Total synthesis and biological evaluation of grassypeptolide A

Article abstract of DOI:10.1002/chem.201203667

Herein, we describe in full our investigations into the synthesis of grassypeptolide A (1) in 17 linear steps with an overall yield of 11.3 %. In particular, this work features the late-stage introduction of sensitive bis(thiazoline) heterocycles and 31-membered macrocyclization conducted at the sterically congested secondary amide site in superb conversion (72 % yield). Biological evaluation indicated that grassypeptolide A significantly inhibited cancer cell proliferation in a dose-dependent manner. It induced cancer cell apoptosis, which was associated with increased cleavage of poly(ADP-ribose) polymerase (PARP) and decreased expression of bcl-2 and bcl-xL. Furthermore, grassypeptolide A also caused cell cycle redistribution by increasing cells in the G1 phase and decreasing cells in the S and G2 phases. In addition, cell cycle arrest was correlated with downregulation of cyclin D and upregulation of p27 and p21. Cytotoxic activity: Synthesis of grassypeptolide A, an anticancer marine cyclodepsipeptide, in 17 linear steps with an overall yield of 11.3 % is described (see figure). Subsequent biological evaluation indicated that grassypeptolide A significantly inhibited cancer-cell proliferation in a dose-dependent manner. Copyright

Full text of DOI:10.1002/chem.201203667

DOI: 10.1002/chem.201203667
Total Synthesis and Biological Evaluation of Grassypeptolide A
Hui Liu,[a] Yuqing Liu,[b] Zhuo Wang,[b] Xiangyou Xing,[a] Anita R. Maguire,[c]
Hendrik Luesch,[d] Hui Zhang,[a] Zhengshuang Xu,*[a, b] and Tao Ye*[a, b]
Abstract: Herein, we describe in full
our investigations into the synthesis of
grassypeptolide A (1) in 17 linear steps
with an overall yield of 11.3%. In par-
ticular, this work features the late-stage
introduction of sensitive bis(thiazoline)
heterocycles and 31-membered macro-
cyclization conducted at the sterically
congested secondary amide site in
superb conversion (72% yield). Biolog-
ical evaluation indicated that grassy-
peptolide significantly inhibited
cancer cell proliferation in a dose-de-
pendent manner. It induced cancer cell
apoptosis, which was associated with
increased cleavage of poly(ADP-
ribose) polymerase (PARP) and de-
creased expression of bcl-2 and bcl-xL.
Furthermore, grassypeptolide A also
caused cell cycle redistribution by in-
creasing cells in the G1 phase and de-
creasing cells in the S and G2 phases.
In addition, cell cycle arrest was corre-
lated with downregulation of cyclin D
and upregulation of p27 and p21.
A
Keywords:
cytotoxicity
cyclodepsipeptides
· grassypeptolide
·
·
thiaACTHNUGRTNEUNGzolines · total synthesis
Introduction
activity (lobocyclamides A–C[9]). Some of the metabolic sig-
natures of cyanobacterial peptides include a high degree of
N-methylated amino acids, d-amino acids, and b-amino
acids. We have been interested in marine secondary metabo-
lites, especially the cyclopeptides and cyclodepsipeptides,[10]
and view their syntheses as playing a key role in structural
confirmation, structural modification, and subsequent activi-
ty control. We recently reported the first convergent total
synthesis of grassypeptolide A.[10e] Herein, we describe our
initial strategy for the total synthesis of grassypeptolide A
and the discoveries that resulted from this approach. A de-
tailed description of the successful route to grassypeptoli-
de A and the establishment of a modality for its biological
evaluation are also provided.
Secondary metabolites produced by marine cyanobacteria
are key synthetic targets in the quest for new leads in the
pharmaceutical industry.[1] One particular species, Lyngbya
confervoides,[2] is responsible for the production of a consid-
erable number of bioactive peptide and depsipeptides.
