Synthesis of polyhalo acridones as pH-sensitive fluorescence probes

Article abstract of DOI:10.1016/j.bmcl.2010.05.101

Polyhalo isophthalonitriles were reacted with substituted anilines and subsequently cyclocondensed in the presence of sulfuric acid to give polyhalo acridones. These polyhalo acridones were proven to be useful as pH-sensitive fluorescent probes for a wide range of acidic and basic conditions.

Full text of DOI:10.1016/j.bmcl.2010.05.101

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4665–4669
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of polyhalo acridones as pH-sensitive fluorescence probes
*
Chao Huang , Sheng-Jiao Yan , Yan-Mei Li, Rong Huang, Jun Lin
Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyhalo isophthalonitriles were reacted with substituted anilines and subsequently cyclocondensed in
the presence of sulfuric acid to give polyhalo acridones. These polyhalo acridones were proven to be use-
ful as pH-sensitive fluorescent probes for a wide range of acidic and basic conditions.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 31 January 2010
Revised 10 May 2010
Accepted 27 May 2010
Available online 2 June 2010
Keywords:
Polyhaloacridone
Polyhalo isophthalonitrile
Fluorescence probe
Fluorescent probes that are pH-sensitive have been widely used
in analytical chemistry, bioanalytical chemistry, cellular biology
(particularly for measuring intracellular pH) and medicine,1 pre-
sumably due to their preferential properties, including nondestruc-
tive character, high sensitivity and specificity, and the availability
of a wide range of indicator dyes.2 To date, a variety of pH-sensitive
probes have been reported,3 most of which can be used in either
acidic or basic conditions.4 In fact, new probes with specific fluo-
rescence over a broad pH range under both acidic and basic condi-
tions are continuously being developed.
Acridone derivatives have received much attention over recent
years because of their perfect fluorescent properties, such as high
quantum yields and excellent photostability,5 and their potential
pharmaceutical applications, possibly being used as antitumor
agents,6 antivirals,7 antimalarials,8 and antibacterials, etc.9 Acri-
done derivatives can be used as fluorescent probes based on their
unique interactions with biomolecules such as DNA and enzymes.
Many documents5a–c,10 regarding the ionization of acridones
including their ammonium salts and neutral molecules in a low
pH medium are widely available. To date, the ionization of acri-
dones in a high pH medium has not been reported. This is probably
because the higher pKa value of acridones limits their ionic disso-
ciation. Therefore, it is crucial to improve the acidity of acridone
derivatives in order to develop pH fluorescent probes that may
be applied to a broad pH range. In this context, the acidity of acri-
done derivatives 3a–e (Scheme 1) could be enhanced by introduc-
ing an acyl group at site 6 of the acridone enatic structure and a
halogen atom at the C-1 position. In this manner, it is envisioned
that compounds 3 could act as potential acid–base dual-purpose
pH fluorescent probes.
Although acridones with 1 or 2 substituents have been synthe-
sized,7a,b,9d access to highly functionalized acridones is still limited,
probably because introducing multiple substituents to the acri-
done ring often requires multistep reactions and complicated prep-
aration procedures. Therefore, the development of efficient and
concise approaches for producing acridones that tolerate a wide
variety of functional groups is desirable.
Polyhalo isophthalonitriles, especially polyfluoro isophthalo-
nitriles, have been widely used as reagents/intermediates in organ-
ic synthesis.11 Halogens and amido groups can be readily derived
and thus provide opportunities for constructing molecule libraries
for screening biological activity. This communication reports the
X
X
Y
i)
NC
X
CN
R
R
NH2
R=H, Cl
NC
X
CN
X
N
H
Y
2a: X=Cl, Y=Cl, R=H yield=78%
2b: X=F, Y=Cl, R=H yield=92%
1a: X=Cl, Y=Cl
1b: X=F, Y=Cl
1c: X=F, Y=F
2c: X=F, Y=F, R=H
yield=92%
2d: X=F, Y=Cl, R=Cl yield=85%
2e: X=F, Y=F, R=Cl yield=89%
O
X
O
ii)
R
3a: X=Cl, Y=Cl, R=H yield=84%
3b: X=F, Y=Cl, R=H yield=76%
H2N
X
N
H
3c: X=F, Y=F, R=H
yield=86%
Y
3d: X=F, Y=Cl, R=Cl yield=85%
3e: X=F, Y=F, R=Cl yield=81%
* Corresponding author. Tel./fax: +86 871 5033215.
