 1 H, 5-Ha), 2.79 (m, 1 H, 5-Hb),
3.85 (br. s, 1 H, 4-H), 4.32 (br. s, 1 H, NH), 4.65 [ddd, 3J14(ax),15(ax)
≈ 3J14(ax),19ax = 10.9, J14(ax),19eq = 4.3 Hz, 1 H, 14-H(ax)], 7.15 (d,
3
3J7,8 = 7.3 Hz, 2 H, 7-H), 7.19 (tt, J9,8 = 7.3, J9,7 = 1.2 Hz, 1 H,
3
4
9-H), 7.26 (dddd, J8,7 ≈ 3J8,9 = 7.3, J8,8Ј ≈ 5J8,7Ј = 1.3 Hz, 2 H, 8-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 16.1, 17.7, 20.8, 22.0,
23.3, 26.2, 28.4, 31.4, 34.3, 36.7, 37.6, 40.8, 41.2, 47.1, 49.9, 74.1,
79.0, 126.3, 128.3, 129.5, 137.9, 155.1, 175.7 ppm.
3
4
Methyl (4R)-4-[((R)-2-{3-[(N-Benzyloxycarbonyl-(S)-isoleucyl)meth-
ylamino]-4-methylpentyl}thiazole-4-carbonyl)amino]-5-phenylpenta-
noate (17): The free acid was obtained as a pale yellow foam
(139 mg, 0.284 mmol, 95%) by saponification of the dipeptide 7
(155 mg, 0.299 mmol) with NaOH (1 , 450 µL, 0.45 mmol). This
acid (49 mg, 100 µmol) was coupled with the hydrochloride 16
(37 mg, 152 µmol) by the General Procedure for mixed anhydride
peptide coupling, with the aid of isobutyl chloroformate (14 µL,
108 µmol) and N-methylmorpholine (2ϫ12 µL, 218 µmol). Purifi-
cation by flash chromatography (hexane/diethyl ether, 3:7, 2:8)
HPLC: Lichrosorb Si-60 5µ, hexane/EtOAc, 95:5, 2 mLmin–1,
tR(epi-14) = 7.83 min, tR(14) = 10.17 min. HRMS (CI): m/z calcd.
for C27H44NO4 [M + H]+ 446.3270; found 446.3280. C27H43NO4
(445.64): calcd. C 72.77, H 9.73, N 3.14; found C 72.42, H 9.49, N
3.16.
Methyl (2S,4R)-4-Amino-2-methyl-5-phenylpentanoate, Hydrochlo-
ride (15):[21] As described in the literature,[8a] the menthyl ester 14
(334 mg, 0.750 mmol) was deprotected and converted into the
methyl ester 15 (138 mg, 0.535 mmol, 71%). [α]2D0 = +11.2 (c = 1.0,
6374
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6367–6378

Synthesis and Biological Evaluation of Pretubulysin and Derivatives
yielded the tripeptide 17 (57 mg, 84.0 µmol, 84%) as a colorless
resin. Rf = 0.24 (Hex/EtOAc, 1:1). [α]2D0 = –10.9 (c = 1.0, CHCl3).
(s, 1 H, 12-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.1, 15.9,
19.5, 20.0, 24.2, 29.1, 29.2, 29.6*, 30.0, 30.2, 30.9, 37.3, 41.4, 45.9,
49.8, 51.5, 53.1, 58.5*, 63.0, 122.3, 126.4, 128.4, 129.4, 137.6, 149.7,
1
3
Major rotamer: H NMR (400 MHz, CDCl3): δ = 0.76 (d, J18,17
3
= 6.5 Hz, 3 H, 18-H), 0.85 (t, J24,23 = 7.4 Hz, 3 H, 24-H), 0.95 (d,
160.8, 169.6, 170.4, 173.0, 173.7 ppm. Selected signals of the minor
3J25,22 = 6.5 Hz, 3 H, 25-H), 0.97 (d, 3J18Ј,17 = 6.3 Hz, 3 H, 18Ј-H), rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.90 (d, J18,17/25,22
=
3
3
3
1.10 (ddq, 2J23a,23b = 13.6, J23a,22 = 9.8, 3J23a,24 = 7.3 Hz, 1 H, 23- 6.8 Hz, 3 H, 18Ј-H/25-H), 1.06 (d, J18Ј,17 = 6.5 Hz, 3 H, 18Ј-H),
2
3
3
Ha), 1.57 (dqd, J23b,23a = 13.6, J23b,24 = 7.6, J23b,22 = 2.9 Hz,
1 H, 23-Hb), 1.65–1.90 (m, 4 H, 3-Ha, 15-Ha, 17-H, 22-H), 1.95 (m,
2.65 (ddd, 3J14a,14b = 15.8, 3J14a,15a/b = 10.9, 3J14,15a/b = 4.6 Hz, 1 H,
3
14-Ha), 2.76 (s, 3 H, 19-H), 3.64 (ddd, J16,15a ≈ 3J16,17 = 10.2,
2
3
3
3
3
1 H, 3-Hb), 2.35 (ddd, J2a,2b = 16.3, J2a,3a/b = 9.3, J2a,3a/b
=
3J16,15b = 3.0 Hz, 1 H, 16-H), 4.92 (dd, J21,NH = 9.8, J21,22
=
2
3
3
6.5 Hz, 1 H, 2-Ha), 2.43 (ddd, J2b,2a = 16.3, J2b,3a/b = 9.8, 6.8 Hz, 1 H, 21-H), 7.30 (d, JNH,4 = 9.5 Hz, 1 H, NH“Phe”), 7.66
3J2b,3a/b = 6.0 Hz, 1 H, 2-Hb), 2.79 (t, J14,15 = 8.2 Hz, 2 H, 14-H), (d, JNH,21 = 9.8 Hz, 1 H, NHIle), 7.86 (s, 1 H, 12-H) ppm. 13C
3
3
2
3
2.82 (dd, J5a,5b = 13.3, J5a,4 = 7.2 Hz, 1 H, 5-Ha), 2.96 (s, 3 H, NMR (100 MHz, CDCl3): δ = 11.2, 16.3, 20.3, 20.4, 23.9, 27.3,
2
3
19-H), 2.97 (dd, J5b,5a = 13.3, J5b,4 = 5.8 Hz, 1 H, 5-Hb), 3.55 (s,
31.3, 37.9, 46.0, 49.9, 52.5, 62.6, 63.1, 122.6, 149.6, 169.8, 170.1,
172.6, 173.7 ppm. HRMS (CI): m/z calcd. for C33H52N5O5S [M +
H]+ 630.3689; found 630.3687.
