Synthesis of dehydroalanine fragments as thiostrepton side chain mimetics

Article abstract of DOI:10.1016/j.bmcl.2005.03.084

Syntheses of dehydroalanine derivatives via a solid-support route, starting from selenocystein, and via conventional solution phase chemistry are described along with initial biological testing. The target compounds were designed as mimetics of the dehydroalanine side chain of the macrocyclic antibiotic thiostrepton that acts on the bacterial ribosome.

Full text of DOI:10.1016/j.bmcl.2005.03.084

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2457–2460
Synthesis of dehydroalanine fragments as thiostrepton
side chain mimetics
Benjamin K. Ayida,a Klaus B. Simonsen,a, Dionisios Vourloumisa and
Thomas Hermannb,*
aDepartment of Medicinal Chemistry, Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
bDepartment of Structural Chemistry, Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
Received 14 February 2005; revised 18 March 2005; accepted 21 March 2005
Available online 12 April 2005
Abstract—Syntheses of dehydroalanine derivatives via a solid-support route, starting from selenocystein, and via conventional solu-
tion phase chemistry are described along with initial biological testing. The target compounds were designed as mimetics of the
dehydroalanine side chain of the macrocyclic antibiotic thiostrepton that acts on the bacterial ribosome.
Ó 2005 Elsevier Ltd. All rights reserved.
Thiostrepton is a cyclic peptide antibiotic that inhibits
bacterial translation by blocking the function of the
ribosomal GTPase center (Fig. 1A).1 The macrocycle
interacts with 23S rRNA at the GTPase-associated do-
main of the 50S ribosomal subunit and shuts down
GTP-dependent reactions during translation. The insol-
uble nature of thiostrepton has prevented its broad use
as an antibiotic and complicated structural studies to
elucidate its interaction with the ribosomal target. De-
spite the fact that a total synthesis of thiostrepton has
been published,2 its complex chemical structure hampers
selective derivatization. Previous synthetic efforts were
focused on fragments of the macrocycle as potential lead
structures for antibacterial discovery targeting the ribo-
some.3 While emerging structural data suggest that the
thiostrepton macrocycle might be interacting with both
ribosomal S23 RNA and the L11 protein,4 the role of
the bis(dehydroalanine) side chain remains elusive. To
provide model compounds for the study of the biologi-
cal activity of the thiostrepton side chain, we synthesized
dehydroalanine fragments 1 and 2 as mimetics of the
bis(dehydroalanine) moiety (Fig. 1).
(A)
O
O
O
H
N
N
H
NH2
S
O
N
O
H
H
N
N
N
O
N
H
S
O
O
N
N
H
N
O
OH
H
NH
S
N
NH
O
HN
HO
O
N
HN
O
N
H
N
OH
S
H
S
O
HO
OH
Thiostrepton
(B)
O
O
O
H
H
N
R1
N
R2
N
R1
N
O
H
H
O
O
1
2
Figure 1. (A) Thiostrepton, a ribosome-binding cyclic peptide antibi-
otic first isolated from Streptomyces azureus. (B) Mimetics 1 and 2 of
the thiostrepton bis(dehydroalanine) side chain (boxed in A), whose
synthesis and preliminary biological testing is described in this
communication.
Keywords: Antibiotics; Dehydroalanine; Ribosome; Thiostrepton;
Translation.
*
Corresponding author. Tel.: +1 858 530 3659; fax: +1 858 527
1540; e-mail: thermann@anadyspharma.com
Established routes for the synthesis of dehydroalanine
and derivatives thereof include (a) conversion of masked
Present address: H. Lundbeck A/S, Ottiliavey 9, 2500 Copenhagen
Valby, Denmark.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.084

