Photoinduced DNA cleavage by anthracene based hydroxamic acids

Article abstract of DOI:10.1016/j.bmcl.2012.05.084

Two different series of naphthalene and anthracene based hydroxamic acids having amino acid derivatives were synthesized. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroxamic acids by UV light (≥350 nm). Both reactive oxy

Full text of DOI:10.1016/j.bmcl.2012.05.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4668–4671
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Bioorganic & Medicinal Chemistry Letters
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Photoinduced DNA cleavage by anthracene based hydroxamic acids
⇑
Nilanjana Chowdhury, Swagata Dasgupta, N. D. Pradeep Singh
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two different series of naphthalene and anthracene based hydroxamic acids having amino acid deriva-
tives were synthesized. Single strand DNA cleavage was achieved on irradiation of newly synthesized
hydroxamic acids by UV light (P350 nm). Both reactive oxygen species (ROS) and generated radicals
from hydroxamic acids were shown to be responsible for the DNA cleavage. Further, DNA cleaving ability
of hydroxamic acids was found to be dependent on its concentration and on its structure.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 31 January 2012
Revised 23 April 2012
Accepted 23 May 2012
Available online 30 May 2012
Keywords:
Hydroxamic acid
DNA cleavage
UV light
Hydroxamic acids are a family of weak organic acids having
general formula RCONR0OH. They are generally considered to be
derivatives of hydroxylamine and carboxylic acids. Hydroxamic
acids and its derivatives have attracted considerable attention
due to their wide spectrum of biological activities such as anti-
inflammatory, antimetastatic, anti-asthmatic, anti-biotic, psycho-
tropic, insecticidal and nematocidal activity.1
During the last few decades, there has been continuous upsurge
of interest in the development of hydroxamic acid derivatives as
potential anticancer agents mainly due to their ability to inhibit
Histone deacetylase (HDAC).2 In 2001, Richon and its co-worker3
reported that suberoylanilide hydroxamic acid (SAHA) as HDAC
inhibitor that induces cell cycle arrest, differentiation and/or apop-
tosis in transformed cultured cell lines. Recently, SAHA has been
approved by the FDA for the treatment of cutaneous T-cell lym-
phoma.4 Further, several groups have also reported potent HDAC
inhibitors based on hydroxamic acid analogous as promising che-
motherapeutic agents such as trichostatin A,5 scriptaid6 and oxa-
mflatin.7 Apart from HDAC inhibitors other types of hydroxamic
acid derivatives are also known which causes DNA damage using
their hydrolytic and oxidative capabilities in the presence of metal
complexes.8
of photodisassociation mechanism has been reported on direct
irradiation of hydroxamic acid (i) homolytic N–O bond cleavage
from the singlet excited state resulting in the formation of anilides
and amides,10 (ii) generation of primary intermediate acylaminyl
oxyl radicals by loss of hydrogen radical from hydroxyl group.11
The promising anticarcinogenic ability and facile photocleavage
property of hydroxamic acids tempted us to investigate their
DNA cleaving ability under light.
Synthesis of hydroxamic acids, 3a–d of series-I were carried out
as depicted in Scheme 1. First, reductive amination of aldehydes
1a–b with amino acid esters furnished desired esters 2a–d. Next,
treatment of esters 2a–d with NH2OHꢀHCl in methanol/1,4
-dioxane mixture provided hydroxamic acids 3a–d in moderate
to good yield.
Second series of hydroxamic acid derivatives, 6a–d, were syn-
thesized using the procedure as shown in Scheme 2. Cinnamic
acids 4a–b were initially converted to corresponding acid chlorides
by using oxalyl chloride in DCM in presence of catalytic amount of
DMF. The freshly prepared acid chlorides were then treated with
various amino acid esters in presence of triethyl amine to produce
corresponding condensation products 5a–d. Treatment of 5a–d
with NH2OHꢀHCl in methanol/1,4-dioxane mixed solvent resulted
hydroxamic acids 6a–d in moderate to good yields. Hydroxamic
acids 3a–d and 6a–d were characterized by NMR, mass spectrum
and elemental analysis.