3
3 H, 32-H), 4.28–4.43 (m, 2 H, 4-H, 16-H), 4.54 (dd, J21,NH = 9.4,
3J21,22 = 7.0 Hz, 1 H, 21-H), 5.06 (d, J27a,27b = 12.5 Hz, 1 H, 27-
2
2
3
Ha), 5.10 (d, J27b,27a = 12.5 Hz, 1 H, 27-Hb), 5.47 (d, JNH,21
=
(4R)-4-[((R)-2-{3-[(N,N-Dimethylglycyl-(S)-isoleucyl)methylamino]-
4-methylpentyl}thiazole-4-carbonyl)amino]-5-phenylpentanoic Acid,
Trifluoroacetic Acid Salt (19): A mixture of the tetrapeptide 18
(52 mg, 82.6 µmol) and NaOH (1 , 165 µL, 165 µmol) in dioxane
(0.8 mL) was stirred at 0 °C until complete saponification. The sol-
vent was evaporated in vacuo, and the residue was dissolved in
water, acidified to pH 2 with trifluoroacetic acid, and extracted
with EtOAc. The combined organic layers were dried with Na2SO4
and the solvent was evaporated in vacuo. Purification by flash
chromatography (CH2Cl2/MeOH, 9:1) gave 19 as a white solid in
quantitative yield. Rf = 0.09 (CH2Cl2/MeOH, 9:1); m.p. 55 °C.
[α]2D0 = –26.1 (c = 1.0, MeOH). Major rotamer: 1H NMR
3
9.4 Hz, 1 H, NHIle), 7.15–7.34 (m, 10 H, arom. H), 7.37 (d, JNH,4
= 9.0 Hz, 1 H, NH“Phe”), 7.89 (s, 1 H, 12-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 11.2, 15.9, 19.5, 20.1, 23.9, 29.1, 29.3, 29.6,
29.9*, 30.2, 31.0, 37.6, 41.5, 49.8, 51.5, 55.8, 58.6*, 66.8, 122.4,
126.5, 127.8, 128.0, 128.4, 128.4, 129.4, 136.4, 137.7, 149.7, 156.5,
160.9, 169.6, 173.3, 173.9 ppm. Selected signals of the minor rot-
1
3
amer: H NMR (400 MHz, CDCl3): δ = 0.86 (t, J24,23 = 7.3 Hz,
3
3
3 H, 24-H), 0.92, 0.93 (2ϫd, J18,17 = J25,22 = 6.8 Hz, 6 H, 18-H,
3
25-H), 1.06 (d, J18Ј,17 = 6.5 Hz, 3 H, 18Ј-H), 2.73 (s, 3 H, 19-H),
3
3
3.56 (s, 3 H, 32-H), 4.64 (dd, J21,NH = 9.8, J21,22 = 6.8 Hz, 1 H,
2
3
21-H), 5.04 (d, J27a,27b = 12.3 Hz, 1 H, 27-Ha), 5.54 (d, JNH,21
=
9.8 Hz, 1 H, NHIle), 7.88 (s, 1 H, 12-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 11.3, 16.2, 20.4, 23.5, 27.4, 29.2, 31.0, 37.9, 50.0, 55.3,
62.7, 66.9, 122.7, 137.8, 169.8, 172.7 ppm. HRMS (CI): m/z calcd.
3
(400 MHz, MeOD): δ = 0.78 (d, J18,17 = 6.5 Hz, 3 H, 18-H), 0.92
3
3
(t, J24,23 = 7.4 Hz, 3 H, 24-H), 0.97 (d, J18Ј,17 = 6.6 Hz, 3 H, 18Ј-
H), 1.02 (d, 3J25,22 = 6.8 Hz, 3 H, 25-H), 1.21 (ddq, 2J23a,23b = 13.8,
for C37H51N4O6S [M
–
CH2]+ 679.3529; found 679.3581.
3J23a,22 = 9.6, J23a,24 = 7.3 Hz, 1 H, 23-Ha), 1.64 (dqd, J23b,23a
=
3
2
C38H53N4O6S (693.92): calcd. C 65.77, H 7.70, N 8.07; found C
65.39, H 7.35, N 8.35.
3
3
13.8, J23b,24 = 7.5, J23b,22 = 2.8 Hz, 1 H, 23-Hb), 1.78 (m, 1 H,
17-H), 1.84–2.06 (m, 4 H, 3-H, 15-Ha, 17-H, 22-H), 2.16 (dtd,
2J15b,15a = 14.5, J15b,14 = 8.1, J15b,16 = 3.3 Hz, 1 H, 15-Hb), 2.32
3
3
Methyl (4R)-4-[((R)-2-{3-[(N,N-Dimethylglycyl-(S)-isoleucyl)meth-
ylamino]-4-methylpentyl}thiazole-4-carbonyl)amino]-5-phenylpenta-
noate (18): Pentafluorophenol (202 mg, 1.1 mmol) and dicyclohexyl
carbodiimide (227 mg, 1.10 mmol) were added to a solution of N-
methylsarcosine (103 mg, 0.999 mmol) in EtOAc, (2.5 mL). After
the system had been stirred overnight, precipitated urea was filtered
off through a pad of celite and the solvent was evaporated in vac-
uo.[7a] The tripeptide 17 (128 mg, 0.189 mmol) was N-deprotected
with HBr in acetic acid (33 wt.-%, 330 µL, 1.91 mmol) at 0 °C and
the resulting hydrobromide was deprotonated with saturated
NaHCO3 to afford the free amine, which was treated with N-meth-
ylsarcosine pentafluorophenol ester (195 mg, 0.724 mmol) in
dichloromethane (1.9 mL) overnight. The tetrapeptide 18 (66 mg,
0.105 mmol, 55%) was obtained by flash chromatography (hexane/
EtOAc, 1:1, 0:100; CH2Cl2/MeOH, 98:2, 95:5) as a pale yellow oil.
Rf = 0.38 (CH2Cl2/MeOH, 9:1). [α]2D0 = –25.7 (c = 1.0, CHCl3).
(dt, 2J2a,2b = 15.9, 3J2a,3 = 7.6 Hz, 1 H, 2-Ha), 2.41 (ddd, 2J2b,2a = 15.9,
3J2b,3a/b = 8.7, J2b,3a/b = 6.2 Hz, 1 H, 2-Hb), 2.72 (s, 6 H, 28-H),
3
2.80–3.03 (m, 4 H, 5-H, 14-H), 3.08 (s, 3 H, 19-H), 3.68 (d, 2J27a,27b
2
= 15.6 Hz, 1 H, 27-Ha), 3.73 (d, J27b,27a = 15.6 Hz, 1 H, 27-Hb),
4.26–4.39 (m, 2 H, 4-H, 16-H), 4.79 (d, J21,22 = 7.8 Hz, 1 H, 21-
3
H), 7.16 (m, 1 H, 9-H), 7.20–7.26 (m, 4 H, 7-H, 8-H), 7.97 (s, 1 H,
12-H) ppm. 13C NMR (100 MHz, MeOD): δ = 11.4, 16.1, 20.2,
20.5, 25.4, 30.3, 30.9*, 30.8, 31.0, 31.3, 32.9, 37.9, 42.1, 44.8, 52.0,
1
55.7, 59.9*, 60.4, 118.2 [q, JC,F = 290.9 Hz], 124.2, 127.4, 129.3,
2
130.4, 139.6, 150.4, 163.1 (q, JC,F = 35.6 Hz), 163.2, 167.6, 171.8,
174.5, 178.9 ppm. Selected signals of the minor rotamer: 1H NMR
(400 MHz, MeOD): δ = 1.11 (d, 3J18Ј,17 = 6.5 Hz, 3 H, 18Ј-H), 2.68
(s, 6 H, 28-H), 2.77 (s, 1 H, 19-H) ppm. 13C NMR (100 MHz,
MeOD): δ = 11.6, 16.5, 21.0, 24.7, 32.5, 38.4, 41.9, 45.0, 54.8,
124.4 ppm. HRMS (CI): m/z calcd. for C32H48N5O4S [M – OH]+
598.3427; found 598.3447.
