Structural development of canthin-5,6-dione moiety as a fluorescent dye and its application to novel fluorescent sensors

Article abstract of DOI:10.1016/j.tet.2016.08.014

Canthin-5,6-dione is a common structure of the fluorescent natural products amarastelline A and nigakinone. Here, we synthesized various derivatives, and studied their fluorescence properties. The effects of substituent groups at the N³-, 4- and 10-positions were clarified. N³-Substitution influenced the solvent-dependent fluorescent change by modulating tautomerization between canthin-5,6-dione and the 5-hydroxy-6-one form. 10-Substitution also influenced the fluorescence, especially in aqueous solution, in combination with N³-substitution. Among the synthesized compounds, 5 showed an ‘OFF-ON-OFF’-type fluorescence change with increase of pH, and therefore served as a novel fluorescent sensor for a specific range of pH. Our findings suggest that canthin-5,6-dione should be useful as a fluorophore moiety for fluorescent labeling of biomolecules and for development of fluorescent probes and sensors.

Full text of DOI:10.1016/j.tet.2016.08.014

Accepted Manuscript
Structural development of canthin-5,6-dione moiety as a fluorescent dye and its
application to novel fluorescent sensors
Hidetomo Yokoo, Ayumi Ohsaki, Hiroyuki Kagechika, Tomoya Hirano
PII:
S0040-4020(16)30772-4
10.1016/j.tet.2016.08.014
TET 27995
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 30 June 2016
Revised Date: 26 July 2016
Accepted Date: 4 August 2016
Please cite this article as: Yokoo H, Ohsaki A, Kagechika H, Hirano T, Structural development of
canthin-5,6-dione moiety as a fluorescent dye and its application to novel fluorescent sensors,
Tetrahedron (2016), doi: 10.1016/j.tet.2016.08.014.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structural development of canthin-5,6-dione moiety as a fluorescent dye and its
application to novel fluorescent sensors
Hidetomo Yokoo,a Ayumi Ohsaki,b Hiroyuki Kagechika,a Tomoya Hiranoa,*
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10
Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
bCollege of Humanities and Sciences, Nihon University, 3–25–40 Sakura–Josui, Setagaya-ku, Tokyo 156-8550,
Japan
*Corresponding author. Tel.: +81-3-5280-8128; fax: +81-3-5280-8127; e-mail: hira.chem@tmd.ac.jp
Supplementary Material
Supplementary data (absorption and fluorescence spectra of some compounds) associated with this article is
available and can be found in the online version at http://xxxxx.
Keywords
Fluorescence; Canthin-5,6-dione; Sensor; pH
Abstract
Canthin-5,6-dione is a common structure of the fluorescent natural products amarastelline A and nigakinone.
Here, we synthesized various derivatives, and studied their fluorescence properties. The effects of substituent
groups at the N3-, 4- and 10-positions were clarified. N3-Substitution influenced the solvent-dependent fluorescent
change by modulating tautomerization between canthin-5,6-dione and the 5-hydroxy-6-one form. 10-Substitution
also influenced the fluorescence, especially in aqueous solution, in combination with N3-substitution. Among the
synthesized compounds, 5 showed an “OFF-ON-OFF”-type fluorescence change with increase of pH, and
therefore served as a novel fluorescent sensor for a specific range of pH. Our findings suggest that
canthin-5,6-dione should be useful as a fluorophore moiety for fluorescent labelling of biomolecules and for
development of fluorescent probes and sensors.
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1. Introduction
Fluorescent dyes have been widely employed for analytical and industrial purposes. They have also been
utilized to develop probes and sensors whose fluorescence properties are changed by binding to or reacting with
specific molecular species, including enzymes, or in response to changes of microenvironment,1 such as pH2 and
viscosity.3 In particular, small molecule-based fluorescent molecules are practically useful, and a number of
well-established fluorophores have been reported.4 However, for example, in the case of multi-color imaging
applications, probes emitting in distinct wavelength regions are needed, and therefore it is important to expand the
range of available fluorophores. For this purpose, our group has constructed a library of fluorescent compounds
obtained by organic synthesis or by isolation of natural products.5 Amarastelline A was isolated as a novel
fluorescent natural product from the bark of Quassia amara.6 It has strong fluorescence and could stain cytoplasm
of living cells (Fig. 1). We also isolated and identified the known compound, named as Nigakinone (1),7 from the
same plant, and found the solvent-dependent fluorescence properties. We considered that these compounds might
be useful as novel fluorophores. Therefore, in the present work, we synthesized various derivatives of the core
canthin-5,6-dione structure to clarify the structure-fluorescence relationship. Among the compounds obtained, one
showed pH-dependent fluorescence change, and could be utilized a fluorescent pH sensor, confirming the
practical value of this fluorescent moiety.
