Synthesis and antiproliferative activity of new cytotoxic tri- and tetraazabenzo[3,2-a]fluorene-5,6-dione derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.08.021

A new series of substituted tri-/tetraazabenzo[3,2-a]fluorene-5,6-diones and their corresponding oxime derivatives have been synthesized and spectroscopically characterized. The antiproliferative activities of all compounds were evaluated on at least thre

Full text of DOI:10.1016/j.bmcl.2013.08.021

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5264–5266
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of new cytotoxic
tri- and tetraazabenzo[3,2-a]fluorene-5,6-dione derivatives
Theerachart Leepasert a,b, Manochehr Shahabi a, Karem Shanab a, Eva Schirmer a, Wolfgang Holzer a,
Helmut Spreitzer a, , Babette Aicher c, Gilbert Müller c, Eckhard Günther c
⇑
a Department of Drug and Natural Product Synthesis, University of Vienna, Althanstraße 14, 1090 Vienna, Austria
b Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c AeternaZentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of substituted tri-/tetraazabenzo[3,2-a]fluorene-5,6-diones and their corresponding oxime
derivatives have been synthesized and spectroscopically characterized. The antiproliferative activities
of all compounds were evaluated on at least three different cell lines.
Received 17 June 2013
Revised 25 July 2013
Accepted 5 August 2013
Available online 13 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keyword:
Anticancer compounds
The large and flat diazobenzo[a]fluorene moiety fulfills all
requirements for DNA intercalation. Therefore, it is an astonishing
fact that only one study focused on cytotoxic effects of dia-
zobenzo[a]fluorene derivatives.1 Instead of that much more exam-
inations focused on properties of this type of compounds as photo
sensitizers2,3 and NOS-inhibitors for the treatment of hyptension,
autoimmune diseases, venous insufficiency and/or inflammation
edema.4–6 Besides that two aza analogues of diazobenzo[a]fluorene
were made accessible and their cytotoxic properties examined.7,8
In continuation of our studies on novel cytotoxic compounds we
report a new series of substituted tri-/tetraazabenzo[3,2-a]fluo-
rene-5,6-diones. The rationale for the synthesis of compounds 1–
9 is the reasonable assumption that the isoquinolinedione moiety
in intercalating agents leads to anticancer compounds with favour-
able properties like reduced cardiotoxic effects.9 Moreover, the iso-
quinolinedione nucleus is the crucial element of promising
anticancer drug candidates like BBR 342210 or BBR 3438,11 respec-
tively, which finally led to the development and market release of
BBR 2778 (Pix antrone, PixuvriÒ) for the treatment of patients with
multiply relapsed aggressive Non Hodgkin0s Lymphoma.12
In consistent further development of our studies13–17 we were
interested in the antiproliferative effects triggered by an isoquino-
line-1,2-dione moiety (instead of an isoquinoline-1,4-dione moi-
ety) thus leading us to a series of compounds 1–9 as shown in
Figure 1.
The synthesis of compounds 1–2 and 6 started from commer-
cially available 5-hydroxyisoquinoline (10), which was converted
into 6,7-dichloroisoquinoline-5,8-dione (11) with HCl/HNO3
instead of the literature method13 with sodium chlorate. The cycli-
zation was accomplished by refluxing 6,7-dichloroisoquinoline-
5,8-dione (11) with the appropriate aminopyridine 12 in ethanol
in presence of potassium carbonate yielding two isomers 1–2
and 6 (Scheme 1).
Oximes 3,4 and 7,8 were prepared by reacting either 1 or 6 with
2-(aminooxy)-ethanol or 2-(aminooxy)-N,N-dimethylethanamine,
respectively in presence of potassium hydroxide in methanol
(Schemes 2 and 3).
When compound 2 was treated with N1,N1,N2-trimethylethane-
1,2-diamine substitution occurred on C-2 to afford compound 5,
leaving chlorine substituted C-9 unaffected. The structure of 5
was confirmed by 2D NMR technique, HMQC (Scheme 4).
