Design, synthesis, and biological properties of highly potent tubulysin D analogues

Article abstract of DOI:10.1002/chem.200701057

Ten analogues of tubulysin D were synthesized and assayed against established mammalian cell lines, including cancer cells measuring inhibition of cell growth by an MTT assay. These experiments establish for the first time the essential features for the potent cytotoxicity of tubulysin D. The activities of analogues 2 to 5 demonstrate that numerous modifications may be introduced at the C-terminus of the natural product with only modest loss in activity, while the activities of analogues 6 to 8 suggest that a basic amine must be present at the N-terminus to maintain activity. Most surprisingly, analogue 10 establishes that replacement of the chemically labile O-acyl N,O-acetal with the stable N-methyl group results in almost no loss in activity. In aggregate, these structure-activity relationships enable the design of analogues such as 11 that are smaller and considerably more stable than tubulysin D but that maintain most of its potent cell-growth inhibitory activity.

Full text of DOI:10.1002/chem.200701057

DOI: 10.1002/chem.200701057
Design, Synthesis, and Biological Properties ofHighly Potent Tubulysin D
Analogues
Andrew W. Patterson,[a] Hillary M. Peltier,[a] Florenz Sasse,*[b] and
Jonathan A. Ellman*[a]
Abstract: Ten analogues of tubulysin D
were synthesized and assayed against
established mammalian cell lines, in-
cluding cancer cells measuring inhibi-
tion of cell growth by an MTT assay.
These experiments establish for the
first time the essential features for the
potent cytotoxicity of tubulysin D. The
activities of analogues 2 to 5 demon-
strate that numerous modifications
may be introduced at the C-terminus
of the natural product with only
modest loss in activity, while the activi-
ties of analogues 6 to 8 suggest that a
basic amine must be present at the N-
terminus to maintain activity. Most sur-
prisingly, analogue 10 establishes that
replacement of the chemically labile
O-acyl N,O-acetal with the stable N-
methyl group results in almost no loss
in activity. In aggregate, these struc-
ture–activity relationships enable the
design of analogues such as 11 that are
smaller and considerably more stable
than tubulysin D but that maintain
most of its potent cell-growth inhibito-
ry activity.
Keywords: cytotoxicity
·
natural
products · total synthesis · tubulin ·
tubulysin
Introduction
teractions, and could have superior properties as anticancer
agents either as isolated entities or as chemical warheads on
targeted antibodies or ligands.
The tubulysins, first isolated by the Hçfle/Reichenbach
group from myxobacterial cultures,[1] are exceptionally
potent cell-growth inhibitors that act by inhibiting tubulin
polymerization and thereby induce apoptosis.[2] The tubuly-
sins, of which tubulysin D is the most potent, have activity
that exceeds other tubulin modifiers including, the epothi-
lones, vinblastine, and paclitaxel (Taxol), by 20- to 1000-
fold.[3] Paclitaxel and vinblastine are current drugs for the
treatments for a variety of cancers, and epothilone deriva-
tives are under active evaluation in clinical trials.[4] Synthetic
derivatives of tubulysin D would provide essential informa-
tion about the mechanism of inhibition and key binding in-
Tubulysin D (1) is a complex tetrapeptide that can be di-
vided into four regions as defined in Figure 1: Mep (d-N-
methyl pipecolinic acid), Ile (l-isoleucine), Tuv (tubuvaline),
and Tup (tubuphenylalanine). All of the more potent deriva-
tives of tubulysin, including tubulysin D, also incorporate
the interesting O-acyl N,O-acetal functionality, which has
rarely been observed in natural products. This reactive func-
tionality is documented to be quite labile to both acidic and
basic reaction conditions, and therefore may play a key role
in the function of the tubulysins.[5]
Recently, we reported the total synthesis of tubulysin D,[6]
which represents the first synthesis of any member of the tu-
bulysin family that incorporates the O-acyl N,O-acetal func-
tionality.[7] Herein, we report the synthesis and for the first
[a] A. W. Patterson, H. M. Peltier, Prof. Dr. J. A. Ellman
Department of Chemistry
University of California–Berkeley
Berkeley, CA 94720-1460 (USA)
Fax : (+1)510-642-8369
E-mail: jellman@uclink.berkeley.edu
[b] Dr. F. Sasse
Department of Chemical Biology
Helmholtz-Zentrum für Infektionsforschung GmbH
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
Fax : (+49)531-6181-3499
E-mail: florenz.sasse@helmholtz-hzi.de
Figure 1. Tubulysin D (1).
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Chem. Eur. J. 2007, 13, 9534 – 9541

FULL PAPER
time the biological evaluation of tubulysin analogues provid-
ing key insights into the structural features necessary for
binding. These studies have also resulted in the identifica-
tion of lower molecular weight and considerably more
stable analogues that maintain the majority of tubulin poly-
merization inhibitory activity.
Results and Discussion
As shown in Figure 2, nine analogues were initially pre-
pared. Analogues 2–5 were designed to probe the Tup posi-
tion at the C-terminus of the peptide natural product, while
analogues 6–8 were designed to probe the Mep position at
the N-terminus. Analogues 9 and 10 were designed to test
the importance of the two most labile sites in the molecule.
Analogue 9 serves to test the importance of the acetyl
group present in the tubulysins. In contrast 10, which incor-
porates a methyl group in place of the reactive O-acyl N,O-
acetal, like the natural product should still be able to access
both cis- and trans-amide conformations because 10 retains
the tertiary amide at the site of modification.
Compounds 2–10 were assayed against established mam-
malian cell lines, including cancer cells, by measuring inhibi-
tion of cell growth by an MTT assay[1] (Table 1). The activi-
Table 1. Biological activity of compounds 1–11.
IC50 [ngmLꢀ1
SW-480[b]
]
Analogue
L929[a]
KB-3-1[c]
1 (Tub D)[d]
2
3
4
5
6
7
0.056[e]
0.24
3.5
0.022
0.30
0.91
0.35
0.35
0.010
15
0.070[f]
0.25
1.8
0.22
1.5
0.30
2.2
0.040[g]
120
0.029
80
8
45
8.8
40
9
10
11
0.25
0.23[h]
1.7
0.057
0.016[g]
0.50
0.22
0.13[g]
1.2
[a] Mouse fibroblasts (DSMZ ACC 2). [b] Human colon adenocarcinoma
(ATCC CCL-228). [c] Human cervix carcinoma (DSMZ ACC 158). [d]
Synthetic tubulysin D prepared previously.[6] [e] The IC50 of isolated tu-
bulysin D was previously determined to be 0.01–0.03 ngmLꢀ1 with cell
line L929.[1,3] [f] The IC50 of isolated tubulysin D was previously deter-
mined to be 0.02 ngmLꢀ1 with cell line KB-3–1.[1,3] [g] Average of two ex-
periments. [h] Average of four experiments.
Figure 2. Tubulysin analogues 2–10.
small as well as hydrophobic and hydrophilic functionality is
of considerable significance because it suggests that this site
would be appropriate for attachment of functionality in the
design of targeted antibodies and for the incorporation of
probe molecules such as fluorescent agents.
ties of the tubulysin analogues varied from 0.05–120 ngmLꢀ1
in L929 mouse fibroblast cells, with a number of simplified
analogues maintaining significant activity.
Upon examination of the Tup position at the C-terminus
of tubulysin D (2–5), we found that a wide range of modifi-
cations were tolerated (Table 1). Analogues designed to
retain only the phenethyl or g-carboxy group showed com-
parable cytoxicity. Even the greatly simplified N-methyl de-
rivative 4 and the truncated tripeptide 5 maintained good
activity. The considerable tolerance at this site for large and
Examination of the Mep position at the N-terminus of the
natural product established the importance of maintaining a
basic amine at this position. Removal of the Mep group (8)
and replacement of the Mep group with a simple acetyl
group (7) caused a drastic decrease in activity (Table 1).
However, compound 6, which retains the tertiary amine
functionality, was essentially equipotent with tubulysin D. In
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9535

J. A. Ellman, F. Sasse et al.
addition to defining the importance of the basic amine, the
simplified nature and lower molecular weight of the N,N-di-
methyl glycine present in 6 is also noteworthy.
the analogues can be attributed to an action on the tubulin/
microtubule system, and is not a result of nonspecific cyto-
toxicity.
The activities observed for analogues 9 and 10 were most
surprising. Analogue 9 showed a minimal drop in cytotoxici-
ty relative to the natural product demonstrating that the O-
acetyl group is not important for activity. The high cytotox-
icity of 10, which is only four-fold less active than tubulysin
D, is even more surprising, and clearly indicates that the
N,O-acetal is not necessary for cytoxicity.