1
3
Major rotamer: H NMR (400 MHz, CDCl3): δ = 0.74 (d, J18,17
3
= 6.5 Hz, 3 H, 18-H), 0.84 (t, J24,23 = 7.3 Hz, 3 H, 24-H), 0.94
Ethyl (R)-2-{3-[(N-Benzyloxycarbonyl-(R)-pipecolyl-(S)-isoleucyl)-
methylamino]-4-methylpentyl}thiazole-4-carboxylate (20): The di-
3
(2ϫd, J18Ј,17
=
3J25,22 = 6.8 Hz, 6 H, 18Ј-H, 25-H), 1.10 (ddq,
3
3
2J23a,23b = 13.8, J23a,22 = 9.8 Hz, J23a,24 = 7.3 Hz, 1 H, 23-Ha), peptide 7 (779 mg, 1.50 mmol) was N-deprotected with HBr in ace-
1.56 (dqd, 2J23b,23a = 13.8, 3J23b,24 = 7.2, 3J23b,22 = 2.8 Hz, 1 H, 23- tic acid (33 wt.-%, 2.59 mL, 15 mmol) at 0 °C and the resulting
Hb), 1.69 (m, 1 H, 17-H), 1.74–1.89 (m, 3 H, 3-Ha, 15-Ha, 22-H),
hydrobromide was deprotonated with saturated NaHCO3 to afford
1.94 (m, 1 H, 3-Hb), 2.09 (dtd, 2J15b,15a = 14.8, 3J15b,14 = 8.5, 3J15,16 the free amine, which was coupled with (R)-Z-pipecolic acid
= 3.4 Hz, 1 H, 15-Hb), 2.24 (s, 6 H, 28-H), 2.34 (ddd, 2J2a,2b = 16.1, (434 mg, 1.65 mmol, Ͼ98 % ee) with the aid of isobutyl chloro-
3
2
3J2a,3a/b = 8.5, J2a,3a/b = 6.7 Hz, 1 H, 2-Ha), 2.40 (ddd, J2b,2a
=
formate (214 µL, 1.65 mmol) and N-methylmorpholine (198 µL,
3
3
16.1, J2b,3a/b = 9.1, J2b,3a/b = 6.4 Hz, 1 H, 2-Hb), 2.75–3.02 (m, 1.80 mmol) by the General Procedure for mixed anhydride peptide
6 H, 5-H, 14-H, 27-H), 2.97 (s, 3 H, 19-H), 3.55 (s, 3 H, 29-H),
coupling to afford the tripeptide 20 (720 mg, 1.15 mmol, 76 %,
Ͼ99% de) as a colorless oil after flash chromatography (hexane/
EtOAc, 6:4, 1:1, 3:7). Rf = 0.18 (hexane/EtOAc, 1:1). HPLC: Lich-
rosorb Si-60 5 µ, hexane/EtOAc, 8:2–6:4, 2 mLmin–1, tR[(R,S,R)-
3
3
4.28–4.43 (m, 2 H, 4-H, 16-H), 4.81 (dd, J21,NH = 9.7, J21,22
=
=
3
7.5 Hz, 1 H, 21-H), 7.13–7.27 (m, 5 H, arom. H), 7.36 (d, JNH,4
3
9.3 Hz, 1 H, NH“Phe”), 7.59 (d, JNH,21 = 9.7 Hz, 1 H, NHIle), 7.87
Eur. J. Org. Chem. 2009, 6367–6378
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6375

A. Ullrich, J. Herrmann, R. Müller, U. Kazmaier
FULL PAPER
20] = 26.04 min, tR[(S,S,R)-20] = 28.22 min. [α]2D0 = +3.8 (c = 1.0,
isobutyl chloroformate (14 µL, 108 µmol) and N-methylmorpholine
CHCl3). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.73 (d, (2ϫ12 µL, 218 µmol) to give the tetrapeptide 22 (65 mg, 82.2 µmol,
3J9,8 = 6.8 Hz, 3 H, 9-H), 0.81 (m, 3 H, 15-H), 0.90 (m, 3 H, 16- 82%) as a colorless resin after column chromatography (hexane/
H), 0.93 (d, 3J9Ј,8 = 6.8 Hz, 3 H, 9Ј-H), 1.01 (m, 1 H, 14-Ha), 1.25–
EtOAc, 1:1, 3:7). Rf = 0.14 (Hex/EtOAc, 1:1). [α]2D0 = +4.8 (c = 1.0,
3
1.41 (m, 2 H, 20-Hax, 21-Hax),1.36 (t, J30,29 = 7.2 Hz, 3 H, 30-H), CHCl3). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.76 (d,
3
1.41–1.63 (m, 4 H, 14-Hb, 19-Hax, 20-Heq, 21-Heq), 1.67 (m, 1 H,
3J18,17 = 6.3 Hz, 3 H, 18-H), 0.83 (m, 3 H, 24-H), 0.91 (d, J25,22
=
3
8-H), 1.73 (m, 1 H, 13-H), 1.87 (m, 1 H, 6-Ha), 2.14 (m, 1 H, 6- 6.3 Hz, 3 H, 25-H), 0.96 (d, J18Ј,17 = 6.5 Hz, 3 H, 18Ј-H), 1.04 (m,
Hb), 2.27 (d, 2J19eq,19ax = 12.8 Hz, 1 H, 19-Heq), 2.77–2.86 (m, 2 H,
1 H, 23-Ha), 1.18–1.89 (m, 10 H, 3-Ha, 15-Ha, 17-H, 22-H, 23-Hb,
2
3
3
5-Ha, 22-Hax), 2.89 (ddd, J5b,5a = 15.1, J5b,6a/b = 6.2, J5b,6a/b
=
28-Hax, 29-H, 30-H), 1.96 (m, 1 H, 3-Hb), 2.12 (m, 1 H, 15-Hb),
2
2
3.3 Hz, 1 H, 5-Hb), 2.96 (s, 3 H, 10-H), 4.07 (br. s, 1 H, 22-Heq), 2.29 (d, J28eq,28ax = 13.3 Hz, 1 H, 28-Hax), 2.35 (dt, J2a,2b = 16.7,
3
2
3
4.32 (m, 1 H, 7-H), 4.37 (q, J29,30 = 7.2 Hz, 2 H, 29-H), 4.78 (dd,
3J2a,3 = 6.8 Hz, 1 H, 2-Ha), 2.42 (dt, J2b,2a = 16.7, J2b,3 = 7.1 Hz,
3J12,NH ≈ 3J12,13 = 8.3 Hz, 1 H, 12-H), 4.87 (br. s, 1 H, 18-H), 5.11 1 H, 2-Hb), 2.73–2.91 (m, 3 H, 14-H, 31-Hax), 2.82 (dd, J5a,5b
=
=
2
2
2
3
2
3
(d, J24a,24b = 11.6 Hz, 1 H, 24-Ha), 5.15 (d, J24b,24a = 12.3 Hz,
1 H, 24-Hb), 6.55 (br. s, 1 H, NHIle), 7.22–7.40 (m, 5 H, arom. H),
8.00 (s, 1 H, 3-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.0,
