Total synthesis and cytotoxicity evaluation of an oxazole analogue of tubulysin U

Article abstract of DOI:10.1055/s-0030-1260806

Tubulysins are strongly cytotoxic natural tetrapeptides with potent antiproliferative, antimitotic, and antiangiogenic activities which might find use in oncology. We herein report the first total synthesis of a stereoisomerically pure oxazole analogue of

Full text of DOI:10.1055/s-0030-1260806

LETTER
1673
Total Synthesis and Cytotoxicity Evaluation of an Oxazole Analogue of
Tubulysin U
Synthesis of
a
r
o
e
A
nalogue
e
of Tubulys
j
in
U
ith Shankar P,a Monica Sani,*b,d, Fiona R Saunders,c Heather M Wallace,c Matteo Zanda*b,c
a
Dipartimento di Chimica, Materiali e Ingegneria Chimica ‘Giulio Natta’, Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy
C. N. R – Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘A. Quilico’, Via Mancinelli 7, 20131 Milano, Italy
Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD,
b
c
Scotland, UK
Website: http://www.abdn.ac.uk/kosterlitz/
E-mail: m.zanda@abdn.ac.uk
d
KemoTech, KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Loc. Piscinamanna, 09010 Pula (CA), Italy
Received 25 March 2011
ficient scalable synthesis of the most potent tubulysins,
and narrow therapeutic window.
Abstract: Tubulysins are strongly cytotoxic natural tetrapeptides
with potent antiproliferative, antimitotic, and antiangiogenic activi-
ties which might find use in oncology. We herein report the first to-
tal synthesis of a stereoisomerically pure oxazole analogue of
tubulysin U, which was found to be more cytotoxic than the thiaz-
ole-containing natural product. Additionally, we have developed an
improved and scalable synthetic route towards the Tup fragment of
the tubulysins.
R1
O
OR2
O
H
N
N
N
N
N
COOH
H
O
R3
S
Key words: tubulysin, oxazole, SAR study, chemotherapy, cyto-
toxicity
Mep
Ile
Tuv
Tup / Tut
1
tubulysin A, R1 = 4-HOC6H4, R2 = Ac, R3 = CH2OCOi-Bu
tubulysin B, R1 = 4-HOC6H4, R2 = Ac, R3 = CH2OCOn-Pr
tubulysin C, R1 = 4-HOC6H4, R2 = Ac, R3 = CH2OCOEt
tubulysin D, R1 = Ph, R2 = Ac, R3 = CH2OCOi-Bu
tubulysin E, R1 = Ph, R2 = Ac, R3 = CH2OCOn-Pr
tubulysin F, R1 = Ph, R2 = Ac, R3 = CH2OCOEt
tubulysin G, R1 = 4-HOC6H4, R2 = Ac, R3 = CH2OCOCH=CMe2
tubulysin H, R1 = Ph, R2 = Ac, R3 = CH2OCOMe
tubulysin I, R1 = 4-HOC6H4, R2 = Ac, R3 = CH2OCOMe
tubulysin V, R1 = Ph, R2 = H, R3 = H
tubulysin U, R1 = Ph, R2 = Ac, R3 = H
tubulysin Y, R1 = 4-HOC6H4, R2 = Ac, R3 = H
tubulysin Z, R1 = 4-HOC6H4, R2 = H, R3 = H
One of the fast developing and major areas of interest in
modern medical and biochemical research has been relat-
ed to the diagnostics and treatment of cancer. Tubulysins
1, which are naturally occurring tetrapeptides produced by
some Myxobacteria strains, are among the most potent
known antimitotic cytotoxic compounds.1 The tubulysins
are active against a wide range of human cancer cell lines
including multidrug resistant tumours, in subnanomolar
concentrations (with IC50 values typically in the range
0.01 to 10 nM).2 Structurally, tubulysins comprise three
nonproteinogenic amino acids [tubuvaline – Tuv, tubu-
phenylalanine – Tup or tubutyrosine – Tut, and N-methyl
(R)-pipecolic acid – Mep; Figure 1] and the proteingenic
amino acid L-isoleucine (Ile).
