Polymer supported synthesis of novel benzoxazole linked benzimidazoles under microwave conditions: In vitro evaluation of VEGFR-3 kinase inhibition activity

Article abstract of DOI:10.1039/c0ob00547a

An efficient soluble polymer-supported method has been developed for the parallel synthesis of substituted benzimidazole linked benzoxazoles using focused microwave irradiation. The key step involves the amidation of 4-hydroxy-3-nitrobenzoic acid with pol

Full text of DOI:10.1039/c0ob00547a

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PAPER
Polymer supported synthesis of novel benzoxazole linked benzimidazoles
under microwave conditions: In vitro evaluation of VEGFR-3 kinase inhibition
activity†
Kaushik Chanda,a Barnali Maiti,a Gorakh S. Yellol,a Ming-Hsien Chien,b Min-Liang Kuoc and
Chung-Ming Sun*a
Received 6th August 2010, Accepted 1st December 2010
DOI: 10.1039/c0ob00547a
An efficient soluble polymer-supported method has been developed for the parallel synthesis of
substituted benzimidazole linked benzoxazoles using focused microwave irradiation. The key step
involves the amidation of 4-hydroxy-3-nitrobenzoic acid with polymer-immobilized
o-phenylenediamine. Application of mild acidic conditions promoted the ring closure to furnish the
benzimidazole ring. After hydrogenation of the nitro-group to amine, the resulted polymer conjugates
underwent efficient ring closure with various alkyl, aryl and heteroaryl isothiocyanates to generate the
polymer-bound benzimidazolyl benzoxazoles. The polymer-bound compounds were finally cleaved
from the support to furnish benzimidazole linked benzoxazole derivatives. The efficacy of the resultant
angular bis-heterocyclic library was studied against vascular endothelial growth factor receptor
(VEGFR-3). The preliminary screening of these novel compounds exhibits moderate to high inhibition
(IC50 = 0.56–1.42 mM). This protocol provides an easy access to novel angular bis-heterocycles which
have potential for the discovery of novel leads for targeted cancer therapeutics.
of inhibiting the blood supply to tumors to deplete the oxygen
Introduction
and nutrients and subsequently arrest tumor cell growth as well
Among many diseases, cancer is still a major cause of death
as proliferation. This approach has been found to be effective
in the world and remains a major focus for ongoing research
and there are presently several antiangiogenic drugs undergoing
various stages of evaluation in clinical trials.2 Recently, two
small molecule inhibitors sunitinib (A) and sorafenib (B) have
been approved, in addition to AMG 706 (C), as antiangiogenic
drugs3 (Fig. 1). In spite of these achievements, it remains an
important challenge to develop new drugs in order to overcome
drug resistance and maintain the steady progress in cancer
research. Hence the discovery of novel lead compounds for
drug development plays an important role in cancer drug re-
search. Concomitantly, coupled with high throughput screening,
development of effective methodologies for rapid synthesis of
diversified libraries of small heterocyclic molecules is of great
importance.4
and development. In recent years a promising approach to the
therapeutic intervention of cancer has focused on antiangio-
genesis therapies.1 This approach takes advantage of the idea
aDepartment of Applied Chemistry, National Chiao Tung University,
Hsinchu, 300-10, Taiwan. E-mail: cmsun@mail.nctu.edu.tw
bGraduate Institute of Clinical Medicine, Taipei Medical University, Wang-
Fang Hospital, Taipei 100, Taiwan
cInstitute of Toxicology, National Taiwan University, Taipei 100, Taiwan
† Electronic supplementary information (ESI) available: General experi-
mental procedures and 1H NMR, 13C NMR, crude HPLC, LRMS, HRMS
and FT-IR spectral data of compounds 15a–p. CCDC reference numbers
783608. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c0ob00547a
Fig. 1 Examples of small molecule kinase inhibitors.
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Fig. 2 Biologically relevant benzoxazole derivatives and the targeted skeleton.
Fig. 3 Reported methods to prepare benzoxazole derivatives.
Motivated by these synthetic challenges, chemists have devel-
oped various solid phase as well as solution phase synthetic
methods for the construction of important heterocycles.5 Due
to the drawbacks such as heterogeneous reaction conditions,
nonlinear kinetics, solvation problems, and difficulty in moni-
toring reactions that solid-phase synthesis experiences, chemists
have sought alternative methodologies such as soluble polymer-
supported synthesis which can be conducted in homogeneous
organic media. Additionally, realizing that organic reactions and
reaction sequences involved in synthesizing the target molecules
should preferably be facile, swift and efficient, there has been a
shift in emphasis towards the use of microwave irradiation in
recent years.6 Microwave-assisted organic synthesis has received
constant attention because the organic reactions can now be
performed in sealed vessels in a temperature and pressure con-
trolled mode, providing reproducible results for producing bio-
logically interesting small heterocyclic molecules. In our previous
studies, we reported the application of microwave techniques
in multi-step liquid phase synthesis to generate heterocyclic
libraries.7
inhibitors 4.14 In an interest to develop biologically promising
bis-heterocyclic molecules, our research focused on developing a
novel benzoxazole linked benzimidazole skeleton 5 and further
extension of this study to biologically evaluate these molecules for
VEGFR-3 kinase inhibition.
A literature survey revealed that only a few methods were
reported for the synthesis of benzoxazole derivatives which
involve the condensation of 2-aminophenol with either carboxylic
acids or aldehydes under high temperature (Fig. 3A and 3B).15
Another method involving metal-catalyzed cross-coupling is also
known (Fig. 3C).16 However, all these methods suffered from
harsh reaction conditions, longer reaction times and limited
diversification. Prompted by the necessity to develop an easy and
efficient method to construct benzoxazole derivatives and our
interest in the benzimidazole linked benzoxazole scaffold, here
we disclose the multidisciplinary synergetic approach comprising
polymer-supported synthesis with microwave synthesis to generate
a bis-heterocyclic skeleton-containing small library and its in vitro
evaluation of VEGFR-3 kinase inhibition activity.
Although the medicinal chemistry fraternity has put up a
brave front to cover a wide range of structurally and func-
tionally derivatized heterocyclic compounds, the libraries of bis-
heterocyclic compounds have not been extensively studied so far.
