Total synthesis of the cyclic depsipeptide YM-280193, a platelet aggregation inhibitor

Article abstract of DOI:10.1021/ol503507g

The first total synthesis of YM-280193, a cyclic depsipeptide that inhibits the ADP-induced aggregation of human platelets, is described. The monomer and dipeptide fragments were prepared using conventional chemistry and subsequently assembled by Fmoc-solid-phase peptide synthesis (Fmoc-SPPS). A late-stage novel bis-alkylation-elimination of cysteine on-resin was employed to introduce the unnatural N-methyldehydroalanine moiety. The final step involved execution of a key macrolactamization reaction between the hindered unnatural N,O-dimethylthreonine and ?2-hydroxyleucine residues.

Full text of DOI:10.1021/ol503507g

Letter
pubs.acs.org/OrgLett
Total Synthesis of the Cyclic Depsipeptide YM-280193, a Platelet
Aggregation Inhibitor
†
†
‡
,†
Harveen Kaur, Paul W. R. Harris, Peter J. Little, and Margaret A. Brimble*
†School of Biological Sciences, Maurice Wilkins Centre for Molecular Biodiscovery and School of Chemical Sciences, The University
of Auckland, 23 Symonds Street, Auckland 1142, New Zealand
‡
Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, Health Innovations Research Institute,
RMIT University, Bundoora, VIC 3083, Australia
*
S Supporting Information
ABSTRACT: The first total synthesis of YM-280193, a cyclic depsipeptide that inhibits the ADP-induced aggregation of human
platelets, is described. The monomer and dipeptide fragments were prepared using conventional chemistry and subsequently
assembled by Fmoc-solid-phase peptide synthesis (Fmoc-SPPS). A late-stage novel bis-alkylation−elimination of cysteine on-
resin was employed to introduce the unnatural N-methyldehydroalanine moiety. The final step involved execution of a key
macrolactamization reaction between the hindered unnatural N,O-dimethylthreonine and β-hydroxyleucine residues.
ardiovascular diseases are a major cause of morbidity and
C
the leading cause of mortality worldwide, accounting for
1
3
0% of total deaths, approximately 17 million annually. Of these
thrombotic complications such as heart attacks and stroke
1
resulted in 7.6 and 5.7 million deaths, respectively. Currently,
platelet aggregation inhibitors such as orally available aspirin and
clopidogrel constitute the largest class of antithrombotic drug2s
that are used to treat and manage thrombotic complications.
The effectiveness of available antithrombotics, however, is
limited by a high degree of interindividual variation in efficacy
and safety, breakthrough thrombosis, and recurrent ischemic
2
,3
events. These limitations and the prevalence of cardiovascular
Figure 1. Structures of YM-280193 (1), YM-254890 (2), YM-254891
3), and YM-254892 (4).
diseases have contributed to the continuous need to develop new
(
2
antithrombotic drugs.
4
Recently, Taniguchi et al. discovered four novel cyclo-
group, respectively. Evaluation of ADP-induced platelet
aggregation inhibitory activity in human platelet-rich plasma
depsipeptides, namely YM-280193 (1), YM-254890 (2), YM-
254891 (3), and YM-254892 (4) with platelet aggregation
revealed that octadepsipeptides 2−4 exhibited potent IC values
inhibitory activity from the Japanese soil microorganism
50
5
4
,5
of 0.26, 0.27, and 0.29 μM, respectively. In contrast,
Chromobacterium sp. QS3666 (Figure 1). The four cyclo-
depsipeptides 1−4 share the common cyclic core of 1, which
consists of an unnatural β-hydroxyleucine (β-Hyleu) residue, D-
phenyllactic acid (D-Pla), three N-methylated residues, namely
heptadepsipeptide 1 displayed reduced potency with an IC of
50
3
.4 μM, which suggests that the additional β-hydroxyleucine
residue is critical for activity; however, steric allowance at the N-
acyl group is also tolerated.
