Synthesis and photophysical properties of 3-[2-(pyridyl)benzoxazol-5-yl]-L-alanine derivatives

Article abstract of DOI:10.1016/S0040-4020(02)00092-3

The syntheses of N-(tert-butyloxycarbonyl)-3-[2-(4-pyridyl)benzoxazol-5-yl]-L-alanine methyl ester and N-(tert-butyloxycarbonyl)-3-[2-(2-pyridyl)benzoxazol-5-yl]-L-alanine methyl ester using different oxidizing agents (NBS, lead tetraacetate, Mitsunobu re

Full text of DOI:10.1016/S0040-4020(02)00092-3

TETRAHEDRON
Pergamon
Tetrahedron 58 )2002) 2201±2209
Synthesis and photophysical properties of
3-[2-ꢀpyridyl)benzoxazol-5-yl]-l-alanine derivatives
Katarzyna Guzow,a,p Mariusz Szabelski,a Joanna Malicka,a Jerzy Karolczakb
and Wiesøaw Wiczka
a
 Â
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland
Â
Quantum Electronics Laboratory, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
b
Received 21 September 2001; revised 17 December 2001; accepted 17 January 2002
AbstractÐThe syntheses of N-)tert-butyloxycarbonyl)-3-[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester and N-)tert-butyloxycar-
bonyl)-3-[2-)2-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester using different oxidizing agents )NBS, lead tetraacetate, Mitsunobu
reaction) in oxidative cyclization of Schiff base are described. The compounds obtained are ¯uorescent. The position of the emission
band and the ¯uorescence quantum yield depend on solvent polarity. The ¯uorescence decays of all the compounds studied are hetero-
geneous. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester )4b)
was synthesized using three different oxidizing agents.
N-)tert-Butyloxycarbonyl)-3-[2-)2-pyridyl)benzoxazol-5-
yl]-l-alanine methyl ester )4a) was prepared using only
one method. Their photophysical properties were also
measured.
Benzoxazoles are a very interesting group of heterocyclic
compounds which are widely used in chemistry, industry
and medicine. The 2-phenylbenzoxazoles are known as
photostable, highly ef®cient UV dyes1 and are used as
organic brightening agents,2 laser dyes,1 and organic plastic
scintillators.3 The benzoxazole moiety is also found in
biologically active compounds.4 Benzoxazoles have been
indicated as 5-HT3 receptor partial agonists,5 HIV protease
inhibitors,6 thrombin inhibitors,7 and a2-antagonist/5-HT
uptake inhibitors.8 Because of their photophysical proper-
ties and biological activity we are developing benzoxazole
derivatives possessing reactive functional groups )amino
and carboxyl) which allow to incorporate them into a
peptide chain. This makes them suitable for use in ¯uores-
cence conformational analysis of peptides or enzymatic
kinetics assays as a ¯uorescence probe incorporated in a
substrate.9
2. Results and discussion
2.1. Synthesis
N-)tert-Butyloxycarbonyl)-3-[2-)2-pyridyl)benzoxazol-5-
yl]-l-alanine methyl ester )4a) and N-)tert-butyloxycar-
bonyl)-3-[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine methyl
ester )4b) were prepared according to the procedures
published previously using as oxidizing agent either lead
tetraacetate or N-bromosuccinimide )NBS).20 In the case
of using lead tetraacetate, the in¯uence of the solvent on
the reaction yield of 4b was checked. Additionally,
cyclization of the Schiff base to 4b was performed by
means of the Mitsunobu reaction using typical reagents,
triphenylphosphine )TPP) and diisopropylazodicarboxylate
)DIAD) )Fig. 1).19,21 Also the removal of the protecting
groups was performed generating compounds 6 and 7
)Fig. 2).
Benzoxazoles can be synthesized according to various
procedures described in the literature.3,10±19 However, not
all of the methods can be used to obtain derivatives
possessing an amino acid moiety. The most suitable
approach is oxidative cyclization of azomethines )Schiff
bases) obtained from aromatic 1-amino-2-hydroxy
compounds )in our case N-Boc-3-nitro-l-tyrosine methyl
ester) and appropriate aldehydes.16±18 To ®nd the most
ef®cient method of synthesis N-)tert-butyloxycarbonyl)-3-
In the case of using NBS the reaction yield was rather low
)about 10%). Furthermore, a side-product was also isolated
)bromo-derivative). A better yield )about 25%), without
by-product, was obtained under the mild conditions of the
Mitsunobu reaction.
Keywords: benzoxazoles; pyridylbenzoxazol-5-yl-l-alanine; Mitsunobu
reaction; ¯uorescence.
Corresponding author. Tel.: 148-58-345-04-13; fax: 148-58-342-03-57;
e-mail: kasiag@chemik.chem.univ.gda.pl
p
Lead tetraacetate was also quite an effective oxidizing
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020)02)00092-3

2202
K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
OH
OH
O2N
H2N
H2/Pd
MeOH
CH2
CH2
Boc NH CH
Boc NH CH
C
O
OMe
C
O
OMe
2
1
2'
X
3'
O
H
1'
EtOHabs
4'
Y
C
6'
5'
Y
X
Y
X
Y
X
CH
OH
O
O
TPP
DIAD
N
3
N
4
N
(CH3COO)4Pb
solvent
5
THF
2
6
1
CH2
Boc NH CH
CH2
Boc NH CH
CH2
Boc NH CH
C
O
OMe
C
O
OMe
C
O
OMe
3a : X = N, Y = CH
3b : X = CH, Y = N
4b : X = CH, Y = N
4b : X = CH, Y = N
NBS
CH2Cl2
Y
X
Y
X
2
O
O
5
8
N
N
Br
9
4
7
+
6
CH2
Boc NH CH
CH2
Boc NH CH
C
O
OMe
C
O
OMe
5a : X =N, Y = CH
5b : X = CH, Y = N
4a : X =N, Y = CH
4b : X = CH, Y = N
Figure 1. Reaction scheme.
agent, but the reaction yield depended on the solvent used.