Compound 9 was prepared in a 5-step reaction starting from
commercially available 2,5-dimethoxybenz aldehyde (13), which
upon treatment with silica gel impregnated with nitric acid gave
14. Compound 14 was reacted with formamide under a HCl gas
stream to yield the desired N,N0-diformamide (15) which was sub-
jected to reductive cyclization with zinc in acetic acid to give 16.
After oxidation and chlorination of 16 to 17 with HCl/HNO3, tetra-
cyclic compound 9 was finally obtained as only isomer by refluxing
17 with 2-aminopyridine in ethanol in presence of potassium
bicarbonate (Scheme 5).
In summary it can be said that in comparison to mitoxantron
and doxorubicin the annelated derivatives 1 and 9, respectively,
exhibit satisfactory actitivities and therefore will serve as starting
⇑
Corresponding author. Tel.: +43 1427755621; fax: +43 142779556.
E-mail address: helmut.spreitzer@univie.ac.at (H. Spreitzer).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.021

T. Leepasert et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5264–5266
5265
Y
O
Y
O
N
O
N
O
N
N
N
N
N
R
X
X
N
N
N
1: R = H, X = H, Y = O
6: X = H, Y = O
7: X = H, Y = N-OCH2CH2OH
9
2
: R = H, X = Cl, Y = O
8: X = H, Y = N-OCH2CH2N(CH3)2
3: R = H, X = H, Y = N-OCH2CH2OH
4
5
: R = H, X = H, Y = N-OCH2CH2N(CH3)2
H3C
: R =
CH3
,
N
N
X = Cl, Y = O
CH3
Figure 1.
O
O
NHCHO
OCH3
OCH3
OCH3
CHO
Cl
Cl
CHO
a
a
b
b
N
N
NHCHO
N
NH2
NO2
OCH3
NO2
OCH3
12
OH
N
OCH3
X
10
11
13
14
15
O
O
c
O
N
O
N
O
OCH3
O
O
N
O
e
Cl
d
X
N
N
N
N
N
N
N
N
Cl
NH2
N
N
1: X = H
2: X = Cl
6
N
OCH3
17
16
9
Scheme 1. Reagents and conditions: (a) HCl/HNO3, 80–90 °C, 1 h; (b) K2CO3, EtOH,
85 °C, 5 h.
Scheme 5. Reagents and conditions: (a) HNO3–SiO2, CH2Cl2, rt, 10 min, 69%; (b)
formamide, HCl, 80 °C, 1 h, 75%; (c) Zn0, CH3COOH, 0 °C, 2.5 h, 54%; (d) HCl/HNO3,
80–90 °C, 15 min, 5%; (e) K2CO3, EtOH, 85 °C, 5 h, 46%.
O
Table 1
3
4
H2N
H2N
OH
N
In vitro cytotoxicity of compounds 1–9 against three human cancer cell lines
1
O
Cells (origin)/
compound
Compound activity (EC50
[l
M]a)18
or
KB/HeLa
(cervix)
SKOV-3
(ovarian)
NCI-H460
(NSCLC)
Scheme 2. Reagents: KOH, MeOH.
1
2
3
4
5
6
7
8
9
0.244 (1)
0.394 (1)
0.532 (1)
0.406 (1)
2.802 (1)
0.917 (1)
4.349 (1)
6.148 (1)
0.145(1)
0.498 0.001 (2)
0.525 0.048 (2)
2.064 (1)
0.931 0.098 (2)
No inhibitionb
1.222 0.294 (2)
14.732 (1)
0.242 0.067 (3)
0.342 0.003 (3)
0.521 0.221 (2)
0.414 0.097 (3)
2.135 1.166 (3)
0.620 0.176 (3)
3.875 1.306 (2)
2.673 0.457 (2)
0.337 0.026 (3)
O
7
8
H2N
H2N
OH
N
6
O
or
2.928 (1)
0.283 0.043 (2)
Scheme 3. Reagents: KOH, MeOH.