13.6, J5a,4 = 7.3 Hz, 1 H, 5-Ha), 2.96 (dd, J5b,5a = 13.6, J5b,4
6.0 Hz, 1 H, 5-Hb), 3.00 (s, 3 H, 19-H), 3.57 (s, 3 H, 38-H), 4.09
(br. s, 1 H, 31-Heq), 4.29–4.43 (m, 2 H, 4-H, 16-H), 4.81 (dd,
14.3, 15.9, 19.5, 19.9, 20.4, 24.1, 24.7*, 25.9*, 29.3, 29.7, 30.1, 30.6, 3J21,NH ≈ 3J21,22 = 8.4 Hz, 1 H, 21-H), 4.90 (br. s, 1 H, 27-H), 5.12
2
2
37.1, 42.2*, 53.6, 55.2*, 58.9, 61.3, 67.6, 126.9, 127.8, 128.0, 128.5,
136.3, 146.9, 156.4, 161.3, 170.3, 170.8, 173.2 ppm. Selected signals
(d, J33a,33b = 12.0 Hz, 1 H, 33-Ha), 5.17 (d, J33b,33a = 12.0 Hz,
1 H, 33-Hb), 6.59 (br. s, 1 H, NHIle), 7.16–7.39 (m, 11 H, arom. H,
NH“Phe”), 7.89 (s, 1 H, 12-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 11.0, 15.8, 19.6, 20.0, 20.4, 24.2, 24.7*, 25.9*, 29.2, 29.3, 29.3*,
30.1, 30.2, 31.0, 37.2, 41.5, 42.1*, 49.8, 51.5, 53.6, 55.1*, 58.7, 67.6,
122.5, 126.5, 127.8, 128.1, 128.4, 128.5, 129.4, 136.3, 137.6, 149.7,
156.4, 160.8, 169.7, 170.5, 173.2, 173.8 ppm. Selected signals of the
1
of the minor rotamer: H NMR (400 MHz, CDCl3): δ = 1.03 (d,
3J9Ј,8 = 6.3 Hz, 3 H, 9Ј-H), 2.73 (s, 3 H, 10-H), 3.61 (m, 1 H, 7-H),
3
3
4.17 (br. s, 1 H, 22-Heq), 4.92 (dd, J12,NH = 9.6, J12,13 = 6.0 Hz,
2
1 H, 12-H), 5.06 (d, J24a,24b = 12.6 Hz, 1 H, 24-Ha), 7.95 (s, 1 H,
3-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.2, 15.2, 16.2, 20.3,
1
3
20.5, 23.4, 27.4, 29.9, 31.3, 53.0, 61.3, 62.7, 67.5, 126.9, 146.8, minor rotamer: H NMR (400 MHz, CDCl3): δ = 1.06 (d, J18Ј,17
171.0 ppm. HRMS (CI): m/z calcd. for C33H49N4O6S [M + H]+ = 6.5 Hz, 3 H, 18Ј-H), 2.77 (s, 3 H, 19-H), 4.19 (br. s, 1 H, 31-Heq),
3
3
629.3373; found 629.3327. C33H48N4O6S (628.83): calcd. C 63.03,
H 7.69, N 8.91; found C 63.07, H 7.36, N 9.16.
4.99 (dd, J21,NH = 9.3, J21,22 = 6.5 Hz, 1 H, 21-H), 5.07 (d,
2J33a,33b = 12.3 Hz, 1 H, 33-Ha), 7.87 (s, 1 H, 12-H) ppm. HRMS
(CI): m/z calcd. for C36H52N5O7S [M – C7H7]+ 698.3587; found
698.3591.
(R)-2-{3-[(N-Benzyloxycarbonyl-(R)-pipecolyl-(S)-isoleucyl)methyl-
amino]-4-methylpentyl}thiazole-4-carboxylic Acid (21): A mixture of
ethyl ester 20 (554 mg, 0.881 mmol) and NaOH (1 , 1.32 mL,
1.32 mmol) in dioxane (9 mL) was stirred at 0 °C until complete
saponification. The solvent was evaporated in vacuo, and the resi-
due was dissolved in water, acidified to pH 2 with HCl (1 ), and
extracted twice with EtOAc. The combined organic layers were
dried with Na2SO4 and the solvent was evaporated in vacuo to give
the free acid 21 (518 mg, 0.862 mmol, 98%) as a colorless glass-
like solid; m.p. 78–80 °C. [α]2D0 = +6.5 (c = 1.0, CHCl3). Major
(4R)-4-[((R)-2-{3-[Methyl-(N-methyl-(R)-pipecolyl-(S)-isoleucyl)-
amino]-4-methylpentyl}thiazole-4-carbonyl)amino]-5-phenylpenta-
noic Acid, Trifluoroacetic Acid Salt (23): The tetrapeptide 22
(64 mg, 75.3 µmol) was N-deprotected with HBr in acetic acid
(33 wt.-%, 110 µL, 637 µmol) at 0 °C and the resulting hydrobro-
mide was deprotonated with saturated NaHCO3 to afford the free
amine. This N-deprotected peptide was dissolved in MeOH
(0.75 mL), and paraformaldehyde (7.0 mg, 77.7 µmol) was added.
After the system had been stirred for 3 h at room temperature, so-
dium cyanoborohydride (5.2 mg, 82.7 µmol) was added. When the
reaction was finished (TLC monitoring), methanol was removed in
vacuo and the residue was dissolved in dichloromethane and
washed with saturated NaHCO3. The aqueous layer was extracted
with dichloromethane and the combined organic layers were dried
with Na2SO4. The N-methylated tetrapeptide (37 mg, 55.2 µmol,
73%) was obtained after flash chromatography (CH2Cl2/MeOH,
95:5, 9:1) as a yellow oil. A mixture of the N-methylated tetrapep-
tide (37 mg, 55.2 µmol) and NaOH (1 , 110 µL, 110 µmol) in di-
oxane (0.55 mL) was stirred at 0 °C until complete saponification.
