Progress toward the synthesis of Urukthapelstatin A and two analogues

Article abstract of DOI:10.1016/j.tetlet.2012.05.105

We report our progress toward the synthesis of Urukthapelstatin A (Ustat A) and two analogues. Our retrosynthetic strategy involved the synthesis of three fragments: a tri-heteroaromatic moiety, a phenyl oxazole fragment, and a dipeptide. Described are the syntheses of three unique tri-heteroaromatic moieties. In addition, the corresponding linear precursors of Ustat A and two analogues are presented.

Full text of DOI:10.1016/j.tetlet.2012.05.105

Tetrahedron Letters 53 (2012) 4065–4069
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Progress toward the synthesis of Urukthapelstatin A and two analogues
Chung-Mao Pan b, Chun-Chieh Lin b, Seong Jong Kim a, Robert P. Sellers b, Shelli R. McAlpine a,
⇑
a School of Chemistry, University of New South Wales, Sydney, NSW 2052 Australia
b Department of Chemistry and Biochemistry, 5500 Campanile Dr., San Diego State University, San Diego, CA 92182-1030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report our progress toward the synthesis of Urukthapelstatin A (Ustat A) and two analogues. Our
retrosynthetic strategy involved the synthesis of three fragments: a tri-heteroaromatic moiety, a phenyl
oxazole fragment, and a dipeptide. Described are the syntheses of three unique tri-heteroaromatic moi-
eties. In addition, the corresponding linear precursors of Ustat A and two analogues are presented.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 8 February 2012
Revised 14 May 2012
Accepted 23 May 2012
Available online 29 May 2012
Keywords:
Urukthapelstatin A
Heterocycles
Thiazole
Oxazole
Hantzsch
Macrocycle
Natural product
Cytotoxicity
Natural products provide original pharmacophores, offering
new scaffolds with novel mechanisms of action. Urukthapelstatin
A (Ustat A) is one such natural product (Fig. 1), with cytotoxicity
against cancer cells in the desirable low nanomolar range (average
GI50 = 15.5 nM).1 It does not share the structural homology with
other classes of marketed cancer therapeutics, and it has a distinct
activity profile compared to structurally similar compounds, indi-
cating that it likely has a unique mechanism of action.1 Given that
O
S
S
Br
O
Proposed
Cyclization
Z
NH2
Z
X
N
N
N
N
O
O
N
N
Y
MeO
N
Y
Fragment 2
O
HN
N
N
X
H2N
NH HN
O
NHBoc
Ot-Bu
O
MeO
HN
O
Peptide residues
O
Attempted
Cyclization
Fragment 1
Fragment 3
10: X=O, Y=O, Z=S
13: X=O, Y=O, Z=O
20: X=S, Y=S, Z=S
Ustat A (Compound 1): X=O, Y=O, Z=S
Compound 2: X=O, Y=O, Z=O
Compound 3: X=S, Y=S, Z=S
Figure 1. Retrosynthetic approach for Ustat A and analogues.
⇑
Corresponding author.
