Synthesis and potent cytotoxic activity of 8- and 9-anilinophenanthridine- 7,10-diones

Article abstract of DOI:10.1016/j.tetlet.2010.10.170

A series of 8-anilino and 9-anilinophenanthridine-7,10-diones was prepared and screened against various cancer cell lines to measure anti-proliferative activity. The compounds tested display potent cytotoxic activity in the micromolar and sub-micromolar r

Full text of DOI:10.1016/j.tetlet.2010.10.170

Tetrahedron Letters 52 (2011) 92–94
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and potent cytotoxic activity of 8-and 9-anilinophenanthridine-7,
10-diones
Paul H. Bernardo a, , Jasmeet K. Khanijou a, Tze Hau Lam b, Joo Chuan Tong b,c, Christina L. L. Chai a,d,
⇑
⇑
a Institute of Chemical & Engineering Sciences, Agency for Science Technology and Research (A*STAR), Chemistry@Neuros, 8 Biomedical Grove, Neuros #07-01,
Singapore 138665, Singapore
b Institute for Infocomm Research, A*STAR, 1 Fusionopolis Way, #21-01 Connexis, Singapore 138632, Singapore
c Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore
d Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2010
Revised 19 October 2010
Accepted 29 October 2010
Available online 4 November 2010
A series of 8-anilino and 9-anilinophenanthridine-7,10-diones was prepared and screened against vari-
ous cancer cell lines to measure anti-proliferative activity. The compounds tested display potent cyto-
toxic activity in the micromolar and sub-micromolar range. These compounds are promising new
leads for developing anticancer compounds.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Quinones
SRB assay
Anticancer
Michael addition
Quinones are a valuable class of compounds with interesting
physicochemical properties.1–3 The redox activity of compounds
such as ubiquinone (1) and menadione (2) play an important role
in electron transport within living cells.4 The redox activity of nat-
urally-occurring and synthetic quinones is important in drug de-
sign. For instance, quinones such as adriamycin (3),5–7 mitomycin
O
O
O
OH
O
OH
H3CO
OH
H3CO
H
O
O
O
O
OH
O
O
6-10
3
1
2
OH
NH2
O
H
N
O
OH
NH
O
O
HN
OH
OH
N
O
O
H
N
O
H2N
O
O
N
O
OH
HN
H2N
N
H
4
6
5
C (4)8,9, and mitoxantrone (5)10,11 possess potent anticancer activ-
ity and are employed as chemotherapeutic drugs.
Recent reports have shown that anilinoquinones such as 6 dis-
play micromolar activity against a range of cancer cell lines.12 In
this work, we present a short synthesis of 8- and 9-anilinophenan-
thridine-7,10-diones starting from commercially available 2,
5-dimethoxybenzoic acid (7). These compounds displayed
⇑
Corresponding authors. Address: Institute of Chemical & Engineering Sciences,
⇑
Agency for Science Technology and Research (A STAR), Chemistry@Neuros,
8
Biomedical Grove, Neuros #07-01, Singapore 138665, Singapore. Tel.: +65 6799
8533; fax: +65 6464 2102 (P.H.B.);tel.: +65 6796 8522; fax: +65 6464 2102 (C.L.L.C.).