The least ef®cient synthesis was that performed in
dimethoxyethane )about 15% of the product), while the
syntheses in propylene carbonate and DMSO were the
most ef®cient )about 80% of the product). The syntheses
in the other solvents, except for DMF )70%), gave the
desired product in about 60%. The reaction was run for
ca. 25 h and monitored by RP-HPLC hold after 1 h and at
the end. The prolongation of the reaction time )above 1 h)
did not substantially change the percentage of the product in
the reaction mixture.
2.2. Photophysical properties
Absorption spectra of compounds 4a and 4b were measured
in MeOH, MeCN and methylcyclohexane and those
obtained for 4b are presented in Fig. 3.
In all solvents studied the absorption maxima are at about
300±310 nm. The vibronic structure of the spectrum is well
resolved only in the case of methylcyclohexane, the least
polar among the solvents used. The molar absorption coef®-
cient values are quite high, about 20 000 dm3 mol21 cm21
.

K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
2203
Y
X
Y
Y
X
X
2
O
5
O
O
1N NaOH
MeOH
8
N
N
N
6.8 M HCl/dioxan
9
4
7
6
CH2
Boc NH CH
CH2
Boc NH CH
CH2
+
C
OMe
C
O
OH
H3N CH
C
O
OH
Cl -
O
4a : X =N, Y = CH
4b : X = CH, Y = N
6a : X =N, Y = CH
6b : X = CH, Y = N
7b : X = CH, Y = N
Figure 2. Scheme for protecting group removal.
Figure 3. Absorption spectra of N-)tert-butyloxycarbonyl)-3-[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester in methanol, acetonitrile and methyl-
cyclohexane.
Figure 4. Emission spectra of N-)tert-butyloxycarbonyl)-3-[2-)2-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester in methanol, acetonitrile and methylcyclo-
hexane.

2204
K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
Figure 5. Emission spectra of N-)tert-butyloxycarbonyl)-3-[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine methyl ester in methanol, acetonitrile and methylcyclo-
hexane.
The ¯uorescence spectra of 4a, 4b and 7b are presented in
Figs. 4±6, respectively. The bromo-derivatives are non-
¯uorescent.
trum of 7b in water shows an emission maximum at 403 nm.
The emission maxima of 4b are at a longer wavelength as
compared with those of 4a in the same solvents. The highest
¯uorescence quantum yield is displayed by 4a in MeOH
)Quantum yield )QY)ˆ0.87), while the lowest quantum
yield are displayed by 4b and 7b in methylcyclohexane
)QYˆ0.01).
Similar to the absorption spectrum, the emission spectra of
all the above-mentioned compounds exhibits well-resolved
vibronic structure only in methylcyclohexane. The position
of the emission band, as well as the ¯uorescence quantum
yield, depends on the solvent polarity. In more polar
solvents, the emission band is shifted to longer wavelength,
and the ¯uorescence quantum yield is higher. The emission
bands of 7b are shifted slightly to shorter wavelength
compared to those of 4b. However, the ¯uorescence spec-
The ¯uorescence lifetimes, pre-exponential factors and the
¯uorescence QY of the compounds studied are presented in
Table 1.
The ¯uorescence intensity decays of the compounds studied
Figure 6. Emission spectra of 3-[2-)4-pyridyl)benzoxazol-5-yl]-l-alanine in methanol, acetonitrile, methylcyclohexane, and water.

K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
2205
Table 1. The ¯uorescence lifetimes )ti), pre-exponential factors )ai) and the ¯uorescence quantum yields )QY) of benzoxazol-5-yl-l-alanine derivatives in
methanol, acetonitrile, methylcyclohexane and water
2
xR
Compound
t
1 )ns)
t
2 )ns)
t3 )ns)
a1
a2
a3
QY
MeOH
4b
2.165
2.188
0.742
0.686
1.189
1.399
1.620
1.0000
0.9355
1.0000
0.9559
1.0000
0.1931
0.1067
1.168
0.40
0.18
0.04
0.686
1.332
0.0645
0.0441
1.099
1.851
0.994
7b
6a
1557
0.276
0.587
0.8069
0.2400
2.692
1.202
0.102
0.6533
MeCN
4b
1.188
1.081
6.095
4.782
1.917
1.314
1.0000
0.7677
1.0000
0.7590
1.0000
0.4472
34.247
1.116
5.045
1.053
440.35
1.093
0.21
0.05
0.20
0.546
0.841
0.120
0.2323
0.2410
0.5528
7b
6a
Methylcyclohexane
4b
0.609
1.615
0.999
3.122
3.032
2.900
1.028
1.0000
0.0061
0.0076
1.0000
0.1557
1.0000
0.1884
37.038
2.225
1.058
58.126
1.065
0.01
0.028
0.013
0.9939
0.9729
0.194
0.0194
7b
6a
0.01
0.20
0.988
0.104
0.8443
0.8116
2072
1.081
Water
7b
0.999
0.443
1.0000
0.2754
21.633
1.112
0.22
1.057
0.7246
in different solvents are mostly two-exponential. Only in the
case of 6a in MeOH and 4b in methylcyclohexane the
decays are three-exponential.