E-mail address: s.mcalpine@unsw.edu.au (S.R. McAlpine).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.105

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C.-M. Pan et al. / Tetrahedron Letters 53 (2012) 4065–4069
there are considerable on-going efforts to expand the arsenal of
drugs that target cancer, the search for new compounds that inhibit
novel cell-survival pathways is of paramount importance to the
evolution of cancer medicine. Herein, we report our synthetic strat-
egy aimed at completing the first synthesis of the natural product
Ustat A and two analogues (Fig. 1). Our efficient, modular approach
O
O
O
OMe
N
1. H2, Pd/C, EtOH
BnO
OMe
BnO
O
O
2. a) DAST, CH2Cl2, -78 o
C
O
NH2
NH
OH
NHBoc
t-BuO
O
b) K2CO3, -78 oC to rt
3. DBU, BrCCl3, CH2Cl2,
-20 oC to rt, 85%, 3 steps
TBTU, DIPEA,
CH2Cl2, 90%
NHBoc
NHBoc
Ot-Bu
Ot-Bu
4
5
O
1. LiOH,
MeOH
2. TBTU
O
1. H2, Pd/C, EtOH
OMe
OMe 2. a) DAST, CH2Cl2, -78 o
b) K2CO3 -78 oC to rt
NH
C
1. NH4OH, MeOH,
ultrasound rt
BnO
O
O
N
N
O
BnO
OMe
N
3. DBU, BrCCl3,
CH2Cl2, -20 oC to rt,
2. Lawesson's reagent,
NH2
O
O
DME, 60 o
C
NHBoc
NHBoc
85%, 3 steps
77%, 2 steps
DIPEA,
CH2Cl2,
Ot-Bu
Ot-Bu
95%, 2 steps
7
6
S
O
S
1. KHCO3, DME
O
NH2
NH2
OEt
S
S
N
N
1. NH4OH, MeOH
ultrasound, rt
O
N
N
Br
OEt
O
N
O
N
O
2. Lawesson's reagent,
DME, 60 oC,
70%, 2 steps
O
2. TFAA, pyridine,
DME, 0 oC, 3 h
then Et3N, rt, 3 h
77%, 2 steps
N
NHBoc
Ot-Bu
N
NHBoc
Ot-Bu
O
NHBoc
Ot-Bu
O
8
Fragment 1
10
9
Scheme 1. Synthetic approach to Ustat A, fragment 1.
OBn
O
O
1. H2, Pd/C, EtOH
2. a) DAST,
CH2Cl2, -78 oC
OMe
OMe
1. LiOH, MeOH
2. TBTU,
O
NH
O
N
O
N
O
b) K2CO3, -78 oC to rt
3. DBU, BrCCl3,
CH2Cl2, -46 oC to rt,
70%, 3 steps
N
O
BnO
OMe
O
N
NHBoc
NH2
DIPEA,
CH2Cl2,
NHBoc
Ot-Bu
Ot-Bu
92%, 2 steps
7
11
O
S
OMe
NH2
N
O
O
O
1. NH4OH, MeOH,
ultrasound, rt
N
N
N
O
O
2. Lawesson's reagent,
DME, 60 oC,
55%, two steps
N
N
NHBoc
Ot-Bu
NHBoc
Ot-Bu
O
Fragment 1
13
12
Scheme 2. Synthetic approach to compound 2, fragment 1.

C.-M. Pan et al. / Tetrahedron Letters 53 (2012) 4065–4069
4067
O
1. TMSD,
benzene, MeOH, rt
O
OEt
N
Br
OEt
O
OH
S
NH2
O
NHBoc
NHBoc
Ot-Bu
S
1. KHCO3, DME
2. TFAA, pyridine,
DME, 0 oC
then Et3N, rt, 3 h
78%, 2 steps
2. NH4OH, MeOH,
ultrasound, rt
3. Lawesson's reagent,
THF, reflux
NHBoc
Ot-Bu
Ot-Bu
15
14
60%, 3 steps
O
O
OEt
Br
OEt
1. NH4OH, MeOH,
ultrasound, rt
1. NH4OH, MeOH,
ultrasound, rt
S
NH2
N
O
N
S
1. KHCO3, DME
S
2. Lawesson's reagent
DME, 60 oC,
53%, 2 steps
2. Lawesson's reagent,
benzene, 60 oC,
73%, 2 steps
N
NHBoc
2. TFAA, pyridine, DME, 0 oC
then Et3N, rt, 3 h
3. NaOEt, EtOH,
S
NHBoc
Ot-Bu
16
Ot-Bu
17
97%, 3 steps
S
O
S
O
NH2
OEt
S
NH2
Br
S
N
1. NH4OH, MeOH,
ultrasound, rt
OEt
N
N
N
S
O
N
S
N
S
1. KHCO3, DME
2. Lawesson's reagent,
benzene, reflux
73%, 2 steps
2. TFAA, pyridine, DME, 0 oC
then Et3N, rt, 3 h
3. NaOEt, EtOH,
N
S
N
NHBoc
NHBoc
Ot-Bu
S
S
NHBoc
Ot-Bu
Ot-Bu
18
87%, 3 steps
19
Fragment 1
20
Scheme 3. Synthetic approach to compound 3, fragment 1.