To discern whether the structure–activity relationships ob-
served for the analogue series were additive, analogue 11
(Figure 3), which combines the truncations present in both
The synthesis of the aforementioned analogues 2–11 was
accomplished in a highly efficient manner. Analogues 2–5
were prepared from intermediate 12 (Scheme 1) that we had
previously reported in the synthesis of tubulysin D.[6] Activa-
tion of the acid as the pentafluorophenyl ester followed by
addition of phenethylamine, 4-aminobutyric acid, or methyl-
amine hydrochloride provided amides 13a–c, respectively.
Acetylation of the Tuv alcohol then provided analogues 2–4.
Compound 5, which is terminated by the Tuv carboxylic
acid, was directly prepared by acetylation of alcohol 12
(Scheme 1).
For compounds 6–8 in which Mep at the N-terminus has
been replaced, an earlier azido intermediate in the synthesis
of tubulysin D (14) was used as the common precursor.[6]
Silyl ether deprotection and then selective cleavage of the
methyl ester over the reactive O-acyl N,O-acetal with
Bu3SnOH provided acid 16 (Scheme 2). Activation of the
carboxylic acid as the pentafluorophenyl ester followed by
addition of tubuphenylalanine hydrochloride (17) and acety-
lation of the Tuv alcohol afforded analogue 8. This analogue
also serves as the penultimate intermediate to analogues 6
Figure 3. Analogue 11.
analogues 4 and 10, was also
prepared. Potent cytotoxicity
(2.0 ngmLꢀ1) was observed for
11 (Table 1). This result is partic-
ularly exciting because 11 at
551 Da is considerably lower in
molecular weight than tubulysin
D (827 Da). In addition, ana-
logue 11 incorporates the stable
N-methyl group in place of the
reactive O-acyl N,O-acetal func-
tionality.
All analogues were also ana-
lyzed in human colon and cervix
cancer lines, with structure–ac-
tivity relationships that largely
parallel SAR for the L929 cell
line. The activities of analogue
6, which possesses a Mep modi-
fication, and analogue 10, which
lacks the N,O-acetal, in the
colon cancer cell line SW-480
were also notable, with both an-
alogues proving to be more
active than tubulysin D.
Scheme 1. Preparation of 2–5. a) pentafluorophenol, 1,3-diisopropylcarbodiimide, CH2Cl2; b) 2-phenylethyla-
mine, 4-aminobutyric acid, or methylamine hydrochloride, iPr2EtN, DMF, 49% (13a), 70% (13b), or 68%
(13c) yield for two steps; c) Ac2O, pyridine, then (for 4 and 6) H2O/dioxane, 99% (3), 81% (4), 90% (5), or
97% (6).
and 7, which were prepared by reduction of the azide in the
To check for the mode of action of the analogues, PtK2
potoroo cells were incubated with the analogues at concen-
trations above the IC50 with L929, and stained for microtu-
bule cytoskeleton by immunofluorescence methods after
18 h. In each case we observed a disturbance in the microtu-
bule system, either an interference with the microtubular
network in non-dividing cells or abnormal mitotic spindles
in dividing cells. These results show that the activity of all of
presence of the pentafluorophenyl ester of N,N-dimethylgly-
cine (18) or acetic acid (19), respectively.
Compound 10, which is directly analogous to tubulysin D
with the O-acyl N,O-acetal being replaced by an N-Me
amide, was synthesized starting from azide 20, which served
as an early intermediate in the synthesis of tubulysin D
(Scheme 3).[6] Deprotonation of the Tuv-amide with
KHMDS followed by addition of methyl iodide provided N-
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Tubulysin D Analogues
FULL PAPER
Conclusion
In conclusion, we have con-
structed a series of analogues of
tubulysin D containing varia-
tions at the Tup, Mep, and N,O-
acetal positions that has for the
first time established the essen-
tial features of these regions of
the natural product necessary
for biological activity against a
series of human and animal
cancer cell lines. The biological
data indicate that a wide range
of modifications at the Tup po-
sition, for example, 2–5, are
well-tolerated indicating that
this could be a key location for
conjugation to antibodies or for
the incorporation of fluorescent
and other probe molecules. The
biological data also indicates
that while a basic amine in the
Mep region of tubulysin is nec-
essary for biological activity,
very simple and low molecular
weight substituents, for exam-
ple, 6, are acceptable at this
site. Notably, neither of the
most labile sites in the natural
product, the O-acetyl group
and the O-acyl N,O-acetal, are
necessary for biological activity
(see analogues 9 and 10, respec-
tively). This finding enables the
design of highly potent tubuly-
sin analogues that are of con-
siderably greater stability than
the natural product. Further
biological characterization of
these new tubulysin analogues
is in progress as are the synthe-
sis and biological evaluation of
additional tubulysin analogues.
Even more efficient and high
Scheme 2. Preparation of 6–8. a) AcOH/THF/H2O, 73%; b) Me3SnOH, ClACHTRE(UGN CH2)2Cl, 608C, 36%; c) pentafluor-
ophenol, 1,3-diisopropylcarbodiimide, CH2Cl2; d) 17, iPr2EtN, DMF; e) Ac2O, pyridine, then H2O/dioxane,
39% yield for three steps; f) 18 or 19, H2, Pd/C, EtOAc then H2O/dioxane, 48% (7) or 77% (8).
Scheme 3. Preparation of 10 and 11. a) potassium bis(trimethylsilyl)amide, THF, ꢀ458C then methyl iodide,
82%; b) 22, H2, Pd/C, EtOAc; c) AcOH/THF/H2O, 87% for two steps; d) Me3SnOH, ClCAHTRE(UNG CH2)2Cl, 608C, 88%;
e) methylamine, THF/MeOH, 1008C, 52%; f) PFP, DIC, CH2Cl2; g) 17, iPr2EtN, DMF; h) Ac2O, pyridine,
then H2O/dioxane, 56% for three steps; i) Ac2O, pyridine, 72%.
Me amide precursor 21. Silyl group deprotection followed
by reductive coupling in the presence of the pentafluoro-
phenyl ester of d-N-methyl pipecolinic acid (22) then pro-
vided Mep-coupled product 23. Cleavage of the methyl
ester, followed by coupling with tubuphenylalanine and ace-
tylation of the Tuv alcohol afforded descarboxy analogue
10.[8] Compound 11 was prepared in a similar manner to 10.
Heating ester 23 in the presence of methylamine directly
provided amide 25 (Scheme 3). Acetylation then afforded
analogue 11.
yielding syntheses of the most promising of the simplified
analogues will also be reported in due course.
Experimental Section
General: Compounds 9, 12, 14, 17, 20, and 22 were prepared as previous-
ly described.[6] Unless otherwise noted, all starting materials were ob-
tained from commercial suppliers and used without further purification.
Methyl iodide was filtered through a plug of basic alumina (Brockman
activity 1) immediately prior to use. Toluene, THF, ether, dioxane, and
CH2Cl2 were dried over alumina under a nitrogen atmosphere. Methanol,
iPr2NH, iPr2EtN, dichloroethane, and pyridine were distilled from CaH2
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J. A. Ellman, F. Sasse et al.
immediately prior to use. Where noted, water and acetic acid were de-
gassed using three consecutive freeze–pump–thaw cycles. Reactions were
carried out in flame or oven-dried glassware under a N2 atmosphere. Ex-
tracts were dried over Na2SO4. Products were concentrated using a Büchi
rotary evaporator under reduced pressure. Chromatography was carried
out either with Merck 60 Š 230–400 mesh silica gel or via HPFC purifica-
tion on a Biotage SP1 instrument (Charlottesville, VA) equipped with a
normal-phase Biotage Si flash column or reverse-phase Biotage C18
column. Where noted, water was removed from samples by lyophilization
using a Labconco Corp. freeze-dry system (Kansas City, MO). Optical ro-
tation measurements were performed on a Perkin-Elmer 241 polarimeter.
Optical rotations ([a]) are measured in deg cm3 gꢀ1 dmꢀ1. Concentration
(c) is measured in gdLꢀ1. IR spectra were recorded on a Nicolet Avatar
360 FTIR spectrometer equipped with an attenuated total reflectance ac-
cessory and only partial data are listed. 1H NMR and 13C NMR spectra
were obtained at room temperature with Bruker AV-400 and DRX-500
spectrometers. Chemical shifts are expressed in ppm relative to internal
solvent. High-resolution mass spectra were performed by the University
of California at Berkeley Micro-Mass Facility.
