Synthesis of heptaoxazole macrocyclic analogues of telomestatin and evaluation of their telomerase inhibitory activities

Article abstract of DOI:10.1246/bcsj.20130198

We have demonstrated various synthetic routes to heptaoxazole macrocyclic analogues of telomestatin and evaluated their inhibitory activities against telomerase. We synthesized three heptaoxazole macrocycles consisting of different numbers of methyloxazole moieties and another heptaoxazole analogue with a bromooxazole moiety instead of one of the two methyloxazole moieties found in the structure of telomestatin. The bromooxazole analogue underwent SuzukiMiyaura coupling leading to six analogues having aromatic substituents on the oxazole moiety. The substituents on the oxazole moiety in the heptaoxazole macrocycles, which include a methyl group, a bromine atom, and aromatic substituents, did not affect the inhibitory activity of the overall molecule. In addition, three amine-linked analogues were synthesized by modification of the S-tert-butyl group in the bromooxazole analogue with amine-linked a-bromoacetamides. Notably, one of the amine-linked heptaoxazole analogues exhibited almost the same inhibitory activity as telomestatin.

Full text of DOI:10.1246/bcsj.20130198

© 2013 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 86, No. 12, 1453-1465 (2013) 1453
Synthesis of Heptaoxazole Macrocyclic Analogues of Telomestatin
and Evaluation of Their Telomerase Inhibitory Activities
Kazuaki Shibata,1 Masahito Yoshida,2 Takashi Takahashi,1,³
Motoki Takagi,3,³³ Kazuo Shin-ya,4 and Takayuki Doi*2
1Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8552
2Graduate School of Pharmaceutical Sciences, Tohoku University,
6-3 Aza-aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578
3Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC),
2-4-7 Aomi, Koto-ku, Tokyo 135-0064
4National Institute of Advanced Industrial Science and Technology (AIST),
2-4-7 Aomi, Koto-ku, Tokyo 135-0064
Received July 14, 2013; E-mail: doi_taka@mail.pharm.tohoku.ac.jp
We have demonstrated various synthetic routes to heptaoxazole macrocyclic analogues of telomestatin and evaluated
their inhibitory activities against telomerase. We synthesized three heptaoxazole macrocycles consisting of different
numbers of methyloxazole moieties and another heptaoxazole analogue with a bromooxazole moiety instead of one of
the two methyloxazole moieties found in the structure of telomestatin. The bromooxazole analogue underwent Suzuki-
Miyaura coupling leading to six analogues having aromatic substituents on the oxazole moiety. The substituents on the
oxazole moiety in the heptaoxazole macrocycles, which include a methyl group, a bromine atom, and aromatic substituents,
did not affect the inhibitory activity of the overall molecule. In addition, three amine-linked analogues were synthesized by
modification of the S-tert-butyl group in the bromooxazole analogue with amine-linked α-bromoacetamides. Notably, one
of the amine-linked heptaoxazole analogues exhibited almost the same inhibitory activity as telomestatin.
Telomestatin (R)-(1) (Figure 1) isolated by Shin-ya et al. is
known to be an extremely potent inhibitor of telomerase.1 It
has been proposed that telomestatin selectively binds to the
G-quadruplex structure produced from the human telomeric
sequence 5¤-TTAGGG-3¤ similar to a sandwich form stabilizing
the G-quadruplex structure.2 Moreover, recent studies showed
that telomestatin interacts with the promoter regions of several
proto-oncogenes as well as the human telomeric G-quadruplex
structure.3 Telomestatin selectively induces apoptosis for can-
cer cells4 and exhibited in vivo efficacy for antitumor activity.5
Recently, telomeres and telomerase represent an extremely
attractive target for cancer therapy.6
In 2006, we reported the asymmetric total synthesis of
telomestatin (R)-(1) and defined the absolute configuration of
the natural product.7 We also synthesized its enantiomer, (S)-1,
which was four-fold as potent as (R)-1; this was evaluated by
determining the Tm values of the complexes with ss-telo24 as
observed in the CD melting curves as well as with the results
of a telomerase repeat amplification protocol (TRAP) assay.8
In addition, we discovered that heptaoxazole macrocycle (R)-2,
which is a synthetic intermediate in the formation of the
thiazoline ring of telomestatin, also has potential inhibitory
activity against telomerase such that its activity is one fourth
that of the natural product.
Since Shin et al. demonstrated the construction of a hexa-
oxazole macrocycle in their synthetic study for telomestatin,9
and a number of studies for the synthesis of hexaoxazole
macrocyclic analogues of telomestatin and the evaluation of
their binding abilities to G-quadruplex structures have been
reported.10-14 Interestingly, amine-linked derivatives including
HXDL,10c L2H2-6OTD,11b L2H2-6OTD-dimer,11g and guani-
dine-linked derivative L2G2-6OTD11b retain their potential
inhibitory activities against telomerase.15 Despite many inten-
sive studies focusing on hexaoxazole macrocycles, only a few
examples of heptaoxazole macrocycles that have planar struc-
tures have been reported.8,16 This is probably because the
construction of the seventh oxazole in a macrocycle will be
difficult due to high strain energy.10a,13 Notably, the Nagasawa
group reported that amine-linked heptaoxazole macrocycle
L1H1-7OTD is a more favorable binder than the corresponding
hexaoxazole macrocycle for strongly inducing a conformational
change of ss-telo24 to an antiparallel G-quadruplex structure.16
We are interested in elucidating the role of the two methyl-
oxazoles in the heptaoxazole macrocycle (R)-2, and develop-
ing various synthetic routes for its heptaoxazole macrocyclic
analogues. We report the synthesis of heptaoxazole macro-
cycles (R)-3b-3d, which have different numbers of methyl-
oxazole moieties, and (R)-3e, which has a bromooxazole
instead of methyloxazole, as well as two methods of diversi-
³
Present address: Yokohama College of Pharmacy, 601
Matanocho, Totsuka-ku, Yokohama, Kanagawa 245-0066
³³ Present address: Translational Research Center, Fukushima
Medical University, 11-23 Sakaemachi, Fukushima 960-8031
Published on the web September 13, 2013; doi:10.1246/bcsj.20130198

1454 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
Synthesis of Heptaoxazole Macrocyclic Analogues
St-Bu
St-Bu
O
O
S
N
O
O
O
R3
N
N
N
N
OBn
O
O
BocHN
H
O
O
N
N
N
O
N
N
N
CH3
CH3
O
N
N
N
R1
3
+
N
O
N
O
N
O
O
O
N
N
N
Boc
N
O
CH3
CH3
O
N
R2
O
O
O
O
O
O
MeO
telomestatin (R)-(1)
(R)–2
6a R1=R2=H
7a R3=H
7b R3=Me
6b R1=Me, R2=H
6c R1=R2=Me
St-Bu
St-Bu
O
O
O
O
R3
6d R1=Br, R2=Me
N
N
H
N
N
H
O
O
Figure 2. Retrosynthesis of heptaoxazole analogues from 3.
St-Bu
O
N
N
N
N
O
N
N
N
N
R1
R
O
O
O
O
N
N
N
N
R2
CH3
H2N OH PyBroP, DIEA
CH2Cl2, rt
BocHN
O
O
O
O
O
O
N
N
O
+
R1
(R)–3a R1=R2=R3=H
(R)–4
(R)–3b R1=Me, R2=R3=H
(R)–3c R1=R3=Me, R2=H
(R)–3d R1=R2=R3=Me
O
MeO
OH
O
8a R1=H
9
(R)–3e R1=Br, R2=Me, R3=H
S
Y
O
N
NH2
8b R1=Me
O
H
N
N
O
H
St-Bu
O
N
N
N
N
Br
BocHN
O
O
O
N
R1
N
N
1) Burgess reagent
THF, 70 °C
CH3
O
O
6a (48%)
6b (52%)
O
HN
(R)–5, Y = linker
2) DBU, BrCCl3
CH2Cl2, rt
O
N
OH
Figure 1. Structures of telomestatin and its heptaoxazole
analogues.
O
MeO
10a R1=H (73%)
10b R1=Me (76%)
fication: 3e to 4 by palladium-catalyzed cross coupling17 and
3e to amine-linked analogues 5a-5c by S-alkylation with
amine-linked α-bromoacetamides. By evaluating the telomer-
ase inhibitory activity of the synthetic heptaoxazole macro-
cycles, amine-linked heptaoxazole macrocycle 5a was found to
be as potent as natural telomestatin (R)-(1).
Scheme 1. Synthesis of trisoxazoles 6a and 6b.
hexaoxazole 14a-14e in 63%-90% yields (Scheme 3). Re-
moval of the Boc group and acetonide in 14a-14d with acid,
followed by basic hydrolysis of the methyl ester yielded the
cyclization precursors (method A). Macrolactamization was
performed using DPPA/HOBt/DMAPO/DIEA (DPPA: di-
phenylphosphoryl azide, DMAPO: 4-(dimethylamino)pyridine
N-oxide, DIEA: N,N-diisopropylethylamine) at room tempera-
ture in CH2Cl2-DMF (1:1) for 3 d.8 After purification by silica
gel chromatography, hexaoxazole macrocycles 15a-15d were
isolated in 7%, 47%, 54%, and 58% overall yields, respec-
tively. Owing to the low yield and the insolubility of 15a, we
could not perform further synthesis of its derivative. Trans-
formation of 14e to 15e was carried out in an alternative way
(method B) because the bromooxazole moiety in 14e decom-
posed under basic conditions. Cleavage of the methyl ester with
Me3SnOH22 followed by acid treatment yielded the cyclization
precursor, which underwent macrolactamization leading to 15e
in 58% overall yield. Dehydration of 15b-15e was performed
via mesylation and β-elimination, as reported previously.7,9
Results and Discussion
Heptaoxazole macrocycles 3 can be synthesized by the
condensation of 6 and 7, followed by macrolactamization and
formation of the seventh oxazole moiety, according to our
reported method (Figure 2).7,8 Amidation of cysteine-derived
monooxazoles 8a and 8b with serine-derived monooxazole
9 using PyBroP18 afforded 10a and 10b (Scheme 1).7 Cyclo-
dehydration of 10a and 10b was performed by treatment with
the Burgess reagent,19 followed by using BrCCl3/DBU20 to
provide trisoxazoles 6a and 6b, respectively. Methylated
trisoxazole 7b was prepared from threonine-derived mono-
oxazole 119 and serine-derived 1221 by a similar method
(Scheme 2).
Removal of the Boc group in 6a-6d7,17 with 4 M HCl in
dioxane and acylation of the resulting amines with the carbox-
ylic acid derived from 7a7 or 7b by hydrogenolysis afforded