Figure 1 Structure and analogues of the tubulysins
Current research in cancer therapeutics is aimed at provid-
ing effective anticancer agents with minimal side effects.
The issues of toxicity and selectivity may be overcome by
the use of immunoconjugates, namely tubulysin-mAb
(monoclonal antibody) conjugates as tumor-activated pro-
drugs,6 or by other targeting vectors, such as folic acid, as
reported by Leamon et al.7
The tubulysins are antimitotic agents that bind to the pep-
tide binding site situated in the Vinca domain of b-tubulin
in a noncompetitive fashion, leading to the destabilisation
of microtubules and decreasing microtubule polymer
mass, eventually causing apoptosis of the cell.3 The potent
biological activity of the tubulysins has encouraged their
preclinical development,4 and it has already been shown
that tubulysin D (the most potent among the natural tubu-
lysins) is superior in cytotoxic activity compared to other
drugs currently used in cancer chemotherapy, such as tax-
ol.5 However, preclinical and clinical development of the
tubulysins has been hindered by low supply, lack of an ef-
The total synthesis of tubulysin analogues was first report-
ed in a patent application in 2001.8 In 2007 we described
the scalable synthesis of tubulysins U and V9 and since
then several reports have been published concerning
chemical modifications of the natural tubulysins that are
intended to provide information about their key binding
interactions and mechanism of inhibition.10 In this regard,
we decided to investigate novel tubulysin U analogues for
carrying out SAR studies, and the first attempt was to re-
assert the significance of the thiazole moiety in the central
Tuv fragment, which is known to be of paramount impor-
tance for the exceptional cytotoxicity of the tubulysins, by
replacing the thiazole heterocycle with an oxazole. To our
SYNLETT 2011, No. 12, pp 1673–1676
x
x
.x
x
.2
0
1
1
Advanced online publication: 21.06.2011
DOI: 10.1055/s-0030-1260806; Art ID: D09311ST
© Georg Thieme Verlag Stuttgart · New York

1674
S. S. P et al.
LETTER
knowledge, there has been no prior report on the total syn- lactic acid 6 followed by coupling with L-serine hydro-
thesis of an oxazole containing tubulysin analogue and chloride ethyl ester using HOBt, EDC·HCl, and Et3N af-
hence no activity data of an oxazole analogue of tubulysin forded the pseudo-dipeptide 8. Cyclisation of 8 to the
are currently available. The only attempt in this area has dihydrooxazole 9 was achieved using diethyl amino sulfur
been from Wipf and co-workers in 2004, who reported the trifluoride and K2CO3 in THF.13 CuBr2-mediated
synthesis of an oxazole-containing Tuv-Tup fragment.11
oxidation14 of 9 in the presence of HMTA and DBU gave
the oxazole 10 in reasonable yield, which, on TBAF-
induced desilylation, followed by Dess–Martin oxidation
gave 4b.15
Our initial attempt was to extend our reported synthetic
strategy for tubulysins U and V to the synthesis of the ox-
azole analogue as well.9 The first step in the synthesis of
tubulysin U and V was the generation of the key 2-acetyl It is worth noting that, although several protocols have
thiazole ethyl ester (4a) from cysteine 2 and further trans- been reported16 for the cyclisation of the pseudo-dipeptide
formations thereof, to afford the Tuv fragment 5a 8 to the oxazoline 9 and its oxidation to the oxazole 10, in-
(Scheme 1).
cluding the one-pot cyclodehydration–oxidation method
of Wipf and Williams,17 the method portrayed in
Scheme 2 proved to be more effective in our hands.
SH
O
H
(b)
(a)
N
COOEt
OH
The 2-acetyl oxazole 4b was then used as a building block
towards the generation of the required Tuv fragment, fol-
lowing exactly the same strategy that we had developed
for the thiazole analogue (Scheme 3).9 One-pot reaction
involving Boc-carbamate, benzene sulfinic acid sodium
salt, and isobutyraldehyde in MeOH–H2O afforded the b-
amino sulfone 12. Enolisation of 4b using NaH followed
by the addition of the amino sulfone 12 to the oxazole eno-
late solution gave the b-amino ketone 13 (81%) as a race-
mic mixture. In order to obtain stereochemically pure Tuv
in its N,C-protected form, we performed an oxazaboroli-
dine mediated reduction7,18 using BH3·Me2S in the pres-
ence of (S)-CBS catalyst at 0 °C, to afford the required
alcohol (R,R)-5b19 along with its diastereoisomer (S,R)-5c
(in the ratio 1:1, 45% overall yield), that could be easily
separated by flash chromatography.