The linked bis-heterocyclic scaffolds with two or more heteroatom-
containing rings have significance to afford additional binding
opportunities as well as improved diversification scope, and hence
have been recognized as the most potent heterocyclic motifs for the
development of novel therapeutics.8 Particularly, benzimidazoles9
and benzoxazoles10 are privileged heterocycles known for a wide
range of biological activities (Fig. 2). Prominently, the benzoxazole
derivatives have been found to be 5-HT3 receptor agonists11 1,
HIV reverse transcriptase inhibitors12 L-697,661 2 and anticancer
agents13 NSC-693638 3. Similarly, the benzoxazole ring is an
intriguing heterocycle embedded in a novel class of potent KDR
Results and discussion
The present strategy commenced with the synthesis of
polymer-immobilized ortho-phenylenediamine 9 from 4-fluoro-3-
nitrobenzoic acid with built-in structural diversity (R1) through
a three-step protocol (Scheme 1).17 In an effort to obtain the
target molecule, compound 9 was N-acylated at the primary
amine functionality with 4-hydroxy-3-nitrobenzoic acid 10 via
DCC activation. Accordingly, anilide conjugates 11 were ob-
tained by the condensation of acid 10 with PEG conjugates 9
through the activated ester generated by DCC and HOBT in
DMF in 12 h reflux. However, the application of microwave
irradiation (90 ◦C) reduced the reaction time to 25 min. For
the construction of the benzimidazole ring, anilide conjugates 11
were subjected to acid catalyzed cyclisation in the presence of
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Scheme 1 General strategy of microwave-assisted synthesis of benzimidazolyl benzoxazoles.
10% trifluoroacetic acid and MgSO4 in 1,2-dichloroethane. The
formation of the conjugate 12 was achieved in 10 h under reflux
conditions.
irradiation at 100 ◦C (5 bar) to furnish the benzoxazole conjugate
14 in 10 min. However, it has been observed that the same reaction
required 10 h under conventional reflux conditions, which in turn
reflects the advantages of microwave irradiation. After completion
of the reaction, the reaction mixture was precipitated with cold
ether and filtered through a fritted funnel to obtain benzoxazole
conjugate 14 in good yields. The formation of the product involves
the nucleophilic attack of the amine group on the isothiocyanate
moiety to form intermediate ‘a’. For mechanistic investigation,
intermediate ‘a’ was isolated before addition of the coupling
reagents and characterized (see the ESI†). The coupling agent
(EDC) promotes further activation of the thiocarbonyl (C S)
moiety of intermediate ‘a’, which after cyclization and electronic
reorganization generate the target compounds 14 as depicted in
Fig. 4. Additionally, the isolated intermediate ‘a’ was also reacted
separately with EDC, providing the same results, which supports
the predicted mechanism.
The benzimidazolyl benzoxazoles were finally cleaved from the
polymer support using a 1% solution of KCN in MeOH at room
temperature within 12 h. The reaction mixture was concentrated
and polymer support was precipitated by ether and removed by
filtration. The filtrates were evaporated and subjected to HPLC
analysis which indicated 76–98% crude purity of the title com-
pounds. Finally, column chromatography purification afforded the
benzimidazolyl benzoxazole derivatives 15 in good overall yields
(Table 1). By utilizing the desired reaction sequence, we have
synthesized various benzimidazolyl benzoxazole derivatives 15
with two diverse substitutions as shown in Table 1. The systematic
In order to achieve the target compound quickly, we applied
the microwave irradiation under sealed vessel conditions (100 ◦C,
8 bar) at this stage, which further reduced the reaction time to
8 min. The polymer conjugate 12 was purified by precipitating out
the reaction mixtures with excess cold ether. For the reduction of
the nitro group, conjugate 12 was treated with ammonium formate
and 10% palladium on activated charcoal in methanol. Formation
of the amine conjugates 13 was achieved under reflux conditions
for 2 h. However, by the application of microwave irradiation at
80 ◦C, the desired conjugates 13 were obtained within 10 min. It
is noteworthy to say that the metal reduction proceeded smoothly
under microwave conditions. After completion, palladium and
excess ammonium formate were removed by filtration. Amine
conjugates 13 were obtained in pure form by further precipitation
in cold ether.
Our main goal was the construction of the benzoxazole ring
together with introduction of additional sets of diversity. It has
been realized that the elaboration of intermediate 13 to the desired
core structure required one carbon electrophile. In an effort to
build the benzoxazole motif to mimic the bioactive compound
as mentioned earlier in Fig. 2 (scaffolds 1–4), we decided to
explore cyclization using various isothiocyanates. Hence, the
amine conjugates 13 were condensed with selective isothiocyanates
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as
an activating agent in anhydrous acetonitrile under microwave
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Fig. 4 Plausible mechanism towards the formation of benzimidazolyl benzoxazoles.
variation of amines and isothiocyanate components led to the
structural diversity in the scaffold.
the identification of the roles of various VEGFRs showed that
VEGFR-3 has been implicated as a key mediator of lymphan-
giogenesis in both normal biology and tumors.20 Consequently,
as a novel VEGFR target, we are intrigued to examine the new
bis-heterocyclic scaffold for the inhibition of VEGFR-3.
Monitoring the progress of a reaction on an insoluble polymer
support throughout a multistep synthetic sequence is a relatively
difficult task. The idea of having a polymer support possessing
conventional solubility properties in organic solvents has been very
well realized in the use of polyethylene glycols (PEG). A significant
advantage of NMR protocols for soluble polymer supports is its
nondestructive nature as compared to the customary “cleave and
characterize” techniques used for solid-supported synthesis.18 The
comparative analysis of NMR spectra (aromatic region) of all the
steps is depicted in Fig. 5. Formation of the anilide conjugates was
confirmed by the appearance of NH signals at 9.18 ppm, whereas
the additional signals emerged from the absorbance of Hd, He
and Hf protons of 4-hydroxy-3-nitrobenzoic acid in spectrum
B. Establishment of the benzimidazole ring was evident from
the disappearance of the NH proton in spectrum C and Ha,
Hb and Hc protons were shifted downfield due to the electron
withdrawing nature of the benzimidazole moiety. Reduction of
the nitro group was noticed from the substantial shifting of Hd
and He protons to the upfield region at 7.00 and 6.81 ppm
in spectrum D. Final cyclization with allylisothicyanate to 14j
generated the electron withdrawing benzoxazole derivatives, which
was observed in spectrum E. Due to the electron withdrawing
effect, the previously apparent upfield protons Hd and He at
7.0 and 6.81 ppm became deshielded and moved to 7.45 and
7.32 ppm. Finally, the cleavage of the product from the polymer
support was confirmed by observing the absence of PEG signals
around 4.4 ppm along with the slight downfield shift of all aromatic
protons of 15j in spectrum F.
