Bifunctional coumarin derivatives in solution and solid phase synthesis of fluorogenic enzyme substrates

Article abstract of DOI:10.1002/jhet.5570380122

The use of the fluorescent bifunctional compounds 7-amino-4-coumarinyl-acetic acid 1, 7-hydroxy-4-coumarinyl-acetic acid 2 and ethyl 7-amino-4-coumarinyl-acetate 3 in solution and solid phase synthesis of fluorogenic enzyme substrates was examined. The in

Full text of DOI:10.1002/jhet.5570380122

Jan-Feb 2001
Bifunctional Coumarin Derivatives in Solution and Solid Phase
Synthesis of Fluorogenic Enzyme Substrates
153
Christos Charitos, George Kokotos and Chryssa Tzougraki*
Department of Chemistry, University of Athens, Panepistimiopolis, Athens 157 71, Greece
Received December 6, 1999
The use of the fluorescent bifunctional compounds 7-amino-4-coumarinyl-acetic acid 1, 7-hydroxy-4-
coumarinyl-acetic acid 2 and ethyl 7-amino-4-coumarinyl-acetate 3 in solution and solid phase synthesis of
fluorogenic enzyme substrates was examined. The intramolecularly quenched fluorogenic substrate N-(7-
amino-4-coumarinyl-acetyl)-L-phenylalanyl-p-nitroanilide 5, and the fluorogenic one ethyl 7-(glutaryl-L-
phenylalanilamido)-4-coumarinyl-acetate 8, both suitable for chymotrypsin and/or chymotrypsin like
enzymes determination, were prepared in solution. The substrates 7-oleyloxy-4-coumarinyl-acetic acid 13
and 7-palmitoyloxy-4-coumarinyl-acetic acid 14, suitable for the enzymatic study of lipases, were prepared
by solid phase technique using 2-chloro-chlorotrityl-resin. The study of the fluorescence properties of the
fluorophores 1, 2, 3, and substrates 5, 8, 13, 14 showed that the examined bifunctional coumarin derivatives
are suitable markers for solution and solid phase synthesis of fluorogenic enzyme substrates.
J. Heterocyclic Chem., 38, 153 (2001).
Introduction.
suitable for fluorometric enzymatic analysis. These com-
pounds were chosen because, in addition to their excellent
fluorescence properties, they allow selective chain elonga-
tion through the two different functional groups they bear.
They also can be used for the preparation of either classical
or intramolecularly quenched fluorogenic substrates.
Moreover, they can be connected through their carboxyl
group to a solid support and serve as fluorescent linkers in
the solid phase synthesis of substrates.
We report here the synthesis and the study of the fluores-
cence properties of the above markers 1, 2, 3 and their
derivatives N-(7-amino-4-coumarinyl-acetyl)-L-phenyl-
alanyl-p-nitroanilide (Aca-Phe-NH-Np, 5), N-(7-amino-4-
coumarinyl-acetyl)-L-phenylalanine (Aca-Phe-OH, 6),
ethyl 7-(glutaryl-L-phenylalanilamido)-4-coumarinyl-
acetate (Glt-Phe-NH-ca-OEt, 8), 7-oleyloxy-4-coum-
arinyl-acetic acid (Ole-Oca, 13) and 7-palmitoyloxy-4-
coumarinyl-acetic acid (Pal-Oca, 14). The last two com-
pounds were prepared by solid phase synthesis technique.
Coumarin derivatives continue to be an interesting area
of research, because of their various and diverse proper-
ties. They exhibit important biological activities, such as
antibiotic [1-3], anticoagulant [4-6], anticancer [7-11],
antiinflammatory [12,13], etc. A considerable number of
natural or synthetic derivatives have pharmaceutical appli-
cation [14-16]. In the last years their strong inhibitory
activity against several enzymes, such as integrase [17,18],
monoaminooxidase (MAO) [19], human leucocyte elas-
tase [12], and HIV protease [17,20-22] has been reported.
