New fluorescent probes for testing combinatorial catalysts with phosphodiesterase and esterase activities

Article abstract of DOI:10.1016/j.tet.2003.12.032

Combinatorial development of new catalysts with phosphodiesterase and esterase activities requires specific fluorescent probes for rapid visual detection of hydrolytic activity. Such fluorescent probes have been synthesized with special attention to solubility in water and stability towards spontaneous hydrolysis at a given pH. The probes reported here include compound 5 based on a fluorescein fluorophore, compound 12 for FRET-detection of phosphodiester hydrolysis and compound 25 based on a quinolinium fluorophore.

Full text of DOI:10.1016/j.tet.2003.12.032

Tetrahedron 60 (2004) 1903–1911
New fluorescent probes for testing combinatorial catalysts with
phosphodiesterase and esterase activities
*
Francisco Caturla, Juan Enjo, M. Carmen Bernabeu and Stephanie Le Serre
Fachbereich Chemie der Philipps-Universit a¨ t Marburg, D-35032 Marburg, Germany
Received 17 April 2003; revised 11 December 2003; accepted 11 December 2003
Abstract—Combinatorial development of new catalysts with phosphodiesterase and esterase activities requires specific fluorescent probes
for rapid visual detection of hydrolytic activity. Such fluorescent probes have been synthesized with special attention to solubility in water
and stability towards spontaneous hydrolysis at a given pH. The probes reported here include compound 5 based on a fluorescein fluorophore,
compound 12 for FRET-detection of phosphodiester hydrolysis and compound 25 based on a quinolinium fluorophore.
q 2003 Elsevier Ltd. All rights reserved.
6
1
. Introduction
serine proteases and water insoluble fluorescein mono-
0
0
esters, for example, 3 - or 6 -laureates or myristates, have
been used for medicinal applications, determining the
activity of pancreas enzymes, lipases or of chymotrypsin
1
Combinatorial chemistry has a major impact on catalyst
2
discovery and optimization. However, the application of
combinatorial techniques to this subject requires new
high throughput screening methods to monitor a large
7
in blood, duodenal fluid, or urine. Moreover, the use of
fluorescein in the preparation of fluorogenic substrates to
continuously monitor the activity of the enzyme phospha-
tidylinositide-specific phospholipase C has been previously
2
a–g,3
number of reactions in a quick and simple way.
While pursuing combinatorial approaches to the develop-
ment of molecular catalysts for the hydrolysis of phospho-
diesters and carboxylic esters, we became interested in
the preparation and characterization of novel substrate
probes that would allow to monitor hydrolysis reactions, by
means of appearance of fluorescence. Our goal was to
provide substrates that allow rapid qualitative visual
screening of compound libraries for catalytic activity in
phosphodiester and carboxylic ester hydrolyses. We were
certainly led by known fluorescent probes for enzyme
assays, but we did not intend to use these substrates for that
purpose.
8
9
described and Scheigetz and co-workers have prepared
0
0
3 ,6 -fluorescein diphosphate and different fluorescein
monophosphates for highly sensitive and continuous protein
tyrosine phosphatase assays.
We therefore wanted to synthesize non-fluorescent esters of
fluorescein, which upon hydrolysis liberate the fluorescent
fluorescein molecule. Fluorescent probes of that nature
could be used in concentrations of up to 102 M, which is
necessary to ensure a reasonable reaction rate on reaction
with a catalyst which is present in even lower concentration.
If one aims at fluorescent probes which could be used at
much lower concentration, one can turn to FRET-based
3
To design these substrates, we focused on the excellent
4
fluorescent properties of fluorescein, which possesses a
relatively high absorptivity and excellent quantum yield.
Fluorescein esters have been used previously as fluorescent
probes to determine esterase activities. Guilbaut and
Kramer reported a study of the hydrolysis of various
fluorescein esters by lipases. Also, p-guanidinobenzoic acid
esters of fluorescein have been used as active site titrants of
10
systems, see for instance the system described by
1
1
Berkessel and co-workers. This methodology requires
the presence of a fluorophore and a quencher group linked
through an ester or phosphodiester bond. Upon irradiation, a
rapid energy transfer occurs between these two parts and the
phosphodiester does not fluoresce. When hydrolysis occurs,
the fluorophore and the quencher are separated and the
internal quenching is disrupted with the consequence that
fluorescence can be observed. In the design of our catalytic
systems, we tried to emulate nature using aqueous media.
Therefore, the fluorescent probes of interest to us should be
soluble in water over a broad concentration range and
should be stable towards spontaneous hydrolysis at a given
pH.
5
Keywords: Combinatorial catalysis; Fluorescence; Ester hydrolysis;
Phosphodiester hydrolysis.
*
Corresponding author at present address: Almirall Prodesfarma, Dpto. de
Qu ´ı mica M e´ dica, Treball 2-4, 08960 Sant Just Desvern, Spain. Tel.: þ34-
9
3-291-35-83; fax: þ34-93-312-86-35;
e-mail address: jfcaturl@almirall.es
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.12.032

1904
F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
2
. Results and discussion
modified the Berkessel system and focused on compound
2 shown in Scheme 2. We envisaged that compound 12
1
2
.1. Phosphodiester probes
would be a suitable substrate, which allows to detect
hydrolysis by simple visual observation of fluorescence
avoiding the use of spectrophotometers. The synthesis of 12
started with the separate preparation of both the fluorophore
7 and the quencher 10 following procedures described in the
Taking into consideration the wide importance of the
phosphodiester linker in nature, we wanted to prepare
fluorogenic substrates to continuously monitor phospho-
diesterase activity of artificial catalysts. We therefore
targeted phosphodiesters of fluorescein. For their prepa-
ration, we used the dichloridite procedure previously
developed for the synthesis of oligonucleotides. In this
vein, dichloridite 2 was reacted sequentially at 278 8C with
methyl-fluorescein 1 and MeOH (Scheme 1). In situ
oxidation at rt with t-BuOOH gave a mixture of two
products that could be separated by column chroma-
tography: the phosphate 3 in 31% yield, and the product 4
resulting from the coupling of two molecules of methyl-
fluorescein 1 to dichloridite 2 in 12% yield (Scheme 1).