5
N,O-dimethylthreonine (N,O-Me Thr), N-methylalanine (N-
2
More importantly, further research proved YM-254890 (2) to
be the first and currently only known selective inhibitor of
MeAla), and N-methyldehydroalanine (N-MeDha), an N-acetyl-
L-threonine residue, and naturally occurring L-alanine.
In addition to the core backbone of 1, octadepsipeptides 2−4
have an extra β-hydroxyleucine residue attached that is N-
acylated with an N-acetyl, N-propionyl, or N-methylthioacetyl
Received: December 4, 2014
©
XXXX American Chemical Society
A
DOI: 10.1021/ol503507g
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Gαq/11, a G protein predominantly expressed in human platelets
that is crucial to platelet activation and aggregation. Hence, in
protected cysteine due to the prevalence of other competing β-
hydroxy amino acids in 1.
6
−8
addition to the search for new antithrombotics, YM-254890 (2)
Choice of an appropriate site to effect the key macrocyclization
step was crucial. Disconnection of 1 at the two macro-
is also an invaluable probe for understanding G -mediated
q
19
biological processes. Unfortunately, despite the biological
importance of YM compounds 1−4, none of these YM molecules
are commercially available to researchers. Demand for the YM
depsipeptides has been highlighted, for example, by a $100000
USD reward for the total synthesis of 1 mg of YM-254890 (2), in
lactonization sites was excluded as well as the three sites that
20
result in an N-methylated terminal linear precursor. The N-
MeDha-Ala junction which results in a masked C-terminal
cysteine residue that is prone to C-terminal racemization and
conversion to Dha during Fmoc-SPPS was also not appro-
9
12,21
which 228 applicants worldwide actively participated. However,
priate.
The initial disconnection site was thus chosen at the
the total synthesis of 2 remains elusive, as the reward was later
withdrawn without announcement of a winning solution.
Intrigued by the platelet aggregation inhibitory activity of these
YM compounds 1−4, their synthetically challenging cyclo-
depsipeptide structure, and the lack of availability of any of these
compounds 1−4 to researchers, we herein report the first total
synthesis of YM-280193 (1), the cyclo-heptadepsipeptide core of
the more potent YM-molecules 2−4.
We chose to construct 1 using a hybrid of solution-phase
synthesis to create the protected building blocks followed by
Fmoc-solid-phase peptide synthesis (Fmoc-SPPS) to assemble
the linear peptide precursor (Scheme 1). The use of Fmoc-SPPS
N-MeAla-Hyleu junction, leading to linear precursor 5
composed of an N-terminal β-Hyleu and a C-terminal N-
MeAla residue (Scheme 1, route a). Moreover, the high number
of N-methylamino acids were anticipated to contribute to
effective cyclization by minimizing the head to tail spatial
proximity through introducing cis-amide bonds and inducing β-
20
turns.
Fmoc-SPPS to access acyclic peptide 5 commenced with
attachment of the C-terminal Fmoc-N-MeAla-COOH (6)
residue onto 2-chlorotrityl chloride (2-CTC) resin (Scheme
α
2). N -Fmoc-deprotection of resin-bound 7 and sequential
Scheme 2. Initial Synthetic Strategy toward Linear Precursor
5
Scheme 1. Initial (Route a) and Final (Route b)
Retrosynthetic Disconnections of YM-280193 (1) and
Building Blocks
for the preparation of the linear precursor eliminates the
necessity for purification and isolation of intermediates during
the synthesis and also overcomes poor solubility issues associated
with protected peptide fragments during solution-phase
elongation with successive residues met with significant
diketopiperazine (DKP) formation despite the use of the
sterically hindered 2-chlorotrityl linker and treatment with the
milder base piperazine (5% in DMF) containing 0.1 M 6-Cl-
1
0
condensation. However, Fmoc-SPPS would render the
dehydroalanine (Dha) residue of 1 susceptible to Michael
addition from nucleophiles such as piperidine which results in the
21−23
HOBt as an acidic additive.
Consequently, the second and
third residues were introduced as the preformed dipeptide Fmoc-
1
1,12
t
formation of β-piperidyl adducts.
Typically, Dha is masked
N- MeCys(S Bu)-Ala-COOH (8) (see the Supporting Informa-
during SPPS and introduced at a late stage via either the
tion).