Intermediate 13c: Acid 12 (30.0 mg, 0.0503 mmol) was added to a solu-
tion of pentafluorophenol (14.0 mg, 0.0754 mmol) and 1,3-diisopropylcar-
bodiimide (8.70 mL, 0.0553 mmol) in CH2Cl2 (0.38 mL) at 08C. The reac-
tion mixture was warmed to RT, stirred for 24 h, and concentrated.
EtOAc (10 mL) was added, and the crude product was filtered, with rins-
ing of the reaction vessel with EtOAc. The filtrate was concentrated, and
the crude material was used without further purification. DMF
(0.201 mL) was added to the crude product at 08C, followed by the hy-
drochloride salt of methylamine (18.0 mg, 0.151 mmol) and iPr2EtN
(44.0 mL, 0.251 mmol). The reaction mixture was allowed to warm to RT,
stirred for 24 h at RT, and concentrated. Normal-phase HPFC purifica-
tion (CH2Cl2/MeOH 100:0
!
90:10) afforded 13c (31.0 mg, 68%).
[a]2D3 =+15.5 (c=1.0 in MeOH); 1H NMR ([D4]methanol, 500 MHz): d
= 0.83 (d, 3H, J=5.9 Hz), 0.90 (app t, 9H, J=7.5 Hz), 0.98 (app t, 6H,
J=7.5 Hz), 1.20–1.30 (m, 3H), 1.49–1.63 (m, 5H), 1.69–1.72 (m, 2H),
1.98–2.06 (m, 5H), 2.13 (s, 3H), 2.14–2.17 (m, 2H), 2.55 (app d, 1H, J=
11.5 Hz), 2.88–2.91 (m, 1H), 2.93 (s, 3H), 4.59 (d, 1H, J=9.9 Hz), 4.75
(d, 1H, J=10.8 Hz), 5.51 (d, 1H, J=12.1 Hz), 6.18 (d, 1H, J=12.1 Hz),
8.08 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 10.6, 16.2,
20.7, 22.7, 22.8, 24.3, 25.9, 26.1, 26.3, 26.6, 31.56, 31.64, 36.9, 37.6, 38.8,
44.4, 44.7, 55.3, 56.6, 69.5, 70.4, 124.4, 150.7, 164.2, 164.9, 173.4, 175.7,
179.1 ppm; IR: n˜ = 1420, 1498, 1654, 1737, 2791, 2872, 2934, 2961, 3322,
3367 cmꢀ1; HRMS (FAB): m/z: calcd for C30H52N5O6S: 610.3638; found:
610.3634 [M+H]+.
Intermediate 13a: Acid 12 (40.0 mg, 0.0670 mmol) was added to a solu-
tion of pentafluorophenol (19.0 mg, 0.101 mmol) and 1,3-diisopropylcar-
bodiimide (11.5 mL, 0.0737 mmol) in CH2Cl2 (0.51 mL) at 08C. The reac-
tion mixture was warmed to RT, stirred for 24 h, and concentrated.
EtOAc (10 mL) was added, and the crude product was filtered, with rins-
ing of the reaction vessel with EtOAc. The filtrate was concentrated, and
the crude material was used without further purification. DMF
(0.270 mL) was added to the crude product at 08C, followed by phene-
thylamine (10.2 mL, 0.0804 mmol) and iPr2EtN (23.0 mL, 0.134 mmol).
The reaction mixture was allowed to warm to RT, stirred for 24 h, and
concentrated. Normal-phase HPFC purification (CH2Cl2/MeOH 100:0 !
90:10) afforded 13a (23.0 mg, 49%). [a]2D3 =+15.0 (c=1.5 in MeOH);
1H NMR ([D4]methanol, 500 MHz): d = 0.80–0.92 (m, 12H), 0.97 (d,
3H, J=6.9 Hz), 1.00 (d, 3H, J=6.7 Hz), 1.19–1.30 (m, 3H), 1.49–1.65 (m,
5H), 1.69–1.75 (m, 2H), 1.98–2.18 (m, 7H), 2.14 (s, 3H), 2.58 (app d, 1H,
J=10.4 Hz), 2.91 (m, 1H), 2.92 (app t, 2H, J=7.5 Hz), 3.55–3.66 (m,
2H), 4.58 (d, 1H, J=9.8 Hz), 4.75 (d, 1H, J=10.2 Hz), 5.51 (d, 1H, J=
12.2 Hz), 6.17 (d, 1H, J=12.2 Hz), 7.18–7.21 (m, 1H), 7.24–7.30 (m, 4H),
8.09 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 10.7, 16.2,
20.7, 22.77, 22,81, 24.3, 25.96, 25.97, 26.1, 26.7, 31.5, 36.71, 36.73, 37.6,
38.8, 42.0, 44.4, 44.7, 55.4, 56.6, 69.5, 70.3, 124.7, 127.5, 129.6, 129.8, 140.3,
150.7, 163.5, 173.4, 175.5, 178.2, 179.1 ppm; IR: n˜ = 1497, 1544, 1661,
1738, 2793, 2874, 2934, 2961, 3301, 3370 cmꢀ1; HRMS (FAB): m/z: calcd
for C37H58N5O6S: 700.4108; found: 700.4105 [M+H]+.
Tubulysin analogue 2: A solution of 13a (15.5 mg, 0.0221 mmol) in pyri-
dine (0.221 mL) was cooled to 08C, and acetic anhydride (10.0 mL,
0.111 mmol) was added. The reaction mixture was allowed to warm to
RT over 2 h and was stirred at RT for 24 h. The solvent was removed
under reduced pressure. Column chromatography (CH2Cl2/MeOH 100:0
! 90:10) afforded 2 as an amorphous solid (16.4 mg, 99%). [a]2D3 =+50.0
1
(c=0.4 in MeOH); H NMR ([D4]methanol, 500 MHz): d = 0.79 (d, 3H,
J=6.3 Hz), 0.83 (d, 3H, J=6.5 Hz), 0.86 (d, 3H, J=6.6 Hz), 0.90 (app t,
3H, J=7.3 Hz), 0.96 (d, 3H, J=7.17 Hz), 1.05 (d, 3H, J=6.4 Hz), 1.16–
1.22 (m, 1H), 1.28–1.36 (m, 2H), 1.53–1.68 (m, 4H), 1.75–1.86 (m, 3H),
1.91–2.08 (m, 3H), 2.10–2.14 (m, 1H), 2.13 (s, 3H), 2.17–2.26 (m, 1H),
2.24 (s, 3H), 2.48 (app t, 1H, J=13.1 Hz), 2.72 (app t, 1H, J=10.8 Hz),
2.91 (app t, 2H, J=7.5 Hz), 2.99 (app d, 1H, J=11.7 Hz), 3.62 (m, 2H),
4.43 (brs, 1H), 4.60 (d, 1H, J=9.3 Hz), 5.40 (d, 1H, J=12.7 Hz), 5.85 (d,
1H, J=11.3 Hz), 6.16 (d, 1H, J=12.5 Hz), 7.18–7.21 (m, 1H), 7.25–7.30
(m, 4H), 8.17 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d
=
10.7, 16.4, 20.3, 20.7, 20.8, 22.71, 22.73, 24.0, 25.6, 25.8, 26.7, 31.4, 32.2,
35.7, 36.7, 37.3, 42.0, 44.2, 44.4, 55.1, 56.5, 70.0, 70.6, 125.7, 127.5, 129.6,
129.9, 140.3, 150.7, 163.1, 170.6, 171.9, 173.2, 174.6, 176.5 ppm; IR: n˜ =
1229, 1370, 1426, 1455, 1497, 1544, 1667, 1742, 2848, 2874, 2934, 2961,
3306, 3383 cmꢀ1; HRMS (FAB): m/z: calcd for C39H60N5O7S: 742.4213;
found: 742.4206 [M+H]+.
Intermediate 13b: Acid 12 (25.0 mg, 0.0419 mmol) was added to a solu-
tion of pentafluorophenol (12.0 mg, 0.0628 mmol) and 1,3-diisopropylcar-
bodiimide (7.22 mL, 0.0461 mmol) in CH2Cl2 (0.31 mL) at 08C. The reac-
tion mixture was warmed to RT, stirred for 24 h, and concentrated.
