Preparation and evaluation of sulfur-containing metal chelators

Article abstract of DOI:10.1039/b307399h

With a view to probe the structure and function of G-protein coupled receptors the synthesis of functionalized 8-mercaptoquinoline derivatives and 2-(2-pyridyl)thiophenol was achieved. A fluorescence-based method for determining the affinity of these metal chelators toward zinc ions was developed.

Full text of DOI:10.1039/b307399h

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Preparation and evaluation of sulfur-containing metal chelators
Sylvain Clavier,a Øystein Rist,b Stina Hansen,b Lars-Ole Gerlach,b Thomas Högbergb and
Jan Bergman*a
a Karolinska Institute, Center for Nutrition and Toxicology, Novum Research Park, SE-141
57 Huddinge, Sweden. E-mail: jabe@biosci.ki.se
b 7TM Pharma A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark
Received 1st July 2003, Accepted 15th September 2003
First published as an Advance Article on the web 21st October 2003
With a view to probe the structure and function of G-protein coupled receptors the synthesis of functionalized
8-mercaptoquinoline derivatives and 2-(2-pyridyl)thiophenol was achieved. A fluorescence-based method for
determining the affinity of these metal chelators toward zinc ions was developed.
Introduction
Organic molecules capable of chelating metal ions are well-
known and used in a variety of disciplines in science, spreading
from biology and medicine, via catalysis, to photo industry.
Amongst the most common chelators are 2,2Ј-bipyridines and
1,10-phenanthrolines, which are well characterized and widely
used to chelate a variety of metal ions.1
G-protein coupled receptors (GPCR) or 7-transmembrane
(7TM) receptors represent the most important class of drug
targets.2 The 7TM receptors are not amenable to conventional
structure-based drug design, but engineered metal binding sites
have proved to be a useful tool to unravel their structure and
function.2 For continued investigations we were in need of vari-
ous functionalized chelators, where the metal chelator would
serve as the bridging moiety between the protein structure con-
taining a metal ion and small molecule fragments capable of
interacting with neighbouring binding epitopes in the receptor
protein.
Previously, we have described the preparation of a broad
selection of 2,2Ј-bipyridines for such a purpose.3 We were, how-
ever, interested in identifying chelators with stronger binding
to zinc ions than 2,2Ј-bipyridine or even 1,10-phenantroline.
Increasing the number of coordinating atoms in the chelator is
one way to increase the complex constant, and indeed, as Fig. 1
indicates, this is the case for e.g. EDTA. It was in our interest,
however, to keep the number of coordinating atoms to two in
order to avoid crowding around the metal centre.
Fig. 1 Binding curves of several zinc-ion chelators.
many metal-ion chelators often are deprotonised upon metal-
ion binding, including the sulfur-containing metal chelators
examined here. However, as the chelators are the subject for
latter biological experiments, we were interested in estimating a
conditional stability constant of the chelators at physiological
pH (pH = 7.4). However, the pH titration is intractable since it
results in stability constants at other pH-values. Alternatively,
spectrophotometric properties of the chelator can be used to
estimate the stability constant, where the complex formation by
metal ions may result in an altered absorption or fluorescent
spectra of the ligands. We have also tested if 8-mercapto-
quinoline resulted in altered absorption or fluorescent spectra
as previously observed for 1,10-phenanthroline and 2,2Ј-bi-
pyridine.8 Unfortunately, we did not observe any difference
in absorption or fluorescent spectra upon addition of Zn2ϩ to
8-mercaptoquinoline. To estimate if 8-mercaptoquinoline was a
stronger Zn2ϩ-chelator than 2,2Ј-bipyridine (logK1(Zn2ϩ) = 5.1)
or 1,10-phenanthroline (logK1(Zn2ϩ) = 6.4),6 we performed a
competition experiment between 8-mercaptoquinoline and
2,2Ј-bipyridine or 1,10-phenanthroline for Zn2ϩ. Upon addition
of 8-mercaptoquinoline to 2,2Ј-bipyridine or 1,10-phen-
anthroline chelated Zn2ϩ the fluorescent signal was terminated
indicating a stronger Zn2ϩ-chelation of 8-mercaptoquinoline
8-Hydroxyquinolineisanothercommonchelator,andreplace-
ment of the oxygen atom with the softer sulfur atom should
increase the strength of the complex. Indeed, 8-mercapto-
quinolines have attracted interest due to their ability to chelate
many metal ions, and the complexation properties of several
alkyl-, aryl-, nitro- and halo-derivatives have been reported.4
Thus, 8-mercaptoquinoline and a close analogue, 2-(2-pyridyl)-
thiophenol, were two of the sulfur-containing chelators we
considered. The first is commercially available, while the latter
needed to be synthesized. A synthesis for this compound has
been published5 and we saw the opportunity to develop a more
efficient route for introduction of the sulfur atom.