Additionally, the structure of the final compounds was unam-
biguously confirmed by the X-ray crystallographic study. Fig. 6
depicts the ORTEP diagram of compound 15n. The single crystal
X-ray analysis of compound 15n indicates that the two rings of
the benzimidazole and benzoxazole moieties are anti-periplanar
to each other and in angular orientation.
After the successful synthesis of the benzoxazole bis-
heterocyclic library, our interest in the biological evaluation
of these compounds deviated towards the VEGFR kinase in-
hibitors due to their structural resemblance with the potent
KDR inhibitor14 and potential VEGFR-3 inhibitor.19 It is a well
established fact in cancer biology that tumor growth depends
on the expression of various growth factors associated with
angiogenesis and lymphangiogenesis. Recent advanced studies on
Accordingly, the new benzimidazolyl linked benzoxazole deriva-
tives 15a–p were examined for in vitro inhibition of VEGFR-
3 kinase in a cell-based assay in the concentration of 3 mM
on H928 cell lines. Interestingly, after preliminary screening it
has been found that all the compounds have shown moderate
to high inhibition from 15% to 87% (Table 2). Gratifyingly,
compounds 15c, 15f, 15h–k, 15m and 15n show higher inhibitory
activity against VEGFR-3. These selective compounds were
further studied for the IC50 values and the respective data are
specified in Table 2. The comparative biological studies point to
compounds 15c, 15m and 15n as the prominent compounds that
inhibit receptor tyrosine kinase VEGFR-3. A generalization of
this study suggests that the new benzimidazole linked benzoxazole
bis-heterocyclic scaffold possesses potential to inhibit receptor
tyrosine kinase VEGFR-3 and further extension of this study can
definitely aid the development of novel cancer therapeutics.
Conclusions
In conclusion, we have developed a new strategy for a rapid
synthesis of novel substituted benzimidazole linked benzoxazole
derivatives in a multistep process on soluble polymer support un-
der focused microwave irradiation. The proposed bis-heterocyclic
system was developed by constructing the benzoxazole ring over
benzimidazole by sequential reactions, specifically condensation,
reduction and cyclization with isothiocyanate. We have signifi-
cantly applied the combination of soluble polymer support and
microwave irradiation to increase the overall efficiency of the
process through rapid reactions as well as purification by simple
precipitation with ether. The efficacy of the novel benzimidazolyl
benzoxazole derivatives was examined for VEGFR-3 tyrosine
kinase inhibition activity. Preliminary screening results have
shown that some of these compounds exhibited moderate to good
inhibition against VEGFR-3, which is related to the invasion
and migration of cancer cells. The bis-heterocyclic structural
framework defined by the benzimidazole linked benzoxazole core
and the ease of its access by microwave assisted soluble supported
synthesis open up new avenues for the development of anti-
angiogenic drugs.
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Table 1 Synthesized benzo[d]oxazol-5-yl-1H-benzo[d]imidazole-5-car-
boxylates 15
Table 2 Percent inhibition of VEGFR-3 and IC50 (mM) value for selected
compounds
Comp.
% inhibition of VEGFR-3a at 3 mM
IC50 (mM)b
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
52
40
60
29
46
69
53
76
86
63
73
15
81
87
81
22
—
—
0.75
—
—
1.19
—
1.03
1.25
1.18
1.42
—
0.56
0.70
—
Comp. R1
R2
Yielda(%) Purityb(%) LRMSc
15a
90
85
93
91
82
88
89
95
97
89
459
441
441
459
511
15b
15c
15d
15e
—
a Results are expressed as the mean percent inhibition at 3 mM level. b For
the selective compounds having inhibition more than 60%; values are an
average of three individual determinations.
Experimental section
General methods
15f
92
95
96
94
493
474
All reactions were performed under an inert atmosphere with
unpurified reagents and dry solvents. Analytical thin-layer chro-
matography (TLC) was performed using 0.25 mm silica gel coated
plates. Flash chromatography was performed using the indicated
solvent and silica gel 60 (230–400 mesh). All the microwave
experiments were performed in a Biotage initiator under optimized
reaction conditions of power and pressure. 1H NMR (300 MHz)
and 13C NMR (75 MHz) spectra were recorded on a 300 MHz
spectrometer. Chemical shifts are reported in parts per million
(ppm) on the scale from an internal standard. High-resolution
mass spectra (HRMS) were recorded on a JEOL TMS-HX 110
mass spectrometer. Normal phase HPLC was performed on a
Shimadzu LC-10AT series machine with a Hypersil (250 ¥ 4.6
mm) analytical column.
15g
15h
15i
82
81
80
82
405
455
15j
89
85
94
80
457
507
15k
General procedure for the preparation of polymer-bound 3-(4-
hydroxy-3-nitrobenzamido)-4-(substituted amino) carboxylates 11.
To a solution of N,N¢-dicyclohexylcarbodiimide (DCC) (139 mg,
0.67 mmol, 3.0 equiv) in N,N¢-dimethylformamide (DMF) was
added 4-hydroxy-3-nitrobenzoic acid 10 (122 g, 0.67 mmol, 3
equiv.) and 1-hydroxybenzotriazole (HOBt) (91 mg, 0.67 mmol,
3.0 equiv) in sequential order. The resulting slurry was stirred
for 5 min at room temperature and then polymer (PEG 4000)
anchored o-phenylene diamine 9 (1.0 g, 0.22 mmol, 1.0 equiv.) in
DMF (5 mL) was added. The reaction mixtures were subsequently
heated with stirring in a 10 mL microwave process vial for 25 min
in the appropriate mode of pressure and temperature to obtain
the polymer conjugate 11. After completion of the reaction, the
suspended byproducts were filtered through filter paper. The
reaction mixtures were precipitated by slow addition of cold
ether (50 mL) and the precipitated amide conjugates 11 were
filtered through a fritted funnel. The crude product was washed
in succession with ether (100 mL ¥ 3) to remove the undesired
impurities and dried for further steps.