The solvent was evaporated in vacuo, and the residue was dissolved
in water, acidified to pH 2 with trifluoroacetic acid, and extracted
twice with EtOAc. The combined organic layers were dried with
Na2SO4 and the solvent was evaporated in vacuo. Purification by
flash chromatography (CH2Cl2/MeOH, 9:1) gave 23 as white solid
in quantitative yield. Rf = 0.11 (CH2Cl2/MeOH, 9:1); m.p. 174–
1
3
rotamer: H NMR (400 MHz, CDCl3): δ = 0.74 (d, J9,8 = 6.5 Hz,
3 H, 9-H), 0.81 (m, 3 H, 15-H), 0.88–0.98 (m, 6 H, 9Ј-H, 16-H),
1.06 (m, 1 H, 14-Ha), 1.24–1.74 (m, 7 H, 8-H, 14-Hb, 19-Hax, 20-
H, 21-H), 1.78–1.95 (m, 2 H, 6-Ha, 13-H), 2.13 (m, 1 H, 6-Hb), 2.29
2
(d, J19eq,19ax = 13.3 Hz, 1 H, 19-Heq), 2.83 (m, 1 H, 22-Hax), 2.87
(t, J5,6 = 7.8 Hz, 2 H, 5-H), 3.02 (s, 3 H, 10-H), 4.07 (br. s, 1 H,
3
3
22-Heq), 4.37 (m, 1 H, 7-H), 4.81 (dd, J12,NH
≈
3J12,13 = 8.7 Hz,
1 H, 12-H), 4.90 (br. s, 1 H, 18-H), 5.12 (d, 2J24a,24b = 12.5 Hz, 1 H,
2
24-Ha), 5.17 (d, J24b,24a = 12.5 Hz, 1 H, 24-Hb), 6.85 (br. s, 1 H,
NH), 7.23–7.37 (m, 5 H, arom. H), 8.10 (s, 1 H, 3-H) ppm. 13C
NMR (100 MHz, CDCl3): δ = 10.8, 15.8, 19.5, 19.9, 20.4, 24.3,
24.8*, 26.0*, 29.6, 30.2, 30.4, 36.7*, 42.1*, 53.8, 55.1*, 59.1, 67.6,
127.6, 127.8, 128.0, 128.4, 136.4, 146.5, 163.2, 170.5, 170.8,
174.0 ppm. Selected signals of the minor rotamer: 1H NMR
(400 MHz, CDCl3): δ = 1.03 (d, 3J9Ј,8 = 6.5 Hz, 3 H, 9Ј-H), 2.73 (s,
3 H, 10-H), 3.64 (m, 1 H, 7-H), 4.14 (br. s, 1 H, 22-Heq), 6.95 (br.
s, 1 H, NH), 8.06 (s, 1 H, 3-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 11.2, 16.1, 20.2, 20.5, 23.6, 27.6, 53.1, 62.8 ppm. C31H44N4O6S
(600.78): calcd. C 61.98, H 7.38, N 9.33; found C 61.92, H 7.47, N
9.39. HRMS (CI): m/z calcd. for C30H45N4O4S [M – CO2 + H]+
557.3162; found 557.3186.
1
178 °C. [α]2D0 = –12.7 (c = 1.0, MeOH). Major rotamer: H NMR
3
(400 MHz, MeOD): δ = 0.78 (d, J18,17 = 6.5 Hz, 3 H, 18-H), 0.91
3
3
(t, J24,23 = 7.5 Hz, 3 H, 24-H), 0.97 (d, J18Ј,17 = 6.5 Hz, 3 H, 18Ј-
H), 1.01 (d, 3J25,22 = 6.8 Hz, 3 H, 25-H), 1.23 (ddq, 2J23a,23b = 13.7,
3
Methyl (4R)-4-[((R)-2-{3-[(N-Benzyloxycarbonyl-(R)-pipecolyl-(S)-
isoleucyl)methylamino]-4-methylpentyl}thiazole-4-carbonyl)amino]-
5-phenylpentanoate (22): The free acid 21 (60 mg, 100 µmol) and
the hydrochloride 16 (27 mg, 111 µmol) were coupled by the Gene-
ral Procedure for mixed anhydride peptide coupling with the aid of
3J23a,22 = 9.0, J23a,24 = 7.3 Hz, 1 H, 23-Ha), 1.48 (ddddd,
2J29ax,29eq ≈ 3J29ax,28ax
≈
3J29ax,30ax = 12.7, J29ax,28eq ≈ 3J29ax,30eq
=
3
3.4 Hz, 1 H, 29-Hax), 1.56–2.06 (m, 11 H, 3-H, 15-Ha, 17-H, 22-H,
2
3
23-Hb, 28-H, 29-Heq, 30-H), 2.16 (dtd, J15b,15a = 14.4, J15b,14
=
=
3
2
3
8.1, J15b,16 = 3.3 Hz, 1 H, 15-Hb), 2.27 (dt, J2a,2b = 15.6, J2a,3
6376
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6367–6378

Synthesis and Biological Evaluation of Pretubulysin and Derivatives
2
3
7.4 Hz, 1 H, 2-Ha), 2.37 (dt, J2b,2a = 15.6, J2b,3 = 7.4 Hz, 1 H, 2-
Hb), 2.51 (s, 3 H, 32-H), 2.68 (dd, 2J31ax,31eq ≈ 3J31ax,30ax = 12.3 Hz,
and washed with saturated NaHCO3. The aqueous layer was ex-
tracted with dichloromethane and the combined organic layers
1 H, 31-Hax), 2.80–3.01 (m, 4 H, 5-H, 14-H), 3.09 (s, 3 H, 19-H), were dried with Na2SO4. The N-methylated tetrapeptide (64 mg,
3.26 (d, 2J31eq,31ax = 12.3 Hz, 1 H, 31-Heq), 3.32 [m, 1 H, 27-H (un-
93.6 µmol, 66 %) was obtained after flash chromatography
(CH2Cl2/MeOH, 99:1, 98:2, 95:5, 9:1) as a yellow oil. A mixture of
3
der MeOD)], 4.26–4.39 (m, 2 H, 4-H, 16-H), 4.72 (d, J21,22
=
8.5 Hz, 1 H, 21-H), 7.15 (m, 1 H, 9-H), 7.18–7.27 (m, 4 H, 7-H, 8- the N-methylated tetrapeptide (39 mg, 57.0 µmol) and NaOH (1 ,
H), 7.99 (s, 1 H, 12-H) ppm. 13C NMR (100 MHz, MeOD): δ =
172 µL, 172 µmol) in dioxane (0.57 mL) was heated to 80 °C until
11.2, 16.1, 20.3, 20.5, 23.1, 24.9, 25.6, 30.3, 29.8*, 30.7, 31.0, 31.3, complete saponification. The solvent was evaporated in vacuo, and
31.8, 34.4, 37.7, 42.0, 43.6, 52.0, 55.6, 56.3, 60.3*, 69.1, 118.2 (q,
1JC,F = 290.9 Hz), 124.4, 127.4, 129.3, 130.4, 139.7, 150.4, 163.1 (q,
2JC,F = 34.3 Hz), 163.2, 171.8, 171.9, 174.7, 181.8 ppm. Selected
the residue was dissolved in water, acidified to pH 2 with trifluoro-
acetic acid, and extracted twice with EtOAc. The combined organic
layers were dried with Na2SO4 and the solvent was evaporated in
signals of the minor rotamer: 1H NMR (400 MHz, MeOD): δ = vacuo. Purification by flash chromatography (CH2Cl2/MeOH, 95:5,
3
1.11 (d, J18Ј,17 = 6.5 Hz, 3 H, 18Ј-H), 2.48 (s, 3 H, 32-H), 2.78 (s,
1 H, 19-H), 4.67 (dd, 3J21,22 = 8.3 Hz, 1 H, 21-H), 7.95 (s, 1 H, 12-
H) ppm. 13C NMR (100 MHz, MeOD): δ = 11.7, 16.5, 21.0, 22.9,
38.6, 41.8, 43.8, 55.3, 68.9, 173.9, 179.8 ppm. HRMS (CI): m/z
calcd. for C35H52N5O4S [M – OH]+ 638.3740; found 638.3708.