K. Shibata et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013) 1455
O
Therefore, neither positive nor negative effects of the additional
aromatic substituents were observed.
O
Me
HO
O
N
OBn
O
Amine linkers were introduced on telomestatin analogue 3e
(Scheme 4). The t-butyl group in 3e was removed by treatment
with DMSO/TMSCl/CH2Cl2/TFA at 0 °C. As the products
were provided as a mixture of thiol and disulfide, reduction of
the disulfide using aqueous dithiothreitol/DIEA followed by
in situ S-alkylation with α-bromoacetamides 17a-17c provided
α-thioacetamides 18a-18c, respectively, in 28%-33% yields
after purification by reversed-phase preparative HPLC. The
products 18a-18c were identified by spectroscopic analysis,
then the Boc group was removed by treatment with 4 M HCl in
1,4-dioxane, and the HCl salts (R)-5a-5c obtained were used
for biological evaluation.
Inhibitory activities of synthetic derivatives (R)-5a-5c
against telomerase were evaluated utilizing a TRAPezeμ XL
Telomerase Detection Kit (Millipore) that enables quantitative
measurements without radioactivity. The IC50 values of (R)-1
and (R)-5a-5c are depicted in Table 2. Compound 5a (IC50
value, 50 « 21 nM) had the same potency as telomestatin (IC50,
49 « 5 nM), while 5b-5c exhibited slightly less potency (IC50,
115-135 nM) than telomestatin. Actually, attachment of the
amine linker with the appropriate length increased the inhibi-
tory activity against telomerase. This result well matched with
the reported results such that the amine-linked HXDL, L2H2-
6OTD, and L1H1-7OTD increased their binding abilities to G-
quadruplex structures.10c,11b,16a
Me
NH2
N
OBn
PyBroP, DIEA
CH2Cl2, rt
11
HO
O
NH
N
+
HO
O
80%
Boc
O
N
N
Boc
O
N
O
13
O
12
O
O
Me
N
OBn
1) Burgess reagent
THF, 70 °C
O
N
N
2) DBU, BrCCl3
CH2Cl2, rt
Boc
N
O
O
7b
Scheme 2. Synthesis of methylated trisoxazole 7b.
Bromomethoxylation, oxazoline formation via intramolecular
etherification, and removal of methanol under acidic conditions
resulted in the complete formation of heptaoxazole macro-
cycles (R)-3b-3e.7 Although the above reactions were success-
fully performed, purification of the final products was quite
difficult owing to their extremely low solubility and adherence
to the silica gel. Therefore, (R)-3b-3e were isolated in overall
15%-22% yields.
Inhibitory activities of (R)-2 and (R)-3b-3e against telomer-
ase were evaluated by a modified TRAP assay with an internal
standard.1,23,24 All synthetic derivatives 3b-3e were found to be
as potent as (R)-2 with IC50 values of 80 nM. Thus, the number
of the methyloxazole moieties, their positions, and bromo-
oxazole in the heptaoxazole macrocyclic analogues do not affect
the inhibitory activity for telomerase; we could not character-
ize an analogue consisting of oxazole moieties that did not
involve any methyloxazole owing to the low yield and lack of
solubility of 15a.
Next, derivatization of (R)-3e was performed by Suzuki-
Miyaura coupling25 of a bromooxazole moiety with six boronic
acids according to our previously reported optimized condi-
tions (Table 1).17 The coupling reactions of 3e with phenyl-, 4-
methoxyphenyl-, and 4-fluorophenylboronic acids were achiev-
ed in the presence of Pd(OAc)2-SPhos26 and KF in 1,4-dioxane
at 80 °C to afford 4a-4c in good yields (Entries 1-3). When
Pd(OAc)2-XPhos26 was used as a catalyst, 3-thienyl deriva-
tive 4d was also afforded in 63% yield (Entry 4). The 4-
(dimethylamino)phenyl and 4-N-morphorylphenyl derivatives
4e and 4f were provided in moderate yields (44% for 4e; 65%
for 4f) when the reactions were performed in 1,2-dimethoxy-
ethane (DME). It should be noted that we could derivatize the
heptaoxazole macrocycles with aromatic and heteroaromatic
substituents in the final stage of the synthesis. Inhibitory
activities of 4a-4f were in the same range as that of 3e.
Conclusion
We have demonstrated various synthetic routes to hepta-
oxazole macrocyclic analogues of telomestatin and evaluated
their telomerase inhibitory activity. The initial examples of
3a-3d involve different numbers of methyloxazole moieties
in the heptaoxazole macrocycles, and 3e has a bromooxazole
moiety instead of a methyloxazole. The synthesis of 3b-3e was
accomplished according to the method we previously utilized
in the total synthesis of telomestatin, although 3a was not
obtained owing to the low yield and lack of solubility of the
synthetic intermediate 15a. The bromooxazole-containing 3e
was synthesized by a modified procedure that avoided decom-
position of the bromooxazole moiety under basic conditions.
The inhibitory activities of these molecules against telomerase
were evaluated by a TRAP assay. As a result, no significant
effect of the methyl group and a bromine atom as a substituent
on the oxazole ring was observed in heptaoxazole macrocycles
although it was reported that the higher increment of methyl-
oxazole moieties induced stronger G-quadruplex stabilizing
activity in hexaoxazole macrocycles.11c It is conceivable that
the heptaoxazole macrocycles have more planar structures than
the hexaoxazole macrocycles, thereby the former could allow
stronger interactions to telomeric G-quadruplex structures by
π-π interactions over the substituent effects of the methyl group
or the bromine atom on the oxazole ring.10a,16a
Next, diversification of 3e was investigated. The Suzuki-
Miyaura coupling of 3e with six aromatic and heteroaromatic
boronic acids was performed in the presence of Pd(OAc)2-
SPhos or Pd(OAc)2-XPhos as a catalyst to provide 4a-4f in
moderate yields. The aromatic rings introduced on the oxazole
ring also did not affect the telomerase inhibitory activity.

1456 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
St-Bu
Synthesis of Heptaoxazole Macrocyclic Analogues
BocHN
O
N
R1
4 M HCl /
1,4-dioxane
rt
N
O
St-Bu
O
O
O
N
R3
O
R2
N
N
O
O
H
MeO
N
N
N
R1
PyBroP, DIEA
CH2Cl2–DMF
rt
6a R1=R2=H
6b R1=Me, R2=H
6c R1=R2=Me
N
O
O
N
6d R1=Br, R2=Me
R2
O
O
NBoc
O
O
O
OMe
R3
N
OBn
14a (63%)
14b (84%)
14c (80%)
14d (90%)
14e (77%)
H2, 10% Pd/C
AcOEt–MeOH
O
N
N
method A
method B
Boc
N
O
1) 4 M HCl / 1,4-dioxane
rt
1) Me3SnOH, (CH2Cl)2,
80 °C
2) 4 M HCl / 1,4-dioxane
rt
3) DPPA, HOBt, DIEA
DMAPO, CH2Cl2–DMF
3 mM, rt, 3 d
2) LiOH•H2O
O
THF–MeOH–H2O, rt
3) DPPA, HOBt, DIEA
DMAPO, CH2Cl2–DMF
3 mM, rt, 3 d
7a R3=H
7b R3=Me
St-Bu
O
St-Bu
O
O
O
R3
R3
N
N
O
H
MsCl, NEt3, CH2Cl2
then DBU
0 °C to rt
N
N
N
H
O
O
N
N
N
R1
O
N
N
R1
N
O
N
H
N
O
H
O
N
N
O
R2
N
R2
O
HO
O
O
O
15a (A; 7%)
15b (A; 47%)
15c (A; 54%)
15d (A; 58%)
15e (B; 58%)
16b (76%)
16c (73%)
16d (78%)
16e (76%)
St-Bu
O
O
1) NBS, MS4A,
CH2Cl2–MeOH
0 °C
2) K2CO3, 1,4-dioxane
60 °C
R3
O
N
N
O
H
R1 R2 R3
N
N
N
R1
a
b
c
d
e
H
Me
Me
H
H
H
H
H
Me
N
O
3) CSA, MS5A
toluene, 70 °C
O
N
N
Me Me Me
Br Me
R2
H
O
O
(R)-3b (15%)
(R)-3c (20%)
(R)-3d (22%)
(R)-3e (22%)
Scheme 3. Synthesis of heptaoxazole macrocycles 3a-3e.
We then synthesized amine-linked heptaoxazole macrocyclic
analogues 5a-5c from 3e by removal of the t-butyl group,
followed by S-alkylation with amine-linked α-bromoacetamides
and removal of the Boc groups. Thus, a combination of Suzuki-
Miyaura coupling and S-alkylation allowed us to access various
telomestatin analogues in the final stage of the synthesis.
Alternative coupling methods we have already reported for the
coupling of a bromooxazole moiety with an alkyl group, amine,
an alkoxy group, and a trifluoromethyl group17 will be applied
to further synthesis of a variety of substituted heptaoxazole

K. Shibata et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013) 1457
Table 1. Preparation of (R)-4 via Suzuki-Miyaura Coupling of (R)-3e with Boronic Acids
St-Bu
St-Bu
O
O
O
O
R–B(OH)2 (6 equiv)
Pd(OAc)2 (20 mol%)
Ligand (40 mol%)
KF (8 equiv)
N
N
N
N
O
O
H
H
O
N
N
N
O
N
N
N
Br
R
80 °C, 24 h
N
O
N
O
O
O
N
N
N
N
CH3
CH3
O
O
O
O
(R)–3e
(R)–4
Entry
R
Liganda)
Solvent
Product
Yield/%
1
2
3
4
5
Ph
SPhos
SPhos
SPhos
XPhos
XPhos
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
DME
4a
4b
4c
4d
4e
73
72
70
63
44
4-C6H4OMe
4-C6H4F
3-thienyl
4-C6H4NMe2
6
XPhos
DME
4f
65
4-C6H4
N
O
a) SPhos: 2-Dicyclohexylphosphino-2¤,6¤-dimethoxybiphenyl, XPhos: 2-Dicyclohexylphos-
phino-2¤,4¤,6¤-triisopropylbiphenyl.
macrocycles. The telomerase inhibitory activities of 5a-5c
and 1 were evaluated utilizing a TRAPezeμ XL Telomerase
Detection Kit (Millipore). Notably, amine-linked heptaoxazole
macrocycle 5a retained almost the same potency as telomestatin
(R)-1, while 5b-5c exhibited slightly less potency than
telomestatin. We noted earlier that various aromatic substituents
{phenyl, 4-methoxyphenyl, 4-fluorophenyl, 3-thienyl, 4-(di-
methylamino)phenyl, and 4-(4-morpholinyl)phenyl} on hepta-
oxazole macrocycles 4 did not affect the telomerase inhibitory
activities. In contrast, the amino-linkers of 5 induced high
potencies that are dependent on their lengths. Probably, the
amino-linker would employ as a cationic functional group and
could be directed toward the phosphate groups of telomere to
stabilize the G-quadruplex structure.11b As a result, 5a that
has the Cys {(4-aminobutyl)aminocarbonylmethyl} moiety in
a rather planar heptaoxazole macrocycle exhibited extremely
potent telomerase inhibitory activity as much as telomestatin.
Further study for elucidation of the binding structures of 5 and
telomere is underway in our laboratories.
Perkin-Elmer Spectrum One FT-IR spectrophotometer. Only the
strongest and/or structurally important absorption is reported as
the IR data given in cm¹1. Optical rotations were measured with
a JASCO P-1020 polarimeter. All reactions were monitored by
thin-layer chromatography carried out on 0.2 mm E. Merck
silica gel plates (60F-254) with UV light, visualized by a p-
anisaldehyde solution, ceric sulfate, or 10% ethanolic phospho-
molybdic acid. Merck silica gel 60 (0.063-0.200 mm) was used
for column chromatography. Gel permeation chromatography
was performed on a Japan Analytical Industry Model LC908
(recycling preparative HPLC) instrument with a polystyrene
gel column (JAIGEL-1H, 20 mm © 600 mm) using chloroform
as the solvent (3.5 mL min¹1). ESI-TOF mass spectra were
measured with a Waters LCT Premier· XE mass spectrometer.
HRMS (ESI-TOF) were calibrated with leucine enkephalin
(SIGMA) as an external standard. Preparative reversed-phase
HPLC was performed on a Gilson 215 system.
Preparation of 8a. To a solution of Boc-L-Cys(t-Bu)-OH
(15.0 g, 54.1 mmol) in CH2Cl2 (162 mL) was added H-L-
Ser-OMe¢HCl (9.30 g, 59.5 mmol), HOBt (8.80 g, 64.9 mmol)
and DIEA (12.0 mL, 70.3 mmol) at room temperature. To the
reaction mixture was added EDCI¢HCl (11.4 g, 59.5 mmol) at
0 °C. After being stirred at room temperature for 2 h, the reac-
tion mixture was poured into 1 M HCl at 0 °C and the aqueous
layer was extracted with ethyl acetate. The combined organic
layer was washed with 3 M HCl, saturated aqueous NaHCO3
and brine, dried over MgSO4 and filtered. After removal of
the solvent, the residue was used for the next reaction without
further purification.
Experimental
General Methods. NMR spectra were recorded on a JEOL
Model EX-270 (270 MHz for 1H, 67.8 MHz for 13C) or a JEOL
1
Model ECP-400 (400 MHz for H, 100 MHz for 13C) instru-
ment in the indicated solvent. Chemical shifts are reported in
parts per million (ppm) relative to the signal for internal tetra-
methylsilane (0 ppm for 1H) for solutions in CDCl3. NMR
spectral data are reported as follows: chloroform (7.26 ppm
1
for H) or chloroform-d (77.1 ppm for 13C), dichloromethane
1
(5.30 ppm for H) or dichloromethane-d2 (53.8 ppm for 13C),
To a solution of the crude dipeptide in THF (162 mL) was
added the Burgess reagent (15.5 g, 64.9 mmol) at room temper-
ature under argon. After being stirred at 70 °C for 14 h, the
reaction mixture was diluted with ethyl acetate and H2O, and
the aqueous layer was extracted with ethyl acetate. The com-
bined organic layer was washed with brine, dried over MgSO4
DMSO (2.50 ppm for 1H) or DMSO-d6 (39.5 ppm for 13C), and
methanol (3.30 ppm for 1H) or methanol-d4 (49.8 ppm for 13C).
Multiplicities are reported by the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; J,
coupling constants in Hertz. IR spectra were recorded on a