15l
95
77
79
98
76
77
457
461
467
15m
15n
15o
15p
87
96
86
98
437
417
a Yields were determined on the weight of purified samples (%). b HPLC
analysis (UV detection at 254 nm) of the crude samples (%). c LRMS were
detected with ESI ionization source.
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Fig. 5 Stepwise monitoring of multistep synthesis of benzimidazolyl benzoxazoles by proton NMR spectroscopy.
General procedure for the preparation of polymer-bound 2-
(4-hydroxy-3-nitrophenyl)-1-alkyl-1H-benzo[d]imidazole carboxy-
lates 12. To a solution of polymer-bound 3-(4-hydroxy-3-
nitrobenzamido)-4-(substituted amino) carboxylates 11 in 1,2-
dichloroethane, trifluoroacetic acid (0.5 mL) and MgSO4 (500
mg) were added and the mixture was subsequently heated with
stirring in a 10 mL microwave process vial for 8 min in the
appropriate mode of pressure and temperature. After completion
of the reaction, the MgSO4 was removed through Celite. The
reaction mixtures were precipitated by slow addition of excess
cold ether (100 mL) and filtered through a fritted funnel to
obtain the polymer-bound 2-(4-hydroxy-3-nitrophenyl)-1-alkyl-
1H-benzo[d]imidazole carboxylates 12 in high purity.
General procedure for the preparation of polymer-bound 2-(3-
amino-4-hydroxyphenyl)-1-alkyl-1H-benzo[d]imidazole carboxy-
lates 13. To a solution of 12 in methanol, Pd on charcoal
(48 mg, 0.45 mmol, 1.0 equiv.) and ammonium formate (132 mg,
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Fig. 6 ORTEP diagram of compound 15n.
2.10 mmol, 10.0 equiv.) were added. The reaction mixture was
subsequently heated with stirring in a 10 mL microwave process
vial for 10 min in the appropriate mode of pressure and tempera-
ture until complete reduction of the nitro group which was evident
from a color change (yellow to greenish blue) upon spotting on
a TLC plate. After completion, the reaction mixtures were then
subjected to centrifugation for removal of the Pd on charcoal and
the supernatant liquid was concentrated by rotary evaporation
to remove methanol. Dichloromethane (10 mL) was then added
to salt out ammonium formate. The reaction mixtures were
filtered through a fritted funnel to remove ammonium formate, to
obtain the polymer-bound 2-(3-amino-4-hydroxyphenyl)-1-alkyl-
1H-benzo[d]imidazole carboxylates 13.
2-(2-(4-Fluorophenylamino)benzo[d]oxazol-5-yl)-1-isobutyl-1H-
benzo[d]imidazole-5-carboxylic acid methyl ester 15a. 1H NMR
(300 MHz, CDCl3) d 8.56 (d, J = 1.5 Hz, 1H), 8.07 (dd, J = 8.5,
1.5 Hz, 1H), 7.98 (brs, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.64 (dd, J =
9.0 Hz, JHF = 4.6 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.45 (dd, J =
8.2, 1.5 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.08 (dd, JHF = 8.8 Hz,
J = 7.4 Hz, 2H), 4.13 (d, J = 7.5 Hz, 2H), 3.98 (s, 3H), 2.11 (sept,
J = 6.6 Hz, 1H), 0.74 (d, J = 6.6 Hz, 6H); 13C NMR (75 MHz,
1
CDCl3) d 168.0, 159.8, 159.4 (d, JCF = 252.9 Hz), 156.5, 149.2,
143.2, 142.7, 139.3, 134.3, 126.7, 125.0, 124.7, 124.1, 122.4, 120.8
3
2
(d, JCF = 7.8 Hz), 118.1, 116.4 (d, JCF = 26.1 Hz), 110.7, 109.8,
52.5, 52.4, 29.4, 20.5, 20.4; IR (cm-1, KBr): 3303, 1708; MS (ESI)
m/z 459 (MH+); HRMS (ESI, m/z) calcd for C26H23FN4O3: m/z
459.1832; Found 459.1834.
General procedure for the preparation of polymer-bound
2-(2-(substituted
benzo[d]imidazole carboxylates 14. To
amino)
benzo[d]oxazol-5-yl)-1-alkyl-1H-
stirred solution
1-Isobutyl-2-(2-(phenylamino)benzo[d]oxazol-5-yl)-1H-benzo-
[d]imidazole-5-carboxylic acid methyl ester 15b. 1H NMR
(300 MHz, CDCl3) d 8.57 (d, J = 1.5 Hz, 1H), 8.07 (dd, J =
8.5, 1.5 Hz, 1H), 7.97 (brs, 1H), 7.74 (d, J = 1.4 Hz, 1H), 7.67
(dd, J = 8.7, 1.2 Hz, 2H), 7.48 (dd, J = 8.3, 1.4 Hz, 1H), 7.47–7.39
(m, 4H), 7.14 (t, J = 7.4 Hz, 1H), 4.13 (d, J = 6.6 Hz, 2H), 3.98
a
of polymer-bound 2-(3-amino-4-hydroxyphenyl)-1-alkyl-1H-
benzo[d]imidazole carboxylates conjugates 13 in CH3CN (5
mL), various isothiocyanates (1.05 mmol, 5.0 equiv.) and N-
3(dimethylaminopropyl)-3-ethyl carbodiimide (EDC) (163 mg,
1.05 mmol, 5.0 equiv) as an activating agent were added. The
reaction mixtures were exposed under pressure to microwave
irradiation for 10 min. Upon completion of cyclization by
checking the NMR, the crude product mixtures were purified by
precipitation with cold ether (100 mL ¥ 3) and dried to obtain the
conjugate 14 in high purity.