9:1) gave 2 (44 mg, 56.1 µmol) as white solid in quantitative yield.
Rf = 0.11 (CH2Cl2/MeOH, 9:1); m.p. 60–62 °C. [α]2D0 = –17.1 (c =
1.0, MeOH). Major rotamer: 1H NMR (400 MHz, MeOD): δ =
3
3
0.81 (d, J19,18 = 6.3 Hz, 3 H, 19-H), 0.93 (t, J25,24 = 7.1 Hz, 3 H,
3
3
25-H), 0.99 (d, J19Ј,18 = 6.3 Hz, 3 H, 19Ј-H), 1.02 (d, J26,23
=
6.5 Hz, 3 H, 26-H), 1.23 (m, 1 H, 24-Ha), 1.53–1.86 (m, 6 H, 3-Ha,
18-H, 24-Hb, 29-Hax, 30-Hax, 31-Hax), 1.86–2.08 (m, 5 H, 3-Hb, 16-
Ha, 23-H, 30-Heq, 31-Heq), 2.11–2.27 (m, 2 H, 16-Hb, 29-Heq), 2.56
(m, 1 H, 2-H), 2.74 (s, 3 H, 33-H), 2.81–3.02 (m, 4 H, 5-H, 15-H),
Methyl (2S,4R)-4-[((R)-2-{3-[(N-Benzyloxycarbonyl-(R)-pipecolyl-
(S)-isoleucyl)methylamino]-4-methylpentyl}thiazole-4-carbonyl)-
amino]-2-methyl-5-phenylpentanoate (24): The free acid 21 (120 mg,
200 µmol) and the hydrochloride 10 (57 mg, 221 µmol) were cou-
pled by the General Procedure for mixed anhydride peptide cou-
pling with the aid of isobutyl chloroformate (28 µL, 216 µmol) and
N-methylmorpholine (2ϫ24 µL, 436 µmol) to give the tetrapeptide
24 (129 mg, 160 µmol, 80 %) as a colorless resin after column
chromatography (hexane/EtOAc, 1:1, 3:7). Rf = 0.19 (Hex/EtOAc,
1:1). [α]2D0 = +19.3 (c = 1.0, CHCl3). Major rotamer: 1H NMR
2
3.09 (dd, J32ax,32eq ≈ 3J32ax,31ax = 12.3 Hz, 1 H, 32-Hax), 3.11 (s,
2
3 H, 20-H), 3.49 (d, J32eq,32ax = 11.5 Hz, 1 H, 32-Heq), 3.76 (d,
3J28,29ax = 10.8 Hz, 1 H, 28-H), 4.24–4.47 (m, 2 H, 4-H, 17-H), 4.70
3
(d, J22,23 = 7.8 Hz, 1 H, 22-H), 7.16 (m, 1 H, 9-H), 7.19–7.29 (m,
4 H, 7-H, 8-H), 7.95 (s, 1 H, 12-H) ppm. 13C NMR (100 MHz,
MeOD): δ = 11.3, 16.0, 18.5, 20.3, 20.5, 22.3, 24.0, 25.5, 30.2, 30.3*,
30.9, 31.4, 37.5, 37.9, 39.2, 42.4, 42.9, 50.7, 56.0, 56.2, 60.6*, 68.1,
118.2 (q, 1JC,F = 290.6 Hz), 124.0, 127.4, 129.3, 130.4, 139.5, 150.5,
3
(400 MHz, CDCl3): δ = 0.76 (d, J19,18 = 6.5 Hz, 3 H, 19-H), 0.82
(m, 3 H, 25-H), 0.92 (d, 3J26,23 = 6.0 Hz, 3 H, 26-H), 0.95 (d, 3J19Ј,18
= 6.5 Hz, 3 H, 19Ј-H), 1.03 (m, 1 H, 24-Ha), 1.12 (d, 3J10,2 = 7.0 Hz,
3 H, 10-H), 1.21–1.65 (m, 7 H, 3-Ha, 24-Hb, 29-Hax, 30-H, 31-H),
2
163.1 (q, JC,F = 32.8 Hz), 163.1, 169.2, 171.9, 174.6, 180.0 ppm.
Selected signals of the minor rotamer: 1H NMR (400 MHz,
MeOD): δ = 2.78 (s, 1 H, 20-H) ppm. 13C NMR (100 MHz,
MeOD): δ = 11.8, 16.4, 18.4, 31.2, 42.1, 43.1, 178.5 ppm. HRMS
(CI): m/z calcd. for C36H56N5O5S [M + H]+ 670.4002; found
670.3984.
2
1.66–1.91 (m, 3 H, 16-Ha, 18-H, 23-H), 1.99 (ddd, J3b,3a = 13.5,
3
3J3b,2/4 = 9.4, J3b,2/4 = 4.0 Hz, 1 H, 3-Hb), 2.11 (m, 1 H, 16-Hb),
2
2.29 (d, J29eq,29ax = 12.8 Hz, 1 H, 29-Heq), 2.58 (m, 1 H, 2-H),
2.69–3.02 (m, 5 H, 5-H, 15-H, 32-Hax), 2.98 (s, 3 H, 20-H), 3.59 (s,
3 H, 39-H), 4.08 (br. s, 1 H, 32-Heq), 4.29–4.44 (m, 2 H, 4-H, 17-
H), 4.80 (dd, 3J22,NH ≈ 3J22,23 = 8.0 Hz, 1 H, 22-H), 4.89 (br. s, 1 H,
((R)-2-{3-[Methyl-(N-methyl-(R)-pipecolyl-(S)-isoleucyl)amino]-4-
methylpentyl}thiazole-4-carbonyl)(S)-phenylalanine, Trifluoroacetic
Acid Salt (25): The free acid 21 (120 mg, 200 µmol) and -phenylal-
anine methyl ester hydrochloride (47 mg, 218 µmol) were coupled
by the General Procedure for mixed anhydride peptide coupling
with the aid of isobutyl chloroformate (28 µL, 216 µmol) and N-
methylmorpholine (2ϫ24 µL, 436 µmol) to give the protected tet-
rapeptide (136 mg, 178 µmol, 89%) as a white foam after column
chromatography (hexane/EtOAc, 1. 1:1, 2. 4:6, 3. 3:7). This tetra-
peptide (99 mg, 0.130 mmol) was N-deprotected with HBr in acetic
acid (33 wt.-%, 180 µL, 1.04 mmol) at 0 °C and the resulting hydro-
bromide was deprotonated with saturated NaHCO3 to afford the
free amine. The N-deprotected peptide was dissolved in MeOH
(1.3 mL) and paraformaldehyde (11.8 mg, 0.131 mmol) was added.
