7924
C. Gao et al. / Tetrahedron 68 (2012) 7920e7925
temperature, water was added with rapid stirring. The resulting
yellow solids were filtered off, washed with water, and further
purified by recrystallization from ethanol. The yellow solids 4
(230 mg) were obtained. The overall yield of the four-step synthesis
v/v) at a concentration of 200 mM and further dilutions were made
freshly in the appropriate aqueous buffer.
4.2.2. Mass spectra The mass spectra were acquired using a Wa-
ters Q-Tof Premier mass spectrometer equipped with an elec-
trospray ionization (ESI) source. The instrument was operated in
the negative-ion mode. Each compound and DNA solution were
diluted with 20:80 (v/v) methanol/100 mm ammonium acetate.
was about 20%. 1H NMR (400 MHz, DMSO-d6)
d (ppm) 3.65 (s, 6H,
OCH3), 5.12 (br s, 4H, NH2), 5.56 (s, 2H, CH2), 6.22 (s, 2H), 6.39 (s, 1H,
ArH), 7.06 (m, 2H, ArH), 7.33 (m, 2H, ArH), 7.46 (m, 2H, ArH); 13C
NMR (100 MHz, DMSO-d6)
d 48.9 (CH2), 55.3 (OCH3), 98.3 (CH),
104.3 (CH), 107.3 (CH), 116.7 (CH), 122.0 (C), 123.4 (CH), 134.2 (C),
140.1 (C), 142.9 (C), 161.1 (C), 175.7 (CO).
The binding assays were performed at 25
50 M compounds. The competitive experiments were carried
out at about 5 M G-quadruplex, 5 M duplex DNA mixture, and
10 M compounds for 6a and 6b (20 M compounds for 7a and
7b). Methanol was added to obtain a good spray.38 The direct
infusion flow rate was 10 L/min. The electrospray source con-
mM DNA and 25 or
m
m
m
4.1.2. General preparation of compound 6 Toa solutionofBoc-amino
acid (1.2 mmol) in dry THF (10 mL) were added HOBt (162 mg,
1.2 mmol), DIC (152 mg, 1.2 mmol), and 2,7-diamino-10-(3,5-
m
m
m
dimethoxybenzyl)-9,10-dihydro-acridone
4
(187 mg, 0.5 mmol).
ditions were spray voltage of 2.4 kV and capillary temperature of
The reaction suspension was stirred under nitrogen overnight at
room temperature. The volatile parts were removed under reduced
pressure and compound 5 was obtained by column chromatography.
Compound 5 was suspended and stirred in hydrochloride 1,4-
dioxane solution (15 mL), The suspension was stirred at room tem-
perature until TLC indicated completion of reaction. Yellow solids
were obtained after filtration.
120 ꢁC.
4.2.3. Circular dichroism spectra The CD spectra of DNA oligonu-
cleotides were carried out at room temperature by using a J-815
spectropolarimeter (JASCO) with a 0.1 cm path-length quartz cell.
The CD spectrum was scanned three times and obtained by taking
the average of them. The scan for buffer was subtracted from the
average scan each time.
4.1.2.1. 2,7-Bis-(6-ammonium-pentyl-carbamoyl)-10-(3,5-
dimethoxybenzyl)-9,10-dihydro-acridinone bis-hydrochlorate
(6a). Yield 169 mg, 56%; yellow solids; mp 263e265 ꢁC; 1H NMR
4.3. Bioassay
(400 MHz, DMSO-d6)
d (ppm) 1.37 (m, 4H, CH2CH2CH2CH2CH2),
4.3.1. Cell culture CCRF-CEM leukemia cells were cultured in RPIM
1.57e1.66 (m, 8H, CH2CH2CH2CH2CH2), 2.36 (m, 4H, COCH2), 2.79
(m, 4H, CH2NH3), 3.66 (s, 6H, OCH3), 5.69 (s, 2H, ArCH2), 6.25 (m,
2H, ArH), 6.41 (m, 1H, ArH), 7.61 (m, 2H, ArH), 7.84 (br s, 6H,
CH2NH3), 7.94 (m, 2H, ArH), 8.66 (m, 2H, ArH), 10.21 (s, 2H, NHCO);
1640 medium (Cibco), containing 10% fetal bovine serum (FBS)
(Hyclone Laboratories Inc.), 100 units/mL penicillin, and 100 mg/mL
streptomycin in a 5% CO2-humidified atmosphere at 37 ꢁC.