(s, 3H), 2.13 (sext, J = 6.6 Hz, 1H), 0.73 (d, J = 6.6 Hz, 6H); 13
C
NMR (75 MHz, CDCl3) d 168.1, 159.4, 156.5, 149.2, 145.4, 143.4,
142.9, 139.4, 138.0, 129.8, 126.9, 124.8, 124.3, 123.9, 122.6, 118.9,
118.3, 110.6, 109.9, 53.2, 52.5, 29.2, 20.5, 20.4; IR (cm-1, KBr):
3434, 1712; MS (ESI) m/z 441 (MH+); HRMS (ESI, m/z) calcd
for C26H24N4O3: m/z 441.1927; Found 441.1925.
General procedure for the cleavage of polymer-bound substituted
benzimidazolylbenzoxazoles 15. To a solution of polymer conju-
gates 14 in methanol (20 mL), KCN (100 mg) was added and stirred
for 12 h at room temperature. After completion of the reaction, the
crude product was precipitated with excess cold ether (100 mL),
the polymer was filtered off and subjected to evaporation. The
residue was dried under vacuum, and subjected to crude HPLC
analysis with UV detection at l = 254 nm (column: Sphereclone
5m Si (250 ¥ 4.6 mm); gradient: 35% ethyl acetate in hexane; flow
rate: 1 mL min-1). The slurry obtained was loaded onto a silica
gel column and eluted with a mixture of ethyl acetate and hexane
(1 : 4) to get the title compounds 15 in 77–96% overall yields.
1-Butyl-2-(2-(phenylamino)benzo[d]oxazol-5-yl)-1H -benzo-
[d]imidazole-5-carboxylic acid methyl ester 15c. 1H NMR
(300 MHz, CDCl3) d 9.48 (s, 1H), 8.58 (s, 1H), 8.08 (d, J = 8.4 Hz,
1H), 7.71 (s, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 8.6 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 7.37–7.29 (m, 3H), 7.07 (t, J =
7.2 Hz, 1H), 4.27 (t, J = 7.1 Hz, 2H), 3.97 (s, 3H), 1.74 (quint,
J = 7.1 Hz, 2H), 1.27–1.22 (m, 2H), 0.83 (t, J = 7.1 Hz, 3H); 13
C
NMR (75 MHz, CDCl3) d 168.1, 159.9, 156.3, 149.3, 143.4, 142.8,
139.1, 138.4, 129.7, 126.2, 124.7, 123.9, 123.7, 123.2, 122.4, 119.1,
117.8, 110.4, 109.8, 52.6, 45.1, 32.1, 20.3, 13.9; IR (cm-1, KBr):
3436, 1714; MS (ESI) m/z 441 (MH+); HRMS (ESI, m/z) calcd
for C26H24N4O3: m/z 441.1927; Found 441.1924.
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1-Butyl-2-(2-(4-fluorophenylamino)benzo[d]oxazol-5-yl)-1H-
benzo[d]imidazole-5-carboxylic acid methyl ester 15d. 1H NMR
(300 MHz, CDCl3) d 9.25 (s, 1H), 8.57 (s, 1H), 8.08 (d, J = 8.5 Hz,
1H), 7.68 (s, 1H), 7.63 (dd, J = 8.5 Hz, JHF = 4.6 Hz, 2H), 7.47 (d,
J = 8.5 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 8.5 Hz, 1H),
7.05 (dd, J = 8.5 Hz, JHF = 8.4 Hz, 2H), 4.27 (t, J = 7.2 Hz, 2H), 3.98
(s, 3H), 1.78 (quint, J = 7.2 Hz, 1H), 1.26–1.19 (m, 2H), 0.84 (t, J =
7.2 Hz, 3H); 19F NMR (282.4 MHz, CDCl3) d -119.0; 13C NMR
(75 MHz, CDCl3) d 168.1, 160.9, 159.9 (d, 1JCF = 260.6 Hz), 156.2,
149.3, 143.3, 142.7, 139.1, 134.5, 126.1, 125.1, 125.0, 124.6, 122.3,
1H), 5.22 (dd, J = 10.1, 1.1 Hz, 1H), 4.12 (m, 4H), 3.96 (s, 3H),
2.10 (sept, J = 6.6 Hz, 1H), 0.71 (d, J = 6.6 Hz, 6H); 13C NMR
(75 MHz, CDCl3) d 168.1, 163.3, 156.6, 149.9, 143.8, 142.9, 139.4,
133.9, 126.6, 124.8, 124.5, 123.3, 122.6, 117.6, 117.4, 110.6, 109.6,
52.5, 52.4, 45.8, 29.1, 20.3; IR (cm-1, KBr): 3278, 1710; MS (ESI)
m/z 405 (MH+); HRMS (ESI, m/z) calcd for C23H24N4O3: m/z
405.1927; Found 405.1929.
2-(2-(Benzylamino)benzo[d]oxazol-5-yl)-1-isobutyl-1H-benzo-
[d]imidazole-5-carboxylic acid methyl ester 15i. 1H NMR
(300 MHz, CDCl3) d 8.53 (d, J = 1.6 Hz, 1H), 8.04 (dd, J =
8.5, 1.6 Hz, 1H), 7.56 (d, J = 1.1 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H),
7.42–7.30 (m, 7H), 5.97 (t, J = 6.0 Hz, 1H), 4.72 (d, J = 6.0 Hz,
2H), 4.13 (d, J = 7.5 Hz, 2H), 3.97 (s, 3H), 2.10 (sept, J = 6.6 Hz,
1H), 0.73 (d, J = 6.6 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 168.1,
163.2, 156.5, 149.9, 143.8, 142.9, 139.4, 137.8, 129.3, 128.4, 128.1,
126.8, 124.8, 124.5, 123.5, 122.6, 117.6, 110.6, 109.0, 52.5, 52.4,
47.6, 29.2, 20.3; IR (cm-1, KBr): 3210, 1705; MS (ESI) m/z 455
(MH+); HRMS (ESI, m/z) calcd for C27H26N4O3: m/z 455.2083;
Found 455.2085.