Fig. 1 Structures of fluorescent natural compounds, amarastelline A and nigakinone (1).
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2. Results and Discussions
2.1. Design and synthesis of canthin-5,6-dione derivatives.
Amarastelline A and nigakinone contain a common canthin-5,6-dione structure, which is a tautomer of
5-hydroxy-canthin-6-one, as shown in Fig. 1 for the case of nigakinone. Tautomerization is expected to alter the
fluorescence properties, and could be hindered by the introduction of functional groups at the N3-position, as well
as by the methoxy group at the 4-position adjacent to the diketone (or α-hydroxyketone) moiety of 1. Functional
groups on the benzene moiety linked to the tautomerizable bicyclic 1,5-naphthyridin-2-one moiety might also
influence the fluorescence properties. Thus, we designed compounds 1 – 10, having a variety of groups at the N3-,
4- and 10-positions (Fig. 2).
Fig. 2 Structures of synthesized canthin-5,6-dione compounds.
Although several canthine compounds have been reported, most of them were extracted from plants,8 and there
have been few synthetic studies.9 Among the compounds shown in Fig. 2, compounds 2 and 7 were synthesized
by reported procedures,9b,10 while the other compounds were synthesized by modified procedures, as shown in
Scheme 1. Tryptamine (11a) and 5-methoxytryptamine (11b) were used as starting materials, and they were
reacted with methoxyacetyl chloride (12a) or acetic anhydride (12b) to obtain acylated tryptamines (13), which
were transformed to 1-alkyl-β-carbolines (14) with Hendrickson reagent11 followed by oxidation. The
construction of the canthin-5,6-dione moiety was performed by the reaction of 14 and dibenzyl oxalate to obtain
compounds 6 - 9, and then reductive debenzylation yielded 1 – 4. Demethylation of the 10-methoxy group of 4
and 9 was performed with tribromoborane, affording 5 and 10, respectively.
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Scheme 1 Synthesis of compounds 1 – 10.
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2.2. Study of fluorescence properties of canthin-5,6-dione compounds.
The fluorescence properties of compounds 1 – 10 were evaluated in chloroform, acetonitrile, DMF, DMSO,
methanol and H2O (100 mM sodium phosphate buffer, pH 7.4), (Fig. S1 – S4), and the results are summarized in
Table 1. As regards the absorption spectra, compounds 1 – 5 with no substituent group at the N3-position, showed
maxima at around 350 – 370 nm, whereas compounds with a N3-benzyl group showed maxima at over 450 nm.
Introduction of a functional group at the N3-position appears to hinder the tautomerization between 5,6-dione and
5-hydroxyl-6-one forms, and shorten the absorption maximum wavelength. The 5,6-dione form might contribute
predominantly to the absorption at over 450 nm.
The parent compound nigakinone (1) showed moderate fluorescence in chloroform with two emission maxima
at 432 and 616 nm; the longer-wavelength emission reached more than 600 nm, and its Stoke’s shift was up to
258 nm. Although its quantum yield was relatively low, emission at longer wavelength would be favorable for
application to living cells or tissues, minimizing the phototoxic effect of the excitation light and interference from
autofluorescence due to biomolecules such as nucleotides. In addition, fluorophores with a large Stoke’s shift
would also be useful in applications such as multicolor imaging and dual-color fluorescence cross-correlation
spectroscopy (FCCS), like a fluorescent protein “Keima”.12 Focusing on the effects of 4-subsituents, removal of
the methoxy group enhanced the fluorescence, as indicated by the quantum yields of 1 vs 2, 3 vs 4, 6 vs 7 and 8 vs
9 (Fig. 3, Table 1). In these compounds, the Stoke’s shift was still large, up to 186 nm in the case of compound 2
(chloroform), and the quantum yield at this wavelength was relatively high (Φ = 0.103). Although further
optimization might be necessary, those compounds appear to be good candidates as novel platforms for
fluorescent dyes.
Fig. 3 The effect of 4-substituents on the fluorescence spectra. (a) The fluorescence spectra in chloroform
(excitation at 360 nm) of 4-OCH3 compounds (1 and 3) are shown as dashed lines and open symbols, and those of
4-H compounds (2 and 4) as solid lines and closed symbols. (b) Fluorescence spectra (excitation at 460 nm) of
N3-benzyl compounds, 6 - 9.