EtOAc (10 mL) was added, and the crude product was filtered, with rins-
ing of the reaction vessel with EtOAc. The filtrate was concentrated and
the crude material was used without further purification. DMF
(0.170 mL) was added to the crude product at 08C, followed by the 4-
aminobutyric acid (5.18 mg, 0.0503 mmol) and iPr2EtN (18.2 mL,
0.105 mmol). The reaction mixture was allowed to warm to RT, stirred
for 24 h, and concentrated. Normal-phase HPFC purification (CH2Cl2/
MeOH 100:0 ! 90:10) afforded 13b (20.0 mg, 70%). [a]2D3 =+10.3 (c=
1.0 in MeOH); 1H NMR ([D4]methanol, 500 MHz): d = 0.81–0.85 (m,
3H), 0.88–0.91 (m, 9H), 0.97 (d, 3H, J=7.0 Hz), 1.00 (d, 3H, J=6.1 Hz),
1.19–1.26 (m, 1H), 1.27–1.34 (m, 1H), 1.53–1.66 (m, 5H), 1.70–1.77 (m,
2H), 1.91 (m, 3H), 1.99–2.06 (m, 4H), 2.13–2.19 (m, 3H), 2.19 (s, 3H),
2.28 (app t, 2H, J=7.3 Hz), 2.70 (app d, 1H, J=11.4 Hz), 2.96 (app d,
1H, J=11.1 Hz), 3.42 (app t, 2H, J=7.2 Hz), 4.59 (d, 1H, J=9.2 Hz),
4.76 (d, 1H, J=9.0 Hz), 5.51 (d, 1H, J=11.9 Hz), 6.16 (d, 1H, J=
Tubulysin analogue 3: A solution of 13b (15.0 mg, 0.0220 mmol) in pyri-
dine (0.220 mL) was cooled to 08C, and acetic anhydride (10.4 mL,
0.110 mmol) was added. The reaction mixture was allowed to warm to
RT over 2 h and was stirred at RT for 24 h. The reaction mixture was
then cooled to 08C, and a 1:1 mixture of dioxane/water (0.630 mL) was
added. The mixture was allowed to warm to RT and was stirred for 12 h
at RT. The solvent was removed under reduced pressure. Column chro-
matography (CH2Cl2/MeOH 100:0 ! 90:10) afforded 3 as an amorphous
solid (13.0 mg, 81%). [a]2D3 =+24.3 (c=0.7 in MeOH); 1H NMR
([D4]methanol, 500 MHz): d = 0.79 (d, 3H, J=6.7 Hz), 0.85 (d, 3H, J=
6.9 Hz), 0.87–0.92 (m, 6H), 0.96 (d, 3H, J=6.6 Hz), 1.05 (d, 3H, J=
6.3 Hz), 1.15–1.23 (m, 1H), 1.35–1.42 (m, 1H), 1.57–1.65 (m, 3H), 1.69–
1.72 (m, 1H), 1.77–1.81 (m, 1H), 1.84–1.93 (m, 4H), 1.95–2.01 (m, 2H),
2.08–2.18 (m, 2H), 2.13 (s, 3H), 2.28–2.39 (m, 4H), 2.33 (s, 3H), 2.48–
2.53 (m, 1H), 2.94 (app d, 1H, J=10.1 Hz), 3.08 (app d, 1H, J=11.3 Hz),
3.40–3.43 (m, 2H), 4.41 (brs, 1H), 4.60 (d, 1H, J=9.5 Hz), 5.40 (d, 1H,
J=12.4 Hz), 5.87 (d, 1H, J=10.8 Hz), 6.13 (d, 1H, J=10.8 Hz), 8.18 ppm
(s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 10.7, 16.4, 20.3, 20.70,
20.76, 22.73, 22.74, 23.7, 25.54, 22.56, 26.7, 26.9, 31.1, 32.2, 34.7, 35.9, 37.4,
40.2, 44.18, 44.25, 55.2, 56.5, 69.7, 70.7, 125.6, 150.8, 163.2, 170.7, 171.9,
173.3, 173.8, 176.4, 180.5 ppm; IR: n˜ = 1371, 1420, 1497, 1544, 1667,
1741, 2342, 2360, 2840, 2872, 2929, 2961, 3293, 3368 cmꢀ1; HRMS (FAB):
m/z: calcd for C35H58N5O9S: 724.3955; found: 724.3937 [M+H]+.
12.2 Hz), 8.08 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d
=
10.7, 16.2, 20.7, 22.77, 22.81, 24.1, 25.9, 26.7, 27.3, 31.4, 35.6, 37.6, 38.89,
38.90, 40.4, 43.0, 44.44, 44.48, 55.4, 56.6, 58.7, 69.9, 70.1, 124.5, 150.9,
163.6, 173.4, 174.92, 174.95, 174.96, 180.9 ppm; IR: n˜ = 1451, 1550, 1643,
1658, 1736, 2795, 2872, 2925, 2961, 3365 cmꢀ1; HRMS (FAB): m/z: calcd
for C33H56N5O8S: 682.3850; found: 682.3861 [M+H]+.
9538
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 9534 – 9541

Tubulysin D Analogues
FULL PAPER
Tubulysin analogue 4: A solution of 13c (10.5 mg, 0.0172 mmol) in pyri-
dine (0.172 mL) was cooled to 08C, and acetic anhydride (8.10 mL,
0.0861 mmol) was added. The reaction mixture was allowed to warm to
RT over 2 h and was stirred at RT for 24 h. The solvent was removed
under reduced pressure. Column chromatography (CH2Cl2/MeOH 100:0
! 90:10) afforded 4 as an amorphous solid (10.1 mg, 90%). [a]2D3 =+60.5
1651, 1735, 2100, 2964 cmꢀ1
;
HRMS (FAB): m/z: calcd for
C22H35N5O6SNa: 520.2206; found: 520.2200 [M+Na]+.
Tubulysin analogue 8: Acid 16 (39.0 mg, 0.0784 mmol) was added to a so-
lution of pentafluorophenol (2.0 mg, 0.118 mmol) and 1,3-diisopropylcar-
bodiimide (13.4 mL, 0.0862 mmol) in CH2Cl2 (1.12 mL) at 08C. The reac-
tion mixture was warmed to RT, stirred for 24 h, and concentrated.
EtOAc (10 mL) was added, and the crude product was filtered with rins-
ing of the reaction vessel with EtOAc. The filtrate was concentrated, and
the crude material was used without further purification. DMF (1.00 mL)
was added to the crude product at 08C, followed by 17 (57.0 mg,
0.235 mmol) and iPr2EtN (68.0 mL, 0.392 mmol). The reaction mixture
was allowed to warm to RT, stirred for 24 h at RT, and concentrated.
Normal-phase HPFC purification (EtOAc/MeOH 100:0 ! 95:5) afford-
ed the product containing trace amounts of iPr2EtN. The product mixture
(32.0 mg, 0.181 mmol) was dissolved in pyridine (1.80 mL), cooled to
08C, and acetic anhydride (0.140 mL, 1.45 mmol) was added. The reac-
tion mixture was allowed to warm to RT over 2 h and was stirred at RT
for 22 h. The reaction mixture was then cooled to 08C, and a 1:1 mixture
of deoxygenated H2O/dioxane (0.5 mL) was added. The mixture was al-
lowed to warm to RT and was stirred for 14 h at RT. The solvent was re-
moved under reduced pressure. Reverse-phase HPFC (MeCN/H2O 20:80
! 100:0) followed by lyophilization afforded 8 as an amorphous solid
(51.0 mg, 39%, over three steps). [a]2D3 =+61.4 (c=1 in MeOH);
1H NMR ([D4]methanol, 500 MHz): d = 0.87 (app t, 6H, J=6.8 Hz),
0.92 (d, 3H, J=6.5 Hz), 0.93 (d, 3H, J=6.5 Hz), 0.98 (t, 3H, J=7.3 Hz),
1.10 (d, 3H, J=6.5 Hz), 1.17 (d, 3H, J=7.0 Hz), 1.25–1.35 (m, 1H), 1.62–
1.69 (m, 1H), 1.72–1.80 (m, 1H), 1.86–1.94 (m, 1H), 1.94–2.04 (m, 2H),
2.06–2.16 (m, 3H), 2.17 (s, 3H), 2.28–2.40 (m, 1H), 2.48–2.58 (m, 2H),
2.91 (d, 2H, J=6.5 Hz), 3.72 (d, 1H, J=9.5 Hz), 4.32–4.41 (m, 1H), 4.41–
4.54 (brs, 1H), 5.46 (d, 1H, J=12.5 Hz), 5.59 (d, 1H, J=12.5 Hz), 5.90
(dd, 1H, J=2.0, 11.0 Hz), 7.16 (app sextet, 1H, J=4.5 Hz), 7.23 (app d,
4H, J=4.5 Hz), 8.10 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d
= 10.9, 16.2, 18.7, 20.1, 20.8, 20.9, 22.8, 22.9, 26.1, 26.8, 32.2, 35.8, 36.3,
38.0, 39.4, 42.3, 44.3, 50.9, 64.6, 70.7, 125.6, 127.5, 129.4, 130.6, 139.5,
150.9, 162.8, 170.9, 172.0, 173.21, 173.24, 180.0 ppm; IR: n˜ = 1218, 1669,
1739, 2099, 2964 cmꢀ1; HRMS (FAB): m/z: calcd for C36H52N6O8NaS:
751.3465; found: 751.3456 [M+Na].