Br
MeO OMe
Br
OH
TBTU,
DIPEA,
CH2Cl2
OMe
1. a) DAST, CH2Cl2, -78 oC
b) pyridine, -78 oC to rt
MeO
Br
OMe
O
OMe
O
O
MeO
NH
OH
+
NH2
N
92%
2. DBU, BrCCl3,
CH2Cl2, -46 oC to rt.
45%, 2 steps
O
O
MeO
OH
MeO
O
22
21
O
Br
formic acid,
50 oC
O
N
O
quantitative yield
MeO
Fragment 2
23
H2N
HN
TFA, anisole, rt,
quantitative yield
BocHN
HN
TBTU, DIPEA,
CH2Cl2, 99%
NH2
NHBoc
MeO
HO
O
+
O
MeO
MeO
O
O
O
O
Fragment 3
25
24
Scheme 4. Synthetic approach to fragments 2 and 3.
is the first report that targets this unique pharmacophore, or that
outlines a method for analogue development. Our approach is a
flexible method intended for building Ustat A derivatives, thereby
providing access to a series of unique molecular scaffolds.
Several natural product peptidomimetic structures that contain
oxazoles and thiazoles have been isolated and synthesized.2–8
Among these natural products, telomestatin is the only natural
product whose mechanism of action has been thoroughly studied.

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C.-M. Pan et al. / Tetrahedron Letters 53 (2012) 4065–4069
Although Ustat A is structurally similar to telomestatin, it does not
appear to target telomerase (using the traditional TRAP assay),1
and thus its highly cytotoxic potency is associated with an as yet
unknown mechanism of action. The isolated natural product was
also tested in numerous anticancer assays, which revealed that it
is also unlikely to be an HDAC, farnesyl transferase, or proteasome
inhibitor.1 The activity profile of Ustat A using COMPARE analysis
resembles that of the known anticancer agent cytarabine, which
is an antimetabolite.1 Indeed, the COMPARE profile suggests that
this molecule acts via a unique mechanism, indicating the value
of further investigation and analogue synthesis.
Herein we outline an efficient synthetic strategy for three frag-
ments. Using a Hantzsch thiazole synthesis to couple fragments 1
and 2, followed by a peptide coupling of fragment 3 completes
the linear precursor. Although cyclization conditions are currently
on-going, we anticipate completing the macrocycle by coupling
between alanine and threonine. Subsequent elimination to the Z-
enamine9 would give the final product. In this Letter, we focus
on developing linear precursors with modified heterocyclic com-
ponents. Oxazoles form stronger hydrogen bonds than thiazoles,
but are significantly more rigid when placed in a macrocyclic con-
formation.10 Thus, syntheses of the natural product and two ana-
logues were designed to investigate how modification of the
heterocycles would alter: (a) the ability of these analogues to
hydrogen bond and (b) the ability of the rings to close. The
synthesis of fragment 1 differed for each of the three macrocycles,
with Schemes 1–3 describing the synthetic approaches for com-
pounds 1, 2, and 3, respectively. However, the synthesis of
fragments 2 and 3 were the same for all three linear precursors.