Results and discussion
Evaluating the chelating properties6
We wanted to investigate the chelating properties of the novel
scaffolds in relation to known chelators. Several methods for
measuring stability constants of metal complexes have pre-
viously been shown to be efficient.7 Such methods include pH
titrations which monitor the mass balances on hydrogen-ions as
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 2 4 8 – 4 2 5 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Scheme 1
Scheme 2
compared to 2,2Ј-bipyridine or 1,10-phenanthroline (data not
Based on these results, we concluded that 8-mercapto-
shown). To further develop this competition assay for Zn2ϩ we
used FluoZin-3 as a Zn2ϩ-probe which upon chelation of Zn2ϩ
becomes a strong fluorophore. The assay was performed as a
competition experiment between a fixed concentration of
FluoZin-3 and the different chelators for Zn2ϩ at physiological
pH, enabling us to rank the affinity of different chelators
according to their Zn2ϩ-affinity.
quinoline fulfilled our requirements from a chelation point of
view. The next step was to develop efficient routes to suitably
functionalized 8-mercaptoquinolines which could be used for
further derivatisations.
Preparation of sulfur-containing chelators
Synthesis of substituted 8-mercaptoquinolines
Results of binding assay
The synthesis of quinolines and their derivatives has been of
considerable interest for many years,10 and methods still con-
tinue to be developed11 as a large number of natural products
and drugs contain this heterocyclic nucleus. Also substituted
8-mercaptoquinolines are known in the literature, e.g. 5-carb-
oxy-8-mercaptoquinolines are described as intermediates for
the synthesis of Hϩ,Kϩ–ATPase inhibitors.12 We now wish to
report several procedures for the preparation of functionalized
8-mercaptoquinolines.
First we focused our interest on the synthesis of 5,7-diamino-
8-mercaptoquinoline 5 which was prepared in five steps starting
from 8-hydroxyquinoline (Scheme 1). Nitration of 8-hydroxy-
quinoline afforded the dinitro derivative 1 which was reacted
with phosphorus oxychloride to give the known chloro-
quinoline 2. Displacement of the chlorine with 2-methyl-2-
propanethiol under basic conditions yielded the quinoline 3,
cf. the synthesis of compound 14. The tert-butylthio group was
then hydrolyzed under acidic conditions and the nitro groups
were reduced with sodium dithionite to gain access to the
expected product 5 in good yield.
The synthesis of 4-hydroxy-8-mercaptoquinoline-3-carb-
oxylic acid 7 was performed by the Gould–Jacobs reaction
sequence13 (Scheme 2). Thus condensation of 2-(methyl-
thio)aniline with diethylethoxymethylenemalonate followed by
intramolecular thermal cyclisation in refluxing diphenyl ether
gave the quinolone 6 in 40% yield. The thiomethyl group was
then deprotected by expanding upon the one-pot procedure
reported by Young et al.,14 involving a Pummerer rearrange-
ment of the corresponding sulfoxide. After hydrolysis of the
As shown in Fig. 1, the order by which the tested chelators
displaced Zn2ϩ from FluoZin-3 was: EDTA > 8-mercapto-
quinoline>1,4,7-triazacyclononane>1,10-phenanthroline>3,4-
toluenedithiol > 2-(2-pyridyl)thiophenol > 8-hydroxyquinoline
= 2,2Ј-bipyridine. These results support the previous data which
indicated that 8-mercaptoquinoline is a stronger Zn2ϩ-chelator
than both 2,2Ј-bipyridine and 1,10-phenanthroline.
Our expectations regarding the suitability of 8-mercapto-
quinoline were confirmed, since the fluorescence data indicated
that it possessed the highest affinity for Zn2ϩ of the tested
bidentate chelators and these results are in agreement with pre-
liminary studies.4 We know from the literature that 1,10-
phenanthroline has a logK1 = 6.4 for Zn2ϩ (at pH 7.4).6 Thus the
current data indicates that 8-mercaptoquinoline is likely to have
a higher logK1 value than 6.4 as it showed stronger Zn2ϩ bind-
ing than 1,10-phenanthroline in the fluorescent assay, yet a
lower affinity than EDTA. Fig. 1 also shows that replacing
both coordinating groups in a bidentate chelator with sulfur,
as in 3,4-toluenedithiolate, did not result in a more potent
chelator. The FluoZin-3 competition assay we have established
here is a simple method as it only requires a conventional
fluorescent plate reader which within few minutes can read a
microtiter plate with 96 or 384 data points. A similar approach
can be applied to other metal ions, simply by identifying
other chelators with fluorescent properties for the applied
metal ion. FluoZin-3 is likely to also respond by fluorescence
upon chelation of metal ions similar to Zn2ϩ, such as Cu2ϩ and
Ni2ϩ 9
.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 2 4 8 – 4 2 5 3
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Scheme 3
Scheme 4
ethyl ester, the 8-mercaptoquinoline 7 was obtained in 63%
Conclusion
overall yield.