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Table 1. Photophysical Properties of Compounds 1 – 10.
Compound 1 (R1 = H; R2 = OCH3)
Compound 6 (R1 = H; R2 = OCH3)
[a]
[a]
Solvent
λabs (nm)
λem (nm)
Φ
Solvent
λabs (nm)
λem (nm)
Φ
fl
fl
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
358
357
432, 616
444
0.021, 0.016
0.147
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
500
499
500
501
488
478
[c]n. d.
[c]n. d.
[c]n. d.
[c]n. d.
[c]n. d.
[c]n. d.
[d]n. d.
[d]n. d.
[d]n. d.
[d]n. d.
[d]n. d.
[d]n. d.
474
[c]n. d.
[c]n. d.
[c]n. d.
[c]n. d.
[d]n. d.
363
[d]n. d.
358
[d]n. d.
358, 457
[d]n. d.
Compound 2 (R1 = H; R2 = H)
Compound 7 (R1 = H; R2 = H)
[a]
[a]
Solvent
λabs (nm)
λem (nm)
Φ
Solvent
λabs (nm)
λem (nm)
Φ
fl
fl
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
356
355
450
360
375
407
378, 542
377, 545
535
0.145, 0.103
0.229, 0.034
0.168
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
456
455
457
460
447
444
513
509
514
518
513
517
0.814
0.688
0.669
0.682
0.711
0.536
404, 533
509
0.053, 0.223
0.229
507
0.313
Compound 3 (R1 = OCH3; R2 = OCH3)
Compound 8 (R1 = OCH3; R2 = OCH3)
[a]
[a]
Solvent
λabs (nm)
λem (nm)
Φ
Solvent
λabs (nm)
λem (nm)
Φ
fl
fl
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
360
358
361
364
362
364
462, 616
467
0.007, 0.012
0.031
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
505
502
504
505
497
484
517
519
0.020
0.016
0.018
0.020
0.016
[d]n. d.
[c]n. d.
478, 635
564
[d]n. d.
524
0.005, 0.003
0.004
539
526
[c]n. d.
[d]n. d.
[c]n. d.
Compound 4 (R1 = OCH3; R2 = H)
Compound 9 (R1 = OCH3; R2 = H)
[a]
[a]
Solvent
λabs (nm)
λem (nm)
Φ
Solvent
λabs (nm)
λem (nm)
Φ
fl
fl
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
357
357
450
361
360
388
461
475
514
505
489
508
0.214
0.093
0.113
0.116
0.214
0.213
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
461
459
463
463
453
395
518
518
521
524
518
532
0.689
0.595
0.596
0.617
0.353
0.022
Compound 5 (R1 = OH; R2 = H)
Compound 10 (R1 = OH; R2 = H)
[a]
[a]
Solvent
λabs (nm)
λem (nm)
Φ
Solvent
λabs (nm)
λem (nm)
Φ
fl
fl
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
357
356
358
362
358
392
485
491
511
511
492
503
0.044
0.035
0.079
0.020
0.112
0.081
CHCl3
CH3CN
DMF
DMSO
CH3OH
[b]H2O
461
456
462
463
400
397
516
516
519
529
515
506
0.467
0.603
0.558
0.588
0.011
[d]n. d.
[a]Quantum yields of fluorescence were determined using that of quinine sulfate (0.577, in 0.1 M H2SO4) or fluorescein (0.90, in 0.1 M
NaOH) as a standard.1a [b]Measured in sodium phosphate buffer (100 mM. pH 7.4). [c]No emission peak was detected. [d]Not determined.
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Next, the effects of 10-substituents on the fluorescence properties were evaluated. In compounds 1 - 5, the
emission maximum wavelengths varied depending upon the 10-substituent, especially in aprotic solvents. For
example, in chloroform, compound 2 (10-H) showed emission maxima at 378 and 542 nm, while 4 (10-OCH3)
showed a maximum at 461 nm, and 5 (10-OH) showed a maximum at 485 nm (Fig. 4a). Similar differences were
seen in other aprotic solvents, acetonitrile, DMF and DMSO, whereas the differences in protic solvents such as
methanol and water were relatively small (Fig. 4b). However, in the case of N3-benzyl compounds, 7 (10-H), 9
(10-OCH3) and 10 (10-OH), the influence of 10-substituents on the emission maximum wavelength was almost
lost in both aprotic and protic solvents (Fig. 4c, 4d).
