N,O-Diacyl-4-benzoyl-N-phenylhydroxylamines as photoinduced DNA cleaving agents

Article abstract of DOI:10.1016/j.bmcl.2010.07.116

Photoinduced homolytic fission of nitrogen-oxygen bond in N,O-diacyl-4-benzoyl-N-phenylhydroxylamines using ≥310 nm UV light for 10 min produced acylaminyl and acyloxy radicals, which resulted in single strand cleavage of DNA at pH 7.0. Further the DNA cl

Full text of DOI:10.1016/j.bmcl.2010.07.116

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5414–5417
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N,O-Diacyl-4-benzoyl-N-phenylhydroxylamines as photoinduced DNA
cleaving agents
*
Nilanjana Chowdhury, Sansa Dutta, Boda Nishitha, Swagata Dasgupta, N. D. Pradeep Singh
Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Photoinduced homolytic fission of nitrogen–oxygen bond in N,O-diacyl-4-benzoyl-N-phenylhydroxylam-
ines using P310 nm UV light for 10 min produced acylaminyl and acyloxy radicals, which resulted in sin-
gle strand cleavage of DNA at pH 7.0. Further the DNA cleaving ability of N,O-diacyl-4-benzoyl-N-
phenylhydroxylamines found to depend both on its concentration and acyl substituents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 May 2010
Revised 21 July 2010
Accepted 26 July 2010
Available online 1 August 2010
Keywords:
Acylaminyl radicals
DNA cleavage
Photolysis
Carbon and oxygen-centered organic radicals are of great
importance in the field of bioorganic and medicinal chemistry
due to its ability to induce DNA cleavage, which is an essential
mechanism in many cancer therapies.1–8
variety of natural products and useful biologically active com-
pounds.16 Moreover, they also exhibit a wide range of pharmaco-
logical and physiological activities.17 Recently, N-arylalkyl-N-
phenylhydroxylamines have been shown to act as efficient DNA
cleaving agents.18,19 The mechanism involves photochemical reac-
tion of N-benzyl-N-phenylhydroxylamines with molecular oxygen
to produce hydroxyl radicals which are responsible for DNA cleav-
age rather than homolytic fission of N–O bond.
Utility of nitrogen-centered stable organic radicals for DNA
cleavage have been less explored, although some examples are re-
cently reported. Hwu et al.,9 showed photolytic fission of weak
N–O bond of oxime esters of anthraquinone generated nitrogen-
centered iminyl (R2C@NÅ) radicals, which resulted in DNA cleavage.
Further, the same group also demonstrated that arylaminyl (R2NÅ)
radicals generated through photoinduced cleavage of N–N single
bond in arylhydrazones posses excellent DNA cleaving ability.10
Among nitrogen-centered aminyl radicals, acylaminyl radicals
(ROC(O)NÅR) are of interest due to their fascinating physical and
chemical properties. Similar to arylaminyls, acylaminyls also exist
On other hand, Sakurai et al.20 showed that triplet sensitized
photolysis of N,O-diacyl-N-phenylhydroxylamines undergoes an
efficient homolytic N–O bond cleavage to generate acylaminyl
(ROC(O)NÅR) and acyloxy (R2COOÅ) radicals, which in polar solvent
escapes from the solvent cage and perform hydrogen abstraction to
yield corresponding arene carboxanilides and carboxylic acids.
Considering N,O-diacyl-N-phenylhydroxylamines ability to pro-
duce acylaminyl radicals under mild irradiation condition, led us
to explore its application as DNA cleaving agents.
In this article, we report for the first time N,O-diacyl-N-phen-
ylhydroxylamines with an inbuilt triplet sensitizer benzophenone
as photoinduced DNA cleaving agents. The results indicated that
acylaminyl, acyloxy and acyl radicals were mostly accountable
for DNA cleavage. In addition, we also showed that acyl substitu-
ents as well as the concentration of N,O-diacyl-N-phenylhydroxyl
amines have great influence on its DNA cleaving ability.
in a p-electronic ground state but with slight delocalization of the
unpaired electrons on the carbonyl group.11,12 In spite of delocal-
ization of unpaired electrons, all the chemical reactions of acylam-
inyls occurs at nitrogen.13 Though, simple acylaminyls are short
lived and sensitive to oxygen12 it undergoes important reactions
like Hofmann–Loffler intramolecular rearrangement,14 facile
hydrogen abstraction15 and addition to unsaturated hydrocar-
bons.14 Consequently, this prompted us to investigate the DNA
cleaving ability of acylaminyl radicals.