In summary, we have devised synthetic routes to introduce the
handles carboxy, amino and hydroxy groups in 8-mercapto-
quinolines, which can be used for further elaborations. We have
also shown the utility of 2-methyl-2-propanethiol for the intro-
duction of sulfur by nucleophilc displacement. Furthermore,
we have developed a method to identify relative binding
affinities toward zinc ions for a variety of chelators.
A modified Doebner–Miller sequence15 between 2-(methyl-
thio)aniline and dimethyl 2-ketoglutaconate16 allowed us to
prepare the diester 8 in 56% yield (Scheme 3). The methylthio
group was then cleaved under conditions previously described
and the esters saponified to afford 8-mercaptoquinoline-2,4-
dicarboxylic acid 9.
2-Methylquinoline-8-thiol 12 was synthesized in three steps
starting from 2-fluoroaniline (Scheme 4). The Doebner–Miller
cyclisation of 2-fluoroaniline with crotonaldehyde was carried
out in a two-phase solvent system without oxidant, accord-
ing to the recent procedure of Matsugi et al.,17 and led to the
fluoroquinoline 10 in 72% yield. Nucleophilic substitution with
2-methyl-2-propanethiol followed by acidic hydrolysis of the
tert-butylthio group gave the desired 8-mercaptoquinoline 12.
These functionalized 8-mercaptoquinolines are now ready
for attaching side-chains by the chemistry disclosed in our
previous publications.3
Experimental
General
Melting points (mp) were determined with a Büchi B-545
apparatus and were uncorrected. IR spectra were recorded on a
Perkin-Elmer FT-IR 1600 instrument or Perkin-Elmer FT-IR
Spectrum One instrument. H and 13C NMR were performed
1
1
on a Bruker Avance DPX300 spectrometer (300 MHz for H,
75 MHz for 13C). All chemical shifts (δ) are given in ppm rel-
ative to the residual deuterated solvent signals. Coupling con-
stants (J) are reported in Hertz. Mass spectra were recorded on
a Perkin-Elmer Sciex API 150 spectrometer in the electro-spray
ionisation mode. LC-MS was recorded on a Agilent 1100 Series,
using a C8 (150 × 4.6 mm) analytical column. Thin-layer
chromatography (TLC) was carried out on Merck silica gel
60F254 precoated plates and flash column chromatography was
performed on silica gel Merck 60 (230–400 mesh). All solvents
were HPLC grade or purified by distillation. All starting
material were commercially available (purchased from Aldrich)
and were used without further purification.
Synthesis of 2-(2-pyridyl)thiophenol
2-(2-Pyridyl)thiophenol 15 was synthesized in a three-step pro-
cedure, starting from 2-fluorophenyl boronic acid (Scheme 5).
This acid was coupled to the pyridyl moiety by a Suzuki cross-
coupling protocol18 to yield the fluorobenzene 13 in high yield.
The fluorine was then displaced by a sulfur-nucleophile in a
nucleophilic aromatic substitution. Various candidates were
tried out, and 2-methyl-2-propanethiol turned out to be the
most reactive, although the reaction needed to be left for three
days for completion to the tert-butyl protected compound 14.
Finally, deprotection under strong acidic conditions resulted in
the free thiophenolic compound 15, although in a modest yield.
Fluorescence based chelator strength assay
For evaluation of the relative chelator strength for Zn2ϩ, a
fluorescence based assay was performed. The tested chelator
was dissolved in 100 µl buffer (20 mM sodiumphosphate buffer
pH 7.4, 100 mM NaCl, 200 µM 2-mercaptoethanol, 20 nM
ZnCl2 and 1 µM FluoZin-3 (Molecular Probes, Eugene, OR)
and incubated for 2 h at room temperature. The 2-mercapto-
ethanol was added to reduce dimerised 8-mercaptoquinoline.
After incubation the fluorescence of fluozin-3 was measured in
a NovoStar reader (BMG, Germany). Each sample in the
microtiter plate was measured for 0.2 s with a 485 nm excitation
filter (slit = 12 nm) and a 520 nm emission filter (slit = 12 nm).
Binding curves was analysed by nonlinear regression using
Prism 3.0 (GraphPad Software, San Diego, CA).
5,7-Dinitroquinolin-8-ol (1)19
To a solution of 65% HNO3 (50 mL) cooled at 0 ЊC was added
portion-wise 8-hydroxyquinoline (5 g, 34.4 mmol). The solution
was stirred at 40 ЊC for 1 h and then poured onto ice with
Scheme 5
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 2 4 8 – 4 2 5 3
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vigorous stirring. The precipitate was filtered off, washed with
water and dried over P2O5 to provide 1 (6.61 g, 82%) as a green-
ish solid; mp 316 ЊC (lit.19 318 ЊC); νmax (KBr)/cmϪ1: 2956, 1644,
114.9 (C), 115.2 (C), 139.0 (CH), 140.6 (CH), 141.3 (C), 147.1
(C), 156.1 (C). m/z 192 (M ϩ 1).