1
3,14
activation and elimination of serine derivatives
or oxidative
Briefly, dipeptide 8 was synthesized by direct N-methylation of
Boc-Cys(S Bu)-COOH followed by HCTU-mediated conden-
15,16
17,18
t
elimination of alkylated cysteines
and selenocysteines.
In
our case, we chose to mask the Dha residue of 1 as a suitably
sation with NH -Ala-CO Me. Ester hydrolysis, Boc-deprotec-
2
2
B
DOI: 10.1021/ol503507g
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 3. Synthesis of YM-280193 (1)
tion, and Fmoc-reprotection furnished the desired dipeptide
fragment 8. Pleasingly, introduction of Fmoc-dipeptide 8 in lieu
of sequential elongation successfully circumvented DKP
formation (Scheme 2).
conveniently removed with TFA during resin cleavage. For our
purposes, the C-terminal Fmoc-β-Hyleu(OH)-COOH (23)
residue was synthesized using Belokon’s nickel complex
chemistry and used herein with its secondary hydroxyl group
unprotected (see the Supporting Information).
26
α
Next, N -Fmoc-deprotection and coupling of TBDMSO-D-
Pla-COOH (9) onto resin-bound N-MeCys 10 using a variety of
peptide coupling reagents and conditions for N-alkylated
residues was slow resulting in incomplete peptide bond
formation. Nevertheless, the poor reactivity of resin-bound N-
MeCys 10 proved advantageous as HO-D-Pla-COOH (11) could
be coupled without protection of its hydroxyl functionality using
HATU and HOAt to give 12, thereby avoiding an extra
protection and deprotection step (Scheme 2). Subsequent
esterification of resin-bound tetrapeptide 12 with the required N-
acetylated derivatives Ac-Thr(TBDMS)-COOH (13) or Ac-
Thr(Bzl)-COOH (14) was then extensively investigated using
Thus, linear precursor 21 was synthesized using our previously
optimized conditions which began by anchoring Fmoc-β-
α
Hyleu(OH)-COOH (23) onto 2-CTC resin (Scheme 3). N -
Fmoc-deprotection using piperidine-DMF followed by HBTU-
mediated coupling of Fmoc-N-MeAla-COOH (6) afforded
α
resin-bound dipeptide 24. N -Fmoc-deprotection of 24 and
t
subsequent coupling of dipeptide Fmoc-N-MeCys(S Bu)-Ala-
COOH (8) was successfully effected using HATU and HOAt,
which are excellent reagents for effecting coupling with N-
11
α
methylated residues. N -Fmoc-deprotection of 25 resulted in
racemization at the N-MeCys residue even when milder
deprotection conditions were used, but this is tolerated in our
synthetic strategy as Cys is eventually converted to the achiral
Dha residue. Subsequent double couplings of unprotected
dipeptide Ac-Thr(OH)-D-Pla-COOH (15) using HATU and
HOAt furnished depsipeptide 26. Next, selective esterification of
24
either Steglich or modified Yamaguchi conditions, acyl halides,
25
traditional mixed anhydride reagents, or the addition of Li salts
with extended reaction time at room temperature or with
microwave irradiation, but all proved fruitless (Scheme 2).
Because of the problematic on-resin esterification of 12, the
fourth and fifth residues were introduced as the preformed side-
chain unprotected dipeptide Ac-Thr(OH)-D-Pla-COOH (15)
Boc-N,O-Me Thr-COOH (22) at the N-AcThr residue of resin-
2
bound depsipeptide 26 using DIC/DMAP provided depsipep-
tide 27 with a masked cysteine residue as a substitute for Dha.