Product 3 was subsequently demethylated by treatment with
1
3,14
literature (Scheme 2).
parts, the phosphoramidite coupling method developed by
For the combination of these two
1
5
Beaucage and Caruthers for the synthesis of oligonucleo-
tides was used. Thus, fluorophore 7 was reacted with
phosphoramidite 8 to afford compound 9 in 61% yield
(Scheme 2). Afterwards, compound 9 could be coupled with
the azo-derivative 10 in the presence of tetrazole, and the
crude product was oxidized in situ with t-BuOOH to give
the phosphate 11 in 64% yield. The last step was the
1
2
1
6
cleavage of the methyl ester group in 11 using Me SiBr to
3
form the desired phosphodiester 12 (Scheme 2).
t-BuNH under reflux, affording phosphodiester 5 in 87%
2
2.2. Carboxylic ester probes
yield after crystallization. The isopropyl group present in
phosphate 4 was cleaved by treatment with BCl to give the
corresponding phosphodiester 6 in 85% yield (Scheme 1).
Aiming at water soluble fluorescent probes capable of
indicating hydrolysis of carboxylic esters, the attachment of
polyol units to fluorescein derivatives was envisioned as a
method to increase solubility in water. This could be done
either through a polyol moiety bound via the phenolic
hydroxyl group to fluorescein or by a polyol group bound
via a carboxyl link to the fluorescein core.
3
For the preparation of a FRET-based system, we aimed at
one which would quickly detect phosphodiesterase activity
of potential catalysts by using a fluorophore, which could be
excited by a simple UV-lamp (labs<360 nm) and which
would produce a visible fluorescence (lem.500 nm) upon
hydrolysis of the phosphodiester bond. We therefore
Regarding the first approach, our synthetic targets were
Scheme 1.

F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
1905
chromatography ketal deprotection was achieved under
essentially neutral conditions with cerium ammonium
1
7
nitrate in 96% yield (Scheme 3).
Unfortunately, despite the presence of a diol group, did the
2
5
solubility in water of neither 16 nor 18 exceed 10 M.
In order to test other possibilities, we turned to carboxy-
fluorescein 19 to introduce a polyol unit attached via the
pendant carboxyl group. Thus, acetylcarboxyfluorescein
2
0 was coupled to the triethanolamine derivative 21 using
0
0
O-benzotriazolyl-N,N,N ,N -tetramethyluronium tetrafluoro-
borate in 54% yield and the tert-butyldimethylsilyl groups
of intermediate 22 were removed by treatment with 5% aq.
HF, yielding compound 23 in 97% yield (Scheme 4).
Scheme 2.
compounds 16 and 18, having a diol moiety to increase
solubility in water (Scheme 3). These compounds were
prepared starting from commercially available fluorescein
1
3 in the following way: the magnesium salt of fluorescein
was reacted with glycidol to afford diol 14 in 44% yield.
After protection of the diol function as a ketal, the phenolic
hydroxyl group of 15 was reacted with acetyl chloride,
affording ester 16 directly in 64% yield (Scheme 3), as ketal
deprotection took place during the work-up. Likewise
compound 15 was treated with chloroacetyl chloride to
give compound 17 in 78% yield. After purification by flash
Scheme 4.
However, again the solubility of compound 23 in water did
not exceed 102 M. Likewise, the solubility of diacetyl-
carboxyfluorescein 20, a potential candidate, was also
around 102 M.
5
5
Due to the difficulties to increase the water solubility of
fluorescein derivatives, we were intrigued by a study of
1
8
Menger and co-workers who tested the activity of some
esterases like acetylcholinesterase and chymotrypsin, using
carboxylic esters derived from 7-hydroxyquinoline 24. The
synthesis of compounds 25 was achieved by acylating
7-hydroxyquinoline 24 with an anhydride or an acid chloride
and treating the resulting esters with MeI (Scheme 5).
Scheme 3.
Scheme 5.

1
906
Table 1. Qualitative characterization of fluorescent probes
Fluorescent probes Solubility
F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
a
b
Stability
Enzymatic assay
Outcome
2
3
c
23
1 d, 10 M, pH¼8.8
c
d
23
c
5
.10 M, pH¼8.8
PDI 10 min, 10 M, pH¼8.8
Green fluorescence
2
5
c
e
e
25
6 h, 10 M, pH¼8.8
c
e
e
d
26
c
1
1
1
2
2
6
8
0
10 M, pH¼8.8
PDI, 10 min, 10 M, pH¼8.8
Green fluorescence
Green fluorescence
Green fluorescence
Green fluorescence
2
5
25
6 h, 10 M, pH¼7.0
f
25
e
10 M, pH¼7.0
PPL, 10 min, 10 M, pH¼7.0
2
5
25
2 h, 10 M, pH¼7.0
f
25
e
10 M, pH¼7.0
PPL, 10 min, 10 M, pH¼7.0
2
5
c
25
2 d, 10 M, pH¼8.8
c
f
25
c
10 M, pH¼8.8
PPL, 10 min, 10 M, pH¼8.8
2
5
c
25
1 d, 10 M, pH¼8.8
c
g
—
g
—
23
10 M, pH¼8.8
h
22
.10 M, pH¼7.0
e
e
e
e
23
2 h, 10 M, pH¼7.0
e
e
g
g
2
2
2
5a
—
—
h
5b
22
.10 M, pH¼7.0
23
3 h, 10 M, pH¼7.0
f
23
e
PPL, 20 min, 10 M, pH¼7.0
Green fluorescence
Green fluorescence
Green fluorescence
i
22
.10 M, pH¼7.0
23
24 h, 10 M, pH¼7.0
e
e
f
23
e
e
5c
PPL, 120 min, 10 M, pH¼7.0
i
22
23
30 h, 10 M, pH¼7.0
f
23
2
5d
.10 M, pH¼7.0
PPL, 240 min, 10 M, pH¼7.0
a
All enzymatic assays were done taking 1 mL of a stock solution of the corresponding fluorescent probe in the buffer and concentration quoted and adding the
enzyme as solid.
b
c
d
e
f
26
The estimated visual detection threshold of all the probes is below 10 M.
0.1 M AMPSO buffer {3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid}.
Phosphodiesterase I (EC 3.1.4.1, type IV from crotalus atrox crude dried venom): 10 mg, 0.1 units.
0
0
.1 M HEPES buffer [N-(2-hydroxyethyl)piperazine-N -(2-ethanesulfonic acid)].
Porcine pancreas lipase (EC 3.1.1.3. type II, crude): 4 mg, 50 units.
Compound not tested because of a lack of enough material.
g
h
i
23
A 10 M solution of this compound in water shows a blue color.