Fig. 4 Effects of 10-substituents on fluorescence spectra. The fluorescence spectra of 2 (10-H, black line), 4
(10-OCH3, blue line) and 5 (10-OH, red line) in (a) chloroform and (b) methanol with excitation at 360 nm are
shown, together with those of N3-benzyl compounds (7 (10-H, black line), 9 (10-OCH3, blue line) and 10 (10-OH,
red line)) with excitation at 460 nm in (c) chloroform and (d) methanol.
The presence of an N3-benzyl group greatly affected the fluorescence properties. For example,
solvent-dependent changes in the florescence spectra of 2 and N 3-benzylated 7 are shown in Fig. 5. Compound 2
showed peaks at around 400 nm in acetonitrile and chloroform, small peaks at this position in DMF and DMSO,
and almost no peak in methanol and water (Fig. 5a). Changes of maximum wavelength, as well as intensity, at
over 500 nm were also observed, that is, 542 nm in chloroform, 535 nm (DMF), 509 nm (methanol) and 507 nm
(water) (Fig. 5a and Table 1). On the other hand, in N3-benzylated 7, only small changes of fluorescence at about
500 nm were induced by change of solvent, with little change of intensity (Fig. 5b). Thus, the fluorescence
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properties of the 5-hydroxy-6-one form appear to be little affected by the solvent. On the other hand, compound 2,
which can undergo tautomerization between 5-hydroxy-6-one and 5,6-dione forms, showed large
solvent-dependent fluorescence changes (Fig. 5c).
Fig. 5 Solvent-dependent fluorescence change of canthin-5,6-dione derivatives. Fluorescence spectra of (a) 2 (Ex.
360 nm) and (b) of 7 (Ex. 460 nm) in various solvents are shown. (c) Proposed relationship of tautomerization and
fluorescence change.
2.3. The sensor function of the compounds.
We expected that the tautomerization equilibrium would be influenced by pH, because of the possibility of
deprotonation of the hydroxyl group of the 5-hydroxy-6-one, and in addition, compound 5 has a hydroxyl group
on the benzene moiety. Therefore, we evaluated the pH-dependent fluorescence changes of compounds 2, 4 and 5,
(Fig. 6). A switch from acidic to neutral conditions increased the fluorescence intensities of all compounds, in
addition to decreasing the absorbance at around 450 nm and 370 nm, and increasing that at around 400 nm (Fig. 6,
S5, S6 and S7), presumably due to deprotonation of the 5-hydroxy-6-one moiety. A change from neutral to basic
conditions had little effect on the spectra of 2 and 4, whereas the 10-hydroxy compound, 5, showed a decrease of
fluorescence as well as an increase of absorption at 390 nm, presumably due to deprotonation of the 10-hydroxy
group. Thus, apparently due to sequential deprotonations of the two hydroxyl groups, compound 5 showed an
“OFF-ON-OFF”-type fluorescence change with increase of pH. So, like our previously reported sensor based on a
coumarin scaffold bearing two hydroxyl groups13 and similar sensors developed by other groups,14 5 could be
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utilized as a fluorescent sensor for a specific range of pH (Fig. 7). Such a sensor should be useful, compared with
most conventional pH sensors, which sense only whether the pH is below or above a specific value.2
Fig. 6 Absorption and fluorescence spectra of 2, 4 and 5 at various pH values. Absorption spectra and
fluorescence spectra of (a and b) 2, (c and d) 4 and (e and f) 5 were measured in a mixture of methanol and 100
mM sodium phosphate buffer (50/50 = v/v).
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Fig. 7 pH-dependent fluorescent change of 5. (a) The pH profile of fluorescence intensity (excitation at 360 nm;
emission at 500 nm) of 5 is shown. Those data were obtained in 100 mM sodium phosphate buffer (pH 3.0 – 12.0)
containing 0.3% DMSO as a co-solvent. (b) Photographs of each solution.