52% in
2 steps
HCl⋅H2N
COOEt
S
2
3
O
steps
N
COOEt
N
COOEt
BocHN
S
S
4a
5a
Scheme 1 Synthesis of Tuv via 2-acetyl thiazole 4a. Reagents and
conditions: (a) pyruvic aldehyde, NaHCO3, EtOH–H2O (1:1), over-
night; (b) MnO2, MeCN, 65 °C, overnight.
We were expecting a similar trend in the reactivity of L-
serine ethyl ester in place of L-cysteine ethyl ester (2), but,
unfortunately, we could not obtain even traces of the ex-
pected 2-acetyl oxazole ethyl ester (4b, Scheme 2). This
led us to explore a different strategy for the synthesis of
4b, inspired by Shioiri et al. who described the synthesis
of the methyl ester analogue of 9 from L-lactic acid.12 Ac-
cordingly, we were able to obtain the required 4b in six
steps. Thus, TBDPS protection of the hydroxy group of L-
(a)
BocNH2 + i-PrCHO + PhSO2Na
95%
BocHN
SO2Ph
12
O
(b)
(c)
N
COOEt
4b
+
12
TBDPSO
BocHN
COOEt
dr = 1:1
81%
45%
H
OH
OTBDPS
COOH
(b)
N
COOEt
OH
(a)
O
13
COOH
84%
73%
O
OH
OH
6
7
8
N
N
COOEt
+
S
R
R
R
TBDPSO
N
TBDPSO
N
BocHN
BocHN
(c)
(d)
COOEt
COOEt
COOEt
COOEt
O
O
5b
5c
59%
76%
O
O
Scheme 3 Synthesis of Tuv with an oxazole moiety. Reagents and
conditions: (a) formic acid, MeOH–H2O, 24 h; (b) NaH, THF, 2–3 h;
(c) (S)-(–)-2-methyl-CBS-oxazaborolidine, BH3·SMe2, THF, 0 °C, 2–
3 h.
9
10
OH
N
O
N
(e)
(f)
97%
86%
O
O
11
4b
The next step was the synthesis of the other nonproteino-
genic amino acid fragment. We have previously reported
the scalable synthesis of Tup starting from L-phenylalani-
nol,9 which was reasonably efficient but required a rather
laborious separation of two epimers of Tup, involving the
formation and chromatographic purification of the (–)-
menthol esters followed by further manipulations of the
Scheme 2 Synthesis of 2-acetyl oxazole ethyl ester. Reagents and
conditions: (a) TBDPSCl, imidazole, DMF–CH2Cl2 (1:3), r.t., 2–3 d;
(b) HOBt, EDC·HCl, then L-serine hydrochloride ethyl ester, Et3N,
CH2Cl2, 0 °C to r.t.; (c) DAST, THF, –78 °C, 90 min, then K2CO3,
–78 °C to r.t.; (d) CuBr2, HMTA, DBU, CH2Cl2, 0 °C to r.t.; (e) TBAF,
THF, 0 °C to r.t., 30 min; (f) Dess–Martin periodane, CH2Cl2, 0 °C to
r.t.
Synlett 2011, No. 12, 1673–1676 © Thieme Stuttgart · New York

LETTER
Synthesis of an Oxazole Analogue of Tubulysin U
1675
drazinolysis of 18b cleaved the phthaloyl protection and
afforded the lactam 19 which, on ring opening by reflux-
ing in 6 M HCl and subsequent esterification of the result-
ing acid 20 led to Tup-OMe 2120 in its stereopure form.