These compounds occur in either linear or cyclic forms with
a variety of significant associated biological activities, in-
cluding cytotoxicity (obyanamide[3]), trypsin inhibition
(pompanopeptin A[4]), carboxypeptidase inhibition (pompa-
nopeptin B[4]), cathepsin E inhibition (grassystatins A–C[5]),
elastase inhibition (lyngbyastatins 5–7,[6] largamides A–C[7]),
chymotrypsin inhibition (lyngbyastatin 4[8]), and antifungal
Grassypeptolide A (1, formerly named grassypeptolide),
isolated from an extract of Lyngbya confervoides collected
off Grassy Key in Florida by the Luesch group, is a potential
anticancer cyclodepsipeptide.[11] It inhibited cancer cell
growth with IC50 values from 1.0 to 4.2 mm. The chemical
structure of 1 was established by using a combination of
chemical and spectral techniques, and subsequently validat-
ed through X-ray analysis. Grassypeptolide A is composed
of a number of unique, nonproteinogenic amino acid resi-
dues, such as the 2-methyl-3-aminobutyric acid (Maba), 2-
aminobutyric acid (Aba), and several D- and N-methylated
amino acids. Grassypeptolide A possesses a 31-membered
macrolactone with a bis(thiazoline)-ring moiety. Although
several bioactive cyclopeptides incorporating a single thiazo-
line have been reported,[12] the closest natural product relat-
ed to 1 is lissoclinamide 7 (2),[13] which also contains two
thiazoline rings (Figure 1). Unlike lissoclinamide 7, grassy-
peptolide A contains a free hydroxy group that is suscepti-
ble to cyclodehydration, leading to an oxazoline.
[a] Dr. H. Liu, Dr. X. Xing, Prof. Dr. H. Zhang, Prof. Dr. Z. Xu,
Prof. Dr. T. Ye
Laboratory of Chemical Genomics, School of Chemical Biology
and Biotechnology, Shenzhen Graduate School of Peking University
Shenzhen 518055 (P.R. China)
Fax : (+86)755-26032290
E-mail: xuzs@pkusz.edu.cn
bctaoye@inet.polyu.edu.hk
[b] Dr. Y. Liu, Z. Wang, Prof. Dr. Z. Xu, Prof. Dr. T. Ye
Department of Applied Biology and Chemical Technology
The Hong Kong Polytechnic University, Kowloon
Hong Kong (P.R. China)
E-mail: bctaoye@inet.polyu.edu.hk
[c] Prof. Dr. A. R. Maguire
Department of Chemistry and School of Pharmacy
Analytical and Biological Chemistry Research Facility
University College Cork, Cork (Ireland)
[d] Prof. Dr. H. Luesch
Department of Medicinal Chemistry, University of Florida
Gainesville, FL 32610 (USA)
Since the first total synthesis of grassypeptolide, reported
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203667.
by us in 2010,[10e] other members of the grassypeptolide
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FULL PAPER
group in 2011. Both 7 and 8 show moderate inhibitory activ-
ity against the transcription factor AP-1.
Results and Discussion
First-generation synthetic strategy for grassypeptolide A:
The principal synthetic challenges associated with the prepa-
ration of grassypeptolide A are the efficient formation of
the macrocyclic depsipeptide and selective assembly of the
delicate bis(thiazoline) heterocycles, in the presence of a
secondary hydroxy group. The
Figure 1. Structures of grassypeptolide A and lissoclinamide 7.
bis(thiazoline)
moiety
was
known to be highly sensitive
and prone to epimerization at
four of its stereogenic sites.[18]
In our retrosynthetic analysis,
we envisioned macrocyclization
and late-stage introduction of
the labile bis(thiazoline) ring
system (Scheme 1). Hence,
grassypeptolide A was envis-
aged to be derived from macro-
cycle 9 by selective cyclodehy-
dration of b-hydroxy thio-
Figure 2. Structures of grassypeptolide A and additional analogues.
Table 1. Substituent and stereochemistry of grassypeptolides.
AHCTUNGTRENGNaUN mides. The regioselectivity is
anticipated to stem from the
chemical reactivity (thioamide vs. amide) and steric effect,
favoring reaction of the primary over the secondary hydrox-
yl group. Macrocycle 9 can be derived from 10 by global de-
protection of the TBS protecting groups. Macrocycle 10
could be prepared either by macrolactonization of 11 or
macrolactamization of 12 (Scheme 1). Further disconnection
of 11 and 12 affords three subunits (13, 14, 15). Both pro-
posed strategies leading to the cyclization precursors (11,
12) would require both intermediates 13 and 15 as the key
coupling partners.
Grassypeptolides
R1
R2
R3
Stereochemistry
A (1)
B (3)
C (4)
D (5)
E (6)
F (7)
G (8)
iBu
iBu
iBu
iBu
iBu
Bn
Et
Me
Et
Et
Et
H
H
H
Me
Me
H
7R,11R,25R,28R
7R,11R,25R,28R
7R,11R,25R,28S
7R,11R,25S,28S
7S,11S,25S,28S
7R,11R,25R,28R
7R,11R,25R,28R
Et
Me
Bn
H
The synthesis of fragment 13 started from the condensa-
tion of N-Cbz-N-methyl-l-valine (16) with l-proline tert-
butyl ester (17) to give rise to dipeptide 18 in 84% yield
(Scheme 2). Hydrogenolytical removal of the Cbz protecting
group in 18 followed by coupling with hydroxy acid 19[19] by
using BOPCl[20] in the presence of triethylamine afforded
the corresponding tripeptide, which was deacetylated to fur-
nish 13 in 95% yield. Further selective acid-catalyzed hy-
drolysis of the tert-butyl ester group in 13 produced the cor-
responding free acid 21.