23
A 10 M solution of this compound in water shows a violet color.
The water solubility of this class of fluorescent probes
3. Testing
2
2
was in all cases, larger than 10 M providing a set of
compounds which should allow easy visual detection of
ester hydrolysis when exposed to potential catalysts.
In order to characterize the new fluorescent probes in a
qualitative manner, these were first subjected to stability

F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
1907
0
spiro(isobenzo-furan-1(3H)-9 (9 H)-xanthen(-3-one (3)
0
tests at pH 7 or 8.8. Lacking active synthetic catalysts at this
stage of our work we tested the viability of our probes by
subjecting them to cleavage by phosphodiesterase I or
porcine pancreas lipase under buffered conditions. The
results reported in Table 1 reflect the appearance of color as
determined by the naked eye.
4.2. 3 -(Isopropoxy(methoxy)phosphoryl(-6 -methoxy-
0
and isopropylbis{6 -methoxy-spiro[isobenzofuran-
0
0
1(3H)-9 (9 H)-xanthen]-3-one-3 -yl}phosphate (4)
0
0
0
Pyridine (1 mmol, 81 mL) was added via syringe to a
i
19
solution of Pr OPCl 2 (0.265 mmol, 43 mg) in dry THF
2
As we can see from the table, the fluorescein derived
phosphodiester 5 showed good solubility in water
(0.35 mL) maintained at 278 8C in a small flask equipped
with a septum cover. To the solution was added methyl-
fluorescein (0.24 mmol, 83 mg) dissolved in 0.65 mL of dry
THF. After a total of 10 min, methanol (0.19 mmol) was
added (via syringe). The solution was maintained for 15 min
at 278 8C. The reaction mixture was warmed to rt and to it
was added a solution of t-BuOOH in CH Cl (7 M, 125 mL).
2
3
(
.10 M) and is stable in aqueous pH 8.8 buffer
safely over one day. Phosphodiester 6 was not tested
because of a lack of enough material. In the case of the
phosphodiester 12, its solubility in water was only around
2
5
1
0
M, but higher concentrations are anyhow not tolerated
2
2
if the FRET technique is to be applied to monitor ester
hydrolysis.
After 2 h at rt, H O was added and the aqueous phase was
2
extracted with CHCl (3£15 mL). The organic layer was
3
dried (MgSO ) and concentrated to afford a residue which
4
Turning to the carboxylic esters 16 and 18, they showed a
2
was purified by flash chromatography (eluent: CH Cl /
2
2
5
low solubility in water (10 M as maximum) and also low
stability towards spontaneous hydrolysis.
EtOAc: 93/7), yielding phosphates 3 and 4 with the yield
mentioned in the text. Compound 3. R ¼0.36 (CHCl /
f
3
1
EtOAc: 9/1). H NMR (200 MHz, CDCl ): d¼1.38 (m, 6H),
3
The problem of insufficient stability was overcome with
compounds 20 and 23 derived from carboxyfluorescein 19,
stability in the buffer system extended to around 2 d for
compound 20. The solubility of compounds 20 and 23 in
3.84 (s, 3H), 3.86 (d, J¼14.0 Hz, 3H), 4.79 (m, 1H), 6.64–
6.79 (m, 5H), 7.19 (m, 2H), 7.66 (m, 2H), and 8.03 (d,
J¼7.0 Hz, 1H). C NMR (50 MHz, CDCl ): d¼23.6, 54.7,
1
3
3
54.9, 55.6, 74.3, 74.4, 82.5, 100.9, 108.5, 111.0, 112.0,
115.8, 116.0, 124.0, 125.1, 126.6, 129.0, 129.4, 129.9,
2
5
water is still low (10 M). The triol moiety therefore did
not contribute too much to increase the solubility in water.
Nevertheless the strong green fluorescence emitted by the
fluorophore can easily be detected, once the carboxylic ester
is hydrolyzed.
3
1
135.1, 152.1, 152.3, 153.1, 161.5, and 169.3. P NMR
(81 MHz, CDCl ): d¼25.98. HR-MS: C H PO –CH
3
24 20
8
3
requires 467.0896; found 467.0903. Compound 4. R ¼0.57
f
1
(CHCl /EtOAc: 9/1). Mp 97–99 8C. H NMR (200 MHz,
3
CDCl ): d¼1.41 (m, 6H), 3.83 (s, 6H), 4.94 (m, 1H), 6.64–
3
In the case of the quinolinium derivatives shown in
Table 1, the solubility was not a problem, being higher
than 102 M. The stability to spontaneous hydrolysis
ranges from 2 h, for compound 25a, to 30 h in the
case of compound 25d. Therefore, compounds 25a and
7.00 (m, 10H), 7.17 (m, 4H), 7.65 (m, 4H), and 8.03
(d, J¼7.0 Hz, 2H). C NMR (50 MHz, CDCl ): d¼23.5,
1
3
3
2
25.8, 55.6, 75.8, 82.3, 100.9, 108.7, 110.9, 112.1, 115.8,
116.5, 123.9, 125.1, 126.5, 129.0, 129.6, 129.9, 135.2,
3
1
151.6, 152.1, 152.2, 153.0, 161.5, and 169.2. P NMR
(81 MHz, CDCl ): d¼213.10. m/z 360 (M 2436, 8%).
þ
HR-MS: C H PO requires 360.0188; found 360.0981.
25b are too labile to be considered useful fluorescent
probes.
3
2
0
10
5
0
4.3. tert-Butylammonium isopropyl-{6 -methoxy-spiro-
(isobenzofuran-1(3H)-9 (9 H)-xanthen(-3-one-3 -
In summary, the best fluorescent probe for monitoring the
cleavage of phosphodiesters is compound 5. Regarding
fluorescent probes for cleavage of carboxylic esters, the best
candidates are compound 20, derived from carboxyfluor-
escein 19 and the quinolinium derivatives 25c and 25d,
showing good solubility in water and a useful stability
against spontaneous hydrolysis.