In search of an organic molecule which can produce acylaminyl
radicals via weak homolytic N–O bond cleavage led us to N-aryl-
hydroxylamine derivatives. N-arylhydroxylamines are important
class of compounds used as key intermediates in the synthesis of
We have synthesized two series of derivatives of 4-benzoyl-N-
phenylhydroxylamine (4-Bz-NPHA) as outlined in Scheme 1. The
series-1 consists of N-benzoyl-O-acyl-4-Bz-NPHA derivatives (3a–
f) and the series-2 has derivatives of N-acyl-O-benzoyl-4-Bz-NPHA
(5a–d). The compounds of series-1 were synthesized, initially by
carrying out N-benzoylation of 4-Bz-NPHA (1) with benzoyl
chloride in presence of NaHCO3 in dry DCM at 0 °C to yield N-ben-
* Corresponding author.
E-mail address: ndpradeep@yahoo.co.in (N.D. Pradeep Singh).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.116

N. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5414–5417
5415
O
O
O
R1COCl, NaH, THF
PhCOCl, DCM
NaHCO3, 0 ºC
R1
0 ºC
O
NHOH
NOH
N
2
3a-f
1
O
Ph
O
O
Ph
Series 1
RCOCl, 0 ºC
NaHCO3, DCM
R1=methyl
R1=phenyl
3a
3b
3c
3d
3e
3f
R1=3,4-dimethoxy phenyl
R1=4-fluoro phenyl
R1=4-chloro phenyl
R1=4-nitro phenyl
O
O
PhCOCl,DCM,Et3N
O
R
Ph
0ºC
N
NOH
R
4a-d
5a-d
O
O
O
Series 2
5a
5b
5c
R = methyl
R = 4-nitro phenyl
R = 4-fluoro phenyl
R = 3,4-dimethoxy phenyl
5d
Scheme 1. Synthesis of N,O-diacyl-4-benzoyl-N-phenylhydroxylamines (3a–j, 5a–d).
Table 1
Table 2
Synthetic yield and UV–vis data of N,O-diacyl-4-benzoyl-N-phenylhydroxylamines
(3a–j, 5a–d)
Single-strand cleavage of supercoiled circular pBR322 DNA (form I) to relaxed circular
DNA (form II) by photolysis of compounds (3a–j, 5a–d) under aerobic conditions at
room temperature with UV light P310 nm for 10 min
Compound Synthetic yield
(%)
UV–vis kmax
Entry
Compounds
Time (10 min)
k1
(nm)
e1
k2
e2
(MÀ1 cmÀ1
)
(nm)
(MÀ1 cmÀ1
)
% Form I
% Form II
(Form II)/(Form I)
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
85
90
92
80
82
75
90
75
82
80
235
219
223
234
241
218
234
228
232
232
27,945
19,184
29,940
35,144
33,041
13,751
37,379
21,409
34,717
25,277
287
291
294
285
281
300
281
295
285
281
18,967
16,852
21,960
24,508
18,078
11,756
27,454
13,556
22,747
20,321
1a
2
3
4
5
6
7
8
9
10
11
12
13
14
15
—
85.47
83.67
25.58
13.16
11.18
37.14
64.95
56.59
76.58
7.83
63.67
59.16
22.92
24.05
26.08
14.53
16.33
73.86
86.84
88.82
62.86
35.05
43.41
23.42
92.17
36.33
40.84
77.08
75.95
72.95
0.17
0.19
2.88
6.59
7.94
1.69
0.54
0.77
0.31
11.77
0.57
0.69
3.36
3.18
2.79
3ab
3a
3b
3c
3d
3e
3f
3ac
5a
5b
5c
zoyl-4-Bz-NPHA (2).21 Later, O-acylation of 2 with various acid
chlorides in presence of sodium hydride in dry THF at 0 °C resulted
in excellent yield of N-benzoyl-O-acyl-4-Bz-NPHA derivatives (3a–
f). While, in the case of series-2, N-acylation of 4-Bz-NPHA (1) was
carried out initially by treating with various acid chlorides to yield
4a–d, which latter was O-benzoylated with benzoyl chloride to
yield N-acyl-O-benzoyl-4-Bz-NPHA derivatives (5a–d). All the
above compounds were characterized by 1H, 13C NMR, IR and mass
spectral analysis.
