New fluorescent trans-dihydrofluoren-3-ones from aldol-Robinson annulation-regioselective addition involved one-pot reaction

Article abstract of DOI:10.1039/c0ob00401d

An unexpected discovery of new trans-4-acetyl-1,9-dimethyl-4,4a-dihydro-3H- fluoren-3-ones from one pot reactions of benzaldehydes and acetylacetone is described. The synthetic mechanism and stereochemistry were discussed. These new derivatives exhibit go

Full text of DOI:10.1039/c0ob00401d

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Organic &
Biomolecular
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Volume 8 | Number 22 | 21 November 2010 | Pages 5021–5248
ISSN 1477-0520
COMMUNICATION
Huo et al.
New fluorescent trans-dihydrofluoren-3-ones from aldol–Robinson
annulation–regioselective addition involved one-pot reaction
1477-0520(2010)8:22;1-Z

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COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
New fluorescent trans-dihydrofluoren-3-ones from aldol–Robinson
annulation–regioselective addition involved one-pot reaction†
Yingpeng Huo, Xu Qiu, Weiyan Shao, Jianing Huang, Yanjun Yu, Yinglin Zuo, Linkun An, Jun Du and
Xianzhang Bu*
Received 8th July 2010, Accepted 10th August 2010
DOI: 10.1039/c0ob00401d
An unexpected discovery of new trans-4-acetyl-1,9-dimethyl-
4,4a-dihydro-3H-fluoren-3-ones from one pot reactions of
benzaldehydes and acetylacetone is described. The synthetic
mechanism and stereochemistry were discussed. These new
derivatives exhibit good fluorescent properties in solutions.
12.80 Hz, 1H) respectively, both of which were further proven to be
adjacent by COSY analysis. In the HMBC spectrum, the signal at
dH 3.32 ppm was found to have correlation to two carbonyl carbons
signals at dC 207.2 and 196.3 ppm respectively. The signal at dC
196.3 ppm showed close correlation with a double bond hydrogen
at dH 5.73 ppm, 1H while the signal at dC 207.2 showed correlation
with a methyl group at dH 2.42 ppm (s, 3H) and suggesting an acetyl
group nearby (Fig. 1).
Fluorescent probes have been of great interest to chemists because
of their wide usage as biological imaging and chemical sensing
reagents1 for various purposes such as chemical species analysis,2
cellular process monitoring3 and tissue visualization.4 Fluorene
derivatives such as 9-fluorenones, which exhibit an extensive
aromatic p system, have attracted much attention owning to their
antiviral and interferon inducing ability5 as well as antitumor
potency;6 besides, 9-fluorenones exhibited excellent fluorescence
properties7 and could be used as fluorescent probes for detecting
amino acids,8 hydrogen binding interactions in cyclodextrin9 and
Ca2+ ions,10 etc. The skeleton of 9-fluorenones could be formed
by intramolecular arylation of biphenyl-2-carbaldehyde11 or 2-
biphenyl carboxylic acid,12 and other cyclization processes.13
Despite all the investigations on synthetic mothedologies and
various properties of 9-fluorenones, the non-extensive aromatic
p system derivatives such as dihydrofluorenones, had rarely been
investigated especially on their fluorescence. We herein describe
an unexpected discovery of a new series of substituted trans-4,4a-
dihydro-3H-fluoren-3-ones as novel fluorophores from one pot
reactions of readily available starting materials.
In an exploration to the synthesis of 3-benzylidenepentane-2,4-
dione derivatives, which acted as intermediates in our ongoing
project on novel curcumin analogues for tumor chemopreventive
agents screening, 2 eq concentrated sulfuric acid was adopted as
the catalyst in the reaction of 3-ethoxy-4-hydroxybenzaldehyde
(1a) and excess actylacetone in cold glacial acetic acid. After
3 h stirring, thin-layer chromatography (TLC) revealed a product
with apparent green-to-yellow fluorescence. This product was
isolated and purified for structural characterization. The MS
and elemental analysis of this product indicated a formula
of C19H20O4, which differed from the targeted 3-(3-ethoxy-4-
hydroxybenzylidene) pentane-2,4-dione (C14H16O4). In the 1H
NMR spectrum, two saturated hydrogen atoms were found at
dH 4.32 ppm (dd, J = 12.79, 1.38 Hz, 1H) and 3.32 ppm (d, J =
Fig. 1 H–C correlations of the product in HMBC.