0
0
0
yl}phosphate (5)
A solution of phosphate 3 (230 mg) in t-BuNH (140 mL)
2
was heated at reflux for 8 h. The solvent was removed at
reduced pressure affording a solid which was recrystallized
from EtOAc/hexane to afford 200 mg of pure phospho-
diester 5 (87%). R ¼0.27 (CHCl /MeOH: 9/1). Mp 205–
f
3
1
2
06 8C. H NMR (200 MHz, CDCl ): d¼1.20 (d, J¼ 6.2 Hz,
3
4
. Experimental
6H), 1.31 (s, 9H), 3.83 (s, 3H), 4.50 (m, 1H), 6.58–7.24 (m,
7H), 7.63 (m, 2H), and 8.01 (m, 1H). C NMR (50 MHz,
1
3
4
.1. General
CDCl ): d¼23.9, 27.7, 51.4, 55.6, 70.5, 83.0, 100.9, 107.7,
3
107.8, 111.1, 111.7, 113.6, 116.0, 116.1, 123.8, 125.0,
126.8, 128.7, 129.0, 129.7, 135.0, 151.9, 152.5, 153.2,
All temperatures quoted are not corrected. Reactions were
1
13
31
carried out under dry nitrogen or argon. H and C NMR
spectra were recorded on Bruker ARX-200 and AC-300
spectrometers. Spectra were recorded for ca. 0.2 mM
154.7, 154.8, 161.4, and 169.4. P NMR (81 MHz, CDCl ):
3
þ
þ
C H O P requires 467.0894; found 467.1537.
t
d¼26.23. m/z 466 (M 2H N Bu -1, 2%). HR-MS:
3
2
4 20 8
solutions in CDCl (99% d), which was also used as an
3
internal standard. Coupling constants are quoted in Hz.
Flash chromatography was run using silica gel Si 60
0
xanthen]-3-one-3 -yl}phosphate (6)
0
0
4.4. Bis{6 -methoxy-spiro[isobenzofuran-1(3H)-9 (9 H)-
0
(
(
40–63 mm, E. Merck AG, Darmstadt). Electron impact
EI, 70 eV) mass spectra were recorded on a Varian CH 7A
To a solution of phosphate 4 (48 mg, 0.06 mmol) in dry
CH Cl (4 mL) was added with stirring a 1 M solution of
instrument.
2
2

1908
F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
BCl in heptane at 210 8C. The stirring was continued
3
under N for 45 min at the same temperature, and then 1 M
2
(50 MHz, CDCl ): d¼40.7, 43.8, 55.6 (d, J¼ 6.0 Hz), 67.7
3
(d, J¼6.0 Hz), 111.8, 115.7, 119.1, 120.6, 120.7, 123.5,
124.0, 125.4, 128.9, 129.8, 129.9, 130.3, 131.0, 135.0,
143.8, 151.2 (d, J¼7.0 Hz), and 152.9.
HCl was added. The resulting mixture was extracted with
EtOAc (3£15 mL). The organic phase was washed with
H O, dried (MgSO ) and concentrated to afford 38 mg (85%
2
4
yield) of phosphodiester 6, essentially pure. R ¼0.24
4.7. 2-(5-Dimethylamino-1-naphthalenesulfonyl-
amide)ethoxy-4-(4-dimethylaminophenylazo)-phenyl-
phosphate (12)
f
1
(
CHCl /MeOH: 85/15). Mp 198–200 8C. H NMR
3
(
200 MHz, CDCl ): d¼3.69 (s, 6H), 6.50–7.11 (m, 14H),
3
1
3
7
.51 (m, 4H), 7.90 (m, 2H), and 8.60 (br s, 1H). C NMR
(
1
1
50 MHz, CDCl ): d¼55.7, 100.7, 108.6, 111.2, 111.2,
To a solution of phosphate 11 (84 mg, 0.14 mmol) in
CH Cl (4 mL), was added Me SiBr (38.2 mL, 0.28 mmol).
3
12.6, 116.2, 116.5, 124.3, 125.4, 126.6, 129.0, 129.4,
29.9, 135.0, 152.2, 152.6, 162.1, and 169.1. P NMR
2
2
3
3
1
The solution was stirred for 3 h at rt and then evaporated
under vacuum (30 8C). The residue was dissolved in acetone
(6.1 mL) followed by addition of water (1.15 mL). The
solution was stirred for 30 min at rt and then evaporated in
vacuo to give a residue that was dissolved in CHCl3
(50 mL), washed with water (20 mL), brine (20 mL), dried
þ
(81 MHz, CDCl ): d¼212.20. m/z 360 (M 2395, 66%).
HR-MS: C H PO requires 360.0188; found 360.0981.
3
2
0
10
5
4
5
.5. N-[2-(Diisopropylamino-methoxyphosphoxy)ethyl]-
-dimethylamino-1-naphthalenesulfonamide (9)
(MgSO ) and concentrated to yield a crude phosphodiester
4
Chloro(diisopropylamino)methoxyphosphine 8 (268 mg,
.35 mmol) was added to a solution of compound 7
200 mg, 0.679 mmol) in CH Cl2 (3 mL), containing
which was purified by chromatography (silica flash, 5 g,
Et O, CHCl /MeOH: 10/1). Concentration of the appro-
priate fractions gave phosphodiester 12 as a red solid (34%
1
(
2
3
2
1
diisopropylethylamine (473 mL, 2.7 mmol). The mixture
was stirred for 35 min at rt. The resulting solution was
diluted with CH Cl (50 mL) and washed with 5% aq.
yield). H NMR (300 MHz, CDCl ): d¼2.76 (s, 6H), 2.87
3
(s, 6H), 2.87 (m, 2H), 4.02 (ddd, J¼9.2, 5.6 Hz, 2H), 6.51
(m, 2H), 6.75 (m, 1H), 6.80 (d, J¼9.2 Hz, 1H), 7.36 (t, J¼
9.0 Hz, 2H), 7.74 (m, 5H), 8.20 (d, J¼7.3 Hz, 1H), 8.37 (d,
2
2
NaHCO3 (2£25 mL), brine (820 mL), dried (MgSO ),
4
3
1
filtered and concentrated. The crude phosphoramidite was
purified by chromatography (5 g of silica gel, prewashed
with a mixture of pentane/EtOAc/Et N: 50/50/1). The
J¼7.4 Hz, 1H), 8.40 (d, J¼8.6 Hz, 1H). P NMR (81 MHz,
3 3
1
3
CDCl ) d¼23.68. C NMR (50 MHz, CDCl ): d¼40.1,
40.4, 42.3, 62.4 (d, J¼9.4 Hz), 118.8, 120.0, 121.8, 122.5,
122.8, 123.2, 123.8, 124.7, 125.4, 126.0, 135.6, 136.6,
140.1, 142.2, 148.1, 153.7 (d, J¼10.1 Hz), and 154.1.
