Acid mediated formation of an N-acyliminium ion from tubulysins: A new methodology for the synthesis of natural tubulysins and their analogs

Article abstract of DOI:10.1016/j.bmcl.2011.09.041

Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been chemically synthesized; however, these efforts have b

Full text of DOI:10.1016/j.bmcl.2011.09.041

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6778–6781
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acid mediated formation of an N-acyliminium ion from tubulysins: A new
methodology for the synthesis of natural tubulysins and their analogs
⇑
Iontcho R. Vlahov , Yu Wang, Marilynn Vetzel, Spencer Hahn, Paul J. Kleindl, Joseph A. Reddy,
Christopher P. Leamon
Endocyte, Inc., 3000 Kent Ave., W. Lafayette, IN 47906, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell
cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been
chemically synthesized; however, these efforts have been hampered by poor yields and arduous purifi-
cations. In contrast, treatment of a mixture of natural tubulysins A, B, C, G, and I, obtained from a fermen-
tation batch with trifluoroacetic acid results in the formation of a single N-acyliminium ion. Subsequent
addition of butyric, isopentyl, or acetic acid results in the formation of tubulysin B, A, or I, respectively, as
a single species. New tubulysin analogs can be formed upon treatment of the acyliminium ion with other
nucleophiles such as alcohols, thiols, and nitriles, resulting in corresponding N-acyl-N,O-acetals, N-acyl-
N,S-thioacetals, and N,N0-diacyl-aminals. Carbon–carbon bond formation is also possible with a modifica-
tion of this protocol. The cytotoxicies of the natural tubulysins and tubulysin analogs synthesized by this
method were evaluated on KB cells.
Received 9 August 2011
Revised 7 September 2011
Accepted 12 September 2011
Available online 17 September 2011
Keywords:
Tubulysin
N-Acyliminium ion
N-Acyl-N,O-acetals
N-Acyl-N,S-thioacetals
N,N0-Diacyl-aminals
C–C bond formation
Ó 2011 Elsevier Ltd. All rights reserved.
The tubulysins are members of a new class of natural products
isolated from myxobacterial species.1 As cytoskeleton interacting
agents, tubulysins are mitotic poisons that inhibit tubulin poly-
merization and lead to cell cycle arrest and apoptosis.2 Tubulysins
are extremely potent cytotoxic molecules, exceeding the cell
growth inhibition of any other microtubule interacting agent, for
example, epothilones, paclitaxel, and vinblastine. Furthermore,
they are potent against multidrug resistant cell lines.3
Structurally, tubulysins are closely related linear tetrapeptides
comprised of distinctive unusual and/or hydrophobic amino acids:
D-N-methyl pipecolinic acid,
L-isoleucine, tubuvaline, and the
c-amino acid tubutyrosine/tubuphenylalanine (
D
-Mep, Ile, Tuv,
and Tut/Tup, respectively, in Fig. 1). In addition, many natural
tubulysins possess an unparalleled acid and a base sensitive
N,O-diacyl N,O-acetal substituent (or a N,O-acetal of formaldehyde)
attached to the amide nitrogen of the Tuv segment.
The antiproliferative activities of tubulysins A–H have been
shown to correlate with their lipophilicities.2a Tup containing
tubulysins of this series are more lipophilic and consequently more
active than their Tut containing counterparts. Likewise, the tubuly-
sins of this series with longer, more lipophilic Tuv—N,O-diacyl
N,O-acetal side-chains possess greater cytotoxicities.
Figure 1. Structures of selected natural tubulysins.
containing tubulysins: A, B, C, G, and I; whereas Angiococcus disci-
formis gives 0.5 mg Lꢀ1 of the Tup tubulysins: D, E, F, and H. In
addition to the low fermentation titer, the isolation of a single nat-
ural tubulysin from culture extracts requires multiple consecutive
chromatographic manipulations and provides only limited quanti-
ties of material.
Natural tubulysins have been isolated from fermentation
mixtures.2a Archangium gephyra produces 2–4 mg Lꢀ1 of the Tut
⇑
Corresponding author.
E-mail address: ivlahov@endocyte.com (I.R. Vlahov).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.041

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6778–6781
6779
Scheme 1. Treatment of stabilized N-acyliminium ion 1 with selected carboxylic acids, alcohols, and thiols.
Figure 2. HPLC profiles of a one-pot conversion of a fermentation batch of tubulysins to a single congener (tubulysin B).