E-mail addresses: paul_bernardo@ices.a-star.edu.sg, paulbernardo@hotmail.com
(P.H. Bernardo), Christina_Chai@ices.a-star.edu.sg (C.L.L. Chai).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.10.170

P. H. Bernardo et al. / Tetrahedron Letters 52 (2011) 92–94
93
O
O
O
O
O
O
O
O
CO2H
R
MOM
iv
i, ii
N
v
N
N
Br
O
O
7
8)
R=H
9) R=MOM
11
iii
10
vi
H
O
7
O
O
O
6
R1
R2
N
R2
6a
5
N
N
N
vii
8
9
+
4a
R1
N
H
4
3
10a
10
O
10b
O
1
13a: R1= R2= H
14a: R1= R2= H
2
12
13b: R1= H, R2= Cl
13c: R1= Br, R2= H
13d: R1= H, R2= Br
14b: R1= H, R2= Cl
14c: R1= Br, R2= H
14d: R1= H, R2= Br
14e: R1= OMe, R2= Br
13e: R1= OMe, R2= Br
Scheme 1. Reagents and conditions: (i) SOCl2, 80 °C, 1 h; (ii) 2-bromoaniline, K2CO3 (2 equiv), THF, rt, 16 h, 97%; (iii) NaH, THF, MOMCl, 40 °C, 16 h, 86%; (iv) 10 mol %
Pd(PPh3)4, K2CO3 (2 equiv), DMF, 140 °C, 92–96%; (v) LiAlH4 in dry Et2O for 1 h, then 1 N HCl, 90%; (vi) CAN, 1:1 CH3CN–H2O, 0 °C for 1 h, 78%; (vii) 0.5 equiv aniline, 1:1
CH3CN–H2O, 1% AcOH, rt, 16 h, 55–90%.
micromolar and sub-micromolar activities against various cancer
H
N
O
H
9.62
N
H 9.72
N
O
cell lines in the sulforhodamine B (SRB) assay. To our knowledge,
this is the first time such compounds have been reported in the lit-
erature, and several of the products presented herein are far more
potent than related anilinoquinones.12–14
6.41
H
183.7
185.0
182.6
186.7
H
N
H
6.48
The synthesis of the anilinophenanthridine-7,10-diones is out-
lined in Scheme 1. Starting from 2,5-dimethoxybenzoic acid (7),
the tertiary amide 9 was rapidly accessed in three steps and in high
yield following the literature procedure.15 Cyclization of the ter-
tiary amide 9 with catalytic Pd(0) in the presence of PPh3 gave
the desired phenanthridinone 10 in 96% yield. Compound 10 was
readily reduced to the corresponding phenanthridine 11 using
LiAlH4,15–17 and subsequently oxidized with cerium(IV) ammo-
nium nitrate at 0 °C to yield the quinone, phenanthridine-7,10-
dione (12), in 78% yield.
The initial addition of aniline to the quinone 12 was carried out
with a 1:1 ratio of reactants and resulted in 50% conversion of the
quinone into the products 13 and 14, and 50% conversion into the
reduced quinone (Scheme 2). This is presumably due to the forma-
tion of the adducts 15 and 16, which in turn rapidly react with 12
resulting in the formation of the anilinoquinones 13 and 14 and the
quinol 17. Upon work-up with aqueous NaHCO3, the quinol 17 is
oxidized by aerial oxygen and the quinone 12 is recovered. How-
ever, an attempt to push the conversion of 12 toward the desired
O
13a
O
14a
3
Figure 1. Key JCH correlations for 13a and 14a obtained by HMBC.
products by bubbling air through the reaction mixture was not
successful. Thus for optimal yields, only 0.5 equiv of the aniline
was required. Stirring the reaction mixture at room temperature
for 16 h resulted in a mixture of 8-anilinophenanthridin-7,10-
diones (13a–e) and the 9-anilino isomers (14a–e, Scheme 1). Com-
parison of the integrations of the singlet quinonic proton signals in
the d 6.3–6.5 range of the 1H NMR spectra showed that roughly a
1:1 mixture was obtained in each reaction. The compounds were
separated and purified using C18 reverse-phase preparatory HPLC.
Assignment of the isomers was accomplished by HMBC (Fig. 1).
3
Specifically, the 8-anilino compounds had JCH signals for H(6)
and H(9) coupled to same carbonyl, C(7). On the other hand, the
9-anilino compounds had distinct non-overlapping JCH signals
for H(6) to C(7) and H(8) to C(10). The JCH couplings were not
3
2
OH
N
N
H
OH
OH
15
12
N
13a 14a
+
+
H
N
OH
12
+ aniline
slow
fast
N
OH
17
OH
16
Scheme 2. Reaction of quinone 12 with aniline.