The 2-methyl-3-amino butyric acid (14) was prepared
from N-Cbz-d-alanine by a three-step sequence involving
the formation of a diazoketone[21] followed by a Wolff rear-
rangement[21,22] and a diastereoselective methylation.[10t,23]
To devise an efficient synthetic approach towards bis-
family were isolated (Figure 2 and Table 1). All of them are
31-membered cyclodepsipetides with a bis(thiazoline)-ring
moiety. The structures of grassypeptolides B (3) and C (4)[14]
are closely related to grassypeptolide A, with only minimal
differences. When the ethyl substituent of 1 is changed to a
methyl substituent in 3, activity is slightly reduced (3–4-
fold). Grassypeptolide C is the epimer of 1 at C28, with a l-
Phe unit flanking the bis(thiazoline) moiety. Unlike trunka-
mide[15] and lissoclinamide 7 (2),[13b] the Phe moiety in both
4 and 1 cannot undergo base-induced interconversion, which
may reflect less overall strain in the macrocycle of grassy-
peptolides.[14] Grassypeptolides D (5) and E (6),[16] which
have identical methyl-substituted thiazoline subunits, were
isolated from the marine cyanobacterium Leptolyngbya sp.
collected from the Red Sea by the McPhail group. Both
compounds were found to have significant cytotoxicity to
HeLa and mouse neuro-2a blastoma cells. Grassypeptoli-
des F (7) and G (8)[17] were isolated from an extract of the
Palauan cyanobacterium Lyngbya majuscula by the McKee
AHCTUNGTREG(NNNU thioamide) 15, we envisioned that 15 should be prepared
from the C- to the N-terminus of the peptide sequence
(Scheme 3). Literature precedent suggested that activation
of the carboxylic acid (15a) adjacent to the thioamide bond
would produce the corresponding thiazolinone (15c),[24]
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6775

Z. Xu and T. Ye et. al.
along with the epimerization of
the alpha stereogenic center
(Scheme 3). Bearing this analy-
sis in mind, we devised a retro-
synthetic strategy towards frag-
ment 15 as illustrated in
Scheme 4.
Thus, d-allo-threonine Tce
ester (23) was condensed with
N-Boc-N-methyl-d-leucine (22)
employing EDCI and HOBt as
the coupling reagent to furnish
dipeptide 24 in 79% yield. Pro-
tection of the secondary hy-
droxy group in 24 as its silyl
ether, followed by deprotection
of the Boc group with
TMSOTf[25] gave the corre-
sponding free amine, which was
then condensed with N-Boc-l-
Ser
G
employing
BOPCl as the coupling reagent
to give tripeptide 25 in 83%
yield. The Boc protecting group
in 25 was cleanly removed and
the resulting free amine was
coupled with the thioacylating
agent 26, prepared by using the
method described by Rapo-
port,[26] to afford the thioamide
27 in 72% yield. To continue
the linear chain elongation,
after Boc deprotection the re-
sulting free amine was con-
Scheme 1. Synthetic strategies to grassypeptolide A. Fmoc=fluorenylmethoxycarbonyl, TBS=tert-butyldime-
thylsilyl, Tce=trichloroethyl.
densed
(OTBS)-OH employing HATU
as the coupling reagent. Inter-
with
N-Boc-l-Ser-
AHCTUNGTRENNUNG
mediate 28 was then elaborated to the key fragment 15 in
82% yield by an identical strategy as described for 27, in-
cluding Boc deprotection and thioacylation with thioacylat-
ing agent 29 (Scheme 4). Further selective deprotection of
Boc afforded the secondary amine 30, which was ready for
further coupling reactions.
Scheme 2. Synthesis of 13: a) 17, EDCI, HOBt, Et3N, CH2Cl2, 08C to RT,
84%; b) 10% Pd/C, H2, MeOH/EtOAc; c) 19, BOPCl, Et3N, CH2Cl2,
08C to RT, 92% from 18; d) K2CO3, MeOH, 08C, 95%; e) H2SO4,
CH2Cl2, 66%. BOPCl=bis(2-oxo-3-oxazolidinyl)phosphinic chloride,
Cbz=carbobenzyloxy, EDCI=N’-(3-dimethylaminopropyl)-N-ethylcar-
bodiimide, HOBt=1-hydroxybenzotriazole.
