Synthesis and characterization of a multi ring-fused 2-pyridone-based fluorescent scaffold

Article abstract of DOI:10.1002/ejoc.201000796

A series of compounds based on a novel fluorescent scaffold have been synthesized. Most of the compounds displayed high quantum yields of fluorescence and unusually long fluorescence lifetimes. HeLa cells were treated with one of the compounds and its use as a fluorescent dye was demonstrated with fluorescence confocal microscopy. A multi ring-fused 2-pyridone-based fluorescent scaffold with good quantum yields of fluorescence and ability to stain HeLa-cells is reported.

Full text of DOI:10.1002/ejoc.201000796

FULL PAPER
DOI: 10.1002/ejoc.201000796
Synthesis and Characterization of a Multi Ring-Fused 2-Pyridone-Based
Fluorescent Scaffold
Magnus Sellstedt,[a] Anders Nyberg,[a] Erik Rosenbaum,[a] Patrik Engström,[b,c]
Malin Wickström,[d] Joachim Gullbo,[d] Sven Bergström,[b,c] Lennart B.-Å. Johansson,[a] and
Fredrik Almqvist*[a]
Keywords: Medicinal chemistry / Heterocycles / Luminescence / Fluorescent probes
A series of compounds based on a novel fluorescent scaffold
have been synthesized. Most of the compounds displayed
high quantum yields of fluorescence and unusually long fluo-
rescence lifetimes. HeLa cells were treated with one of the
compounds and its use as a fluorescent dye was demon-
strated with fluorescence confocal microscopy.
its biological properties. Here, a novel fluorescent 2-pyr-
idone-based scaffold is presented. In addition to function
as a fluorescent probe, the scaffold can easily be substituted
in various ways, potentially allowing the fluorophore itself
to interact with different biological targets.
There exists many naturally occurring ring fused 2-pyr-
idones with interesting biological activity, e.g. fredericamy-
cin A,[2] 1, and camptotecin,[3] 2, which both are antitumor
agents (Figure 1). Carbostyrils,[4] 3, and naphthalimides,[5]
4, are well known fluorescent probes with versatile bio-
logical applications. Even the simple isoquinolone 5 has flu-
orescent properties,[6] although not as prominent.
Introduction
Small organic compounds with fluorescent properties
have found wide application in molecular biology, e.g. as
DNA-staining dyes or as intracellular probes for calcium
ion concentrations. Fluorescent reporter molecules can also
be covalently linked to proteins, antibodies, or known in-
hibitors of a specific target, to give information about cellu-
lar distribution and mode of action.[1] However, linking a
fluorescent probe to a small inhibitor can significantly alter
Results and Discussion
The synthesis of bicyclic 2-pyridones like 6a (Figure 2)
has previously been described.[7] By changing the substitu-
tion pattern, these compounds become active either as anti-
bacterial agents,[8] or as inhibitors of the formation of Alz-
heimer’s amyloids.[9] The same compounds can also inhibit
formation of a bacterial amyloid, curli, which is a virulence
factor in Gram-negative bacteria.[10] Now, given the fluores-
cent structures 3–5, we sought to ring-close the formylated
derivative 7a,[11] to form an extended aromatic scaffold with
potential fluorescent properties. A related ring-closure of 4-
formyldihydropyridones have been described using TiCl4 in
Figure 1. Examples of naturally occurring cytotoxic compounds (1,
2), and fluorescent probes (3–5), containing 2-pyridone-like motifs.
[a] Department of Chemistry, Umeå University,
90187 Umeå, Sweden
[b] Department of Molecular Biology, Umeå University,
90187 Umeå, Sweden
[c] Laboratory for Molecular Infection Medicine Sweden (MIMS),
Umeå University,
90187 Umeå, Sweden
E-mail: fredrik.almqvist@chem.umu.se
[d] Department of Medicinal Sciences, Division of Clinical
Pharmacology, Uppsala University Hospital,
75185 Uppsala, Sweden
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000796.
Figure 2. A route to prepare polyaromatic 2-pyridones.
View this journal online at
wileyonlinelibrary.com
Eur. J. Org. Chem. 2010, 6171–6178
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6171

F. Almqvist et al.
FULL PAPER
dichloromethane.[12] Initial attempts to transform 7a under
The fluorescent properties of the polyaromatic com-
similar conditions were promising, and the generated com- pounds 8 were then examined. The absorption and fluores-
pound 8a was indeed fluorescent. cence spectra of the compounds exhibit similar shapes over
A series of formylated 2-pyridones were synthesized in an identical wavelength region. The absorption spectra are
analogy with 7a to examine the applicability of the syn- quite broad, which enables a wide range of excitation wave-
thetic route for preparation of differently substituted com- lengths for measuring fluorescence (Figure 3). The fluores-
pounds (Table 1). This offers an opportunity to fine-tune cence spectra cover a substantial part of the visible region.
both their fluorescent properties and their potential interac- In addition, the small overlap between the lowest absorp-
tion with a biological target.
tion transition and the fluorescence spectrum enables the
use of relatively high dye concentrations with a negligible
probability of reabsorption.
Table 1. Synthesis of formylated bicyclic 2-pyridones.
Entry R1
R2
Product
% Yield Product
% Yield
Figure 3. The shapes of the absorption (thin line) and corrected
fluorescence spectrum (thick line) of 8b, when dissolved in glycerol,
at 277 K.
1
2
3
4
5
Ph
Ph
o-tolyl
o-MeOPh
p-FPh
H
Me
H
H
H
6b
6c[a]
6d
6e
6f
88
63
92
96
93
7b
7c[a]
7d
7e
7f
76
41
62
87
69
With the exception of compound 8a, the fluorescence
quantum yields of are quite high, and the fluorescence life-
times are unusually long (ca. 8–20 ns) (Table 3). Compound
8a has a more typical lifetime (ca. 1 ns).[1] The influence of
solvent viscosity is not of great importance for the fluores-
cence lifetimes of these compounds. The fluorescence
quenching caused by dissolved O2 is not dramatically in-
creased, despite the fact that the viscosity is lower by three
orders of magnitude in ethanol, as compared to glycerol.
Assuming that the O2 solubility is known (0.56 mmol/L),
the quenching constant can be estimated from the measured
fluorescence lifetimes of the substances in the presence and
absence of dissolved O2. The obtained values are reason-
able,[1] typically in the range of 1010–1011 mol–1 Ls–1. Sim-
ilar results are observed for these compounds, when dis-
solved in dichloromethane (data not shown).
[a] Obtained as a mixture of diastereomers.
The formylated species 7 were heated with microwave ir-
radiation in the presence of excess TiCl4 to obtain the ring-
closed products 8 (Table 2). The reaction worked well for
most compounds by using microwave heating at 100 °C for
10–70 min. However, the electron poor fluorobenzyl-substi-
tuted compound 7f proved more difficult to react and re-
quired heating at 160 °C for 120 min. These conditions also
hydrolyzed the methyl ester, and prior to purification, the
crude reaction mixture was re-methylated using TMS chlo-
ride in methanol.
Table 2. Ring closure to prepare the fluorescent compounds 8a–f.
Table 3. The quantum yield of fluorescence (Φf) and fluorescence
lifetime (τf) of different polyaromatic 2-pyridones when dissolved
in glycerol (g) at 277 K. 1,2-propanediol (p) was chosen as solvent
for compound 8a, due to its poor solubility in glycerol. Ethanol (e)
was used to exemplify a solvent of low viscosity at 293 K.