1
1582, 1547, 1515, 1340, 1252, 1202, 1011, 830, 743; H NMR
Ethyl 8-(methylthio)-4-oxo-1,4-dihydroquinoline-3-carboxylate
(6)21
(DMSO-d6): δH 4.99 (1H, br s, OH), 8.25 (1H, dd, J = 8.7, 5.0,
H-3), 8.92 (1H, dd, J = 5.0, 1.4, H-4), 9.21 (1H, s, H-6), 9.77
(1H, dd, J = 8.7, 1.4, H-2); 13C NMR (DMSO-d6): δC 123.1(C),
127.5 (C), 128.1 (CH), 128.2 (CH), 130.4 (CH), 131.1 (C), 138.2
(C), 142.2 (CH), 142.6 (CH). m/z 236 (M ϩ 1).
To a solution of 2-(methylthio)aniline (1 g, 7.18 mmol) in
toluene (80 mL) was added diethyl ethoxymethylenemalonate
(2.33 g, 10.78 mmol) and the mixture was refluxed for 2 h. The
solvent was evaporated and the residue was added over 30 min
to a solution of diphenyl ether (40 mL) heated at 250 ЊC. The
mixture was kept under reflux for 1 h, quickly cooled to 50 ЊC
and diluted with hexane. The resulting brown precipitate was
filtered off, washed with hexane and dried under reduced pres-
sure to provide 6 as a pale brown solid (765 mg, 40%); mp 202
ЊC (lit.21 206–207 ЊC); νmax (KBr)/cmϪ1: 3149, 2980, 1711, 1604,
8-Chloro-5,7-dinitroquinoline (2)20
A solution of 1 (1 g, 4.25 mmol) in excess phosphorus oxy-
chloride (50 mL) was refluxed for 2 h. After removing of the
excess phosphorous oxychloride under reduced pressure, the
residue was poured onto ice and the obtained precipitate was
filtered off, washed with water and dried over P2O5 to provide 2
(840 mg, 78%) as a yellow solid; mp 152 ЊC (lit.20 152–153 ЊC);
νmax (KBr)/cmϪ1: 3064, 1561, 1531, 1401, 1332, 1009, 964, 820,
809, 786; 1H NMR (CDCl3): δH 7.90 (1H, dd, J = 8.9, 4.1, H-3),
8.80 (1H, s, H-6), 9.19 (1H, dd, J = 8.9, 1.5, H-4), 9.31 (1H, dd,
J = 4.1, 1.5, H-2); 13C NMR (CDCl3): δC 119.1 (CH), 122.6 (C),
122.8 (C), 125.4 (C), 126.0 (CH), 132.3 (CH), 134.3 (C), 144.3
(C), 153.2 (CH). m/z 254 (M ϩ 1).
1
1529, 1437, 1380, 1283, 1190, 1110, 1025, 930, 773, 742; H
NMR (DMSO-d6): δH 1.27 (3H, t, CH3, J = 7.0), 2.53 (3H, s,
SCH3), 4.21 (2H, q, CH2, J = 7), 7.39 (1H, m), 7.79 (1H, m),
8.06 (1H, m), 8.45 (1H, m, H-2), 11.6 (1H, br s, NH); 13C NMR
(DMSO-d6): δC 14.2 (CH3), 17.3 (SCH3), 59.6 (CH2), 110.0 (C),
118.5 (C), 124.6 (CH), 126.5 (C), 127.7 (C), 129.9 (CH), 133.9
(CH). 137.6 (C), 145.1 (CH), 164.4 (CO), 173.3 (CO). m/z 264
(M ϩ 1).
8-(tert-Butylthio)-5,7-dinitroquinoline (3)
4-Hydroxy-8-mercaptoquinoline-3-carboxylic acid (7)
To a solution of 2 (200 mg, 0.79 mmol) in THF (20 mL) was
added 2-methyl-2-propanethiol (0.18 mL, 1.58 mmol) and tri-
ethylamine (0.22 mL, 1.58 mmol). The resulting mixture
was refluxed for 12 h. After evaporation of the solvent, flash
chromatography (hexane–EtOAc (98 : 2)) provided 5 (159 mg,
66%) as an orange solid; mp 138 ЊC; Found: C, 50.96; H, 4.38;
N, 13.81; S, 10,66. C13H13N3O4S requires C, 50.81; H, 4.26; N,
13.67; S, 10.43%; νmax (KBr)/cmϪ1: 2963, 1526, 1401, 1367,
1333, 1162, 821, 792; 1H NMR (CDCl3): δH 1.37 (9H, s,
SC(CH3)3), 7.81 (1H, dd, J = 8.8, 4.1, H-3), 8.47 (1H, s, H-6),
9.05 (1H, m, H-4), 9.30 (1H, dd, J = 3.8, 1.1, H-2); 13C NMR
(CDCl3): δC 31.6 (CH3); 52.8 (C); 117.3 (CH), 121.4 (C), 121.6
(C), 124.8 (CH), 132.1 (CH), 145.4 (C), 149.6 (C), 149.8 (C),
152.8 (CH). m/z 308 (M ϩ 1).