There have been an increasing number of reports concerning
light-induced DNA scission by a variety of metal complexes having
hydroxamic acid groups attached to the ligand system.8 But, till
date there has been no report on the photoinduced DNA cleaving
ability of hydroxamic acids in the absence of metal complexes.
After 1980s, significant progress has been made to understand
the photochemical behavior of hydroxamic acids.9 So far two types
The UV/vis absorption and emission spectra of degassed 4 ꢁ
10ꢂ6 M solution of hydroxamic acids (3a–d and 6a–d) in absolute
ethanol were recorded. The absorption and emission maxima,
molar absorptivities (e) and fluorescence quantum yield (UF) of
above hydroxamic acids were summarized in Table 1. Fluorescence
quantum yields were calculated using anthracene as standard
(
⇑
Corresponding author. Tel.: +91 3222 282324; fax: +91 3222 282252.
E-mail address: ndpradeep@chem.iitkgp.ernet.in (N.D. Pradeep Singh).
UF = 0.27 in ethanol).12
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.084

N. Chowdhury et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4668–4671
4669
R2
dium phosphate buffer (pH 7.0, 10 mM) with 50% DMF
containing the pBR322 plasmid DNA (form I; 62.5 g/ml) for 1 h
NH R2(CH)CO Me,
O
DCM, reflux, 24 h
1.
2
2
l
R1
N
CO2Me
R1
1a-b
H
H
under aerobic condition. By analyzing the results from gel electro-
phoresis on 1.0% agarose gel with ethidium bromide staining, we
found that compound 3b caused the single strand cleavage of
DNA (form I) to give the relaxed circular DNA (form II) and the
DNA cleaving ability of 3b enhanced with the increase of its con-
centration (Fig. 1). Based on the above concentration study, the
DNA cleaving ability of all hydroxamic acids were carried out using
2. NaBH4, MeOH, 30 min
2a-d
R1 = 1-C10H7, 9-C14H9
R2 = C6H5, CH2C6H5, CH2C8H6N
R2
R1
N
H
3a-d
CONHOH
NH
NH2OH.HCl, (2.0 equiv), KOH (4.0 equiv)
MeOH/1,4-Dioxane (1:1), 0 °C -rt, 3 h
100 lM concentration for a period of 1 h and the results are
Ph
Ph
CONHOH
2
4
10
25 67
97 % of form II
HOHNOC
CONHOH Ph
NH
CONHOH
NH
form II
NH
NH
form I
lane
1
2
3
4
5
6
7
3a; 72%
3b; 83%
3c; 84%
3d; 66%
Figure 1. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to
relaxed circular DNA (form II) was carried out by compound 3b at different
concentration (10–100 lM) in a sodium phosphate buffer (pH 7.0, 10 mM) upon
Scheme 1. Synthesis of naphthalene and anthracene based hydroxamic acids 3a–d.
irradiation of UV light (P350 nm) under aerobic condition at room temperature for
1 h. The resultant products were subjected to electrophoresis on 1% agarose gel
followed by ethidium bromide staining under UV light. Lane 1, DNA alone; lane 2,
To find out the optimum concentration for DNA cleavage by the
above mentioned hydroxamic acids, initially we irradiated com-
pound 3b at different concentration (10–100 lM) dissolved in so-
DNA + 3b (10
DNA + 3b (50
l
M); lane 3, DNA + 3b (25
l
M); lane 4, DNA + 3b (40
l
l
M); lane 5,
M).
lM); lane 6, DNA + 3b (75
lM); lane 7, DNA + 3b (100
1. (COCl)2 (1.2 equiv), DMF (0.1 equiv),
DCM, 0 °C -rt, 3 h
O
R2
COOH
4a-b
R1
R1
N
CO2Me
NH R2(CH)CO Me,
2.