1458 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
Synthesis of Heptaoxazole Macrocyclic Analogues
O
St-Bu
O
O
S
R
O
N
O
1) TFA, TMSCl
DMSO, CH2Cl2,
0 °C, 1 h
H
N
N
O
H
N
N
O
O
N
N
N
H
Br
O
N
N
N
Br
2) DTT, DIEA
CH3CN–H2O;
then rt, 3 h
N
O
N
O
O
N
N
O
CH3
O
N
N
CH3
O
O
Br
R
O
N
O
H
(R)–3e
17a, R = (CH2)2–NHBoc
17b, R = (CH2)6–NHBoc
(R)–18a, R = (CH2)2–NHBoc, 33%
(R)–18b, R = (CH2)6–NHBoc, 29%
17c, R = (OCH2CH2)2–NHBoc (R)–18c, R = (OCH2CH2)2–NHBoc, 28%
4 M HCl /
quant.
1,4-dioxane
O
O
•
S
NH2 HCl
3
•
NH2 HCl
S
O
O
N
O
N
O
H
H
N
N
N
N
H
O
O
H
O
N
N
N
O
N
N
N
N
Br
Br
N
O
O
O
O
N
N
N
N
CH3
CH3
O
O
O
O
(R)–5a
(R)–5b
O
S
O
O
•
N
NH2 HCl
O
2
H
N
N
O
H
O
N
N
N
Br
N
O
O
N
N
CH3
O
O
(R)–5c
Scheme 4. Synthesis of amine-linked heptaoxazole macrocycles 5a-5c.
and filtered. After removal of the solvent, the residue was used
for the next reaction without further purification.
Table 2. Telomerase Inhibitory Activities of (R)-1 and (R)-
5a-5ca)
To a solution of the crude oxazoline in CH2Cl2 (162 mL) was
added DBU (24.0 mL, 162 mmol) and BrCCl3 (16.0 mL, 162
mmol) at 0 °C under argon. After being stirred at room tem-
perature for 8 h, the reaction mixture was poured into 3 M HCl
at 0 °C and the aqueous layer was extracted with ethyl acetate.
The combined organic layer was washed with 3 M HCl,
saturated aqueous NaHCO3 and brine, dried over MgSO4 and
filtered. After removal of the solvent, the residue was purified
by column chromatography on silica gel (0% to 20% ethyl
Compound
telomestatin (R)-1 (R)-5a
(R)-5b
(R)-5c
IC50/nM
49 « 5 50 « 21 115 « 29 135 « 59
a) The activity was measured using a TRAPeze XL Telomerase
Detection Kit. Data are mean « S.D. derived from three
separate experiments.

K. Shibata et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013) 1459
20
acetate in hexane) to give the methyl ester of 8a (28.7 g, 80.1
13: 80% yield. ½ꢀꢀD ¹76.2 (c 1.41, CHCl3). 1H NMR
19
mmol, 53% in 3 steps). ½ꢀꢀD ¹8.69 (c 1.09, CHCl3). 1H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.73 (s, 1H), 8.58 (s, 1H), 7.86
(d, J = 8.70 Hz, 1H), 7.42-7.33 (m, 5H), 5.31 (s, 2H), 5.11 (dd,
J = 6.77, 2.90 Hz, 1H), 5.08 (dd, J = 8.70, 3.87 Hz, 1H), 4.27
(dd, J = 9.18, 6.77 Hz, 1H), 4.29-4.23 (m, 1H), 4.06 (dd, J =
9.18, 2.90 Hz, 1H), 1.64 (s, 3H), 1.52 (s, 3H), 1.30 (brs, 9H),
1.13 (d, J = 6.28 Hz, 3H). 13C NMR (100 MHz, DMSO-d6,
80 °C): ¤ 162.9, 162.7, 160.1, 159.5, 145.1, 141.7, 135.5, 135.0,
132.1, 128.0, 127.7, 127.6, 93.8, 78.7, 66.4, 66.3, 65.5, 54.2,
52.5, 27.5, 19.5. FT-IR (KBr, cm¹1): 3405, 2980, 1704, 1598,
1504, 1379, 1267, 1108, 848, 756, 699. HRMS (ESI-TOF):
Calcd for C28H35N4O9 [M + H]+: 571.2404, Found: 571.2401.
(400 MHz, CDCl3): ¤ 8.20 (s, 1H), 5.54 (brd, J = 6.77 Hz, 1H),
5.16 (brs, 1H), 3.91 (s, 3H), 3.11 (dd, J = 12.6, 5.80 Hz, 1H),
3.05 (dd, J = 12.6, 6.28 Hz, 1H), 1.44 (s, 9H), 1.30 (s, 9H).
13C NMR (100 MHz, CDCl3): ¤ 163.9, 161.2, 154.7, 144.0,
133.1, 80.0, 52.0, 49.0, 42.6, 32.2, 30.7, 28.1. FT-IR (KBr,
cm¹1): 3349, 2974, 1717, 1584, 1511, 1164, 1111, 1002, 772.
To a solution of the above methyl ester (5.00 g, 13.9 mmol)
in THF (13.9 mL), MeOH (13.9 mL) and H2O (3.5 mL) was
added LiOH¢H2O (1.30 g, 31.0 mmol) at 0 °C. After being
stirred at room temperature for 3 h, the reaction mixture was
poured into 3 M HCl at 0 °C and the aqueous layer was
extracted with ethyl acetate. The combined organic layer was
washed with brine, dried over MgSO4 and filtered. After
removal of the solvent, the residue was purified by column
chromatography on silica gel (0% to 4% MeOH in CHCl3) to
General Procedure for Formation of Trisoxazole. To a
solution of bisoxazole amide (1 equiv) in THF (10 mL mmol
¹1
)
was added Burgess reagent (1.2 equiv) at room temperature
under argon. After being stirred at 70 °C for 13 h, the reac-
tion mixture was diluted with ethyl acetate and H2O, and the
aqueous layer was extracted with ethyl acetate. The combined
organic layer was washed with brine, dried over MgSO4 and
filtered. After removal of the solvent, the residue was used for
the next reaction without further purification.
To a solution of the crude oxazoline in CH2Cl2 (5.0
mL mmol¹1) was added DBU (5.0 equiv) and BrCCl3 (5.0
equiv) at 0 °C under argon. After being stirred at room tem-
perature for 12 h, the reaction mixture was poured into 3 M
HCl at 0 °C and the aqueous layer was extracted with
ethyl acetate. The combined organic layer was washed with
3 M HCl, saturated aqueous NaHCO3 and brine, dried over
MgSO4 and filtered. After removal of the solvent, the residue
was purified by column chromatography on silica gel to give
trisoxazole.
24
give carboxylic acid 8a (4.31 g, 12.5 mmol, 90%). ½ꢀꢀD ¹6.28
1
(c 0.920, CHCl3). H NMR (400 MHz, CDCl3): ¤ 8.28 (s, 1H),
5.79 (brs, 1H), 5.20 (brs, 1H), 3.12 (dd, J = 13.2, 5.80 Hz, 1H),
3.07 (dd, J = 13.2, 6.28 Hz, 1H), 1.44 (s, 9H), 1.30 (s, 9H).
13C NMR (100 MHz, CDCl3): ¤ 165.1, 164.2, 155.2, 145.0,
133.2, 80.5, 49.3, 42.9, 32.5, 30.9, 28.3. FT-IR (KBr, cm¹1):
3319, 2976, 1716, 1515, 1368, 1163, 1112, 757, 597.
General Procedure for the Condensation Utilizing
PyBroP. To a solution of the mono-oxazole amino alcohol
in CH2Cl2 (5.0 mL mmol¹1) was added monooxazole carbox-
ylic acid (1 equiv) and DIEA (3.0 equiv) at room temperature
under argon. To the reaction mixture was added PyBroP
(1.3 equiv) at 0 °C. After being stirred at room temperature for
3 h, the reaction mixture was poured into 1 M HCl at 0 °C
and the aqueous layer was extracted with ethyl acetate. The
combined organic layer was washed with 3 M HCl, saturated
aqueous NaHCO3 and brine, dried over MgSO4 and filtered.
After removal of the solvent, the residue was purified by
column chromatography on silica gel to give bisoxazole amide.
22
6a: 48% yield in 2 steps. ½ꢀꢀD ¹2.80 (c 0.760, CHCl3).
1H NMR (400 MHz, CDCl3): ¤ 8.43 (s, 1H), 8.33 (s, 1H), 8.32
(s, 1H), 5.53 (brd, J = 7.25 Hz, 1H), 5.22 (brs, 1H), 3.96
(s, 3H), 3.16 (dd, J = 13.1, 5.32 Hz, 1H), 3.09 (dd, J = 13.1,
6.28 Hz, 1H), 1.46 (s, 9H), 1.31 (s, 9H). 13C NMR (100 MHz,
CDCl3): ¤ 164.7, 161.4, 156.1, 155.5, 155.0, 144.0, 139.7,
139.4, 134.5, 130.9, 129.9, 80.5, 52.4, 49.2, 43.0, 32.6, 30.9,
28.4. FT-IR (KBr, cm¹1): 3370, 2965, 1705, 1519, 1324, 1249,
1164, 1115, 972, 728. HRMS (ESI-TOF): Calcd for C22H28-
N4O7SNa [M + Na]+: 515.1576, Found: 515.1575.
23
10a: 73% yield. ½ꢀꢀD ¹3.85 (c 1.06, CHCl3). 1H NMR (400
MHz, CDCl3): ¤ 9.17 (brd, J = 7.26 Hz, 1H), 8.20 (s, 1H), 7.80
(s, 1H), 6.53 (brd, J = 9.18 Hz, 1H), 5.45 (m, 1H), 4.89 (m, 1H),
4.24 (dd, J = 12.1, 7.25 Hz, 1H), 3.96 (dd, J = 12.1, 3.87 Hz,
1H), 3.83 (s, 3H), 2.57 (dd, J = 13.5, 5.80 Hz, 1H), 2.44 (dd,
J = 13.5, 9.18 Hz, 1H), 1.43 (s, 9H), 1.24 (s, 9H). 13C NMR
(100 MHz, CDCl3): ¤ 164.0, 162.9, 162.0, 161.9, 155.1, 144.2,
141.5, 135.3, 133.2, 80.2, 62.7, 52.5, 50.7, 49.4, 42.9, 31.0,
30.8, 28.5. FT-IR (KBr, cm¹1): 3474, 3264, 2974, 1717, 1657,
1600, 1519, 1367, 1165, 1113, 757. HRMS (ESI-TOF): Calcd
for C22H33N4O8S [M + H]+: 513.2019, Found: 513.2020.
22
6b: 52% yield in 2 steps. ½ꢀꢀD ¹17.0 (c 1.41, CHCl3).
1H NMR (400 MHz, CDCl3): ¤ 8.41 (s, 1H), 8.32 (s, 1H),
5.50 (brd, J = 6.77 Hz, 1H), 5.15 (brs, 1H), 3.96 (s, 3H), 3.14
(dd, J = 13.1, 5.32 Hz, 1H), 3.07 (dd, J = 13.1, 5.80 Hz, 1H),
2.74 (s, 3H), 1.46 (s, 9H), 1.31 (s, 9H). 13C NMR (100 MHz,
CDCl3): ¤ 162.0, 161.3, 157.0, 155.7, 154.9, 151.6, 143.8,
139.0, 134.3, 130.7, 124.6, 80.2, 52.3, 49.0, 42.8, 32.4, 30.9,
28.3, 11.8. FT-IR (KBr, cm¹1): 3352, 2976, 1693, 1519, 1327,
1278, 1163, 1050, 970, 758. HRMS (ESI-TOF): Calcd for
C23H30N4O7SNa [M + Na]+: 529.1733, Found: 529.1734.
21
1
10b: 76% yield. ½ꢀꢀD ¹32.7 (c 0.830, CHCl3). H NMR
(400 MHz, CDCl3): ¤ 8.31 (brd, J = 7.25 Hz, 1H), 8.20 (s, 1H),
5.81 (brd, J = 7.73 Hz, 1H), 5.48 (m, 1H), 4.94 (brs, 1H), 4.21
(dd, J = 11.6, 5.32 Hz, 1H), 4.01 (dd, J = 11.6, 3.87 Hz, 1H),
3.87 (s, 3H), 2.83 (dd, J = 13.1, 5.80 Hz, 1H), 2.74 (dd, J =
13.1, 7.25 Hz, 1H), 2.50 (s, 3H), 1.43 (s, 9H), 1.27 (s, 9H).
13C NMR (100 MHz, CDCl3): ¤ 163.5, 162.2, 161.5, 159.7,
155.0, 153.8, 144.3, 133.0, 128.5, 80.0, 62.9, 52.2, 49.5, 49.1,
42.7, 31.6, 30.8, 28.3, 11.6. FT-IR (KBr, cm¹1): 3405, 3286,
2974, 1712, 1656, 1584, 1517, 1367, 1166, 1114, 757. HRMS
(ESI-TOF): Calcd for C23H35N4O8S [M + H]+: 527.2176,
Found: 527.2173.
20
7b: 61% yield in 2 steps. ½ꢀꢀD ¹54.3 (c 1.26, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.88 (s, 1H), 8.79
(s, 1H), 7.46-7.33 (m, 5H), 5.36 (s, 2H), 5.13 (dd, J = 6.77,
2.90 Hz, 1H), 4.29 (dd, J = 9.18, 6.77 Hz, 1H), 4.07 (dd, J =
9.18, 2.90 Hz, 1H), 2.72 (s, 3H), 1.66 (s, 3H), 1.53 (s, 3H), 1.32
(brs, 9H). 13C NMR (100 MHz, DMSO-d6, 80 °C): ¤ 163.9,
159.9, 155.5, 153.1, 150.9, 144.7, 139.8, 135.5, 133.0, 129.0,
128.0, 127.7, 127.5, 124.6, 93.8, 79.5, 78.7, 66.4, 65.6, 54.2,