Ph
Ph
H
O
O
OH
(a)
O
N
N
(b) 89%
97%
O
O
With the required fragments in hand, the assembling se-
quence started with the Boc-deprotection of 5b
(Scheme 5) with 20% TFA in CH2Cl2 and coupling of the
resulting amine 22 with Boc-Ile-OH in the presence of
HOBt, EDC·HCl, and DIPEA. The reaction afforded the
corresponding Boc-dipeptide 23 in 53% yield. Hydrolytic
cleavage of the ethyl ester under mild alkaline conditions
afforded the acid 24, which on subsequent coupling with
Tup-OMe 21 using HOAt and HATU in the presence of
Et3N generated the tripeptide 25. Coupling between N-
methyl-(R)-pipecolic acid 27 and the tripeptide 26, ob-
tained by treatment of 25 with TFA, proceeded smoothly
in the presence of HOAt, HATU, and Et3N, affording the
oxa-tubulysin U methyl ester (28) in good yield and with-
out any detectable loss in stereochemical purity. The me-
thyl ester function was then hydrolysed using 1 M
aqueous LiOH in THF and eventually treated with acetic
anhydride in pyridine affording the final tubulysin U ana-
logue oxa-121 in stereopure form.
14
15
Ph3P
COOEt
16
Ph
Ph
O
O
N
COOEt
N
COOEt
(c)
dr = 1:1
90%
O
O
17
18a
+
Ph
Ph
O
(d)
HN
O
N
COOEt
quant.
O
18b
19
(e)
98%
Ph
Ph
(f)
The oxazole analogue oxa-1 of tubulysin U thus synthe-
sised was assayed for its cytotoxic activity and compared
with the anticancer drug docetaxel (Table 1). Human pro-
myelogenous leukaemic cells (HL-60) were grown under
standard conditions for 48 hours before exposure to in-
creasing concentration of the compounds for 48 hours.
Cytotoxicity was then measured by MTT assay.22
86%
HCl⋅H2N
COOH
HCl⋅H2N
COOMe
20
21
Scheme 4 Novel synthesis of Tup-OMe. Reagents and conditions:
(a) Dess–Martin periodinane, NaHCO3, CH2Cl2, 90 min; (b) CH2Cl2,
overnight; (c) H2, Pd/C, EtOAc, overnight, then chromatographic se-
paration; (d) H2NNH2·H2O, EtOH, reflux; (e) 6 N HCl, 145 °C; (f)
2,2-DMP, concd HCl (cat.), MeOH, reflux.
Table 1 Biological Test on Human Leukaemic Cell Line (HL-60)
Compound
tubulysin U
oxa-1
IC50 (nM)
42.5 3.5
23.8 2.5
1.5 0.2
protecting groups. We therefore decided to develop an im-
proved synthesis of orthogonally protected and stereopure
Tup (Scheme 4).
N-Phthalimido L-phenylalaninol (14) was oxidised with
Dess–Martin periodinane to the aldehyde 15. The latter tubulysin A
was subjected to a Wittig reaction with the phosphorane
docetaxel
57.5 1.0
16, and the resulting a,b-unsaturated ester 17 was hydro-
genated affording an equimolar mixture of epimeric esters a Values are mean SEM (n = 12).
18, which were separated by flash chromatography. Hy-
Ph
OH
O
OH
O
OH
O
(b)
(d)
N
COOEt
BocHN
N
COOR
RHN
N
BocHN
N
N
N
H
COOMe
53%
H
69%
H
O
O
O
5b R = Boc
22 R = H⋅TFA
23R = Et
24 R = H
25 R = Boc
(a)
(c)
(a)
26 R = H⋅TFA
O
O
Ph
Ph
(e) 89%
(f)
O
OH
O
O
O
H
H
N
N
N
N
81%
N
N
N
H
COOMe
N
N
N
COOH
H
H
H
O
O
O
O
N
COOH
oxa-1
28
27
Scheme 5 Completion of the synthesis. Reagents and conditions: (a) TFA–CH2Cl2 (1:4), 0 °C to r.t.; (b) HOBt, EDC·HCl, then Boc-Ile,
DIPEA, CH2Cl2, 0 °C to r.t.; (c) LiOH·H2O, THF–H2O (4:1), 0 °C to r.t.; (d) HOAt, HATU, then Tup-OMe 21, Et3N, CH2Cl2, 0 °C to r.t.; (e)
HOAt, HATU, then Mep (27), Et3N, 0 °C to r.t.; (f) (i) 1 N LiOH, THF, 0 °C to r.t., 2–3 d; (ii) Ac2O, pyridine.