3
2
120.9 (d, JCF = 4.0 Hz), 118.1, 116.5 (d, JCF = 23.1 Hz), 111.5,
110.4, 52.6, 45.1, 32.1, 20.3, 13.9; IR (cm-1, KBr): 3303, 1709; MS
(ESI) m/z 459 (MH+); HRMS (ESI, m/z) calcd for C26H23FN4O3:
m/z 459.1832; Found 459.1831.
1-(2-Cyclohexenylethyl)-2-(2-(4-fluorophenylamino)benzo[d]-
oxazol-5-yl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
15e. 1H NMR (300 MHz, CDCl3) d 8.56 (brs, 2H), 8.08 (d, J =
8.5 Hz, 1H), 7.72 (s, 1H), 7.62 (dd, J = 8.6 Hz, JHF = 4.5 Hz, 2H),
7.49 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 1H), 7.09 (dd, J =
8.6 Hz, JHF = 8.5 Hz, 2H), 5.19 (m, 1H), 4.39 (t, J = 7.2 Hz, 2H),
3.99 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.83–1.75 (m, 4H), 1.44–
1.34 (m, 4H); 19F NMR (282.4 MHz, CDCl3) d -118.9; 13C NMR
(75 MHz, CDCl3) d 168.1, 161.1, 159.7, 159.4 (d, 1JCF = 262.2 Hz),
156.2, 149.3, 143.3, 142.8, 139.2, 134.2, 133.4, 126.5, 125.1, 124.9,
2-(2-(Allylamino)benzo[d]oxazol-5-yl)-1-(2-Cyclohexenylethyl)-
1H-benzo[d]imidazole-5-carboxylic acid methyl ester 15j. 1H
NMR (300 MHz, CDCl3) d 8.53 (d, J = 1.5 Hz, 1H), 8.05 (dd,
J = 8.5, 1.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.46 (dd, J = 8.2,
1.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H),
6.00 (m, 1H), 5.86 (m, 1H), 5.35 (dd, J = 17.1, 1.3 Hz, 1H), 5.24
(dd, J = 10.3, 1.3 Hz, 1H), 5.18 (m, 1H), 4.36 (t, J = 7.2 Hz, 2H),
4.17–4.14 (m, 2H), 3.97 (s, 3H), 2.33 (t, J = 7.2 Hz, 2H), 1.81–1.74
(m, 4H), 1.45–1.43 (m, 4H); 13C NMR (75 MHz, CDCl3) d 168.1,
163.2, 156.3, 150.1, 143.9, 142.9, 139.3, 133.9, 133.4, 126.4, 124.9,
124.8, 124.5, 123.3, 122.6, 117.4, 117.3, 110.3, 109.6, 52.5, 45.8,
44.1, 38.1, 28.6, 25.5, 22.9, 22.3; IR (cm-1, KBr): 3424, 1714; MS
(ESI) m/z 457 (MH+); HRMS (ESI, m/z) calcd for C27H28N4O3:
m/z 457.2240; Found 457.2239.
124.7, 124.2, 122.5, 120.9 (d, 3JCF = 7.5 Hz), 118.0, 116.4 (d, 2JCF
=
30.0 Hz), 110.4, 109.8, 52.6, 44.2, 38.1, 28.6, 25.5, 22.9, 22.3; IR
(cm-1, KBr): 3446, 1716; MS (ESI) m/z 511 (MH+); HRMS (ESI,
m/z) calcd for C30H27FN4O3: m/z 511.2145; Found 511.2142.
1-(2-Cyclohexenylethyl)-2-(2-(phenylamino)benzo[d]oxazol-5-
yl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester 15f. 1H
NMR (300 MHz, CDCl3) d 9.00 (brs, 1H), 8.57 (d, J = 1.5 Hz, 1H),
8.08 (dd, J = 8.5, 1.5 Hz, 1H), 7.73 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H),
7.45 (d, J = 8.5 Hz, 2H), 7.44–7.30 (m, 3H), 7.10 (t, J = 8.2 Hz,
1H), 5.18 (m, 1H), 4.37 (t, J = 7.2 Hz, 2H), 3.98 (s, 3H), 2.34 (t,
J = 7.2 Hz, 2H), 1.81–1.73 (m, 4H), 1.44–1.41 (m, 4H); 13C NMR
(75 MHz, CDCl3) d 168.1, 159.8, 156.3, 149.2, 143.3, 142.8, 139.2,
138.3, 133.4, 129.7, 126.4, 125.1, 124.9, 124.7, 123.8, 123.6, 122.4,
119.0, 117.9, 110.5, 109.8, 52.6, 43.1, 38.1, 28.6, 25.5, 22.9, 21.8;
IR (cm-1, KBr): 3421, 1712; MS (ESI) m/z 493 (MH+); HRMS
(ESI, m/z) calcd for C30H28N4O3: m/z 493.2240; Found 493.2242.
2-(2-(Benzylamino)benzo[d]oxazol-5-yl)-1-(2-Cyclohexenyl-
ethyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester 15k.
1H NMR (300 MHz, CDCl3) d 8.51 (d, J = 1.4 Hz, 1H), 8.05
(dd, J = 8.5, 1.4 Hz, 1H), 7.54 (d, J = 1.3 Hz, 1H), 7.43 (d, J =
8.5 Hz, 1H), 7.40–7.23 (m, 7H), 6.65 (m, 1H), 5.18 (m, 1H), 4.68
(d, J = 4.9 Hz, 2H), 4.34 (t, J = 7.2 Hz, 2H), 3.96 (s, 3H), 2.32
(t, J = 7.2 Hz, 2H), 1.81–1.73 (m, 4H), 1.45–1.42 (m, 4H); 13C
NMR (75 MHz, CDCl3) d 168.2, 163.4, 156.3, 150.1, 143.9, 142.9,
139.3, 137.9, 133.4, 129.2, 128.2, 128.0, 126.3, 125.0, 124.8, 124.5,
123.2, 122.5, 117.2, 110.3, 109.6, 52.5, 47.5, 44.1, 38.1, 28.6, 25.5,
23.0, 22.3; IR (cm-1, KBr): 3209, 1714; MS (ESI) m/z 507 (MH+);
HRMS (ESI, m/z) calcd for C31H30N4O3: m/z 507.2396; Found
507.2395.