Mitoxantrone
Doxorubicin
0.420 0.060 (2)
0.250 0.140 (3)
n.d.
0.290 0.160 (10)
0.030 0.010 (2)
0.040 0.010 (5)
O
a
The data presented are EC50 values of cytotoxicity assessments with resazurin
O
as detection reagent performed in quadruplicate measurements. EC50 values are
depicted as mean values standard deviation with the number of replicates
indicated in round brackets.
N
N
O
N
b
N
No inhibition is defined as less than 30% inhibition in the highest final
N
O
N
N
compound concentration analyzed (31.6 lM).
O
H
N
N
O
N
Cl
N
References and notes
N
N
N
Cl
N
1. Tapia, R. A.; Cantuarias, L.; Cuellar, M.; Villena, J. Braz. Chem. Soc. 2009, 20, 999.
2. Podemska, K.; Podsiadly, R.; Szymczak, A. M.; Chrzescijanska, E.; Sokolowska, J.
Dyes Pigm. 2012, 95, 74.
2
5
Scheme 4. Reagents and conditions: THF, EtOH, 85 °C.
3. Podemska, K.; Podsiadly, R.; Szymczak, A. M.; Chrzescijanska, E.; Sokolowska, J.
Dyes Pigm. 2012, 94, 113.
4. Mijalli, A.M.M.; Sarshar, S.; Zhang, Ch. U.S. 1998, US5723451 A 19980303.
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Torregrosa, J.-L.; Yannic-Art PCT Int. Appl. 1997, WO 9721709 A 19970619.
points for a new series of cytotoxic compounds with a tri- and tet-
raazabenzo[3,2-a]fluorene-5,6-dione core (Table 1)19
.

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T. Leepasert et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5264–5266
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19970220.
7. Puetter, J. Zeitschr. f. Phys. Chem. 1963, 332, 1.
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1829, 11.
9. Krapcho, A. P.; Menta, E. Drugs Fut. 1997, 22, 641.
10. Xu, L.-N.; Liang, G.-Z.; Li, Z.-L.; Wang, J.; Zhou, P. J. Mol. Graph. Model 2008, 26,
1252.
11. Hofheinz, R.-D.; Porta, C.; Hartung, G.; Santoro, A.; Hanauske, A.-R.; Kutz, K.;
Stern, A.; Barbieri, P.; Verdi, E.; Hehlmann, R. Invest. New Drugs 2005, 23, 363.
12. EMA/CHMP/102366/2012.
13. Shahabi, M.; Schirmer, E.; Shanab, K.; Leepasert, T.; Ruzicka, J.; Holzer, W.;
Spreitzer, H.; Aicher, B.; Schmidt, P.; Blumenstein, L.; Mueller, G.; Guenther, E.
Bioorg. Med. Chem. Lett. 2013, 1846, 23.
14. Shanab, K.; Schirmer, E.; Wulz, E.; Weissenbacher, B.; Lassnig, S.; Slanz, R.;
Foesleitner, G.; Holzer, W.; Spreitzer, H.; Schmidt, P.; Aicher, B.; Mueller, G.;
Guenther, E. Bioorg. Med. Chem. Lett. 2011, 21, 3117.
15. Shanab, K.; Schirmer, E.; Knafl, H.; Wulz, E.; Holzer, W.; Spreitzer, H.; Schmidt,
P.; Aicher, B.; Mueller, G.; Guenther, E. Bioorg. Med. Chem. Lett. 2010, 20, 3950.
16. Pongprom, N.; Mueller, G.; Schmidt, P.; Holzer, W.; Spreitzer, H. Chem. Monthly
2009, 140, 309.
17. Shanab, K.; Pongprom, N.; Wulz, E.; Holzer, W.; Spreitzer, H.; Schmidt, P.;
Aicher, B.; Mueller, G.; Guenther, E. Bioorg. Med. Chem. Lett. 2007, 17, 6091.
