Synthesis and cytotoxicity evaluation of diastereoisomers and N-terminal analogues of tubulysin-U

Article abstract of DOI:10.1016/j.tetlet.2013.09.010

Tubulysins are potent anti-mitotic natural compounds and a scalable and efficient synthetic route for generation of its analogues has been developed and extended to the synthesis of diastereoisomers and N-terminal analogues of tubulysin-U. Structure-activ

Full text of DOI:10.1016/j.tetlet.2013.09.010

Tetrahedron Letters 54 (2013) 6137–6141
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and cytotoxicity evaluation of diastereoisomers and
N-terminal analogues of tubulysin-U
P. Sreejith Shankar a, Serena Bigotti a, Paolo Lazzari b,c, Ilaria Manca d, Marco Spiga b,
Monica Sani b,e, , Matteo Zanda e,f,
⇑
⇑
a Dipartimento di Chimica, Materiali e Ingegneria Chimica ‘Giulio Natta’, Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy
b KemoTech, KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Loc. Piscinamanna, 09010 Pula (CA), Italy
c Dipartimento di Chimica e Farmacia dell’Università degli Studi di Sassari, Via F. Muroni 23/A, 07100 Sassari (SS), Italy
d C.N.R.—Istituto di Farmacologia Traslazionale, Parco Scientifico della Sardegna, Edificio 5, Loc. Piscinamanna, 09010 Pula (CA), Italy
e C.N.R.—Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘A. Quilico’, Via Mancinelli 7, 20131 Milano, Italy
f Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubulysins are potent anti-mitotic natural compounds and a scalable and efficient synthetic route for
generation of its analogues has been developed and extended to the synthesis of diastereoisomers and
N-terminal analogues of tubulysin-U. Structure–activity-relationship studies on these synthetic ana-
logues reaffirmed the significance of native stereochemistry of tubulysins for optimal biological activity
and cytotoxicity. However, while modification of Tup stereochemistry has only minor effect on the
tubulysins cytotoxicity, Tuv stereochemistry is critically important and modification of either Tuv stereo-
centre produced a dramatic drop in cytotoxicity.
Received 2 August 2013
Accepted 4 September 2013
Available online 11 September 2013
Keywords:
Tubulysin
Diastereoisomers
Structure–activity-relationship
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Anti-cancer
Introduction
functional group differences in the non-proteinogenic Tuv and
Tup/Tut fragments give rise to the majority of the analogues—
Diagnosis and treatment of cancer has been arguably the fastest
developing area of modern day biomedical research. Selectivity of
anti-cancer drugs towards tumour cells over normal cells is a key
factor for achieving therapeutic efficacy, and highly potent tubuly-
sins may turn out to be extremely effective tools in this regard,
particularly if coupled to a monoclonal antibody (MAb)—the es-
sence of targeted drug delivery. Tubulysins are tetrapeptide natu-
ral products produced in rather small quantities by two different
species of Myxobacteria1 (Fig. 1).
Tubulysins are amongst the most potent known anti-mitotic
compounds. Mechanistically, they bind to tubulin and disintegrate
microtubules of dividing cells, thereby inducing apoptosis.2 Struc-
turally, they are composed of three non-proteinogenic amino acids
(Tubuvaline/Tuv, Tubuphenylalanine/Tup or Tubutyrosine/Tut and
N-methyl pipecolic acid/Mep) and proteinogenic Isoleucine/Ile.
Several natural structural variants of tubulysins 1 have been de-
scribed—tubulysin D being the most cytotoxic. All these tubulysin
derivatives possess the same N-terminal amino acid—N-methyl
pipecolic acid, followed by the natural isoleucine fragment. The
Tubulysins A–I, U, V, Y and Z (Table 1).