94
P. H. Bernardo et al. / Tetrahedron Letters 52 (2011) 92–94
Table 1
Growth inhibition (GI50) of test compounds against cancer cell lines. NT = Not tested due to poor solubility
GI50 SEM ( M) determined via SRB assay
l
Substrate
MCF-7
NCI-H460
SF268
A549
DU145
A8
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
Taxol
0.094 0.008
0.697 0.014
0.678 0.029
0.676 0.014
0.065 0.002
0.338 0.003
0.396 0.003
NT
0.062 0.001
0.561 0.017
0.265 0.004
0.554 0.012
0.060 0.002
0.933 0.021
0.633 0.002
NT
0.633 0.043
6.283 1.322
0.858 0.020
1.289 0.413
0.098 0.015
0.557 0.032
0.553 0.024
NT
0.184 0.006
0.848 0.026
0.642 0.021
0.667 0.034
0.077 0.004
2.013 0.776
0.910 0.119
NT
0.086 0.000
0.680 0.016
0.569 0.035
0.671 0.022
0.061 0.007
2.430 0.193
0.722 0.025
NT
0.070 0.011
0.586 0.048
0.498 0.072
0.584 0.05
0.061 0.006
0.490 0.107
0.546 0.139
NT
2.981 0.029
0.400 0.037
0.008 0.000
4.477 1.249
0.686 0.79
0.007 0.000
3.947 0.588
0.552 0.013
0.050 0.010
5.139 0.114
0.821 0.006
0.041 0.012
3.149 0.728
0.691 0.023
0.036 0.012
1.905 0.161
0.520 0.074
0.058 0.001
2. Abegaz, B. M. Phytochem. Rev. 2002, 1, 299–310.
3. Bolton, J. L.; Trush, M. A.; Penning, T. M.; Dryhurst, G.; Monks, T. J. Chem. Res.
Toxicol. 2000, 13, 135–160.
observed. Interestingly, the quinonic H(8) chemical shifts were
0.05–0.07 ppm lower than the H(9) chemical shifts.
The purified compounds 13a–e and 14a–e were screened
against various cancer cells lines using the SRB assay to determine
the growth inhibition.18,19 The human cancer cell lines assayed
were breast (MCF-7), lung (NCI-H460, A549), brain (SF268), pros-
tate (DU145), and epothilone-resistant ovarian cancer cells (A8).
All compounds were tested in triplicate. In this assay, the cells
were incubated in a 96-well plate with varying concentrations of
the test compounds (10 lM down to 0.001 lM) for 48 h, then fixed
with 50% TFA. The cells were washed with distilled water and fixed
with SRB. After washing away excess SRB, the remaining protein-
bound SRB was solubilized with Tris base (100 lL, 10 mM) and
4. Monks, T. J.; Hanzlik, R. P.; Cohen, G. M.; Ross, D.; Graham, D. G. Toxicol. Appl.
Pharmacol. 1992, 112, 2–16.
5. Swenton, J. S.; Raynolds, P. W. J. Am. Chem. Soc. 1978, 100, 6188–6195.
6. Keizer, H. G.; Pinedo, H. M.; Schuurhuis, G. J.; Joenje, H. Pharmacol. Ther. 1990,
47, 219–231.
7. Tritton, T. R. Curr. Commun. Cell Mol. Biol. 1991, 3, 121–137.
8. Tomasz, M. Chem. Biol. 1995, 2, 575–579.
9. Bradner, W. T. Cancer Treat. Rev. 2001, 27, 35–50.
10. Hagemeister, F.; Cabanillas, F.; Coleman, M.; Gregory, S. A.; Zinzani, P. L.
Oncologist 2005, 10, 150–159.