E-mail address: linjun@ynu.edu.cn (J. Lin).
These authors contributed equally to this Letter.
Scheme 1. Synthesis of polyhalo acridones. Reagents and conditions: (i) DMF,
K2CO3, rt; (ii) H2SO4, 90 °C.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.101

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C. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4665–4669
synthesis of acid–base dual-purpose pH fluorescent probes with
specific fluorescence based on a new class of polyhalo acridones.
The synthetic pathway is depicted in Scheme 1. Polyhalo isopht-
halonitrile 1 was reacted with the substituted aniline in combina-
tion with potassium carbonate in N,N-dimethylformamide at room
temperature to produce compound 2, which easily run intra-cycli-
zation and sequential hydrolysis in the presence of sulfuric acid at
90 °C for 1 h to give the target product polyhalo acridone 3 with an
excellent yield. All compounds were fully characterized by 1H
NMR, 13C NMR, 19F NMR, and HRMS.12,13
Then, the polyhalo acridones 3a–e were analyzed by UV spec-
troscopy at a fixed concentration and at various phosphate buffer
pH values. The UV spectrum of the polyhalo acridone was found
to be dependent on pH. The UV absorption of compounds 3a–e
were red-shift and strengthened with increasing of pH values in
acid environments. While the UV absorption were blue-shift and
weakened with increasing of pH values in basic conditions
(Fig. 1A and B). At neutral condition, the maximum absorption
peaks of compounds 3a–e were 277, 266, 264, 267 and 266 nm,
respectively (see the Supplementary data data). The absorption
spectra of 3a and 3b are shown in Figure 1A and B.
the published method14 and was found to be 0.73 (Fig. 2). Com-
pounds 3a–e had different fluorescence at different pH values in
a solvent of H2O/DMSO = 1:1 (10À5 M) (Fig. 3).
The emission maxima of 3aH+ was lower than that of the neu-
tral 3a, presumably due to the electronic effects of the protonated
amino group in 3aH+. Under acidic conditions, 3a was reversibly
converted to 3aH+, as shown in the ionization equilibrium between
3a and 3aH+ (Scheme 2).
The fluorescent properties of compounds 3a–e can be seen in
Figure 4A–E. Equimolar solutions of the compounds were made
up in phosphate buffers over a broad pH range of ca. 0.5–14.6.
The relative fluorescent intensities of the probes were greatly re-
duced as the buffers became more basic. Accordingly, the fluores-
cence intensity of 3a was quickly reduced at 4.6 < pH < 7.0,
whereas there is a little change in the range of pH >7.0, indicating
that the optimum pH value for 3a as a fluorescence indicator
would be between pH 4.6 and 7.0. The fluorescence intensity of
3a was almost unchanged within 0.8 < pH < 5.6. The emission peak
position was only affected by pH (Fig. 4A). These results suggest
that there were two kinds of molecular morphologies present as
luminescence species: the neutral molecule 3a and the acidic salt
3aH+.
Next, the pH-dependent fluorescence response of compounds 3
in different buffer solutions was investigated. At pH <4.6, the exci-
tation and emission maxima of protonated 3aH+ were found to be
277 and 456 nm, respectively. The emission wavelength of 3a was
480 nm. The fluorescence quantum yield was tested according to
All of the polyhalo-substituted derivatives 3a–e, compound 3b
gave the highest fluorescence quantum yield (up to 0.98). The or-
der
of
the
maximum
excitation
wavelength
was
3a > 3d > 3e = 3b > 3c. The emission maxima of protonated 3aH+,
3bH+, 3cH+, 3dH+, and 3eH+ were 456 nm, 463 nm, 462 nm,
450 nm and 470 nm, respectively. The emission maxima of the
neutral molecules 3a–e were 480 nm, 490 nm, 487 nm, 496 nm
and 492 nm, respectively (Figs. 2 and 4A–E). Accordingly, the emis-
sion maxima of protonated 3aH+–3eH+ were lower than those of
neutral 3a–e because of the strong electron-withdrawing proper-
ties of the protonated nitrogen of the amino group in 3aH+–3eH+.