Scheme 3. Analysis of the thioamide effect in peptide coupling processes.
Boc=tert-butoxycarbonyl.
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Total Synthesis and Biological Evaluation of Grassypeptolide A
FULL PAPER
To further explore the use of
key intermediate 31, we decid-
ed to install the ester moiety
prior to the macrolactamization
(Scheme 6). However, treat-
ment of 31 with acid chloride
36 (prepared from 14) under
various conditions yielded only
a trace amount of product 12.
This might be explained by as-
suming that the secondary hy-
droxy group of 31 formed a
strong complex with the pep-
tide backbone, which reduces
its reactivity towards the esteri-
fication.
Second-generation
synthetic
strategy for grassypeptolide A:
The failure to synthesize macro-
cycle 10 caused us to revise our
strategy towards the macrocyc-
lization. Conformational analy-
sis of grassypeptolide A in solu-
tion and solid state by NMR
spectroscopy and X-ray crystal-
lography suggested that all the
amide bonds were trans and the
Scheme 4. Synthesis of 15: a) 23, EDCI, HOBt, Et3N, CH2Cl2, 08C to RT, 79%; b) TBSCl, imidazole, CH2Cl2,
08C to RT, 85%; c) TMSOTf, NMM, CH2Cl2, RT; then Boc-l-Ser
to RT, 83% (2 steps); d) TMSOTf, 2,6-lutidine, CH2Cl2, RT; e) 26, THF, 08C, 72% from 25; f) TMSOTf, 2,6-
lutidine, CH2Cl2, RT; g) Boc-l-Ser(OTBS)-OH, HATU, DIPEA, CH2Cl2, 08C to RT, 60% from 27;
ACHTUGNTERN(NUGN OTBS)-OH, BOPCl, DIPEA, CH2Cl2, 08C
AHCTUNGTRENNUNG
h) TMSOTf, 2,6-lutidine, CH2Cl2, RT; i) 29, THF, 08C, 90% from 28; j) TMSOTf, 2,6-lutidine, CH2Cl2, RT,
73%. DIPEA=N,N-diisopropylethylamine, HATU=2-(1-H-7-azabenzotriazol)-1,1,3,3-methyluronium hexa-
fluorophosphate, NMM=N-methyl morpholine, TMSOTf=trimethylsilyl trifluoromethanesulfonate.
To our surprise, coupling of hydroxy acid 21 with the sec-
ondary amine 30 gave amide 31 in a low yield (44%), even
when using an excess amount of 21. Examination of this
coupling reaction unveiled that cyclodepsipeptide 32 was
the major side product (Scheme 5). Formation of medium-
sized rings has been considered as the inherent challenge.[27]
Because the proline and N-Me amino acid could adopt the
cis-amide configuration,[28] the hydroxy and carboxy termini
in 21 would then reside in close proximity to each other, fa-
cilitating an intramolecular cyclization. To remedy the low-
yield problem, we decided to employ acetyl-protected acid
33 as the coupling partner. Thus, treatment of 30 and 33
with BOPCl in the presence of diisopropylethylamine fur-
nished the corresponding peptide 34 in 78% yield. Reduc-
tive removal of the trichloroethyl ester under zinc-mediat-
ed[29] buffered conditions afforded the corresponding free
acid, which was then condensed with b-amino acid methyl
ester 35 (derived from 14) to give linear precursor 11 in
21% yield. It should be noted that both of the above reac-
tions were sluggish, probably due to the steric effect of the
adjacent TBS protecting group on the secondary hydroxy in
theronine. Saponification of both the acetyl and methyl
ester gave the corresponding seco-acid and set the stage for
macrolactonization. Unfortunately, all attempts at this point
to effect macrocyclization, by employing a variety of proto-
cols, including EDCI/DMAP, N,N-dicyclohexylcarbodiimide
(DCC)/DMAP/DMAP·HCl,[30] BOPCl,[20] and the Yamagu-
chi method[31] were unsuccessful.
molecule had hydrogen bonds between the NH of Maba
(C1–5) to the N of threonine (C6–9) and ester O of phenyl-
lactic acid (C48–56), and an additional hydrogen bond be-
tween the NH of threonine (C6–9) to the carbonyl of
Aba-thn-ca (C17–23).[11] We hypothesized that these afore-
mentioned hydrogen bonds might facilitate the preorganiza-
tion of a potential cyclization precursor that would in turn
facilitate the final macrocyclization. According to this ra-
tionale, it was decided to close the macrocycle via the pep-
tide bond between proline (C37–41) and N-Me-Phe-thn-ca
(C24–36), despite the known problem of slower acylation of
secondary amines. Further retrosynthetic analysis revealed
that the linear precursor 9 may be most conveniently con-
structed by the assembly of the two fragments 38 and 39
with approximately equal complexity (Scheme 7).