Et3N (3 equiv), DCM, 0 °C -rt, 0.5 h
H
2
2
5a-d
R1 = 1- C10H7, 9-C14H9 R2 = C6H5, CH2C6H5, CH2C8H6N
O
R2
NH2OH.HCl, (2.0 equiv), KOH (4.0 equiv),
R1
N
CONHOH
HN
H
MeOH/1,4-Dioxane (1:1)
0 °C -rt, 3 h
6a-d
Ph
H
Ph
H
H
H
O
N
O
N
O
N
Ph
O
N
CONHOH
CONHOH
CONHOH
CONHOH
6a; 75%
6b; 77%
6d; 67%
6c; 81%
Scheme 2. Synthesis of naphthalene and anthracene based hydroxamic acids 6a–d.
Table 1
UV/vis and fluorescence data of hydroxamic acids (3a–d and 6a–d) in absolute ethanol
Entry
Compound
UV/vis
Fluorescence
a
c
e
kmax (nm)
log eb
kmax (nm)
Stokes’ shiftd (nm)
UF
1
2
3
4
5
6
7
8
3a
3b
3c
3d
6a
6b
6c
6d
315
384
385
387
317
390
390
391
4.00
3.84
3.98
3.75
4.06
3.93
3.84
3.72
—
—
28
28
25
—
412
413
412
—
503
502
504
0.012
0.010
0.008
—
0.015
0.018
0.010
—
113
112
113
a
b
c
Maximum absorption wavelength.
Molar absorption coefficient (in molꢂ1 L cmꢂ1) at the maximum absorption wavelength.
Maximum emission wavelength.
d
e
Difference between maximum absorption wavelength and maximum emission wavelength.
Fluorescence quantum yield (error limit within 5%).

4670
N. Chowdhury et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4668–4671
16
97
98
91
15
98
99
89 % of form II
form II
form I
lane
1
2
3
4
5
6
7
8
9
Figure 2. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to relaxed circular DNA (form II) was carried out by compounds 3a–d and 6a–d (100 lM) upon
irradiation of UV light (P350 nm) in a sodium phosphate buffer (pH 7.0, 10 mM) under aerobic condition at room temperature for 1 h. The resultant products were subjected
to electrophoresis on 1% agarose gel followed by ethidium bromide staining under UV light. Lane 1, DNA alone; lane 2, DNA + 3a; lane 3, DNA + 3b; lane 4, DNA + 3c; lane 5,
DNA + 3d; lane 6, DNA + 6a; lane 7, DNA + 6b; lane 8, DNA + 6c; lane 9, DNA + 6d.
presented in Figure 2. All hydroxamic acids showed remarkable
DNA cleaving ability except naphthalene based hydroxamic acids
3a and 6a. Anthracene based hydroxamic acids containing phenyl-
alanine and phenylglycine amino acid residue showed better DNA
cleaving ability. Both series I and series II compounds exhibited al-
most similar DNA cleaving ability.
photocleavage of DNA. The singlet oxygen formation was also con-
firmed from the enhancement of the DNA cleavage activity in D2O
due to longer lifetime of 1O2 in D2O medium15 (Fig. 4).
To investigate the role of radicals generated from hydroxamic
acid in the DNA photocleavage process, we carried out control
experiment in presence of ascorbic acid, a known radical scaven-
ger. Figure 4 showed that addition of ascorbic acid to supercoiled
circular DNA partially inhibited the photoinduced DNA cleavage
ability of 3b.
Finally, To understand the essential role of hydroxamic acid
moiety in the photoinduced DNA cleavage process, we have syn-
thesized compound 7 and 8 [which are analogous to compound
3b and 6b except hydroxamic acid (CONHOH) moiety was replaced
by carboxylic acid (COOH)] as shown in Scheme 3 (for synthetic
procedure see Supplementary data).