Many canthine compounds, mostly natural products, have been reported, but only limited information is
available about their biological properties, such as cytotoxicity.15 Here, we synthesized several canthin-5,6-dione
derivatives and evaluated their structure-fluorescence relationship. We found that the fluorescence properties of
these compounds are influenced by substitution at the N3-, 4- and 10-positions. In particular, N3-benzylated
compounds showed little solvent-dependent fluorescent change. Many fluorescent dyes with solvent-dependent
fluorescent change, such as dansyl and NBD,16 show a longer emission maximum wavelength in more polar
solvents such as DMSO and water. Among our compounds, 4 showed such behavior (emission at 461 nm in
chloroform and 508 nm in water); this change is thought to be due to solvent relaxation. On the other hand, 2
showed two emission maximum wavelengths, at 378 nm and 542 nm in less polar chloroform, and one at 507 nm
in water (Fig. 8). The longer-wavelength maximum in chloroform is thought to involve a state other than a locally
excited (LE) state, such as an internal charge transfer (ICT) state and excited-state intramolecular proton transfer
(ESIPT),1a,17 and the difference between 2 and 4 should be due to the presence of the 10-methoxy group. Another
interesting effect of the 10-methoxy group was seen on comparing N3-benzyl compounds 7 and 9. In aqueous
solution, fluorescence of 9 was weak (quantum yield 0.022), whereas 7 showed strong fluorescence (0.536). As
we previously reported, amarastelline A showed similar properties to compound 9, that is, its quantum yield
values were 0.78 in CHCl3, 0.44 in CH3OH and <0.001 in H2O.6 Interestingly, amarastelline A does not possess a
10-methoxy group, and its N3-group, 9-methoxy-β-carboline, is different from those of 7 and 9, which have a
benzyl group. Thus the combination of substituent groups at the N3- and 10-positions can regulate the
fluorescence properties of canthin-5,6-diones, especially in aqueous solution. For example, we showed that 5
could be used as a sensor responding to a specific range of pH, based on its “OFF-ON-OFF”-type fluorescence
change with increase of pH. The insights presented here should be valuable for the development of a range of
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novel fluorescent sensors by the introduction of recognition sites for various analytes into the canthin-5,6-dione
structure. Work along this line is in progress.
Fig. 8 Effects of a 10-methoxy group.
3. Conclusion
We synthesized several derivatives of canthin-5,6-dione, which is a common structural component of the
fluorescent natural products amarastelline A and nigakinone, to evaluate substituent effects on the fluorescence
properties. We found that the combination of N3- and 10-substituted groups can regulate the fluorescence
properties, and we showed that this behavior could be utilized to obtain a fluorescent sensor responding to a
specific range of pH. Our findings indicate that the canthin-5,6-dione moiety could be a valuable scaffold for a
range of fluorescent sensors and probes.
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4. Experimental Section
4.1. General
All reagents were purchased from Sigma-Aldrich Chemical, Tokyo Kasei Kogyo, Wako Pure Chemical
Industries, and Kanto Kagaku. Silica gel for column chromatography was purchased from Kanto Kagaku. NMR
spectra were recorded on Bruker AVANCE 400 or Bruker Advance 500 spectrometer. Mass spectral data was
obtained on Brucker Daltonics microTOF-2focus in the positive and negative ion detection modes. Melting points
were taken on a Yanagimoto micro melting point apparatus and are uncorrected. UV spectra were recorded with
JASCO V-550, and fluorescence spectra were recorded with JASCO FP-6600.
4.2. Synthesis
4.2.1. Preparation of 13
A solution of triptamine (11a) (3.0 g, 19 mmol), methoxyacetyl chloride (12a) (2.6 ml, 28 mmol) and pyridine
(4.7 mL, 56 mmol) in dry dichloromethane (90 ml) was stirred for 23h at room temperature, and saturated
hydrochloric acid was added. The mixture was extracted with dichloromethane. The organic solution was
washed with brine and concentrated. Purification of the residue by column chromatography (silica gel, AcOEt)
gave 13a (3.3 g, 75%) as brown oil. 1H NMR (400 MHz, CDCl3)
δ 8.20 (brs, 1 H), 7.62 (d, J = 8.0, 1.2 Hz, 1 H),
7.38 (dd, J = 8.0, 1.2 Hz, 1 H), 7.21 (td, J = 8.0, 1.2 Hz, 1 H), 7.13 (td, J = 8.0, 1.2 Hz, 1 H), 7.04 (s, 1 H), 6.66
(brs, 1 H), 3.88 (s, 2H), 3.65 (q, J = 6.8 Hz, 2 H), 3.32 (s, 3 H), 3.01 (t, J = 6.8, 0.8 Hz, 2 H); 13C NMR: (125
MHz, CDCl3): δ 169.8, 136.5, 127.4, 122.3, 122.1, 119.6, 118.8, 113.0, 111.4, 72.1, 59.3, 39.2, 25.5; HRMS (ESI)
calcd for C13H17N2O2 (M+H)+ 233.1285. Found 233.1280.