Several reports on the total synthesis of tubulysins and their
analogues3 including structurally simplified ‘Tubugis’ compounds,
N-methyl tubulysins, pre-tubulysins, etc., have been reported, all
due to overwhelming interest in their potential as novel anticancer
agents. The development of a library of stereoisomers of tubulysins
could shed light on the key binding interactions of tubulysins with
tubulin as a function of the overall stereochemistry of the mole-
cule. Tamura and co-workers have demonstrated that stereoiso-
mers of tubulysin D possessing different configurations at the
Tuv stereogenic centres are at least three orders less cytotoxic,
R1
O
OR2
S
O
H
N
N
1
N
N
N
H
COOH
O
R3
Mep
Ile
Tuv
Tup / Tut
⇑
Corresponding authors. Tel.: +44 1224 437513; fax: +44 1224 437465.
E-mail addresses: m.zanda@abdn.ac.uk, matteo.zanda@polimi.it (M. Zanda).
Figure 1. Structure of tubulysins 1.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.09.010

6138
P. S. Shankar et al. / Tetrahedron Letters 54 (2013) 6137–6141
Table 1
Natural tubulysins
SH
(a)
O
O
H
N
(b)
COOEt
N
COOEt
HCl.H2N
COOEt
52% in
2 steps
R1
R2
R3
S
S
Tubulysin
A
B
C
D
E
F
G
H
I
U
V
Y
Z
p-OH-Ph
p-OH-Ph
p-OH-Ph
Ph
Ph
Ph
p-OH-Ph
Ph
p-OH-Ph
Ph
Ph
p-OH-Ph
p-OH-Ph
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
–CH2OCO-i-Butyl
–CH2OCO-n-Propyl
–CH2OCOEt
–CH2OCO-i-Butyl
–CH2OCO-n-Propyl
–CH2OCOEt
–CH2OCOCH@C(CH3)2
–CH2OCOMe
–CH2OCOMe
H
2
3
4
Scheme 1. Synthesis of 2-acyl thiazole ethyl ester. Reagents and conditions: (a)
methyl glyoxal aqueous solution, NaHCO3, EtOH:H2O (1:1), overnight; (b) MnO2,
MeCN, 65 °C, overnight.
(a)
97%
BocNH2 + iPr-CHO + PhSO2Na
H
H
H
BocHN
SO2Ph
SO2Ph
Ac
H
5
O
N
COOEt
even though the inhibition of tubulin polymerization is
comparable to that of the native analogue.4 The significance of nat-
ural (R)-configuration of the Tuv O-acetate functionality and its
influence on the binding with tubulin was reported also by Wipf
et al.5 More recently, while this Letter was in preparation, Yang
et al. reported that the epimer of tubulysin U having (S)-stereo-
chemistry at the Tup C-4 maintained significant potency against
several cell lines.6 It would be justifiably prominent to obtain a
reliable full picture of the structure–activity-relationship (SAR)
profile of different stereoisomers of tubulysins, with the potential
of developing more active and stable analogues and hence to reaf-
firm the significance of native stereochemistry in active tubulysins.
Herein, we report the total synthesis of different diastereoiso-
mers and N-terminal analogues of tubulysin U for implementing
a SAR study designed to investigate the importance of native ste-
reochemistry and N-terminus of the molecule for its optimal cyto-
toxic activity.
BocHN
(b)
S
52%
5
4a
O
N
COOEt
BocHN
S
racemic
6
(d)
(c)
OH
OH
N
COOEt
N
COOEt
BocHN
BocHN
S
S
92% ee
90% ee
7a (39%)
(36%)
7c
Results and discussion
+
+
We have reported a scalable synthesis of Tubulysin U and V in
20077 and we very recently demonstrated the effect of variations
in functional groups in the central Tuv fragment3k,8 on the cytotox-
icity of various tubulysin U analogues. Even though tubulysins
have attracted the interests of the medicinal chemistry community
ever since their discovery and quite a few reports have been ded-
icated to studies on structural variations in Tuv and Tup/Tut frag-
ments, only less than a handful surveyed the significance of the
tubulysins’ native stereochemistry.4–6
With a reliable and scalable synthetic strategy in hand, we
decided to probe the effect on cytotoxicity of systematically alter-
ing the stereochemistry of different carbon centres in two of the
non-proteinogenic amino acids (Tuv and Tup) in tubulysin U,
alongside a couple of N-terminal analogues.