1
(c=0.6 in MeOH); H NMR ([D4]methanol, 500 MHz): d = 0.78 (d, 3H,
J=7.0 Hz), 0.84 (d, 3H, J=6.7 Hz), 0.87–0.91 (m, 6H), 0.96 (d, 3H, J=
6.7 Hz), 1.04 (d, 3H, J=6.3 Hz), 1.25–1.15 (m, 1H), 1.24–132 (m, 1H),
1.48–1.65 (m, 4H), 1.72–1.75 (m, 2H), 1.79–1.86 (m, 1H), 1.93–2.18 (m,
5H), 2.13 (s, 3H), 2.15 (s, 3H), 2.22–2.29 (m, 1H), 2.47–2.57 (m, 2H),
2.89–2.94 (m, 1H), 2.92 (s, 3H), 4.43 (brs, 1H), 4.60 (d, 1H, J=10.1 Hz),
5.39 (d, 1H, J=12.2 Hz), 5.87 (d, 1H, J=12.0 Hz), 6.18 (d, 1H, J=
12.5 Hz), 8.16 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d
=
10.7, 16.4, 20.3, 20.69, 20.74, 22.68, 22.69, 24.3, 25.6, 26.1, 26.3, 26.6, 31.5,
32.2, 35.9, 37.3, 44.2, 44.7, 55.0, 56.6, 70.4, 70.7, 125.5, 150.7, 163.8, 170.8,
171.9, 173.3, 175.5, 176.7 ppm; IR: n˜ = 1229, 1371, 1420, 1466, 1499,
1549, 1665, 1742, 2792, 2848, 2875, 2934, 2961, 3305, 3380 cmꢀ1; HRMS
(FAB): m/z: calcd for C32H54N5O7S: 652.3744; found: 652.3719 [M+H]+.
Tubulysin analogue 5: A solution of 12 (25.0 mg, 0.0419 mmol) in pyri-
dine (0.420 mL) was cooled to 08C, and acetic anhydride (19.8 mL,
0.209 mmol) was added. The reaction mixture was allowed to warm to
RT over 2 h and was stirred at RT for 24 h. The reaction mixture was
then cooled to 08C, and a 1:1 mixture of dioxane/water (1.50 mL) was
added. The mixture was allowed to warm to RT and was stirred for 12 h
at RT. The solvent was removed under reduced pressure. Column chro-
matography (CH2Cl2/MeOH 100:0 ! 90:10) afforded 5 as an amorphous
solid (26.0 mg, 97%). [a]2D3 =+12.0 (c=2.6 in MeOH); 1H NMR
([D4]methanol, 500 MHz): d = 0.82–0.84 (m, 3H), 0.88–0.92 (m, 9H),
0.96 (d, 3H, J=6.7 Hz), 1.01 (d, 3H, J=6.3 Hz), 1.15–1.22 (m, 1H), 1.52–
1.70 (m, 4H), 1.75–1.82 (m, 2H), 1.93–2.05 (m, 4H), 2.12 (s, 3H), 2.20–
2.23 (m, 4H), 2.39–2.55 (m, 6H), 3.15–3.22 (m, 1H), 4.64 (d, 1H, J=
9.6 Hz), 5.39 (d, 1H, J=12.1 Hz), 5.83 (d, 1H, J=11.7 Hz), 5.98 (brs,
1H), 8.02 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 10.9,
16.3, 20.6, 20.8, 20.9, 22.8, 23.2, 25.1, 25.4, 26.6, 30.9, 31.8, 36.1, 37.5, 44.0,
44.1, 54.8, 55.5, 56.3, 69.1, 71.0, 125.3, 155.1, 169.0, 171.8, 172.0, 173.5,
175.7, 178.4 ppm; IR: n˜ = 1371, 1422, 1471, 1499, 1597, 1666, 1743, 2874,
2934, 2962, 3384 cmꢀ1
; HRMS (FAB): m/z: calcd for C31H51N4O8S:
639.3428; found: 639.3439 [M+H]+.
N,N-dimethylglycine pentafluorophenyl ester (18): To a solution of N,N-
dimethylglycine (82.0 mg, 0.800 mmol) in EtOAc (2.00 mL, filtered
through activated alumina) were added pentafluorophenol (162 mg,
0.880 mmol) and 1,3-dicyclohexylcarbodiimide (182 mg, 0.88 mmol). The
reaction mixture was stirred for 12 h at RT at which time it was filtered
(washing with EtOAc) and concentrated. Ester 18 was used immediately
without further purification.
Intermediate 15: A solution of 14 (475 mg, 0.759 mmol) in deoxygenated
AcOH/H2O/THF (3:1:1, 38.0 mL) was stirred at RT for 27 h. Addition of
toluene (400 mL) followed by concentration and normal-phase HPFC pu-
rification (hexanes/EtOAc 95:5 ! 60:40) afforded 15 as an amorphous
solid (283 mg, 73%). [a]D23
([D4]methanol, 500 MHz): d
=
=
+55.1 (c=1.0 in MeOH); 1H NMR
0.88–0.99 (m, 15H), 1.02 (d, 3H, J=
Tubulysin analogue 6: Pd/C (10 wt%, 8.7 mg) and azide 8 (18.0 mg,
0.0247 mmol) were added to a solution of 18 (0.0988 mmol) in EtOAc
(0.40 mL, filtered through activated alumina). The reaction mixture was
stirred under a hydrogen atmosphere for 26 h and then filtered through a
plug of Celite with washing of the filter pad with EtOAc. The filtrate was
concentrated, and a 1:1 mixture of deoxygenated H2O/dioxane (4.0 mL)
was added. The mixture was stirred for 20 h at RT and concentrated. Re-
verse-phase HPFC (MeCN/H2O 20:80 ! 100:0) followed by lyophiliza-
tion afforded 6 as an amorphous solid (9.3 mg, 48%). [a]2D3 =ꢀ2.0 (c=0.6
in MeOH); 1H NMR ([D4]methanol, 500 MHz): d = 0.81 (d, 3H, J=
6.5 Hz), 0.87 (d, 3H, J=6.5 Hz), 0.89 (d, 3H, J=7.0 Hz), 0.92 (t, 3H, J=
7.5 Hz), 0.98 (d, 3H, J=6.5 Hz), 1.06 (d, 3H, J=6.5 Hz), 1.12–1.21 (m,
1H), 1.17 (d, 3H, J=7.0 Hz), 1.58–1.70 (m, 2H), 1.77–1.91 (m, 2H),
1.92–2.05 (m, 3H), 2.06–2.17 (m, 2H), 2.16 (s, 3H), 2.35 (s, 6H), 2.46–
2.57 (m, 2H), 2.92 (d, 2H, J=5.5 Hz), 3.11 (q, 2H, J=16.2 Hz), 4.28–4.50
(brs, 1H), 4.32–4.38 (m, 1H), 4.70 (d, 1H, J=8.5 Hz), 5.46 (d, 1H, J=
12.0 Hz), 5.87 (d, 1H, J=11.0 Hz), 6.05 (d, 1H, J=12.0 Hz), 7.12–7.18
(m, 1H), 7.19–7.26 (m, 4H), 8.09 ppm (s, 1H); 13C NMR ([D4]methanol,
125 MHz): d = 11.1, 16.6, 19.0, 20.3, 20.8, 20.9, 22.9, 25.5, 26.2, 26.9, 32.4,
35.9, 37.6, 39.1, 39.7, 42.1, 44.5, 45.8, 51.2, 55.2, 62.7, 70.8, 125.6, 127.5,
129.4, 130.6, 139.7, 150.9, 162.7, 170.8, 171.7, 172.0, 173.3, 176.5,
181.8 ppm; IR: n˜ = 1226, 1496, 1542, 1741, 2964 cmꢀ1; HRMS (FAB):
m/z: calcd for C40H62N5O9S: 788.4268; found: 788.4256 [M+H]+.