The spots were revealed using a UV lamp )254 nm,
366 nm). All solvent ratios are in volume parts. The puri®-
cation was carried out by means of column chromatography
)Merck, Silica gel 60, 0.040±0.063 mm) and semi-prepara-
tive RP-HPLC )Kromasil column, C-8, 5 mm, 250mm long,
IDˆ20mm). The purity of the obtained compounds was
checked by means of analytical RP-HPLC )Kromasil
column, C-18, 5 mm, 250mm long, ID ˆ4.5 mm) with
detection at 223 nm. Melting points )mp) were determined
3. Conclusion
All the described methods of synthesis of benzoxazol-5-yl-
l-alanine were effective and gave the desired product.
However, the worst method was that using NBS as the
oxidizing agent because of the presence of side-product.
The Mitsunobu reaction gave better yields, but it was the
most time-consuming method. So the best approach seems
to be method using lead tetraacetate in DMSO or propylene
carbonate.
1
in capillary tubes and are uncorrected. H NMR, 13C NMR
and 1H±1H COSY spectra were recorded on a Varian,
Mercury-400 BB spectrometer )400 MHz). They were
taken in CDCl3 or DMSO-d6. d was referenced internally
to the residual proton resonance of CDCl3 )7.27 ppm) or
DMSO-d6 )3.29 ppm), respectively, or to SiMe4
)ˆ0.00 ppm) for 1H and to DMSO-d6 )39.51 ppm for center-
line) for 13C, respectively. Infrared spectra were recorded on
a Bruker IFS-66 instrument. Mass spectra were recorded on
a MASSLAB TRIO-3 instrument. Elemental analyses were
taken on a Carlo Erba CNSO Eager 200 instrument. Absorp-
tion spectra were measured on a Perkin±Elmer Lambda 18
spectrophotometer. Fluorescence spectra were measured on
a Perkin±Elmer LS-50B spectro¯uorimeter. The solvents
used in our studies: MeOH, MeCN, methylcyclohexane
were of either spectroscopic or HPLC grade. QY were
calculated using as a reference quinine sulphate in 0.5 M
H2SO4 )QYˆ0.53^0.0224) or tryptophan in water
)QYˆ0.1425). The ¯uorescence lifetimes were measured
using a time-correlated single-photon counting apparatus
)the pico/femtosecond laser system, Ti:Sapphire `Tsunami'
laser pumped with an argon ion laser `Beammlok' 2060 and
R3809U-05 MCP-PTM; a half-width of the response func-
tion was about 35 ps) at the Laboratory of Ultrafast Laser
The properties of the obtained compounds are very interest-
ing and make them suitable as the ¯uorescence probes to
study the micro-environmental polarity of peptides.
4. Experimental
4.1. General
2-Pyridinecarboxaldehyde, 4-pyridinecarboxaldehyde, tri-
phenylphosphine )TPP) and diisopropylazodicarboxylate
)DIAD) were purchased from Aldrich, lead tetraacetate
and NBS from Lancaster and 3-nitro-l-tyrosine from
Fluka. The following compounds were prepared according
to literature procedures: 3-nitro-l-tyrosine methyl ester22
and N-Boc-3-nitro-l-tyrosine methyl ester.23 TLC was
carried out on Merck silica gel plates )Kieselgel 60F 254).

2206
K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
Â
Spectroscopy, Adam Mickiewicz University, Poznan,
Poland.26 Fluorescence decay data were ®tted by the
iterative convolution to the sum of exponents
X
compounds was checked by means of analytical RP-
HPLC. The mobile phase was a gradient running from 0
to 80% of an aqueous solution of acetonitrile with addition
of 0.1% tri¯uoroacetic acid )TFA) over 60 min
)tRˆ43.14 min )4a), tRˆ48.94 min )5a)).
Iꢀt† ˆ
ai expꢀ2t=ti†;
ꢀ1†
i
Compound 4a: mp 102±103.58C; )Found: C, 63.35; N,
10.34; H, 5.70. C21H23N3O5 requires C, 63.46; N, 10.57;
H, 5.83%); Rf )67% AcOEt/petroleum ether) 0.14; nmax
)liquid ®lm) 3345.8 )w), 2979.1 )m), 1744.3 )s), 1710.4
where ai is the pre-exponential factor obtained from the
¯uorescence intensity decay analysis and ti is the decay
time of the ith component. The adequacy of the exponential
decay ®tting was judged by visual inspection of the plots of
weighted residuals and by the statistical parameter xR2 and
shape of the autocorrelation function of the weighted
residuals and serial variance ratio )SVR).
)s), 1250.0 )s), 1167.3 )s), 754.2 )s) cm21; dH )400 MHz,
0
CDCl3) 8.81 )1H, d, Jˆ4.4 Hz, C3 H), 8.33 )1H, d,
0
0
Jˆ8.0Hz, C 6 H), 7.89 )1H, t, Jˆ8.0Hz, C 5 H), 7.58 )2H,
0
d, Jˆ8.4 Hz, C4 H, C7H), 7.45 )1H, dd, Jˆ5.2, 7.2 Hz, C4H),
7.17 )1H, d, Jˆ8.8 Hz, C6H), 5.06 )1H, d, Jˆ8.0Hz, NH),
4.64 )1H, dd, Jˆ5.6, 13.2 Hz, CaH), 3.72 )3H, s, OCH3),
3.17±3.29 )2H, m, CbH2), 1.41 )9H, s, )CH3)3); dC
)100 MHz, DMSO-d6) 172.40)C aCOO), 161.47
4.2. N-ꢀtert-Butyloxycarbonyl)-3-[2-ꢀ2-pyridyl)-
benzoxazol-5-yl]-l-alanine methyl ester ꢀ4a)
4.2.1. N-Boc-3-amino-l-tyrosine methyl ester ꢀ2). A
mixture of N-Boc-3-nitro-l-tyrosine methyl ester )1)
)1.02 g, 3 mmol, mp 90±918C) and 5% palladium on active
carbon in MeOH )30mL) was stirred under a hydrogen
atmosphere at room temperature for 90min )TLC monitor-
ing )CH2Cl2/MeOH/AcOH 100:10:1), Rfˆ0.9 )1), Rfˆ0.72
)2)). The catalyst was ®ltered off and the solvent evaporated
in vacuo to give the brownish, oily title compound 2, which
was used at once without any additional puri®cation in the
next step of the synthesis.
