Structure-activity relationships of ustiloxin analogues

Article abstract of DOI:10.1016/j.tetlet.2010.11.165

Four novel ustiloxin D analogues were synthesized focusing on the size of the macrocyclic core, the stereochemistry at the bridgehead ether, and the enantiomer of ustiloxin D. All four were subjected to biological evaluation testing the inhibition of tubu

Full text of DOI:10.1016/j.tetlet.2010.11.165

Tetrahedron Letters 52 (2011) 2136–2139
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Structure–activity relationships of ustiloxin analogues
Madeleine M. Joullié a, , Simon Berritt a, Ernest Hamel b
⇑
a Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
b Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 13 January 2011
Four novel ustiloxin D analogues were synthesized focusing on the size of the macrocyclic core, the ste-
reochemistry at the bridgehead ether, and the enantiomer of ustiloxin D. All four were subjected to bio-
logical evaluation testing the inhibition of tubulin polymerization. Only 2,2-dimethyl-ustiloxin D
retained any activity.
Dedicated to Professor Harry Wasserman on
the occasion of his 90th birthday.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Ustiloxin
Tubulin polymerization
Aziridine ring opening
Structure–activity relationships
1. Introduction
pounds universally inhibit the assembly of the a,b-tubulin dimer
into microtubules, thus resulting in mitotic arrest of eukaryotic
cells. Many antimitotic agents are known, ranging from the vinca
alkaloids and taxoids to chalcones,2 but the mechanism of action
of the naturally occurring peptides that inhibit tubulin polymeriza-
tion is still not fully elucidated.3
The ustiloxins A–F (Fig. 1) are a class of antimitotic natural
products isolated from the water extracts of false smut balls,
caused by the parasitic fungus Ustilaginoidea virens.1 These com-
Recent syntheses of ustiloxin D4 and F5 have been published,
and the syntheses of eight analogues focusing on structural varia-
O
OH
OH
O
O
N
H
O
O
HN
O
OH
OH
R1
HO
OH
O
OH
O
O
N
H
O
O
N
H
10
6
9
N
H
R2
O
O
HN
HN
HN
O
O
NH2 OH
O
S
HO
N
H
IC50/µM
HO
N
H
HN
Ustiloxin A
Ustiloxin B
1.0
HN
OH
HO2C
HO2C
R1 R2 CH(CH3)2
NH2 OH
O
S
ent-ustiloxin D 1
2,2-dimethyl-ustiloxin D 2
1.8
4.4
R1 R2 CH3
OH
O
S
O
O
NH
O
Ustiloxin C
O
HO
R1 R2 CH3
OH
OH
O
O
N
H
O
O
R1 R2 CH2(CH3)2
HN
Ustiloxin D
Ustiloxin F
H
H
2.5
O
HN
R1 R2 CH3
O
10.3
NH
HO
N
H
Figure 1. Ustiloxins A–F with IC50 values based on inhibition of porcine brain
HO
N
H
HN
microtubule assembly.
O
HN
(2S)-epi-ustiloxin D 3
7-N-Gly-ustiloxin D 4
⇑
Corresponding author. Tel.: +1 215 898 3158; fax: +1 215 573 9711.
E-mail address: mjoullie@sas.upenn.edu (M.M. Joullié).
Figure 2. Four ustiloxin D analogues.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.165