Entry
Φf[a]
τf [ns][b] Φf[a]
τf [ns][b] τf [ns][b,c]
1
2
3
4
5
6
8a
8b
8c
8d
8e
8f
0.06(p)
0.72(g)
0.46(g)
0.66(g)
0.56(g)
0.57(g)
1.6(p)
0.11(e)
1.1(e)
14.0(e)
9.0(e)
14.4(e)
8.1(e)
14.3(e)
1.0(e)
Entry
1
R
R2 Reaction time [min] Product
% Yield
79
17.0(g)
11.3(g)
17.1(g)
14.0(g)
16.2(g)
0.44(e)
0.29(e)
0.38(e)
0.34(e)
0.27(e)
19.0(e)
11.0(e)
19.5(e)
10.1(e)
17.9(e)
9,10 ring-fused
Ph
H
H
10-Me
10-MeO
8-F
H
10
8a
2
3
4
H
Me
H
H
H
50
20
20
70
120
8b
8c
8d
8e
8f
89
85
79
64
51
[a] The fluorescence reference used is described elsewhere[13] and
λex = 410 nm. [b] λex = 404 nm. [c] Degassed with argon.
5
6[a]
The photo stability of compounds 8a and 8b were also
investigated. Samples dissolved in chloroform were exposed
to high-energy white light (approximately 770 mW/cm2). It
[a] Reaction performed at 160 °C. The product was obtained after
re-methylation (TMSCl, MeOH) of the initially obtained carbox-
ylic acid.
6172
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6171–6178

Multi Ring-Fused 2-Pyridone-Based Fluorescent Scaffold
was found that after 44 min, the absorption spectra of the
In addition to being used as a dye, the scaffold can po-
first two bands of substance 8a had decreased by 50%. The tentially be used as a fluorescent tag, using the methyl ester
same effect could be seen after 54 min for sample 8b. This as a linking point. However, then the scaffold must show a
value can be compared to a half-life of 12 min for a defined limited cytotoxicity profile. Hence, the cytotoxic effects of
and used BODIPY probe dissolved in chloroform, upon ex- 8b were evaluated for three different human cell types com-
posure to approximately eight times higher light intensity monly used in cytotoxicity tests, T-cell leukemia cell line
(6 W/cm2), in the same wavelength range.[14] The BODIPY CCRF-CEM, U937 lymphoma cells, and normal peripheral
probe used in the reference has a quantum yield Ͼ 95%. blood mononuclear cells (PBMC). The LD50 values (i.e.
For both 8a and 8b, the absorbance increased at wave- concentrations leading to 50% survival) after 72 h exposure,
lengths shorter than 300 nm following the photon-bleach- was determined to 37 and 23 µ for U937 and PBMC cells,
ing, which is compatible with the generation of smaller aro- respectively. The LD50 for the CCRF-CEM cells was esti-
matic systems produced by photo-degradation. The inten- mated to be Ͼ 100 µ (Figure 5). Similar survival data were
sities of the used high-energy light sources are at least one obtained for cells treated for 24 h. In these rather sensitive
order of magnitude greater than those used in conventional cell systems this is considered to be moderate cytotoxic po-
spectroscopic experiments. A similar experiment was per- tential, considerably lower than that of, for example, the
formed where two samples containing 8a and 8b in chloro- lipophilic fluorescent probe calcein/AM with a LD50 of
form were exposed to white light from a desktop lamp, 0.25 µ in U937.[15] These data indicate that 8b might be
which exposed the samples to an intensity of approximately useful as a fluorescent probe in biological systems.
2 mW/cm2. Then, the absorption decrease due to sample
degradation was less than 5% after an exposure time of 24
hours.
The high quantum yields of fluorescence of these com-
pounds facilitate their use as fluorescent markers in bio-
logical systems. This was demonstrated by the use of one of
the most fluorescent compounds (8b) for staining HeLa
cells. Fluorescence microscopy shows that the compound
was taken up and distributed in almost the entire cell (Fig-
ure 4, A–B). The fluorophore appeared to be concentrated
in areas rich in microtubule. However, cross staining with
antibodies against α-tubulin revealed only partial overlap,
with accumulation of the compound around the cellular nu-
cleus (Figure 4, C–E). Neither could any clear effect on
microtubule polymerization be detected (see Supporting In-
formation). Nonetheless, this exemplifies the use of this
compound as a cell staining dye.
Figure 5. Cell survival after 72 h in presence of compound 8b at
different concentrations. Three different human cell lines were
tested: T-cell leukemia cell line CCRF-CEM, lymphoma cell line
U-937, and normal peripheral blood mononuclear cells (PBMC).
Error bars indicate the standard deviation of duplicates.
Conclusions
Here we present a new scaffold with useful fluorescent
properties. The scaffold can hold independently variable
substituents, which allows for tuning of its biological prop-
erties. A high quantum yield of fluorescence was obtained
for most compounds, regardless of the electronic nature of
their substituents. Unusually long fluorescence lifetimes
were also observed. HeLa cells were stained with one of the
synthesized compounds and fluorescence microscopy sug-
gests that the compound is located primarily in the vicinity
of microtubules and surrounding the cell nucleus. In ad-
dition to functioning as a dye in itself, the limited cytotoxi-
city of the compound enables its potential use as a fluores-
cent tag, utilizing the methyl ester as a linking point.
Experimental Section
Figure 4. Fluorescence confocal microscopy of HeLa cells. Scale
bars are 10 µm. A and B) Cells incubated presence of 100 µ 8b
before fixation with methanol. C) Cells treated with 100 µ 8b after
fixation with methanol. The wavelength used for excitation was
408 nm (violet diode laser). D) α-tubulin stained cells. The exci-
tation wavelength used was 543 nm. E) Picture C and D merged.
General: All reactions were carried out under an inert atmosphere
with dry solvents under anhydrous conditions, unless otherwise
stated. Microwave heated reactions were conducted in sealed Em-
rysTM process vials with an InitiatorTM 2.0 microwave instrument
Eur. J. Org. Chem. 2010, 6171–6178
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6173

F. Almqvist et al.
FULL PAPER
from Biotage. Temperatures were monitored with an IR probe.
TLC was performed on Silica Gel 60 F254 using UV light detection.
Silica gel chromatography employed normal phase silica gel (60 Å,
230–400 mesh, Merck, grade 9385). Optical rotations were mea-
sured with a Perkin–Elmer 343 polarimeter at 20 °C and 589 nm.
IR spectra were recorded on a Bruker Equinox 55 spectrometer
equipped with an ATR-device. The 1H and 13C NMR spectra were
recorded at 298 K with a Bruker DRX-400 or DRX-360 spectrom-
eter and calibrated using the residual peak of CHCl3 as internal
standard (CHCl3 δH = 7.26 ppm, CDCl3 δC = 77.16 ppm). HRMS
was conducted using a Micromass Q-Tof UltimaTM mass spectrom-
eter with electrospray (ES+) ionization. Polyethylenglycol PEG-400
was used as calibration chemical. LRMS was conducted on a
Micromass ZQ mass spectrometer with ES+ ionization. The ab-
sorption spectra were recorded by a Varian Cary, 5000 UV/Vis
spectrometer. The fluorescence spectra were transcribed by means
of a Fluorolog®-3 (Jobin Yvon) spectrometer equipped with Glan–
Thompson polarisers. The bandwidth of the excitation and emis-
sion light was 2 nm. All fluorescence spectra were corrected. The
time-resolved fluorescence decays were measured by means of the
time-correlated single photon-counting technique using a Fluo-
rolog® TCSPC (Horiba) spectrometer. For the pulsed excitation, a
NanoLED (Horiba) was used in combination with an interference
filter, centred at 398.8 nm (HBW = 11.4 nm). The fluorescence life-
time was calculated by using a deconvolution method[1] based on
the Levenberg–Maquardt algorithm.[16] Cell images were aquired
using a Nikon e-C1plus fluorescence confocal microscope. The pic-
tures were analysed with Nikon E2-C1 software and processed in
Adobe® Photoshop CS4.