Inspired by the solution-phase conversion of cysteine to Dha
(
see the Supporting Information). Briefly, solution-phase
esterification of Boc-Thr(Bzl)-COOH with HO-D-Pla-CO Bn
2
27
using DCC/DMAP afforded the terminally protected depsipep-
via bis-alkylation−elimination reported by Davis et al. and
tide in almost quantitative yield. Global deprotection via Pd/C
previous work on the elimination of labile sulfonium ions to
α
28,29
catalyzed hydrogenolysis and acid-mediated N -Boc-removal
dehydroalanine,
we applied this strategy to the solid-phase
followed by N-acetylation of the free amine furnished the desired
ester fragment 15. Gratifyingly, coupling of fragment 15 onto
resin-bound N-MeCys 10 using HATU and HOAt afforded
pentadepsipeptide 16 (Scheme 2). Consequent on-resin
synthesis of dehydroalanyl peptide 28. To the best of our
knowledge, this is the first application of this methodology to the
solid-phase synthesis of dehydroalanine-containing peptides.
Reduction of the S-tert-butylthio group of heptapeptide 27 with
dithiothreitol (DTT) and DIPEA afforded the free thiol
peptide. Pleasingly, bis-alkylation of the sulfhydryl function-
ality with 1,4-dibromobutane and elimination of the labile
sulfonium salt with potassium carbonate afforded resin-bound
dehydropeptide 28.
Next, dehydropeptide 21 was released from 2-CTC resin by
treatment of resin-bound 28 with trifluoroacetic acid, which
concomitantly removed the N-terminal Boc group. RP-HPLC
purification of unprotected depsipeptide 21 was then followed by
a difficult macrocyclisation between the N-terminally methylated
esterification of pentapeptide 16 with Fmoc-N,O-Me Thr-
2
30
COOH (17) using DIC/DMAP afforded Fmoc-hexadepsipep-
α
tide 18 (Scheme 2). N -Fmoc-deprotection of depsipeptide 18,
however, led to complete α,β-elimination at the N-AcThr residue
forming the dehydrobutyrine-pentapeptide byproduct 19 and no
12,21
desired Fmoc-deprotected 20 (Scheme 2).
Having established that depsipeptide 18 was stable to acid but
unstable to base, the disconnection site was repositioned to the
N,O-Me Thr-β-Hyleu junction, forming linear precursor 21
2
(
Scheme 1, route b). This allows the use of Boc-N,O-Me Thr-
2
COOH (22) (see the Supporting Information) instead of Fmoc-
N,O-Me Thr residue and the C-terminal sterically hindered β-
2
N,O-Me Thr-COOH (17), as the N-terminal Boc group is
Hyleu residue. The key macrolactamisation was conducted
2
C
DOI: 10.1021/ol503507g
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
under high dilution solution phase conditions by slowly adding a
AUTHOR INFORMATION
Corresponding Author
■
*
mixture of linear precursor 21, HATU, and HOAt in CH Cl /
2
2
DMF (9:1) to a stirring solution of DIPEA in CH Cl . LC-MS
2
2
E-mail: m.brimble@auckland.ac.nz.
analysis of a crude aliquot after addition of the coupling mixture
indicated rapid consumption of the linear peptide and the
formation of two closely eluting products in a 1:2 ratio, both of
which exhibited the correct mass of the cyclic product. Prolonged
heating at 50 °C of an aqueous acetonitrile solution of these
products with monitoring by LC−MS indicated that the two
products were stable to interconversion. This suggested that the
cyclic products were likely to be epimers resulting from C-
terminal epimerization during macrocyclisation rather than cis−
trans rotational isomers at the N-methylated amide bonds.
However, our attempts to improve this ratio by modifying either
the base, coupling reagents or solvent were all unsuccessful.
Subsequent RP-HPLC purification of the reaction mixture
suggested the earlier eluting product to be YM-280193 (1), as its
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Maurice Wilkins Centre for Molecular
Biodiscovery for financial support.
■
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■
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4
(
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COOH (23) allowed us to incorporate these residues with
minimal protection during Fmoc-SPPS. Pleasingly, the N-
MeDha residue was installed selectively demonstrating the first
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Fmoc-SPPS. The powerful coupling reagent HATU was used for
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5
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■
*
S
Supporting Information
Experimental procedures and spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
Org. Biomol. Chem. 2010, 8, 1351.
D
DOI: 10.1021/ol503507g
Org. Lett. XXXX, XXX, XXX−XXX