M. M. Joullié et al. / Tetrahedron Letters 52 (2011) 2136–2139
2137
R3
O
R2
O
R2
R3
OH
OH
OH
OH
N
H
6
OH
O
O
N
H
N
H
O
O
HN
O
O
O
H2N
HO
OH
HO
N
H
R4
OH
NH
HN
R4
5
7
Figure 3. Retrosynthetic analysis of the ustiloxins.
tions at R2, stereochemistry at C9 and C10, and methylation of the
phenol were also reported by our group.6 To complete the SAR
analysis we wished to probe the stereochemistry and the substitu-
ents at the tertiary aryl-ether bridge, the size of the macrocyclic
core, and the enantiomer of ustiloxin D. The four analogues are
shown in Figure 2.
dol7 reaction, respectively. This convergent route allows for rapid
derivation of any of the three components.
To access 2,2-dimethyl ustiloxin D, oxazolidine 8 was utilized to
form the requisite dimethylaziridine 11 (Scheme 1).
Thus, the 2,2-dimethyl aziridine was synthesized by treatment
of the tertiary alcohol 8 with aqueous HCl and subsequent
ortho-nitrobenzenesulfonamide protection of the amine afforded
diol 9. TEMPO oxidation, followed by EDCI-mediated coupling of
glycine benzyl ester gave the precursor to aziridine 10, and a Mits-
unobu reaction afforded the dimethyl aziridine 11 in good yield.
The (2S,3R)-ethynyl aziridine 15 was synthesized by Grignard
addition to ketone 12 which resulted in a 11:1 diastereomeric ra-
tio; the minor diastereomer (13) being utilized within this route.
The synthesis of 15 was continued supra vida to afford the second
aziridine.
2. Synthesis of ustiloxin D analogues
The convergent synthesis of the ustiloxins developed in our lab-
oratory allowed for expeditious synthesis of the four ustiloxin ana-
logues. In a retrosynthetic manner, the ustiloxins can be divided
into three constituent parts: b-hydroxytyrosine 5, aziridine 6,
and commercially available amino acid 7 (Fig. 3).
The aziridine 6 and b-hydroxytyrosine moiety 5 can be quickly
-valine,4e and a (S)-salen-aluminium catalyzed al-
The requisite b-hydroxytyrosine moiety (16) was synthesized as
previously reported6 and coupled to aziridines 11 and 15 via a reg-
ioselective 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) mediated ring
opening reaction developed in our laboratory,8 to afford the nosyl
protected amines 17 and 18 (Scheme 2).
accessed from
D
O
HO
O
OBn
b,c
The synthesis of ent-ustiloxin D followed the previous6 synthe-
sis of ustiloxin D and will not be reported in detail here. An (R)-
salen-aluminium catalyzed aldol reaction afforded the b-hydrox-
a
HO
d
OH
HO
N
H
BocN
NHNs
9
NHNs
O
8
10
O
ytyrosine, L-serine provided the aziridine and D-valine was used
OBn
to complete the protected linear precursor. The synthesis of 7-N-
Gly-ustiloxin D (4) also followed the previous synthesis to give
the nosyl protected amine 21 (Scheme 3).
N
H
N
O
Ns
11
Removal of the nosyl protecting group on each of the three ana-
logues with benzene thiol proceeded without incident to provide
the free amine. EDCI-mediated coupling of tripeptides 17 and 18
with N-Boc-D-valine gave compounds 19 and 20, respectively. Cou-
pling of amine 21 with N-Boc-glycine-valine afforded 22 (Scheme
3).
O
e
O
+
HO
HO
O
O
BocN
BocN
13
BocN
13a
12
O
With all three analogues and the ent-ustiloxin D protected
linear precursor in hand (not shown), the synthesis was concluded
b,c
a
OBn
N
H
HO
OH
N
O
with
(Scheme 4).
Treatment of the linear precursors with TFA/Et3SiH afforded the
a
deprotection/macrocyclization/deprotection sequence
Ns
NHNs
14
15
Scheme 1. Synthesis of aziridines 11 and 15. Reagents and conditions: (a) HCl, THF,
0 °C, 1 h, then NsCl, Na2CO3, THF/H2O, rt, 16 h, 58% for 9, 90% for 14; (b) TEMPO,
NaClO2, NaClO, Na2HPO4, MeCN, 40 °C, 18 h; (c) H-GlyOBnÁHCl, EDCI, HOBt,
NaHCO3, DMF, 12 h, rt, 95% for 10, 90% for alkynyl precursor; (d) DIAD, PPh3,
CH2Cl2, 18 h, rt, 77% for 11, 68% for 15; (e) ethynylMgBr, THF, rt, 80% ds = 11:1
(13a:13).
TFA salts, which were subsequently converted into the hydrochlo-
ride salts. Subjection of each amino acid salt to EDCI-mediated
macrocyclization and concomitant Pd/C catalyzed hydrogenolysis
gave ent-ustiloxin D (1), 2,2-dimethyl ustiloxin D (2), (2S)-epi-usti-
loxin D (3), and 7-N-Gly-ustiloxin D (4).
R1 R2
O
OBn
R1
O
OBn
OBn
OH
O
OBn
R2
N
H
O
O
N
H
a
O
N
+
NHNs
O
Ns
TBSO
OPMB
11 R1= R2= Me
15 R1= Ethyne; R2= Me
TBSO
OPMB
NCbz
CbzN
17 R1= R2= Me (72%)
18 R1= Ethyne; R2= Me (78%)
16
Scheme 2. Ring opening of aziridines 11 and 15. Reagents and conditions: (a) TBD, toluene, 0 °C to rt, 3 h.