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I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6778–6781
The difficulties associated with fermentation-based approaches
have spurred efforts toward the total synthesis of selected natural
tubulysins and some structurally simplified analogs.4 Among the
multiple challenging synthetic and stereochemical issues, most
crucial is the generation of the labile N,O-diacyl N,O-acetal.
Herein, we report a new and simple route to a variety of natural
tubulysins and tubulysin analogs that is far more efficient than any
previously reported approach. The key to this novel one-pot proce-
dure is the in situ generation of a conjugation-stabilized N-acyli-
minium ion (1, Scheme 1) under acidic conditions from
a
mixture of multiple natural tubulysins derived from culture ex-
tracts. A nucleophile is then added to 1, effectively transforming
the mixture into a single derivative.
Reactions between N-acyliminium species and nucleophiles
(Mannich-type condensations) have been utilized to introduce
substituents to carbon atoms adjacent to amide nitrogens.5 In
our hands, simple treatment of a mixture of tubulysins (A, B, C,
G, and I) with trifluoroacetic acid (TFA) followed by addition of bu-
tyric acid produced tubulysin B (2a) in excellent yield (Scheme 1
andFig. 2).6a Tubulysins A and I (2b and 2c) were also prepared (re-
sults not shown).
Under similar conditions, alcohols serve as O-nucleophiles, gen-
erating variety of novel tubulysin-like N-acyl-N,O-acetals (3a–d,
Scheme 1) in excellent yield.6b
Next, the synthetic potential of 1 was greatly expanded using S-
nucleophiles (Scheme 1).6c The resulting N-acyl-N,S-thioacetals (
4a–f) represent an easily accessible new class of tubulysins.
Remarkably, a literature survey on the synthesis of N-acyl-N,S-
thioacetals indicates that there is only one reliable method re-
ported for the synthesis of trivial N-methythiomethyl-amides,
which takes advantage of the terminal rearrangement of poorly
accessible O-methylthiomethyl-imidates.7
Further, the nitrogen atom of nitriles can also attack as an N-
nucleophile on the N-acyliminium cation 1 (Scheme 2).8 In this
simple, one-step, Ritter-type reaction, the intermediate nitrilium
ion, upon interacting with sulfuric acid and water, gives rise to
N,N0-diacyl-aminal derivative 5.
Scheme 3. C–C bond formation via N-acyliminium intermediate 1.
Table 1
IC50’s of tubulysins and tubulysin analogsa
Drug
IC50 (nM)
Drug
IC50 (nM)
2b (Tubulysin A)
1.0
1.7
2.0
2.7
9.0
1.4
12
4a
4b
4c
4d
4e
4f
5
2.0
1.4
0.7
0.8
26
26
8.0
1.3
Tubulysin Ab
2a (Tubulysin B)
Tubulysin Bb
Finally, we extended the synthetic utility of the N-acyliminium
intermediate 1 to reactions leading to C–C bond formation and
3a
3b
3c
3d
generated
a new type of tubulysin, one which possesses a
18
6
chemically stable N-allyl fragment in the Tuv side chain (6 in
Scheme 3). 3-Trimethylsilyl-1-propene (allyl-TMS) was treated
with 1 in presence of Lewis acids, such as boron trifluoride etherate
(BF3ꢁEt2O) and trimethylsilyl triflate (TMS-OTf), but gave only
IC50 values derived from [3H]-thymidine incorporation assay using KB cells.
Literature values as reported in Ref. 2a for the KB-V1 cell line.
a
b
inferior results. In contrast, the reaction path involving 3a is
smooth and high yielding.9
When tested in vitro, all tubulysins and their analogs synthe-
sized as reported in this Letter, inhibit the growth of KB cells in a
dose-responsive manner with IC50 values (Table 1) comparable to
those of tubulysins derived from natural origin. Sulfur containing
analogs 4c and 4d were found to have the lowest IC50 values in
the sub-nM range.
In summary, we developed a universal, simple, and easily scal-
able one-pot approach for synthesis of naturally occurring tubuly-
sins and their analogs via N-acyliminium ion 1. Intermediate 1 can
be generated in situ from any mixture of natural tubulysins. Natu-
rally, this methodology allows for the interconversion of single
tubulysin species as well. Tubulysins and analogs produced by this
method were found to be very potent, all having measured IC50 val-
ues of less than 30 nM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.041.
Scheme 2. One-pot synthesis of N,N0-diacyl-aminal derivative.