13b18 and 13d19 were prepared according to a reported procedure. 13c were similarly prepared from 11b and
12a according to the procedure described for 13a. Analytical data are given below.
13c: 1H NMR (400 MHz, CDCl3)
1 H), 6.87 (dd, J = 8.8, 2.4 Hz, 1 H), 6.69 (brs, 1 H), 3.89 (s, 2H), 3.87 (s, 3H), 3.64 (q, J = 6.8 Hz, 2 H), 3.32 (s, 3
H), 2.97 (t, J = 6.8 Hz, 2 H); 13C NMR: (125 MHz, CDCl3):
169.9, 154.2, 131.6, 127.8, 122.9, 112.7, 112.5,
δ 8.11 (brs, 1 H), 7.26 (d, J = 8.8 Hz, 1 H), 7.05 (d, J = 2.4 Hz, 1 H), 7.02 (s,
δ
112.1, 100.6, 72.1, 59.3, 56.0, 39.2, 25.5; HRMS (ESI) calcd for C14H19N2O3 (M+H)+ 263.1390. Found 263.1384.
4.2.2. Preparation of 14
Triflic anhydride (1.1 ml, 6.6 mmol) was added to a solution of triphenylphosphine oxide (3.6 g, 13 mmol) in
anhyd chlorobenzene (30 ml) at 0°C. The reaction mixture was stirred for 15 min at 0°C, and a solution of 13a
(1.0 g, 4.4 mmol) in dry chlorobenzene (4 ml) was added. The reaction mixture was stirred for 1 h at room
tempreture, and then the mixture was allowed to warm to 130°C. Stirring was continued for another 1h, then
manganese dioxide (1.1 g, 13 mmol) was added. Stirring was continued for 4 h, then the reaction mixture was
cooled to room temperature, and was diluted with chloroform (100 mL). The resulting mixture was filtered
through celite. The filtrate was washed with saturated aqueous ammonium chloride and brine and concentrated.
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Purification of the residue by column chromatography (silica gel, CHCl3/AcOEt = 1/1) gave 14a (0.12 g, 13%) as
brown crystals. Mp 119.7-120.5°C (hexane/CH2Cl2); 1H NMR (400 MHz, DMSO-d6)
= 5.2 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1 H), 8.07 (d, J = 5.2 Hz, 1 H), 7.65 (d, J = 8.0 Hz, 1 H), 7.54 (t, J = 8.0, 1.2
Hz, 1 H), 7.24 (t, J = 8.0, 1.2 Hz, 1H), 4.88 (s, 2 H), 3.91 (s, 3 H); 13C NMR: (125 MHz, CDCl3):
141.6, 140.5,
δ 11.4 (s, 1 H), 8.27 (d, J
δ
137.8, 134.1, 129.7, 128.7, 121.9, 121.3, 120.1, 114.2, 111.8, 76.1, 59.1; HRMS (ESI) calcd for C13H13N2O
(M+H)+ 213.1022. Found 213.1025.
14b9a and 14d20 were prepared according to a reported procedure. 14c were similarly prepared from 13c
according to the procedure described for 14a, and analytical data is given below.
1
14c: Mp 122.3-123.9°C; H NMR (400 MHz, CDCl3)
δ 9.02 (brs, 1 H), 8.32 (d, J = 5.4 Hz, 1 H), 7.88 (d, J =
5.4 Hz, 1 H), 7.56 (d, J = 2.5 Hz, 1 H), 7.46 (d, 1 H, J = 8.8 Hz), 7.23 (dd, J = 8.8, 2.5, 1 H), 5.10 (s, 2H), 3.94 (s,
3H), 3.57 (s, 3H); 13C NMR: (125 MHz, CDCl3):
154.3, 141.8, 137.4, 135.4, 134.7, 129.5, 121.6, 118.9, 114.1,
δ
112.6, 103.6, 59.1, 56.2, 29.9; HRMS (ESI) calcd for C14H15N2O2 (M+H)+ 243.1128. Found 243.1127.
4.2.3. Preparation of 6 - 9
14a (57 mg, 0.27 mmol) and dibenzyl oxalate (0.32 g, 1.2 mmol) was stirred at 160°C for 7.5 h. Purification of
the reaction mixture by column chromatography (silica gel, AcOEt/MeOH = 10/1, CHCl3/MeOH = 10/1) gave 6
(65 mg, 77%) as red crystals. A small amount of the compound was recrystallized to give an analytical sample.