3
product was eluted with the same mixture and evaporation
of appropriate fractions gave the phosphoramidite as a
yellow oil (71% yield). R ¼0.53 (pentane/EtOAc/Et N: 50/
f
3
1
0 0
(isobenzofuran-1(3H)-9 (9 H)-xanthen(-3-one (14)
5
6
1
7
0/1). H NMR (300 MHz, CDCl ): d¼1.00 (d, J¼6.8 Hz,
4.8. 3 -Hydroxy-6 -(2,3-dihydroxypropoxy)spiro-
3
0
0
H), 2.78 (s, 6H), 2.99 (t, J¼5.83 Hz, 2H), 3.16 (d, J¼
2.8 Hz, 3H), 3.39–3.48 (m, 4H), 6.56 (t, J¼9.8 Hz, 1H),
.16 (m, 1H), 7.47–7.55 (m, 2H), and 8.11–8.48 (m, 2H).
Fluorescein 13 (332 mg, 1 mmol) was added in small
portions under stirring at rt to a methanol solution of
Mg(OCH ) (0.25 M, 8 mL). After stirring for 40 min the
3 2
3
1
13
P NMR (81 MHz, CDCl3) d¼149.5. C NMR (50 MHz,
CDCl ): d¼25.0, 43.2, 45.3 (d, J¼13.5 Hz), 50.5 (d, J¼
3
1
1
0.06 Hz), 62.5 (d, J¼17.1 Hz), 115.4, 119.2, 123.5, 128.6,
solvents were evaporated to dryness in vacuum. The
resulting solid was powdered and added to a solution of
glycidol (271 mL, 4 mmol) in DMF (8 mL). The mixture
was stirred at 120 8C for 16 h. The resulting solution was
diluted with 1 M HCl to pH¼2 and extracted by continuous
extraction with CH Cl during 1 d. The organic layer was
29.7, 130.0, 130.3, 130.4, 135.1, 137.0.
4
.6. 2-(5-Dimethylamino-1-naphthalenesulfonyl-
amide)ethoxy-4-(4-dimethyl-aminophenylazo)-phenyl-
methylphosphate (11)
2
2
dried (MgSO ) and evaporated to give a residue which was
4
A solution of phosphoramidite 9 (196 mg, 0.43 mmol) in
dry and acid free CH Cl (2 mL) was added to a solution of
purified by flash chromatography (CHCl /MeOH: 85/15) on
3
silica gel, affording 179 mg (44%) of compound 14 as a
yellow solid (mixture of diastereomers). R ¼0.25 (CHCl /
2
2
the azo-compound 10 (103 mg, 0.43 mmol) in CH Cl
2
2
f
3
1
(
1 mL) containing tetrazole (1.9 mL, 0.45 M in CH CN).
3
CH OH: 85/15). H NMR (300 MHz, CD OD): d¼3.52–
3
3
The mixture was stirred for 2 h and then t-BuOOH (3 M
solution in isooctane, 430 mL) was added and this mixture
was stirred for an additional hour. The resulting solution
was diluted with CH Cl (30 mL) and washed with 5% aq.
3.74 (m, 2H), 3.89–4.02 (m, 3H), 4.78 (broad s, 2H), 6.39–
6.25 (m, 5H), 6.74 (d, J¼2.2 Hz, 1H), 7.04 (m, 1H, ArH),
1
3
7.59 (m, 2H), and 7.86 (m, 1H). C NMR (75 MHz,
CD OD): d¼61.1, 63.3, 69.9, 70.8, 78.6, 80.2, 85.5, 101.9,
2
2
4
NaHCO (2£15 mL), brine (20 mL), and dried (MgSO ).
102.9, 110.4, 110.7, 112.2, 112.9, 124.4, 124.6, 125.0,
125.1, 127.2, 127.5, 129.2, 129.3, 129.4, 130.3, 135.7,
135.8, 153.1, 153.3, 153.5, 160.3, 160.5, 161.4, 170.8, and
3
4
The solution was filtered and evaporated under vacuum and
the residue was chromatographed (silica flash, 5 g, CH Cl /
EtOAc: 10/1 and 7/3). Evaporation of appropriate fractions
2
2
þ
170.9. m/z 406 (M , 1%). HR-MS: C H O requires
2
3 18 7
1
gave the phosphotriester 11 as an oil (64% yield). H NMR
(
406.1053; found 406.1052.
300 MHz, CDCl ): d¼2.74 (s, 6H), 2.97 (s, 6H), 3.36 (dd,
3
0 0
4.9. 6 -Hydroxy-3 -(2,2-dimethyl-1,3-dioxolan-4-yl-
methoxy)spiro(isobenzo-furan-1(3H),9 (9 H)-xanthen(-3-
J¼5.5, 5.4 Hz, 2H), 3.65 (d, J¼11.4 Hz, 3H), 4.03 (ddd,
0
0
J¼9.2, 5.6 Hz, 2H), 6.73 (d, J¼9.2 Hz, 1H), 7.16 (m, 4H),
7
1
1
.43–7.47 (m, 2H), 7.69 (t J¼9.2 Hz, 4H), 8.12 (dd, J¼7.3,
one (15)
.3 Hz, 1H), 8.26 (d, J¼8.3 Hz, 1H), 8.44 (d, J¼8.6 Hz,
3
1
13
H). P NMR (81 MHz, CDCl3) d¼24.36. C NMR
Anhydrous FeCl (33 mg, 0.20 mmol) was added at rt to a
3

F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
1909
þ
C H ClO requires 521.1003; found 521.0989.