The total synthesis of all the analogues started with the gener-
ation of the thiazole ring of the Tubuvaline fragment. Condensation
of L-cysteine hydrochloride ethyl ester 2 with methyl glyoxal in the
presence of sodium bicarbonate gave a thermally unstable thiazo-
line 3, which on MnO2 mediated oxidation afforded the 2-acyl thi-
azole ester 4 in 52% overall yield (Scheme 1).
OH
OH
N
N
COOEt
COOEt
BocHN
94% ee
(38%)
BocHN
96 % ee
(37%)
S
S
7d
7b
Scheme 2. Synthesis of the four Tuv stereoisomers. Reagents and conditions: (a)
formic acid, THF:H2O, 24 h.; (b) NaH, THF, 2–3 h; (c) (S)-(À)-2-methyl-CBS-
oxazaborolidine, BH3ÁMe2S, THF, 0 °C, 2–3 h; (d) (R)-(+)-2-methyl-CBS-oxazabor-
olidine, BH3ÁMe2S, THF, 0 °C, 2–3 h.
The relative configuration of the two stereocentres was con-
firmed by single crystal X-ray diffraction8 of 7b and further corre-
lation with enantiopure O-Ac-Cbz-Tuv-OEt as reported by Wipf
et al.9
The next step was the synthesis of the second non-proteino-
genic amino acid fragment Tup. We have earlier reported the syn-
thesis of Tup bearing its natural stereochemistry,3k,7 but that was
not found to be quite efficient in generating the diastereoisomers
in good yields and stereopurity, presumably due to the challenging
chromatographic separations and protecting group manipulations
involved. Hence, we decided to extend the strategy that was re-
ported more recently8 and, satisfactorily, we were able generate
the desired isomers of Tup in their stereopure form (Scheme 3).
Acylation of (À)-menthol using bromoacetyl bromide followed
by treatment with triphenylphosphine generated ylide 9 which
was methylated using MeI to afford 10 and the stage was set for
The enol derivative of thiazole 4 was coupled with a-amino sul-
fone 5 obtained by a one-pot reaction involving Boc-carbamate,
isobutyraldehyde and benzene sulfinic acid sodium salt in the
presence of formic acid, to give the corresponding b-amino ketone
6 as a racemate. The stereopure precursors of Tuv 7a–d were ob-
tained by the (S)- or (R)-CBS-oxazoborolidine/BH3ÁMe2S mediated
reduction of ketone 6, followed by chromatographic separation of
the diastereoisomers (ratio 1:1). The ee of each stereoisomer was
determined by HPLC (Scheme 2).
a
Wittig reaction involving the corresponding aldehyde 11
obtained by the oxidation of protected - or -phenylalaninol.
L
D

P. S. Shankar et al. / Tetrahedron Letters 54 (2013) 6137–6141
6139
O
OH
O
OH
(a)
72%
(b)
95%
O
O
(b)
BocHN
N
COOR
+
Br
N
H
N
COOEt
PPh3
Br
*
*
Br
RHN
55-94%
O
O
O
OH
*
*
S
S
9
8
17a-d R = Et
18a-d R = H
7a-d
16a-d
R = Boc
R = H.TFA
(a)
(c)
O
(c)
PPh3
96%
Ph
O
OH
O
10
(d)
(e)
60-95%
BocHN
N
N
H
N
COOMe
Ph
*
*
*
*
57-94%
Ph
BocHN
H
Ph
BocHN
S
(d)
O
10
(e)
70-78%
H
OH
BocHN
60-86%
*
*
*
19a-k
20a-k
R = Boc
R = H.TFA
O
O
(a)
11a-b
12a-b
Ph
Ph
Ph
O
OH
O
(f)
( )n
O
H
N
O
O
(f)
48-98%
+
BocHN
N
BocHN
87-90%
*
*
N
R
N
*
N
COOMe
O
*
*
*
*
H
H
O
S
13c-d
13a-b
21a-m
(
g)
90-95%
(
g)
90-95%
R = H, Me
n = 1, 2
O
Ph
Ph
Ph
O
H
O
O
( )
n
-
+
.