5d
3ad
3ae
a
b
c
Refers to DNA alone.
Refers to DNA + compound (3a) in the dark.
Refers to DNA + compound (3a) + ascorbic acid.
Refers to DNA + compound (3a) + (0.1 M) NaN3 solution.
Refers to DNA + compound (3a) bubbling with argon gas.
d
e
The UV–vis absorption spectra of degassed 2.5 Â 10À5 M solu-
tion of compounds (3a–j, 5a–d) in methanol were recorded and
the results were summarized in Table 1. The results clearly showed
that all the compounds exhibited strong absorption maxima be-
tween 280–300 nm, thus makes them suitable for DNA cleavage.
1
2
3 4 5 6
7
8
9 10 11 12 13 14 15
Figure 1. Single strand cleavage of supercoiled circular pBR322 DNA (form I) to
relaxed circular DNA (form II) was carried out by irradiation of compounds (3a–j,
5a–d) (250 lM) with 310 nm UV light in a sodium phosphate buffer (pH 7.0) under
aerobic condition at room temperature for 10 min. The resultant products were
Figure 2. Effect of concentration versus DNA cleaving ability of compound 5a in a
sodium phosphate buffer (pH 7.0) upon irradiation of 310 nm UV light for 10 min.
subjected to electrophoresis on 1% agarose gel followed by ethidium bromide
staining under UV light. (a) Series-1: lane 1, DNA alone; lane 2, DNA + 3a (250 lM)
in the dark; lane 3, DNA + 3a; lane 4, DNA + 3b; lane 5, DNA + 3c; lane 6, DNA + 3d;
lane 7, DNA + 3e; lane 8, DNA + 3f; (b) series-2: lane 9, DNA + ascorbic acid; lane 10,
DNA + 5a; lane 11, DNA + 5b; lane 12, DNA + 5c; lane 13, DNA + 5d; lane 14,
DNA + 3a + sodium azide; lane 15, DNA + 3a bubbling with argon gas.
In order to assess the DNA cleaving ability of N,O-diacyl-4-ben-
zoyl-N-phenylhydroxylamines, we irradiated the compounds (3a–
j, 5a–d) with UV light (P310 nm, 125 W medium pressure mer-

5416
N. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5414–5417
O
O
hυ
O
Ph
used for DNA cleavage
O
N
Ph
+
N
3b
310nm
O
O
Ph
O
O
Ph
O
Ph
hυ, benzene
1,4-cyclohexadiene
NH
Ph
6
O
Scheme 2. Mechanism for the photolysis of N,O-diacyl-4-benzoyl-N-phenylhydroxylamine (3b).
cury lamp) at the concentration of 250
phosphate buffer (pH 7.0, 10 mM) with 10% DMSO containing the
circular supercoiled pBR322 plasmid DNA (form I; 62.5
room temperature for 10 min under aerobic condition.
By analyzing the results from gel electrophoresis on 1.0% aga-
rose gel with ethidium bromide staining, we found that com-
pounds (3a–j, 5a–d) caused the single strand cleavage of DNA to
give the relaxed circular DNA (form II). The ratios of (form II)/(form
I) ranged from 0.54 to 11.77 (Fig. 1, Table 2).
To find out the requirement of UV light by N,O-diacyl-4-Bz-
NPHA for DNA cleavage, we carried out a control experiment in
the dark with 250 lM of 3a with DNA. We observed the ratio of
l
M dissolved in a sodium
enhanced with the increase of its concentration. For example, the
ratio of (form II)/(form I) was 1.4 at 125 M whereas it is 11.77
and 34.74 at 250 and 500 M concentration.