Similarly, compounds 3b–e and 3bH+–3eH+ reached ionization
equilibrium under acidic conditions as depicted in Scheme 2.
Figure 5A and B illustrates the pH responses of probes 3a–e as a
function of I/Imax versus pH, where I is the measured fluorescent
emission and Imax is the maximum output of the probe. Figure 5A
indicates that the most suitable condition for 3b as a fluorescence
indictor was 2.9 < pH < 8.0, while the working pH of 3c–e was low-
er than that of 3b. Unlike 3a, compounds 3b–e reached ionization
equilibrium under basic conditions. For example, the peak profile
of 3b changed at pH >6.8, becoming higher toward the right than
the left with increasing pH, thus showing the ionization equilib-
rium of 3b and 3b- (Scheme 2). At pH >10.5, the fluorescence inten-
sity rapidly decreased with increasing pH, indicating that 3b is a
suitable fluorescence indicator for 10.5 < pH < 14.1. (Fig. 5A). In
short, 3a can only be used as a pH fluorescence probe in acidic con-
ditions, while 3b–e can be applied to either acidic or basic environ-
1.0
0.8
0.6
0.4
0.2
0.0
pH 0.8
pH 1.2
pH 4.4
pH 5.8
pH 7.3
pH 10.7
pH 11.8
pH 12.2
pH 13.8
pH 14.0
1.0
0.8
0.6
0.4
0.2
0.0
a (acid)
7.3
5.8
4.4
1.2
0.8
240 260 280 300 320 340
1.0
0.8
0.6
0.4
0.2
0.0
b (base)
7.3
10.7
11.8
12.2
13.8
14.0
240 260 280 300 320 340
240
260
280
300
320
340
Wavelength, nm
Figure 1A. UV spectrum of 3a under different pH values (ethanol/buffers) 1:4. (a)
UV spectrum of 3a in acidic conditions. (b) UV spectrum of 3a in basic conditions.
1.0
1.0
0.8
0.6
0.4
0.2
0.0
pH 0.3
pH 1.0
pH 3.7
pH 5.3
pH 6.8
pH 8.0
pH 10.5
pH 12.3
pH 13.3
pH 13.7
a (acid)
6.8
5.3
3.7
1.0
0.3
0.8
0.6
O
Cl
Cl
O
O
F
F
O
O
F
F
F
O
240 260 280 300 320 340
H2N
Cl
3a
H2N
H2N
1.0
0.8
0.6
0.4
0.2
0.0
N
H
N
H
N
H
6.8
8.0
10.5
12.3
13.3
13.7
b (base)
Cl
3c
3b
λ
Φ = 0.73
ex = 277 nm
em = 456, 480 nm
Φ = 0.98
ex = 266 nm
Φ = 0.73
0.4
0.2
0.0
λ
ex = 264 nm
λ
λ
em= 463, 490 nm
λ
em = 462, 487 nm
λ
O
F
O
240 260 280 300 320 340
O
F
F
O
Cl
Cl
H2N
H2N
F
N
H
Φ = 0.93
N
H
Φ = 0.81
ex = 267 nm
em = 450, 496 nm
Cl
F
3e
λ
ex = 266 nm
λ
240
260
280
300
320
340
3d
λ
em = 470, 492 nm
λ
Wavelength, nm
Figure 1B. UV spectrum of 3b under different pH values (ethanol/buffers = 1:4). (a)
UV spectrum of 3b in acidic conditions. (b) UV spectrum of 3b in basic conditions.
Figure 2. Polyhalo acridones and their fluorescence quantum yield (U), and
excitation and emission maxima in H2O/DMSO = 1:1 (10À5 M).

C. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4665–4669
4667
Figure 3. Photo of fluorescence of 3a–3e (from left to right) taken under a hand-held UV (365 nm) lamp.