Coumarins, however, are also interesting because of their
fluorescence properties and have been shown to be impor-
tant in fluorimetry applications. Some very well known fluo-
rescent markers, such as the 7-amino-4-methyl-coumarin
(Amec) [23], have been widely used in synthetic substrates,
fluorogenic or intramolecularly quenched fluorogenic ones.
Both types of substrates have been successfully used for the
sensitive determination of several proteases and the study of
their active site and secondary specificity as well.
Results and Discussion.
During the last ten years we have developed a number of
fluorescent markers and fluorogenic substrates for various
proteases [24,25]. We have also prepared platinum and
copper complexes of some coumarins and tested them for
anticancer activity [26,27]. Continuing our research in this
area, we have examined the use of the compounds 7-
amino-4-coumarinyl-acetic acid (Aca, 1), 7-hydroxy-4-
coumarinyl-acetic acid (Hca, 2) and the ethyl ester of 1
(Aca-OEt, 3) (Scheme 1) as markers in synthetic substrates
The synthesis of Aca (1) and the intramolecularly
quenched fluorogenic substrate 5 is outlined in Scheme 2.
The preparation of 1 was achieved by the condensation of
carbethoxyaminophenol (4) [28] with acetone dicarboxylic
acid and acidolysis of the intermediate derivative.
Compound 1 was converted to its ethyl ester by treatment
with thionylchloride in anhydrous ethanol. The substrate 5
was prepared by the condensation of Aca with phenylalanyl-
p-nitroanilide (H-Phe-NH-Np) [29] using 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride (WSCI)
as the coupling agent in the presence of 1-hydroxy-
benzotriazole (HOBt) [30] with a 65% yield. The
p-nitroanilide group, widely used in chromogenic sub-
strates, was chosen as the quencher. The same coupling
Scheme 1
t
method was applied for the preparation of Aca-Phe-OBu ,
acidolysis of which afforded the compound Aca-Phe-OH
(6) (Scheme 2). This compound was synthesized for com-
parison reasons. Both couplings took place without previous

154
C. Charitos, G. Kokotos and C. Tzougraki
Vol. 38
Scheme 2
trypsin-like enzymes. The substrate 5, which is a p-nitro-
anilide derivative, can be used in colorimetry as well.
In order to examine the use of the bifunctional com-
pounds 1 and 2 in the solid phase synthesis of fluorogenic
substrates, they were successfully linked through their car-
boxyl group with 2-chloro-chlorotrityl-resin [33] to pro-
duce the resin derivatives 9 and 10 (Scheme 4). However,
efforts for acylation of the amino group of 9 with Fmoc-
protected amino acids, using several coupling methods,
resulted in very low yields. On the contrary the resin deriv-
ative 10 was successfully acylated with oleic or palmitic
acid using dicyclohexylcarbodiimide (DCC) as a coupling
agent in the presence of a catalytic amount of dimethyl-
aminopyridine (DMAP) [34]. The substrates Ole-Oca (13)
and Pal-Oca (14) (Scheme 4), suitable for the enzymatic
study of lipases, were obtained after cleavage from the
resin with a mixture of trifluoroethanol (TFE),
dichloromethane (DCM), acetic acid (AcOH)
(TFE:DCM:AcOH 2:7:1).
protection of the 7-amino group of Aca, due to its low nucle-
ophilicity, as is also mentioned in the literature [31]. Indeed,
efforts to protect the 7-amino group with t-butyloxycarbonyl
(Boc) or benzyloxycarbonyl (Z) group resulted in very low
yields, while reaction with 9-fluorenyl-methyloxycarbonyl
chloride (Fmoc-Cl) produced the dimer Fmoc-NH-ca-NH-
ca. Finally, Fmoc-NH-ca was obtained by acidolysis of
Fmoc-Aca-OEt, prepared from Aca-OEt (3) by treatment
with Fmoc-Cl. It seems that esterification of the carboxyl
group increases the nucleophilicity of the aromatic 7-amino
group, permitting its acylation in solution. Thus, for the syn-
thesis of the fluorogenic substrate Glt-Phe-NH-ca-OEt (8),
outlined in Scheme 3, compound 3 was coupled with Boc-
Phe-OH by the mixed anhydride method [32], to produce
compound 7. After removal of the Boc group, the free inter-
mediate reacted with glutaric anhydride to give the substrate
in good yield. Both substrates, 5 and 8, are suitable for the
fluorometric determination of chymotrypsin or chymo-
Scheme 4
The fluorescence properties of the intramolecularly
quenched fluorogenic substrate 5 and the fluorescent com-
pound 6 are shown in Table 1, while their fluorescence
spectra are shown in Figure 1. Efficient quenching (88.9%)
of fluorescence was observed at the excitation and emission
maxima of compound 6.