1.74 [s, 6 H, C(CH3)2] ppm. 13C NMR (100 MHz, CDCl3): δ =
195.3, 170.5, 160.3, 137.0, 132.7, 130.4, 129.9, 127.6, 126.1, 104.9,
91.6, 38.9, 26.7 (2 C), 19.7 ppm.
5-[1-Hydroxy-2-(2-methoxyphenyl)ethylidene]-2,2-dimethyl-1,3-
dioxane-4,6-dione (9e): By first following the general procedure for
synthesis of acyl Meldrum’s acids, 1663 mg (10.01 mmol) o-meth-
oxyphenylacetic acid was converted to a crude product that was
further purified by dissolving it in CH2Cl2 and extract it with satu-
rated NaHCO3(aq.) three times. The aqueous phase was washed
once with a small amount of CH2Cl2, acidified with aqueous HCl
(to pH ≈ 2) and then extracted with CH2Cl2. The organic phase
was dried with anhydrous Na2SO4, filtered and concentrated to
1
2189 mg (75%) product 9e. IR: ν
= 1737, 1655 cm–1. H NMR
˜
max
(400 MHz, CDCl3): δ = 15.37 (br. s, 1 H, OH), 7.29–7.23 (m, 1 H,
Ar), 7.17 (d, J = 7.3 Hz, 1 H, HAr), 6.94–6.84 (m, 2 H, HAr), 4.45
H
(s, 2 H, CH2), 3.77 (s, 3 H, OCH3), 1.73 [s, 6 H, C(CH3)2] ppm.
13C NMR (100 MHz, CDCl3): δ = 195.8, 170.5, 160.3, 157.4, 131.0,
128.8, 122.6, 120.5, 110.4, 104.8, 91.4, 55.4, 36.6, 26.6 (2 C) ppm.
5-[2-(4-Fluorophenyl)-1-hydroxyethylidene]-2,2-dimethyl-1,3-di-
oxane-4,6-dione (9f): By following the general procedure for synthe-
sis of acyl Meldrum’s acids, 1546 mg (10.03 mmol) p-fluorophen-
ylacetic acid was converted to 1840 mg (65%) crude product 9f. IR:
1
ν
= 1738, 1646 cm–1. H NMR (400 MHz, CDCl3): δ = 15.33
˜
max
(br. s, 1 H, OH), 7.39–7.33 (m, 2 H, HAr), 7.04–6.96 (m, 2 H, HAr),
4.38 (s, 2 H, CH2), 1.71 [s, 6 H, C(CH3)2] ppm. 13C NMR
(100 MHz, CDCl3): δ = 194.4, 170.6, 162.4 (d, J = 246.0 Hz), 131.4
(d, J = 8.0 Hz, 2 C), 129.8 (d, J = 3.5 Hz), 115.7 (d, J = 21.4 Hz,
2 C), 105.2, 91.5, 40.1, 27.0 (2 C) ppm.
General Procedure for Synthesis of Bicyclic Pyridones 6: Thiazoline
10[7] (1.0 equiv.) and acyl Meldrum’s acid 9 (2.5 equiv.) were dis-
solved in 1,2-dichloroetane (1.6 mL/mmol 10). Trifluoroacetic acid
(2.0 equiv.) was added and the solution was heated with microwave
irradiation for 3 min at 125 °C. The reaction was diluted with
CH2Cl2, quenched with saturated NaHCO3(aq.), and extracted
three times with CH2Cl2. The combined organic phases were dried
with anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified with silica gel chromatography
using heptane/ethyl acetate 2:1Ǟ1:4 as mobile phase to afford 6
as white foam.
General Synthesis of Acyl Meldrum’s Acids 9: The carboxylic acid
(I) to be reacted (1.0 equiv.), Meldrum’s acid (1.1 equiv.), and 4-
(dimethylamino)pyridine (1.6 equiv.) were dissolved in CH2Cl2
(6.0 mL/mmol I) and cooled to 0 °C. Dicyclohexylcarbodiimide
(1.15 equiv.) in CH2Cl2 (1.5 mL/mmol I) was added dropwise over
60 min. The reaction was allowed to attain room temp. while stir-
ring overnight. The resulting mixture was cooled to 0 °C, filtered
and washed three times with 6% KHSO4(aq.). The combined aque-
ous phases were extracted once with CH2Cl2. The combined or-
ganic phases were washed once with brine, dried with anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The res-
idue was taken up in ethyl acetate, which made 9f precipitate, this
precipitate was collected and used without further purification. In
all other cases undissolved substance was filtered off and the sol-
vent was concentrated under reduced pressure. The crude products
were used without further purification.
Compound 6a was prepared as described previously.[7]
(R)-Methyl 7-Benzyl-2,3-dihydro-8-phenylthiazolo[3,2-a]pyridine-5-
one-3-carboxylate (6b): By following the general synthesis of bicy-
clic pyridones, 708 mg (3.01 mmol) thiazoline 10 and 1974 mg
(7.53 mmol) 9b was converted to 995 mg (88%) 6b. [α]D = –166 (c
Compound 9a and 9b was synthesized as described previously.[7,17]
1
= 1.0, CHCl ). IR: ν
= 1750, 1650 cm–1. H NMR (400 MHz,
˜
3
max
CDCl3): δ = 7.38–7.31 (m, 3 H, HAr), 7.21–7.08 (m, 5 H, HAr),
6.92–6.86 (m, 2 H, HAr), 6.13 (s, 1 H, 6-H), 5.62 (dd, J = 8.5,
2.5 Hz, 1 H, Hα), 3.81 (s, 3 H, CO2CH3), 3.61 (dd, J = 11.7, 8.5 Hz,
1 H, Hβ), 3.57 (s, 2 H, ArCH2), 3.42 (dd, J = 11.7, 2.5 Hz, 1 H,
Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ = 168.6, 161.4, 154.6,
146.8, 137.7, 136.3, 130.3, 129.9, 129.1 (2 C), 128.8, 128.7, 128.4 (2
C), 128.3, 126.5, 116.3, 115.5, 63.6, 53.3, 39.6, 31.7 ppm. HRMS:
m/z = 378.1167 [M + H], C22H20NO3S requires 378.1164.
5-(1-Hydroxy-2-phenylpropylidene)-2,2-dimethyl-1,3-dioxane-4,6-di-
one (9c): By following the general procedure for synthesis of acyl
Meldrum’s acids, 1502 mg (10.00 mmol) racemic 2-phenylpropionic
acid was converted to 2732 mg (99%) crude product 9c. IR: ν
˜
max
= 1736, 1662 cm–1. H NMR (400 MHz, CDCl3): δ = 15.70 (br. s,
1 H, OH), 7.46–7.41 (m, 2 H, HAr), 7.36–7.23 (m, 3 H, HAr), 5.46
(q, J = 7.1 Hz, 1 H, CHCH3), 1.73 (s, 3 H, CH3), 1.62 (s, 3 H,
CH3), 1.59 (d, J = 7.1 Hz, 3 H, CHCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 198.5, 171.0, 160.2, 140.1, 128.7 (2 C),
128.2 (2 C), 127.6, 104.9, 90.8, 42.7, 26.9, 26.6, 18.1 ppm.