þ
solution of compound 14 (100 mg, 0.25 mmol) in dry
acetone (13 mL), stirring the mixture at 36 8C. After 2 h,
evaporation of solvent left a residue, which was purified by
m/z 521 (M 21, 2%), and 523 (M , 2%). HR-MS:
2
8
22
8
0 0
4.12. 6 -(2-Chloroacetoxy)-3 -(2,3-dihydroxypropoxy)-
spiro-(isobenzofuran-1(3H),9 (9 H)-xanthen(-3-one (18)
flash chromatography (CHCl /MeOH: 95/5) to afford the
3
0
0
ketal 15 as a yellow solid (94 mg, 84%, mixture of
1
diastereomers). R ¼0.20 (CHCl /CH OH: 95/5). H NMR
f
3
3
(
300 MHz, CDCl ): d¼1.41, 1.47 (2 s, 6H), 3.90 (dd, J¼8.3,
A solution of CAN (123 mg, 0.22 mmol) in 6 mL of H O
2
3
5
5
7
.8 Hz, 1H), 3.97 (dd, J¼9.6, 5.7 Hz, 1H), 4.06 (dd, J¼9.6,
.4 Hz, 1H), 4.17 (m, 1H), 4.50 (m, 1H), 6.60–6.76 (m, 6H),
.17 (d, J¼7.2 Hz, 1H), 7.64 (m, 2H), and 8.02 (d,
was added at 70 8C under inert atmosphere to a stirred
solution of the protected ester 17 (45 mg, 0.09 mmol) in
3 mL of CH CN. The mixture was stirred at 70 8C during
3
1
3
J¼6.9 Hz, 1H). C NMR (75 MHz, CDCl ): d¼25.3,
5 min. Then, H O was added (15 mL) and this mixture was
2
3
2
1
1
6.7, 66.6, 69.0, 73.8, 84.5, 101.6, 103.1, 110.0, 110.7,
11.5, 111.9, 112.5, 124.0, 125.0, 126.7, 129.1, 129.2,
29.7, 135.1, 152.4, 153.1, 158.3, 160.2, and 170.1. m/z 446
extracted with CHCl (3£15 mL). The combined organic
3
layers were dried over MgSO and the solvent was
4
evaporated under reduced pressure affording 40 mg of
pure product 18 (96%) as mixture of diastereomers. R ¼0.32
þ
þ
(M , 2%), and 431 (M 215, 12%). HR-MS: C H O
22
requires 446.1366; found 446.1372.
2
6
7
f
1
(pentane/EtOAc: 1/4). H NMR (500 MHz, CDCl ): d¼
3
2
.04, 2.61 (2 broad s, 2H), 3.75 (dd, J¼11.4, 5.4 Hz, 1H),
0
isobenzo-furan-1(3H),9 (9 H)-xanthen(-3-one (16)
0
4
(
.10. 6 -Acetoxy-3 -(2,3-dihydroxypropoxy)spiro-
3.85 (dd, J¼11.4, 3.8 Hz, 1H), 4.05–4.16 (m, 3H, CH ),
2
0
0
4.31 (s, 2H), 6.62–6.84 (m, 5H), 7.12–7.17 (m, 2H), 7.67
1
3
(
m, 2H), and 8.03 (d, J¼7.5 Hz, 1H). C NMR (100 MHz,
A solution of ketal 15 (118 mg, 0.26 mmol), acetyl chloride
21 mL, 0.29 mmol) and an excess of 4-dimethylamino-
pyridine (DMAP) in dry CH Cl (4 mL) was heated under
CDCl ): d¼40.8, 63.5, 69.4, 70.2, 82.1, 101.7, 110.0, 111.5,
3
(
112.3, 117.0, 117.4, 123.9, 125.2, 126.4, 129.2, 130.0,
135.2, 151.4, 151.8, 152.1, 152.9, 160.3, 165.4, and 169.2.
2
2
þ
reflux for 3 h. Then, one more portion of acetyl chloride
21 mL, 0.29 mmol) was added, and the mixture was
refluxed for 4 h and kept overnight at rt H O was added,
m/z 439 (M 244, ,1%).
(
0 0
4.13. 3 ,6 -Diacetyl-5(6)-carboxyspiro-(isobenzofuran-
1(3H),9 (9 H)-xanthen(-3-one (20)
2
0
0
the phases were separated and the aqueous layer was
extracted with CHCl (3£15 mL). The combined organic
3
layers were dried (MgSO ) and evaporated in vacuum to
4
Acetic anhydride (1.08 g, 10.58 mmol) was added dropwise
to a solution of carboxyfluorescein (2.00 g, 5.31 mmol) in
dry pyridine (25 mL) and dry Et N (14 mL). The yellow
give a residue which was purified by silica flash chroma-
tography eluting with CHCl /MeOH: 95/5 to give the ester
3
3
1
6 (72 mg, 64%) as a mixture of diastereomers. R ¼0.20
solution was stirred for 36 h and then HCCl (30 mL) was
3
f
1
(
CHCl /CH OH: 95/5). H NMR (300 MHz, CDCl ):
3
added, washed with HCl 10% (4 x 20 mL) and dried
(Na SO ). The residue was crystallized in EtOAc–pentane
to obtain the desired diacetyl carboxyfluorescein 20 (1.75 g,
3
3
d¼1.67 (broad s, 1H), 2.31 (s, 3H), 2.67 (m, 1H), 4.06 (m,
2
4
2
7
H), 4.14–4.34 (m, 3H), 6.58–6.83 (m, 5H), 7.08 (m, 1H),
.17 (m, 1H), 7.66 (m, 2H), and 8.03 (m, 1H). C NMR
1
3
71%) as a white solid.
(
75 MHz, CDCl ): d¼21.1, 65.2, 68.4, 69.1, 84.5, 101.7,
3
1
1
1
10.3, 112.2, 116.7, 117.5, 124.0, 125.1, 126.5, 129.0,
29.1, 129.9, 135.1, 151.8, 152.0, 152.2, 153.0, 160.1,
The observed spectral data were in accord with those
2
reported in literature.
0
þ
68.8, and 171.1. m/z 446 (M 22, 4%). C H O : requires
2
5 20 8
0
oxymethyl]methylcarbamoyl}spiro-(isobenzo-furan-
0
C, 66.96; H, 4.50; found C, 66.85; H, 4.66.
4.14. 3 ,6 -Diacetyl-5(6)-{tris[(tert-butyldimethyl)-silyl-
0
lan-4-yl-methoxy)spiro-(isobenzofuran-1(3H),9 (9 H)-
0
0
0
4
.11. 6 -(2-Chloroacetoxy)-3 -(2,2-dimethyl-1,3-dioxo-
1(3H),9 (9 H)-xanthen(-3-one (22)
0
0
xanthen(-3-one (17)
To a solution of compound 20 (200 mg, 0.43 mmol), amine
2
4 (202 mg, 0.43 mmol), and triethylamine (0.13 mL,
A solution of compound 15 (64 mg, 0.14 mmol), chloro-
acetyl chloride (13 mL, 0.16 mmol) and an excess of
0.93 mmol) in dry acetonitrile (10 mL) was added TBTU
(167 mg, 0.52 mmol). After stirring at rt for 25 min, brine
(20 mL) was added and the aqueous layer extracted with
EtOAc (3£15 mL). Organic layers were washed succes-
4
-dimethylaminopyridine (DMAP) in dry CH Cl (3 mL)
2 2
was heated under reflux for 6 h. The volatile components
were removed under reduced pressure and the residue was
purified by flash chromatography (pentane/EtOAc: 1/1) on
silica gel to give the ester 17 as almost colorless solid
sively with HCl 10% (20 mL), water (20 mL), NaHCO 5%
3
(2£25 mL), and water (20 mL), and dried with Na SO .