1000
900
800
700
600
500
400
300
200
100
0
O
X
X
Y
O
O
X
X
Y
OH
0.26
0.97
1.75
2.94
3.67
4.44
5.30
5.66
5.82
6.13
6.79
R
OH-
H+
7.97
9.18
R
H2N
H2N
N
H
N
10.51
11.28
11.47
11.80
12.25
12.57
12.94
13.26
13.37
13.70
14.13
H+
3aH+: X=Cl, Y=Cl, R=H
3bH+: X=F, Y=Cl, R=H
3cH+: X=F, Y=F, R=H
3dH+: X=F, Y=Cl, R=Cl
3eH+: X=F, Y=F, R=Cl
3a: X=Cl, Y=Cl, R=H
3b: X=F, Y=Cl, R=H
3c: X=F, Y=F, R=H
3d: X=F, Y=Cl, R=Cl
3e: X=F, Y=F, R=Cl
O
X
X
O-
3b-: X=F, Y=Cl, R=H
3c-: X=F, Y=F, R=H
3d-: X=F, Y=Cl, R=Cl
3e-: X=F, Y=F, R=Cl
OH-
H+
R
H2N
N
Y
Scheme 2. Ionization equilibrium of 3 and 3H+.
350 375 400 425 450 475 500 525 550 575 600 625 650
wavelength
700
600
500
400
300
200
100
0
Figure 4B. Fluorescence characteristics of 3b under different pH values (ethanol/
buffers = 1:4).
0.77
1.88
3.08
3.76
4.63
4.76
4.97
5.57
5.70
5.98
6.22
6.71
7.01
7.72
8.98
9.66
10.92
11.20
12.07
12.85
13.26
13.67
14.01
800
700
600
500
400
300
200
100
0
0.23
1.41
2.34
2.94
4.36
4.81
5.22
5.70
5.82
6.04
6.22
6.43
6.56
7.26
8.26
9.26
10.73
11.72
11.95
12.12
12.60
13.02
13.40
13.68
14.23
14.61
350 375 400 425 450 475 500 525 550 575 600 625 650
wavelength
Figure 4A. Fluorescence characteristics of 3a under different pH values (ethanol/
buffers = 1:4).
350 375 400 425 450 475 500 525 550 575 600 625 650
ments (Fig. 5B). As such, compounds 3b–e have high potential for
application as pH sensors for acidic and basic conditions compared
with the current commercially available pH-sensitive fluorescent
probes. The presence of fluorine atom is crucial to the fluorescence
properties of acridones, as the electron-withdrawing effects of the
fluorine atom. However, compound 3a showed different fluores-
cence characteristics and only can use as a base to accept proton
to produce 3aH+ in acidic environments. The presence of fluorine
atom would be crucial to the fluorescence properties of acridones,
as the electron-withdrawing effects of the fluorine atom could ac-
count for higher acidic properties in fluorine-containing acridones
3b–e. Therefore, 3b–e could easily release proton and thus form
3bÀ–3eÀ under basic conditions. However, compound 3a showed
different fluorescence characteristics, and only can be used as a
base to accept hydrogen proton to produce 3aH+ in acidic
environments.
Wavelength
Figure 4C. Fluorescence characteristics of 3c under different pH values (ethanol/
buffers = 1:4).
In conclusion, a novel type of pH fluorescent probe was pre-
pared and proven to have excellent fluorescent properties, such
as broad pH application range, intense fluorescence and high fluo-
rescence quantum yield. The preliminary results for biological
activity using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tet-
razolium bromide (MTT) colorimetric assay15 showed that some
of the title compounds 3a–e possessed moderate anti-cancer activ-
ities against the K562, HL60, A431, HepG2 and Skov-3 cell lines
(see Supplementary data). Consequently, this simple process pre-

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C. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4665–4669
1000
900
800
700
600
500
400
300
200
100
0
0.76
3b
3c
3d
3e
1.0
0.8
0.6
0.4
0.2
0.0
7.20
8.20
9.61
1.71
2.81
3.36
3.73
3.90
4.07
4.36
5.45
6.72
10.95
11.27
11.97
12.03
12.51
12.79
13.60
13.93
14.30
14.63
0
2
4
6
8
10
12
14
350 375 400 425 450 475 500 525 550 575 600 625 650
pH
Wavelength
Figure 5B. pH dependence of 3b, 3c, 3d and 3e (I/Imax) at 295 K in phosphate
buffers of differing pH values (3b, kexc = 266 nm; 3c, kexc = 264 nm; 3d,
kexc = 267 nm; 3d, kexc = 266 nm).
Figure 4D. Fluorescence characteristics of 3d under different pH values (ethanol/
buffers = 1:4).