To understand the mechanistic aspects of the photoinduced
DNA cleavage by hydroxamic acids, compound 3b was studied un-
der different conditions. The DNA photocleavage reactions involv-
ing molecular oxygen could proceed via two major pathways, viz
the type-II process forming singlet oxygen (1O2) species or a
photo-redox pathway forming reactive hydroxyl radicals. To
understand the involvement of the singlet oxygen during the
course of DNA cleavage, control experiments were carried out
using NaN3, a known singlet oxygen quencher.13 The experiments
were carried out by adding increasing amounts of NaN3 in the
phosphate buffer solution containing compound 3b and super-
coiled circular pBR322 DNA. The percentage of form II DNA de-
creased from 97% to 64% on increasing the amount of NaN3 from
0 to150 mM (Fig. 3). Further increase in the amount of NaN3 from
150 to 600 mM has no effect on the percentage of form II DNA.
Thus the contribution resulting from singlet oxygen to DNA cleav-
age is ꢃ33%.
To compare the DNA cleaving ability of carboxylic acids 7 and 8
with corresponding hydroxamic acids 3b and 6b, we irradiated
supercoiled circular pBR322 DNA for 1 h in presence of 100 lM
of above said acids individually and the results were presented in
Figure 5. The results revealed that hydroxamic acids 3b and 6b
showed better DNA cleaving ability compared to their correspond-
ing acid analogous 7 and 8 indicating the importance of hydroxa-
mic acid moiety in DNA cleavage process.
In conclusion, we have reported the simple, convenient and
high yielding synthesis of naphthalene and anthracene based
hydroxamic acids of amino acid derivatives. The gel electrophore-
sis data showed concentration and substrate dependent DNA
Further, hydroxyl radical scavengers, viz DMSO also showed
partial inhibition in the DNA cleavage activity14 (Fig. 4). The above
results suggested that formation of both singlet oxygen and hydro-
xyl radicals as reactive oxygen species (ROS) are responsible for the
100
80
60
40
20
0
100
80
60
40
20
0
Figure 3. Bar diagram displaying the single strand cleavage of supercoiled circular
Figure 4. Bar diagram displaying the single strand cleavage of supercoiled circular
pBR322 DNA (form I) to relaxed circular DNA (form II) by compounds 3b (100
lM)
pBR322 DNA (form I) to relaxed circular DNA (form II) by compounds 3b (100 lM)
in presence of increasing amount of NaN3 (20–600 mM) in a sodium phosphate
buffer (pH 7.0, 10 mM) upon irradiation of UV light (P350 nm) under aerobic
condition at room temperature for 1 h. The resultant products were subjected to
electrophoresis on 1% agarose gel followed by ethidium bromide staining under UV
light.
in presence of various additives in a sodium phosphate buffer (pH 7.0, 10 mM) upon
irradiation of UV light (P350 nm) under aerobic condition at room temperature for
1 h. The resultant products were subjected to electrophoresis on 1% agarose gel
followed by ethidium bromide staining under UV light. The additive concentra-
tions/quantities are: D2O, 4 lL; DABCO, 50 mM; DMSO, 4 lL, Ascorbic acid, 50 mM.

N. Chowdhury et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4668–4671
4671
Ph
Ph
Supplementary data
CO2Me
NH
COOH
NH
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
05.084.
NaOH(4.0 equiv)
MeOH/H2O
(5:1)
References and notes
2b
7
1. (a) Neilands, J. B. Science 1967, 156, 1443; (b) Weber, G. Cancer Res. 1983, 43,
3466; (c) Miller, M. J. Chem. Rev. 1989, 89, 1563.
Ph
Ph
H
H
2. (a) Hockly, E.; Richon, V. M.; Woodman, B.; Smith, D. L.; Zhou, X.; Rosa, E.;
Sathasivam, K.; Ghazi-Noori, S.; Mahal, A.; Lowden, P. A. S.; Steffan, J. S.; Marsh,
J. L.; Thompson, L. M.; Lewis, C. M.; Marks, P. A.; Bates, G. P. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 2041; (b) Kelly, W. K.; Marks, P. A. Nat. Rev. Clin. Oncol. 2005, 2,
150; (c) Bieliauskas, A. V.; Weerasinghe, S. V. W.; Pflum, M. K. H. Bioorg. Med.