1-Butyl-2-(2-(3-chlorophenylamino)benzo[d]oxazol-5-yl)-1H-
benzo[d]imidazole-5-carboxylic acid methyl ester 15g. 1H NMR
(300 MHz, CDCl3) d 9.20 (brs, NH), 8.56 (s, 1H), 8.09 (d, J =
8.5 Hz, 1H), 7.80 (s, 1H), 7.72 (s, 1H), 7.55 (d, J = 8.2 Hz, 1H),
7.48 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 8.2, 1H), 7.30–7.25 (m, 2H),
7.05 (d, J = 7.8 Hz, 1H), 4.29 (t, J = 7.4 Hz, 2H), 3.99 (s, 3H), 1.80
(quint, J = 7.4 Hz, 2H), 1.30–1.22 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H);
13C NMR (75 MHz, CDCl3) d 168.0, 159.1, 156.2, 149.2, 143.2,
142.7, 139.6, 139.1, 135.3, 130.7, 126.2, 125.1, 124.8, 124.3, 123.6,
122.3, 118.8, 118.2, 116.8, 110.4, 109.9, 52.6, 45.1, 32.1, 20.3, 13.9;
IR (cm-1, KBr): 3399, 1747; MS (EI) m/z 474 (M+); HRMS (ESI,
m/z) calcd for C26H23ClN4O3: m/z 475.1537; Found 475.1533.
1-Cyclopentyl-2-(2-(furan-2-ylmethylamino)benzo[d]oxazol-5-
yl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester 15l. 1H
NMR (300 MHz, CDCl3) d 8.54 (s, 1H), 8.00 (d, J = 8.6 Hz, 1H),
7.61 (s, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.45–7.39 (m, 3H), 6.42–6.32
(m, 2H), 5.62 (t, J = 5.5 Hz, 1H), 4.99 (quint, J = 8.9 Hz, 1H),
4.71 (d, J = 5.5 Hz, 2H), 3.98 (s, 3H), 2.36–2.29 (m, 2H), 2.14–2.06
(m, 4H), 1.75–1.73 (m, 2H); 13C NMR (75 MHz, CDCl3) d 168.0,
163.0, 156.7, 150.9, 150.1, 143.6, 143.0, 142.9, 136.9, 126.6, 124.6,
124.0, 123.6, 122.9, 117.5, 111.9, 110.9, 109.7, 108.4, 58.1, 52.5,
40.4, 30.8, 25.6; IR (cm-1, KBr): 3423, 1714; MS (ESI) m/z 457
2-(2-(Allylamino)benzo[d]oxazol-5-yl)-1-isobutyl-1H-benzo[d]-
imidazole-5-carboxylic acid methyl ester 15h. 1H NMR
(300 MHz, CDCl3) d 8.54 (d, J = 1.4 Hz, 1H), 8.04 (dd, J =
8.5, 1.4 Hz, 1H), 7.60 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.38–7.34
(m, 2H), 6.11 (brs, 1H), 5.99 (m, 1H), 5.33 (dd, J = 17.0, 1.1 Hz,
1924 | Org. Biomol. Chem., 2011, 9, 1917–1926
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(MH+); HRMS (ESI, m/z) calcd for C26H24N4O4: m/z 457.1876;
Found 457.1878.
overnight at 37 ◦C in 5% CO2. After the supernatants were
removed, the cells were serum-starved for 24 h. A medium
containing a test compound was added into each well and the
cell culture was incubated for 30 min before it was stimulated by
recombinant VEGF-C for 15 min. After the supernatants were
removed, 100 ml of a lysis buffer were added into each well to lyse
the cells and solubilize the VEGFR-3. The lysis buffer included
150 mM NaCl containing 50 mM Hepes (Genentech media prep),
0.5% Triton-X100 (Genentech media prep), 0.01% thimerosol, 30
kIU/ml aprotinin (ICN Biochemicals, Aurora, Ohio), 1 mM 4-(2-
aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF; ICN
Biochemicals), and 2 mM sodium orthovanadate. The plate was
then put on a plate shaker (Bellco Instruments Vineland, N.J.)
and the substance in each well of the plate underwent mixing for
60 minutes at room temperature. While the cells get soluble, an
ELISA microtiter plate (Nunc Maxisorp, Inter Med, Denmark)
coated overnight at 4 ◦C with the affinity-purified polyclonal anti-
VEGFR-3 (2.5 mg/well block buffer (PBS containing 0.5% BSA
and 0.01% thimerosol)) for 60 min at room temperature with gentle
agitation. The anti-VEGFR-3 coated plate was subsequently
washed twice with a wash buffer (PBS containing 0.05% Tween 20
and 0.01% thimerosol). The lysate containing solubilized VEGFR-
3 from the cell culture microtiter well was transferred (85 ml/well)
to the anti-VEGFR-3 coated ELISA plate and incubated for
2 h at room temperature with gentle agitation. The unbound
receptors were removed by washing with a wash buffer. 100 ml of
biotinylated 4G10 (antiphosphotyrosine) diluted to 0.2 mg ml-1 in
dilution buffer (PBS containing 0.5% BSA, 0.05% Tween 20, 5 mM
EDTA, and 0.01% thimerosol) were added into each well. After
incubation for 2 h at room temperature, the plates were washed
and 100 ml HRP-conjugated streptavidin (Zymed Laboratories,
San Francisco, Calif.) diluted 1 : 2000 in dilution buffer was further
added. After the free avidin conjugate was washed away, 100 ml
freshly prepared substrate solution (tetramethyl benzidine, TMB)
was added to each well. The reaction was allowed to proceed for
10 min and the color development was stopped by the addition
of 100 ml/well 1.0 M H3PO4. The absorbance at 450 nm and the
absorbance at a reference wavelength of 650 nm (A450/650) were
measured using an ELISA reader and the data were repeated
3 times. The inhibition efficacy of each test compound is expressed
as an inhibition percentage calculated according to following
formula: 1 - [(C - A)/(B - A)] ¥ 100. In this formula, A is the
basal amount of phosphotyrosine detected in a blank control,
B is the amount of phosphotyrosine detected with VEGFR-C
only, and C is the amount of phosphotyrosine detected with a test
compound and VEGF-C. For IC50 generation, the final compound
concentrations ranged from 0.1 mM to 10 mM.