0
0
)HNCOO), 155.34 )C2), 150.14 )C1 ), 149.30)C 3 ), 145.21
0
)C8), 141.20)C ), 137.602 )C5 ), 134.73 )C5), 127.41 )C6),
9
0
126.11 )C6 ), 123.48 )C4 ), 120.67 )C4), 110.71 )C7), 78.23
)C)CH3)3), 55.39 )Ca), 51.75 )OCH3), 36.28 )Cb), 28.01
)C)CH3)3); m/z )FAB) 398 )25, MH1), 342 )100), 298
)22), 238 )68), 210)70%).
Compound 5a: mp 173±1758C; )Found: C, 52.92; N, 8.75;
H, 4.62. C21H22N3O5Br requires C, 52.95; N, 8.82; H,
4.66%); nmax )KBr) 3351.3 )m), 2983.0)w), 1737.7 )s),
1686.4 )s), 1526.4 )s), 1250.3 )s), 1166.6 0 )s) cm21; dH
)400 MHz, DMSO-d6) 8.81±8.83 )1H, m, C3 H), 8.33 )1H,
4.2.2. N-Boc-3-[ꢀpyrid-2-ylmethylene)amino]-l-tyrosine
methyl ester ꢀ3a). To a solution of 2 )3 mmol) in absolute
EtOH )6 mL), 2-pyridinecarboxaldehyde )0.321 g, 0.29 mL,
3 mmol) was added. The mixture was stirred at rt overnight
)TLC monitoring )AcOEt), Rfˆ0.62; no substrate left).
After this time the solvent was removed by evaporation
and the brown, oily title compound 3a was used at once
without any additional puri®cation in the next step of the
synthesis.
0
0
d, Jˆ8.0Hz, C 6 H), 8.07 )1H, td, Jˆ1.6, 7.6, 7.8 Hz, C5 H),
7.76 )1H, d, Jˆ1.2 Hz, C6H), 7.61±7.69 )2H, m, C4H,
0
C4 H), 7.34 )1H, d, Jˆ8.0Hz, NH), 4.26±4.32 )1H, m,
CaH), 3.65 )3H, s, OCH3), 2.96±3.23 )2H, m, CbH2), 1.29
)9H, s, )CH3)3); each proton signal was con®rmed by 1H±1H
COSY spectrum; dC )100 MHz, DMSO-d6) 172.16
0
)CaCOO), 161.59 )HNCOO), 155.30)C ), 150.23 )C1 ),
2
0
0
148.96 )C3 ), 147.35 )C8), 141.81 )C90), 137.71 )C5 ),
0
136.72 )C5), 130.02 )C6), 126.48 )C6 ), 123.79 )C4 ),
120.27 )C4), 101.28 )C7), 78.26 )C)CH3)3), 55.00 )Ca),
51.82 )OCH3), 35.86 )Cb), 27.97 )C)CH3)3); m/z )FAB)
477 and 479 )28 and 30, MH1), 420and 422 )76 and 72),
399 )50), 376 )20), 342 )100), 316 )37%).
4.2.3. N-ꢀtert-Butyloxycarbonyl)-3-[2-ꢀ2-pyridyl)benz-
oxazol-5-yl]-l-alanine methyl ester ꢀ4a) and N-ꢀtert-
butyloxycarbonyl)-3-[7-bromo-2-ꢀ2-pyridyl)benzoxazol-
5-yl]-l-alanine methyl ester ꢀ5a)Ðcyclization of 3a in
the presence of NBS. To a solution of 3a )2.5 mmol) in
CH2Cl2 )10mL) NBS )0.49 g, 2.75 mmol) was added and
the reaction mixture was stirred for about 30min )TLC
monitoring )AcOEt/petroleum ether 2:3), Rfˆ0.14 )4a)).
Then the solvent was removed by evaporation. The
brown, oily residue was dissolved in ethyl acetate )30mL)
and washed with water )2£10mL), a 5% solution of
NaHCO3 )2£10mL) and a saturated aqueous solution of
NaCl )2£10mL), dried over anhydrous MgSO 4 and the
solvent evaporated in vacuo. Recrystallizations of the
crude mixture from AcOEt/petroleum ether 2:3 and then
from ethanol gave the title compound 5a )208.4 mg,
0.44 mmol, 18%) as a white solid. The ®ltrates were
collected, evaporated and the residue was puri®ed twice
by means of column chromatography using as eluents
AcOEt/petroleum ether )2:3) and AcOEt/petroleum ether
)1:1). Then 4a was isolated by means of semi-preparative
HPLC. The mobile phase was a gradient running from 0to
80% of an aqueous solution of acetonitrile over 120 min.