Coupling of acid 40 (prepared from 14) with the afore-
mentioned alcohol 13, promoted by various agents, including
EDCI/DMAP, DCC/DMAP, and the Mukaiyama reagent,[32]
failed to provide any useful quantities of product, presuma-
bly due to the steric bulk of the coupling partners. After ex-
tensive experimentation, we were delighted to find that the
acyl chloride, derived from acid 40, was superior in acceler-
ating this sluggish reaction, and the key amide 38 was isolat-
ed in 90% yield (Scheme 8).
With the key fragment 38 in hand, we next turned our at-
tention to the synthesis of thioamide 39 (Scheme 9). The
synthetic route is identical to the one employed previously
for intermediate 15 (Scheme 4). Thus, the Boc group in 41
was removed with TMSOTf in the presence of 2,6-lutidine,
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Z. Xu and T. Ye et. al.
the thioacylating agent 26 to
give rise to thioamide 43 in
88% yield.[33] After removal of
the Boc protecting group in 43,
the free amine was condensed
with
N-Boc-l-serineACHTUNGTRENNUNG(OTBS)-
OH by employing various cou-
pling reagents. Surprisingly, the
condensation resulted generally
in low yields when coupling re-
agents, such as EDCI/1-hy-
droxy-7-aza-benzotriazole
(HOAt), HATU, BOPCl, and
Mukaiyama reagent were em-
ployed.
Gratifyingly,
when
PyAOP[34] was employed as a
coupling agent, amide 44 was
obtained in 88% yield.[33] Inter-
mediate 44 was then elaborated
to the key fragment 39 in 82%
yield[33] by an identical strategy
as described for 43, including
Boc deprotection and thiolation
of the resulting free amine with
thioacylating agent 29.
To test the feasibility of the
selective construction of the
bis(thiazoline) ring moiety from
b-hydroxy thioamides 9 without
the interfering oxazoline forma-
tion, a model study was under-
taken by using segment 39 in
the projected cyclodehydration
(Scheme 10). Regioselectivity
should be readily achieved
when the alcohol moieties are
in drastically different electron-
ic or steric environments. This
led to a simple hypothesis: if
activation of two primary alco-
hols was truly occurring before
the activation of a secondary al-
cohol, then selective formation
of bis(thiazolines) in the pres-
ence of a secondary b-hydroxy
amide might be achievable. It
was with this hypothesis in
mind that substrate 45 was syn-
thesized. In the event, desilyla-
tion of 39 turned out to be
somewhat problematic due to
the sensitivity of the thioamide
functionalities and protecting
Scheme 5. Attempted macrolactonization of 11: a) BOPCl, Et3N, CH2Cl2, 30, 08C to RT, 44%; b) BOPCl,
DIPEA, CH2Cl2, 30, 78%; c) Zn, NH4OAc, THF; d) 35, HATU, DIPEA, 21%; e) LiOH, MeOH/THF; f) vari-
ous conditions for macrolactonization.
Scheme 6. Attempted synthesis of the macrolactamization precursor.
and the resulting free amine underwent a BOPCl-mediated
groups present in 39. After some experimentation, it was
found that the desired desilylated product 45 could be ob-
tained in high yield by treatment of 39 with TBAF buffered
with acetic acid. To our delight, treatment of 45 with the
coupling reaction with N-Cbz-l-serineACTHNUTRGNE(NUG OTBS)-OH to afford
tripeptide 42 in 89% yield.[33] Upon hydrogenolysis of the
Cbz group in 42, the resulting free amine was coupled with
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Total Synthesis and Biological Evaluation of Grassypeptolide A
FULL PAPER
mation of some unidentified by-
products might have been due
to the presence of triethylamine
derived from the decomposition
over time of the Burgess re-
agent.
Encouraged by the success of
the model study yielding the
bis(thiazoline) derivative 46,
which is closely related to the
grassypeptolide target molecule,
we turned our attention to the
required macrocycle 9 for the
natural product. Thus, hydroge-
nolysis of the Cbz protecting
group in 38 afforded the corre-
sponding amine and saponifica-
tion of the methyl ester of 39
gave the free acid, which under-
went a PyAOP-mediated cou-
Scheme 7. Retrosynthetic analysis of grassypeptolide A.