1
(3R)-Methyl 2,3-Dihydro-8-phenyl-7-(1-phenylethyl)thiazolo[3,2-a]-
pyridine-5-one-3-carboxylate (6c): By following the general synthe-
sis of bicyclic pyridones, 353 mg (1.50 mmol) thiazoline 10 and
5-(1-Hydroxy-2-o-tolylethylidene)-2,2-dimethyl-1,3-dioxane-4,6-
dione (9d): By following the general procedure for synthesis of acyl
Meldrum’s acids, 1502 mg (10.00 mmol) o-tolylacetic acid was con-
1038 mg (3.76 mmol) 9c was converted to 371 mg (63%) 6c. [α]D
–143 (c = 1.0, CHCl ). IR: ν
= 1752, 1649 cm–1 1H NMR
(400 MHz, CDCl3): δ = 7.44–7.36 (m, 1 H, HAr), 7.34–7.28 (m, 1
cm–1. 1H NMR (400 MHz, CDCl3): δ = 15.49 (br. s, 1 H, OH), H, HAr), 7.27–7.22 (m, 1 H, HAr), 7.20–7.04 (m, 4 H, HAr), 6.36
7.23–7.11 (m, 4 H, HAr), 4.49 (s, 2 H, CH2), 2.33 (s, 3 H, ArCH3), and 6.35 (s, 1 H, 6-H), 5.64–5.58 (m, 1 H, Hα), 3.88–3.78 (m, 1 H,
=
.
˜
3
max
verted to 1426 mg (52%) crude product 9d. IR: ν
= 1726, 1656
˜
max
6174
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6171–6178

Multi Ring-Fused 2-Pyridone-Based Fluorescent Scaffold
CHCH3), 3.81 and 3.80 (s, 3 H, CO2CH3), 3.59 (dd, J = 11.7,
8.6 Hz, 1 H, Hβ), 3.42–3.36 (m, 1 H, Hβ), 1.40 and 1.39 (d, J =
7.1 Hz, 3 H, CHCH3) ppm. 13C NMR (100 MHz, CDCl3): δ =
with anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified with silica gel chromatog-
raphy using heptane/ethyl acetate 4:1Ǟ1:4 as mobile phase to af-
168.5, 161.2, 159.1, 146.8 and 146.7 (1 C), 143.8 and 143.7 (1 C), ford 7 as yellow foam.
136.4 and 136.2 (1 C), 130.8, 130.5 and 130.2 (1 C), 129.6, 128.6,
Preparation of Arnold’s Reagent in Acetonitrile: Oxalyl chloride
128.6 and 128.5 (1 C), 128.3 (2C, split), 128.2 and 128.1 (1 C),
127.5 (split), 126.3 (split), 116.4 and 116.3 (1 C), 113.2 and 113.1
(1 C), 63.6, 53.2, 41.4 (split), 31.5, 21.6 (split) ppm. HRMS: m/z =
392.1323 [M + H], C23H22NO3S requires 392.1320.
(1.0 equiv.) was added dropwise to DMF (1.0 equiv.) in acetonitrile
(6.25 mL/mmol dmF). The solution was stirred 15 min at room
temp. and then used directly.
Compound 7a was prepared as described previously.[11]
(R)-Methyl 2,3-Dihydro-8-phenyl-7-(o-tolylmethyl)thiazolo[3,2-a]-
pyridine-5-one-3-carboxylate (6d): By following the general synthe-
sis of bicyclic pyridones, 361 mg (1.53 mmol) thiazoline 10 and
(R)-Methyl 7-Benzyl-6-formyl-2,3-dihydro-8-phenylthiazolo[3,2-a]-
pyridine-5-one-3-carboxylate (7b): By following the general pro-
cedure for formylation, 283 mg (0.75 mmol) 6b was converted to
232 mg (76%) 7b. Four equivalents of freshly prepared Arnold’s
1028 mg (3.72 mmol) 9d was converted to 552 mg (92%) 6b. [α]D
=
–130 (c = 1.0, CHCl ). IR: ν
= 1752, 1650 cm–1 1H NMR
.
˜
3
max
reagent was used. [α]D = –284 (c = 1.0, CHCl ). IR: ν
= 1751,
˜
(400 MHz, CDCl3): δ = 7.43–7.33 (m, 3 H, HAr), 7.25–7.17 (m, 2
H, HAr), 7.13–7.04 (m, 3 H, HAr), 7.02–6.95 (m, 1 H, HAr), 5.87 (s,
1 H, 6-H), 6.62 (dd, J = 8.7, 2.6 Hz, 1 H, Hα), 3.80 (s, 3 H,
CO2CH3), 3.63 (dd, J = 11.6, 8.7 Hz, 1 H, Hβ), 3.57–3.44 (m, 2 H,
ArCH2), 3.42 (dd, J = 11.6, 2.6 Hz, 1 H, Hβ), 2.02 (s, 3 H, ArCH3)
ppm. 13C NMR (100 MHz, CDCl3): δ = 168.5, 161.3, 154.3, 146.5,
136.4, 136.3, 136.0, 130.3, 130.2, 130.0, 129.7, 128.9 (2C, split),
128.3, 126.9, 126.1, 116.2, 114.7, 63.5, 53.3, 37.3, 31.7, 19.4 ppm.
HRMS: m/z = 392.1327 [M + H], C23H22NO3S requires 392.1320.
3
max
1674, 1639 cm–1. H NMR (360 MHz, CDCl3): δ = 10.44 (s, 1 H,
CHO), 7.35–7.22 (m, 3 H, HAr), 7.12–7.04 (m, 3 H, HAr), 7.03–
6.91 (m, 2 H, HAr), 6.77–6.70 (m, 2 H, HAr), 5.70 (dd, J = 9.0,
2.6 Hz, 1 H, Hα), 4.35–4.15 (m, 2 H, ArCH2), 3.82 (s, 3 H,
CO2CH3), 3.64 (dd, J = 12.1, 9.0 Hz, 1 H, Hβ), 3.42 (dd, J = 12.1,
2.6 Hz, 1 H, Hβ) ppm. 13C NMR (90 MHz, CDCl3): δ = 191.0,
167.9, 162.4, 157.9, 156.5, 137.8, 135.0, 130.3, 130.0, 128.7 (3 C),
128.3 (2 C), 128.0 (2 C), 125.9, 117.9 (2C, split), 64.1, 53.4, 34.9,
31.4 ppm. LRMS: m/z = 406 [M + H], C23H20NO4S requires 406.
1
(R)-Methyl 7-(2-Methoxybenzyl)-2,3-dihydro-8-phenylthiazolo[3,2-
a]pyridine-5-one-3-carboxylate (6e): By following the general syn-
thesis of bicyclic pyridones, 353 mg (1.50 mmol) thiazoline 10 and
(3R)-Methyl 6-Formyl-2,3-dihydro-8-phenyl-7-(1-phenylethyl)-
thiazolo[3,2-a]pyridine-5-one-3-carboxylate (7c): By following the
general procedure for formylation, 230 mg (0.59 mmol) 6c was con-
verted to 101 mg (41%) 7c. In total 12 equiv. of commercially avail-
able Arnold’s reagent was used, 6c was however never fully con-
1096 mg (3.75 mmol) 9e was converted to 589 mg (96%) 6e. [α]D
=
–145 (c = 1.0, CHCl ). IR: ν
= 1752, 1650 cm–1 1H NMR
.