2
4
Solvent was removed to obtain a yellow foam which was
purified by silica gel flash chromatography (Et O/pentane:
(
57 mg, 78%, mixture of diastereomers). R ¼0.38 (CHCl /
f
3
2
1
CH OH: 10/1). H NMR (3£00 MHz, CDCl ): d¼1.41,
1/1) affording fluorescein derivative 22 as a white foam
1
3
3
1
5
6
6
.47 (2s, 6H), 3.90 (dd, J¼8.3, 6.0 Hz, 1H), 3.98 (dd, J¼9.6,
.8 Hz, 1H), 4.08 (dd, J¼9.6, 5.5 Hz, 1H), 4.18 (dd, J¼8.3,
.7 Hz, 1H), 4.32 (s, 2H), 4.49 (q, J¼ 5.8 Hz, 1H), 6.61–
.84 (m, 5H), 7.10–7.20 (m, 2H), 7.67 (m, 2H), and 8.04
(210 mg, 54%). H NMR (200 MHz, CDCl , mixture of two
3
compounds) d¼20.03 (s, 18H ), 0.07 (s, 18H ), 0.78 (s,
B
A
27H ), 0.89 (s, 27H ), 2.32 (s, 6H þ6H ), 3.88 (s, 6H ),
B
A
A
B
B
3.98 (s, 6H ), 6.28 (s, 1H ), 6.77 (s, 4H þ4H ), 7.07 (s,
A
B
A
B
1
3
(
m, 1H). C NMR (50 MHz, CDCl ): d¼25.3, 26.7, 40.7,
2H þ2H ), 7.20 (m, 1H ), 7.36 (s, 1H ), 7.95 (d, J¼
3
A
B
A
B
6
1
1
6.6, 69.1, 73.7, 82.2, 101.6, 109.9, 110.0, 111.3, 112.3,
16.9, 117.3, 123.9, 125.2, 126.4, 129.1, 129.3, 129.9,
35.2, 151.3, 151.8, 152.0, 152.9, 160.4, 165.4, and 169.2.
4.2 Hz, 1H ), 8.05 (m, 1H þ1H ), 8.29 (s, 1H ). m/z 890
B A B A
þ
(MH 2Me–2Ac, 4%).
þ
(M 2Me, 52%), 848 (MH 2Me–Ac, 100%), 806
þ

1910
F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
0
carbamoyl]spiro-(isobenzofuran-1(3H),9 (9 H)-
0
4
.15. 3 ,6 -Diacetyl-5(6)-[tris[(hydroxymethyl)methyl-
and benzoyl chloride (0.24 mL, 2.06 mmol) in dry CH Cl
2 2
0
0
(2 mL) was heated at reflux for 29 h. Water (5 mL) was
added, the aqueous layer extracted with CH Cl (3£5 mL),
xanthen(-3-one (23)
2
2
and the combined organic layers were washed with sat.
NaHCO (1£15 mL) and dried (Na SO ). The crude was
To a solution of fluorescein derivative 22 (100 mg,
.11 mmol) in acetonitrile (2 mL) was added 1 mL of a
3
2
4
0
solved in dry CH Cl (5 mL) and methyl iodide (1.00 mL,
2 2
solution of HF 5% (acetonitrile/water). Ten minutes later,
CH Cl (10 mL) and water (10 mL) were added. The
16.06 mmol) was added. The mixture was stirred at 50 8C
for 24 h and a solid was formed. Ethyl ether was added to
ensure complete precipitation, and the solution was filtered.
The crude solid was crystallized twice from methanol–ethyl
2
2
aqueous layer was extracted with CH Cl (3£10 mL), and
2
2
organic layers were washed with brine (2£10 mL), and
dried with Na SO . Solvent was removed to obtain
4
ether to obtain 85 mg (64%) of yellow crystals. Mp 213–
1
2
1
fluoresceintriol 23 as a white solid (60 mg, 97%).
H
214 8C (decomp.). H NMR (200 MHz, CDCl ) d¼4.87 (s,
3
NMR (200 MHz, CDCl , mixture of two compounds) d¼
3H), 7.59 (tt, J¼7.5, 1.5 Hz, 2H), 7.74 (tt, J¼7.5, 1.5 Hz,
1H), 7.93 (dd, J¼9.0, 2.0 Hz, 1H), 8.16 (dd, J¼8.3, 5.8 Hz,
1H), 8.25 (t, J¼1.8 Hz, 2H), 8.28 (dd, J¼2.0, 1.0 Hz, 1H),
3
2
3
7
8
.19 (s, 6H þ6H ), 3.60 (s, 6H ), 3.68 (s, 6H ), 6.60 (m,
A B A B A
A
B
B
A
H þ4H ), 6.98 (m, 2H þ2H ), 7.15 (d, J¼4.0 Hz, 1H ),
.24 (s, 1H ), 7.42 (s, 1H ), 7.56 (s, 1H ), 7.95 (m, 1H ),
B
8.40 (d, J¼8.8 Hz, 1H), 9.06 (dm, J¼8.0 Hz, 1H), 10.46
A
B
A
þ
13
.08 (d, J¼4.2 Hz, 1H ), 8.39 (s, 1H ). m/z 564 (MH , 8%).
(dm, J¼5.7 Hz, 1H). C NMR (50 MHz, MeOH-d ) d¼
B
A
4
4
6.55, 112.37, 122.73, 127.43, 129.37, 129.77, 130.14,
4
.16. 7-Acetoxy-N-methylquinolinium iodide (25a)
131.49, 133.49, 135.73, 141.49, 148.59, 151.64, 158.02,
þ þ
65.71. m/z 249 (M 2Me, 8%), 142 (M 2PhCO H, 26%),
2
1
þ þ
105 (PhCO , 100%), 77 (Ph , 34%). HR-MS: C H NO
16 11 2
A solution of 7-hydroxyquinoline 24 (60 mg, 0.41 mmol) in
acetic anhydride (3 mL, 31.8 mmol) was stirred for 40 h at
requires 249.0790; found 249.0789.