This gave the title compound 4a )108.3 mg, 0.27 mmol,
11%) as a cream solid. The purity of the obtained
4.3. N-ꢀtert-Butyloxycarbonyl)-3-[2-ꢀ4-pyridyl)-
benzoxazol-5-yl]-l-alanine methyl ester ꢀ4b)
4.3.1. N-Boc-3-amino-l-tyrosine methyl ester ꢀ2). This
was synthesized according to the above-mentioned
procedure )Section 4.2.1) using 4 mmol )1.36 g) of
N-Boc-3-nitro-l-tyrosine methyl ester )1).
4.3.2. N-Boc-3-[ꢀpyrid-4-ylmethylene)amino]-l-tyrosine
methyl ester ꢀ3b). To a solution of 2 )4 mmol) in absolute
EtOH )6 mL) 4-pyridinecarboxaldehyde )0.428 g, 0.38 mL,
4 mmol) was added. After about 30min, a yellow precipi-
tate formed. The mixture was stirred at rt overnight )TLC
monitoring )AcOEt): no substrate left). The precipitate was
®ltered off, washed with cold ethanol and used without any
additional puri®cation in the next step of the synthesis
)0.95 g, 2.38 mmol, 59.5%, tRˆ27.96 min).
Compound 3b: mp 164±1658C; )Found: C, 63.31; N, 10.32;

K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
2207
H, 6.27. C21H25N3O5 requires C, 63.14; N, 10.52; H,
6.31%); Rf )AcOEt) 0.63; nmax )KBr) 3376.7 )m), 2978.5
)w), 1751.1 )s), 1680.4 )s), 1599.5 )m), 1502.5 )s), 1219.0
)s), 1165.9 )s) cm21; dH )400 MHz, CDCl3) 8.78 )2H, dd,
)1H, br. d, Jˆ8.4 Hz, NH), 4.25±4.31 )1H, m, CaH), 3.65
)3H, s, OCH3), 2.98±3.25 )2H, m, CbH2), 1.29 )9H, s,
)CH3)3); each proton signal was con®rmed by 1H±1H
COSY spectrum; dC )100 MHz, DMSO-d6) 172.13
0
0
0
Jˆ1.6, 4.4 Hz, C3 H, C5 H), 8.66 )1H, s, NvCH), 7.74 )2H,
)Ca0 COO), 160.61 )HNCOO), 155.30 )C2), 150.90 )C30 ,
0
0
dd, Jˆ1.6, 4.4 Hz, C2 H, C6 H), 7.13 )1H, d, Jˆ1.2 Hz, OH),
7.02 )2H, br. d, Jˆ8.4 Hz, C2H, C6H), 6.96 )1H, d,
Jˆ8.4 Hz, C5H), 5.00 )1H, br. s, NH), 4.58 )1H, br. s,
CaH), 3.72 )3H, s, OCH3), 3.01±3.12 )2H, m, CbH2), 1.42
)9H, s, )CH3)3); m/z )FAB) 401 )100, )M12)1), 344 )95),
302 )30), 212 )78%).
C5 ), 147.24 )C8), 141.76 )C90), 136.95 )C5), 132.91 )C1 ),
0
130.33 )C6), 120.83 )C2 , C6 ), 120.34 )C4), 101.26 )C7),
78.28 )C)CH3)3), 54.99 )Ca), 51.82 )OCH3), 35.83 )Cb),
27.98 )C)CH3)3); m/z )FAB) 477 and 479 )75 and 60,
MH1), 420and 422 )78 and 86), 376 )25), 319 )30), 290
)35), 221 )100%).
4.3.3.2. Cyclization of 3b by means of the Mitsunobu
reaction. To a solution of 3b )0.798 g, 2 mmol) in
anhydrous THF )30mL) cooled to 0 8C under argon,
triphenylphospine )1.05 g, 4 mmol) was added. After
stirring for about 5 min, diisopropylazodicarboxylate
)0.81 g, 0.79 mL, 4 mmol) was added dropwise. The
reaction mixture was stirred for about 20min. Afterwards
the ice bath was removed and the mixture was stirred under
argon at rt for 3 days )TLC monitoring )AcOEt), Rfˆ0.65
)4b)). Then the solvent was removed by evaporation. The
brown, oily residue was dissolved in diethyl ether )30mL).
The obtained white precipitate )triphenylphosphine oxide)
was ®ltered off and washed with diethyl ether. The ®ltrate
was evaporated and puri®ed by means of column
chromatography using as an eluent AcOEt. After
recrystallization from ethanol 196.1 mg of 4b )0.49 mmol,
25%) was isolated.
4.3.3. N-ꢀtert-Butyloxycarbonyl)-3-[2-ꢀ4-pyridyl)benz-
oxazol-5-yl]-l-alanine methyl ester ꢀ4b) and N-ꢀtert-
butyloxycarbonyl)-3-[7-bromo-2-ꢀ4-pyridyl)benzoxazol-
5-yl]-l-alanine methyl ester ꢀ5b)
4.3.3.1. Cyclization of 3b in the presence of NBS. To a
solution of 3b )0.798 g, 2 mmol) in CH2Cl2 )8 mL) NBS
)0.39 g, 2.2 mmol) was added and the reaction mixture
was stirred for about 30min )TLC monitoring )AcOEt),
Rfˆ0.65 )4b)). Then the solvent was removed by evapora-
tion. The brown, oily residue was dissolved in ethyl acetate
)30mL) and washed with water )2 £10mL), a 5% solution
of NaHCO3 )2£10mL) and a saturated aqueous solution of
NaCl )2£10mL), dried over anhydrous MgSO 4 and evapo-
rated. Recrystallizations of the crude mixture from AcOEt
gave 5b as a white solid )830mg, 1.7 mmol, 87%). The
®ltrates were collected, evaporated and the residue was puri-
®ed by means of column chromatography using as an eluent
AcOEt. Recrystallization from ethanol gave the title
compound 4b as a dark beige solid )77.8 mg, 0.19 mmol,
10%). The purity of obtained compounds was checked by
means of analytical RP-HPLC. The mobile phase was a
gradient running from 0to 80% of aqueous solution of
acetonitrile with addition of 0.1% TFA over 60 min
)tRˆ35.96 min )4b), tRˆ41.88 min )5b)).