Table 1
Fluorescence Properties of Compounds 6 and 5
Compound [a]
λ
/nm
λ
/nm
em
F/a.u.[b]
q.e.[c] %
88.9
ex
6
5
364
364
458
458
1674
186
[a] 1 µM solution in 0.05 M Tris-HCl pH 7.8, containing 1% DMSO.
[b] Arbitrary units. [c] q.e. = (F -F)/F ; F and F are the fluorescence
0
0
0
intensities of the donor in the absence and presence of the acceptor.
Table 2 lists the fluorescence properties of markers 2 and 3
and substrates 8, 13 and 14. The emission maxima and fluo-
rescence intensities of compounds 3 and 8 are distinctly dif-
ferent (Figure 2). Their fluorescence intensities at various λ
ex

Jan-Feb 2001
Bifunctional Coumarin Derivatives in Solution and Solid Phase Synthesis
155
maximum fluorescence. At these wavelengths the presence
of substrate 8 does not interfere with the fluorescence of
marker 3. As shown in Table 2 compound 2 has λ
em-
max=453 nm at λ max=350 nm, while compounds 13 and
ex
14 have λ max=458 nm at λ max=368 nm (Figure 3). The
em
ex
fluorescence intensity of marker 2 at its maxima wavelengths
was 24-fold and 28-fold greater than that of equimolar solu-
tions of substrates 13 and 14, respectively.
Figure 1. Fluorescence spectra of (a) Aca-Phe-OH (6), (b) Aca-Phe-NH-
Np (5); λ =364 nm. 1 µM solutions in 0.05 M Tris-HCl, pH 7.8,
ex
containing 1% DMSO.
Table 2
Fluorescence Properties of Compounds 2, 3, 8, 13, 14
Compound [a]
λ
/nm
λ
/nm
em
F/a.u.[b]
ex
Figure 3. Fluorescence spectra of (a) Hca (2); λ =350 nm, (b) Ole-Oca
ex
(13), (c) Pal-Oca (14); λ =368 nm. 1 µM solutions in 0.05 M Tris-HCl,
pH 7.8, containing 1% DMSO.
3
8
2
13
14
366
341
350
368
368
460
408
453
458
458
665
453
360
15
ex
13
From the above results it is concluded that the proposed
compounds 1 and 3 are suitable markers in the solution syn-
thesis of fluorogenic or intramolecularly quenched fluoro-
genic substrates for proteases, whereas compound 2 can be
used for the development of fluorogenic enzyme substrates
on a solid support. It should also be mentioned that marker
1 can be used for the conversion of peptide nitroanilide sub-
strates into intramolecularly quenched fluorogenic ones,
which by retaining their chromogenic properties, may be
applied in both colorimetric and fluorometric assays.
[a] 1 µM solution in 0.05 M Tris-HCl pH 7.8, containing 1% DMSO.