900
800
700
600
500
400
300
200
100
0
0.92
1.61
2.67
3.20
3.57
3.76
4.36
4.66
5.92
6.57
8.20
9.03
9.36
Acknowledgments
10.36
11.27
11.90
12.26
12.67
13.03
13.42
14.11
14.52
14.96
This work was supported by the National Natural Science Foun-
dation of China (Nos. 30860342, 20762013) and the Natural Sci-
ence Foundation of Yunnan Province (2009CC017 and 2008CD063).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.101.
References and notes
350 375 400 425 450 475 500 525 550 575 600 625 650
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Magnano, A.; Gatto, B.; Palumbo, M.; Menta, E.; Pescalli, N.; Martelli, S. J. Med.
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Blanca, M.; Pietrob, A. D. Bioorg. Med. Chem. 2007, 15, 2892; (e) Dodic, N.;
Dumaitre, B.; Daugan, M.; Pianetti, P. J. Med. Chem. 1995, 38, 2418; (f) Chao, H.-
J.; Jun, M.-J.; Kwonb, Y.; Na, Y. Bioorg. Med. Chem. Lett. 2009, 19, 6766; (g)
Boutefnouchet, S.; Gaboriaud-Kolar, N.; Minh, N. T.; Depauw, S.; David-
Cordonnier, M. H.; Pfeiffer, B.; Léonce, S.; Pierré, A.; Tillequin, F.; Lallemand,
M. C.; Michel, S. J. Med. Chem. 2008, 51, 7287.
with water to give a crude product, which was further recrystallized by ethyl
acetate to form the final products 2.
13. General procedure for the synthesis of polyhalo acridone 3: Polyhalo
isophthalonitrile 2 (2 mmol) was suspended in 6 mL of 95–98% sulfuric acid.
The reaction mixture was stirred at 90 °C for 1 h. After the reaction, the
mixture was cooled to room temperature, put into a beaker (100 mL) with
50 mL iced water, and then neutralized with solid Na2CO3 to a pH of 9–10. The
solid was filtered off and washed with water to give a crude product that was
purified by flash column chromatography, to afford polyhalo acridone 3 in 76–
86% yield. 1,3,4-Trichloro-9-oxo-9,10-dihydroacridine-2-carbox-amide 3a:
yellow solid, mp: 251–252 °C. IR (KBr): 3478, 3416, 1665, 1568, 1398, 1326,
1164, 755, 612 cmÀ1 1H NMR (500 MHz, DMSO-d6): d 8.48–8.18 (m, 3H, PhH),
.
7.95–7.80 (m, 3H, NH2, PhH), 7.48 (br, 1H, NH). 13C NMR (125 MHz, DMSO-d6):
d 162.7, 152.5, 148.5, 145.5, 132.2, 131.9, 130.7, 130.0, 129.2, 125.4, 124.4,
123.4, 115.0, 108.4. HRMS (TOF ES+): m/z calcd for C14H8Cl3N2O2 [(M+H)+],
340.9646; found, 340.9644. 4-Chloro-1,3-difluoro-9-oxo-9,10-dihydroacrid- ine-
2-carboxamide 3b: yellow solid, mp: >300 °C. IR (KBr): 3486, 3371, 3246, 1679,
1559, 1382, 1260, 834, 759, 601 cmÀ1 1H NMR (500 MHz, DMSO-d6): d 8.54–
.
7.78 (m, 6H, PhH, NH2), 7.46–7.43 (br, 1H, NH). 13C NMR (125 MHz, DMSO-d6):
d 161.3, 156.2 (d, J = 267.5 Hz), 154.8 (d, J = 258.8 Hz), 151.8, 149.5, 145.2,
132.5, 129.1, 123.8, 123.4, 114.1, 111.6, 109.1 (t, J = 27.5 Hz), 101.2 (d,
J = 32.5 Hz). 19F NMR (470 MHz, DMSO-d6):
d
À111.3 (d, J = 4.7 Hz, 1F),
À111.7 (d, J = 4.7 Hz, 1F). HRMS (TOF ES+): m/z calcd for C14H8ClF2N2O2
[(M+H)+],
309.0237;
found,
309.0233.