Meanwhile, we found the signal at dH 5.77 ppm was correlated
with another methyl group at dH 2.37 (d, J = 1.17 Hz, 3H) in the
H–H COSY spectrum, which meant that a structural fragment of
–CH C(CH3)– existed.14 It is obvious that a methyl and acetyl
substituted cyclohex-2-enone moiety presented based on above
analysis. On the other hand, only two independent Ar–H were
found at dH 7.00 (s, 1H) and 6.74 (s, 1H) ppm, implying that
the third Ar–H of 1a had been substituted. Furthermore, signal
at dH 4.32 ppm was found to exhibit an additional correlation
with an aromatic carbon at 140.2 ppm. Combining all of the
informations, the product was finally identified as 4-acetyl-
6-ethoxy-7-hydroxy-1,9-dimethyl-4,4a-dihydro-3H-fluoren-3-one
(2a) (Table 1, Scheme 1).
Scheme 1
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou,
510006, China. E-mail: phsbxzh@mail.sysu.edu.cn; Fax: 8620-39943054;
Tel: 8620-39943054
† Electronic supplementary information (ESI) available: Synthesis, char-
acterization data of new compounds, stereochemistry discussion and the
crystallographic data for 2b. CCDC reference number 762211. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0ob00401d
The revealed structure has two chiral carbons present in 4a and
4 position respectively; according to the Karplus equation (J =
7cosq + 5cos2q,15 see the ESI†), the dihedral angel q(H-C4a-C4-H)
can be calculated from coupling constant between the H4 and
H4a signals. We therefore caculated the q(H–C4a–C4–H) of 2a, the
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Table 1 Structure information, yields, UV/vis and fluorescence data for compounds 2a–ia
H–C4a–C4–H
Fluorescence
Dihedral UV/vis
Stoke’s
shift/nm
Entry Benzaldehyde R1
R2
R3
H
Compd Yield (%) M.P./◦C
J/Hz angle qi
Ex/nm
l
l
Em/nm fe
1
2
1a
1b
OH
H
OC2H5
OCH3 OCH3 2b
2a
15
20
177.5–180.0 12.80 ª180◦
404
360
360
381
382
398
401
398
396
392
396
392
542
481
484
516
497
534
539
533
531
536
532
537
0.036 138
165.0–166.2 11.79 162◦
0.20f 121
0.26g 124
0.34 135
0.77h 115
0.18 136
0.033 138
0.12 135
0.22 135
0.065 142
0.15 136
0.16 145
3
4
5
6
7
8
9
1c
OC2H5
OH
OCH3
OCH2CH CH2 OCH3
OCH(CH3)2 OCH3
OCH2CH CH2 OC2H5
OCH(CH3)2 OC2H5
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
2c
2d
2e
2f
2g
2h
2i
16
18
15
18
15
11
14
157.2–160.4 12.79 ª180◦
163.9–165.5 12.87 ª180◦
159.2–161.4 12.79 ª180◦
131.8–133.2 12.79 ª180◦
138.9–140.8 12.81 ª180◦
126.9–130.4 12.80 ª180◦
101.0–103.7 12.82 ª180◦
1d
1e
1fa
1gb
1hc
1id
a For synthesis details and structure characterization data, please see the ESI;† a,c: aldehydes 1f and 1h were prepared from allylation of 1d and 1a
respectively; b,d: aldehydes 1f and 1h were prepared by isopropylation of 1d and 1a respectively; e: determined using quinine sulfate in 1 N H2SO4 as
a standard (1 mmol L-1), and compounds 2a–i were dissolved in EtOH as 1 mmol L-1 solution except when mentioned alternatively; f : in acetone; g: in
CH2Cl2; h; in DMSO; i: calculated based on Karplus equation.