2-(2-(Furan-2-ylmethylamino)benzo[d]oxazol-5-yl)-1-(3-metho-
xypropyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
15m. 1H NMR (300 MHz, CDCl3) d 8.54 (d, J = 1.5 Hz, 1H),
8.05 (dd, J = 8.5, 1.5 Hz, 1H), 7.67 (d, J = 1.4 Hz, 1H), 7.50 (d,
J = 8.5 Hz, 1H), 7.46–7.36 (m, 3H), 6.35 (dd, J = 5.6, 4.9 Hz, 2H),
6.13 (brs, 1H), 4.70 (s, 2H), 4.42 (t, J = 7.1 Hz, 2H), 3.97 (s, 3H),
3.24 (t, J = 6.0 Hz, 2H), 3.21 (s, 3H), 1.99 (quint, J = 6.2 Hz, 2H);
13C NMR (75 MHz, CDCl3) d 168.1, 162.9, 156.1, 150.9, 150.1,
143.7, 143.0, 142.8, 139.4, 125.3, 124.7, 124.6, 123.4, 122.5, 117.4,
110.9, 110.3, 109.7, 108.5, 69.1, 59.0, 52.5, 42.2, 40.4, 30.4; IR
(cm-1, KBr): 3211, 1708; MS (ESI) m/z 461 (MH+); HRMS (ESI,
m/z) calcd for C25H24N4O5: m/z 461.1825; Found 461.1822.
2-(2-(Benzylamino)benzo[d ]oxazol-5-yl)-1-cyclopentyl-1H-
benzo[d]imidazole-5-carboxylic acid methyl ester 15n. 1H NMR
(300 MHz, CDCl3) d 8.51 (d, J = 1.5 Hz, 1H), 8.00 (dd, J = 8.6,
1.5 Hz, 1H), 7.54 (d, J = 1.0 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.42–
7.34 (m, 7H), 6.16 (t, J = 5.4 Hz, 1H), 4.98 (quint, J = 8.5 Hz, 1H),
4.71 (d, J = 5.4 Hz, 2H), 3.97 (s, 3H), 2.35–2.26 (m, 2H), 2.13–2.03
(m, 4H), 1.77–1.73 (m, 2H); 13C NMR (75 MHz, CDCl3) d 168.1,
163.3, 156.7, 150.1, 143.7, 143.6, 137.8, 136.9, 129.3, 128.3, 128.0,
126.6, 124.5, 124.0, 123.5, 122.9, 117.4, 111.9, 109.6, 58.1, 52.5,
47.5, 30.8, 25.6; IR (cm-1, KBr): 3266, 1712; MS (ESI) m/z 467
(MH+); HRMS (ESI, m/z) calcd for C28H26N4O3: m/z 467.2083;
Found 467.2080. Elemental analysis calcd (%) for C28H26N4O3: C
72.09, H 5.62, N 12.01; found: C 72.05, H 5.68, N 11.81, (O calcd
10.29, found 10.41).
2-(2-(Isobutylamino)benzo[d]oxazol-5-yl)-1-(3-methoxypropyl)-
1H-benzo[d]imidazole-5-carboxylic acid methyl ester 15o. 1H
NMR (300 MHz, CDCl3) d 8.54 (d, J = 1.5 Hz, 1H), 8.05 (dd, J =
8.5, 1.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H),
7.47 (dd, J = 8.2, 1.5 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 5.50 (m,
1H), 4.42 (t, J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.34 (m, 2H), 3.27 (t, J =
6.2 Hz, 2H), 3.21 (s, 3H), 2.18 (m, 1H), 2.02–1.97 (m, 2H), 1.03
(d, J = 6.7 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 168.2, 163.5,
156.2, 150.0, 145.4, 144.0, 142.9, 139.5, 124.9, 124.6, 123.1, 122.5,
117.1, 110.2, 109.5, 69.2, 59.0, 52.5, 51.0, 42.2, 30.3, 28.9, 20.4; IR
(cm-1, KBr): 3426, 1712; MS (ESI) m/z 437 (MH+); HRMS (ESI,
m/z) calcd for C24H28N4O4: m/z 437.2189; Found 437.2190.
2-(2-(Allylamino)benzo[d ]oxazol-5-yl)-1-Cyclopentyl-1H-
benzo[d]imidazole-5-carboxylic acid methyl ester 15p. 1H NMR
(300 MHz, CDCl3) d 8.54 (d, J = 1.5 Hz, 1H), 8.00 (dd, J = 8.6,
1.5 Hz, 1H), 7.58 (d, J = 1.0 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H),
7.42–7.37 (m, 2H), 6.01 (m, 1H), 5.64 (m, 1H), 5.36 (dd, J = 17.1,
1.2 Hz, 1H), 5.26 (dd, J = 10.3, 1.2 Hz, 1H), 4.98 (quint, J = 8.5 Hz,
1H), 4.16 (t, J = 5.5 Hz, 2H), 3.97 (s, 3H), 2.39–2.26 (m, 2H), 2.14–
2.06 (m, 4H), 1.77–1.73 (m, 2H); 13C NMR (75 MHz, CDCl3) d
168.1, 163.1, 156.7, 150.0, 143.8, 143.7, 136.9, 133.9, 126.7, 125.0,
124.0, 123.6, 122.9, 117.5, 117.4, 111.9, 109.7, 58.1, 52.5, 45.8,
30.8, 25.6; IR (cm-1, KBr): 3413, 1714; MS (ESI) m/z 417 (MH+);
HRMS (ESI, m/z) calcd for C24H24N4O3: m/z 417.1927; Found
417.1926.
Acknowledgements
The authors thank the National Science Council of Taiwan for
the financial assistance and the authorities of the National Chiao
Tung University for providing the laboratory facilities.
References
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