Mp 223-231°C (decomp.) (hexane/CH2Cl2); 1H NMR:(400 MHz, DMSO-d6)
δ 8.44 (d, J = 8.0 Hz, 1 H), 8.23 (d, J
= 8.0 Hz, 1 H), 8.12 (d, J = 6.8 Hz, 1 H), 7.69 (t, J = 8.0 Hz, 1 H), 7.60 (d, J = 6.8 Hz, 1 H), 7.54 (t, J = 8.0 Hz, 1
13
H), 7.37 (t, J = 7.6 Hz, 2 H), 7.29 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1 H), 5.95 (s, 2H), 3.51 (s, 3H); C
NMR: (125 MHz, DMSO-d6): δ 165.4, 157.2, 139.5, 138.7, 137.5, 132.9, 132.8, 130.3, 128.8, 127.6, 126.0, 126.0,
125.8, 124.8, 122.9, 122.7, 116.1, 105.2, 59.2, 58.3; HRMS (ESI) calcd for C22H17N2O3 (M+H)+ 357.1234. Found
357.1227.
7 was prepared according to a reported procedure.9b,10 8 and 9 were similarly reported from the corresponding
1-alkyl-β-carboline moieties, 14c and 14d, respectively, and analytical data are given below.
1
8: Mp 250°C (decomp.) (hexane/CH2Cl2/MeOH); H NMR (500 MHz, DMSO-d6)
δ 8.30 (d, J = 9.0 Hz, 1 H),
8.11 (d, J = 6.5 Hz, 1 H), 7.84 (d, J = 2.5 Hz, 1 H), 7.59 (d, J = 6.5 Hz, 1 H), 7.37 (t, J = 7.5 Hz, 2 H), 7.29 (t, J =
7.5 Hz, 1 H), 7.26 (dd, J = 9.0, 2.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 2 H), 5.93 (s, 2H), 3.89 (s, 3H), 3.49 (s, 3H); 13C
NMR (125 MHz, DMSO-d6):
δ 165.5, 157.6, 156.8, 138.5, 137.5, 133.8, 132.8, 132.6, 128.9, 128.8, 127.6, 126.0,
123.0, 117.7, 116.8, 106.5, 105.3, 59.2, 58.3, 55.8; HRMS (ESI) calcd for C23H19N2O4 (M+H)+ 387.1339. Found
387.1336.
9: Mp >300°C (CH2Cl2); 1H NMR (400 MHz, DMSO-d6)
δ 8.32 (d, J = 8.8 Hz, 1 H), 8.24 (d, J = 6.8 Hz, 1 H),
7.87 (d, J = 2.4 Hz, 1 H), 7.59 (d, J = 6.8 Hz, 1 H), 7.39 (m, 2 H), 7.30 (m, 4 H), 5.96 (s, 1 H), 5.58 (s, 2 H), 3.90
13
(s, 3 H); C NMR (125 MHz, DMSO-d6)
δ 171.1, 157.9, 156.9, 140.5, 136.8, 135.7, 134.2, 129.3, 128.4, 127.4,
13

ACCEPTED MANUSCRIPT
126.2, 125.9, 125.5, 118.2, 117.3, 107.0, 105.2, 94.4, 56.3, 56.2; HRMS (ESI) calcd for C22H17N2O3 (M+H)+
357.1234. Found 357.1224.
4.2.4. Preparation of 2 - 4
8 (15.9 mg, 0.04 mmol) and hydrogenated over palladium on carbon (8.1 mg) in ethanol (6 ml) was stirred for
23h. The reaction mixture was filtrated, and concentrated. Purification of the residue mixture by column
chromatography (silica gel, AcOEt/MeOH/NH3 aq. = 10/1/0.1, CH2Cl2/MeOH/NH3 aq. = 10/1/0.1) gave 3 (11.0
1
mg, 93%) as a brown solid. Mp 230°C (decomp.) (AcOEt/MeOH); H NMR (500 MHz, DMSO-d6)
δ 9.98 (s,
1H), 8.77 (d, J = 5.0 Hz, 1 H), 8.37 (d, J = 9.0 Hz, 1 H), 8.17 (d, J = 5.0 Hz, 1 H), 7.97 (d, J = 2.5 Hz, 1 H), 7.33
13
(dd, J = 9.0, 2.5 Hz 1 H), 4.19 (s, 3 H), 3.91 (s, 3H); C NMR (125 MHz, DMSO)
δ 157.4, 156.9, 145.0, 143.2,
140.6, 134.4, 132.5, 129.0, 126.4, 126.0, 117.9, 116.7, 114.9, 107.4, 60.9, 55.9; HRMS (ESI) calcd for
C16H13N2O4 (M+H)+ 297.0870. Found 297.0863.