2458
B. K. Ayida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2457–2460
b, c
a
Se
Se
O
R1CO2H
O
O
BocHN
R1
N
H
SeLi
O
5
O
3
7
MsO
Boc
O
N
H
O
d,e
R2NH2
4
Se
O
O
H
N
H
f
N
R1
N
H
R2
R1
N
H
R2
O
O
1
9
R1CO2H =
R2NH2
=
CO2H
CO2H
O
O
N
N
O
NH2
S
S
N
H
O
N
6a
6b
8e
N
t-BuO
N
Boc
CO2H
CO2H
N
O
H2N
N
N
6c
6d
8f
8g
O
Scheme 1. Reagents and conditions: (a) 4 (2.0 equiv), THF (0.22 M), 2 h, 23 ! 50 °C; (b) 55% TFA in CH2Cl2, 2 h, 23 °C; (c) DIC (6.0 equiv), HOBt
(7.0 equiv), R1CO2H (6a–d, 4.0 equiv), DMF (0.07 M), 20 h, 23 °C; (d) LiOH (10.0 equiv), THF/H2O (5:1, 0.47 M); (e) DIC (5.0 equiv), HOBt
(5.0 equiv), R2NH2 (8e–g, 3.0 equiv), DMF (0.07 M), 20 h, 23 °C; (f) H2O2 (30% aq, 6.0 equiv), Me2S (10.0 equiv), 1 h, 23 °C. THF = tetra-
hydrofuran; TFA = trifluoroacetic acid; HOBt = 1-hydroxybenzotriazole; DMF = N,N-dimethyl-formamide; DIC = 1,3-diisoproylcarbodiimide.
OTBDPS
OH
serine and threonine residues, either as mesyloxy inter-
mediates or halides,5 (b) direct elimination of b-hydroxy
groups,6 via asparagine residues, and (c) Hoffmann elimi-
nation of 2,3-diaminopropionic acids.7 Few examples
have been reported using phenyl selenocysteine to pro-
mote site specific and chemoselective introduction of
dehydroalanine residues.8 In this communication, we re-
port the use of selenocysteine as a solid phase selenium
linker for peptide synthesis that, upon oxidative cleav-
age from the solid support, yields the dehydroalanine
functionality by elimination.
OH
O
a-c
R1
N
H
O
H2N
6a-d
O
O
10
11
d
OTBDPS
O
O
O
O
Selenium-functionalized resin 3, obtained following a
protocol reported by Nicolaou and co-workers,9 was
alkylated with 2-amino-3-methanesulfonyloxy-propi-
onic acid methyl ester 4 to yield solid-supported seleno-
cysteine precursor 5 (Scheme 1).
H
H
N
e-g
N
R1
N
H
O
R1
N
H
O
O
O
2
OH
12
Scheme 2. Reagents and conditions: (a) R1CO2H (1.0 equiv), BOP
(1.1 equiv), i-Pr2NEt (2.2 equiv), CH2Cl2 (0.12 M), 3 h, 23 °C; (b)
TBDPSCl (1.2 equiv), imidazol (2.2 equiv), THF (0.07 M), 3 h,
0 ! 23 °C; (c) LiOH (2.0 equiv), MeOH (0.11 M), 1.5 h, 23 °C; (d)
10 (1.2 equiv), BOP (1.2 equiv), i-Pr2NEt (2.2 equiv), CH2Cl2 (0.12 M),
3 h, 23 °C; (e) MsCl (6.0 equiv), Et3N (10.0 equiv), CH2Cl2 (0.04 M),
3 h, 23 °C; (f) TBAF (1.2 equiv), THF (0.05 M), 3 h, 23 °C; (g)
MsCl (6.0 equiv), Et3N (10.0 equiv), CH2Cl2 (0.04 M), 3 h, 23 °C.
BOP = benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-
fluorophosphate; TBDPSCl = 1,1-dimethylethyl-diphenylsilyl chlo-
ride; TBAF = tetrabutylammonium fluoride; for reagent abbreviations
see also legend to Scheme 1.
Methyl ester 4 was synthesized from L-serine methyl es-
ter 10 (Scheme 2) by treatment with di-tert-butyl dicar-
bonate followed by mesylation of the serine hydroxy
group.10 The efficiency of resin loading was determined
by oxidative cleavage (H2O2/Me2S) of an aliquot of
the alkylated intermediate 5, followed by NMR quanti-
tation using 2,5-dimethylfuran as internal reference.11
Acidic removal of the Boc protecting group, followed
by coupling with various acids (6a–d) proceeded under
standard conditions (DIC, HOBt) to produce amides