After the system had been stirred for 3 h at room temperature,
sodium cyanoborohydride (9.0 mg, 0.143 mmol) was added. When
2
2
28-H), 5.11 (d, J34a,34b = 12.0 Hz, 1 H, 34-Ha), 5.16 (d, J34b,34a
=
12.0 Hz, 1 H, 34-Hb), 6.56 (br. s, 1 H, NHIle), 7.10–7.38 (m, 11 H,
arom. H, NHTup), 7.88 (s, 1 H, 13-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 11.0, 15.8, 17.7, 19.6, 20.0, 20.4, 24.1, 24.8*, 25.5*,
29.3, 29.3*, 30.1, 30.2, 36.4, 37.2, 37.8, 41.2, 42.2*, 48.4, 51.6, 53.6,
54.9*, 58.8*, 67.6, 122.3, 126.4, 127.8, 128.1, 128.3, 128.5, 129.4,
136.3, 137.6, 149.8, 156.2*, 160.6, 169.6, 170.4, 173.2, 176.5 ppm.
Selected signals of the minor rotamer: 1H NMR (400 MHz,
CDCl3): δ = 1.05 (d, 3J19Ј,18 = 6.5 Hz, 3 H, 19Ј-H), 2.77 (s, 3 H, 20-
3
3
H), 4.18 (br. s, 1 H, 32-Heq), 4.97 (dd, J22,NH = 9.0, J22,23
=
2
7.0 Hz, 1 H, 22-H), 5.06 (d, J34a,34b = 12.6 Hz, 1 H, 34-Ha), 7.86
(s, 1 H, 13-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.2, 16.2,
20.3, 20.5, 23.5, 27.3, 62.7, 122.6 ppm. HRMS (CI): m/z calcd. for
C44H62N5O7S [M + H]+ 804.4370; found 804.4331.
(2S,4R)-4-[((R)-2-{3-[Methyl-(N-methyl-(R)-pipecolyl-(S)-isoleucyl)- the reaction was finished (TLC monitoring), methanol was re-
amino]-4-methylpentyl}thiazole-4-carbonyl)amino]-2-methyl-5-phen-
ylpentanoic Acid, Trifluoroacetic Acid Salt (2): The tetrapeptide 24
(114 mg, 0.142 mmol) was N-deprotected with HBr in acetic acid
(33 wt.-%, 240 µL, 1.39 mmol) at 0 °C and the resulting hydrobro-
mide was deprotonated with saturated NaHCO3 to afford the free
amine. This N-deprotected peptide was dissolved in MeOH
moved in vacuo and the residue was dissolved in dichloromethane
and washed with saturated NaHCO3. The aqueous layer was ex-
tracted with dichloromethane and the combined organic layers
were dried with Na2SO4. The N-methylated tetrapeptide (40 mg,
62.3 µmol, 48%) was obtained after flash chromatography
(CH2Cl2/MeOH, 1. 99:1, 2. 98:2, 3. 9:1) as a yellow oil. A mixture
(1.4 mL), and paraformaldehyde (12.8 mg, 0.142 mmol) was added. of the N-methylated tetrapeptide (40 mg, 62.3 µmol) and NaOH
After the system had been stirred for 3 h at room temperature, so- (1 , 100 µL, 100 µmol) in dioxane (0.62 mL) was stirred at 0 °C
dium cyanoborohydride (9.4 mg, 0.150 mmol) was added. When until complete saponification. The solvent was evaporated in vacuo,
the reaction was finished (TLC monitoring), methanol was re-
moved in vacuo and the residue was dissolved in dichloromethane
and the residue was dissolved in water, acidified to pH 2 with tri-
fluoroacetic acid, and extracted twice with EtOAc. The combined
Eur. J. Org. Chem. 2009, 6367–6378
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6377

A. Ullrich, J. Herrmann, R. Müller, U. Kazmaier
FULL PAPER
[2]
a) M. W. Khalil, F. Sasse, H. Lünsdorf, Y. A. Elnakady, H.
Reichenbach, ChemBioChem 2006, 7, 678–683; b) G. Kaur, M.
Hollingshead, S. Holbeck, V. Schauer-Vukasinovic, R. F. Cam-
alier, A. Dömling, S. Agarwal, Biochem. J. 2006, 396, 235–242.
H. Steinmetz, N. Glaser, E. Herdtweck, F. Sasse, H. Reichen-
bach, G. Höfle, Angew. Chem. 2004 116, 4996–5000; Angew.
Chem. Int. Ed. 2004, 43, 4888–4892.
a) A. Dömling, B. Beck, U. Eichelberger, S. Sakamuri, S.
Menon, Q.-Z. Chen, Y. Lu, L. A. Wessjohann, Angew. Chem.
2006, 118, 45, 7393–7397; Angew. Chem. Int. Ed. 2006, 45,
7235–7239; b) A. Dömling, W. Richter, Mol. Diversity 2005, 9,
141–147.
H. M. Peltier, J. P. McMahon, A. W. Patterson, J. A. Ellman, J.
Am. Chem. Soc. 2006, 128, 16018–16019.
a) P. Wipf, Z. Wang, Org. Lett. 2007, 9, 1605–1607; b) Z. Wang,
P. A. McPherson, B. S. Raccor, R. Balachandran, G. Zhu,
B. W. Day, A. Vogt, P. Wipf, Chem. Biol. Drug Des. 2007, 70,
75–86.
a) A. W. Patterson, H. M. Peltier, F. Sasse, J. A. Ellman, Chem.
Eur. J. 2007, 13, 9534–9541; b) A. W. Patterson, H. M. Peltier,
J. A. Ellman, J. Org. Chem. 2008, 73, 4362–4369.
a) M. Sani, G. Fossati, F. Huguenot, M. Zanda, Angew. Chem.
2007, 119, 3596–3599; Angew. Chem. Int. Ed. 2007, 46, 3526–
3529; b) B. Raghavan, R. Balasubramanian, J. C. Steele, D. L.
Sackett, R. A. Fecik, J. Med. Chem. 2008, 51, 1530–1533.
A. Ullrich, Y. Chai, D. Pistorius, Y. A. Elnakady, J. E. Herrm-
ann, K. J. Weissman, U. Kazmaier, R. Müller, Angew. Chem.
2009, 121, 4486–4489; Angew. Chem. Int. Ed. 2009, 48, 4422–
4425.
organic layers were dried with Na2SO4 and the solvent was evapo-
rated in vacuo. Purification by flash chromatography (CH2Cl2/
MeOH, 9:1) gave 25 as a white solid in quantitative yield. Rf = 0.10
(CH2Cl2/MeOH, 9:1); m.p. 88 °C. [α]2D0 = –1.0 (c = 1.0, MeOH).