Synlett 2011, No. 12, 1673–1676 © Thieme Stuttgart · New York

1676
S. S. P et al.
LETTER
2009, 6367. (j) Peltier, H. M.; McMahon, J. P.; Patterson,
A. W.; Ellman, J. A. J. Am. Chem. Soc. 2006, 128, 16018.
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Azumaya, I.; Tamura, O. Chem. Eur. J. 2010, 16, 11678.
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Hirasawa, Y.; Hosoya, T.; Tamura, O. Bioorg. Med. Chem.
Lett. 2011, 21, 431. (m) Wang, Z.; McPherson, P. A.;
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A.; Wipf, P. Chem. Biol. Drug Des. 2007, 70, 75.
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D. R. Org. Lett. 2000, 2, 1165.
The results showed that oxa-1 is more potent than doce-
taxel and its natural counterpart tubulysin U but, as ex-
pected, is less effective than tubulysin A in these cells.
An efficient and scalable synthetic strategy for oxazole
analogues of the tubulysins has been developed. The re-
placement of the thiazole sulfur atom with an oxygen in
tubulysin U brings about an unexpected increase of cyto-
toxicity on the HL-60 cell line. We are currently investi-
gating on other tubulysins whether this is a general effect,
as this would open up new perspectives in the area of tu-
bulysin analogues.
(14) Barrish, J. C.; Singh, J.; Spergel, S. H.; Han, W.-C.; Kissick,
T. P.; Kronenthal, D. R.; Mueller, R. H. J. Org. Chem. 1993,
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Acknowledgment
(15) Characterisation Data for Compound 4b
Rf = 0.35 (EtOAc–hexane = 3:7). 1H NMR (400 MHz,
CDCl3): d = 8.21 (s, 1 H), 4.22 (q, J = 7.1 Hz, 2 H), 2.51 (s,
3 H), 1.19 (t, J = 7.1 Hz, 3 H). 13C NMR (100.5 MHz,
CDCl3): d = 185.1, 160.1, 157.6, 146.0, 134.7, 61.5, 26.6,
14.1. ESI-LCMS: m/z = 183.9 [M + H]+, 205.8 [M + Na]+.
(16) For other methods accomplishing the mentioned transfor-
mation, see: (a) Williams, D. R.; Lowder, P. D.; Gu, Y.-G.;
Brooks, D. A. Tetrahedron Lett. 1997, 38, 331.
We thank Martin Van (University of Aberdeen) for performing in-
itial cytotoxicity tests. Financial support from the project EU-FP7
ADAMANT (HEALTH-F2-2008-201342) is gratefully acknowled-
ged.
References and Notes
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4888; Angew. Chem. 2004, 116, 4996.
(3) For a recent study on the tubulin-bound structure of
tubulysin A, see: Kubicek, K.; Grimm, S. K.; Orts, J.; Sasse,
F.; Carlomagno, T. Angew. Chem. Int. Ed. 2010, 49, 4809.
(4) Jackson, J. R.; Patrick, D. R.; Dar, M. M.; Huang, P. S. Nat.
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Steinmetz, H. Pure Appl.Chem. 2003, 75, 167.
(b) Chattopadhyay, S. K.; Kempson, J.; McNeil, A.;
Pattenden, G.; Reader, M.; Rippon, D. E.; Waite, D.
J. Chem. Soc., Perkin Trans. 1 2000, 2415.
(17) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams,
D. R. Org. Lett. 2000, 2, 1165.
(18) Corey, E. J.; Helal, C. J. Angew. Chem. Int. Ed. 1998, 37,
1986; Angew. Chem. 1998, 110, 2092.