Scheme 8. Synthesis of fragment 38: a) 40, (COCl)2, cat. DMF, CH2Cl2,
08C to RT; then 13, cat. DMAP, NMM, CH2Cl2, 08C to RT, 90% from
13. DMAP=4-dimethylaminopyridine.
Scheme 10. Model studies on the selective formation of thiazolines:
a) TBAF, HOAc, THF, 08C to RT, 97%; b) Burgess reagent, THF, 60–
708C, 45%. Burgess reagent=methyl-N-(triethylammoniumsulfonyl)car-
bamate, TBAF=tetra-n-butylammonium fluoride.
Burgess reagent (inner salt)[35] at elevated temperature pro-
vided bis(thiazoline) 46 in 45% yield (Scheme 10). It was
found that reagent purity and carefully controlled reaction
times were critical to achieve optimal reaction yields, as for-
pling reaction to provide the linear precursor 37 in 81%
yield (Scheme 11). Simultaneous removal of the tert-butyl
ester[36] and Boc protecting group was achieved by treatment
of 37 with TMSOTf/2,6-lutidine
at room temperature to pro-
duce the desired amino acid,
which was immediately activat-
ed by BOPCl[20] in the presence
of 2,6-lutidine under high-dilu-
tion conditions (0.001m in
CH2Cl2) to afford cyclodepsi-
peptide 47 in 72% yield[37] and
without noticeable epimeriza-
tion evident by NMR spectro-
scopic analysis. The primary
TBS protecting groups in 47
were removed by using HOAc-
buffered TBAF to give the triol
Scheme 9. Synthesis of fragment 39: a) TMSOTf, 2,6-lutidine, CH2Cl2, RT; b) Cbz-l-Ser
DIPEA, CH2Cl2, 08C to RT, 89% from 41; c) Pd/C, H2, MeOH, RT; d) 26, THF, 08C, 88% from 42;
e) TMSOTf, 2,6-lutidine, CH2Cl2, RT; f) Boc-l-Ser(OTBS)-OH, PyAOP, DIPEA, CH2Cl2, 08C to RT, 88%
from 43; g) TMSOTf, 2,6-lutidine, CH2Cl2, RT; h) 29, THF, 08C; then acidic workup, 82% from 44. PyAOP=
(7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate)
ACHTUNGTREN(NUGN OTBS)-OH, BOPCl,
9. This set the stage for our en-
visioned selective cyclodehydra-
tion of b-hydroxy thioamides
that was expected to afford the
AHCTUNGTRENNUNG
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Z. Xu and T. Ye et. al.
ed as a Troc ester, and the ex-
isting two primary TBS protect-
ing groups were selectively re-
moved by using HOAc-buf-
fered TBAF to give b-hydroxy
thioamide 49. Activation of the
primary hydroxy groups of thio-
amide 49 with DAST[39] in
CH2Cl2 at À78 to À508C led to
the cyclized product 50, which
was then treated with activated
zinc and aqueous NH4OAc in
THF to afford the fully synthet-
ic grassypeptolide A (1), in
37% yield (Scheme 12). Cyclo-
dehydration of b-hydroxy thio-
AHCTUNGTERGaNNUN mide 49 with Burgess reagent
under the same conditions as
for 46 (Scheme 10) was also at-
tempted. In this case, only trace
amounts of the desired product
were observed by TLC analysis.
The spectral data for the syn-
thetic material (1H, 13C NMR
spectra, and HRMS) were iden-
tical to those published for the
natural product and the optical
rotation
[a]2D0 = +
(c=0.12 in
87 cm3 gÀ1 dmÀ1
CH2Cl2) was in close agreement
with values reported in the lit-
erature for natural grassypepto-
lide A, [a]2D0 = +76 cm3 gÀ1 dmÀ1
(c=0.1 in CH2Cl2), thus con-
firming the structure of the nat-
ural product. The synthetic
method presented in this full
Scheme 11. Synthesis of cyclodepsipeptides 47 and 9 and attempts to complete the total synthesis: a) 10% Pd/
C, H2, EtOAc, RT; b) LiOH (1n), tBuOH/THF, 08C; c) PyAOP, DIPEA, CH2Cl2, 08C to RT, 81% from 39;
d) TMSOTf, 2,6-lutidine, CH2Cl2, RT; e) BOPCl, 2,6-lutidine, CH2Cl2 (0.001m), 08C to RT, 72% from 37;
f) TBAF, HOAc, THF, 98%; g) Burgess reagent, THF, 60–708C, 20%.
bis(thiazoline) of grassypeptolide A. Unfortunately, expo-
sure of triol 9 to the same conditions as those shown in
Scheme 10 failed to produce grassypeptolide A; instead, the
major product isolated was the fully dehydrated product 48
along with trace amounts of monothiazoline-containing cy-
clodepsipeptide (Scheme 11). The lack of chemoselectivity
in the cyclodehydration of b-hydroxy amide/thioamides
might have been due to the increased conformational con-
straints of the macrocycle imposed by the formation of the
first thiazoline skeleton. Several reaction conditions using
DAST and various equivalents of reagents and reaction tem-
peratures also failed to give rise to grassypeptolide A.