18. Cytotoxic/antiproliferative activity.
121.8, 124.2, 125.0, 125.8, 132.9, 137.5, 147.4, 149.4, 149.7, 155.4, 167.0, 180.3.
IR (KBr): mmax 3101, 2923, 2852, 1700, 1651, 1595, 1487, 1408, 821 cmꢀ1. MS:
m/z (% relative intensity) 283 (M+, 23), 255 (100), 227 (16), 220 (8), 192 (23),
165 (17), 138 (8), 114 (24), 100 (16), 88 (27), 76 (45), 57 (28), 43 (25). HRMS:
m/z calcd for C14H6ClN3NaO2: 306.0046 found: 306.0047.
Compound 3 Mp = 199–201 °C. 1H NMR (DMSO-d6): d (ppm): 3.82 (m, 2H),
4.57 (m, 2H), 5.05 (s, 1H), 7.38 (m, 1H), 7.77 (m, 1H), 7.94 (m, 1H), 8.09 (d,
J = 4.90 Hz, 1H), 8.76 (d, J = 4.90 Hz, 1H), 9.23, (d, J = 6.61 Hz, 1H), 9.91 (s, 1H).
13C NMR (DMSO-d6): d (ppm): 59.4, 79.7, 116.8, 117.6, 117.9, 119.7, 121.9,
128.3, 132.0, 134.0, 144.0, 149.1, 149.3, 151.5, 151.6, 169. IR (KBr): mmax 3250,
3038, 2919, 2847, 1651, 1630, 1604, 1501, 1423, 1400, 1253, 1077, 1041,
1005 cmꢀ1. MS: m/z (% relative intensity) 308 (M+, 9), 260 (53), 248 (64), 247
(77), 220 (100), 219 (43), 208 (30), 192 (23), 165 (21), 100 (18), 88 (32), 78
(100), 63 (29), 51 (66), 45 (48), 43 (49). HRMS: m/z calcd for C16H12N4Na O3:
331.0807 found: 331.0800.
Compound 4 Mp = 140–142 °C. 1H NMR (CDCl3): d (ppm): 2.34 (s, 6H), 2.80–
2.86 (t, J = 5.81 Hz, 2H), 4.69–4.75 (t, J = 5.81 Hz, 2H), 7.13–7.20 (m, 1H), 7.55–
7.63 (m, 1H), 7.77–7.82 (m, 1H), 8.12–8.14 (d, J = 4.92 Hz, 1H), 8.72–8.74 (d,
J = 5.04 Hz, 1H), 9.33–9.36 (m,1H), 10.00 (s, 1H). 13C NMR (CDCl3): d (ppm):
45.8, 57.8, 76.4, 115.9, 117.7, 117.9, 120.0, 122.2, 128.7, 131.1, 134.2, 144.2,
149.8, 150.1, 151.3, 152.2, 170.9. IR (KBr): mmax 3457, 3080, 3023, 2966, 2945,
2816, 2759, 1648, 1604, 1501, 1423, 1400, 1250, 1000 cmꢀ1. HRMS: m/z calcd
for C18H18N5O2: 336.1460 found: 336.1469.
Compound 5 Mp = 230–231 °C. 1H NMR (CDCl3): d (ppm): 2.34 (s, 6H), 2.62 (t,
J = 6.9 Hz, 2H), 3.31 (s, 3H), 3.92 (s, 2H), 7.18 (s,1H), 7.58 (d, J = 9.5 Hz, 1H), 7.75
(d, J = 9.5 Hz, 1H), 8.90 (s, 1H), 9.37 (s, 1H). 13C NMR (CDCl3): d (ppm): 37.3,
45.6, 48.3, 56.7, 99.7, 114.7, 118.3, 121.7, 124.8, 126.6, 132.7, 138.1, 147.8,
151.3, 153.8, 160.9, 170.1, 178.4. IR (KBr): mmax 2924, 2853, 1649, 1600, 1540,
730 cmꢀ1. HRMS: m/z calcd for C19H19ClN5O2 383.1149 found [MH+] 384.1222.