[3]
[4]
1
3
Major rotamer: H NMR (400 MHz, MeOD): δ = 0.79 (d, J16,15
3
= 6.5 Hz, 3 H, 16-H), 0.91 (t, J22,21 = 7.4 Hz, 3 H, 22-H), 0.97 (d,
3
3J16Ј,15 = 6.5 Hz, 3 H, 16Ј-H), 1.01 (d, J23,20 = 6.8 Hz, 3 H, 23-H),
3
1.21 (ddq, 2J21a,21b = 13.6, J21a,20 = 9.4, 3J21a,22 = 7.3 Hz, 1 H, 21-
Ha), 1.52–1.69 (m, 2 H, 21-Hb, 27-Hax), 1.71–2.05 (m, 7 H, 13-Ha,
15-H, 20-H, 26-Hax, 27-Heq, 28-H), 2.10–2.27 (m, 2 H, 26-Heq, 13-
Hb), 2.73 (s, 3 H, 30-H), 2.80–3.00 (m, 2 H, 12-Ha, 12-Hb), 3.06
(m, 1 H, 29-Hax), 3.07 (s, 3 H, 17-H), 3.20 (dd, 2J3a,3b = 13.8, 3J3a,2
= 6.8 Hz, 1 H, 3-Ha), 3.33 [m, 1 H, 3-Hb (under MeOD)], 3.48 (d,
2J29eq,29ax = 12.3 Hz, 1 H, 29-Heq), 3.78 (d, 3J25,26ax = 11.6 Hz, 1 H,
25-H), 4.21 (br. s, 1 H, 14-H), 4.69 (d, 3J19,20 = 7.5 Hz, 1 H, 19-H),
4.85 [m, 1 H, 2-H (under H2O)], 7.15–7.30 (m, 5 H, arom. H), 8.04
(s, 1 H, 10-H) ppm. 13C NMR (100 MHz, MeOD): δ = 11.4, 16.1,
20.3, 20.5, 22.3, 24.0, 25.4, 30.0, 30.2, 30.4*, 31.0, 31.2, 37.5, 38.5,
[5]
[6]
[7]
[8]
1
42.8, 55.2, 56.1, 60.9*, 68.1, 118.2 (q, JC,F = 289.5 Hz), 124.6,
2
127.9, 129.4, 130.5, 138.2, 150.0, 162.7, 163.0 (q, JC,F = 32.8 Hz),
169.2, 172.2, 174.4, 174.7 ppm. Selected signals of the minor rot-
1
3
amer: H NMR (400 MHz, MeOD): δ = 1.09 (d, J16Ј,15 = 6.3 Hz,
3 H, 16Ј-H), 2.34 (m, 1 H, 13-Hb), 2.55 (s, 3 H, 30-H), 2.65 (s, 3 H,
17-H), 3.70 (dd, J14,13a/b ≈ 3J14,15 = 9.0 Hz, 1 H, 14-H), 3.84 (d,
3
[9]
3J25,26ax = 12.0 Hz, 1 H, 25-H), 7.97 (s, 1 H, 10-H) ppm. 13C NMR
(100 MHz, MeOD): δ = 11.7, 16.5, 20.6, 20.9, 24.5, 28.3, 38.7, 43.0,
55.3, 64.8, 124.8, 127.8, 129.3, 130.6, 138.5, 150.2, 169.4, 172.6,
173.7 ppm. HRMS (CI): m/z calcd. for C24H42N5O3S [M –
C9H7O]+ 480.3008; found 480.2983.
[10]
[11]
A. Sandmann, F. Sasse, R. Müller, Chem. Biol. 2004, 11, 1071–
1079.
U. Schmidt, R. Utz, A. Lieberknecht, H. Griesser, B. Potzolli,
J. Bahr, K. Wagner, P. Fischer, Synthesis 1987, 236–241.
MTT Assay: Cells were seeded at 6ϫ103 cells per well on 96-well
plates (Corning CellBind®) in complete medium (180 µL) and di-
rectly treated with tubulysins and pretubulysins dissolved in meth-
anol in serial dilution. Each compound, as well as the internal
methanol control, was tested in duplicate. After 5 d incubation,
MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro-
mide, 5 mgmL–1, 20 µL] in PBS (phosphate-buffered saline, pH 7.4)
was added to each well and incubation was continued for a further
2 h at 37 °C. The medium was then discarded and cells were washed
with PBS (100 µL) before addition of propan-2-ol/10  HCl (250:1,
100 µL) in order to dissolve formazan granules. The absorbance at
570 nm was measured with a microplate reader (EL808, Bio-Tek
Instruments Inc.), and cell viability was expressed as a percentage
relative to the corresponding methanol control. IC50 values were
obtained by sigmoidal curve fitting.
[12] a) U. Schmidt, P. Gleich, H. Griesser, R. Utz, Synthesis 1986,
992–998; b) P. Wipf, T. Takada, M. J. Rishel, Org. Lett. 2004,
6, 4057–4060.
[13] P. Li, J. C. Xu, J. Org. Chem. 2000, 65, 2951–2958.
[14] D. A. Evans, Aldrichimica Acta 1982, 15, 23–32.
[15] For comparable reductions see: a) H. Kizu, M. Koshijima, T.
Tomimori, Chem. Pharm. Bull. 1985, 33, 3176–3181; b) K. D.
Croft, E. L. Ghisalberti, P. R. Jefferies, C. L. Raston, A. H.
White, S. R. Hall, Tetrahedron 1977, 33, 1475–1480; c) K. Tak-
ahashi, M. Takani, Chem. Pharm. Bull. 1975, 23, 538–542.
[16] D. Davidson, S. A. Bernhard, J. Am. Chem. Soc. 1948, 70,
3426–3428.
[17] M. Smrcina, P. Majer, E. Majerová, T. A. Guerassina, M. A.
Eissenstat, Tetrahedron 1997, 53, 12867–12874.
[18] W. V. Murray, S. Sun, I. J. Turchi, F. K. Brown, A. D. Gauthier,
J. Org. Chem. 1999, 64, 5930–5940.
[19] J. C. A. Hunt, C. Lloyd, C. J. Moody, A. M. Z. Slawin, A. K.
Takle, J. Chem. Soc. Perkin Trans. 1 1999, 23, 3443–3454.
[20] B. Hin, P. Majer, T. Tsukamoto, J. Org. Chem. 2002, 67, 7365–
7368.
[21] A. Dömling, B. Beck, U. Eichelberger, S. Sakamuri, S. Menon,
Q.-Z. Chen, Y. Lu, L. A. Wessjohann, Angew. Chem. 2007, 119,
2389–2400; Angew. Chem. Int. Ed. 2007, 46, 2347–2348.
Received: September 1, 2009
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) and by the Fonds der Chemischen Industrie.
[1] F. Sasse, H. Steinmetz, J. Heil, G. Höfle, H. Reichenbach, J.
Antibiot. 2000, 53, 879–885.
Published Online: November 5, 2009
6378
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6367–6378