-
+
.
Cl H3N
COOX
(X = H)
Cl H3N
COOX
(X = H)
N
N
N
R
N
H
N
H
COOH
14c
15c
*
*
*
14a
(h)
(h)
>98%
O
S
>98%
15a
(X = Me)
(X = Me)
1a-m
R = Ac, Me
n = 1, 2
Ph
Ph
-
+
.
Cl H3N
COOX
(X = H)
-
+
.
Cl H3N
COOX
(X = H)
Scheme 4. Final assembly of fragments. Reagents and conditions: (a) TFA:CH2Cl2
(1:4), 0 °C to rt, 1 h; (b) HOBt, EDC HCl, then Boc-Ile, DIPEA, CH2Cl2, 0 °C to rt, 3 h; (c)
LiOHÁH2O, THF:H2O (4:1), 0 °C to rt, 5 h; (d) HOAt, HATU, then Tup-OMe 15, Et3N,
CH2Cl2, 0 °C to rt, 3 h; (e) HOAt, HATU, then N-methyl pipecolic acid or pipecolic
acid or N-methyl proline, Et3N, 0 °C to rt, 3 h; (f) (i) 1 N LiOH, THF, 0 °C to rt, 2–
3 days; (ii) Ac2O, pyridine, overnight.
14d
15d
14b
15b
(h)
(h)
>98%
>98%
(X = Me)
(X = Me)
Scheme 3. Synthesis of diastereoisomers of Tup. Reagents and conditions: (a) Et3N,
dry THF, 0 °C to rt, 2 h; (b) PPh3, THF, 2 h, 0.38 N NaOH, toluene, 3 h; (c) MeI, CH2Cl2,
0 °C to rt, overnight; (d) Dess–Martin periodinane, CH2Cl2, 6 h; (e) CH2Cl2, 0 °C to rt,
8 h; (f) H2, Pd/C, EtOAc, overnight; (g) 6 N HCl, 130 °C, 1.5 h; (h) 2,2-dimethoxy-
propane, concd HCl, MeOH, 60 °C, overnight.
21a–m in good yields and with no detectable loss of sterochemical
purity. LiOH mediated hydrolysis of the methyl ester followed by
acetylation using acetic anhydride in pyridine afforded the final
tubulysin analogues 1a–m in their stereopure form (Scheme 4
and Fig. 2).
The
a,b-unsaturated aminoester 12 thus obtained was further
hydrogenolyzed using Pd–C (10%) to give N,C-protected Tup pre-
cursors 13a–d as a mixture of their corresponding diastereoiso-
mers (2:1 ratio, evidenced by 1H NMR measurements), separable
by simple flash chromatography. Separation of the isomers fol-
lowed by full deprotection to the free Tup stereoisomers 14a–d
and subsequent carboxyl group methylation using 2,2-dimethoxy
propane afforded the corresponding Tup-methyl esters 15a–d as
hydrochloride salts in good yields. The relative stereochemistry
was assigned based on the single crystal X-ray analysis of one of
the diastereoisomers.8
With all the stereoisomers of both fragments in hand, the final
assembly of tubulysins via standard solution phase peptide synthe-
sis was initiated by Boc deprotection of the stereoisomers 7a–d to
16a–d with 20% TFA:CH2Cl2, followed by a subsequent coupling
with N-Boc isoleucine (Boc-Ile) affording the corresponding dipep-
tides 17a–d. Saponification of the ethyl ester under mild alkaline
conditions to afford 18a–d and subsequent coupling with the re-
quired isomer of Tup-OMe 15a–d gave the tripeptides 19a–k in