11. Martincic, D.; Hande, K. R. Cancer Chemother. Biol. Response Modif. 2005, 22,
101–121.
12. Valderrama, J. A.; Ibacache, J. A. Tetrahedron Lett. 2009, 50, 4361–4363.
13. Valderrama, J. A.; Ibacache, J. A.; Arancibia, V.; Rodriguez, J.; Theoduloz, C.
Bioorg. Med. Chem. 2009, 17, 2894–2901.
14. Vásquez, D.; Rodríguez, J. A.; Theoduloz, C.; Verrax, J.; Calderon, P. B.;
Valderrama, J. A. Bioorg. Med. Chem. Lett. 2009, 19, 5060–5062.
15. Bernardo, P. H.; Fitriyanto, W.; Chai, C. L. L. Synlett 2007, 1935–1939.
16. Bernardo, P. H.; Wan, K.-F.; Sivaraman, T.; Xu, J.; Moore, F. K.; Hung, A. W.;
Mok, H. Y. K.; Yu, V. C.; Chai, C. L. L. J. Med. Chem. 2008, 51, 6699–6710.
17. Harayama, T.; Akamatsu, H.; Okamura, K.; Miyagoe, T.; Akiyama, T.; Abe, H.;
Takeuchi, Y. J. Chem. Soc., Perkin Trans. 1 2001, 523–528.
18. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107–1112.
19. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757–766.
the relative protein concentrations were determined spectrometri-
cally at 515 nm. The results, reported as the concentration required
for 50% growth inhibition (GI50), are shown in Table 1.
The results show that most of the compounds were active at
single-digit and sub-micromolar ranges against a variety of cancer
cells. Compound 14c could not be tested due to its poor solubility.
Overall, compound 13e was the most potent synthetic compound
against all the cell lines, while compound 14d was the least active.
Compound 13a was the second best inhibitor. Compounds 13a20
and 13e21 show similar activity against the A8 cell line as Taxol,
but were ten-fold less effective against SF268. Within the 8-anilino
series, product 13e was the most active followed by 13a while
compounds 13b–d shared similar activities. No such trend was
observed with the 9-anilino series in which 14a,22 14b, and
14e23 all had very similar activities within the standard deviations
observed. Only the bromo compound 14d displayed strong attenu-
ation of activity within the series.
In summary, we have outlined a brief synthesis of 8- and 9-ani-
linophenanthridine-7,10-diones starting from 2,5-dimethoxyben-
zoic acid and various anilines. These compounds display
promising cytotoxic activity against several cancer cell lines and
may be useful as leads for anticancer compounds. Further work is
underway to determine the mechanism of action of these new qui-
nones as well as to optimize desirable physicochemical properties.
20. Compound 13a: mp 211–212 °C. Rf 0.41 (1:3 EtOAc/light petroleum). 1H NMR
(CDCl3, 400 MHz): d 6.48 (s, 1H, C–H), 7.30 (m, 3H, Ar–H), 7.46 (m, 2H, Ar–H),
7.52 (s, 1H, N–H), 7.75 (app. t, J = 7 Hz, 1H, Ar–H), 7.87 (app. t, J = 8 Hz, 1H, Ar–
H), 8.18 (d, J = 8 Hz, 1H, Ar–H), 9.61 (d, J = 6 Hz, 1H, Ar–H), 9.62 (s, 1H, Ar–H).
13C NMR (CDCl3, 100 MHz): d 105.1, 122.0, 122.6, 123.0, 125.9, 129.1, 129.8,
129.9, 130.1, 132.3, 133.7, 137.1, 142.9, 147.0, 152.8, 182.6 (CO), 186.7 (CO).
ESI-MS m/z 301 ([M+H]+, 100). HRMS calcd for C19H13N2O2: 301.0972 [M+H]+;
found: 301.0982.