6.5 Hz), 1.25–1.35 (m, 1H), 1.72–1.79 (m, 1H), 1.80–1.90 (m, 1H), 1.98–
2.09 (m, 2H), 2.10–2.25 (m, 2H), 2.34 (d, 2H, J=7.0 Hz), 3.74 (d, 1H,
J=9.5 Hz), 3.89 (s, 3H), 4.47–4.70 (brs, 1H), 4.79 (d, 1H, J=10.5 Hz),
5.48 (d, 1H, J=12.5 Hz), 5.58 (d, 1H, J=10.5 Hz), 8.30 ppm (s, 1H);
13C NMR ([D4]methanol, 125 MHz): d
=
11.0, 16.1, 20.4, 20.8, 22.88,
22.91, 26.1, 26.7, 32.0, 36.7, 39.3, 44.2, 52.8, 64.6, 69.6, 129.3, 147.7, 163.3,
173.6, 173.7, 180.2 ppm; IR: n˜ = 1095, 1212, 1652, 1735, 2099, 2963 cmꢀ1
;
HRMS (FAB): m/z: calcd for C23H37N5O6SNa: 534.2362; found: 534.2367
[M+Na]+.
Intermediate 16: Me3SnOH (736 mg, 4.07 mmol) was added to a solution
of methyl ester 15 (260 mg, 0.509 mmol) in dichloroethane (25.0 mL).
The reaction mixture was heated to 558C for 22 h and then concentrated.
Normal-phase HPFC (CH2Cl2/MeOH/AcOH 100:0 ! 90:10:1) followed
by reverse-phase HPFC (MeCN/H2O 20:80 ! 100:0) and lyophilization
afforded 16 as an amorphous solid (90.0 mg, 36%). [a]2D3 =+51.5 (c=1.0
in MeOH); 1H NMR ([D4]methanol, 500 MHz): d = 0.88–0.94 (m, 9H),
0.97 (app t, 6H, J=6.8), 1.03 (d, 3H, J=6.5), 1.31 (sept, 1H, J=7.4),
1.72–1.81 (m, 1H), 1.82–1.90 (brs, 1H), 2.00–2.09 (m, 2H), 2.10–2.17 (m,
1H), 2.18–2.28 (m, 1H), 2.31 (app t, 2H, J=7.5), 3.75 (d, 1H, J=9.5),
4.44–4.68 (brs, 1H), 4.77 (d, 1H, J=9.5), 5.48 (d, 1H, J=12.5), 5.58 (d,
1H, J=12.5), 8.24 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d =
10.8, 16.0, 20.3, 20.6, 22.71, 22.73, 26.0, 26.5, 32.0, 36.7, 39.1, 44.1, 64.6,
69.5, 128.5, 149.2, 164.7, 173.58, 173.61, 179.5 ppm; IR: n˜ = 1088, 1217,
Chem. Eur. J. 2007, 13, 9534 – 9541
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9539

J. A. Ellman, F. Sasse et al.
Acetic acid pentafluorophenyl ester (19): To a solution of acetic acid
(46 mL, 0.800 mmol) in EtOAc (2.00 mL, filtered through activated alumi-
na) were added pentafluorophenol (162 mg, 0.880 mmol) and 1,3-dicyclo-
hexylcarbodiimide (182 mg, 0.88 mmol). The reaction mixture was stirred
for 12 h at RT at which time it was filtered (washing with EtOAc) and
concentrated. Ester 19 was used immediately without further purifica-
tion.
2.57 (d, 1H, J=9.0 Hz), 2.85–2.95 (m, 1H), 3.17 (s, 3H), 3.91 (s, 3H),
4.40–4.55 (brs, 1H), 4.68 (d, 1H, J=9.5 Hz), 4.75 (d, 1H, J=9.0 Hz),
8.32 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 11.2, 16.2,
20.5, 20.7, 24.4, 25.9, 26.3, 31.1, 31.6, 32.1, 37.7, 38.8, 44.9, 52.8, 55.0, 56.7,
69.9, 70.6, 129.3, 147.5, 163.2, 175.4, 175.7, 180.6; IR: n˜ = 1095, 1212,
1238, 1495, 1622, 1722, 2936 cmꢀ1
; HRMS (FAB): m/z: calcd for
C25H43N4O5S: 511.2954; found: 511.2947 [M+H]+.
Tubulysin analogue 7: Pd/C (10 wt%, 7.0 mg) and azide 8 (15.0 mg,
0.021 mmol) were added to a solution of 19 (0.082 mmol) in EtOAc
(0.40 mL, filtered through activated alumina). The reaction mixture was
stirred under a hydrogen atmosphere for 21 h and then filtered through a
plug of Celite with washing of the filter pad with EtOAc. The filtrate was
concentrated, and a 1:1 mixture of deoxygenated H2O/dioxane (2.0 mL)
was added. The mixture was stirred for 7 h at RT and concentrated.
Normal-phase HPFC (CH2Cl2/MeOH 99:1 ! 90:10) afforded 7 as an
amorphous solid (12.0 mg, 77%). [a]2D3 =ꢀ7.0 (c=0.4 in MeOH);
1H NMR ([D4]methanol, 500 MHz): d = 0.81 (d, 3H, J=7.0 Hz), 0.86 (d,
3H, J=7.0 Hz), 0.88 (d, 3H, J=7.0 Hz), 0.92 (t, 3H, J=7.5 Hz), 0.95 (d,
3H, J=7.0 Hz), 1.07 (d, 3H, J=6.5 Hz), 1.17 (d, 3H, J=7.5 Hz), 1.12–
1.22 (m, 1H), 1.26–1.36 (m, 1H), 1.57–1.69 (m, 2H), 1.80–1.91 (m, 1H),
1.95 (s, 3H), 1.96–2.08 (m, 4H), 2.09–2.16 (m, 1H), 2.16 (s, 3H), 2.25–
2.34 (m, 1H), 2.45–2.57 (m, 2H), 2.91 (d, 2H, J=7.0 Hz), 4.32–4.50 (brs,
1H), 4.34–4.42 (m, 1H), 4.62 (d, 1H, J=9.5 Hz), 5.42 (d, 1H, J=
12.0 Hz), 5.89 (d, 1H, J=9.5 Hz), 6.13 (d, 1H, J=12.0 Hz), 7.12–7.18 (m,
1H), 7.19–7.25 (m, 4H), 8.10 ppm (s, 1H); 13C NMR ([D4]methanol,
125 MHz): d = 11.0, 16.4, 18.8, 20.0, 20.8, 20.9, 22.2, 22.87, 22.88, 25.6,
26.9, 32.5, 35.8, 37.2, 38.1, 39.5, 42.3, 44.5, 50.9, 55.6, 70.8, 125.7, 127.5,
129.4, 130.6, 139.5, 150.7, 162.7, 170.9, 172.0, 173.0, 173.3, 176.6,
Intermediate 24: Me3SnOH (496 mg, 2.74 mmol) was added to a solution
of methyl ester 23 (175 mg, 0.343 mmol) in dichloroethane (17.0 mL).
The reaction mixture was heated to 608C for 20 h and then concentrated.
Column chromatography (100% CH2Cl2 to elute tin containing materials
followed by CH2Cl2/MeOH/NH4OH 80:20:1 to elute the product) afford-
1
ed 24 as an amorphous solid (150 mg, 88%). The H NMR corresponded
to a 6:1 mixture of rotamers, with the major isomer reported. [a]D23
=
1
ꢀ17.4 (c=1.0 in MeOH); H NMR ([D6]DMSO, 500 MHz): d = 0.70 (m,
3H), 0.75–0.82 (m, 3H), 0.83–0.90 (m, 6H), 1.04–1.16 (m, 1H), 1.17–1.28
(m, 2H), 1.37–1.55 (m, 3H), 1.56–1.72 (m, 3H), 1.73–1.91 (m, 3H), 2.00–
2.24 (m, 2H), 2.22 (s, 3H), 2.84 (brs, 1H), 2.94–3.00 (m, 1H), 3.04 (s,
3H), 4.16–4.60 (brs, 1H), 4.49 (d, 1H, J=10.5), 4.56 (app t, 1H, J=9.0),
5.93–6.40 (brs, 1H), 8.05 (s, 1H), 8.25 ppm (s, 1H); 13C NMR
([D6]DMSO, 125 MHz): d = 10.0, 14.7, 19.1, 19.5, 19.6, 21.8, 23.58, 23.62,
28.75, 28.8, 35.2, 36.7, 42.6, 52.3, 54.1, 67.1, 67.5, 126.7, 147.8, 162.2, 170.7,
172.0, 177.6 ppm; IR: n˜
= ;
1276, 1368, 1471, 1616, 2874, 2961 cmꢀ1
HRMS (FAB): m/z: calcd for C24H41N4O5S: 497.2798; found: 497.2793
[M+H]+.