1460 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
Synthesis of Heptaoxazole Macrocyclic Analogues
18
27.5, 10.9. FT-IR (KBr, cm¹1): 3133, 2977, 1707, 1576, 1378,
1159, 1094, 994, 850, 755. HRMS (ESI-TOF): Calcd for
C28H31N4O8 [M + H]+: 551.2142, Found: 551.2145.
General Procedure for the Formation of Hexaoxazole
Amides 14. To 10% Pd/C (20 wt %) was added ethyl acetate
(10 mL mmol¹1) and MeOH (10 mL mmol¹1) under argon. The
reaction mixture was purged with H2 three times. To the
reaction mixture was added a solution of trisoxazole benzyl
ester 7 (1 equiv) in ethyl acetate (10 mL mmol¹1) and MeOH
(10 mL mmol¹1) at room temperature. After being stirred at
the same temperature for 2 h, the reaction mixture was filter-
ed through a pad of Celiteμ. The filtrate was concentrated
in vacuo. The residue was used for the next reaction without
further purification.
To a solution of N-Boc-trisoxazole 6 (1.2 equiv) in 1,4-
dioxane (1.0 mL mmol¹1) was added 4 M HCl/1,4-dioxane
(5.0 mL mmol¹1) at 0 °C. After being stirred at room temper-
ature for 6 h, the reaction mixture was concentrated in vacuo.
The residue was used for the next reaction without further
purification.
14c: 80% yield in 2 steps. ½ꢀꢀD ¹27.5 (c 0.875, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.91 (s, 1H), 8.86 (s,
1H), 8.78 (s, 1H), 8.70 (d, J = 8.22 Hz, 1H), 8.69 (s, 1H), 5.38
(dt, J = 8.22, 7.25 Hz, 1H), 5.13 (dd, J = 6.28, 2.90 Hz, 1H),
4.29 (dd, J = 9.18, 6.28 Hz, 1H), 4.07 (dd, J = 9.18, 2.90 Hz,
1H), 3.85 (s, 3H), 3.31 (dd, J = 13.5, 7.25 Hz, 1H), 3.23 (dd,
J = 13.5, 7.25 Hz, 1H), 2.76 (s, 3H), 2.69 (s, 3H), 1.65 (s, 3H),
1.53 (s, 3H), 1.32 (brs, 18H). 13C NMR (100 MHz, DMSO-d6,
80 °C): ¤ 164.0, 161.0, 160.4, 159.2, 156.1, 154.9, 154.7,
153.1, 151.0, 150.8, 144.9, 141.6, 139.9, 139.7, 136.0, 133.1,
129.6, 124.7, 123.6, 93.8, 78.7, 66.4, 54.2, 51.3, 47.3, 42.1,
30.4, 30.2, 27.5, 10.9. FT-IR (KBr, cm¹1): 3306, 3120, 2975,
1705, 1662, 1592, 1367, 1153, 1101, 1052, 756, 728. HRMS
(ESI-TOF): Calcd for C39H44N8O12SNa [M + Na]+: 871.2697,
Found: 871.2685.
19
14d: 90% yield in 2 steps. ½ꢀꢀD ¹24.3 (c 0.745, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.82 (s, 1H), 8.77 (s,
1H), 8.69 (s, 1H), 8.68 (d, J = 8.22 Hz, 1H), 5.37 (dt, J = 8.22,
7.25 Hz, 1H), 5.13 (dd, J = 6.28, 2.90 Hz, 1H), 4.29 (dd, J =
9.18, 6.28 Hz, 1H), 4.07 (dd, J = 9.18, 2.90 Hz, 1H), 3.85 (s,
3H), 3.30 (dd, J = 13.5, 7.25 Hz, 1H), 3.22 (dd, J = 13.5, 7.25
Hz, 1H), 2.76 (s, 3H), 2.72 (s, 3H), 2.67 (s, 3H), 1.66 (s, 3H),
1.53 (s, 3H), 1.32 (brs, 18H). 13C NMR (100 MHz, DMSO-d6,
80 °C): ¤ 164.0, 160.9, 160.5, 159.2, 155.5, 154.9, 153.9,
153.1, 150.8, 150.4, 150.3, 144.4, 141.6, 139.7, 136.0, 133.0,
129.0, 124.7, 124.5, 123.7, 93.8, 78.7, 66.4, 54.2, 51.3, 47.3,
42.1, 30.4, 30.2, 27.5, 11.0, 10.9, 10.8. FT-IR (KBr, cm¹1):
3310, 3153, 2976, 1705, 1654, 1592, 1367, 1168, 1108,
1047, 756. HRMS (ESI-TOF): Calcd for C40H46N8O12SNa
[M + Na]+: 885.2854, Found: 885.2834.
To a solution of the above ammonium salt in CH2Cl2
(5.0 mL mmol¹1) was added the carboxylic acid and DIEA
(3.0 equiv) at room temperature under argon. To the reaction
mixture was added PyBroP (1.3 equiv) at 0 °C. After being
stirred at room temperature for 3 h, the reaction mixture was
poured into 1 M HCl at 0 °C and the aqueous layer was
extracted with ethyl acetate. The combined organic layer was
washed with 3 M HCl, saturated aqueous NaHCO3 and brine,
dried over MgSO4 and filtered. After removal of the solvent,
the residue was purified by column chromatography on silica
gel to give hexaoxazole amide 14.
14e: 77% yield in 2 steps, recrystallized from hexane/ethyl
acetate. Mp 170 °C. ½ꢀꢀD ¹17.3 (c 1.30, CHCl3). 1H NMR
26
27
14a: 63% yield in 2 steps. ½ꢀꢀD ¹27.2 (c 0.515, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.94 (s, 1H), 8.90 (s,
1H), 8.87 (s, 1H), 8.86 (s, 1H), 8.84 (s, 1H), 8.81 (d, J = 8.22
Hz, 1H), 8.72 (s, 1H), 5.42 (dt, J = 8.22, 7.25 Hz, 1H), 5.14
(dd, J = 6.28, 2.90 Hz, 1H), 4.30 (dd, J = 9.18, 6.28 Hz,
1H), 4.08 (dd, J = 9.18, 2.90 Hz, 1H), 3.85 (s, 3H), 3.33 (dd,
J = 13.5, 7.25 Hz, 1H), 3.26 (dd, J = 13.5, 7.25 Hz, 1H), 1.66
(s, 3H), 1.53 (s, 3H), 1.32 (brs, 18H). 13C NMR (100 MHz,
DMSO-d6, 80 °C): ¤ 164.1, 163.5, 160.4, 159.2, 155.3, 154.6,
154.0, 144.9, 142.2, 140.6, 140.4, 140.3, 136.2, 133.1, 130.1,
129.8, 129.7, 128.9, 128.7, 93.8, 78.7, 66.4, 54.2, 51.4, 47.6,
42.1, 30.4, 30.2, 27.5. FT-IR (KBr, cm¹1): 3406, 3143, 2976,
1711, 1675, 1593, 1367, 1148, 1097, 976, 756, 725.
(400 MHz, DMSO-d6, 80 °C): ¤ 8.91 (d, J = 8.22 Hz, 1H), 8.90
(s, 1H), 8.85 (s, 2H), 8.74 (s, 1H), 5.40 (ddd, J = 8.22, 7.73,
7.25 Hz, 1H), 5.16 (dd, J = 6.77, 2.90 Hz, 1H), 4.31 (dd, J =
9.18, 6.77 Hz, 1H), 4.09 (dd, J = 9.18, 2.90 Hz, 1H), 3.87 (s,
3H), 3.32 (dd, J = 13.5, 7.25 Hz, 1H), 3.25 (dd, J = 13.5,
7.73 Hz, 1H), 2.75 (s, 3H), 1.67 (s, 3H), 1.54 (s, 3H), 1.33 (s,
18H). 13C NMR (100 MHz, DMSO-d6, 80 °C): ¤ 164.1, 160.5,
159.2, 155.3, 154.0, 151.8, 151.2, 144.5, 142.2, 140.3 © 2,
136.1, 133.0, 129.7, 128.8, 127.2, 124.9, 122.7, 93.8, 78.7,
66.4, 54.2, 51.3, 47.7, 42.2, 30.4, 29.8, 27.5, 10.9. FT-IR (KBr,
cm¹1): 3307, 3145, 2977, 1714, 1660, 1367, 1159, 1104, 1054,
756. HRMS (ESI-TOF): Calcd for C38H41N8O12S79BrNa
[M + Na]+: 935.1646, Found: 935.1679, and C38H41N8O12-
S81BrNa [M + Na]+: 937.1625, Found: 937.1613. Anal. Calcd
for C38H41N8O12SBr: C, 49.95; H, 4.52; N, 12.26%. Found: C,
50.49; H, 4.66; N, 12.30%.
26
14b: 84% yield in 2 steps. ½ꢀꢀD ¹23.2 (c 0.705, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.91 (s, 1H), 8.90 (s,
1H), 8.86 (s, 1H), 8.84 (s, 1H), 8.74 (d, J = 8.22 Hz, 1H), 8.72
(s, 1H), 5.37 (dt, J = 8.22, 7.25 Hz, 1H), 5.14 (dd, J = 6.28,
2.90 Hz, 1H), 4.30 (dd, J = 9.18, 6.28 Hz, 1H), 4.08 (dd, J =
9.18, 2.90 Hz, 1H), 3.85 (s, 3H), 3.29 (dd, J = 13.5, 7.25 Hz,
1H), 3.23 (dd, J = 13.5, 7.25 Hz, 1H), 2.69 (s, 3H), 1.66 (s,
3H), 1.53 (s, 3H), 1.32 (brs, 18H). 13C NMR (100 MHz,
DMSO-d6, 80 °C): ¤ 164.1, 161.0, 160.4, 159.1, 156.1, 155.3,
154.7, 154.0, 151.0, 144.9, 142.1, 140.4, 140.2, 139.9, 136.2,
133.1, 129.7, 129.6, 128.8, 123.6, 93.8, 78.7, 66.4, 54.2, 51.3,
47.4, 42.0, 30.4, 30.2, 27.5, 10.9. FT-IR (KBr, cm¹1): 3399,
3133, 2976, 1705, 1592, 1367, 1155, 1101, 968, 756, 725.
HRMS (ESI-TOF): Calcd for C38H42N8O12SNa [M + Na]+:
857.2541, Found: 857.2543.
General Procedure for the Preparation of Macrolactams
15a-15d (Method A). To a solution of hexaoxazole amide
14a-14d (1 equiv) in 1,4-dioxane (10 mL mmol¹1) was added
4 M HCl/1,4-dioxane (60 mL mmol¹1) at 0 °C. After being
stirred at room temperature for 2 h, the reaction mixture was
concentrated in vacuo. The residue was used for the next
reaction without further purification.
To a solution of the residue in THF (50 mL mmol¹1), MeOH
(50 mL mmol¹1), and H2O (16 mL mmol¹1) was added LiOH¢
H2O (0.1 M) at 0 °C, and then warmed up to room temperature.
After being stirred at the same temperature for 2 h, the reaction