determined dihedral angel is about 180◦ (Table 1), indicating a
trans-conformation of 2a.
other benzaldehydes failed during the synthesis (data not shown),
indicating the electron density of the phenyl ring played a critical
role during the final production formation. Based on this infor-
mation and the structure of products, the synthetic mechanism
was proposed as Scheme 2. In this procedure, an aldol reaction
followed by Robinson annulation occurred, and the final products
were formed through a carbonyl-selective intramolecular aromatic
electrophilic addition–elimination process, the regioselectivity was
achieved probably owning to the possible hydrogen bond between
the enol form of the b-diketone moiety during the aromatic
electrophilic addition process.
It should be pointed out that though the reaction takes place
in an achiral environment, only the trans products were found
in current study. We reasoned that this phenomenon would
result from the low-level energy state of reaction intermediate
during the Robinson annulation step, the cyclohexanone-like
intermediate presented a chair form during the attacking; the
energy and bulk effect would be lower when large groups were
at e-position, resulting in the trans conformation between H4 and
H4a (Scheme 2).
The above finding indicated that the skeleton of trans-4,4a-
dihydro-3H-fluoren-3-one formed in an acceptable isolated yield
from readily available starting materials. Though in low yields,
the product 2a can be easily distinguished and purified by
column chromatography from the reaction mixture, which is
mainly constituted of unreacted starting materials and polymer
by-products; moreover, we found 2a showed strong fluorescence in
solution. We therefore adopted this procedure for more derivatives
synthesis. Using various benzaldehydes instead of 1a, another 8
new compounds were obtained successfully in 11–20% isolated
yields, structure characterization indicated all of them have the
same skeleton as 2a (Table 1, Scheme 1 2b–i), the dihedral angel
q(H-C4a-C4-H) calculation also revealed a trans-conformation of 2b–i
(Table 1); besides, the skeleton of trans-4,4a-dihydro-3H-fluoren-
3-one was further supported by the X-ray data of 2b (Fig. 2).16
To further verify the mechanism, we prepared the interme-
diate 1d¢ (R1 = OH, R2 = OCH3 R3 = H) and the Robinson
annulation product 2b¢ (R1 = H, R2 = R3 = OCH3) (Scheme 2)
respectively. Both 1d¢ and 2b¢ cannot be obtained in the current
acid catalysis condition and therefore were synthesized under
piperidine catalysis. Noticeably, the intermediate 2b¢ would be
trans- and cis-isomer mixture in the base catalysis condition.17
After that, the acetylacetone was mixed with 1d¢ under the same
condition as in the synthesis of 2d (Scheme 3). The second reaction
started from the intermediate 2b¢, which was treated directly
with acetic acid and 2 eq concentrated sulfuric acid. Expectedly,
intermediates 1d¢ resulted in the target product 2d with higher
yield (45%); in the case of intermediate 2b¢, the cis- and trans-
forms of 4-acetyl-5,6-dimethoxy-1,9-dimethyl-4,4a-dihydro-3H-
fluoren-3-one (2b-cis and 2b) were obtained in 28% and 16% yield
respectively (total yield, 44%). NMR revealed 6.11 Hz of JH4a-H4 in
2b-cis, the q(H–C4a-C4–H) of 2b-cis was therefore calculated to be
46◦, which match the cis H-C4a-C4-H form in 2b-cis. Regardless
Fig. 2 X-Ray structure of 2b (CCDC no. 762211).
Noticeably, we found only benzaldehydes containing electron
donating R2 could transformed into the final products 2 while
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Scheme 2 Proposed mechanism of 2a–i synthesis.
of the other ones, indicating that the hydroxyl group at the 7
position would decrease fluorescence.
Remarkably, the 5,6-dimethyoxyl derivative 2b showed the
strongest fluorescence intensity and highest quantum yield in
ethanol. The quantum yield of 2b in various solutions (Fig. 4) were
therefore measured and the result showed that U of 2b depended
on the polarity of solvent (U = 0.77 in DMSO, and 0.20, 0.26, 0.34
in acetone, CH2Cl2 and ethanol respectively, Table 1).