2 was prepared according to a reported procedure.9b,10 1 was similarly prepared from 6, and it was identified by
the comparison with reported NMR data. 4 was similarly prepared from 9, and its analytical data is given below.
4: Mp 254°C (decomp.) (CH2Cl2/MeOH); 1H NMR (500 MHz, DMSO-d6)
J = 9.0 Hz, 1 H), 8.03 (d, J = 5.0 Hz, 1 H), 7.95 (d, J = 2.5 Hz, 1 H), 7.33 (dd, J = 9.0, 2.5 Hz 1 H), 7.11 (s, 1 H),
3.92 (s, 3 H); 13C NMR (125 MHz, DMSO-d6)
158.0, 156.3, 153.8, 145.7, 138.3, 133.0, 128.5, 127.2, 126.9,
δ 8.67 (d, J = 5.0 Hz, 1 H), 8.40 (d,
δ
118.0, 117.4, 113.8, 110.7, 107.7, 56.2; HRMS (ESI) calcd for C15H11N2O3 (M+H)+ 267.0764. Found 267.0760.
4.2.5. Preparation of 5 and 10
In a microwave vaial equipped a stir bar, boron tribromide (1.0 M solution in dichloromethane, 1.4 ml, 1.4
mmol) was added to a solution of 4 (49 mg, 0.14 mmol) in dry dichloromethane (2.3 ml) at room tempreture
under an atmosphere of argon. The reaction mixture was stirred in a microwave resctor at for 10 min at 100°C,
and added to iced water and sturated aqueous sodium bicarbonate was added. The mixture was extracted with
dichloromethane. The organic phase was washed with brine and concentrated. Purification of the reaction mixture
by column chromatography (silica gel, CH2Cl2/MeOH = 10/1) gave 5 (25 mg, 70%) as yellow crystals. A small
amount of the compound was recrystallized to give an analytical sample. Mp >300°C (EtOH/H2O); 1H NMR (400
MHz, MeOD)
δ
8.59 (d, J = 5.2 Hz, 1 H), 8.39 (d, J = 8.8 Hz, 1 H), 7.95(d, J = 5.2 Hz, 1 H), 7.59 (d, J = 2.4 Hz,
156.1, 155.6, 152.8, 145.4,
1 H), 7.18 (dd, J = 8.8, 2.4 Hz, 1 H), 7.11 (s, 1 H); 13C NMR (125 MHz, DMSO-d6)
δ
137.4, 131.7, 128.6, 127.0, 126.7, 118.2, 117.2, 113.9, 110.6, 109.2; HRMS (ESI) calcd for C14H9N2O3 (M+H)+
253.0608. Found 253.0607.
10 was similarly prepared from 9 according to the procedure described for 5, and analytical data is given
below.
1
10: Mp >300°C (MeOH); H NMR (500 MHz, DMSO-d6)
δ 9.91 (s, 1 H), 8.24 (d, J = 8.5 Hz, 1 H), 8.22 (d, J =
6.5 Hz, 1 H), 7.56 (d, J = 6.5 Hz, 1 H), 7.55 (d, J = 2.5 Hz, 1 H), 7.39 (t, J = 7.5 Hz, 2 H), 7.33 (t, J = 7.5 Hz, 1
14

ACCEPTED MANUSCRIPT
H), 7.28 (d, J = 7.5 Hz, 2 H), 7.12 (dd, J = 8.5, 2.5 Hz, 1 H), 5.94 (s, 1 H), 5.58 (s, 2 H); 13C NMR (125 MHz,
DMSO-d6)δ 170.8, 156.5, 155.7, 140.1, 136.3, 135.4, 132.9, 128.9, 128.0, 127.0, 125.9, 125.4, 125.2, 118.2,
117.0, 108.4, 104.8, 94.0, 55.9; HRMS (ESI) calcd for C21H15N2O3 (M+H)+ 343.1077. Found 343.1071.
Acknowledgments
The work described in this paper was partially supported by Grants-in-Aid for Scientific Research from The
Ministry of Education, Science, Sports and Culture, Japan (Grants No 15K08019 to TH, 26670052 to HK,
24510293 to AO). TH also gratefully acknowledges funding from Terumo Life Science Foundation. This work
was also partially supported by Project for Development of Innovative Research on Cancer Therapeutics, the
Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT, Japan), and JSPS Core-to-Core
Program A, Advanced Research Networks.
23 – 23
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