3
7 8C. Methyl iodide (1.50 mL, 24.09 mmol) was added and
the mixture was stirred at 50 8C for 24 h. A solid was
formed. Ethyl ether was added to ensure complete
precipitation, and the solution was filtered. The crude
solid was crystallized three times from methanol–ethyl
4.19. 7-(tert-Butylcarbonyloxy)-N-methylquinolinium
iodide (25d)
A mixture of 7-hydroxyquinoline 24 (50 mg, 0.34 mmol)
and pivaloyl chloride (0.26 mL, 2.13 mmol) in dry CH Cl
ether to obtain 115 mg (85%) of yellow crystals. Mp 202–
1
2
2
2
3
03 8C (decomp.). H NMR (200 MHz, CDCl ) d¼2.43 (s,
(2 mL) was heated at reflux for 29 h. Water (5 mL) was
added and the aqueous layer was extracted with CH Cl
3
H), 4.73 (s, 3H), 7.74 (dd, J¼9.0, 2.0 Hz, 1H), 8.09 (dd,
2
2
J¼8.3, 5.8 Hz, 1H), 8.17 (d, J¼1.5 Hz, 1H), 8.33 (d, J¼
(3£5 mL). The combined organic layers were washed with
9
1
1
1
.0 Hz, 1H), 9.18 (d, J¼8.3 Hz, 1H), 9.94 (d, J¼6.0 Hz,
sat. NaHCO (1£15 mL), dried (Na SO ) and concentrated.
3
2
4
1
3
H). C NMR (50 MHz, MeOH-d ) d¼21.11, 46.59,
The residue was dissolved in dry CH Cl (5 mL) and methyl
2 2
4
12.20, 122.63, 127.36, 129.19, 133.35, 141.39, 148.50,
51.57, 157.77, 170.06. m/z 187 (M 2Me, 5%), 160
iodide (1.00 mL, 16.06 mmol) was added. Upon stirring the
mixture at 50 8C for 24 h a solid formed. Ethyl ether was
added to ensure complete precipitation, and the solution was
filtered. The crude material was crystallized twice from
methanol–ethyl ether to obtain 66 mg (52%) of yellow
þ
þ
M 2AcOH, 43%). HR-MS: C H NO requires
þ
(
MH 2Ac, 7%), 145 (MH 2Me–Ac, 100%), 142
þ
87.0633; found 187.0637.
(
1
1
1
9
2
1
crystals. Mp 180–181 8C (Decomp.). H NMR (200 MHz,
CDCl ) d¼1.44 (s, 9H), 4.84 (s, 3H), 7.72 (dd, J¼9.0,
4
.17. 7-Propionyloxy-N-methylquinolinium iodide (25b)
3
2
.0 Hz, 1H), 8.10 (s, 1H), 8.14 (dd, J¼8.3, 5.8 Hz, 1H), 8.35
A mixture of 7-hydroxyquinoline 24 (48 mg, 0.33 mmol) in
propionic anhydride (3 mL, 23.4 mmol) was stirred at 40 8C
for 48 h. Methyl iodide (1.50 mL, 24.09 mmol) was added
and the mixture was stirred at reflux for 24 h. Ethyl ether
was added and the yellow precipitate was filtered. The crude
solid was crystallized twice from methanol–ethyl ether to
(dd, J¼9.0, 1.5 Hz, 1H), 9.08 (dm, J¼8.0 Hz, 1H), 10.35
1
3
(dm, J¼6.2 Hz, 1H). C NMR (50 MHz, MeOH-d )
4
d¼27.22, 40.32, 111.87, 122.45, 127.08, 129.04, 133.24,
þ
0.5%), 229 (M 2Me, 11%), 186 (M 2Me– BuH, 1%),
141.27, 148.33, 151.37, 158.03, 177.34. m/z 244 (M ,
þ
159 (M 2 BuCO, 6%), 145 (MH 2 BuCO , 94%), 142
þ
t
t
þ
(M 2 BuCO H, 60%), 57 ( Bu , 100%). HR-MS:
t
þ
þ
2
þ
C H NO requires 229.1103; found 229.1105.
t
t
obtain 47 mg (43%) of dark yellow crystals. Mp 159–
1
2
1
60 8C (decomp.). H NMR (200 MHz, CDCl ) d¼1.30 (t,
3
14 15
2
J¼7.6 Hz, 3H), 2.75 (q, J¼7.5 Hz, 2H), 4.80 (s, 3H), 7.73
(
(
dd, J¼9.0, 2.0 Hz, 1H), 8.10 (dd, J¼8.3, 5.7 Hz, 1H), 8.22
d, J¼1.5 Hz, 1H), 8.42 (d, J¼9.0 Hz, 1H), 9.04 (d,
Acknowledgements
1
3
J¼8.3 Hz, 1H), 10.16 (d, J¼5.7 Hz, 1H). C NMR
(
50 MHz, MeOH-d ) d¼9.12, 28.51, 46.65, 112.16,
4
We thank Professor Dr. R. W. Hoffmann for his continuous
support and advice during the realization of this work. This
work has been supported by the European Commission
through the TMR network ERB FMRX CT-960011.
1
1
1
5
22.61, 127.36, 129.16, 133.35, 141.41, 148.49, 151.55,
57.89, 173.59. m/z 201 (M 2Me, 12%), 159 (M 2EtCO,
þ
1%), 145 (MH 2Me–EtCO, 100%), 142 (M 2EtCO H,
þ
þ
þ
2%), 57 (EtCO , 30%), 29 (Et , 49%). HR-MS:
2
þ
þ
C H NO requires 201.0790; found. 201.0784.
1
2
11
2
4
.18. N-Methyl-7-benzoyloxyquinolinium iodide (25c)
References and notes
A mixture of 7-hydroxyquinoline 24 (50 mg, 0.34 mmol)
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F. Caturla et al. / Tetrahedron 60 (2004) 1903–1911
1911
4
11. (b) In Combinatorial chemistry; Wilson, S. R., Czarnik,
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2
288.
2
3
4
. (a) Dahmen, S.; Br a¨ se, S. Synthesis 2001, 1431. (b) Shimizu,
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1
0. (a) Brand, L.; Witholt, B. Meth. Enzymol. 1967, 11, 776.
(
b) Steinberg, I. Z. Annu. Rev. Biochem. 1971, 40, 83. (c) Styer,
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