m-Chloroperbenzoic acid (180 mg, 1.04 mmol) was added, at
0 ЊC, to a stirred solution of 6 (250 mg, 0.95 mmol) in chloro-
form (50 mL) over 30 min. The mixture was then allowed to
warm to room temperature and stirred for 1 h. A saturated
NaHCO3 solution was added, the organic layer was separated,
dried over MgSO4, filtered and evaporated. The crude sulfoxide
was dissolved in an excess of trifluoroacetic anhydride (10 mL)
and the solution refluxed for 30 min. After evaporation of the
anhydride, the residue was dissolved in methanol (30 mL) and a
solution of NaOH 1M (3 mL) was added. The mixture was
refluxed for 1 h, cooled to room temperature and the methanol
evaporated. A 33% HCl solution was added dropwise to the
aqueous layer at 0 ЊC. The white precipitate which was collected
was filtered off, washed with water and dried over P205 to
provide 7 (132 mg, 63%) as a white solid; mp 278–280 ЊC;
Found : C, 54.40; H, 3.22; N, 6.39; S, 14.62. C10H7NO3S
requires C, 54.29; H, 3.19; N, 6.33; S, 14.49%; νmax (KBr)/cmϪ1:
3060, 2516, 1709, 1606, 1553, 1470, 1376, 1332, 1271, 1202,
5,7-Dinitroquinoline-8-thiol (4)
A solution of 3 (300 mg, 0.98 mmol) in 35% HCl was heated at
100 ЊC for 15 h. The mixture was then cooled to 0 ЊC and the
pH adjusted to 8 by addition of aqueous 6 M NaOH. After
extraction with EtOAc, the organic layer was dried on MgSO4,
filtered and evaporated. Purification by flash chromatography
(dichloromethane–EtOAc (9 : 1)) provided 4 (188 mg, 77%) as
an orange solid; mp 148 ЊC; Found: C, 43.25; H, 2.18; N, 16.81;
S, 12.98. C9H5N3O4S requires C, 43.03; H, 2.01; N, 16.73; S,
12.76%; νmax (KBr)/cmϪ1: 3183, 2504, 1639, 1589, 1526, 1446,
1330, 890, 854, 801, 756; 1H NMR (DMSO-d6): δH 3.10 (1H, br
s, SH), 7.37 (1H, m, H-3), 7.94 (1H, s, H-6), 8.58 (1H, m, H-4),
8.78 (1H, m, H-2); 13C NMR (DMSO-d6): δC 112.3 (C), 112.7
(CH), 116.0 (CH), 119.2 (CH), 145.6 (C), 148.0 (C), 148.2 (C),
149.4 (C), 151.5 (CH). m/z 252 (M ϩ 1).
1
1066, 937, 781, 685; H NMR (DMSO-d6): δH 3.24 (1H, br s,
SH), 7.41 (1H, m, H-6), 7.54 (1H, m, H-7), 8.36 (1H, m, H-5),
8.56 (1H, s, H-2), 12.6 (1H, br s, OH); 13C NMR (DMSO-d6):
δC 108.2 (C), 124.5 (C), 125.7 (C), 125.9 (CH), 128.2 (CH),
139.7 (C), 141.5 (CH). 145.2 (CH), 165.7 (C), 178.3 (CO). m/z
222 (M ϩ 1).
Dimethyl 8-(methylthio)quinoline-2,4-dicarboxylate (8)
To a stirred solution of 2-(methylthio)aniline (200 mg, 1.44
mmol) and dimethyl 2-ketoglutaconate16 (297 mg, 1.72 mmol)
in toluene (30 mL), p-toluenesulfonic acid (55 mg, 0,29 mmol)
was added and the mixture was refluxed for 4 h. After cool-
ing to room temperature and evaporation of the solvent, the
residue was dissolved in EtOAc, washed with saturated
NaHCO3 followed by 0.1 M HCl. The organic layer was
separated, dried over MgSO4 and concentrated under reduced
pressure. Purification by flash chromatography (dichloro-
methane–EtOAc (8 : 2)) provided the diester 8 as a white solid
(235 mg, 56%); mp 128 ЊC; Found: C, 57.84; H, 4.62; N, 4.93; S,
11.22. C14H13NO4S requires C, 57.72; H, 4.50; N, 4.81; S,
11.01%; νmax (KBr)/cmϪ1: 2944, 1719, 1434, 1239, 1154, 1137,
5,7-Diaminoquinoline-8-thiol (5)
Sodium dithionite (1.11 g, 6.40 mmol) was added in small
portions over 30 min to a solution of 4 (160 mg, 0.64 mmol) in
methanol–water (30 mL, 1 : 1). The resulting red precipitate was
filtered off and washed with water. After recrystallisation from
ethanol, 5 was obtained as a dark red solid (188 mg, 77%); mp
260 ЊC (decomp.); Found: C, 56.70; H, 4.80; N, 22.12; S, 16,91.