K. Shibata et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013) 1461
mixture was quenched with 4 M HCl/1,4-dioxane at 0 °C, and
concentrated in vacuo. The residue was dried under reduced
pressure for 12 h and used for the next reaction without further
purification.
¤ 163.0, 161.5, 161.1, 160.0, 156.6, 156.3, 155.7, 154.4, 151.5,
151.4, 151.1, 141.1, 140.9, 139.9, 137.0, 136.8, 130.2, 126.3,
126.0, 125.1, 65.1, 51.7, 48.6, 42.9, 33.1, 31.0, 12.0, 11.9. FT-
IR (KBr, cm¹1): 3391, 2926, 1667, 1596, 1511, 1170, 1107,
1071, 930, 746, 725, 574. HRMS (ESI-TOF): Calcd for
C31H31N8O9S [M + H]+: 691.1935, Found: 691.1923.
To a solution of DPPA/HOBt (30 equiv), DIEA (40 equiv)
and DMAPO (1.0 equiv) in CH2Cl2 (160 mL mmol¹1) and DMF
(80 mL mmol¹1) was added dropwise a solution of the residue in
DMF (80 mL mmol¹1) at room temperature under argon. After
being stirred at room temperature for 3 d, the reaction mixture
was poured into 3 M HCl at 0 °C and the aqueous layer was
extracted with ethyl acetate. The combined organic layer was
washed with 3 M HCl, saturated aqueous NaHCO3 and brine,
dried over MgSO4 and filtered. After removal of the solvent, the
residue was purified by column chromatography on silica gel
and by GPC eluted with CHCl3 to give macrolactam 15a-15d.
Preparation of Macrolactam 15e (Method B).
To a
solution of hexaoxazole amide 15e (202 mg, 0.221 mmol) in
1,2-dichloroethane (6.6 mL) was added trimethyltin hydroxide
(277 mg, 1.55 mmol) at room temperature under argon. After
being stirred at 80 °C for 8 h, the reaction mixture was diluted
with CHCl3 and 3 M HCl, and the aqueous layer was extracted
with CHCl3. The combined organic layer was washed with 3 M
HCl and brine, dried over Na2SO4 and filtered. After removal
of the solvent, the residue was used for the next reaction
without further purification.
To a solution of the residue in 1,4-dioxane (4.0 mL) was
added 4 M HCl/1,4-dioxane (22.0 mL) at 0 °C. After being
stirred at room temperature for 4 h, the reaction mixture was
concentrated in vacuo. The residue was used for the next reac-
tion without further purification.
28
15a: 7% yield in 3 steps. ½ꢀꢀD +4.39 (c 0.230, CHCl3).
1H NMR (400 MHz, CD2Cl2): ¤ 8.55 (d, J = 5.80 Hz, 1H), 8.51
(d, J = 6.77 Hz, 1H), 8.31-8.24 (m, 6H), 5.52 (m, 1H), 5.40 (m,
1H), 4.19 (m, 1H), 4.01 (m, 1H), 3.25 (dd, J = 13.1, 5.80 Hz,
1H), 3.20 (dd, J = 13.1, 4.83 Hz, 1H), 1.23 (s, 9H). 13C NMR
(100 MHz, CD2Cl2): ¤ 164.2, 162.9, 161.0, 159.8, 156.8, 156.6,
155.3, 155.1, 141.4, 141.2, 140.7, 140.3, 139.3 © 2, 137.3,
137.1, 131.4 © 2, 130.1, 130.0, 65.3, 52.0, 48.9, 43.0, 33.0,
31.0. FT-IR (KBr, cm¹1): 3388, 3123, 2927, 1668, 1597, 1512,
1189, 1108, 1069, 916, 760, 724, 550. HRMS (ESI-TOF): Calcd
for C28H25N8O9S [M + H]+: 649.1465, Found: 649.1466.
To a solution of HOBt (299 mg, 2.21 mmol) in CH2Cl2 (37
mL) and DMF (17 mL) was added DIEA (0.28 mL, 1.7 mmol),
DMAPO (30.5 mg, 0.221 mmol), and DPPA (0.48 mL, 2.2
mmol) at room temperature under argon. To the reaction mixture
was added dropwise a solution of the residue in DMF (20.0 mL)
and DIEA (0.28 mL, 1.7 mmol) at the same temperature. After
being stirred at the same temperature for 3 d, the reaction mix-
ture was concentrated in vacuo. The residue was diluted with
ethyl acetate and 3 M HCl, and the aqueous layer was extracted
with ethyl acetate. The combined organic layer was washed with
3 M HCl, saturated aqueous NaHCO3 and brine, dried over
MgSO4 and filtered. After removal of the solvent, the residue
was purified by column chromatography on silica gel (0% to
40% ethyl acetate in hexane, then 1% to 8% MeOH in CHCl3)
and by GPC eluted with CHCl3 to give macrolactam 15e (95.2
28
15b: 47% yield in 3 steps. ½ꢀꢀD +1.45 (c 0.445, CHCl3).
1H NMR (400 MHz, CD2Cl2): ¤ 8.55 (d, J = 6.28 Hz, 1H), 8.45
(d, J = 7.25 Hz, 1H), 8.30 (s, 1H), 8.29 (s, 1H), 8.26 (s, 1H),
8.25 (s, 1H), 8.23 (s, 1H), 5.46 (m, 1H), 5.40 (m, 1H), 4.18 (m,
1H), 4.01 (m, 1H), 3.26 (dd, J = 13.1, 5.80 Hz, 1H), 3.17 (dd,
J = 13.1, 4.35 Hz, 1H), 2.70 (s, 3H), 1.24 (s, 9H). 13C NMR
(100 MHz, CD2Cl2): ¤ 163.0, 161.6, 161.1, 159.8, 158.0, 156.6,
155.5, 155.1, 152.3, 141.4, 141.1, 140.6, 139.2, 139.1, 137.3,
137.1, 131.4, 131.1, 130.1, 125.0, 65.3, 51.9, 48.7, 42.9, 33.0,
31.0, 12.0. FT-IR (KBr, cm¹1): 3375, 3103, 2928, 1662, 1598,
1511, 1191, 1104, 1070, 916, 755, 725, 565. HRMS (ESI-
TOF): Calcd for C29H27N8O9S [M + H]+: 663.1622, Found:
663.1624.
27
mg, 0.128 mmol, 58% in 3 steps). ½ꢀꢀD ¹3.8 (c 0.095, CHCl3).
1H NMR (400 MHz, DMSO-d6, 80 °C): ¤ 8.98 (s, 1H), 8.96 (s,
1H), 8.82 (s, 1H), 8.79 (s, 1H), 8.30 (brd, J = 7.73 Hz, 2H), 5.63
(m, 1H), 5.37 (ddd, J = 7.73, 3.87, 3.87 Hz, 1H), 5.07 (brs, 1H),
3.92 (m, 2H), 3.34 (dd, J = 13.5, 5.80 Hz, 1H), 3.20 (dd, J =
13.5, 4.35 Hz, 1H), 2.77 (s, 3H), 1.23 (s, 9H). 13C NMR (100
MHz, DMSO-d6): ¤ 163.8, 163.3, 158.8, 158.7, 155.7, 155.2,
154.6, 152.4, 151.7, 142.6, 142.3, 141.7, 141.0, 136.0, 135.8,
129.7, 128.4, 127.3, 125.2, 124.1, 62.0, 50.3, 47.9, 42.4, 31.4,
30.4, 11.6. FT-IR (KBr, cm¹1): 3358, 3101, 2925, 1660, 1595,
1506, 1356, 1107, 1071, 915, 723. HRMS (ESI-TOF): Calcd for
C29H26N8O9S79Br [M + H]+: 741.0727, Found: 741.0729, and
C29H26N8O9S81Br [M + H]+: 743.0706, Found: 743.0709.
General Procedure for the Preparation of Macrocyclic
Enamides (R)-16b-16e. To a solution of macrolactam 15b-
15e (1 equiv) in CH2Cl2 (20 mL mmol¹1) was added Et3N
(15 equiv) and MsCl (7.5 equiv) at 0 °C under argon. After
being stirred at room temperature for 30 min, to the reaction
mixture was added DBU (5.0 equiv) at 0 °C. After being stirred
at 0 °C to room temperature for 2 h, the reaction mixture was
diluted with ethyl acetate and 1 M HCl, and the aqueous layer
was extracted with ethyl acetate. The combined organic layer
was washed with 1 M HCl, saturated aqueous NaHCO3 and
18
15c: 54% yield in 3 steps. ½ꢀꢀD ¹6.61 (c 0.510, CHCl3).
1H NMR (400 MHz, CD2Cl2): ¤ 8.54 (d, J = 6.28 Hz, 1H), 8.41
(d, J = 7.25 Hz, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.24 (s, 1H),
8.23 (s, 1H), 5.47 (m, 1H), 5.38 (m, 1H), 4.17 (dd, J = 11.6,
3.38 Hz, 1H), 4.00 (dd, J = 11.6, 4.83 Hz, 1H), 3.24 (dd, J =
13.1, 5.80 Hz, 1H), 3.16 (dd, J = 13.1, 4.35 Hz, 1H), 2.72 (s,
3H), 2.70 (s, 3H), 1.24 (s, 9H). 13C NMR (100 MHz, CD2Cl2):
¤ 162.8, 161.6, 161.0, 160.0, 158.0, 156.3, 155.4, 154.3, 152.2,
151.4, 141.3, 140.9, 140.0, 139.1, 137.0, 131.1, 130.2, 126.4,
125.0, 65.3, 48.6, 42.9, 33.0, 31.0, 12.0, 11.9. FT-IR (KBr,
cm¹1): 3387, 2925, 1667, 1598, 1513, 1190, 1109, 1073, 931,
758, 726, 576. HRMS (ESI-TOF): Calcd for C30H29N8O9S
[M + H]+: 677.1778, Found: 677.1778.
27
15d: 58% yield in 3 steps. ½ꢀꢀD ¹4.21 (c 0.395, CHCl3).
1H NMR (400 MHz, CD2Cl2): ¤ 8.50 (d, J = 6.77 Hz, 1H), 8.39
(d, J = 7.25 Hz, 1H), 8.25 (s, 1H), 8.24 (s, 1H), 8.22 (s, 1H),
5.45 (m, 1H), 5.38 (m, 1H), 4.17 (dd, J = 11.6, 2.90 Hz, 1H),
4.00 (dd, J = 11.6, 4.83 Hz, 1H), 3.23 (dd, J = 13.1, 5.80
Hz, 1H), 3.15 (dd, J = 13.1, 4.35 Hz, 1H), 2.72 (s, 3H), 2.71 (s,
3H), 2.68 (s, 3H), 1.24 (s, 9H). 13C NMR (100 MHz, CD2Cl2):