satisfactory yields. A second Boc-deprotection of 19 to 20a–k fol-
lowed by further coupling to N-methyl pipecolic acid, or N-acetyl
pipecolic acid or N-methyl proline (the last two were used in case
of tubulysin U precursors bearing the natural stereochemistry on
all carbons) using HOAT-HATU-Et3N yielded the tetrapeptides
Biological tests
Most of the tubulysins 1a–m, with the exception of 1b and 1i
(whose samples were accidentally lost after characterization),
were subjected to cytotoxicity assays on two different cell lines:
HL60 (human promyelocytic leukaemia) and C6 (rat glial tu-
mour). Results are summarized in Table 2. Modification of Tup
stereochemistry has a minor influence on the cytotoxicity of
tubulysins, as witnessed by the fact that 1a, which is the C-2
Tup epimer, is nearly as potent as tubulysin U (1c), whereas 1d,
which is the C-4 Tup epimer, is only ca. 10 times less potent than
1c. These results are in line with those published by Yang et al.,
who observed that 1d is only 3-times less potent than 1c on
HL-60 cells.6 In contrast, modification of either Tuv stereocenter
has a dramatic effect on the cytotoxicity of tubulysins, as demon-
strated by the fact that all of the derivatives 1e–k are at least
1000 times less potent than 1c. Finally, replacement of the Mep
N-methyl group with an acetyl in 1l also results in a dramatic
drop of cytotoxicity, whereas the Mep ring contraction to N-
methyl proline is quite well tolerated as seen with 1m which is
only ca. 10 times less potent than 1c.

6140
P. S. Shankar et al. / Tetrahedron Letters 54 (2013) 6137–6141
Ph
Ph
Ph
(R) CO2H
O
OAc
O
O
OAc
O
H
N
O
O
OAc
O
O
H
N
H
N
N
N
N
N
N
CO2H
CO2H
NH (R)
H (R)
(R)
N
N (R)
H
N (S) (R) CO2H
H
(S)
(R)
N
N
N ( R)
H
NH (R)
(R)
O
S
O
S
O
O
S
1c
(tubulysin U)
1a
1b
Ph
(S) CO2H
Ph
Ph
OAc
O
OAc
O
O
OAc
O
H
N
H
H
N
N
N
N
N
N
H
(R)
N (S)
H
N
N (S)
H
N (R)
H
CO2H
N
N (S)
H
N (S)
H
(R)
(R)
(R)
(R)
(R)
S
O
S
O
S
1e
1d
1f
Ph
Ph
Ph
(R) CO2H
O
OAc
O
O
O
O
OAc
O
O
O
O
OAc
O
O
H
N
H
N
H
N
N
N
N
N
N
N
CO2H
CO2H
N
N
N
N (S)
H
(R) CO2H
(S)
(S)
N
N
N
H
N
H
(S)
( S)
(S)
(S)
(R)
(R)
H
H (S)
H
O
O
S
O
O
S
O
O
S
1i
1h
1g
Ph
Ph
(R) CO2H
(S)
Ph
O
OAc
S
OAc
OAc
H
N
H
N
H
N
N
N
N
N
Ac
N
H
N
H
N (R)
H
N
H
N
H
N
CO2H
(S)
(S)
(S)
(S)
H (S)
S
S
1l
1k
1j
Ph
OAc
S
O
O
H
N
N
N
N
H
N
CO2H
H
O
1m
Figure 2. Library of tubulysin derivatives.
Table 2
Biological tests on the tubulysin analogues
Project ‘DOL.IMMU.CHEM.: Nuove entità chimiche per la terapia
del DOLore, per il trattamento di patologie del sistema IMMUnitar-
io o per la CHEMioterapia’).