C9H9N3S requires C, 56.52; H, 4.74; N, 21.97; S, 16.76%; νmax
(KBr)/cmϪ1: 3470, 3330, 2904, 1630, 1585, 1536, 1443, 1233,
1
818, 761, 712; H NMR (CDCl3): δH 2.60 (3H, s, SCH3), 4.07
1
1154, 824, 776; H NMR (DMSO-d6): δH 3.29 (1H, br s, SH),
(3H, s, OCH3), 4.10 (3H, s, OCH3), 7.35 (1H, m, H-6), 7.70 (1H,
m, H-7), 8.55 (1H, m, H-5), 8.72 (1H, s, H-3); 13C NMR
(CDCl3): δC 13.8 (SCH3), 52.4 (OCH3), 52.7 (OCH3), 61.6 (C),
6.16 (1H, s, H-6), 7.10 (1H, m, H-3), 8.32 (1H, m, H-4), 8.70
(1H, m, H-2); 13C NMR (DMSO-d6): δC 98.0 (CH), 111.8 (CH),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 2 4 8 – 4 2 5 3
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120.1 (CH), 122.2 (CH), 123.1 (CH), 126.1 (C), 129.7 (CH),
135.6 (C), 142.4 (C), 145.1 (C), 145.3 (C), 164.7 (CO), 165.4
(CO). m/z 292 (M ϩ 1).
2-Methylquinoline-8-thiol (12)
A solution of 11 (170 mg, 0.73 mmol) in 35% HCl solution
(50 mL) was heated at 90 ЊC for 12 h. The mixture was then
allowed to cool to room temperature and the pH adjusted to 8
by addition of aqueous 6 M NaOH. After extraction with
EtOAc, the organic layer was dried over MgSO4, filtered and
evaporated. Purification by flash chromatography (dichloro-
methane–EtOAc (95 : 5)) afforded 12 (113 mg, 88%) as a yellow
solid; mp 260–262 ЊC; Found: C, 68.62; H, 5.23; N, 8.11; S,
18.52. C10H9NS requires C, 68.54; H, 5.18; N, 7.99; S, 18.30%;
νmax (KBr)/cmϪ1: 2919, 1597, 1497, 1418, 1370, 1315, 1242,
8-Mercaptoquinoline-2,4-dicarboxylic acid (9)
To a stirred solution of 8 (200 mg, 0.69 mmol) in chloroform
(80 ml) at 0 ЊC was added m-chloroperbenzoic acid (130 mg,
0.76 mmmol) over 30 min. The mixture was then allowed to
warm to room temperature and stirred for 1 h. A saturated
NaHCO3 solution was added, the organic layer was separated,
dried on MgSO4 and evaporated. The crude sulfoxide was dis-
solved in excess of trifluoroacetic anhydride (5 mL) and the
solution refluxed for 30 min. After evaporation of the excess
anhydride, the residue was dissolved in methanol (30 mL) and a
solution of 1 M NaOH (3 ml) was added. The mixture was
refluxed for 1 h, cooled to room temperature and the methanol
evaporated. A 33% HCl solution was added dropwise to the
aqueous phase at 0 ЊC. The white precipitate obtained was
collected by filtration, washed with water and dried over P2O5
to provide 9 (112 mg, 65%) as a white solid; mp 310 ЊC
(decomp.); Found : C, 53.14; H, 2.90; N, 5.78; S, 13,02;
C11H7NO4S requires C, 53.01; H, 2.83; N, 5.62; S, 12.86%;
νmax (KBr)/cmϪ1: 2923, 2580, 1704, 1597, 1445, 1261, 1203,
1
1196, 1134, 1013, 974, 823, 785, 760, 663; H NMR (CDCl3):
δH 2.85 (3H, s, CH3), 7.31–7.39 (2H, m), 7.57 (1H, m), 7.86
(1H, m, H-7), 8.06 (1H, d, J = 8.4, H-4); 13C NMR (CDCl3):
δC 24.8 (CH3), 122.1 (CH), 124.0 (CH), 124.3 (CH), 125.4 (CH),
125.9 (C), 134.1 (C), 135.7 (CH). 145.0 (C), 158.0 (C); m/z 176
(M ϩ 1).
2-(2-Pyridyl)fluorobenzene (13)
2-Fluorophenylboronic acid (3 g, 21.4 mmol) and 2-bromo-
pyridine (1.64 mL, 17.2 mmol) were dissolved in aqueous 2 M
K2CO3 (20 mL) and DME (40 mL). The mixture was degassed
for approximately 30 min. Pd(PPh3)2Cl2 was added and the mix-
ture was heated to 80 ЊC overnight. The reaction mixture was
allowed to cool to room temperature, filtered through Celite,
and partioned between H2O (200 mL) and EtOAc (200 mL).