(19) Characterisation Data for Compound 5b
Rf = 0.43 (EtOAc–hexane = 45:55). FT-IR (film): nmax
=
3374.8, 1734.5, 1660.4, 1505.4, 1391.9, 757.4 cm–1. 1H
NMR (400 MHz, CDCl3): d = 8.14 (s, 1 H), 4.84 (d, J = 10.8
Hz, 1 H), 4.72 (br s, 1 H), 4.55 (d, J = 9.3 Hz, 1 H), 4.31 (q,
J = 7.1 Hz, 2 H), 3.73–3.63 (m, 1 H), 2.02 (ddd, J = 13.7,
11.3, 2.5 Hz, 1 H), 1.80–1.65 (m, 2 H), 1.38 (s, 9 H), 1.30 (t,
J = 7.1 Hz, 3 H), 0.90 (d, J = 6.9 Hz, 3 H), 0.88 (d, J = 6.9
Hz, 3 H). 13C NMR (100.5 MHz, CDCl3): d = 165.6, 161.1,
157.6, 143.9, 133.2, 80.1, 64.6, 61.0, 52.1, 39.1, 32.1, 28.2,
19.2, 18.2, 14.2. ESI-LCMS: m/z = 379.1 [M + Na]+.
(20) Characterisation Data for Compound 21
(6) For a review on drug-conjugated monoclonal antibodies for
the treatment of cancer, see: Lambert, J. M. Curr. Opin.
Pharmacol. 2005, 5, 543.
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Leamon, C. P. Bioorg. Med. Chem. Lett. 2008, 18, 4558.
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(d) Kularatne, S. A.; Wang, K.; Santhapuram, H.-K. R.;
Low, P. S. Mol. Pharm. 2009, 6, 780.
Rf = 0.3 (MeOH–CH2Cl2 = 1:9). [a]D23 +8.6 (c 1.0, MeOH).
1H NMR (400 MHz, CD3OD): d = 7.46–7.15 (m, 5 H), 3.63
(s, 3 H), 3.59–3.47 (m, 1 H), 3.04 (dd, J = 19.9, 6.2 Hz, 1 H),
2.91 (dd, J = 13.7, 7.7 Hz, 1 H), 2.79–2.65 (m, 1 H), 2.08–
1.95 (m, 1 H), 1.16 (d, J = 6.9 Hz, 3 H). 13C NMR (100.5
MHz, CD3OD): d = 178.1, 137.8, 131.3, 130.9, 129.3, 53.3,
41.1, 37.9, 37.7, 18.7. ESI-LCMS: m/z = 221.9 [M + H]+.
(21) Characterisation Data for the Tubulysin U Analogue
oxa-1
(8) (a) Höfle, G.; Leibold, T.; Steinmetz, H. DE 10008089,
2001. See also: (b) Neri, D.; Fossati, G.; Zanda, M.
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Chem. Int. Ed. 2007, 46, 3526.
Rf = 0.53 (MeOH–CHCl3 = 12:88). 1H NMR (250 MHz,
CD3OD): d = 8.14 (s, 1 H), 7.19–7.00 (m, 5 H), 5.61 (dd,
J = 11.1, 2.4 Hz, 1 H), 4.24–4.22 (m, 1 H), 4.10 (d, J = 8.3
Hz, 1 H), 3.90–3.75 (m, 1 H), 3.13–2.88 (m, 2 H), 2.77 (d,
J = 6.6 Hz, 2 H), 2.52–2.36 (m, 2 H), 2.35 (s, 3 H), 2.27–2.09
(m, 1 H), 2.02 (s, 3 H), 1.99–1.10 (m, 13 H), 1.05 (d, J = 6.8
Hz, 3 H), 0.92–0.74 (m, 12 H). 13C NMR (63 MHz, CD3OD):
d = 176.7, 173.7, 173.1, 171.6, 163.4, 162.1, 143.0, 139.7,
137.5, 130.5, 129.3, 127.3, 69.5, 66.9, 59.6, 56.3, 51.8, 50.7,
44.0, 42.0, 39.3, 37.5, 35.7, 33.8, 32.7, 31.0, 25.9, 25.3, 23.5,
20.6, 19.5, 18.6, 16.2, 14.4, 11.1. ESI-LCMS: m/z = 698.2
[M + H]+, 720.2 [M + Na]+.
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