Tubulysin analogue
IC50 on HL60 cells (nM)
IC50 on C6 cells (nM)
1a
1.7
2.4
1c (Tubulysin U)
0.8
9.1
1.2
19
Supplementary data
1d
1e
1f
1g
1h
1j
1k
1l
1m
>10,000
6,200
1200
>10,000
>10,000
3600
>10,000
23
>10,000
>10,000
3400
>10,000
>10,000
4400
Supplementary data (general experimental procedures and
spectral data) associated with this article can be found, in the on-
line version, at http://dx.doi.org/10.1016/j.tetlet.2013.09.010.
References and notes
>10,000
140
1. (a) Sasse, F.; Steinmetz, H.; Höfle, H.; Reichembach, H. J. Antibiot. 2000, 53, 879;
(b) Steinmetz, H.; Glaser, N.; Herdtweck, E.; Sasse, F.; Reichenbach, H.; Höfle, G.
Angew. Chem., Int. Ed. 2004, 43, 4888.
2. Khalil, M. W.; Sasse, F.; Lünsdorf, H.; Elnakady, Y. A.; Reichenbach, H.
ChemBioChem 2006, 7, 678.
Conclusions
3. (a) Balasubramanian, R.; Raghavan, B.; Begaye, A.; Sackett, D. L.; Fecik, R. A. J.
Med. Chem. 2009, 52, 238; (b) Balasubramanian, R.; Raghavan, B.; Steele, J. C.;
Sackett, D. L.; Fecik, R. A. Bioorg. Med. Chem. Lett. 2008, 18, 2996; (c) Raghavan,
B.; Balasubramanian, R.; Steele, J. C.; Sackett, D. L.; Fecik, R. A. J. Med. Chem. 2008,
51, 1530; (d) Patterson, A. W.; Peltier, H. M.; Ellman, J. A. J. Org. Chem. 2008, 73,
4362; (e) Patterson, A. W.; Peltier, H. M.; Sasse, H.; Ellman, J. A. Chem. Eur. J.
2007, 13, 9534; (f) Wipf, P.; Wang, Z. Org. Lett. 2007, 9, 1605; (g) Chandrasekhar,
S.; Mahipal, B.; Kavitha, M. J. Org. Chem. 2009, 74, 9531; (h) Pando, O.; Dörner, S.;
Preusentanz, R.; Denkert, A.; Porzel, A.; Richter, W.; Wessjohann, L. Org. Lett.
2009, 11, 5567; (i) Ullrich, A.; Herrmann, J.; Müller, R.; Kazmaier, U. Eur. J. Org.
Chem. 2009, 6367; (j) Peltier, H. M.; McMahon, J. P.; Patterson, A. W.; Ellman, J. A.
J. Am. Chem. Soc. 2006, 128, 16018; (k) Shankar, S. P.; Sani, M.; Saunders, F. R.;
Wallace, H. M.; Zanda, M. Synlett 2011, 1673; (l) Shibue, T.; Hirai, T.; Okamoto, I.;
Morita, N.; Masu, H.; Azumaya, I.; Tamura, O. Chem. Eur. J. 2010, 16, 11678; (m)
Pando, O.; Stark, S.; Denkert, A.; Porzel, A.; Preusentanz, R.; Wessjohann, L. A. J.
Am. Chem. Soc. 2011, 133, 7692; (n) Eirich, J.; Burkhart, J. L.; Ullrich, A.; Rudolf, G.
C.; Vollmar, A.; Zahler, S.; Kazmaier, U.; Sieber, S. A. Mol. BioSyst. 2012, 8, 2067;
(o) Burkhart, J. L.; Kazmaier, U. RSC Adv. 2012, 2, 3785; (p) Rath, S.; Liebl, J.; Fürst,
R.; Ullrich, A.; Burkhart, J. L.; Kazmaier, U.; Herrmann, J.; Müller, R.; Günther, M.;
Schreiner, L.; Wagner, E.; Vollmar, A. M.; Zahler, S. Br. J. Pharmacol. 2012, 167,
1048; (q) Herrmann, J.; Elnakady, Y. A.; Wiedmann, R. M.; Ullrich, A.; Rohde, M.;
Kazmaier, U.; Vollmar, A. M.; Müller, R. PLoS One 2012, 7, e37416; (r) Floyd, W.