4.3.3.3. The in¯uence of a solvent on the reaction yield
of cyclization 3b in the presence of lead tetraacetate:
general procedure. To a solution of 3b )15.9 mg,
40 mmol) in a solvent )4 mL of toluene or CH2Cl2 or THF
or dimethoxyethane or propylene carbonate or acetic acid)
lead tetraacetate )26.8 mg, 60 mmol) was added and the
reaction mixture was stirred at rt. In the case of DMF and
DMSO )1 mL) 4 mg )10 mmol) of 3b and 6.7 mg )15 mmol)
of lead tetraacetate were used in the reaction. The progress
of the reaction was monitored by means of analytical RP-
HPLC after 60min and about 25 h. The mobile phase was a
gradient running from 0to 80% of aqueous solution of
acetonitrile with addition of 0.1% TFA over 60 min
)tRˆ27.96 min )3b), tRˆ35.96 min )4b)).
Compound 4b: mp 137±1398C; )Found: C, 63.25; N, 10.67;
H, 5.88. C21H23N3O5 requires C, 63.46; N, 10.57; H,
5.83%); Rf )AcOEt) 0.65; nmax )KBr) 3354.1 )m), 2980.6
)m), 1737.7 )s), 1689.2 )s), 1637.4 )m), 1524.8 )m), 1254.1
)m), 1169.1 )s), 1059.3 )m), 721.8 )m) c0m21; dH )400 MHz,
0
CDCl3) 8.81 )2H, dd, Jˆ1.2, 4.6 Hz, C3 H, C5 H), 8.06 )2H,
0
0
dd, Jˆ1.2, 4.6 Hz, C2 H, C6 H), 7.57 )1H, s, C4H), 7.55 )1H,
d, Jˆ8.4 Hz, C7H), 7.21 )1H, dd, Jˆ1.6, 8.8 Hz, C6H), 5.14
)1H, d, Jˆ8.0Hz, NH), 4.66 )1H, dd, Jˆ5.6, 13.6 Hz, CaH),
3.74 )3H, s, OCH3), 3.18±3.31 )2H, m, CbH2), 1.42 )9H, s,
)CH3)3); dC )100 MHz, DMSO-d6) 172.56 )CaCOO),
The percentage of the product in the reaction mixture was
determined basing on analytical RP-HPLC )10 mL of the
each reaction mixture was dissolved in 100 mL of EtOH
and 50 mL of the prepared solution was injected) and was
as follows: toluene )58%), CH2Cl2 )60%), THF )58%),
dimethoxyethane )15%), propylene carbonate )80%), acetic
acid )59%), DMF )70%), DMSO )80%).
0
0
160.43 )HNCOO), 154.82 )C2), 150.84 )C30 , C5 ), 144.02
)C8), 141.45 )C9), 135.01 )C5), 133.12 )C1 ), 127.78 )C6),
0
0
120.71 )C2 , C6 ), 120.68 )C4), 110.72 )C7), 79.10) C)CH3)3),
55.35 )Ca), 51.75 )OCH3), 36.21 )Cb), 28.00 )C)CH3)3); m/z
)FAB) 398 )85, MH1), 342 )100), 298 )35), 258 )24), 237
)58%).
4.4. The removal of protecting groups: general
procedure
Compound 5b: mp 186±1878C; )Found: C, 52.82; N, 8.76;
H, 4.62. C21H22N3O5Br requires C, 52.95; N, 8.82; H,
4.66%); nmax )KBr) 3334.0)m), 2985.7 )w), 1741.0)s),
1683.4 )s), 1529.5 )m), 1254.4 )m), 1164.6 )m), 1057.3
)m) cm21; dH )400 MHz, DMSO-d6) 8.86 )1H, dd, Jˆ1.6,
4.4.1. Methyl ester group removal: N-ꢀtert-butyloxycar-
bonyl)-3-[2-ꢀ2-pyridyl)benzoxazol-5-yl]-l-alanine ꢀ6a)
and N-ꢀtert-butyloxycarbonyl)-3-[2-ꢀ4-pyridyl)benzoxa-
zol-5-yl]-l-alanine ꢀ6b). 4a or 4b was dissolved in a 1 M
solution of NaOH in MeOH and stirred at rt for about an
hour )TLC monitoring )AcOEt): no substrate left). Then the
solvent was removed by evaporation. The residue was
0
0
0
4.4 Hz, C3 H, C5 H), 8.08 )2H, dd, Jˆ1.6, 4.4 Hz, C2 H,
0
C6 H), 7.77 )1H, br. s, C6H), 7.64 )1H, br. s, C4H), 7.34

2208
K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
dissolved in AcOEt and washed twice with a 1 M solution of
KHSO4 and three times with a saturated aqueous solution of
NaCl, dried over anhydrous MgSO4 and evaporated.
Recrystallization of the crude mixture from EtOH gave
the product )6a as a white solid: 28%; 6b as a beige solid:
25%). The purity of the obtained compounds was checked
by means of analytical RP-HPLC. The mobile phase was a
gradient running from 0to 80% of aqueous solution of
acetonitrile with addition of 0.1% TFA over 60 min
)tRˆ36.64 min )6a), tRˆ29.96 min )6b)).