Re-evaluation of our synthetic strategy was required. To
circumvent the difficulties associated with selective intro-
duction of the labile bis(thiazoline) ring moiety, we elected
to protect the secondary alcohol prior to cyclodehydration
of b-hydroxy thioamides by taking advantage of the known
propensity of the 2,2,2-trichloroethoxycarbonyl (Troc)[38]
protecting group to undergo cleavage under neutral condi-
tions. Thus, the secondary hydroxy group of 47 was protect-
paper gives the cyclodepsipeptide grassypeptolide A in 17
steps for the longest linear sequence, and an overall 11.3%
yield (average of 88% per step).
Biological evaluation of grassypeptolide A and synthetic an-
alogues: The anticancer effect of the synthetic grassypeptoli-
de A (1) and its analogues 9, 47, and 48 was evaluated by
cell proliferation in vitro by using cervical cancer cell line
HeLa and colon cancer cell line HT29. Grassypeptolide A
(1) significantly inhibited proliferation of HT29 and HeLa
cells in a dose-dependent manner. The IC50 values of HeLa
and of HT29 were 1.9 and 2.3 mm, respectively. Compound
48 also inhibited the growth of HeLa and HT29 with IC50
values of 9 and 3.6 mm, respectively (Figure 3). Compound 9
inhibited HeLa cell proliferation with an IC50 value at
23 mm, but it did not suppress the growth of HT29 cells at
concentrations below 30 mm. In addition, compound 47 was
not active towards the tested cell lines (Figure 3), which in-
dicated the importance of the bis(thiazoline) system, which
had been hypothesized to be responsible for the Zn2+ and
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Total Synthesis and Biological Evaluation of Grassypeptolide A
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Scheme 12. Completion of the total synthesis: a) TrocCl, Py, CH2Cl2,
08C; b) TBAF/HOAc, THF, 08C to RT; c) DAST, CH2Cl2, À78 to
À508C; d) Zn, NH4OAc (1 m), THF, 08C, 37% from 47. DAST=diethy-
laminosulfur trifluoride, Troc=2,2,2-trichlorolethoxycarbonyl, TrocCl=
trichloroethylchloroformate.
Cu2+ binding ability of grassypeptolides and to play a role
in the antiproliferative activity.[14]
Among other hallmarks, cancer is characterized by the
ability to evade apoptosis and aberrant cell cycle progres-
sion, leading to uncontrolled proliferation.[40] These acquired
capabilities of cancer cells are the target of standard anti-
cancer chemotherapy, and therefore we wanted to probe the
effects of grassypeptolide A on related biochemical parame-
ters and markers. For example, a desirable and standard
downstream mechanism of action includes forcing cancer
cells to undergo apoptosis. Furthermore, effective anticancer
agents also commonly induce phase-specific cell cycle arrest,
depending on the target and upstream mechanism. Antimi-
totic agents cause G2/M arrest, whereas agents interfering
with growth factor signaling or DNA synthesis usually
induce G1 or S arrest, respectively.
First, we validated the effect of grassypeptolide A (1) on
cell cycle progression. As shown in Figure 4A, at concentra-
tions near the IC50 1 triggered a significant increase in the
number of cells in the G0–G1 phase, with a corresponding
decrease in the number of cells in the S and G2/M phase,
consistent with previous observations for the natural prod-
uct.[14] To elucidate the molecular mechanisms of 1 induction
of cell cycle arrest in cancer cells, we measured the expres-
sion of cyclin D, which regulates the G1 to S transition, as
well as cyclin-dependent kinase inhibitors p27 and p21 that
suppress cell cycle progression in response to numerous
stimuli. We observed for the first time a strong induction of
p27 and p21 expression by the administration of 1 (Fig-
ure 4B). During tumor development, a lack of p21 and p27
expression was reported.[40] Therefore, up-regulation of p21
Figure 3. Effect of grassypeptolide A (1) on cell proliferation in A) HeLa
~
&
*
^
and B) HT29 cell lines ( : 48, : 1, : 9, : 47). Cells were cultured for
48 h in the presence of various concentrations of grassypeptolide A (1)
and its analogues. Proliferation was measured by MTS assay. Each point
represents the meanÆSE from four determinations.
and p27 by 1 may contribute to its antitumor effect. Overex-
pression of cyclin D1 is another common feature in malig-
nancy;[41] reduced cyclin D1 expression by 1 may be an im-
portant mechanism of its anticancer activity.