C., III; Datta, G. K.; Imamura, S.; Kieler-Ferguson, H. M.; Jerger, K.; Patterson, A.
W.; Fox, M. E.; Szoka, F. C.; Fréchet, J. M. J.; Ellman, J. A. ChemMedChem 2011, 6,
49; (s) Leamon, C. P.; Reddy, J. A.; Vetzel, M.; Dorton, R.; Westrick, E.; Parker, N.;
Wang, Y.; Vlahov, I. Cancer Res. 2008, 68, 9839; (t) Vlahov, I. R.; Wang, Y.; Kleindl,
P. J.; Leamon, C. P. Bioorg. Med. Chem. Lett. 2008, 18, 4558; (u) Reddy, J. A.;
Dorton, R.; Dawson, A.; Vetzel, M.; Parker, N.; Nicoson, J. S.; Westrick, E.; Klein, P.
J.; Wang, Y.; Vlahov, I. R.; Leamon, C. P. Mol. Pharm. 2009, 6, 1518; (v) Kularatne,
S. A.; Wang, K.; Santhapuram, H.-K. R.; Low, P. S. Mol. Pharm. 2009, 6, 780; (x)
The stereochemistry of the Tup fragment has a minor effect on
the cytotoxicity of tubulysins, whereas the configuration of the
central Tuv fragment is critically important and modification of
either Tuv stereocentre resulted in a dramatic loss of cytotoxicity,
namely 1000 times or more relative to the natural (R,R)-Tuv stereo-
chemistry. The N-methyl group of Mep also appears to play a key
role, and its replacement with an acetyl group causes a very strong
loss of cytotoxicity, suggesting that the piperidine nitrogen basicity
is essential. Conversely, the Mep ring tolerates contraction to N-
methyl proline, which gave rise to a relatively modest loss of po-
tency. Overall, these results confirm the importance of the natural
stereochemistry of tubulysins and provide strong additional evi-
dence that only the C-terminus, that is, the Tup fragment, is readily
amenable to structural changes, whereas modification of either the
N-terminus, that is, the Mep fragment, or the central part of
tubulysins, that is, the Tuv, is much more likely to induce a signif-
icant loss of potency.
Acknowledgments
We thank Regione Autonoma della Sardegna (Italy), Assessorato
all’Industria, for economic support (BANDO 2011 L.R.37, ART.4;

P. S. Shankar et al. / Tetrahedron Letters 54 (2013) 6137–6141
6141
Yang, X.; Dong, C.; Chen, J.; Liu, Q.; Han, B.; Zhang, Q.; Chen, Y. Tetrahedron Lett.
2013, 54, 2986.
7. Sani, M.; Fossati, G.; Huguenot, F.; Zanda, M. Angew. Chem., Int. Ed. 2007, 46,
3526.
4. Shibue, T.; Okamoto, I.; Morita, N.; Morita, H.; Hirasawa, Y.; Hosoya, T.; Tamura,
O. Bioorg. Med. Chem. Lett. 2011, 21, 431.
5. Wang, Z.; McPherson, P. A.; Raccor, B. S.; Balachandran, R.; Zhu, G.; Day, B. W.;
Vogt, A.; Wipf, P. Chem. Biol. Drug Des. 2007, 70, 75.
8. Shankar, S. P.; Jagodzinska, M.; Malpezzi, L.; Lazzari, P.; Manca, I.; Greig, I. R.;
Sani, M.; Zanda, M. Org. Biomol. Chem. 2013, 11, 2273.
9. Wipf, P.; Takada, T.; Rishel, M. J. Org. Lett. 2004, 6, 4057.
6. Yang, X.; Dong, C.; Chen, J.; Ding, Y.; Liu, Q.; Ma, X.; Zhang, Q.; Chen, Y. Chem.
Asian J. 2013, 8, 1213.