Total synthesis and biological activity of natural product Urukthapelstatin A

Article abstract of DOI:10.1021/ol401412v

Herein we report the first total synthesis of the natural product Urkuthaplestatin A (Ustat A) utilizing a convergent synthetic strategy. The characterization and biological activity match those of the previously published natural product. Interestingly,

Full text of DOI:10.1021/ol401412v

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Total Synthesis and Biological Activity
of Natural Product Urukthapelstatin A
Chun-Chieh Lin,‡ Worawan Tantisantisom,† and Shelli R. McAlpine*,†
School of Chemistry, University of New South Wales, Kensington, NSW 2052 Australia,
and Department of Chemistry and Biochemistry, 5500 Campanile Drive, San Diego State
University, San Diego, California 92182-1030, United States
s.mcalpine@unsw.edu.au
Received May 18, 2013
ABSTRACT
Herein we report the first total synthesis of the natural product Urkuthaplestatin A (Ustat A) utilizing a convergent synthetic strategy. The char-
acterization and biological activity match those of the previously published natural product. Interestingly, several intermediates, including the
linear and serine cyclized precursors, show a 100-fold decrease in cytotoxicity, with IC50’s in the low micromolar range. These data indicate that
the rigidity and the consecutive aromatic heterocyclic system are responsible for the biological activity.
Reports on oxazole and thiazole containing natural
products have stimulated interest in this class of molecules
for decades.1 A large number of these natural products
come from marine creatures, and they possess promis-
ing biological activity,2 with many becoming drug candi-
dates.3 A series of biologically relevant compounds that
contain a mixture of oxazole and thiazoles within their
backbone include merchercharmycin A (IB-01211), ascidia-
cyclamide, patellamide D, and telomestatin (Figure 1). These
four compounds have all shown significant anticancer
activity. Merchercharmycin A shows an average IC50 =
43 nM4 against multiple cancer cell lines, while ascidiacy-
clamide has an average of 29 μM.5,6 Patellamide D is
clinically used in combination with vinblastine,7 and telo-
mestatin is currently in clinical trials (average GI50
=
5 nM).8,9 These four molecules resemble the structure of
a novel natural product, urukthapelstatin A (Ustat A),
which contains a sequential series of heterocycles. Impor-
tantly, Ustat A has an IC50 value of 15.5 nM10 (average
over 39cancercells). Despite its highly cytotoxic properties,
and the similarity to current biologically active drugs,
‡ San Diego State University.
† University of New South Wales.
(4) Romero, P.; Malet, L.; Canedo, L. M.; Cuevas, C.; Fernando
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r
10.1021/ol401412v
XXXX American Chemical Society