[b] Arbitrary units.
and λ were determined. At chosen λ =380 nm and
em
ex
λ
=490 nm the fluorescence intensity of the marker 3 was
em
approximately 350-fold greater than that of an equimolar
solution of the substrate 8, while retaining 40% of its
EXPERIMENTAL
Melting points are all uncorrected and were determined in cap-
illary tubes using Buchi apparatus. Thin layer chromatography
was performed on precoated plates of silica gel 60 F (Merck,
254
Darmstand, Germany) using the following solvent systems: chlo-
roform/methanol (5:1); chloroform/methanol (9:1); chloroform/
methanol (95:5); chloroform/methanol/acetic acid (3:1:1); chlo-
roform/methanol/acetic acid (90:10:0.3); chloroform/methanol/
acetic acid (50:10:0.3); 1-butanol/acetic acid/water (4:1:1). The
crude products were purified using column chromatography on
silica gel 60 (70-230 mesh, Merck). Elemental analyses were
carried out on a Perkin-Elmer CHN 2400 instrument. Fast Atom
Bombardment (FAB) mass spectra were obtained on a VG
Analytical ZAB-SE instrument using positive ion mode and
1
m-nitrobenzyl alchohol as matrix. The H nmr spectra were
Figure 2. Fluorescence spectra of (a) Glt-Phe-NH-ca-OEt (8); λ =341
ex
recorded on a Varian 200 spectrometer in dimethyl sulfoxide-
nm, (b) Aca-OEt (3); λ =366 nm. 1 µM solutions in 0.05 M Tris-HCl,
ex
pH 7.8, containing 1% DMSO.
d . The solvent signal at 2.54 ppm was used as reference.
6

156
C. Charitos, G. Kokotos and C. Tzougraki
Vol. 38
The fluorescence spectra were recorded on a Perkin-Elmer 512
fluorescence spectrophotometer. Phenylalanine was of L-configu-
ration and purchased from Fluka and 7-hydroxy-4-coumarinyl-
acetic acid (Hca) from Aldrich.
aqueous (0.17 g, 1 mmol) were added consecutively. The reaction
mixture was stirred for one hour at 0 °C and at room temperature
overnight. After evaporation in vacuo the residue was dissolved
in ethyl acetate and washed consecutively with water, a solution
of 1 M potassium bicarbonate, brine, a solution of 5% sodium
bicarbonate and brine. The organic phase was dried with sodium
sulfate and evaporated in vacuo to yield a crude solid which was
7-Amino-4-coumarinyl-acetic Acid (1).
In a procedure similar to this of Benson et al. [31] a solution of
citric acid (10.51 g, 50.05 mmol) in concentrated sulfuric acid
(2.32 ml) was stirred at room temperature for one hour and at 70
°C for thirty minutes. The solution then was cooled to 0 °C and 3-
carbethoxyaminophenol (4) (7.24 g, 40 mmol) and concentrated
sulfuric acid (5.61 ml) were added in three equal portions. The
mixture was stirred at 0 °C for one hour and at room temperature
overnight. The reaction mixture was poured into ice (100 g) and
the resulted solid was filtered and washed several times with
water. The crude product, 7-carbethoxyamino-4-coumarinyl-
acetic acid, was triturated with 50 ml of 1 N sodium carbonate
solution and the resulting mixture was filtered. The filtrate was
acidified with concentrated hydrochloric acid. The resulting
white precipitate was collected by filtration and recrystallized
from methanol. The obtained pure 7-carbethoxyamino-4-
coumarinyl-acetic acid (2.86 g, 9.8 mmol) was dissolved in a
mixture of glacial acetic acid (8.43 ml) and concentrated sulfuric
acid (4.80 ml). The mixture was heated at 100 °C for one hour
and then at 120 °C for four hours. The mixture was allowed to
cool to room temperature and then water (40 ml) was added, fol-
lowed by a 25% solution of ammonium hydroxide until pH 2.
The yellow precipitate was filtered and dissolved in 5% sodium
carbonate solution. The solution was extracted with chloroform
and the aqueous phase was acidified with concentrated
hydrochloric acid until pH 2. The resulting yellow solid was fil-
tered. The crude product was purified by recrystallization from
1
triturated with ether twice, yield 0.08 g (63%); H nmr: δ 2.9 (dd,
J
= 13 Hz, J = 14 Hz, 1H, CHHC H ), 3.1 (dd, J
= 13
gem
6
5
gem
Hz, J = 5 Hz, 1H, CHHC H ), 3.6 (s, 2H, CH ), 4.7 (m, 1H,
6
5
2
CH), 5.9 (s, 1H, H ), 6.1 (s, 2H, NH ), 6.4 (d, J = 2 Hz, 1H,
3
2
8,6
H ), 6.5 (dd, J
= 9 Hz, J = 2 Hz, 1H, H ), 7.3 (m, 6H, C H ,
8
6,5
6 6 5
H ), 7.8 (d, J = 10 Hz, 2H, H , H Np), 8.2 (d, J = 10 Hz, 2H, H ,
5
2
6
3
H Np), 8.8 (d, J = 8 Hz, 1H, NH), 10.8 (s, 1H, NH); ms: m/z
5
+
+
509(M +23), 487(M +1), 349, 321, 202.