1,3,4-Trifluoro-9-oxo-9,10-
dihydroacridine-2-carboxamide 3c: yellow solid, mp: >300 °C. IR (KBr): 3526,
3421, 3151, 1685, 1557, 1388, 1265, 967, 758, 602 cmÀ1 1H NMR (500 MHz,
DMSO-d6):
8.53–7.78 (m, 6H, PhH, NH2), 7.44 (br, 1H, NH). 13C NMR
.
d
(125 MHz, DMSO-d6): d 161.0, 152.6 (d, J = 261.3 Hz), 151.2, 149.4, 145.3 (d,
J = 252.5 Hz), 140.5 (d, J = 248.8 Hz), 140.4, 132.4, 129.1, 123.7, 123.5, 114.1,
108.4 (t, J = 25.0 Hz), 101.2. HRMS (TOF ES+): m/z calcd for C14H8F3N2O2
[(M+H)+], 293.0532; found, 293.0529. 4,7-Dichloro-1,3- difluoro-9-oxo-9,10-
dihydro-acridine-2-carboxamide 3d: yellow solid, mp: >300 °C. IR (KBr): 3433,
10. (a) Bahr, N.; Tiemey, E.; Reymond, J. L. Tetrahedron Lett. 1997, 38, 1489; (b)
White, E.; Roswell, D. F.; Dupont, A. C.; Wilson, A. A. J. Med. Chem. 1987, 109,
5189; (c) Lunardi, C. N.; Bonilha, J. B. S.; Tedesco, A. C. J. Lumin. 2002, 99, 61; (d)
Qiu, B.; Guo, L.-H.; Chen, Z.-T.; Chi, Y.-W.; Zhang, L.; Chen, G.-N. Biosens.
Bioelectron. 2009, 24, 1281.
3353, 3245, 1650, 1554, 1376, 1251, 1102, 834, 630 cmÀ1 1H NMR (500 MHz,
.
DMSO-d6):
d
8.67 (br, 1H, NH), 8.28–7.74 (m, 6H, PhH, NH2). 13C NMR
11. (a) Yan, S.-J.; Huang, C.; Su, C.-X.; Ni, Y.-F.; Lin, J. J. Comb. Chem. 2010, 12, 91; (b)
Yan, S.-J.; Huang, C.; Zheng, X.-H.; Huang, R.; Lin, J. Bioorg. Med. Chem. Lett.
2010, 20, 48; (c) Shu, L.-J.; Mayor, M. Chem. Commun. 2006, 4134; (d) Kitazume,
T.; Nakajima, S. J. Fluorine Chem. 2004, 125, 1447; (e) Chan, P. K.; Leong, W. K.
Organometallics 2008, 27, 1247.
12. General procedure for the synthesis of polyhalo isophthalonitrile 2: A 50 mL
round-bottomed flask was charged with polyhalo isophthalonitrile 1 (5 mmol),
DMF (30 mL), aniline derivatives (6.0 mmol), and potassium carbonate 1.4 g
(10 mmol). The solution was stirred for 0.5–18 h at room temperature until the
substrate 1 was completely consumed. The mixture was put into a beaker
(100 mL) and quenched by the addition of water (30 mL). The reaction mixture
was stirred for another 15 min, and then filtered off. The residue was washed
(125 MHz, DMSO-d6): d 161.0, 156.0 (d, J = 255.0 Hz), 155.0 (d, J = 247.5 Hz),
151.2, 147.9, 145.4, 132.9, 131.3, 128.2, 122.4, 114.6, 111.8, 109.9 (t,
J = 26.3 Hz), 101.2. HRMS (TOF ES+): m/z calcd for C14H7Cl2F2N2O2 [(M+H)+],
342.9847; found, 342.9845. 7-Chloro-1,3,4-trifluoro-9,10-dihydro-9-oxoacri-
dine-2-carboxamide 3e: yellow solid, mp: 181–185 °C. 19F NMR (467 MHz,
DMSO-d6): d À115.4 (d, J = 14.1 Hz, 1F), À139.3 (s, 1F), À155.9 (d, J = 14.1 Hz,
1F). HRMS (TOF ES+): m/z calcd for C14H7ClF3N2O2 [(M+H)+], 327.0143; found,
327.0140.
14. (a) Eaton, D. J. Photochem. Photobiol., B 1988, 2, 523; (b) Ci, Y.-X.; Jia, X. Chin. J.
Anal. Chem. 1986, 14, 616.
15. Mossman, T. J. Immunol. Methods 1983, 65, 55.