)m), 1200.0 )m), 1058.6 )m), 825.5 )m), 702.1 )m), 511.6
)m) cm21; dH )400 MHz, DMSO-d6) 8.83 )2H, dd, Jˆ1.6,
0
0
4.6 Hz, C3 H, C5 H), 8.15 )3H, br. s, NH31), 8.10)2H, dd,
0
0
Jˆ1.6, 4.4 Hz, C2 H, C6 H), 7.75 )2H, s, C4H), 7.44 )1H, d,
Jˆ8.4 Hz, C7H), 7.16 )1H, d, Jˆ8.4 Hz, C6H), 4.12±4.19
)1H, m, CaH), 2.96±3.22 )2H, m, CbH2); dC )100 MHz,
0
DMSO-d6) 172.97 )CaCOO), 155.06 )C2), 150.45 )C30 ,
0
C5 ), 148.84 )C8), 140.77 )C90), 135.04 )C5), 133.10)C 1 ),
0
127.44 )C6), 120.42 )C2 , C6 ), 120.35 )C4), 110.31 )C7),
55.06 )Ca), 35.91 )Cb); m/z )FAB) 284 )5, MH1), 277
)100), 259 )14), 241 )100), 223 )18), 207 )40%).
Compound 6a: mp 145±1478C; )Found: C, 62.45; N, 10.74;
H, 5.60. C20H21N3O5 requires C, 62.65; N, 10.96; H,
5.52%); nmax )KBr) 3372.7 )m), 2972.5 )w), 2936.4 )w),
1725.5 )s), 1679.2 )s), 1521.1 )s), 1465.6 )m), 1442.6 )m),
1290.4 )m), 1252.7 )m), 1163.7 )m), 1043.7 )w) cm21; dH
Acknowledgements
This work was supported by the State Committee for
Scienti®c Research )KBN) under grant DS-8351-4-0132-0.
)400 MHz, CDCl3) 11.78 )1H, br. s, OH),0 8.82 )1H, d,
0
Jˆ4.4 Hz, C3 H), 8.31 )1H, d, Jˆ8.0Hz, C 6 H), 7.90)1H,
0
0
t, Jˆ8.0Hz, C 5 H), 7.57 )2H, d, Jˆ8.4 Hz, C4 H, C7H), 7.48
)1H, dd, Jˆ5.2, 7.2 Hz, C4H), 7.19 )1H, d, Jˆ8.8 Hz, C6H),
5.10)1H, d, Jˆ8.0Hz, NH), 4.63 )1H, dd, Jˆ5.6, 13.2 Hz,
CaH), 3.15±3.29 )2H, m, CbH2), 1.41 )9H, s, )CH3)3); dC
)100 MHz, DMSO-d6) 172.41 )CaCOO), 161.44 )HNCOO),
References
1. Kanegae, Y.; Peariso, K.; Martinez, S. S. Appl. Spectrosc.
1996, 50, 316±319.
0
0
155.40)C 2), 150.11 )C1 ), 149.35 )C3 ), 145.24 )C8), 141.22
2. Krasovitskii, B. M.; Bolotin, B. M. Organic Luminescent
Materials; VCH: Weinheim, 1988 pp 169±211.
3. Pla-Dalmau, A. J. Org. Chem. 1995, 60, 5468±5473.
Â
4. Rodriguez, A. D.; Ramirez, C.; Rodriguez, I. I.; Gonzalez, E.
Org. Lett. 1999, 1, 527±530.
0
0
)C9), 137.61 )C5 ), 134.75 )C5), 127.43 )C6), 126.14 )C6 ),
0
123.46 )C4 ), 120.66 )C4), 110.73 )C7), 78.21 )C)CH3)3),
55.36 )Ca), 36.26 )Cb), 28.00 )C)CH3)3); m/z )FAB) 384
)25, MH1), 329 )100), 284 )52), 238 )68), 210 )50%).
5. Sato, Y.; Yamada, M.; Yoshida, S.; Soneda, T.; Ishikawa, M.;
Nizato, T.; Suzuki, K.; Konno, F. J. Med. Chem. 1998, 41,
3015±3021.
Compound 6b: mp 252±2538C; )Found: C, 62.47; N, 10.84;
H, 5.66. C20H21N3O5 requires C, 62.65; N, 10.96; H,
5.52%); nmax )KBr) 3325.2 )m), 3075.4 )w), 2976.0 )m),
2931.6 )m), 2455.0)br), 1912.4 )br), 1708.0)s), 1618.0
)m), 1519.6 )s), 1365.3 )m), 1294.2 )m), 1234.7 )s),
1173.3 )s), 1058.5 )m), 1010.8 )m), 832.3 )m), 707.9
)m) cm21; dH )400 MHz, DMSO-d6) 8.84 )2H, dd, Jˆ1.6,
6. Chen, P.; Cheng, P. T. W.; Alam, M.; Beyer, B. D.; Bisacchi,
G. S.; Dejneka, T.; Evans, A. J.; Greytok, J. A.; Hermsmeier,
M. A.; Humphreys, W. G.; Jacobs, G. A.; Kocy, O.; Lin, P.-F.;
Lis, K. A.; Marella, M. A.; Ryono, D. E.; Sheaffer, A. K.;
Spergel, S. H.; Sun, C.-Q.; Tino, J. A.; Vite, G.; Colonno,
R. J.; Zahler, R.; Barrish, J. C. J. Med. Chem. 1996, 39,
1991±2007.