Compound 6 Mp = 283–285 °C. 1H NMR (CDCl3): d (ppm): 7.42 (m, 1H), 7.82
(m, 1H), 8.01 (m, 1H), 8.87 (m, 1H), 9.23 (m, 1H), 9.31(s, 1H). 13C NMR (CDCl3):
d (ppm): 117.5, 118.1, 120.7, 120.9, 124.7, 128.4, 132.7, 136.8, 145.3, 149.3,
150.6, 152.6, 166.8, 180.7. IR (KBr): mmax 3121, 3028, 2919, 1700, 1646, 1622,
1566, 1493, 1478, 1382, 1250, 1137, 770 cmꢀ1. MS: m/z (% relative intensity)
249 (M+, 11), 222 (11), 221 (45), 193 (15), 166 (8), 125 (13), 97 (25), 83 (35), 71
(33), 69 (33), 57 (68), 55 (49), 43 (100), 41 (36). HRMS: m/z calcd for
Assessment of cytotoxic/antiproliferative activity was conducted with human
cancer cell lines KB/HeLa (ATCC CCL17, cervix carcinoma), SKOV3 (ATCC HTB-
77, ovarian carcinoma) and NCI-H460 (NCI 503473, large cell lung cancer).
Measurement of the cellular cytotoxic/antiproliferative activity is based on the
dye Resazurin (Sigma, cat. no. R7017), which exhibits fluorescence change in
the appropriate oxidation–reduction range relating to cellular metabolic
reduction [Nociari et al., J. Immunol. Methods 1998 213, 157] yielding
a
fluorescence signal at 590 nm. The increase of fluorescence at 590 nM is an
indicator of cellular viability/cell number.
The cells were seeded in the respective growth medium recommended by the
supplier (media and reagents purchased from Gibco-BRL) in 125 ll per 96 well
and were grown for 24 h at 37 °C/5%CO2. Cell numbers were adapted for each
cell line to generate signals in the linear detection range under the
experimental conditions applied. After 45 h of compound incubation at
C14H7N3NaO2: 272.0436 found: 272.0433.
Compound 7 Mp = 216–218 °C. 1H NMR (DMSO-d6): d (ppm): 3.80–3.82 (m,
2H), 4.57–4.60 (m, 2H), 5.03 (s, 1H), 7.33–7.40 (m, 1H), 7.73–7.81 (m, 1H),
7.92–7.96 (m, 1H), 8.61–8.64 (m, 1H), 8.76–8.79 (m, 1H), 9.22–9.26 (m, 1H),
9.37 (s, 1H). 13C NMR (DMSO-d6): d (ppm): 59.2, 80.1, 116.5, 117.6, 121.5,
37 °C/5%CO2 15 ll of the Resazurin detection reagent (0.2 mg/mL in DPBS
(Gibco, 14190), steril filtered) was added for additional 3 h and after a total of
48 h of compound incubation cellular metabolic activity was quantified by
measurement of fluorescence at 590 nm. Non-treated cells and blank controls
w/o cells were set as reference values.
124.2, 128.3, 131.8, 131.9, 144.4, 145.5, 149.5, 151.7, 169.4. IR (KBr): mmax 3385,
2919, 2847, 1726, 1640, 1501, 1431, 1390, 1281, 1088, 1028, 889 cmꢀ1. MS: m/
z (% relative intensity) 308 (M+, 18), 264 (16), 260 (29), 248 (66), 247 (100), 220
(78), 219 (46), 206 (16), 192 (15), 165 (16), 88 (25), 78 (84), 51 (48), 45 (31).
HRMS: m/z calcd for C16H12N4O3Na: 331.0807 found: 331.0803.