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6778–6781
6781
6. General procedure: Conversion of a mixture of natural tubulysins to N-acylimium
ion 1 and its subsequent treatment with: (a) carboxy-; (b) hydroxyl-; and (c)
References and notes
mercapto-nucleophiles.
A solution of the fermentation mixture, containing
1. Sasse, F.; Steinmetz, H.; Höfle, G.; Reichenbach, H. J. Antibiot. 2000, 53, 879.
2. (a) Steinmetz, H.; Glasser, N.; Herdtweck, E.; Sasse, F.; Reichenbach, H.; Höfle, G.
Angew. Chem., Int. Ed. 2004, 43, 4888. Angew. Chem., Int. Ed. 2004, 116, 4996; (b)
Khalil, M.; Sasse, F.; Lünsdorf, H.; Elnakady, Y.; Reichenbach, H. Chem. Bio. Chem.
tubulysins A, B, C, G, and I in TFA/CH2Cl2 was stirred at rt under argon for 1 h
(every 100 mg of tubulysin mixture requires 1.0 mL TFA and 4.0 mL anhydrous
CH2Cl2). LC/MS indicated a single N-hydroxymethyl-tubulysin peak, which was
the result of the addition of water to 1 during the LC/MS process. The reaction
mixture was treated respectively with: (a) butyric acid (20% v/v); (b) anhydrous
1-propanol (50% v/v); or (c) 1-propanethiol (20% v/v). In all three cases, the
solution was stirred at room temperature under argon for 10 min, and
concentrated ivac to oil. The crude product was purified by HPLC to afford: (a)
68 mg of tubulysin B 2a (70% yield w/w from 105 mg of fermentation mixture)
as a white solid; (b) 11 mg of N-acyl-N,O-acetal 3b (60% yield w/w from 17 mg of
fermentation mixture) as a white solid; or (c) 12 mg of N-acyl-N,S-thioacetal 4b
(67% yield w/w from 18 mg of fermentation mixture) as a white solid.
7. Vilsmaier, E.; Bayer, R. Synthesis 1976, 1, 46.
´
2006, 7, 678; (c) Kaur, G.; Hollingshead, M.; Holbeck, S.; Schauer-Vikašinovic;
Camalier, R.; Dömling, A.; Agarwal, S. Biochem. J. 2006, 396, 235.
3. For a review on tubulysins see: Dömling, A.; Richter, W. Mol. Diversity 2005, 9,
141. and references therein.
4. (a) Höfle, G.; Glaser, N.; Leibold, T.; Karama, U.; Sasse, F.; Steinmetz, H. Pure Appl.
Chem. 2003, 75, 167; (b) Friestad, G.; Marié, J. C.; Deveau, A. Org. Lett. 2004, 6,
3249; (c) Wipf, P.; Takada, T.; Rishel, M. Org. Lett. 2004, 6, 4057; (d) Wipf, P.;
Wang, Z. Org. Lett. 2007, 9, 1605; (e) Peltier, H. M.; McMahon, J. P.; Patterson, A.
W.; Ellman, J. A. J. Am. Chem. Soc. 2006, 128, 16018; (f) Patterson, A.; Peltier, H.;
Sasse, F.; Ellman, J. Chem. Eur. J. 2007, 13, 9534; (g) Wang, Z.; McPherson, P.;
Raccor, B.; Balachandran, R.; Zhu, G.; Day, B.; Vogt, A.; Wipf, P. Chem. Biol. Drug
Des. 2007, 70, 75; (h) Sani, M.; Fossati, G.; Huguenot, F.; Zanda, M. Angew. Chem.,
Int. Ed. 2007, 46, 3526. Agnew. Chem., Int. Ed. 2007, 119, 3596; (i) Patterson, A.;
Peltier, H.; Ellman, J. J. Org. Chem. 2008, 73, 4362; (j) Dömling, A.; Beck, B.;
Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen, Q. Z.; Lu, Y.; Wessjohann, L.
Angew. Chem., Int. Ed. 2006, 45, 7235. reported the total synthesis of natural
tubulysins U and V but actually a correction stated that epimeric compounds
were synthesized: Angew. Chem., Int. Ed. 2007, 46, 2337; (k) Pando, O.; Dörner,
S.; Preusentanz, R.; Denkert, A.; Porzel, A.; Richter, W.; Wessjohann, L. Org. Lett.
2009, 11, 5567; (l) Shibue, T.; Hirai, T.; Okamoto, I.; Morita, N.; Masu, H.;
Azumaya, I.; Tamura, O. Chem. Eur. J. 2010, 16, 11678.
5. For excellent reviews on chemistry of N-acyliminium ions and their application
see the contributions of one of the pioneers in the field W. Nico Speckamp:
Speckamp, W. N.; Moolenaar, M. Tetrahedron 2000, 56, 3817; Speckamp, W. N.;
Hiemstra, H. Ibid 1985, 41, 4367; De Koning, H.; Moolenaar, M.; Hiemstra, H.;
Speckamp, W. N. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.;
Elsevier: Amsterdam, 1993; Vol. 13, pp 473–518; Hiemstra, H.; Speckamp, W. N.
In Comprehensive Organic Synthesis; Trost, B., Fleming, I., Eds.; Pergamon: Oxford,
1991; Vol. 2, pp 1047–1082; Hiemstra, H.; Speckamp, W. N. In The Alkaloids;
Brossi, A., Ed.; Academic: Oxford, 1988; Vol. 32, pp 271–339. See also; (e) Petrini,
M.; Torregiani, E. Synthesis 2007, 2, 159.
8. One-pot conversion of a mixture of natural tubulysins to N,N0-diacyl-aminal 5: To a
solution of a tubulysin mixture containing tubulysins A, B, C, G, and I (25 mg) in
isovaleronitrile (150
l
L) was added a solution of TFA (30
lL) and concentrated
H2SO4 (20 L) in isovaleronitrile (150
l
l
L). After stirring at room temperature for
22 h, the reaction was quenched with 2.0 mM sodium phosphate buffer (pH 7.0,
15 mL) and purified by HPLC to give 16 mg of 5 (64% yield w/w) as a white solid.
9. One-pot conversion of a mixture of natural tubulysins to N-allyl tubulysin analog 6:
TFA (0.15 mL) was added to a solution of a tubulysin mixture (19 mg) containing
tubulysins A, B, C, G, and I in anhydrous CH2Cl2 (0.60 mL) at rt. After stirring for
40 min at rt under argon, the reaction was quenched with anhydrous MeOH
(0.50 mL). The solution was concentrated ivac, co-evaporated with anhydrous
MeOH (2ꢂ) and anhydrous CH2Cl2 (2ꢂ), vacuumed for 30 min, co-evaporated
again with anhydrous MeOH and anhydrous CH2Cl2 (2ꢂ), and vacuumed for an
additional 1.5 h. The residue was dissolved in anhydrous CH2Cl2 (0.75 mL) and
allyltrimethylsilane (0.30 mL) was added. The mixture was cooled in an ice bath
and BF3ꢁEt2O (0.23 mL) was added. After 30 min, the cooling was removed and
the reaction mixture was stirred for an additional 3 h, concentrated and the
residue was purified by HPLC to afford 10 mg of 6 (52% yield w/w) as a white
solid.