Utilizing a peptide approach to generate the heterocycles pro-
vided the fragments in the fewest number of steps (vs a cross-cou-
pling approach-theoretical calculation) and gave high overall
S
1. KHCO3, DME
NH2
O
Z
S
O
N
O
N
N
Br
O
Z
MeO
N
N
N
N
Y
O
N
MeO
Fragment 2 (23)
N
Y
NHBoc
OtBu
X
NHBoc
OtBu
2. TFAA, pyridine,
DME, 0 oC, 3 h
then Et3N, rt, 3 h,
69-88% 2 steps
X
Fragment 1
10: X=O, Y=O, Z=S
13: X=O, Y=O, Z=O
20: X=S, Y=S, Z=S
26a: X=O, Y=O, Z=S
26b: X=O, Y=O, Z=O
26c: X=S, Y=S, Z=S
O
N
S
N
1. LiOH, MeOH,
quantitative yield
2. TBTU, DIPEA,
O
Z
N
HN
HN
1. LiOH, MeOH,
quantitative yield
O
H2N
N
Y
2. TFA, CH2Cl2,
anisole,
quantitative yield
MeO
HN
O
N
MeO
O
NHBoc
X
O
Fragment 3 (25)
CH2Cl2, 47-65%,
2 steps
OtBu
27a: X=O, Y=O, Z=S
27b: X=O, Y=O, Z=O
27c: X=S, Y=S, Z=S
O
N
S
N
O
Z
N
HN
HN
O
N
Y
HO
N
O
NH2
X
OH
28a: X=O, Y=O, Z=S
28b: X=O, Y=O, Z=O
28c: X=S, Y=S, Z=S
Scheme 5. Synthetic approach to the linear precursors.

C.-M. Pan et al. / Tetrahedron Letters 53 (2012) 4065–4069
4069
yields. The synthesis of fragment 1 for compound 1 began with the
construction of the dipeptide 4 (Scheme 1). This dipeptide was
generated by coupling free amine H2N-Ser(Bn)-OMe and free acid
Boc-Thr(OtBu)-OH using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate (TBTU) and diisopropylethyl-
amine (DIPEA) in anhydrous methylene chloride (CH2Cl2)
yielding 4 in 90% yield. Removal of the benzyl ether protecting
group was accomplished via hydrogenolysis using Pd/C (10%) as
catalyst. A two-step procedure involving an intramolecular cycliza-
tion using the fluorinating agent diethylaminosulfur trifluoride
(DAST) and K2CO3 to yield the intermediate oxazoline, which was
oxidized using bromochloroform (BrCCl3) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU), generated 5 in 85% overall yield over
three steps. Hydrolysis of the ester 5 using LiOH and subsequent
coupling between the free acid and free amine H2N-Ser(Bn)-OMe
was performed using TBTU and DIPEA. This yielded compound 6
in 95% yield. Hydrogenolysis of 6 and subsequent conversion of
the free serine into an oxazole using DAST/BrCCl3 resulted in 7
(85% overall yield over three steps). The formation of thioamide
8 was carried out using ammonium hydroxide in methanol fol-
lowed by conversion of the resulting amide into the thioamide
using Lawesson’s reagent (77% yield over two steps). A base-
induced Hantzsch thiazole synthesis11 was performed using an
excess of ethyl bromopyruvate and KHCO3 to afford the intermedi-
ate thiazoline, which was dehydrated using trifluoroacetic anhy-
dride (TFAA) and pyridine to give compound 9 (77% yield over
two steps). Compound 10 was generated using ammonium
hydroxide in methanol followed by treatment with Lawesson’s re-
agent (70% yield over two steps).
induced by DAST and then pyridine in CH2Cl2 to afford the inter-
mediate phenyloxazoline. This intermediate was then advanced
to phenyloxazole through oxidation using BrCCl3 and DBU (45%
overall yield 2 steps). Deprotection of ketone was achieved using
formic acid induced hydrolysis at 50 °C for 20 min yielding 23
(quantitative yield). Fragment 3 (25), was generated via a peptide
coupling reaction between free acid Boc-d-allo-Ile-OH and free
amine H2N-Ala-OMe using TBTU and DIPEA (99% yield). The dipep-
tide was then amine deprotected using TFA, providing 25 (quanti-
tative yield).