˜
3
max
(400 MHz, CDCl3): δ = 7.40–7.30 (m, 3 H, HAr), 7.25–7.19 (m, 2
H, HAr), 7.17–7.10 (m, 1 H, HAr), 6.91–6.86 (m, 1 H, HAr), 6.82–
6.77 (m, 1 H, HAr), 6.74 (d, J = 8.3 Hz, 1 H, HAr), 5.98 (s, 1 H, 6-
H), 5.58 (dd, J = 8.5, 2.5 Hz, 1 H, Hα), 3.77 (s, 3 H, CO2CH3),
3.65 (s, 3 H, ArOCH3), 3.63–3.44 (m, 3 H, Hβ and ArCH2), 3.38
(dd, J = 11.7, 2.5 Hz, 1 H, Hβ) ppm. 13C NMR (100 MHz, CDCl3):
δ = 168.5, 161.3, 157.1, 154.8, 146.0, 136.4, 130.7, 130.2, 129.8,
128.6 (2C, split), 128.0 (2C, split), 126.2, 120.3, 116.3, 114.6, 110.3,
63.4, 55.1, 53.1, 33.6, 31.5 ppm. HRMS: m/z = 408.1281 [M + H],
C23H22NO4S requires 408.1270.
sumed. [α]D = –159 (c = 1.0, CHCl ). IR: ν
= 1752, 1673, 1633
˜
3
max
1
cm–1. H NMR (400 MHz, CDCl3): δ = 10.37 and 10.33 (s, 1 H,
CHO), 7.36–7.29 (m, 1 H, HAr), 7.28–7.18 (m, 2 H, HAr), 7.13–
6.97 (m, 4 H, HAr), 6.90–6.82 (m, 2 H, HAr), 6.56–6.33 (m, 1 H,
HAr), 5.73–5.68 (m, 1 H, Hα), 5.34–5.07 (m, 1 H, CHCH3), 3.87 (s,
3 H, CO2CH3), 3.69–3.60 (m, 1 H, Hβ), 3.45–3.39 (m, 1 H, Hβ),
1.55 and 1.51 (d, J = 7.2 Hz, 3 H, CHCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 191.5 and 191.2 (1 C), 168.2, 164.6 and
164.4 (1 C), 162.3 and 162.2 (1 C), 157.0 and 156.8 (1 C), 142.6
and 142.5 (1 C), 136.1 (split), 130.9 (split), 130.6 and 130.2 (1 C),
128.8 and 128.6 (1 C), 128.5 (split), 128.4, 128.0 (2C, split), 127.0
(2C, split), 125.8 (split), 118.3 and 118.2 (1 C), 117.7 (split), 64.5
(split), 53.7, 38.4 and 37.8 (1 C), 31.4 (split), 17.7 and 17.6 (1 C)
ppm. LRMS: m/z = 420 [M + H] C24H22NO4S requires 420.
(R)-Methyl 7-(4-Fluorobenzyl)-2,3-dihydro-8-phenylthiazolo[3,2-a]-
pyridine-5-one-3-carboxylate (6f): By following the general synthe-
sis of bicyclic pyridones, 118 mg (0.50 mmol) thiazoline 10 and
350 mg (1.25 mmol) 9f was converted to 185 mg (93%) 6f. [α]D
=
–142 (c = 1.0, CHCl ). IR: ν
= 1752, 1651 cm–1 1H NMR
.
˜
3
max
(R)-Methyl 6-Formyl-2,3-dihydro-8-phenyl-7-(o-tolylmethyl)-
thiazolo[3,2-a]pyridine-5-one-3-carboxylate (7d): By following the
general procedure for formylation, 256 mg (0.65 mmol) 6d was con-
verted to 171 mg (62%) 7d. In total 9 equiv. of commercially avail-
able Arnold’s reagent was used. [α]D = –232 (c = 1.0, CHCl3). IR:
(400 MHz, CDCl3): δ = 7.34–7.27 (m, 3 H, HAr), 7.12–7.02 (m, 2
H, HAr), 6.84–6.72 (m, 4 H, HAr), 6.06 (s, 1 H, 6-H), 5.58 (dd, J =
8.7, 2.6 Hz, 1 H, Hα), 3.75 (s, 3 H, CO2CH3), 3.57 (dd, J = 11.8,
8.7 Hz, 1 H, Hβ), 3.50 (s, 2 H, ArCH2), 3.37 (dd, J = 11.8, 2.6 Hz,
1 H, Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ = 168.3, 161.4 (d,
J = 245.0 Hz), 161.1, 154.1, 146.9, 136.0, 133.3 (split), 130.4 (d, J
= 8.0 Hz, 2 C), 130.1, 129.7, 128.6 (2C, split), 128.1, 115.8, 115.1,
115.0 (d, J = 21.3 Hz, 2 C), 63.4, 53.1, 38.6, 31.5 ppm. HRMS: m/z
= 396.1070 [M + H], C22H19FNO3S requires 396.1070.
ν
= 1755, 1675, 1644 cm–1. 1H NMR (400 MHz, CDCl3): δ =
˜
max
10.43 (s, 1 H, CHO), 7.34–7.18 (m, 3 H, HAr), 7.06–6.96 (m, 4 H,
HAr), 6.90–6.85 (m, 1 H, HAr), 6.77–6.72 (m, 1 H, HAr), 5.77 (dd,
J = 9.1, 2.7 Hz, 1 H, Hα), 4.23–3.97 (m, 2 H, ArCH2), 3.89 (s, 3
H, CO2CH3), 3.72 (dd, J = 11.8, 9.1 Hz, 1 H, Hβ), 3.49 (dd, J =
11.8, 2.7 Hz, 1 H, Hβ), 1.86 (s, 1 H, ArCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 191.0, 168.1, 162.5, 158.8, 156.2, 136.7,
136.4, 135.2, 130.2, 129.8 (2 C, split), 128.9, 128.8 (2 C), 126.6,
126.1, 125.3, 118.7, 118.3, 64.2, 53.7, 32.7, 31.6, 19.5 ppm. LRMS:
m/z = 420 [M + H], C24H22NO4S requires 420.
General Procedure for Formylation of 6: Pyridone 6 (1.0 equiv.) was
added to a mixture of Arnold’s reagent (4.0 equiv.) in acetonitrile
(25 mL/mmol 6). (The Arnold’s reagent were either freshly pre-
pared or used as the commercially available salt.) The mixture was
then refluxed for 3 h. If necessary, (judged by LC-MS), additional
Arnold’s reagent was added and the = mixture was further refluxed
until 6 was completely consumed. The solvent was removed under
reduced pressure and the residue was taken up in CH2Cl2 and
washed with saturated NaHCO3(aq.). The aqueous phase was ex-
tracted with CH2Cl2 and the combined organic phases were dried
(R)-Methyl 7-(2-Methoxybenzyl)-6-formyl-2,3-dihydro-8-phenyl-
thiazolo[3,2-a]pyridine-5-one-3-carboxylate (7e): By following the
general procedure for formylation, 244 mg (0.60 mmol) 6e was con-
verted to 227 mg (87%) 7e. In total 18 equiv. of commercially avail-
Eur. J. Org. Chem. 2010, 6171–6178
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6175

F. Almqvist et al.
FULL PAPER
able Arnold’s reagent was used. [α]D = –276 (c = 1.0, CHCl3). IR:
H, CO2CH3), 3.68 (dd, J = 11.5, 8.0 Hz, 1 H, Hβ), 3.49 (dd, J =
11.5, 2.0 Hz, 1 H, Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ =
169.1, 161.6, 137.5, 136.7, 135.7, 134.1, 131.0, 130.7, 130.4, 129.7,
ν
= 1752, 1680, 1641 cm–1. 1H NMR (400 MHz, CDCl3): δ =
˜
max
10.43 (s, 1 H, CHO), 7.31–7.18 (m, 3 H, HAr), 7.11–7.04 (m, 1 H,
HAr), 7.01–6.89 (m, 2 H, HAr), 6.78–6.69 (m, 2 H, HAr), 6.65 (d, J 129.4, 129.2, 129.0, 128.4, 128.3, 127.9, 125.9, 122.8, 121.8, 112.8,
= 8.2 Hz, 1 H, HAr), 5.73 (dd, J = 8.9, 2.9 Hz, 1 H, Hα), 4.26–4.15 62.9, 53.3, 31.6 ppm. HRMS: m/z = 388.1013 [M + H],
(m, 2 H, ArCH2), 3.87 (s, 3 H, CO2CH3), 3.67 (dd, J = 11.8, 8.9 Hz,
1 H, Hβ), 3.55 (s, 3 H, ArOCH3), 3.44 (dd, J = 11.8, 2.9 Hz, 1 H,
Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ = 190.7, 168.0, 162.1,
158.9, 156.7, 156.0, 135.1, 130.0, 129.7, 128.5 (2C, split), 128.4,
127.9, 127.0, 126.7, 120.1, 118.5, 118.2, 109.8, 64.0, 55.0, 53.4, 31.3,
29.1 ppm. LRMS: m/z = 436 [M + H], C24H22NO5S requires 436.
C23H18NO3S requires 388.1007.
(R)-Methyl 2,3-Dihydro-11-methyl-12-phenylbenzo[g]thiazolo[3,2-
b]isoquinoline-5-one-3-carboxylate (8c): By following the general
procedure for synthesis of 8, 63 mg (0.15 mmol) 7c was converted
to 51 mg (85%) 8c. In total, 2 equiv. of TiCl4 and 20 min of heating
were used. [α]D = –215 (c = 1.0, CHCl ). IR: ν
= 1749, 1648
˜
3
max
(R)-Methyl 7-(4-Fluorobenzyl)-6-formyl-2,3-dihydro-8-phenyl- cm–1. H NMR (400 MHz, CDCl3): δ = 8.99 (s, 1 H, 6-H), 8.06–
1
thiazolo[3,2-a]pyridine-5-one-3-carboxylate (7f): By following the
general procedure for formylation, 178 mg (0.45 mmol) 6f was con-
verted to 131 mg (69%) 7f. In total 16 equiv. of commercially avail-
able Arnold’s reagent was used. [α]D = –225 (c = 1.0, CHCl3). IR:
7.96 (m, 2 H, HAr), 7.61–7.54 (m, 1 H, HAr), 7.53–7.36 (m, 5 H,
HAr), 7.22–7.15 (m, 1 H, HAr), 5.85 (dd, J = 8.0, 2.1 Hz, 1 H, Hα),
3.84 (s, 3 H, CO2CH3), 3.59 (dd, J = 11.5, 8.0 Hz, 1 H, Hβ), 3.45
(dd, J = 11.5, 2.1 Hz, 1 H, Hβ), 2.18 (s, 3 H, ArCH3) ppm. 13C
NMR (100 MHz, CDCl3): δ = 169.2, 161.7, 141.9, 139.7, 136.3,
131.8, 131.0, 130.2, 130.0 (2C, split), 129.1, 128.9 (2C, split), 128.6,
ν
= 1752, 1673, 1640 cm–1. 1H NMR (400 MHz, CDCl3): δ =
˜
max
10.45 (s, 1 H, CHO), 7.39–7.27 (m, 3 H, HAr), 7.03–6.98 (m, 1 H,
HAr), 6.96–6.91 (m, 1 H, HAr), 6.82–6.74 (m, 2 H, HAr), 6.71–6.64 128.4, 127.7, 125.6, 124.4, 123.6, 112.7, 62.9, 53.4, 31.3, 18.8 ppm.
(m, 2 H, HAr), 5.73 (dd, J = 9.1, 2.9 Hz, 1 H, Hα), 4.31–4.13 (m, 2
H, ArCH2), 3.86 (s, 3 H, CO2CH3), 3.70 (dd, J = 12.0, 9.1 Hz, 1
H, Hβ), 3.47 (dd, J = 12.0, 2.9 Hz, 1 H, Hβ) ppm. 13C NMR
(100 MHz, CDCl3): δ = 191.4, 168.0, 162.5, 161.3 (d, J = 244.5 Hz),
157.9, 156.6, 135.1, 133.5 (split), 130.5, 130.2, 129.9 (2C, split),
129.0 (2C, split), 128.9, 118.1, 117.9, 114.9 (d, J = 21.3 Hz, 2 C),
64.2, 53.6, 34.2, 31.5 ppm. LRMS: m/z = 424 [M + H],
C23H19FNO4S requires 424.
HRMS: m/z = 402.1170 [M + H], C24H20NO3S requires 402.1164.
(R)-Methyl 2,3-Dihydro-10-methyl-12-phenyl-benzo[g]thiazolo[3,2-
b]isoquinoline-5-one-3-carboxylate (8d): By following the general
procedure for synthesis of 8, 105 mg (0.25 mmol) 7d was converted
to 79 mg (79%) 8d. In total, 2 equiv. of TiCl4 and 20 min of heating
were used. [α]D = –255 (c = 1.0, CHCl ). IR: ν
= 1751, 1655
˜
3
max
cm–1. H NMR (400 MHz, CDCl3): δ = 8.99 (s, 1 H, 6-H), 7.91–
7.84 (m, 1 H, HAr), 7.76 (s, 1 H, 11-H), 7.58–7.43 (m, 5 H, HAr),
7.39–7.30 (m, 2 H, HAr), 5.80 (dd, J = 7.8, 2.1 Hz, 1 H, Hα), 3.84
(s, 3 H, CO2CH3), 3.67 (dd, J = 11.6, 7.8 Hz, 1 H, Hβ), 3.49 (dd,
J = 11.6, 2.1 Hz, 1 H, Hβ), 2.47 (s, 3 H, ArCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 169.1, 161.6, 137.4, 136.8, 135.1, 134.3,
134.0, 131.2, 130.7, 130.3, 130.1, 129.1, 128.9, 128.8, 128.3, 127.8,
125.7, 122.4, 118.4, 113.3, 62.9, 53.3, 31.6, 19.4 ppm. HRMS: m/z
= 402.1170 [M + H], C24H20NO3S requires 402.1164.
1
General Procedure for Synthesis of Multicyclic Pyridones 8a–e: A
pyridone of general structure 7 (1.0 equiv.) was dissolved in CH2Cl2
(10 mL/mmol 7) 1.0  TiCl4 in CH2Cl2 (2.0 mL/mmol 7, 2.0 equiv.)
was added and the solution was heated with microwave irradiation
for 10 (8a) or 20 (8b–e) minutes at 100 °C. Unless full conversion
of 7 was indicated by TLC, more 1.0  TiCl4 in CH2Cl2 was added
and the mixture was heated with microwave irradiation at 100 °C.