1462 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
Synthesis of Heptaoxazole Macrocyclic Analogues
brine, dried over Na2SO4 and filtered. After removal of the
solvent, the residue was purified by column chromatography on
silica gel to give macrocyclic enamide (R)-16b-16e.
ed with 10% aqueous Na2S2O3 and the aqueous layer was
extracted with ethyl acetate. The combined organic layer
was washed with brine, dried over MgSO4 and filtered. After
removal of the solvent, the residue was purified by column
chromatography on silica gel and by GPC eluted with CHCl3
to give bromide.
To a solution of the bromide (1 equiv) in 1,4-dioxane (0.10
mL mg¹1) was added K2CO3 (3.0 equiv) at room temperature
under argon. After being stirred at 60 °C for 8 h, the reaction
mixture was filtered through a pad of Celite. The filtrate was
concentrated in vacuo. The residue was purified by column
chromatography on silica gel to give oxazoline.
27
(R)-16b: 76% yield. ½ꢀꢀD +2.09 (c 0.390, CHCl3). 1H NMR
(400 MHz, CD2Cl2): ¤ 9.61 (brs, 1H), 8.46 (brd, J = 6.77 Hz,
1H), 8.32 (s, 2H), 8.30 (s, 1H), 8.28 (s, 1H), 8.24 (s, 1H), 6.75
(s, 1H), 5.85 (s, 1H), 5.44 (m, 1H), 3.30 (dd, J = 13.0, 5.80 Hz,
1H), 3.18 (dd, J = 13.0, 3.87 Hz, 1H), 2.71 (s, 3H), 1.23 (s,
9H). 13C NMR (100 MHz, CD2Cl2): ¤ 161.5, 159.9, 159.6,
159.5, 156.2, 155.1, 152.5, 152.4, 146.6, 142.0, 141.1, 140.5,
139.5, 139.3, 137.7, 137.3, 130.9, 128.7, 124.2, 104.0, 48.9,
42.8, 31.0, 30.1, 12.0. FT-IR (KBr, cm¹1): 3363, 3120, 2925,
1661, 1589, 1516, 1354, 1186, 1116, 916, 725. HRMS (ESI-
TOF): Calcd for C29H25N8O8S [M + H]+: 645.1516, Found:
645.1490.
A solution of the oxazoline (1 equiv) and 5 ¡ molecular
sieves in dry toluene (150 mL mmol¹1) was stirred at room
temperature for 1 h under argon to remove a trace amount of
water. To the mixture was added camphorsulfonic acid (CSA)
(5.0 equiv) (azeotroped with toluene) at room temperature
under argon. After being stirred at 70 °C for 14 h, the reac-
tion mixture was filtered through a pad of Celite. The filtrate
was quenched with saturated aqueous NaHCO3 at 0 °C and the
aqueous layer was extracted with ethyl acetate and CHCl3. The
combined organic layer was washed with brine, dried over
Na2SO4 and filtered. After removal of the solvent, the residue
was purified by silica gel column chromatography to give
heptaoxazole macrocycle (R)-3b-3e.
27
(R)-16c: 73% yield. ½ꢀꢀD +2.20 (c 0.605, CHCl3). 1H NMR
(400 MHz, CD2Cl2): ¤ 9.59 (brs, 1H), 8.41 (brd, J = 6.77 Hz,
1H), 8.31 © 2 (each s, 2H), 8.26 (s, 1H), 8.24 (s, 1H), 6.73
(s, 1H), 5.82 (s, 1H), 5.44 (m, 1H), 3.28 (dd, J = 13.0, 5.80
Hz, 1H), 3.17 (dd, J = 13.0, 4.35 Hz, 1H), 2.73 (s, 3H), 2.70
(s, 3H), 1.22 (s, 9H). 13C NMR (100 MHz, CD2Cl2): ¤ 161.5,
160.1, 159.6, 159.3, 158.0, 156.3, 155.4, 154.2, 152.4, 151.4,
142.0, 140.8, 140.0, 139.4, 137.6, 137.0, 131.1, 131.0, 128.8,
126.4, 125.0, 103.8, 48.8, 42.8, 32.7, 30.9, 12.0, 11.9. FT-IR
(KBr, cm¹1): 3378, 3123, 2926, 1662, 1589, 1520, 1356, 1193,
1113, 1074, 931, 702. HRMS (ESI-TOF): Calcd for C30H27-
N8O8S [M + H]+: 659.1673, Found: 659.1642.
26
1
(R)-3b: 15% yield. ½ꢀꢀD +11.9 (c 0.100, CHCl3). H NMR
(400 MHz, CD2Cl2): ¤ 8.22 (s, 1H), 8.20 (s, 1H), 8.18 (s, 1H),
8.15 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 8.07 (brs, 1H), 5.45 (m,
1H), 3.26 (dd, J = 13.5, 5.80 Hz, 1H), 3.12 (dd, J = 13.5,
4.35 Hz, 1H), 2.67 (s, 3H), 1.19 (s, 9H). FT-IR (KBr, cm¹1):
3420, 3138, 2925, 1667, 1590, 1496, 1365, 1262, 1103, 917,
716. HRMS (ESI-TOF): Calcd for C29H23N8O8S [M + H]+:
643.1360, Found: 643.1359.
18
(R)-16d: 78% yield. ½ꢀꢀD +2.52 (c 0.275, CHCl3). 1H NMR
(400 MHz, CD2Cl2): ¤ 9.54 (brs, 1H), 8.41 (brd, J = 6.77 Hz,
1H), 8.34 (s, 1H), 8.25 (s, 1H), 8.23 (s, 1H), 6.74 (s, 1H), 5.83
(s, 1H), 5.42 (m, 1H), 3.26 (dd, J = 13.5, 5.80 Hz, 1H), 3.16
(dd, J = 13.5, 4.35 Hz, 1H), 2.75 (s, 3H), 2.73 (s, 3H), 2.69
(s, 3H), 1.22 (s, 9H). 13C NMR (100 MHz, CD2Cl2): ¤ 161.4,
160.0, 159.8, 159.3, 156.7, 156.3, 155.7, 154.3, 151.7, 151.4,
151.3, 141.8, 140.7, 139.9, 137.4, 137.1, 131.0, 128.8, 126.5,
126.0, 125.0, 103.9, 48.7, 42.8, 32.8, 31.0, 12.1, 12.0, 11.9.
FT-IR (KBr, cm¹1): 3378, 3120, 2961, 1664, 1590, 1519, 1365,
1169, 1111, 1072, 872, 727, 703. HRMS (ESI-TOF): Calcd for
C31H29N8O8S [M + H]+: 673.1829, Found: 673.1830.
26
1
(R)-3c: 20% yield. ½ꢀꢀD +4.4 (c 0.065, CHCl3). H NMR
(400 MHz, CD2Cl2): ¤ 8.21 (s, 1H), 8.14 (s, 1H), 8.10 (s, 1H),
8.09 (s, 1H), 8.08 (s, 1H), 7.98 (brs, 1H), 5.42 (m, 1H), 3.24
(dd, J = 13.5, 6.28 Hz, 1H), 3.10 (dd, J = 13.5, 4.35 Hz, 1H),
2.67 (s, 6H), 1.17 (s, 9H). FT-IR (KBr, cm¹1): 3419, 3158,
2925, 1662, 1592, 1505, 1362, 1191, 1080, 914, 715. HRMS
(ESI-TOF): Calcd for C30H25N8O8S [M + H]+: 657.1516,
Found: 657.1530.
(R)-16e: 76% yield as a white solid. Mp >300 °C (decomp.).
26
1
½ꢀꢀD +4.36 (c 0.270, CH2Cl2). H NMR (400 MHz, DMSO-
d6): ¤ 9.42 (s, 1H), 9.19 (s, 1H), 9.15 (s, 1H), 9.05 (s, 1H), 8.96
(s, 1H), 8.29 (d, J = 6.77 Hz, 1H), 6.73 (s, 1H), 5.88 (s, 1H),
5.66 (m, 1H), 3.40 (dd, J = 13.5, 4.83 Hz, 1H), 3.17 (dd, J =
13.5, 3.38 Hz, 1H), 2.78 (s, 3H), 1.17 (s, 9H). 13C NMR (100
MHz, DMSO-d6): ¤ 163.7, 158.8, 158.7, 158.1, 155.5, 155.2,
154.5, 152.4, 152.3, 143.6, 142.5, 141.3, 135.8 © 2, 129.7,
129.4, 127.8, 127.2, 125.1, 124.5, 103.7, 48.1, 42.4, 31.1, 30.4,
11.8. FT-IR (KBr, cm¹1): 3380, 3123, 2925, 1682, 1589, 1519,
1355, 1118, 1075, 1032, 916, 725. HRMS (ESI-TOF): Calcd for
C29H24N8O8S79Br [M + H]+: 723.0621, Found: 723.0623, and
C29H24N8O8S81Br [M + H]+: 725.0601, Found: 725.0607.
General Procedure for the Synthesis of Heptaoxazole
Macrocycles 3b-3e. To a solution of macrocyclic enamide
(R)-16b-16e (1 equiv) and 4 ¡ molecular sieves in CH2Cl2
(0.10 mL mg¹1) and MeOH (1.0 mL) was added a solution of
NBS (1.05 equiv) in CH2Cl2 at 0 °C under argon. After being
stirred at the same temperature for 2 h, the reaction mixture
was filtered through a pad of Celiteμ. The filtrate was quench-
26
1
(R)-3d: 22% yield. ½ꢀꢀD +8.07 (c 0.310, CHCl3). H NMR
(400 MHz, CDCl3): ¤ 8.19 (s, 3H), 8.15 (s, 1H), 8.03 (brs, 1H),
5.28 (brs, 1H), 3.20 (dd, J = 13.1, 5.32 Hz, 1H), 3.09 (brd,
J = 13.1 Hz, 1H), 2.66 (brs, 9H), 1.16 (s, 9H). FT-IR (KBr,
cm¹1): 3409, 3138, 2925, 1673, 1591, 1497, 1365, 1176, 1076,
915, 801, 716. HRMS (ESI-TOF): Calcd for C31H27N8O8S
[M + H]+: 671.1673, Found: 671.1674.
(R)-3e: 28% yield as a pale yellow solid. Mp >300 °C
27
1
(decomp.). ½ꢀꢀD +14.7 (c 0.120, CHCl3). H NMR (400 MHz,
DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H), 8.88 (s, 2H),
8.81 (s, 1H), 8.54 (brd, J = 8.22 Hz, 1H), 5.64 (m, 1H), 3.28
(dd, J = 13.5, 6.28 Hz, 1H), 3.14 (dd, J = 13.5, 4.83 Hz, 1H),
2.76 (s, 3H), 1.23 (s, 9H). 13C NMR (100 MHz, CD2Cl2): ¤
163.9, 160.3, 157.1, 156.8, 156.7, 156.4, 155.4, 153.7, 150.2,
142.3, 139.3, 138.3, 137.5, 137.3, 137.0, 131.2, 130.7, 130.6,
130.5 © 2, 126.4, 49.2, 43.1, 33.1, 30.9, 11.7. FT-IR (KBr,
cm¹1): 3413, 3144, 2924, 1668, 1587, 1495, 1359, 1268,
1122, 1084, 1027, 918, 716. HRMS (ESI-TOF): Calcd for