l
l
g/ml) at
l
Based on the literature precedence,20 mechanism for the ob-
served photochemistry of the N,O-diacyl-4-benzoyl-N-phenylhydr-
oxylamines is outlined in Scheme 2. The mechanism proceeds
through photoinduced homolytic N–O bond cleavage to generate
acylaminyl and acyloxy radicals. The above generated radicals,
then escape from the solvent cage to induce significant DNA cleav-
age. Further the acyloxy radicals produced can also undergo effec-
tive decarboxylation to yield aryl radicals, which are also known to
posses’ DNA cleaving ability.24
(form II)/(form I) to be 0.17 in the dark (entry 2, Table 2) compared
to 2.88 with UV light (Table 2, entry 3), which clearly suggest that
UV light is an essential requirement to initiate the DNA scission
process. Secondly, to understand the role of molecular oxygen in
the DNA cleavage process, we carried out the irradiation of DNA
with compound 3a under argon (argon was bubbled for a period
of 15 min to reduce the concentration of dissolved oxygen), the ra-
tio of (form II)/(form I) was found to be 2.79 (Table 2, entry 15)
which is similar to the result obtained under aerobic condition (Ta-
ble 2, entry 3) which rules out the involvement of oxygen in the
DNA nicking process. In another experiment, we also carried out
the irradiation of DNA with 3a by adding sodium azide22,23 (scav-
enger of singlet oxygen) and the ratio of (form II)/(form I) was
found to be 3.18 (Table 2, entry 14), which excludes the possibility
of DNA cleavage by singlet oxygen.
Analytical results from gel electrophoresis showed that acyl
substituents have great influence on the DNA cleaving ability of
N,O-diacyl-4-Bz-NPHA derivatives. Among series-1, compound 3c
having electron donating methoxy group attached to the O-ben-
zoyl substituent exhibited best DNA cleaving ability while, com-
pound 3f with electron withdrawing nitro group showed poor
performance. The above fact can be attributed to the stability of
aryloxy radicals.9
To confirm the DNA cleavage is due to the radicals generated
from N,O-diacyl-4-Bz-NPHA, we irradiated DNA with 3a in the
presence of radical quencher ascorbic acid.1a,25 We found that
ascorbic acid completely inhibited the cleavage of DNA (lane 9,
Fig. 1) and the ratio of (form II)/(form I) was 0.31 (Table 2, entry 9).
In order to trap the generated acylaminyl radicals, we irradiated
3b (0.1 mmol) in benzene containing 1,4-cyclohexadiene26
(1.1 mmol) using 310 nm UV light for 30 min. After completion
of the reaction the photoproducts were isolated using column
chromatography. We obtained 4-benzoylbenzanilide27 (6) in 48%
yield, which should have came from the abstraction of hydrogen
atom by acylaminyl radicals from 1,4-cyclohexadiene.28
In summary, acylaminyl radicals produced via photo induced
homolytic fission of weak N–O bond of N,O-diacyl-4-benzoyl-N-
phenyl hydroxyl amines resulted in efficient DNA cleavage. Further
strong electron withdrawing acyl substituent reduces the DNA
cleaving ability of acylaminyl radicals. Among the derivatives, N-
acetyl-O-benzoyl-4-benzoyl-N-phenylhydroxylamine 5a showed
best DNA cleaving ability at 250
irradiation.
lM concentration at 10 min of
Acknowledgments
In the case of series-2, compound 5a having N-acetyl substitu-
ent showed excellent DNA cleaving ability compared to compound
3a (series-1) with N-benzoyl substituent. Since, stronger electron
withdrawing acyl substituent delocalize the unpaired electron
more into carbonyl group, which reduces the spin density on the
nitrogen atom of acyl aminyl radicals.11,12 This is further confirmed
by the fact that compound 5b with electron withdrawing nitro
group attached to the N-benzoyl substituent showed inferior cleav-
ing ability compared to compound 5d with electron donating
methoxy substituent. Among both the series compound 5a exhib-
ited the best DNA cleaving ability.
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.
Supplementary data
Supplementary data (general synthetic procedure, spectral data
of compounds and HPLC chromatogram of the photolysis of com-
pound 3b) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.07.116.
To find out the optimum concentration required for DNA cleav-
age by N,O-diacyl-4-benzoyl-N-phenyl hydroxylamines, we carried
out irradiation of DNA with different concentrations of 5a (50–
250 lM) for a period of 10 min. Results from the concentration
study of 5a showed that cleavage of DNA is depended upon its con-
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5 lm)mobile phase, acetonitrile/water = 80:20, flow rate 1 mL/ min,
detection: UV at 254 nm). The HPLC chromatogram showed only two
significant photoproducts namely 4-benzoylbenzanilide (6) and benzoic acid.