Figure 2. Retrosynthetic strategy for Ustat A.
Figure 1. Ustat A and related structures: merchercharmycin A
(IB-01211)1 ascidiacyclamide,4 patellamide D, and telomestatin.12
Formation of Ustat A (1) involved intramolecular cycli-
zation of cyclic precursor 2 and subsequent oxidation of
the oxazoline intermediate. Structure 2 was formed via
cyclization of the linear precursor 3 between the serine
N-terminus and the free acid on the oxazole moiety.
Compound 3 was synthesized by coupling Fragment
I (4) and Fragment II (5). Fragment I was obtained by
condensing the thioamide 6 and the bromoketone oxazole
7 via the Hantzsch thiazole synthesis, followed by an acid
deprotection. The thiazole in 6 was also generated by the
Hantzsch thiazole synthesis, and the phenyloxazole of
7 was obtained from the cyclization and oxidation of a
β-hydroxyl Phe residue. Fragment II was derived from
peptide coupling of the oxazole 8 to Ala, followed by the
installation of the enamide moiety. Subsequent N-terminal
extension of the oxazole-enamide pseudopeptide by pep-
tide coupling to D-allo-Ile delivered Fragment II.
Synthesis of Fragment I (Scheme 1) started with protec-
tion of serine as a dimethyloxazolidine and was accom-
plished using 2,2-dimethoxypropane (DMP) and pyridinium
p-toluenesulfonate (PPTS) in THF. The free acid of Ser was
then converted to a methyl ester using trimethylsilyl diazo-
methane (TMSD) in a mixture of methanol and benzene,
affording the ester 9. Amide conversion of 9 was completed
using ammonium hydroxide and methanol. The amide was
subjected to Lawesson’s reagent to furnish the thioamide 10.
Next, modified Hantzsch thiazole conditions were applied
to the thioamide 10 and ethyl bromopyruvate. Potassium
bicarbonate (KHCO3) was used in the first step to generate a
hydroxyl thiazoline intermediate, which was subsequently
dehydrated using trifluoroacetic anhydride (TFAA) and
mechanistic studies have not been successful in identifying
how Ustat A functions within the cell. Further, the synthe-
tically challenging aspects of the molecule have made
extensive mechanistic studies unfeasible. Indeed, several
mechanisms have been investigated for Ustat A, but to date
there has been no success in identifying its biological
pathway.9 Thus, this interesting molecule is an ideal syn-
thetic target and an outstanding medicinal chemistry lead.
Herein, we report the first total synthesis of Ustat A
using a convergent synthetic strategy (Figure 2). This
report details the only successful route for synthesizing
this class of compounds. We have published several other
attempts at forming this macrocycle13 including a Hantzsch
cyclization ring closure, cyclization between the threonine
and alanine, and solid phase synthesis whereupon the
oxazoles and thiazoles were installed after macrocyclization.
None of these approaches were successful. We believe the
downfall of these strategies was the attempt to cyclize a rigid
structure that was unable to connect the two ends with all
heterocycles intact. Thus, this strategy maintained a single
open oxazole as a serine until macrocyclization was com-
plete (2, Figure 2).
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S. R. Tetrahedron Lett. 2012, 53, 4065–4069. (b) Kim, S. J.; McAlpine,
S. R. Molecules 2013, 18, 1111–1121.
B
Org. Lett., Vol. XX, No. XX, XXXX

pyridine, generating the desired thiazole 11. Conversion of
the ethyl ester into a thioamide using ammonium hydro-
xide and Lawesson’s reagent resulted in the formation of
thioamide 6. The bromoketone oxazole 7 was prepared
using previously reported conditions.13a15a Reacting the
thioamide 6 with the oxazole 7 under the presence of
KHCO3 gave a thiazoline intermediate, whereupon sub-
sequent dehydration was performed to give triazole 12 in
an excellent yield. Finally, methyl ester hydrolysis of 12
was accomplished using lithium hydroxide (LiOH) in
methanol and furnished the desired Fragment I (4).
Scheme 2. Synthesis of Fragment II
Scheme 1. Synthesis of Fragment I
The macrocyclization of Ustat A is summarized in
Scheme 3. Peptide coupling of the free acid Fragment I
(4) and the free amine Fragment II (5) was carried out with
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), DMTMM, bromo-tris-
pyrrolidino-phosphonium hexafluorophosphate (PyBroP),
and DIPEA to furnish the protected linear precursor 3.
Subsequently acid and base deprotection prepared the
linear precursor 17 ready for cyclization. Macrocycliza-
tion of 17 was performed using HATU, DMTMM, 2,4,6-
tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide
(T3P), and DIPEA in a dilute condition to afford the cyclic
precursor 2.
The final oxazole formation began with fluorination of
the free serine side chain on 2 (Scheme 4) using diethyl-
aminosulfur trifluoride (DAST), followed by base-induced
cyclodehydration to yield an oxazoline intermediate.
Oxazoline oxidation with 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) and bromotrichloromethane furnished
Ustat A as an inseparable mixture of 2:1 Z/E isomers.15
During the oxazoline formation step using DAST, HF is
formed. We believe that Z/E isomerization of the enamide
Fragment II (5) (Scheme 2) was generated by starting
with oxazole 8, which we have previously reported.13a15a
Simultaneous alcohol/amine deprotection of 8 using 50%
trifluoroacetic acid (TFA) in dichloromethane generated
H-Thr-oxazole-OMe (13). Coupling Boc-Ala-OH to
the free amine 13 with 4-(4,6-dimethoxy-1,3, 5-triazin-
2-yl)-4-methylmorpholinium chloride (DMTMM) and
N,N-diisopropylethylamine (DIPEA) produced the pseu-
dopeptide 14 with a free Thr residue. Methanesulfonyl
chloride (MsCl) was used to convert the free alcohol of
14 into a leaving group, whereupon E2 elimination using
triethylamine (TEA) installed only the desired Z-enamide
(as determined by NOESY)14 moiety of 15. The Boc
protecting group of 15 was removed using 25% TFA in
dichloromethane; the resulting amine was coupled to Boc-
D-allo-Ile-OH using DMTMM and DIPEA in dichloro-
methane to yield the pseudopeptide 16. Fragment II (5)
was then generated utilizing 4 M hydrochloric acid solu-
tion in dioxane to remove the Boc group of 16.
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(14) See Supporting Information for synthetic details
Org. Lett., Vol. XX, No. XX, XXXX
C