13C NMR (100 MHz, DMSO-d6)
d 25.1 (CH2CH2CH2CH2CH2), 26.0
4.3.2. Cell growth inhibition The cells were suspended at a con-
centration of 2ꢂ105 cells/mL and seeded in 96-well microtiter
plates. Various concentrations of compound dissolved in DMSO
were added to each well in quintuplet followed by incubation for
48 h. After treatment, the cells were incubated with 10 mL of MTT
(3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
from Sigma) solution (5 mg/mL) for 4 h. The formazan precipitate
(COCH2CH2), 27.3 (COCH2CH2CH2CH2), 36.5 (COCH2), 39.1
(CH2NH3), 49.3 (ArCH2), 55.6 (OCH3), 98.8 (CH), 104.6 (CH), 115.8
(CH), 117.1 (CH), 121.7 (C), 127.0 (CH), 133.9 (C), 138.4 (C), 139.5 (C),
161.4 (C), 171.5 (CO),176.6 (CO); HRMS calcd for C34H44N5O5
[MꢀHCl2]þ 602.3342, found 602.3342.
4.1.2.2. 2,7-Bis-(2,6-diammonium-2-pentyl-carbamoyl)-10-(3,5-
dimethoxybenzyl)-9,10-dihydro-acridinone tetra-hydrochlorate
(6b). Yield 161 mg, 51%; yellow solids; mp 92e94 ꢁC; 1H NMR
was dissolved in 100 mL DMSO and the absorbance at 490 nm was
measured by a Benchmark microplate reader (Molecular Devices
Corporation). IC50 values are the concentration at which cell growth
was inhibited by 50%.
(400 MHz, DMSO-d6) d (ppm) 1.47 (m, 4H, CHCH2CH2CH2CH2), 1.63
(m, 4H, CHCH2CH2CH2CH2), 1.91 (m, 4H, CHCH2CH2CH2CH2), 2.78
(m, 4H, CHCH2CH2CH2CH2), 3.66 (s, 6H, OCH3), 4.11 (m, 2H, CHCH2),
5.74 (s, 2H, ArCH2), 6.27 (m, 2H, ArH), 6.42 (m,1H, ArH), 7.69 (m, 2H,
ArH), 8.01e8.04 (m, 8H, ArH and CH2NH3), 8.52 (br s, 6H, CHNH3),
8.74 (m, 2H, ArH), 11.3 (s, 2H, NHCO); 13C NMR (100 MHz, DMSO-d6)
Acknowledgements
The authors would like to thank the financial supports from the
Ministry of Science and Technology of China (2012ZX09506001-
010, 2012CB722605, 2012AA020305 and 2011DFA30620), the Chi-
nese National Natural Science Foundation (21172129 and
20902053), The Science Industry Trade and Information Technol-
ogy Commission of Shenzhen Municipality (JC200903180526A and
JC201005280602A).
d
21.4 (CHCH2CH2), 26.5 (CHCH2), 30.6 (CHCH2CH2CH2), 38.4
(CHCH2CH2CH2CH2), 49.5 (ArCH2), 52.8 (CHCH2), 55.4 (OCH3), 98.5
(CH), 104.4 (CH), 116.2 (CH), 117.2 (CH), 121.5 (C), 126.9 (CH), 132.7
(C), 138.7 (C), 139.1 (C), 161.4 (C), 167.6 (CO), 176.2 (CO) HRMS calcd
for C34H46N7O5 [MꢀHCl2]þ 632.3560, found 632.3556.
Supplementary data
4.2. Biophysical evaluation
HRMS spectra of compounds 6a and 6b. The mass spectra of
compounds 7b with G-quadruplex DNA. Supplementary data as-
sociated with this article can be found in the online version, at
4.2.1. Materials Single-stranded oligonucleotides (TTAGGG)2,
(CCCTAA)2 and (TTAGGG)4 were purchased from Invitrogen
(Guangdong, China). First, the oligonucleotides were denatured by
heating to 90 ꢁC for 10 min in a buffer consisting of NH4AC (pH 7.0).
The samples were then allowed to cool slowly to room tempera-
ture (over 4 h) to allow time for complete formation of the G-
quadruplex.38 The concentration of quadruplex was determined
spectroscopically. The stock solutions of the G-quadruplexes and
References and notes
1. Nathalie, P.; Nathalie, L. J.; Delphine, D.; Nathalie, P.; Dubois, F. Curr. Med.
Imaging Rev. 2006, 2, 193e203.
2. Reddy, M. V. R.; Mallireddigari, M. R.; Cosenza, S. C.; Pallela, V. R.; Iqbal, N. M.;
Robell, K. A.; Kang, A. D.; Reddy, E. P. J. Med. Chem. 2008, 51, 86e100.
duplex DNA containing single DNA are 200
mM. The synthesized
compounds were dissolved in a mixture of methanol/water (50:50,