Furthermore, we investigated preliminarily the distribution of
2b in lung cancer cell line A549 by laser confocal microscopy
imaging. The result indicates that 2b distributed in the cytoplasm
equably but kept out of the nucleus (Fig. 5). These results suggested
the structure of 2b might be an useful model for novel fluorescent
probes discovery based on dihydrofluoren-3-one.
Scheme 3 Synthesis of 2b, 2b-cis and 2d from intermediates.
In summary, we found 4,4a-dihydro-3H-fluoren-3-one deriva-
tives can be synthesized under simple conditions with readily avail-
able starting materials and 9 new analogues were obtained success-
fully in acceptable isolated yield. The mechanism was proposed;
to the best of our knowledge, only Anastassiou et al. reported
the preparation of single compound 4,4a-dihydro-3H-fluoren-
3-one by acid catalyzed pericyclization of benzo[9]annulenone
in 198119 and no further investigation on this skeleton was
found ever since. Compared with the reported method which
needed expensive starting material, long synthetic steps and low
diversity, our finding afforded a simple, economical procedure to
of the 2b-cis obtained from a racemic mixture 2b¢, the obtained
results provided evidences to the mechanism shown in Scheme 2.
We next investigated the fluorescence properties of 2a–i. The
results are summarized in Table 1. In ethanol, lEx and lEm of
2a, 2c–i were all round 395 nm and 530 nm respectively, while
those of 2b were slightly different, with lEx = 381 nm and lEm
=
516 nm (Fig. 3). The Stoke’s shifts of all compounds were as
large as 130 nm approximately, showing a broad emission peak
(see Fig. 3). Among all compounds, the fluorescence intensity and
quantum yield18 of 2a and 2d were comparatively lower than those
Fig. 3 UV-vis (left) and emission (right) spectrum of 10 mmol L-1 2a–i in ethanol.
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Fig. 4 UV-vis (left) and emission (right) spectrum of 1 mmol L-1 2b in different solvents.
Fig. 5 Confocal imaging of 2b treated A549 cells. a, without 2b; b, with 0.5 mM 2b, 2 h; c, with 0.5 mM 2b, 6 h.
generate more 4-acetyl-1,9-dimethyl-4,4a-dihydro-3H-fluoren-3-
one diversities though the final yields are relative low. Remarkably,
we found the obtained compounds 2a–i, especially 2b, exhibit
good fluorescence properties. The high fluorescence quantum
yield, large Stoke’s shift and lack of ionic charge for fluorescence
emission of them predicated the significance of our finding in
searching for novel fluorophores based on dihydrofluoren-3-one,
distinguishing from the well studied 9-fluorenone with extensive
p-aromatic system, and such investigations are now in progress in
our group.
30973619) for financial support. We also thanks Prof. Xiao Peng
Hu of Sun Yat–Sen University for his kind help in X-ray data
analysis.
Notes and references
1 (a) M. S. T. Gonc¸alves, Chem. Rev., 2009, 109, 190–212; (b) M.
Leopoldo, E. Lacivita, F. Berardi and R. Perrone, Drug Discovery
Today, 2009, 14(13–14), 706–712; (c) M. Ferna´ndez-Sua´rez and A. Y.
Ting, Nat. Rev. Mol. Cell Biol., 2008, 9(12), 929–943.
2 (a) N. Inoue, Y. Suzuki, K. Yokoyama and I. Karube, Biosci., Biotech-
nol., Biochem., 2009, 73(5), 1215–1217; (b) Z. Cao, P. Nandhikonda
and M. D. Heagy, J. Org. Chem., 2009, 74, 3544–3546.
3 (a) T. Kihara, C. Nakamura, M. Suzuki, S.-W. Han, K. Fukazawa, K.
Ishihara and Miyake, J. Biosens. Bioelectron., 2009, 25, 22–27; (b) S.
Fakih, M. Podinovskaia, X.-L. Kong, U. E. Schaible, H. L. Collins and
R. C. Hider, J. Pharm. Sci., 2009, 98(6), 2212–2226.
Acknowledgements
We are indebted to the National High-tech R&D 863 Program
(No. 2008AA02Z304), and National Science Fund of China (No.