K. Shibata et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013) 1463
C29H22N8O8S79Br [M + H]+: 721.0465 Found: 721.0449, and
C29H22N8O8S81Br [M + H]+: 723.0444 Found: 723.0446.
General Procedure for Palladium-Catalyzed Suzuki-
J = 2.90, 0.97 Hz, 1H), 7.79 (dd, J = 4.83, 2.90 Hz, 1H), 7.73
(dd, J = 4.83, 0.97 Hz, 1H), 5.63-5.68 (m, 1H), 3.30 (dd,
J = 13.0, 6.77 Hz, 1H), 3.21 (dd, J = 13.0, 5.32 Hz, 1H),
2.78 (s, 3H), 1.24 (s, 9H). 13C NMR (67.8 MHz, CD2Cl2): ¤
160.8, 160.3, 157.3, 156.8, 156.6, 156.4, 155.3, 149.8, 147.4,
146.4, 142.4, 139.3, 138.3, 137.5, 137.4, 137.1, 131.1, 130.5,
130.3 © 2, 128.1, 127.8, 127.7, 127.2, 126.8, 126.4, 49.0, 43.0,
33.6, 31.0, 11.7. FT-IR (KBr, cm¹1): 3410, 3139, 2925, 1667,
1584, 1505, 1364, 1269, 1178, 1120, 1087, 917, 716. HRMS
(ESI-TOF): Calcd for C33H25N8O8S2 [M + H]+: 725.1237,
Found: 725.1235.
Miyaura Coupling of (R)-3e with Arylboronic Acids.
A
screw cap test tube was charged with Pd(OAc)2 (20 mol %)
and ligand (SPhos or XPhos, 40 mol %) under argon. To the
reaction vessel was added bromooxazole-containing hepta-
oxazole macrocycle (R)-3e (1 equiv), solvent (1,4-dioxane or
1,2-dimethoxyethane, 0.03 M), KF (8.0 equiv) and arylboronic
acid (6.0 equiv). After being stirred at 80 °C for 24 h, the
reaction mixture was diluted with CHCl3 and brine, and the
aqueous layer was extracted with CHCl3. The combined
organic layer was washed with saturated aqueous NaHCO3
and brine, dried over Na2SO4 and filtered. After removal of the
solvent, the residue was purified by column chromatography on
silica gel to give the coupling product (R)-4.
26
1
(R)-4e: 44% yield. ½ꢀꢀD +2.76 (c 0.250, CH2Cl2). H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H), 8.88
(s, 1H), 8.87 (s, 1H), 8.80 (s, 1H), 8.62 (brd, J = 8.22 Hz, 1H),
7.81 (d, J = 9.18 Hz, 2H), 6.89 (d, J = 9.18 Hz, 2H), 5.63 (m,
1H), 3.28 (dd, J = 13.5, 7.25 Hz, 1H), 3.19 (dd, J = 13.5,
4.83 Hz, 1H), 3.04 (s, 6H), 2.72 (s, 3H), 1.25 (s, 9H). 13C NMR
(100 MHz, CD2Cl2): ¤ 160.3, 160.0, 157.5, 156.8, 156.6, 156.5,
156.1, 155.3, 152.4, 151.9, 149.2, 142.3, 139.2, 138.2, 137.5,
137.4, 136.9, 131.2, 130.6, 129.2, 126.2, 121.4, 114.0, 111.9,
49.0, 43.0, 40.3, 33.5, 31.0, 11.6. FT-IR (KBr, cm¹1): 3419,
3148, 2926, 1667, 1607, 1500, 1367, 1200, 1171, 1088,
918, 819, 715. HRMS (ESI-TOF): Calcd for C37H32N9O8S
[M + H]+: 762.2095, Found: 762.2095.
28
(R)-4a: 73% yield. ½ꢀꢀD +3.74 (c 0.255, CH2Cl2). 1H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H),
8.88 © 2 (each s, 2H), 8.81 (s, 1H), 8.70 (brd, J = 8.22 Hz,
1H), 7.97 (d, J = 6.77 Hz, 2H), 7.63-7.55 (m, 3H), 5.68 (m,
1H), 3.30 (dd, J = 13.5, 6.77 Hz, 1H), 3.21 (dd, J = 13.5,
5.32 Hz, 1H), 2.71 (s, 3H), 1.25 (s, 9H). 13C NMR (100 MHz,
CD2Cl2): ¤ 161.7, 160.3, 157.3, 156.8, 156.6, 156.4, 155.3,
151.1, 150.0, 142.4, 139.4, 138.4, 137.7, 137.4, 137.1, 131.1,
130.8, 130.4, 130.2, 129.2, 128.0, 127.0 © 2, 126.2, 123.9,
49.0, 43.1, 33.6, 31.0, 11.6. FT-IR (KBr, cm¹1): 3428, 3153,
2927, 1667, 1585, 1504, 1364, 1269, 1181, 1120, 1089, 918,
716. HRMS (ESI-TOF): Calcd for C35H27N8O8S [M + H]+:
719.1673, Found: 719.1671.
26
1
(R)-4f: 65% yield. ½ꢀꢀD ¹7.26 (c 0.125, CHCl3). H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H), 8.88
(s, 1H), 8.87 (s, 1H), 8.81 (s, 1H), 8.64 (brd, J = 7.73 Hz, 1H),
7.85 (d, J = 9.18 Hz, 2H), 7.12 (d, J = 9.18 Hz, 2H), 5.66-5.61
(m, 1H), 3.79 (t, J = 4.83 Hz, 4H), 3.30 (t, J = 4.83 Hz, 4H),
3.28 (m, 1H), 3.19 (dd, J = 13.5, 4.83 Hz, 1H), 2.72 (s, 3H),
1.25 (s, 9H). 13C NMR (100 MHz, DMSO-d6): ¤ 160.4, 159.6,
156.1, 155.9, 155.7, 155.5, 154.8, 154.7, 154.4, 151.9, 149.1,
143.4, 140.7, 139.7, 138.9, 138.4, 136.5, 130.0, 129.7 © 2,
129.5, 128.5, 121.5, 116.0, 114.1, 65.9, 47.9, 47.2, 42.5, 32.7,
30.6, 11.2. FT-IR (KBr, cm¹1): 3421, 3136, 2926, 1730, 1668,
1607, 1495, 1364, 1270, 1122, 1078, 919, 832, 716. HRMS
(ESI-TOF): Calcd for C39H34N9O9S [M + H]+: 804.2200,
Found: 804.2189.
28
1
(R)-4b: 72% yield. ½ꢀꢀD +9.3 (c 0.080, CHCl3). H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H), 8.88
(s, 1H), 8.87 (s, 1H), 8.81 (s, 1H), 8.65 (brd, J = 8.22 Hz, 1H),
7.92 (d, J = 9.18 Hz, 2H), 7.17 (d, J = 9.18 Hz, 2H), 5.65 (m,
1H), 3.89 (s, 3H), 3.29 (dd, J = 13.5, 7.25 Hz, 1H), 3.20 (dd,
J = 13.5, 4.83 Hz, 1H), 2.71 (s, 3H), 1.25 (s, 9H). 13C NMR
(67.8 MHz, CD2Cl2): ¤ 161.7, 161.0, 160.3, 157.3, 156.8,
156.6, 156.4, 155.6, 155.3, 152.0, 151.2, 149.5, 142.3, 139.2,
138.2, 137.4, 137.0, 131.2, 130.6, 130.4, 129.7, 126.3, 123.1,
122.9, 119.5, 114.6, 55.9, 49.0, 43.0, 33.6, 31.0, 11.6. FT-IR
(KBr, cm¹1): 3419, 3152, 2927, 1667, 1586, 1495, 1363, 1263,
1182, 1119, 1087, 918, 838, 715. HRMS (ESI-TOF): Calcd for
C36H29N8O9S [M + H]+: 749.1778, Found: 749.1788.
General Procedure for S-Alkylation of (R)-3e with
α-Bromoacetamides 17. To a solution of (R)-3e (1 equiv)
in CH2Cl2 (40 mL mmol¹1) was added trifluoroacetic acid (60
mL mmol¹1), dimethyl sulfoxide (40 equiv), and trimethylsilyl
chloride (100 equiv) at 0 °C. After being stirred at the same
temperature for 1 h, the reaction mixture was concentrated
in vacuo. The residue was quenched with toluene and Et3N at
0 °C. After removal of the solvent, the residue was used for the
next reaction without further purification.
To a solution of the crude disulfide in CH3CN (50
mL mmol¹1) and H2O (25 mL mmol¹1) was added DIEA (20
equiv) at 0 °C. To the reaction mixture was added dithiothreitol
(1.5 equiv) in H2O (25 mL mmol¹1) at room temperature, and
the reaction mixture was sonicated for 2 min. To the reaction
mixture was added α-bromoacetamide 17 (5.0 equiv) at room
temperature, and the reaction mixture was sonicated for 2 min.
After being stirred at the same temperature for 3 h, the reaction
mixture was concentrated in vacuo. The residue was purified by
preparative reversed-phase HPLC (Senshu Pak PEGASIL-ODS,
5 ¯m, 20 © 250 mm, UV detection at 254 nm, 10 mL min¹1) to
give sulfide (R)-18.
27
1
(R)-4c: 70% yield. ½ꢀꢀD ¹3.81 (c 0.255, CH2Cl2). H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H),
8.88 © 2 (each s, 2H), 8.81 (s, 1H), 8.70 (brd, J = 8.22 Hz,
1H), 8.03 (dd, J = 9.18, 5.32 Hz, 2H), 7.44 (dd, J = 9.18,
8.70 Hz, 2H), 5.67 (m, 1H), 3.30 (dd, J = 13.5, 6.77 Hz, 1H),
3.20 (dd, J = 13.5, 5.32 Hz, 1H), 2.71 (s, 3H), 1.25 (s, 9H).
13C NMR (67.8 MHz, CD2Cl2): ¤ 164.1 (d, J = 251 Hz), 161.8,
160.4, 157.3, 156.9, 156.7, 156.5, 155.4, 155.2, 149.9, 149.8,
142.3, 139.1, 138.1, 137.5, 137.4, 137.0, 131.3, 130.8, 130.6,
130.5, 130.3 (d, J = 8.4 Hz), 126.6, 124.1, 123.6, 116.3 (d,
J = 22.3 Hz), 49.1, 43.1, 33.8, 31.0, 11.7. FT-IR (KBr, cm¹1):
3486, 3154, 2926, 1667, 1585, 1494, 1364, 1240, 1164, 1086,
918, 841, 715. HRMS (ESI-TOF): Calcd for C35H26N8O8SF
[M + H]+: 737.1578, Found: 737.1597.
28
(R)-4d: 63% yield. ½ꢀꢀD ¹1.72 (c 0.165, CH2Cl2). 1H NMR
(400 MHz, DMSO-d6, 80 °C): ¤ 8.97 (s, 1H), 8.93 (s, 1H), 8.88
(s, 2H), 8.81 (s, 1H), 8.65 (brd, J = 8.22 Hz, 1H), 8.32 (dd,