The mixture was then quenched with 1  HCl (aq.) and extracted
three times with CH2Cl2. The combined organic phases were dried
with anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified with silica gel chromatography
using heptane/ethyl acetate 2:1Ǟ1:2 as mobile phase. The products
were obtained as yellow foams.
(R)-Methyl 2,3-Dihydro-10-methoxy-12-phenylbenzo[g]thiazolo[3,2-
b]isoquinoline-5-one-3-carboxylate (8e): By following the general
procedure for synthesis of 8, 109 mg (0.25 mmol) 7e was converted
to 67 mg (64%) 8e. In total, 8 equiv. of TiCl4 and 70 min of heating
were used. [α]D = –269 (c = 1.0, CHCl ). IR: ν
= 1744, 1658
˜
3
max
cm–1. 1H NMR (400 MHz, CDCl3): δ = 8.96 (s, 1 H, 6-H), 8.01 (s,
1 H, 11-H), 7.59 (d, J = 8.3 Hz, 1 H, HAr), 7.57–7.43 (m, 5 H,
HAr), 7.39–7.32 (m, 1 H, HAr), 6.79 (d, J = 7.6 Hz, 1 H, HAr), 5.78
(dd, J = 8.0, 2.0 Hz, 1 H, Hα), 3.88 (s, 3 H, CH3), 3.83 (s, 3 H,
CH3), 3.65 (dd, J = 11.5, 8.0 Hz, 1 H, Hβ), 3.47 (dd, J = 11.5,
2.0 Hz, 1 H, Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ = 169.1,
161.6, 155.3, 137.2, 136.9, 133.6, 132.0, 130.7, 130.4, 129.1 (2C,
split), 129.0, 128.5, 128.2, 126.0, 123.1, 121.5, 116.6, 113.4, 105.3,
63.0, 55.6, 53.3, 31.6 ppm. HRMS: m/z = 418.1119 [M + H],
C24H20NO4S requires 418.1113.
(R)-Methyl 10,11-Dihydro-13-phenylnaphtho[1,2-g]thiazolo[3,2-b]-
isoquinoline-8-one-10-carboxylate (8a): By following the general
procedure for synthesis of 8, 124 mg (0.27 mmol) 7a was converted
to 95 mg (79%) 8a. In total, 2 equiv. of TiCl4 and 10 min of heating
were used. [α]D = –88 (c = 1.0, CHCl ). IR: νmax = 1745, 1652 cm–1.
˜
3
1H NMR (400 MHz, CDCl3): δ = 9.95–8.90 (m, 1 H, 7-H), 8.44 (s,
1 H, 14-H), 8.29 (d, J = 8.3 Hz, 1 H, HAr), 7.83–7.76 (m, 2 H,
HAr), 7.64 (d, J = 8.9 Hz, 1 H, HAr), 7.61–7.46 (m, 7 H, HAr), 5.84–
5.78 (m, 1 H, Hα), 3.85 (s, 3 H, CO2CH3), 3.72–3.64 (m, 1 H, Hβ),
3.53–3.47 (m, 1 H, Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ =
169.0, 161.3, 138.3, 136.6, 135.0 (split), 133.6, 133.1, 130.8, 130.4,
129.8, 129.7, 129.3, 129.1, 129.0, 128.7, 128.4, 127.9, 127.3, 127.2,
126.8, 123.4, 122.6, 117.1, 113.1, 63.1, 53.3, 31.6 ppm. HRMS: m/z
= 438.1170 [M + H], C27H20NO3S requires 438.1164.
(R)-Methyl 8-Fluoro-2,3-dihydro-12-phenylbenzo[g]thiazolo[3,2-b]-
isoquinoline-5-one-3-carboxylate (8f): 64 mg (0.15 mmol) 7f was dis-
solved in 1.5 mL of CH2Cl2, 1.5 mL of 1.0  TiCl4 in CH2Cl2
(1.5 mmol) was added and the mixture was heated with microwave
irradiation for 2 h at 160 °C. The reaction was quenched with 1 
(R)-Methyl 2,3-Dihydro-12-phenylbenzo[g]thiazolo[3,2-b]isoquinol- HCl (aq.) and extracted three times with CH2Cl2. The combined
ine-5-one-3-carboxylate (8b): By following the general procedure for
synthesis of 8, 61 mg (0.15 mmol) 7b was converted to 52 mg (89%)
8b. In total, 5 equiv. of TiCl4 and 50 min of heating were used.
organic phases were dried with anhydrous Na2SO4, filtered and
concentrated under reduced pressure. As LC-MS indicated signifi-
cant hydrolysis of the methyl ester of 8f, the residue was dissolved
[α]D = –291 (c = 1.0, CHCl ). IR: νmax = 1749, 1654 cm–1. 1H NMR in 3.2 mL of methanol and 0.56 mL (4.4 mmol) chlorotrimethylsil-
˜
3
(400 MHz, CDCl3): δ = 9.02 (s, 1 H, 6-H), 8.01 (d, J = 8.1 Hz, 1
H, HAr), 7.73 (d, J = 8.2 Hz, 1 H, HAr), 7.62 (s, 1 H, 11-H), 7.57– and then concentrated under reduced pressure. The residue was
7.39 (m, 7 H, HAr), 5.80 (dd, J = 8.0, 2.0 Hz, 1 H, Hα), 3.84 (s, 3 purified with silica gel chromatography using heptane/ethyl acetate
ane was added. The mixture was stirred overnight at room temp.
6176
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6171–6178

Multi Ring-Fused 2-Pyridone-Based Fluorescent Scaffold
2:1Ǟ1:2 as mobile phase. 31 mg (51%) 8f was obtained as yellow
(1:1000 in BSA-PBS). To visualize α-tubulin, a secondary anti-
mouse Rhodamine (TRITC)-conjugated antibody (diluted 1:500 in
BSA-PBS) was used (incubated at 37 °C for 1 h). Thereafter, cells
were stained with compound 8b (100 µ compound and 0.5 %
DMSO in PBS) at 37 °C for 1 h and subsequently washed twice in
foam. [α]D = –110 (c = 1.0, CHCl ). IR: ν
= 1749, 1655 cm–1.
max
˜
3
1H NMR (400 MHz, CDCl3): δ = 8.94 (s, 1 H, 6-H), 7.76–7.70 (m,
1 H, HAr), 7.65–7.40 (m, 6 H, HAr), 7.62 (s, 1 H, 11-H), 7.33–7.26
(m, 1 H, HAr), 5.79 (dd, J = 7.9, 2.2 Hz, 1 H, Hα), 3.84 (s, 3 H,
CO2CH3), 3.72–3.64 (m, 1 H, Hβ), 3.50 (dd, J = 11.6, 2.2 Hz, 1 H, PBS.