With fragments 1 and 2 in hand, we performed the Hantzsch
thiazole synthesis (Scheme 5) to generate the heterocyclic hemi-
sphere of Ustat A and analogues (overall yield for two steps 26a,
b, and c = 69%, 72%, and 88%, respectively). Upon gaining access
to penta-heterocycle fragments 26a–c, we performed an acid
deprotection and coupled the resulting free acids to fragment 3
using TBTU and DIPEA in CH2Cl2, thereby generating 27a–c (61%,
65%, and 47% yields, respectively). The ester group in compounds
27a–c was deprotected using LiOH, and the Boc and tert-butyl
groups were removed using TFA, generating compounds 28a–c.
NMR spectroscopic studies and high resolution mass spectroscopy
confirmed the structures of the three linear precursors 28a–c.
In summary, we have outlined an efficient and viable syn-
thetic approach to three different rigid penta-heterocycles. We
then converted these into the linear precursors in preparation
for cyclization into Urukthapelstatin A (1) and two analogues 2
and 3. We are currently investigating cyclization strategies,12
which will be reported in due course. These linear analogues will
be tested for their ability to inhibit colon and pancreatic cancer
cell growth.13,14
The synthesis of 13 en route to the Ustat A analogue utilized
dioxazole 7, which was converted into the free acid using LiOH
in methanol (Scheme 2). Subsequent peptide bond formation be-
tween the free acid Boc-Thr(OtBu)-dioxazole-OH and the free
amine H2N-Ser(Bn)-OMe using TBTU and DIPEA in anhydrous
CH2Cl2 gave 11 (92% yield, 2 steps). The benzyl ether of 11 was re-
moved via hydrogenolysis followed by the formation of the third
oxazole moiety, 12, using DAST/BrCCl3 in 70% yield over the three
steps. Conversion of the ester into a thioamide to generate 13 was
achieved using ammonium hydroxide followed by sulfur installa-
tion using Lawesson’s reagent (55% yield, 2 steps).
Acknowledgements
We thank the University of New South Wales for the support of
S.R.M. and S.J.K., the Frasch foundation (658-HF07) for support for
C.M.P. and C.C.L. We thank Dr. Lafferty for his assistance in NMR
experiments.
Supplementary data
The construction of Ustat A analogue 20 is summarized in
Scheme 3. Starting with protection of the free acid Boc-Thr(OtBu)-
OH using trimethylsilyl diazomethane (TMSD) in methanol and
then conversion into the thioamide via ammonium hydroxide and
Lawesson’s reagent resulted in the formation of 14 (60% yield over
three steps). The Hantzsch thiazole synthesis was then performed
reacting 14, ethyl bromopyruvate, and KHCO3 to yield the interme-
diate thiazoline, whereupon subsequent dehydration was facili-
tated using TFAA and pyridine at 0 °C to give 15 (78% over two
steps). Generation of 16 was accomplished using a two-step proce-
dure involving ammonium hydroxide and Lawesson’s reagent (53%
yield, two steps). The next two thiazole moieties were installed by
repeating the Hantzsch thiazole synthesis process, whereby the thi-
oamide was reacted with ethyl bromopyruvate and KHCO3. Dehy-
dration using TFAA and pyridine and then treatment with NaOEt
in EtOH gave 17 (97% yield over three steps). This approach was re-
peated on 17 to generate thioamide 18 (73% yield over two steps),
and then tri-thiazole 19 (87% yield over three steps). Conversion
of 19 into a thioamide using ammonium hydroxide and Lawesson’s
reagent provided 20 (73% yield over two steps).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.05.
105.
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Fragments 2 (23) and 3 (25) were synthesized next (Scheme 4).
Phenylserine 21 was generated by coupling 3-bromo-2,2-dimeth-
oxypyruvic acid with (2R,3S)/(2S,3R)-racemic H2N-b-hydroxy-
Phe-OMe using TBTU and DIPEA in anhydrous CH2Cl2 (92% yield).
Racemic 21 was then subjected to intramolecular cyclization