2138
M. M. Joullié et al. / Tetrahedron Letters 52 (2011) 2136–2139
OBn
O
R2
R1 R2
R1
O
NH
O
OBn
OBn
OBn
O
O
N
H
O
O
HN
O
O
NHNs
a,b
BocHN
OPMB
TBSO
OPMB
TBSO
CbzN
CbzN
17 R1= R2= Me
19 R1= R2= Me (96%)
18 R1= Ethyne; R2= Me
20 R1= Ethyne; R2= Me (76%)
OBn
O
O
O
NH
OBn
OBn
OBn
O
O
N
H
O
HN
a,c
O
O
O
NHNs
HN
TBSO
OPMB
TBSO
OPMB
CbzN
O
NHBoc
CbzN
21
22
Scheme 3. Deprotection and coupling of amino acid residues. Reagents and
conditions: (a) PhSH, Cs2CO3, DMF, 0 °C, 4 h; (b) N-Boc-Gly, EDCI, HOBt, NaHCO3,
DMF, 12 h, rt; (c) N-Boc-Val-Gly, EDCI, HOBt, NaHCO3, DMF, 12 h, 0 °C to rt, 79%.
Figure 4. SpartanÒ’03 energy minimized molecular models at HF/AM1 level of
theory. Ustiloxin D (A), 2,2-dimethyl-ustiloxin D (B), and (2S)-epi-ustiloxin D (C).
The difference in inhibitory activity of ustiloxin D, (2S)-epi, and
the dimethyl species was investigated using molecular modeling
(Fig. 4). Comparison of ustiloxin D (A) to 2,2-dimethyl ustiloxin
(B) and (2S)-epi-ustiloxin (C) quite clearly shows distortion of the
glycine sidechain, progressively pushing it toward the valine resi-
due. The out-of-plane glycine sidechain may lessen the ability of
the molecule to bind at this position, but it may also block the
binding ability of the valine residue, which was shown to play an
important role in the ustiloxins inhibitory effect.6,9
OBn
O
R1 R2
O
R2
OH
R1
O
OH
NH
O
OBn
O
N
H
a,b,c
O
HN
HN
O
O
O
O
BocHN
OPMB
HO
N
H
TBSO
HN
CbzN
19 R1= R2= Me
2 R1= R2= Me
20 R1= Ethyne; R2= Me
3 R1= Ethyne; R2= Me
4. Conclusions
OBn
OH
O
Novel ustiloxin analogues were synthesized to investigate the
effect of changes at the C2 center, the macrocycle size, and the
enantiomeric series on tubulin inhibition. The structural modifica-
tions produced significant changes in the biological activity sug-
gesting the importance of the valine residue in the binding process.
O
O
NH
HN
NH
O
O
OH
OBn
O
O
HN
HN
O
O
a,b,c
O
NH
HO
N
H
TBSO
OPMB
O
NHBoc
Acknowledgments
O
HN
CbzN
22
4
We thank NIH (CA-40081) and NSF (CHE-0951394) for support
of this work. Financial support for the departmental instrumenta-
tion was provided by NIH (1S10RR23444-1). We also thank Dr.
George Furst and Dr. Rakesh Kohli for NMR and MS assistance,
respectively.
Scheme 4. Reagents and conditions: (a) TFA, Et3SiH, CH2Cl2, 0 °C, 4 h; (b) EDCI,
HOBt, NaHCO3, DMF, 0 °C to rt, 24 h, 46% for R1 = R2 = Me, 30% for R1 = Me
R2 = ethyne, 34% for N-Gly macrocycle; (c) Pd/C, H2, THF/H2O, 24 h, 80% for 2, 60%
for 3, and 55% for 4.
References and notes
3. Bioactivity
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The effect of the changes present in the four analogues on the
polymerization of purified tubulin was investigated using the
IC50 value of ustiloxin D (2.5
evaluation of ent-ustiloxin D (1) showed no inhibition with an
IC50 value >40 M. Similarly, 7-N-Gly-ustiloxin D (4) showed no
inhibition (IC50 >40 M), suggesting that the naturally occurring
lM) as the benchmark. Biological
2. Hamel, E. Med. Res. Rev. 1996, 16, 207.
l
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l
ustiloxin D macrocycle size is optimum. (2S)-epi-Ustiloxin D (3)
exhibited a very slight inhibitory effect, but overall had an IC50
>40
ether bridge afforded 2,2-dimethyl ustiloxin
IC50 = 9.2 M.
l
M. Interestingly, removal of the stereogenic carbon at the
D
(2) with an
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