It was previously reported that at higher concentration
this compound induces G2/M arrest.[14] This concentration-
dependent effect on the cell cycle has precedence; for exam-
ple, class I histone deacetylase (HDAC) inhibitors with Zn2+
chelating ability show the same effect.[42] In both cases, the
IC50 correlates with the G1 arrest G1–S phase transition that
is positively regulated by complexes of cyclins and cyclin-de-
pendent kinases (CDKs) and negatively regulated by the en-
dogenous inhibitors, that is to say, cyclin-dependent kinase
inhibitors p21 and p27. CDKs themselves are promising an-
ticancer targets[43] and the success of HDAC inhibitors in
the clinic is partially attributed to cyclin D downregulation
and the potent induction of p21, which serves as a biomark-
er.[44]
We further examined by flow cytometry whether grassy-
peptolide A (1) exhibits apoptosis-inducing activity. Apop-
totic cells were defined as those stained with Annexin V but
not with propidium iodide (PI). As shown in Figure 5A,
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Z. Xu and T. Ye et. al.
Figure 4. A) Effect of grassypeptolide A (1) on cell cycle distribution. HT29 and HeLa cells were pretreated with 1 at incremental concentrations for
24 h, and then stained with PI, followed by flow cytometry analysis. Relative to the control untreated cells, grassypeptolide A led to accumulation of
cells in the G0–G1 phase and diminished cells in the S and G2M phases. b) The effect of grassypeptolide A treatment on cyclin D1, p27, and p21 protein
expression in HT29 and HeLa cells. Expression of the proteins in cells treated with 1 at different concentrations for 24 h was analyzed by western blot.
The results of a representative study are shown. Two additional experiments yielded similar results.
grassypeptolide A induced tumor cell apoptosis in a dose-
dependent manner. To gain insight into the molecular mech-
anisms in conjunction with 1 induction of apoptosis, we in-
vestigated the expression of the key regulators of apoptosis,
poly(ADP-ribose) polymerase (PARP), bcl-2, and bcl-xL, in
vitro. Our study provided the first data demonstrating that
the expression levels of both antiapoptotic bcl-2 and bcl-xL
significantly decreased after treatment with 1 (Figure 5B).
Both bcl-2 and bcl-xL have been regarded as potent thera-
peutic targets of cancer therapy based on their ability to dis-
rupt apoptosis and confer resistance to chemotherapy and
radiotherapy in cancer cells.[45,46] After grassypeptolide A
administration, we also detected increased cleavage of
PARP, a prominent marker of apoptosis. PARP cleavage is
effected by caspase 3 and could be a sign that cells should
undergo apoptosis because they were unable to repair the
cellular injury triggered by the apoptosis inducers.[47]
formation is noteworthy when viewed in the context of pre-
viously reported results for depsipeptide macrocyclization.
Pre-organization of the cyclization precursor by potential
hydrogen bonds resulting in a more favorable configuration
could be attributed to the success of this transformation.
The synthesis will provide a convenient access to a variety
of grassypeptolide derivatives. Synthesis of other members
of the grassypeptolide family and further structural modifi-
cations of grassypeptolides are currently in progress.
Experimental Section
Detailed experimental procedures have been moved into the Supporting
Information.
Acknowledgements
We acknowledge financial support from the Hong Kong Research Grants
Council (Projects: PolyU 5040/10P, PolyU 5037/11P, PolyU 5020/12P),
Hong Kong Polytechnic University (PolyU 5636/08M, PolyU 5634/09M),
Fong Shu Fook Tong Foundation, Joyce M. Kuok Foundation, National
Science Foundation of China (21072007, 21133002, 21272011), and Shenz-
hen Bureau of Science, Technology, Information (JC200903160367A,
JC201005260102A, JC201005260220A, ZYC201105170351A).
Conclusions
We have developed a convergent total synthesis of grassy-
peptolide A (1). Key to the success of our synthetic route
was the late-stage introduction of the sensitive tandem thia-
ACHTUNGTRENNUNGzoline heterocycles and 31-membered macrocyclization con-
ducted at the sterically congested secondary amide site in
superb conversion (72% yield). The efficiency of this trans-
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Total Synthesis and Biological Evaluation of Grassypeptolide A
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Received: October 14, 2012
Revised: February 18, 2013
Published online: March 27, 2013
6784
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