B. K. Ayida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2457–2460
2459
Table 1. Activity of dehydroalanine fragments as in vitro translation
inhibitors
7. After the C-terminal ester was hydrolyzed with lith-
ium hydroxide, the resulting carboxylic acids were re-
acted with amines 8e–g to furnish the corresponding
dipeptides 9. The final dehydroalanine products 1 were
liberated from the resin by oxidative cleavage with
H2O2/Me2S. This method resulted in the best overall
yields (8–32%) among all others tested, including
NaIO4, tert-butyl peroxide, and aqueous H2O2. The
dehydroalanine derivatives 1 exhibited satisfactory
NMR and LC–MS spectra.
Compounds 1
% Inhibitiona Concentration
(lM)
R1
R2
6a
6b
6b
6b
8f
5
150
400
400
8f 30
8e 20
8g
O
O
H
N
N
H
O
S
N
O
20
15
The relatively low overall yields of the solid-phase syn-
thesis were attributed to the sensitivity of the double
bond in 1 under the oxidative cleavage conditions. This
effect became more profound in the synthesis of the
bis(dehydroalanine) analogs 2 under the same condi-
tions, as expected from the presence of two olefinic func-
tionalities. Specifically, a variety of oxidized products
were identified by LS–MS after removal of the final
compounds from the solid support. In view of these re-
sults, we decided to proceed with the synthesis of 2
through a stepwise solution phase synthesis. Conse-
quently, the route to the bis(dehydroalanine) com-
pounds 2 commenced with the commercially available
L-serine methyl ester 10 (Scheme 2). O-Silylation of 10
with TBDPSCl, followed by standard coupling (BOP,
i-Pr2NEt) with acids 6a–d (Scheme 1) and subsequent
hydrolysis of the ester functionality gave access to the
carboxylic acids 11. Standard peptide coupling, as de-
scribed above, afforded dipeptides 12 as the sole prod-
uct. Attempts were made to subject 12, after TBAF-
mediated silyl deprotection, to a one-pot per-O-mesyla-
tion followed by double b-elimination of the resulting
mesyloxy groups to furnish 12. This resulted in various
combinations of the mono- and di-mesylated as well as
b-elimination products, which were difficult to isolate
using conventional methods. Eventually, O-mesylation
of 12 in the presence of a slight excess of weak base
(Et3N) resulted in b-elimination of the corresponding
mesyloxy group. Cleavage of the silyloxy group with
TBAF, followed by mesylation and b-elimination affor-
ded the final products 2 in good overall yield. The
bis(dehydroalanine) derivatives 2 exhibited satisfactory
NMR and LC–MS spectra.
NH
N
6c
6c
6c
6d
6d
6d
8e
4
400
400
150
400
400
150
8f 40
8g 20
8e 25
8f 15
8g 10
Compound 2, R1 = 6d
Thiostrepton
30
50
400
0.17
a Inhibition calculated as means of three replicate experiments for each
compound ( 10%).
1–(6d/8g) showed about 20-fold less activity. The fact
that relatively subtle variations in the substituent at R1
cause significant changes in compound activity suggests
that further chemical exploration at this site might lead
to more potent dehydroalanine translation inhibitors.
In conclusion, we have described the synthesis of two
series of dehydroalanine fragment series, 1 and 2, as
mimetics of the linear side chain of the peptide antibiotic
thiostrepton. A solid-supported synthetic route, starting
from selenocysteine, was used to produce the dehydro-
alanine derivatives 1, and conventional solution-phase
synthesis afforded the bis(dehydroalanine) series 2. Sim-
ilar to thiostrepton itself, the dehydroalanine derivatives
displayed low solubility in aqueous media, which inter-
fered with a full assessment of their biological activity.
Nevertheless, one compound was discovered that
showed activity as an inhibitor of bacterial translation.
Future efforts might aim at improving solubility of the
thiostrepton side chain mimetics starting with the active
translation inhibitor 1–(6b/8g) as a lead structure.
To assess the biological activity of the synthesized dehy-
droalanine derivatives, they were tested for their activity
as inhibitors of bacterial in vitro translation (Table 1).12
All but one of the bis(dehydroalanine) compounds 2 had
very poor solubility in 5% DMSO, which prevented their
testing in the translation assay. Ten of the dehydroala-
nine derivatives 1 were soluble enough to allow testing
of their inhibitory activity at one compound concentra-
tion. The limited solubility prevented collection of dose–
response data at higher concentrations for the determi-
nation of IC50 values.
Acknowledgements
We thank Ms. S. Fish for help with the in vitro transla-
tion assay. This work was supported by the National
Institutes of Health (Grant AI51104 to T.H.).
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