Anal. Calcd. for C N O ·0.25H O: C, 63.60; H, 4.62; N,
H
26 22
4
6
2
11.41. Found: C, 63.72; H, 4.65; N, 11.21.
7-Amino-4-coumarinyl-acetyl-2-chlorotrityl-resin Ester (9).
2-Chloro-chlorotrityl-resin (1 g) was triturated with 2 ml of
dichloromethane for 5 minutes. Then, diisopropylethylamine
(0.69 ml, 4 mmol) was added followed by the addition of a solu-
tion of 7-amino-4-coumarinyl-acetic acid (0.263 g, 1.2 mmol) in
N,N-dimethylformamide (3 ml). The mixture was stirred for thirty
minutes and then was filtered. A solution of dichloromethane:
methanol:diisopropylethylamine 85:10:5 was added to the resin
and the mixture was stirred for ten minutes. The mixture was then
filtered and the resin was washed first three times with the previ-
ous solution and then with N,N-dimethylformamide, 2-propanol
and ether (four times each). Finally the resin was dried in vacuo
over phosphorous pentoxide.
7-Hydroxy-4-coumarinyl-acetyl-2-chlorotrityl-resin Ester (10).
1
methanol, yield 0.94 g (44%), mp 225-227 °C; H nmr: δ 3.8 (s,
2H, CH ), 6.1 (s, 1H, H ), 6.6 (d, J
= 1 Hz, 1H, H ), 6.7 (dd,
Resin derivative 10 was prepared in a similar manner to that
described for the preparation of 9 starting with 7-hydroxy-4-
coumarinyl-acetic acid (0.264 g, 1.2 mmol).
2
3
8,6
8
J
= 9 Hz, J = 2 Hz, 1H, H ), 7.4 (d, J = 9 Hz, 1H, H ); ms:
6,5
6
5
+
+
m/z 242(M +23), 220(M +1).
Anal. Calcd. for C H NO ·0.5H O: C, 57.89; H, 4.42; N,
6.14. Found: C, 58.09; H, 4.26; N, 6.08.
11
9
4
2
7-Oleyloxy-4-coumarinyl-acetic Acid (13).
Resin derivative 10 (0.12 g, equivalent to 0.12 mmol of
Hca) was triturated with N,N-dimethylformamide (0.4 ml) for
5 minutes. Then, dimethylaminopyridine (2.5 mg, 0.024 mmol)
and a solution of oleic acid (0.102 g, 0.36 mmol) and dicyclo-
hexylcarbodiimide (0.082 g, 0.4 mmol) in N,N-dimethylfor-
mamide (0.76 ml) were added. The reaction mixture was stirred for
three hours. The esterification was repeated one more time using
oleic acid (0.034g, 0.12 mmol). The mixture was filtered and the
resin was washed with N,N-dimethylformamide, 2-propanol and
ether (four times each) and dried in vacuo over phosphorous
pentoxide. A solution of trifluoroethanol:dichloromethane:acetic
acid 2:7:1 (10 ml) was added to the dry resin and the mixture was
stirred for one hour. The reaction mixture was filtered and the resin
was washed with dichloromethane. The filtrate was evaporated in
vacuo to give the crude product, which was purified by column
chromatography using chloroform:methanol (9:1) containing 4
Ethyl 7-Amino-4-coumarinyl-acetate (3), Hydrochloric Salt.