Hβ) ppm. 13C NMR (100 MHz, CDCl3): δ = 169.0, 161.3, 160.5
Toxicity Evaluation: The fluorometric microculture cytotoxicity as-
(d, J = 247.8 Hz), 137.5, 136.6, 133.7 (split), 132.9, 131.5 (d, J =
9.4 Hz), 130.8, 130.6 (d, J = 8.8 Hz), 130.4, 129.3, 129.1, 128.8 (d,
J = 6.3 Hz), 128.5, 123.6, 123.6, 122.1, 119.5 (d, J = 26.3 Hz),
112.7, 111.8 (d, J = 20.5 Hz), 63.0, 53.4, 31.6 ppm. HRMS: m/z =
406.0921 [M + H], C23H17FNO3S requires 406.0913.
say (FMCA) is a total cell kill assay, based on the ability of cells
with intact cell membranes to convert non-fluorescent diacetate
(FDA) to fluorescent fluorescein. The details of the method has
been presented previously,[18] and briefly, cell suspensions are ex-
posed to varying concentrations of the test substance (in dupli-
cates) in 96-well microtiter plates for 72 h whereafter the cells are
washed and FDA is added. Fluorescence is measured after 30 min
incubation. The fluorescence is proportional to the number of liv-
ing cells, and results presented as survival index (fraction of surviv-
ing cells vs. untreated control wells). Two different human cell lines
were used, the lymphoma cell line U-937 GTB and the T-cell leuke-
mia cell line CCRF-CEM, both of which are considered generally
“sensitive” to xenobiotics. In addition normal peripheral blood
mononuclear cells (PBMC) from two healthy donors were included
in the analysis.
Fluorescence Quantum Yield Determination: The fluorescence quan-
tum yields (Φ) were calculated by comparison with a reference (ref)
substance,[1] by means of the equation given below.
Φ = Φrefn2F{1 – exp[–Aref(λex)ln10]}/nref Fref{1 – exp[–A(λex)ln10]}
2
F and Fref are the integrated fluorescence spectra of the unknown
and the reference sample, respectively. A and Aref denote the corre-
sponding absorbance at the excitation wavelength (λex), and n is
the refractive index for the media in which the substances are dis-
solved. The used refractive indices were 1.47 (glycerol), 1.43 (1,2-
propanediol) and 1.42 (dichloromethane). Compound 11 (Figure 6)
dissolved in chloroform was used as fluorescence reference sam-
ple.[13]
Supporting Information (see also the footnote on the first page of
this article): NMR spectra for all new compounds and data from
the evaluation of compound 8b in a microtubule polymerization
assay.
Acknowledgments
We thank the Swedish Natural Research Council, the Knut and
Alice Wallenberg Foundation, and JC Kempe Foundation
(SJCKMS) for financial support. The work was performed within
the Umeå Center for Microbial Research (UCMR).
[1] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2006,
Springer, Singapore.
[2] R. Misra, R. C. Pandey, J. V. Silverton, J. Am. Chem. Soc.
1982, 104, 4478–4479.
Figure 6. Structure of the fluorescence reference compound 11.
[3] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T.
McPhail, G. A. Sim, J. Am. Chem. Soc. 1966, 88, 3888–3890.
[4] a) M. V. Kulkarni, G. M. Kulkarni, C. H. Lin, C. M. Sun, Curr.
Med. Chem. 2006, 13, 2795–2818; b) G. A. Strohmeier,
W. M. F. Fabian, G. Uray, Helv. Chim. Acta 2004, 87, 215–226.
[5] B. M. Aveline, S. Matsugo, R. W. Redmond, J. Am. Chem. Soc.
1997, 119, 11785–11795.
Photo Stability Measurement: The photo stability of compounds
8a and 8b were investigated when dissolved in chloroform. The
samples were exposed to approximately 770 mW/cm2, by means of
a high intensity Xe-light source. A water-cooled long pass filter was
used to selectively remove all wavelengths below 330 nm. In order
to prevent evaporation, a water-cooled cuvette holder was used and
quartz lenses were implemented to focus the light into the cuvette.
[6] R. A. Henry, C. A. Heller, D. W. Moore, J. Org. Chem. 1975,
40, 1760–1766.
Fluorescence Confocal Microscopy of HeLa-cells Treated with Com-
pound 8b: HeLa cells (DSMZ) were grown in RPMI supplemented
with 10% fetal calf serum (PromoCell), 20 m HEPES and 2 m
-glutamine. The incubations were performed at 37 °C in the pres-
ence of 5% CO2. HeLa cells were seeded on glass cover slips (Ø =
12 mm) in 24-wells tissue culture plate and incubated for 24 h prior
to addition of compound.
[7] a) H. Emtenäs, L. Alderin, F. Almqvist, J. Org. Chem. 2001,
66, 6756–6761; b) H. Emtenäs, K. Åhlin, J. S. Pinkner, S. J.
Hultgren, F. Almqvist, J. Comb. Chem. 2002, 4, 630–639; c) H.
Emtenäs, C. Taflin, F. Almqvist, Mol. Div. 2003, 7, 165–169;
d) E. Chorell, S. Edvinsson, F. Almqvist, Tetrahedron Lett.
2010, 51, 2461–2463.
[8] a) V. Åberg, F. Almqvist, Org. Biomol. Chem. 2007, 5, 1827–
1834; b) J. S. Pinkner, H. Remaut, F. Buelens, E. Miller, V.
Åberg, N. Pemberton, M. Hedenström, A. Larsson, P. Seed,
G. Waksman, D. J. Hultgren, F. Almqvist, Proc. Natl. Acad.
Sci. USA 2006, 103, 17897–17902.
[9] V. Åberg, F. Norman, E. Chorell, A. Westermark, A. Olofsson,
A. E. Sauer-Eriksson, F. Almqvist, Org. Biomol. Chem. 2005,
3, 2817–2823.
[10] L. Cegelski, J. S. Pinkner, N. D. Hammer, C. K. Cusumano,
C. S. Hung, E. Chorell, V. Åberg, J. N. Walker, P. C. Seed, F.
Almqvist, M. R. Chapman, S. J. Hultgren, Nat. Chem. Biol.
2009, 5, 913–919.
HeLa-cells Incubated in Presence of 8b: Medium supplemented with
compound 8b (100 µ compound in 0.5% DMSO), or only 0.5%
DMSO, were added to the cells. After 20 h at 37 °C, the cells were
washed twice with PBS and fixed in methanol for 5 min. For fluo-
rescence confocal microscopy, DMSO treated cells were used to set
the background.
Cross-Staining of α-Tubulin: HeLa cells were grown in the absence
of compound 8b and fixed as described above. α-Tubulin staining
was performed overnight (4 °C) with a monoclonal antibody
Eur. J. Org. Chem. 2010, 6171–6178
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6177

F. Almqvist et al.
FULL PAPER
[11] V. Åberg, M. Sellstedt, M. Hedenström, J. S. Pinkner, S. J.
Hultgren, F. Almqvist, Bioorg. Med. Chem. 2006, 14, 7563–
7581.
[12] M. R. Tsai, T. C. Hung, B. F. Chen, C. C. Cheng, N. C. Chang,
Tetrahedron 2004, 60, 10637–10644.
[15] G. Liminga, P. Nygren, S. Dhar, K. Nilsson, R. Larsson, Anti-
cancer Drugs 1995, 6, 578–585.
[16] K. Levenberg, Quart. Appl. Math. 1944, 2, 164.
[17] U. S. Sørensen, E. Falch, P. J. Krogsgaard-Larsen, J. Org.
Chem. 2000, 65, 1003–1007.
[13] S. Kalinin, M. Speckbacher, H. Langhals, L. B.-Å. Johansson,
Phys. Chem. Chem. Phys. 2001, 3, 172–174.
[14] D. Marushchak, S. Kalinin, I. Mikhalyov, N. Gretskaya, L. B.-
Å. Johansson, Spectrochim. Acta A 2006, 65, 113–122.
[18] E. Lindhagen, P. Nygren, R. Larsson, Nat. Protoc. 2008, 3,
1364–1369.
Received: June 3, 2010
Published Online: September 24, 2010
6178
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6171–6178