The organic layer was separated and dried over MgSO4, filtered,
and evaporated under vacuum. Purification by flash chromato-
graphy (dichloromethane–10% NH3 in methanol (10 : 0.2))
provided 13 as a light orange oil (2.4 g, 80%); Found: C, 76.42;
H, 4.78; F, 11.12; N, 8.27. C11H8FN requires C, 76.29; H, 4.66;
F, 10.97; N, 8.09%; νmax (film)/cmϪ1: 3054, 3011, 1615, 1588,
1494, 1464, 1456, 1427, 1303, 1248, 1220, 1208, 1154, 1111,
1
1154, 821, 764; H NMR (DMSO-d6): δH 3.19 (1H, br s, SH),
7.62 (1H, m, H-6), 7.78 (1H, m, H-7), 8.53 (1H, m, H-5), 8.85
(1H, s, H-3), 12.7 (1H, br s, OH), 12.9 (1H, br s, OH); 13C NMR
(DMSO-d6): δC 122.0 (CH), 123.2 (CH), 126.1 (C), 126.5 (CH),
130.0 (CH), 136.3 (C), 137.7 (C), 145.0 (C), 147.7 (C), 165.4
(CO), 166.7 (CO); m/z 250 (M ϩ 1).
8-Fluoro-2-methylquinoline (10)
Toluene (30 mL) and crotonaldehyde (3.71 mL, 45 mmol) were
added to a solution of 2-fluoroaniline (2.5 g, 22.50 mmol) in
aqueous 6 M HCl (100 mL) and heated at 100 ЊC. Heating
was continued for 2 h and the mixture was then allowed to cool
to room temperature. The aqueous layer was separated and
neutralized with 6 M NaOH solution. After extraction with
dichloromethane (2 × 50 mL), the organic layer was separated,
dried over MgSO4, filtered and evaporated. Purification by flash
chromatography (hexane–EtOAc (8 : 2)) provided 10 (2.6 g,
82%) as a colourless oil; Found: C, 74.68; H, 5.18; F, 11.94; N,
8.88. C10H8FN requires C, 74.52; H, 5.00; F, 11.79; N, 8.69%;
νmax (film)/cmϪ1: 3056, 2970, 1607, 1567, 1504, 1476, 1430, 1327,
1
1095, 1061, 1025, 990, 947, 862, 827, 792, 757, 719, 611; H
NMR (CDCl3): δH 7.13–7.23 (1H, ddd, J = 1.3, 8.1, 11.5), 7.24–
7.33 (2H, m), 7.34–7.45 (1H, m), 7.73–7.85 (2H, m), 7.95–8.04
(1H, dt, J = 2.0, 7.9) and 8.71–8.78 (1H, m, H-2); 13C NMR
(CDCl3): δC 116.1 (d, J = 22.5, CH), 122.3 (CH), 124.4 (d,
J = 2.7, CH), 124.5 (d, J = 2.8, CH), 127.4 (d, J = 11.5, C), 130.4
(d, J = 8.2, CH), 131.0 (d, J = 2.7, CH), 136.3 (CH), 149.7 (CH),
153.3 (d, J = 2.2, C), 160.4 (d, J = 249.7, C); m/z 174 (M ϩ 1).
2-(2-(tert-Butylthio)phenyl)pyridine (14)
1
1235, 1083, 833, 755; H NMR (CDCl3): δH 2.75 (3H, s, CH3),
DMF (10 mL) was degassed for approximately 1 h. Sodium
hydride (231 mg, 5.77 mmol) and 2-methyl-2-propanethiol (715
µL, 5.77 mmol) were added and formation of gas was observed.
The mixture was stirred for about 10 min. 2-(2-Pyridyl)fluoro-
benzene, 13, (500 mg, 2.89 mmol) was added, and the reaction
mixture turned orange. The mixture was heated to 120 ЊC for
3 days under N2 atmosphere. The mixture was allowed to
cool to room temperature and extracted with H2O (50 mL)
and EtOAc (70 mL). The organic layer was washed with H2O
(50 mL), dried over MgSO4, filtered and evaporated under
vacuum at 60 ЊC providing 14 as an orange oil (747 mg, quant.);
Found: C, 74.32; H, 7.26; N, 5.82; S, 13.33. C15H17NS requires
C, 74.03; H, 7.04; N, 5.76; S, 13.17%; νmax (film)/cmϪ1: 3412 (br),
2257, 2130, 1658 (br), 1204, 1135, 1049, 1026, 1002, 827, 765;
1H NMR (CDCl3): δH 1.05 (9H, s), 7.22–7.27 (1H, ddd, J = 1.5,
4.9, 7.0), 7.35–7.43 (1H, dt, J = 1.7, 7.5), 7.46–7.53 (1H, dt,
J = 1.3, 7.5), 7.64–7.76 (4H, m), 8.67–8.72 (1H, m, H-2); 13C
NMR (CDCl3): δC 30.9 (CH3), 47.5 (C(CH3)3), 121.7 (CH),
126.8 (CH), 128.1 (CH), 129.2 (CH), 130.7 (C), 130.8 (CH),
134.8 (CH), 139.5 (CH), 147.1 (C), 148.9 (CH), 159.3 (C); m/z
244 (M ϩ 1).