Thionylchloride (0.08 ml, 1.10 mmol) was added dropwise in
absolute ethanol (0.5 ml) at -5 °C, under stirring. After the addi-
tion was completed, 7-amino-4-coumarinyl-acetic acid (1) (0.22
g, 1 mmol) was added. The reaction mixture was kept under stir-
ring at 35-40 °C for two hours and then evaporated in vacuo. The
residue was triturated with absolute ether and the resulting solid
was filtered and purified by recrystallization from methanol/
1
absolute ether, yield 0.20 g (71%); H nmr: δ 1.3 (t, J = 7 Hz,
3H, CH ), 4.0 (s, 2H, CH CO), 4.2 (q, J = 7 Hz, 2H, CH CH ),
3
2
3
2
6.4 (s, 1H, H ), 7.3 (m, 2H, H , H ), 7.8 (d, J
ms: m/z 248(M +1).
= 9 Hz, 1H, H );
3
6
8
5,6
5
+
Anal. Calcd. for C
H NO ·HCl·0.5H O: C, 53.34; H, 4.82;
13 13 4 2
N, 4.78. Found: C, 53.34; H, 5.00; N, 4.70.
N-(7-Amino-4-coumarinyl-acetyl)-L-phenylalanyl-p-nitro-
anilide (5).
1
drops of acetic acid as the eluent, yield 0.025 g (45%); H nmr: δ
0.8 (t, J = 7 Hz, 3H, CH ), 1.3 (m, 20H, CH (CH ) ,
3
3
2 6
To a stirred and cooled at 0 °C solution of phenylalanyl-p-
nitroanilide in N,N-dimethylformamide (5 ml), 7-amino-4-
coumarinyl-acetic acid (0.219 g, 1 mmol), triethylamine (0.15
ml, 1.1 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (0.21 g, 1.1 mmol) and 1-hydroxybenzotriazole
(CH ) CH CH CO), 1.7 (m, 2H, CH CH CO), 2.0 (m, 4H,
2 4
2
2
2
2
CH CH=CH=CH ), 2.6 (m, 2H, CH CH CO), 5.3 (m, 2H,
2
2
2
2
CH=CH), 6.4 (s, 1H, H ), 7.2 (dd, J = 9 Hz, J = 2 Hz, 1H,
3
6,5
6,8
H ), 7.3 (d, J = 2 Hz, 1H, H ), 7.9 (d, J = 9 Hz, 1H, H ); ms: m/z
6
8
5
+
+
508(M +23), 486(M +1), 266, 221.

Jan-Feb 2001
Bifunctional Coumarin Derivatives in Solution and Solid Phase Synthesis
157
[2] E. Bergogne-Berezin, Presse Med, 28 Suppl 3, 25, Review
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Anal. Calcd. for C
Found: C, 71.37; H, 8.42.
H O ·0.25H O: C, 71.21; H, 8.34.
29 40 6 2
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2400 (1996).
7-Palmitoyloxy-4-coumarinyl-acetic Acid (14).
Compound 14 was prepared and purified in a similar manner
to that described for the preparation of 13 starting with resin
derivative 10 (0.12 g) and palmitic acid (0.092 g, 0.36 mmol),
1
yield 0.025 g (47%); H nmr: δ 0.8 (t, J = 7 Hz, 3H, CH ), 1.2
3
(m, 24H, CH (CH ) CH ), 1.6 (m, 2H, CH CH CO), 2.6 (m,
3
2 12
2
2
2
2H,CH CH CO), 6.4 (s, 1H, H ), 7.2 (dd, J
= 9 Hz, J
=
2
2
3
6,5
6,8
2 Hz, 1H, H ), 7.3 (d, J = 2 Hz, 1H, H ), 7.9 (d, J = 9 Hz, 1H,
6
8
5
+
+
H ); ms: m/z 482(M +23), 460(M +1), 239, 221.
Anal. Calcd. for C O ·0.25H O: C, 69.95; H, 8.37.
H
27 38
6
2
Found: C, 70.06; H, 8.42.
Ethyl N-(7-tert-butyloxycarbonyl-L-phenylalanylamido)-4-
coumarinyl-acetate (7).