MS EXCEL was used for formating and analysis of data. All data were calculated
as % efficacy compared to the mean of the respective negative (non-treated
cells) and positive control wells (blank) on each assay plate. EC50 values were
calculated by using non-linear regression software GraphPad Prism.
19. All of the final structures were confirmed by 1H NMR, 13C NMR, IR and MS as
the following.
Compound 8 Mp = 149–151 °C. 1H NMR (CDCl3): d (ppm): 2.3 (s, 6H), 2.78–2.84
(t, J = 5.81Hz, 2H), 4.68–4.74 (m, 2H), 7.10–7.16 (m, 1H), 7.57–7.61 (m, 1H),
7.75–7.61 (m, 1H), 8.56–8.59 (m, 1H), 8.70–8.72 (m, 1H), 9.29–9.32 (m, 1H),
9.50 (s, 1H). 13C NMR (CDCl3): d (ppm): 45.8, 57.9, 76.7, 115.6, 117.7, 119.1,
121.8, 124.2, 128.6, 131.1, 132.1, 144.6, 146.4, 150.0, 151.3, 151.7), 170.3. IR
Compound 1 Mp = 300–302 °C (decomp.). 1H NMR (CDCl3): d (ppm): 7.44 (m,
1H), 7.83 (m, 1H), 8.00 (d, J = 4.9 Hz, 1H), 8.02 (d, J = 4.9 Hz, 1H), 8.90 (d, J = 4.9
Hz, 1H), 9.03 (s, 1H), 9.23 (d, J = 6.6 Hz, 1H). 13C NMR (CDCl3): d (ppm): 117.6,
117.8, 118.4, 121.8, 125.3, 128.4, 132.7, 138.0, 149.2, 149.3, 149.6, 155.5, 166.9,
180.8. IR (KBr): mmax 3121, 2950, 2919, 2847, 1690, 1659, 1599, 1488, 1405,
1253, 899 cmꢀ1. MS: m/z (% relative intensity) 249 (M+, 17), 222 (15), 221
(100), 193 (39), 166 (23), 139 (19), 88 (39), 87 (19), 78 (41), 76 (22), 63 (26), 62
(27), 51 (56), 50 (26). HRMS: m/z calcd for C14H7N3NaO2: 272.0436 found:
272.0434.
(KBr): mmax 3405, 3131, 3033, 2940, 2852, 2816, 2764, 1651, 1633, 1498, 1429,
1256, 1026, 1000 cmꢀ1. HRMS: m/z calcd for C18H18N5O2: 336.1461 found:
336.1467.
Compound 9 Mp = 273 °C (decomp.). 1H NMR (CDCl3): d (ppm): 7.48 (m, 1H),
7.85 (m, 1H), 8.07 (m, 1H), 9.20 (s, 1H), 9.29 (m, 1H), 9.46 (s, 1H). 13C NMR
(CDCl3): d (ppm): 118.2, 118.9, 123.9, 124.7, 128.2, 132.6, 149.0, 149.1, 156.0,
156.2, 161.9, 166.7, 179.4. IR (KBr): mmax 3123, 3029, 2923, 1709, 1669, 1653,
1646, 1363 cmꢀ1. MS: m/z (% relative intensity) 250 (M+, 18), 222 (100), 194
(19), 167 (43), 84 (19), 78 (36), 51 (37). HRMS: m/z calcd for C13H6N4NaO2:
273.0388 found: 273.0387.
Compound 2 Mp = 309–310 °C. 1H NMR (CDCl3): d (ppm): 7.88 (dd, J = 2.1, 9.5
Hz, 1H), 8.02 (d, J = 5.0 Hz,1H), 8.05 (d, J = 9.5 Hz, 1H), 8.92 (d, J = 5.1 Hz, 1H),
9.07 (s, 1H), 9.24 (m, J = 2.1 Hz, 1H). 13C NMR (CDCl3): d (ppm): 117.5, 119.1,