7.30–7.36 (3H, m), 7.49 (1H, m), 7.99 (1H, d, J = 9.9, H-4); 13
C
NMR (CDCl3): δC 25.0 (CH3), 112.8 (CH), 122.5 (CH), 122.6
(CH), 124.8 (CH), 127.6 (C), 135.3 (CH), 137.4 (C). 155.3 (C),
158.8 (C). m/z 162 (M ϩ 1).
8-(tert-Butylthio)-2-methylquinoline (11)
2-Methyl-2-propanethiol (0.42 ml, 3.72 mmol) and sodium
hydride (149 mg, 3.72 mmol) were added to a solution of 10
(300 mg, 1.86 mmol) in DMF (30 mL). The resulting mixture
was refluxed for 12 h. After evaporation of the solvent, the
residue was dissolved in EtOAc and washed with water (2 ×
50 mL). The organic layer was separated, dried over MgSO4,
filtered and evaporated. Purification by flash chromatography
(dichloromethane–EtOAc (95 : 5)) provided 11 (392 mg, 91%)
as a yellow solid; mp 32 ЊC; Found: C, 72.82; H, 7.53; N, 6.11; S,
14.02. C14H17NS requires C, 72.68; H, 7.41; N, 6.05; S, 13.86%;
νmax (KBr)/cmϪ1: 2962, 1597, 1547, 1494, 1421, 1360, 1313,
1
1237, 1154, 1013, 976, 838, 797, 771, 662; H NMR (CDCl3):
δH 1.38 (9H, s, C(CH3)3), 2.79 (3H, s, CH3), 7.29 (1H, d, J = 8.0,
H-3), 7.41 (1H, dd, J = 7.7, 7.8, H-6), 7.73 (1H, d, J = 8.0, H-4),
7.97–8.06 (2H, m); 13C NMR (CDCl3): δC 25.1 (CH3), 30.9
(3CH3), 46.4 (C), 121.6 (CH), 124.5 (CH), 126.5 (C), 128.0
(CH), 132.7 (C), 136.0 (CH), 137.6 (CH), 148.3 (C), 158.7 (C).
m/z 232 (M ϩ 1).
2-(2-Pyridyl)thiophenol (15)
A solution of tert-butyl-2-(2-pyridyl)thiophenol 14 (200 mg,
0.82 mmol) in conc. HCl (4 mL) was heated at 110 ЊC overnight.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 2 4 8 – 4 2 5 3
4252

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Selected Stability Constants of Metal Complexes Database. (6.0),
The mixture was allowed to cool to room temperature and
extracted with H2O (15 mL) and EtOAc (20 mL). The aqueous
layer was separated and the pH was adjusted to approximately
7, and extracted with EtOAc (2 × 20 mL). The organic layer was
dried over MgSO4, filtered and evaporated under vacuum at
40 ЊC. The product was purified by chromatography on Silica
(EtOAc–heptane (1 : 1)) providing 15 as a pale wax (43 mg,
28%); Found: C, 70.02; H, 4.21; N, 7.52; S, 16.87. C11H9NS
requires C, 70.55; H, 4.84; N, 7.48; S, 17.12%; νmax (film)/cmϪ1:
3053, 3007, 1583, 1568, 1478, 1461, 1432, 1421, 1296, 1257,
1154, 1131, 1095, 1076, 1036, 1021, 990, 945, 894, 870, 794,
National Institute of Standards and Technology, 2001.
7 W. A. E. McBryde, IUPAC Chemical Data Series, Pergamon Press,
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Tetrahedron Lett., 2003, 44, 2033–2035; (b) A. Arcadi, M. Chiarini,
S. Di Giuseppe and F. Marinelli, Synlett, 2003, 2, 203–206; (c) H. Z.
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1
751, 679, 621; H NMR (CDCl3): δH 7.25–7.35 (3H, m), 7.50–
7.55 (1H, m), 7.59–7.65 (1H, m), 7.76–7.85 (2H, m) and 8.73–
8.77 (1H, m, H-2); 13C NMR (CDCl3): δC 122.4 (CH), 123.5
(CH), 126.3 (CH), 127.7 (CH), 129.4 (CH), 129.4 (CH), 136.6
(CH), 136.9 (C), 139.2 (C), 148.7 (CH), 157.8 (C); m/z 186
(M-1).
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