Tubulysin analogue 10: Acid 24 (34.0 mg, 0.0684 mmol) was added to a
solution of pentafluorophenol (19.0 mg, 0.103 mmol) and 1,3-diisopropyl-
carbodiimide (12.0 mL, 0.0752 mmol) in CH2Cl2 (0.52 mL) at 08C. The re-
action mixture was warmed to RT, stirred for 24 h, and concentrated.
EtOAc (10 mL) was added, and the crude product was filtered with rins-
ing of the reaction vessel with EtOAc. The filtrate was concentrated, and
the crude material was used without further purification. DMF
(0.270 mL) was added to the crude product at 08C, followed by 17
(50.0 mg, 0.205 mmol) and iPr2EtN (60.0 mL, 0.342 mmol). The reaction
mixture was allowed to warm to RT, stirred for 24 h at RT, and concen-
trated. Normal-phase HPFC purification (CH2Cl2/MeOH 98:2 ! 80:20)
followed by reverse-phase HPFC (MeCN/H2O 20:80 ! 100:0) afforded
34.0 mg of product containing trace amounts of iPr2EtN. The product
mixture (34.0 mg, 0.496 mmol) was dissolved in pyridine (0.50 mL),
cooled to 08C, and acetic anhydride (38.0 mL, 0.397 mmol) was added.
The reaction mixture was allowed to warm to RT over 2 h and was stirred
at RT for 22 h. The reaction mixture was then cooled to 08C, and a 1:1
mixture of deoxygenated H2O/dioxane (1.6 mL) was added. The mixture
was allowed to warm to RT and was stirred for 20 h at RT. The solvent
was removed under reduced pressure. Normal-phase HPFC (CH2Cl2/
MeOH 95:5 ! 80:20) followed by lyophilization afforded 10 as an amor-
180.3 ppm; IR: n˜
=
1225, 1554, 1647, 1740, 2876, 2963 cmꢀ1; HRMS
(FAB): m/z: calcd for C38H56N4O9NaS: 767.3666; found: 767.3648
[M+Na]+.
Intermediate 21: A solution of 20 (905 mg, 1.77 mmol) in THF (6.0 mL)
was cooled to ꢀ458C and KHMDS (6.02 mL, 3.01 mmol, 0.50m in tolu-
ene) was added. The resulting mixture was stirred for 20 min at ꢀ458C.
Methyl iodide (754 mg, 5.31 mmol, filtered through activated alumina)
was added, and the reaction mixture was allowed to warm to RT over
4.5 h at which time the reaction was quenched with MeOH (5.0 mL). The
crude product was diluted with EtOAc (250 mL) and washed with brine
(100 mL). The aqueous layer was extracted with EtOAc (2”100 mL).
The organic portions were dried, filtered, and concentrated. Normal-
phase HPFC (hexanes/EtOAc 95:5 ! 60:40) yielded 21 as an amorphous
1
solid (761 mg, 82%). The H NMR corresponded to a 10:1 mixture of ro-
tamers, with the major isomer reported. [a]2D3 =+67.5 (c=1.0 in CHCl3);
1H NMR (CDCl3, 400 MHz): d = 0.55–0.70 (m, 6H), 0.84 (d, 3H, J=
6.8 Hz), 0.85–0.93 (m, 15H), 0.95 (d, 3H, J=6.8 Hz), 1.17–1.29 (m, 1H),
1.60–1.79 (m, 2H), 2.00–2.20 (m, 3H), 2.92 (s, 3H), 3.50 (d, 1H, J=
9.6 Hz), 3.89 (s, 3H), 4.37–4.45 (m, 1H), 4.90 (dd, 1H, J=3.6, 6.4 Hz),
8.07 ppm (s, 1H); 13C NMR (CDCl3, 100 MHz): d 4.6, 6.7, 10.6, 15.9, 19.1,
20.0, 25.0, 30.2, 34.9, 40.1, 52.2, 57.3, 63.9, 71.0, 127.4, 146.4, 161.8, 169.5,
1
phous solid (28.0 mg, 56%, over three steps). The H NMR corresponded
to a 16:1 mixture of rotamers, with the major isomer reported. [a]D23
=
ꢀ19.2 (c=0.9 in MeOH); 1H NMR ([D4]methanol, 500 MHz): d = 0.81
(d, 3H, J=6.5 Hz), 0.92 (t, 3H, J=7.3 Hz), 0.98 (d, 3H, J=6.5 Hz), 1.03
(d, 3H, J=6.5 Hz), 1.16 (d, 3H, J=7.0 Hz), 1.09–1.23 (m, 1H), 1.37–1.41
(m, 1H), 1.56–1.74 (m, 5H), 1.75–1.92 (m, 4H), 1.96–2.05 (m, 1H), 2.15
(s, 3H), 2.31 (s, 3H), 2.23–2.41 (m, 3H), 2.51 (brs, 1H), 2.85 (d, 1H, J=
10.5 Hz), 2.92 (d, 2H, J=6.5 Hz), 3.05 (d, 1H, J=11.5 Hz), 3.10 (s, 3H),
4.30–4.50 (m, 2H), 4.73 (d, 1H, J=8.0 Hz), 5.71 (dd, 1H, J=2.5,
11.0 Hz), 7.13–7.18 (m, 1H), 7.19–7.25 (m, 4H), 8.08 ppm (s, 1H);
13C NMR ([D4]methanol, 125 MHz): d = 11.3, 16.4, 19.1, 20.4, 20.6, 20.9,
23.7, 25.5, 25.5, 30.9, 31.0, 31.1, 35.6, 37.6, 39.5, 39.6, 42.0, 44.2, 51.2, 55.2,
56.4, 69.7, 71.2, 125.1, 127.4, 129.3, 130.6, 139.8, 151.1, 162.7, 171.6, 171.8,
178.3 ppm; IR: n˜ = 1094, 1210, 1238, 1645, 1736, 2098, 2877, 2960 cmꢀ1
;
HRMS (FAB): m/z: calcd for C24H44N5O4SiS: 526.2883; found: 526.2877
[M+H]+.
Intermediate 23: Pd/C (10 wt%, 242 mg) and azide 21 (359 mg,
0.683 mmol) were added to a solution of 22 (2.17 mmol) in EtOAc
(6.80 mL, filtered through activated alumina). The reaction mixture was
stirred under a hydrogen atmosphere for 26 h and then filtered through a
plug of Celite, with washing of the filter pad with EtOAc. Normal-phase
HPFC purification (EtOAc/MeOH 99:1 ! 95:5) provided 483 mg of
Mep-coupled product. The product was dissolved in deoxygenated
AcOH/H2O/THF (35.0 mL, 3:1:1) and stirred at RT for 28 h. Concentra-
tion followed by normal-phase HPFC purification (EtOAc/MeOH 98:2
! 85:15) afforded 23 as an amorphous solid (302 mg, 87%, over two
steps). The 1H NMR corresponded to a 7.5:1 mixture of rotamers, with
the major isomer reported. [a]2D3 =ꢀ4.8 (c=1.0 in MeOH); 1H NMR
([D4]methanol, 500 MHz): d = 0.81 (d, 3H, J=6.5 Hz), 0.91 (t, 3H, J=
7.5 Hz), 0.97 (d, 3H, J=6.5 Hz), 0.99 (d, 3H, J=6.5 Hz), 1.16–1.34 (m,
2H), 1.49–1.66 (m, 4H), 1.75 (d, 2H, J=10.5 Hz), 1.77–1.86 (brs, 1H),
1.89–2.01 (m, 2H), 2.02–2.09 (m, 1H), 2.17 (s, 3H), 2.18–2.26 (m, 1H),
173.6, 175.0, 182.5 ppm; IR: n˜ = 1220, 1495, 1541, 1643, 1712, 2964 cmꢀ1
;
HRMS (FAB): m/z: calcd for C38H57N5O7S: 728.4057; found: 728.4053
[M+H]+.
Intermediate 25: In a sealed tube, a solution of methylamine (5.00 mmol,
2m in THF) was added to a solution of 23 (27.0 mg, 0.0529 mmol) in
MeOH (2.50 mL). The reaction solution was heated to 1008C for 21 h.