0
0
0
4.6 Hz, C3 H, C5 H), 8.09 )2H, dd, Jˆ1.6, 4.4 Hz, C2 H,
0
C6 H), 7.76 )2H, s, C4H, NH), 7.41 )1H, d, Jˆ8.4 Hz,
C7H), 7.14 )1H, d, Jˆ8.4 Hz, C6H), 4.13±4.19 )1H, m,
CaH), 2.95±3.21 )2H, m, CbH2), 1.29 )9H, s, )CH3)3); dC
7. Costanzo, M. J.; Maryanoff, B. E.; Hecker, L. R.; Schott,
M. R.; Yabut, S. C.; Zhang, H.-C.; Andrade-Gordon, P.;
Kauffman, J. A.; Lewis, J. M.; Krishnan, R.; Tulinski, A.
J. Med. Chem. 1996, 39, 3039±3043.
)100 MHz, DMSO-d6) 172.990)CaCOO), 160.01 )HNCOO),
0
155.04 )C2), 150.48 )C3 , C5 ), 148.82 )C8), 140.76 )C90),
0
0
135.04 )C5), 133.12 )C1 ), 127.44 )C6), 120.42 )C2 , C6 ),
120.35 )C4), 110.32 )C7), 77.65 )C)CH3)3), 55.02 )Ca),
35.93 )Cb), 27.69 )C)CH3)3); m/z )FAB) 384 )45, MH1),
329 )100), 284 )58), 238 )61), 210 )52), 168 )50), 148
)100%).
8. Meyer, M. D.; Hancock, A. A.; Tietje, K.; Sippy, K. B.;
Prasad, R.; Stout, D. M.; Arendsen, D. L.; Donner, B. G.;
Carroll, W. A. J. Med. Chem. 1997, 40, 1049±1062.
9. Szabelski, M.; Guzow, K.; Stachowiak, K.; Wiczk, W. In
Natural Science, Proceedings of the Third International
Conferernce of PhD Students, University of Miskolc, Innova-
4.4.2. Boc group removal: 3-[2-ꢀ4-pyridyl)benzoxazol-5-
yl]-l-alanine ꢀ7b). 6b was dissolved in 6.8 M HCl in dioxan
and stirred at rt for about 30min )TLC monitoring )CH 2Cl2/
EtOH/AcOH 100:5:1): no substrate left). Then the solvent
was removed by evaporation. Recrystallization from EtOH
gave the title compound 7b as a light beige solid )40%). The
purity of the obtained compound was checked by means of
analytical RP-HPLC. The mobile phase was a gradient
running from 0to 80% of aqueous solution of acetonitrile
with addition of 0.1% TFA over 60 min )7b: tRˆ15.68 min).
Â
tion and Technology Transfer Centre, Lehoczky, L., Kalmar,
L., Eds., 2001, pp 299±303.
10. Krasovitskii, B. M.; Bolotin, B. M. Organic Luminescent
Materials; VCH: Weinheim, 1988 pp 89±91.
11. So, Y.-H.; Heeschen, J. P. J. Org. Chem. 1997, 62, 3552±
3561.
12. DeLuca, M. R.; Kerwin, S. M. Tetrahedron 1997, 53, 457±
464.
13. DeLuca, M. R.; Tarapolewala, I. B.; Kerwin, S. M. Hetero-
cycles 1999, 51, 979±982.
14. Grellmann, K. H.; Tauer, E. J. Am. Chem. Soc. 1973, 95,
3104±3108.
Compound 7b: mp 253±2548C; )Found: C, 63.74; N, 14.85;
H, 4.67. C20H21N3O5 requires C, 63.60; N, 14.83; H,
4.63%); nmax )KBr) 3439.0)br), 2936.7 )br), 2095.7 )w),
1595.2 )s), 1545.7 )m), 1416.9 )m), 1346.1 )m), 1318.5
15. Hein, D. W.; Alheim, R. J.; Leavitt, J. J. J. Am. Chem. Soc.
1957, 79, 427±429.
16. Stephens, F. F.; Bower, J. D. J. Chem. Soc. 1949, 2971±2972.

K. Guzow et al. / Tetrahedron 58 22002) 2201±2209
2209
17. Stephens, F. F.; Bower, J. D. J. Chem. Soc. 1950, 1722±1726.
18. Srivastava, R. G.; Venkataramani, P. S. Synth. Commun. 1988,
18, 1537±1544.
Houben-Weyl, Methoden der Organischen Chemie; Georg
Thieme Verlag: Stuttgart, 1974; Vol. 15, p. 317.
23. Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis;
2nd ed.; Pierce Chemical Company: Rockford, IL, 1984 pp 63.
24. Adams, M. J.; High®eld, J. G.; Kirkbright, G. F. Anal. Chem.
1977, 49, 1850±1852.
19. Wang, F.; Hauske, J. R. Tetrahedron Lett. 1997, 38, 6529±
6532.
20. Guzow, K.; Szabelski, M.; Malicka, J.; Wiczk, W. Helv. Chim.
Acta 2001, 84, 1086±1092.
Â
21. Wisniewski, K.; Koøodziejczyk, A. S.; Falkiewicz, B.
25. Chen, R. F. Anal. Lett. 1967, 1, 35±42.
26. Karolczak, J.; Komar, D.; Kubicki, J.; Szymanski, M.;
Â
Â
WrozÇowa, T.; Maciejewski, A. Bull. Pol. Acad. Sci. Chem.
1999, 47, 361±380.
J. Peptide Sci. 1998, 4, 1±14.
22. WuÈnsch, E. In Synthese von Peptiden, Mhller, E., Ed.;