Chem. Lett. 2007, 17, 2216; (d) Yin, D.; Ong, J. M.; Hu, J.; Desmond, J. C.;
Kawamata, N.; Konda, B. M.; Black, K. L.; Koeffler, H. P. Clin. Cancer Res. 2007, 13,
1045; (e) Fiskus, W.; Ren, Y.; Mohapatra, A.; Bali, P.; Mandawat, A.; Rao, R.;
Herger, B.; Yang, Y.; Atadja, P.; Wu, J.; Bhalla, K. Clin. Cancer Res. 2007, 13, 4882;
(f) Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R. Clin. Cancer Res. 2001, 7,
759; (g) Hebbel, R. P.; Vercellotti, G. M.; Pace, B. S.; Solovey, A. N.; Kollander, R.;
Abanonu, C. F.; Nguyen, J.; Vineyard, J. V.; Belcher, J. D.; Abdulla, F.; Osifuye, S.;
Eaton, J. W.; Kelm, R. J., Jr.; Slungaard, A. Blood 2010, 115, 2483; (h) Acharya, M.
R.; Sparreboom, A.; Venitz, J.; Figg, W. D. Mol. Pharmacol. 2005, 68, 917.
3. Butler, L. M.; Zhou, X.; Xu, W. –S.; Scher, H. I.; Rifkind, R. A.; Marks, P. A.;
Richon, V. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11700.
O
N
O
N
COOH
CO2Me
NaOH(4.0 equiv)
MeOH/H2O
(5:1)
8
5b
Scheme 3. Synthesis of naphthalene and anthracene based carboxylic acids 7 and 8.
100
80
60
40
20
0
4. (a) Mann, B. S.; Johnson, J. R.; Cohen, M. H.; Justiceand, R.; Pazdur, R. Oncologist
2007, 12, 1247; (b) Saharan, R.; Singh, R.; Nagar, N.; Verma, S.; Sharma, V.;
Yadav, S.; Nagori, B. P. Pharmacophore 2011, 2, 104.
5. (a) Vigushin, D. M.; Ali, S.; Pace, P. E.; Mirsaidi, N.; Ito, K.; Adcock, I.; Coombes,
R. C. Clin. Cancer Res. 2001, 7, 971; (b) Platta, C. S.; Greenblatt, D. Y.;
Kunnimalaiyaan, M.; Chen, H. J. Surg. Res. 2007, 168, 197; (c) Zhang, X.; Yashiro,
M.; Ren, J.; Hirakawa, K. Oncol. Rep. 2006, 16, 563; (d) Ailenberg, M.; Silverman,
M. Biochem. Biophys. Res. Commun. 2002, 298, 110; (e) Yoshida, M.; Horinouchi,
S.; Beppu, T. BioEssays 1995, 17, 423.
6. (a) Kuribyashi, T.; Maki ohara, M.; Sora, S.; Kubota, N. Int. J. Mol. Med. 2010, 25,
25; (b) Thuan, N. V.; Bui, H. T.; Kim, J. H.; Hikichi, T.; Wakayama, S.; Kishigami,
S.; Mizutani, E.; Wakayama, T. Reproduction 2009, 138, 309; (c) Lee, E. J.; Lee, B.
B.; Kim, S. J.; Park, Y. D.; Park, J.; Kim, D. H. Int. J. Oncol. 2008, 33, 767; (d) Ying,
H.; Zhang, Y.; Lin, S.; Han, Y.; Zhu, Z. H. Int. J. Mol. Med. 2010, 26, 265; (e) Strobl,
J. S.; Cassell, M.; Mitchell, S. M.; Reilly, C. M.; Lindsay, D. S. J. Parasitol. 2007, 93,
694.