After the solution cooled to RT, the solvent was removed under reduced
pressure. Reverse-phase HPFC (MeCN/H2O 20:80 ! 100:0) followed by
lyophilization provided compound 25 (14.0 mg, 52%) as an amorphous
9540
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 9534 – 9541

Tubulysin D Analogues
FULL PAPER
solid. The 1H NMR corresponded to a 6:1 mixture of rotamers, with the
major isomer reported. [a]2D3 =ꢀ2.9 (c=1.0 in MeOH); 1H NMR
([D4]methanol, 500 MHz): d = 0.83 (d, 3H, J=6.5 Hz), 0.90 (t, 3H, J=
7.5 Hz), 0.966 (d, 3H, J=6.5 Hz), 0.974 (d, 3H, J=6.5 Hz), 1.15–1.25 (m,
1H), 1.25–1.32 (m, 1H), 1.48–1.66 (m, 4H), 1.72 (d, 2H, J=10.5 Hz),
1.81–1.99 (m, 3H), 2.00–2.10 (m, 1H), 2.16 (s, 3H), 2.19–2.37 (m, 1H),
2.53–2.58 (m, 1H), 2.87–2.94 (m, 1H), 2.92 (s, 3H), 3.17 (s, 3H), 4.16–
4.58 (brs, 1H), 4.64 (dd, 1H, J=2.3, 10.3 Hz), 4.72 (d, 1H, J=9.0 Hz),
8.06 ppm (s, 1H); 13C NMR ([D4]methanol, 125 MHz): d = 11.1, 16.1,
20.5, 20.6, 24.4, 25.9, 26.3, 26.4, 31.3, 31.7, 37.8, 38.8, 44.8, 55.4, 56.7, 70.0,
70.6, 124.2, 151.0, 164.4, 175.3, 175.7, 179.4 ppm; IR: n˜ = 1070, 1499,
1551, 1646, 2876, 2961 cmꢀ1; HRMS (FAB): m/z: calcd for C25H44N5O4S:
510.3114; found: 510.3098 [M+H]+.
IgG antibody conjugated with Alexa Fluor 488 (1:200; Molecular Probes)
at 378C for 45 min. Nuclei and chromosomes were stained with DAPI
(1 mgmLꢀ1). The cells were washed with PBS between all incubations.
The coverslips were mounted using Prolong Antifade Gold (Molecular
Probes), and viewed with a Zeiss Axiophot fluorescence microscope
using appropriate filter sets.
Acknowledgement
J.A.E., H.M.P., and A.W.P. gratefully acknowledge the NSF (CHE-
0446173) for support of this work. A.W.P. gratefully acknowledges an
American Chemical Society Medicinal Chemistry Graduate Student Fel-
lowship sponsored by Bristol-Myers Squibb, and H.M.P. gratefully ac-
knowledges a graduate fellowship from Eli Lilly. F.S. gratefully acknowl-
edges the technical assistance of Bettina Hinkelmann and Birte Engel-
hardt.
Tubulysin analogue 11: A solution of 25 (12.0 mg, 0.0235 mmol) in pyri-
dine (0.500 mL) was cooled to 08C, and acetic anhydride (18.0 mL,
0.188 mmol) was added. The reaction mixture was allowed to warm to
RT over 2 h and was stirred at RT for 21 h. The solvent was removed
under reduced pressure. Reverse-phase HPFC (MeCN/H2O 20:80
!
100:0) followed by lyophilization afforded 11 as an amorphous solid
1
(9.3 mg, 72%). The H NMR corresponded to a 23:1 mixture of rotamers,
1
with the major isomer reported. [a]2D3 =ꢀ2.2 (c=0.6 in MeOH); H NMR
([D4]methanol, 500 MHz): d = 0.80 (d, 3H, J=7.0 Hz), 0.92 (t, 3H, J=
7.5 Hz), 0.98 (d, 3H, J=7.0 Hz), 1.02 (d, 3H, J=6.5 Hz), 1.13–1.22 (m,
1H), 1.24–1.34 (m, 1H), 1.49–1.67 (m, 4H), 1.72–1.78 (m, 2H), 1.79–1.91
(m, 2H), 2.07 (dt, 1, J=3.0, 11.5 Hz), 2.15 (s, 3H), 2.18 (s, 3H), 2.22–2.31
(m, 1H), 2.34–2.41 (m, 1H), 2.56 (dd, 1H, J=2.5, 11.0 Hz), 2.90–2.95 (m,
1H), 2.94 (s, 3H), 3.11 (s, 3H), 4.40–4.51 (brs, 1H), 4.74 (d, 1H, J=
8.0 Hz), 5.70 (dd, 1H, J=2.5, 11.5 Hz), 8.14 ppm (s, 1H); 13C NMR
([D4]methanol, 125 MHz): d = 11.2, 16.4, 20.4, 20.6, 20.9, 24.4, 25.6, 26.3,
26.4, 31.1, 31.7, 35.7, 37.7, 44.9, 54.9, 56.7, 70.6, 71.2, 125.0, 150.9, 163.9,
171.82, 171.83, 175.3, 175.6 ppm; IR: n˜ = 1221, 1498, 1549, 1643, 1755,
2937 cmꢀ1; HRMS (FAB): m/z: calcd for C27H46N5O5S: 552.3220; found:
552.3218 [M+H]+.
[1] F. Sasse, H. Steinmetz, G. Hçfle, H. Reichenbach, J. Antibiot. 2000,
53, 879–885.
[2] a) M. W. Khalil, F. Sasse, H. Lünsdorf, Y. A. Elnakady, H. Reichen-
bach, ChemBioChem 2006, 7, 678–683; b) G. Kaur, M. Hollingshead,
ˇ
´
S. Holbeck, V. Schauer-Vukasinovic, R. F. Camalier, A. Dçmling, S.
Agarwal, Biochem. J. 2006, 396, 235–242.
[3] a) H. Steinmetz, N. Glaser, E. Herdtweck, F. Sasse, H. Reichenbach,
G. Hçfle, Angew. Chem. 2004, 116, 4996–5000; Angew. Chem. Int.
Ed. 2004, 43, 4888–4892; b) G. Hçfle, N. Glaser, T. Leibold, U.
Karama, F. Sasse, H. Steinmetz, Pure Appl. Chem. 2003, 75, 167–178.
[4] A list of all approved cancer drugs can be found at http://www.fda.-
gov/cder/cancer/druglistframe.htm.
[5] J. Iley, R. Moreira, T. Calheiros, E. Mendes, Pharm. Res. 1997, 14,
1634–1639.
[6] H. M. Peltier, J. P. McMahon, A. W. Patterson, J. A. Ellman, J. Am.
Chem. Soc. 2006, 128, 16018–16019.
[7] The total synthesis but not the bioactivities of simple tubulysins or tu-
bulysin derivatives lacking the N,O-acetal have been reported: a) A.
Dçmling, B. Beck, U. Eichelberger, S. Sakamuri, S. Menon, Q.-Z.
Chen, Y. Lu, L. A. Wessjohann, Angew. Chem. 2006, 118, 7393–7397;
Angew. Chem. Int. Ed. 2006, 45, 7235–7239; b) P. Wipf, Z. Wang,
Org. Lett. 2007, 9, 1605–1607; c) M. Sani, G. Fossati, F. Huguenot, M.
Zanda, Angew. Chem. 2007, 119, 3596–3599; Angew. Chem. Int. Ed.
2007, 46, 3526–3529.
Biological assays
Cell culture and growth inhibition assay: Cell lines were obtained from
the American Type Culture Collection (ATCC) and the German Collec-
tion of Microorganisms and Cell Cultures (DSMZ). All cell lines were
cultivated under conditions recommended by their respective depositors.
Growth inhibition was measured in microtiter plates. Aliquots of 120 mL
of the suspended cells (50000 mLꢀ1) were given to 60 mL of a serial dilu-
tion of the inhibitor and incubated at 378C and 10% CO2. After 5 d,
when control cells had grown to confluence state, the metabolic activity
in each well was determined using an MTT assay.[9] IC50 values were de-
fined as the analogue concentration that showed only 50% of the activity
of the control wells.
Fluorescence staining: PtK2 cells (ATCC CCL-56) were grown on glass
coverslips (13 mm diameter) in four-well plates. Exponentially growing
cells were incubated with the analogues for 18 h. Cells were then fixed
with cold (ꢀ208C) acetone/methanol 1:1 for 10 min. For labeling the mi-
crotubules, cells were incubated with a primary monoclonal antibody
against a-tubulin (1:500; Sigma), then with a secondary goat anti-mouse
[8] This analogue has recently been reported by Wipf and Wang and was
prepared in 20 steps and 2.1% overall yield. See ref. [7c].
[9] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
Received: July 10, 2007
Published online: September 7, 2007
Chem. Eur. J. 2007, 13, 9534 – 9541
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9541