21. Compound 13e: mp: 230–232 °C. 1H NMR (CDCl3, 400 MHz): d 3.94 (s, 3H, CH3),
6.45 (s, 1H, C–H), 6.81 (m, 2H, Ar–H), 7.49 (s, 1H, N–H), 7.59 (d, J = 8 Hz, 1H, Ar–
H), 7.75 (app. t, J = 8 Hz, 1H, Ar–H), 7.88 (app. t, J = 8 Hz, 1H, Ar–H), 8.17 (d,
J = 8 Hz, 1H, Ar–H), 9.59 (d, J = 8 Hz, 1H, Ar–H), 9.61 (s, 1H, Ar–H). 13C NMR
(CDCl3, 100 MHz): d 56.4, 105.8, 106.5, 108.4, 115.6, 120.2, 121.9, 122.9, 129.0,
130.0, 130.1, 132.4, 134.1, 137.6, 142.5, 147.0, 152.8, 156.8, 182.3 (CO), 186.7
(CO). ESI-MS m/z 409 ([M+H]+, 79Br, 100), 411 ([M+H]+, 81Br, 98). HRMS calcd
for C20H1479BrN2O3: 409.0182 [M+H]+; found: 409.0186.
22. Compound 14a: mp 243–244 °C. Rf 0.41 (1:3 EtOAc/light petroleum). 1H NMR
(CDCl3, 400 MHz): d 6.41 (s, 1H, C–H), 7.31 (m, 3H, Ar–H), 7.47 (m, 2H, Ar–H),
7.64 (s, 1H, N–H), 7.82 (app. t, J = 9 Hz, 1H, Ar–H), 7.88 (app. t, J = 8 Hz, 1H, Ar–
H), 8.23 (d, J = 8 Hz, 1H, Ar–H), 9.45 (d, J = 9 Hz, 1H, Ar–H), 9.72 (s, 1H, Ar–H).
13C NMR (CDCl3, 100 MHz): d 101.4, 122.0, 123.0, 123.6, 126.1, 126.8, 129.8,
129.9, 130.6, 130.7, 131.4, 137.1, 145.3, 148.0, 151.4, 183.7 (CO), 185.0 (CO).
ESI-MS m/z 301 ([M+H]+, 100). HRMS calcd for C19H13N2O2: 301.0972 [M+H]+;
found: 301.0983.
Acknowledgments
We thank the Agency for Science, Technology and Research
(A*STAR), Singapore, for funding this project. We also thank Sum
Rongji and Jessie Lim of the Chemical Synthesis Laboratory@ Biop-
olis (A*STAR) for carrying out the biological assays.
23. Compound 14e: mp: 240–241 °C. Rf 0.33 (1:3 EtOAc/hexanes). 1H NMR (CDCl3,
400 MHz): d 3.94 (s, 3H, CH3), 6.40 (s, 1H, C–H), 6.84 (m, 2H, Ar–H), 7.60 (m,
2H, Ar–H, N–H), 7.83 (app. t, J = 8 Hz, 1H, Ar–H), 7.89 (app. t, J = 8 Hz, 1H,
Ar–H), 8.24 (d, J = 8 Hz, 1H, Ar–H), 9.43 (d, J = 8 Hz, 1H, Ar–H), 9.72 (s, 1H, Ar–
H). 13C NMR (CDCl3, 100 MHz): d 56.5, 102.1, 106.8, 108.7, 116.0, 122.0, 123.4,
126.8, 129.8, 130.6, 130.8, 131.5, 134.1, 137.6, 144.9, 148.0, 151.5, 156.9, 183.7
(CO), 184.7 (CO). ESI-MS m/z 409 ([M+H]+, 79Br, 100), 411 ([M+H]+, 81Br, 100).
HRMS calcd for C20H1479BrN2O3: 409.0182 [M+H]+; found: 409.0192.
References and notes
1. Thomson, R. H. In Naturally Occurring Quinones; Thomson, R. H., Ed.; Academic
Press: London, 1971; pp 1–38.