7. (a) Kim, Y. B.; Lee, K. H.; Sugita, K.; Yoshida, M.; Horinouchi, S. Oncogene 1999,
18, 2461; (b) Dear, A. E.; Medcalf, R. L. Biochim. Biophys. Acta 2000, 1492, 15; (c)
Sonoda, H.; Nishida, K.; Yoshioka, T.; Ohtani, M.; Sugita, K. Oncogene 1996, 13,
143; (d) Dear, A. E.; Liu, H. B.; Mayes, P. A.; Perlmutter, P. Org. Biomol. Chem.
2006, 4, 3778.
6b
3b
8
7
8. (a) Hashimoto, S.; Nakamura, Y. J. Chem. Soc., Chem. Commun. 1995, 1413; (b)
Hashimoto, S.; Nakamura, Y. J. Chem. Soc., Perkin Trans. 1 1996, 2623; (c)
Hashimoto, S.; Itai, K.; Takeuchi, Y.; Nakamura, Y. Nucleic Acids Symp. Ser. 1997,
37, 7; (d) Hashimoto, S.; Nakamura, Y. Chem. Pharm. Bull. 1998, 46, 1941; (e)
Hashimoto, S.; Inui, T.; Nakamura, Y. Chem. Pharm. Bull. 2000, 48, 603.
9. (a) Kochany, E. L. Chem. Rev. 1991, 91, 477; (b) Sałdyka, M.; Mielke, Z. Phys.
Chem. Chem. Phys. 2003, 5, 4790; (c) Kaczor, A.; Szczepanski, J.; Vala, M.;
Proniewicz, L. M. Phys. Chem. Chem. Phys. 2005, 7, 1960.
Figure 5. Bar diagram displaying the single strand cleavage of supercoiled circular
pBR322 DNA (form I) to relaxed circular DNA (form II) by compounds 3b, 7, 6b and
8 (100 lM) in a sodium phosphate buffer (pH 7.0, 10 mM) upon irradiation of UV
light (P350 nm) under aerobic condition at room temperature for 1 h. The resultant
products were subjected to electrophoresis on 1% agarose gel followed by ethidium
bromide staining under UV light.
10. Walling, C.; Naglieri, A. N. J. Am. Chem. Soc. 1960, 82, 1820.
11. (a) Lipczynska-Kochany, E.; Kochany, J. J. Photochem. 1987, 38, 331; (b)
Lipczynska-Kochany, E.; Kochany, J. J. Photochem. Photobiol. 1988, 44, 317.
12. Endo, A.; Sujki, K.; Yoshihara, T.; Tobita, S.; Yahiro, M.; Adachi, C. Chem. Phys.
Lett. 2008, 460, 155.
cleaving ability of hydroxamic acids in the presence of UV light
(P350 nm).
13. (a) Deby-Dupont, G.; Deby, C.; Mouithys-Mickalad, A.; Hoebeke, M.; Jadoul, L.;
Vandenberghe, A.; Lamy, M. Biochim. Biophys. Acta 1998, 1379, 61; (b) Hwu, J.
R.; Lin, C. C.; Chuang, C. H.; King, K. Y.; Su, T. R.; Tsay, S. C. Bioorg. Med. Chem.
2004, 12, 2509.
14. Hussain, A.; Gadadhar, S.; Goswami, T. K.; Karande, A. A.; Chakravarty, A. R.
Dalton Trans. 2012, 41, 885.
Acknowledgments
We thank DST (SERC Fast Track Scheme) for financial support,
DST-FIST for 400 MHz NMR. Nilanjana is thankful to UGC for re-
search fellowship.
15. (a) Khan, A. U. J. Phys. Chem. 1976, 80, 2219; (b) Merkel, P. B.; Kearns, D. R. J. Am.
Chem. Soc. 1972, 94, 1029.