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The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 5048–5052 | 5051
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4 (a) Y. Urano, Anal. Sci., 2008, 24, 51–53; (b) M. Sawa, T.-L. Hsu, T.
Itoh, M. Sugiyama, S. R. Hanson, P. K. Vogt and C.-H. Wong, Proc.
Natl. Acad. Sci. U. S. A., 2006, 103(33), 12371–12376.
5 (a) S. A. Lyakhov, E. A. Lyakhova, A. S. Karpenko, G. V. Mal’tsev, I. V.
Vel’cheva, L. A. Litvinova, M. N. Lebedyuk, G. A. Khorokhorina and
V. P. Fedchuk, Pharm. Chem. J., 2004, 38(3), 128–131; (b) S. Alcaro,
A. Artese, J. N. Iley, R. Maccari, S. Missailidis, F. Ortuso, R. Ottana`,
P. Ragazzon and M. G. Vigorita, Bioorg. Med. Chem. Lett., 2007, 17,
2509–2514.
12 (a) D. Tilly, S. S. Samanta, D. Asish, A. Castanet and J. Mortier, Org.
Lett., 2005, 7, 827–830; (b) D. Tilly, S. S. Samanta, F. Faigl and J.
Mortier, Tetrahedron Lett., 2002, 43, 8347–8350.
13 (a) X.-X. Zhang and R. C. Larock, Org. Lett., 2005, 7, 3973–3976; (b) J.
Zhao, D.-W. Yue, M. A. Campo and R. C. Larock, J. Am. Chem. Soc.,
2007, 129, 5288–5295; (c) V. S. Thirunavukkarasu, K. Parthasarathy
and C.-H. Cheng, Angew. Chem., Int. Ed., 2008, 47, 9462–9465.
14 N. E. Jacobsen, University of Arizona, NMR Spectroscopy Explained-
Simplified Theory, Applications and Examples for Organic Chemistry
and Structural Biology, John Wiley & Sons, Inc., Hoboken, New Jersey,
chapter 9.
15 N. E. Jacobsen, University of Arizona, NMR Spectroscopy Explained-
Simplified Theory, Applications and Examples for Organic Chemistry
and Structural Biology, John Wiley & Sons, Inc., Hoboken, New Jersey,
chapter 2.
6 N. S. Youssef, E. A. El Zahany, M. M. Anwar and S. A.
Hassan, Phosphorus, Sulfur Silicon Relat. Elem., 2009, 184, 103–
125.
´
7 (a) J. R. Heldt, J. Heldt, M. Jo´zefowicz and J. Kamihski1, J. Fluoresc.,
2001, 11(1), 65–73; (b) H.-X. Shao, X.-P Chen, Z.-X Wang and P. Lu,
J. Lumin., 2007, 127, 349–354.
8 T.-J. Hsieh and S. Chen, Amino Acids, 2007, 33, 97–104.
9 L. J. Biczok, J. Inclusion Phenom. Mol. Recognit. Chem., 1994, 18,
237–245.
16 CCDC reference number 762211.
17 A. Sharma, J. Pandey and R. P. Tripathi, Tetrahedron Lett., 2009,
50(16), 1812–1816.
10 N. T. Player, S. Shinoda and H. Tsukube, Org. Biomol. Chem., 2005, 3,
18 Franklyn G. Prendergast, Michael Meyer, Gerald L. Carlson, Shozo
Iida and Jame D. Potter, J. Biol. Chem., 1983, 258(12), 7541–7544.
19 A. G. Anastassiou, H. S. Kasmai and M. R. Saadein, Helv. Chim. Acta,
1981, 64(2), 617–619.
1615–1616.
11 J. Barluenga, M. Trincado, E. Rubio and Jose M. Gonzalez, Angew.
Chem., Int. Ed., 2006, 45, 3140–3143.
5052 | Org. Biomol. Chem., 2010, 8, 5048–5052
This journal is
The Royal Society of Chemistry 2010
©