A solution of tert-butyloxycarbonyl-phenylalanine (0.08 g, 0.3
mmol) in tetrahydrofuran (1.5 ml) was cooled to -10 °C and
N-methylmorpholine (0.033 m1, 0.3 mmol) was added.
Subsequently isobutyl chloroformate (0.039 ml, 0.3 mmol) was
added dropwise. After stirring for 5 minutes at -10 °C, an ice
cooled solution of 3 (0.07 g , 0.3 mmol) in tetrahydrofuran (2.5
ml) was added dropwise, so that the temperature did not exceed -
5 °C. The reaction mixture was stirred at -10 °C for one hour and
at room temperature overnight. After evaporation of the solvent,
the residue was dissolved in ethyl acetate and washed consecu-
tively with water, a 0.5 N solution of hydrochloric acid, water, a
5% solution of sodium bicarbonate, water, brine and dried with
sodium sulfate. The solvent was evaporated in vacuo and the
residue was purified by column chromatography using petroleum
ether (40-60 °C):ethyl acetate 1:1 as the eluent.
Ethyl 7-(glutaryl-L-phenylalanilamido)-4-coumarinyl-acetate (8).
The N-protected compound 7 (0.032 g, 0.064 mmol) was treated
with a solution of 4 N hydrochloric acid in tetrahydrofuran (1.06 ml,
4.25 mmol) for one hour. The reaction mixture was evaporated in
vacuo and the residue was triturated with ether to give a white solid,
that was filtered and dried in vacuo. This solid was dissolved in ethyl
acetate and washed three times with a 5% solution of sodium bicar-
bonate. The organic phase was evaporated until a small volume
remained (approximately 2 ml). Then, glutaric anhydride (0.011g,
0.096 mmol) was added and the mixture was heated at 45 °C for two
hours. The reaction mixture was allowed to cool to room tempera-
ture and then cooled to 0 °C. The precipitated solid was filtered and
1
washed with cool ethyl acetate, yield 0.022 g (66%); H nmr : δ 1.2
(t, J = 7 Hz, 3H, CH ), 1.6 (m, 2H, CH CH CH ), 1.9-2.2 (m, 4H,
3
2
2
2
CH CH CH ), 2.8 (dd, J
= 13 Hz, J = 14 Hz, 1H, CHH), 3.0
2
2
2
gem
(dd, J
= 13 Hz, J = 5 Hz, 1H, CHH), 3.9 (s, 2H, CH CO), 4.1 (q,
gem
2
J = 7 Hz, 2H, CH O), 4.6 (m, 1H, CH), 6.4 (s, 1H, H ), 7.2 (m, 5H,
[22] S. Thaisrivongs, K. D. Watenpaugh, W. J. Howe, P. K.
Tomich, L. A. Dolak, K. T. Chong, C. S. C. Tomich, A. Tomaseli, S.
R. Turner, J. W. Strohbach, A. M. Mulichak, M. N. Janakiraman, J.
B. Moon, J. C. Lynn, M. M. Horng, R. R. Hinshaw, K. A. Curry and
D. L. Rothrock, J. Med. Chem., 38, 3624 (1995).
[23] M. Zimmerman, E. Yurewicz and G. Patel, Anal.
Biochem., 70, 258 (1976).
2
3
C H ), 7.4 (dd, J = 9 Hz, J = 2 Hz, 1H, H ), 7.6 (d, J = 9 Hz,
6
5
6,5
6,8
6
1H, H ), 7.8 (d, J = 2 Hz, 1H, H ), 8.4 (d, J = 8 Hz, 1H, NH), 10.6 (s,
5
8
+
+
1H, NH); ms: m/z 531(M +23), 509(M +1), 395, 377, 248.
Anal. Calcd. for C N O ·0.5H O: C, 62.66; H, 5.64; N,
H
27 28
2
8
2
5.41. Found: C, 62.94; H, 5.72; N, 5.31.
[24] G. Kokotos and C. Tzougraki, J. Chem. Soc. Perkin Trans.
2, 495 (1991).
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th
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