1464 Bull. Chem. Soc. Jpn. Vol. 86, No. 12 (2013)
Synthesis of Heptaoxazole Macrocyclic Analogues
27
(R)-18a: 33% yield. ½ꢀꢀD +1.95 (c 0.235, CHCl3). 1H NMR
1.39-1.21 (m, 10H). HRMS (ESI-TOF): Calcd for C35H34-
N10O9S79Br [M + H]+: 849.1414, Found: 849.1414, and
C35H34N10O9S81Br [M + H]+: 851.1394, Found 851.1400.
(R)-5c: 1H NMR (400 MHz, CD3OD): ¤ 8.77 (s, 1H), 8.76 (s,
1H), 8.70 (s, 2H), 8.61 (s, 1H), 5.72 (brs, 1H), 4.66 (brs, 1H),
3.99-3.90 (m, 2H), 3.55-3.47 (m, 3H), 3.34 (m, 1H), 3.29-2.99
(m, 8H), 2.79 (s, 3H). HRMS (ESI-TOF): Calcd for C33H30-
N10O11S79Br [M + H]+: 853.1000, Found: 853.0997, and
C33H30N10O11S81Br [M + H]+: 855.0979, Found: 855.0976.
TRAP Assay. The oligonucleotides, Ts (5¤-AATCCGTC-
GAGCAGAGTT-3¤) and CX primer (5¤-CCCTTACCCTTA-
CCCTAACCCTAA-3¤), were synthesized by Sigma Genosys.
The TRAP reaction was carried out in two steps (elongation and
amplification). Elongation reactions were carried out at 30 °C
for 30 min in a final volume of 40 mL containing 1 © 104 cells
of CHAPS extract from Namalva cells. The buffer contained
20 mM Tris¢HCl (pH 8.3), 63 mM KCl, 1.5 mM MgCl2, 1 mM
(400 MHz, CD2Cl2): ¤ 8.57 (brs, 1H), 8.28 (s, 1H), 8.24 (s, 1H),
8.22 (s, 1H), 8.17 (s, 1H), 8.16 (s, 1H), 7.20 (brs, 1H), 5.56 (brs,
1H), 4.80 (brs, 1H), 3.28 (dd, J = 14.5, 3.87 Hz, 1H), 3.15-3.12
(m, 5H), 3.00-2.95 (m, 2H), 2.70 (s, 3H), 1.29-1.24 (m, 13H).
13C NMR (67.8 MHz, CDCl3): ¤ 168.3, 163.2, 160.4, 156.6,
156.3, 156.2, 156.1, 155.9, 155.0, 153.2, 149.9, 142.4, 139.0,
138.0, 137.3, 136.7, 130.8, 130.2, 130.0 © 2, 128.5, 126.0,
122.8, 70.7, 48.9, 40.2, 39.5, 32.0, 29.8, 28.5, 27.4, 26.6, 11.6.
FT-IR (KBr, cm¹1): 3349, 3089, 2928, 1668, 1586, 1506, 1365,
1266, 1173, 1123, 919, 717. HRMS (ESI-TOF): Calcd for
C36H34N10O11S79Br [M + H]+: 893.1313, Found: 893.1318,
and C36H34N10O11S81Br [M + H]+: 895.1292, Found:
895.1285.
27
(R)-18b: 29% yield. ½ꢀꢀD +4.66 (c 0.240, CHCl3). 1H NMR
(400 MHz, CD2Cl2): ¤ 8.50 (brs, 1H), 8.25 (s, 2H), 8.22 (s,
1H), 8.17 (s, 1H), 8.16 (s, 1H), 6.99 (brs, 1H), 5.55 (brs, 1H),
4.56 (brs, 1H), 3.27 (dd, J = 14.0, 5.32 Hz, 1H), 3.16-3.08 (m,
5H), 3.00 (dt, J = 6.77, 6.28 Hz, 2H), 2.70 (s, 3H), 1.41-1.33
(m, 13H), 1.29-1.24 (m, 8H). 13C NMR (100 MHz, CD2Cl2): ¤
168.3, 164.0, 160.5, 157.2, 156.8, 156.7, 156.5, 155.4, 153.7,
150.6, 142.7, 139.5, 138.5, 137.8, 137.2, 130.6, 130.5, 130.4,
130.3, 128.8, 126.2, 123.1, 70.9, 49.0, 40.9, 40.1, 32.3, 30.4,
30.1, 29.7, 29.5, 28.5, 27.2, 27.1, 11.7. FT-IR (KBr, cm¹1):
3326, 3096, 2926, 1668, 1586, 1516, 1365, 1251, 1173, 1124,
918, 717. HRMS (ESI-TOF): Calcd for C40H42N10O11S79Br
[M + H]+: 949.1939, Found: 949.1936, and C40H42N10O11-
S81Br [M + H]+: 951.1918, Found: 951.1917.
¹1
EGTA, 0.005% Tween 20, 0.05 mg mL T4 gene 32 protein,
¹1
and 0.1 mg mL BSA supplemented with 50 mM dNTP and
0.3 mM Ts primer. Reactions were stopped by heating for 2 min
at 94 °C, and the samples were placed on ice. The amplification
step was performed on ice by adding to the samples 5 mL of a
PCR solution containing 0.3 mM CX primer and 0.2 units/mL
Taq polymerase. The PCRs were then performed for amplifi-
cation as follows: 94 °C for 2 min and then 28 cycles as follows:
94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s. Telomerase
extension products were analyzed by 10% PAGE in 1X TBE,
and the gels were scanned with a Typhoon (GE Healthcare). The
extension products were quantified by using ImageQuant 5.2
software and normalized to the signal of the extension products
in the control solvent.
27
(R)-18c: 28% yield. ½ꢀꢀD +11.6 (c 0.225, CHCl3). 1H NMR
(400 MHz, CD2Cl2): ¤ 8.78 (brs, 1H), 8.29 (s, 1H), 8.25 (s, 1H),
8.24 (s, 1H), 8.20 (s, 1H), 8.18 (s, 1H), 7.36 (brs, 1H), 5.60
(brs, 1H), 5.05 (brs, 1H), 3.49-3.11 (m, 16H), 2.69 (s, 3H), 1.36
(s, 9H). 13C NMR (67.8 MHz, CD2Cl2): ¤ 168.3, 164.1, 160.6,
157.2, 156.8, 156.7, 156.5, 156.3, 155.3, 153.8, 150.7, 142.9,
139.5, 138.6, 137.9, 137.3, 137.2, 130.9, 130.2 © 2, 130.1,
128.6, 126.1, 123.9, 70.9, 70.5, 70.4, 69.9 © 2, 48.8, 40.8,
39.9, 32.3, 30.1, 28.5, 11.7. FT-IR (KBr, cm¹1): 3407, 3094,
2925, 1668, 1586, 1505, 1365, 1252, 1173, 1124, 918, 717.
HRMS (ESI-TOF): Calcd for C38H38N10O13S79Br [M + H]+:
953.1524, Found: 953.1533, and C38H38N10O13S81Br
[M + H]+: 955.1503, Found: 955.1491.
The IC50 values in Table 2 were detected using the
μ
TRAPEZE XL Telomerase Detection Kit (Millipore, S7707),
which is an in vitro assay for the fluorometric detection of
telomerase activity with fluorescence resonance energy primers
transfer, according to the manufacturer’s protocol. Briefly,
elongation reactions were carried out at 30 °C for 30 min in
a final volume of 20 ¯L containing the Namalva cell extract
(same as the above). The amplification step was carried out by
PCR (36 cycles involving 94 °C for 30 s, 59 °C for 30 s, and
72 °C for 60 s, followed by an extension step at 72 °C for 3 min
and then at 55 °C for 25 min, concluding with 4 °C incubation).
The fluorescence (excitation: 495 nm, emission: 516 nm) was
measured using an EnSpire microplate reader (PerkinElmer).
General Procedure for Removal of the Boc Group of
(R)-18.
To a solution of (R)-18 (1 equiv) in 1,4-dioxane
(100 mL mmol¹1) was added 4 M HCl/1,4-dioxane (200
mL mmol¹1) at 0 °C under argon. After being stirred at room
temperature for 2 h, the reaction mixture was concentrated
in vacuo. The residue was azeotroped with 1,4-dioxane twice,
and then concentrated in vacuo to give quantitatively the HCl
salt of amine-linked heptaoxazole macrocycle (R)-5.
We thank Prof. Kazuo Nagasawa (TUAT) for his helpful
discussion. This study was supported by a grant from the New
Energy and Industrial Technology Development Organization
(NEDO) of Japan and a Grant-in-Aid (No. 23310145) from
MEXT (T.D.).
1
(R)-5a: H NMR (400 MHz, CD3OD): ¤ 8.78 (s, 1H), 8.76
(s, 1H), 8.70 (s, 1H), 8.69 (s, 1H), 8.57 (s, 1H), 5.64 (brs, 1H),
4.29 (brs, 2H), 3.47 (m, 1H), 3.34 (m, 1H), 3.12-2.93 (m, 4H),
2.78 (s, 3H), 1.69-1.51 (m, 4H). HRMS (ESI-TOF): Calcd for
C31H26N10O9S79Br [M + H]+: 793.0788, Found: 793.0786, and
C31H26N10O9S81Br [M + H]+: 795.0768, Found: 795.0778.
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24 We always compare the activity of the compounds with
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