assays against the HCT-116 colon cancer cell line. GI50
(growth inhibition value for inhibiting 50% of cell growth)
of compound 12 was 8 μM, a surprising result given that both
16 and 3showed no biological activity (Table 1). These results
suggest that the potency comes from the upper hemisphere of
the molecule. However, as shown in numerous reports,15
conformation of the macrocycle plays a key role in biological
activity, where 2 is not as active as the small fragment 12.
Finally, the natural product, 1, demonstrated a GI50 similar
to published reports in the low nanomolar range.4
Scheme 3. Synthesis of Linear and Cyclic Precursors
Table 1. Cytotoxicity Values of Compounds against HCT-116
Colon Cancer Cell Line
compd
%GIa
12
16
3
2
1
99
10
N/A
0
100
100
b
IC50
8 ( 0.7
μM
N/A
30 ( 2.0
μM
20 ( 1.7
nM
a % growth inhibition at 40 μM. b IC50 values were only determined
for compounds that showed greater than 50% growth inhibition at
40 μM. The experiments were run in triplicates with each data point
performed in quadruplicates. Each IC50 value is an average value of
three independent cytotoxicity assays with the standard deviations
representing errors.
Insummary, wehavedescribedthefirst total synthesisof
the natural product Urukthapelstatin A (1) and verified
the structure thatwas proposedby Matsuo etal.10 Wehave
also identified the (E)-enamide analog of Ustat A. It
appears that the configuration of the enamide has little
impact on the biological activity as the GI50 of the mixture
matches that of the published natural product. These data
suggest that the upper hemisphere of the molecule is re-
sponsible for the biological activity of Ustat A. Mechanistic
studies of Ustat A, analogs, and fragments used to build this
natural product are currently underway in our lab.
Scheme 4. Synthesis of Urukthapelstatin A
Acknowledgment. We thank the University of New
South Wales for the support of S.R.M. and C.C.L. We
thank Dr. Macro Ciufolini (UBC) and Dr. David Lefranc
(UBC) for their helpful discussions on this project. We also
thank Dr. Donald Thomas at the NMR facility UNSW for
his help in collecting NMR data.
was induced at this step, as compound 2 was the Z-enamide,
while NMR confirmed that the final product after
oxazole formation generated the natural product 1 with
the Z-enamide as well as the E-enamide.
Supporting Information Available. General experimen-
tal procedures, NMR and mass spectral data for com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Compounds 12, 16, 3, 2, and 1 (Fragments I, II, linear,
cyclized precursors, and Ustat A) were tested in cytotoxicity
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX