Aminyl and iminyl radicals from arylhydrazones in the photo-induced DNA cleavage

Article abstract of DOI:10.1016/j.bmc.2004.03.037

Photolytic cleavage of the nitrogen-nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R₂N^·) and iminyl (R₂C=N^·) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-cleaving agents. Irradiation with 350nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer solution containing the supercoiled circular φX174 RFI DNA at pH6.0 resulted in single-strand cleavage of DNA. Attachment of the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.

Full text of DOI:10.1016/j.bmc.2004.03.037

Bioorganic & Medicinal Chemistry 12 (2004) 2509–2515
Aminyl and iminyl radicals from arylhydrazones in the
photo-induced DNA cleavage
a,c
Jih Ru Hwu,a,b,c, Chun Chieh Lin, Shih Hsien Chuang,a,b Ke Yung King,a,c
*
Tzu-Rong Suc and Shwu-Chen Tsayd
aDepartment of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC
bCenter for Nano-Science and Technology, University System of Taiwan, Taiwan, ROC
cInstitute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC
dWell-being Biochemical Corporation, Taipei 10468, Taiwan, ROC
Received 3 February 2004; revised 17 March 2004; accepted 18 March 2004
Available online 10 April 2004
Abstract—Photolytic cleavage of the nitrogen–nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R2NÅ) and
iminyl (R2C@NÅ) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-
cleaving agents. Irradiation with 350 nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer
solution containing the supercoiled circular /X174 RFI DNA at pH 6.0 resulted in single-strand cleavage of DNA. Attachment of
the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate
that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Hydrazones are an important class of organic com-
pounds, some of which show significant biological
activities. For example, Heinisch and co-workers19
developed a series of N-heteroaryl hydrazones as
potential novel antitumor agents.
Cleavage of DNA by chemical methods often involves
carboradicals and the hydroxyl radical HOÅ.1–13
Recently 1,5-azulenequinones have also been devel-
oped as photo-induced DNA cleaving agents.14;15 In
a systematic study of DNA cleavage, we made
efforts to understand the cleaving ability of different
species.
Alvarez-Builla and co-workers20 reported that 2,6-di-
tert-butyl-4-(2-azinylhydrazonomethyl)phenol deriva-
tives possess potential as 5-lipoxygenase inhibitors.
5-Lipoxygenase metabolites of arachidonic acid are
implicated as mediators in a diversity of diseases,
including asthma and many other inflammatory pathol-
ogies. Moreover, Barreiro and co-workers21 utilized
Utility of nitrogen-centered radical species as ‘DNA
nucleases’ has recently been documented.16;17 Binkley18
reported the photolytic cleavage of the nitrogen–nitro-
gen single bond in benzaldehyde phenylhydrazone. An
aminyl radical (R2NÅ) and an iminyl radical (R2C@NÅ)
are generated, which can escape from solvent cage and
perform hydrogen abstraction.
arylcarbaldehyde
4-(1-phenyl-3-methylpyrazolo-[3,4-
b]pyridine) hydrazone derivatives as a new pharmaco-
phoric tool for the development of more efficacious
analgesics.
Two factors associated with phenylhydrazones must be
taken into account for their development as DNA-
cleaving agents. First, the size and shape of these ‘guests’
should allow themselves to fit well into the host (i.e.,
DNA). Performance of graphic molecular modeling of
oligodeoxyribonucleotides and hydrazones provided us
valuable information on the molecular design of
Keywords: Aminyl radicals; Iminyl radicals; Photo-induced; DNA
cleavage.
* Corresponding author. Tel.: +886-35-725813; fax: +886-35-721594;
e-mail: jrhwu@mx.nthu.edu.tw
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.037

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J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
gen-centered radicals R2NÅ and R2C@NÅ can function as
the DNA-cleaving species.
2. Results
2.1. Synthesis of arylhydrazones
We prepared various substituted arylhydrazones 1–15 in
71–92% yields by condensation of the corresponding
carbonyl compounds with different arylhydrazines in a
mixture of glacial acetic acid and ethanol (Scheme 1).26
The electrical effects27 resulting from substituents
attached to the aromatic rings of arylhydrazones
could play vital roles in the DNA-cleaving process.
Consequently, we used arylhydrazines containing an
electron-donating (+M) group, such as OMe, and elec-
tron-withdrawing ()M) group, such as NO2, in investi-
gation. Given the same reason, carbonyl compounds
containing an OMe, NHCOMe, or NO2 substituted aryl
group were allowed to react with hydrazines. Further-
more, we introduced special moieties, including naph-
thyl, 3-indolyl, purin-6-yl, and 2-chloropyrimidin-4-yl
groups, into the arylhydrazones (e.g., 11, 12, 14, and 15).
We listed the UV spectroscopic data of arylhydrazones
1–15 in Table 1. Most of the compounds possessed a
strong UV absorption between 320 and 360 nm, which
fits into an ideal range of wavelengths in the photo-
chemical activation of chemical agents for DNA scis-
sion.
2.2. Photo-induced DNA cleavage by hydrazones
Scheme 1. Synthesis of arylhydrazones.
To investigate their DNA-cleaving potency, we dis-
solved hydrazones 1–15 (250 lM) individually in a so-
dium phosphate buffer (pH 6.0) and 10% ethanol
containing the supercoiled circular /X174 RFI DNA
(form I; 50 lM/base pair). These solutions were irradi-
ated with UV light (350 nm, 16 W) under aerobic con-
ditions at room temperature for 2.0 h. By analyzing the
the cleaving agents 1–15 shown in Scheme 1. Second,
stability and lifetime of the radical intermediates
generated photochemically are essential to their cleaving
ability.
Ingold and co-workers22 found that the second-order
decay of dimethylaminyl and diethylaminyl radicals
occurs with the range (2 1) · 109 Mꢀ1 sꢀ1 by the ESR
technique during photolysis. The rate constants for the
bimolecular self-reactions are 4 · 107 and 4 · 109 Mꢀ1 sꢀ1
for diisopropyliminyl and ditrifluoromethyliminyl radi-
cals, respectively.23 Lusztyk and co-workers24 reported
that the lifetimes of diethylaminyl and piperidinyl radi-
cals are 1.6 and 2.5 ls in 0.05 M malonic acid/aceto-
nitrile solution. The corresponding phenyl substituted
aminyl radicals are more stable than the dialkylaminyl
radicals; lifetimes of PhMeNÅ and Ph2NÅ are 50 and
80 ls, respectively. Consequently, we planned to syn-
thesize arylhydrazones bearing various substituents on
both aryl rings. Their influence on DNA cleavage could
result from the inductive and the field effects.25
Table 1. UV spectroscopic data of arylhydrazones 1–15
Arylhydrazone UV–vis kmax (EtOH)/nma
1
236 (18,057), 296 (16,129), 344 (29,187)
243 (22,344), 302 (30,679), 345 (47,771)
250 (11,787), 304 (9930), 354 (25,694)
264 (26,607), 424 (42,350)
2
3
4
5
236 (11,672), 312 (14,530), 346 (20,189)
236 (13,662), 312 (14,924), 346 (20,235)
299 (3061), 328 (2493), 404 (17,942)
275 (11,392), 444 (22,394)
6
7
8
9
235 (10,471), 292 (9014), 329 (5133), 416 (29,035)
246 (11,995), 314 (17,733), 354 (19,260)
233 (24,477), 293 (7862), 354 (23,791)
339 (22,537)
10
11
12
13
14
15
236 (16,733), 298 (10,025), 340 (19,148)
211 (25,000), 268 (18,173)
224 (12,309), 326 (23,183)
Herein we report our new findings that cleavage of
DNA took place upon irradiation of arylhydrazones
with UV light. Our results indicate that both the nitro-
a Numbers marked with bold face represent the absorption between
320 and 360 nm.

J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
2511
results from gel electrophoresis on 1% agarose gel with
ethidium bromide staining, we found that arylhydraz-
ones 1–15 caused the single-strand cleavage of DNA to
give the relaxed circular DNA (form II). The ratios of
(form II)/(form I) ranged from 0.19 to 24 (see Fig. 1 and
Table 2). The cleaving efficacy of 1 in phosphate buffers
over the pH range between 5.0 and 8.0 was found to be
slightly pH dependent.
the dark (the third row in Table 2) and 1.3 with UV light
(the second row). Under photolytic conditions for 2.0 h
without addition of 1, the ratio was 0.091 (the last row).
Thus the UV light functioned as a ‘trigger’ to initiate an
arylhydrazone for the DNA strand scission.
Sodium azide can function as a scavenger of singlet
oxygen,28;29 of which presence may contribute to DNA
damage.30;31 We investigated the percentages of DNA
scission resulting from singlet oxygen during the pho-
tolysis of arylhydrazones. A series of control experi-
ments were performed by addition of sodium azide into
the photolytic solution containing hydrazone 1 and
DNA. The results are summarized in Table 3.
We found that the UV light was essential for arylhyd-
razones to cleave DNA. Under aerobic conditions with
250 lM of 1, the ratios of (form II)/(form I) were 0.14 in
The percentage of form II DNA decreased from 83% to
67% upon addition of sodium azide (rows 1 and 2 in
Table 3). Furthermore, we detected the same percentage
of form II DNA (i.e., 67%) by increasing the amount of
sodium azide from 100 to 500 mM. These results indi-
cate that singlet oxygen in the solution was destroyed
Figure 1. Single-strand cleavage of supercoiled circular /X174 RFI
DNA (form I, 50 lM/base pair, molecular weight 3.50 · 106, 5386 base
pairs in length) to relaxed circular DNA (form II) was carried out by
irradiation of a hydrazone (250 lM) with 350 nm UV light in a sodium
phosphate buffer (pH 6.0) under aerobic conditions at room tempera-
ture for 2.0 h. The resultant products were subjected to electrophoresis
on 1% agarose gel followed by ethidium bromide staining: lane 1,
DNA alone; lane 2, 1 in the dark; lane 3, 1; lane 4, 2; lane 5, 3; lane 6,
4; lane 7, 5; lane 8, 6; lane 9, 7; lane 10, 8; lane 11, 9; lane 12, 10; lane
13, 11; lane 14, 12; lane 15, 13; lane 16, 14; lane 17, 15.
completely by sodium azide in
a concentration
P100 mM. In comparison with the results from the
experiment in the absence of hydrazone 1 (i.e., 9.9%, the
last row in Table 3), the percentage of DNA cleavage
resulting from 1 was 63% [ ¼ (67% ) 9.9%)/(33% +
67% ) 9.9%)]. On the other hand, the percentage of
DNA scission caused by both 1 and singlet oxygen was
calculated as 81% [ ¼ (83% ) 9.9%)/(17% + 83% ) 9.9%)].
Thus the contribution resulting from singlet oxygen to
DNA cleavage was ꢁ18% ( ¼ 81% ) 63%).
Table 2. Single-strand cleavage of supercoiled circular /X174 RFI
DNA (form I) to relaxed circular DNA (form II) by photolysis of
hydrazones 1–15 under aerobic conditions at room temperature with
350 nm UV light for 2.0 h
Among various substituents onto the benzene ring in an
arylhydrazone, our results from gel electrophoresis
indicate that attachment of the electron-donating group
OMe could enhance its DNA-cleaving activity. As
shown in Figure 1 and Table 2, the methoxy-containing
hydrazones (e.g., 2, 6, and 10) exhibited appealing
potency. Moreover, in a direct comparison of (form II)/
(form I), we found that ratios were 5.4 for 2 and 16 for
6. Thus, under the photolytic conditions, the corre-
sponding methoxy-containing aminyl radical generated
in situ acted more potent than the iminyl radical for
DNA cleavage. Replacement of a benzene nucleus in a
Hydrazonea pH
% Form Ib
% Form IIb (Form II)/
(Form I)
1
5.0
6.0
6.0
7.0
8.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
37
43
88
45
50
16
13
70
45
6.0
76
75
76
4.0
33
45
45
84
83
92
63
57
12
55
50
84
87
30
55
94
24
25
24
96
67
55
55
16
17
1.7
1.3
0.14
1.2
1.0
5.4
6.7
0.42
1.2
1
1c
1
1
2
3
4
5
6
16
7
0.31
0.33
0.31
Table 3. Influence of sodium azide on single-strand cleavage of
supercoiled circular /X174 RFI DNA (form I) to relaxed circular
DNA (form II) by photolysis of hydrazone 1 under aerobic conditions
at room temperature with 350 nm UV light for 2.0 h
8
9
10
11
12
13
14
15
None
24
2.0
1.2
Hydrazonea
NaN3
(mM)
% Form Ib % Form IIb (Form II)/
(Form I)
1.2
1
0
100
500
0
17
33
33
90
83
67
67
4.9
2.0
2.0
0.11
0.19
0.20
0.091
1
1
None
8.3
9.9
a A 0.10 M sodium phosphate buffer (Na2HPO4 and NaH2PO4) con-
taining 250 lM of a hydrazone, 50 lM/base pair of form I DNA
(molecular weight 3.50 · 106, 5386 base pairs in length), and 10%
EtOH.
b Analyzed by gel electrophoresis with 1% agarose and ethidium bro-
mide staining.
c In the dark.
a A 0.10 M sodium phosphate buffer (Na2HPO4 and NaH2PO4) con-
taining 500 lM of a hydrazone, 50 lM/base pair of form I DNA
(molecular weight 3.50 · 106, 5386 base pairs in length), and 10%
EtOH.
b Analyzed by gel electrophoresis with 1% agarose and ethidium bro-
mide staining.

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J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
benzaldehyde arylhydrazone with a naphthyl (cf. 11),
indolyl (cf. 12), purinyl (cf. 14), or chloropyrimidinyl (cf.
15) unit did not perform superior potency (see Table 2).
Their low solubility (34.8 mg/mL for 14 and 21.6 mg/mL
for 15) in aqueous solution caused the poor efficacy.
Compound 10, in which the two aromatic rings on each
side of @NANA unit possess a methoxy group, exhib-
ited the greatest cleaving activity (lane 12 of Fig. 1)
among all of the tested arylhydrazones. Results from
dose measurements of 10 (10–250 lM) reveal that
cleavage of DNA depended upon its concentration (Fig.
2); it was able to cleave DNA with (form II)/(form
I) ¼ 1.4 at the concentration even as low as 50 lM.
Furthermore, we used polyacrylamide gel electropho-
resis to analyze the DNA cleavage pattern resulting
from arylhydrazones under photolytic conditions. We
preincubated a mixture of 50-32P-end-labeled SalI–SphI
double-helical DNA fragment of pBR322 and arylhydr-
azone 10 (250–1000 lM) in a sodium phosphate buffer
(pH 6.0) containing 10% ethanol at room temperature
for 30 min. The 93 base pair DNA fragment was
obtained enzymically.32;33 Photo-excitation (312 and
350 nm) of the mixtures at room temperature for 2.0 h
followed by piperidine treatment at 95 ꢁC for 30 min
generated a sequence independent pattern on an auto-
radiogram of a 20% polyacrylamide/8.0 M urea gel (see
Fig. 3). Use of piperidine as an external base assisted the
vision of the fragmentation pattern. Its use caused DNA
scission in the dark but to a limited extent. The results
indicate that arylhydrazones could be useful for DNA
footprinting.34
Figure 3. Autoradiogram of a denaturing polyacrylamide gel repre-
senting the cleavage of 50-32P-end-labeled SalI–SphI fragment of
pBR322 by photolysis of arylhydrazone 10 (250–1000 lM): –, DNA
alone; p: DNA fragments with piperidine treatment; G, A + G, T + C,
C, nucleotide residues in sequence determined by Maxam–Gilbert
chemical sequencing method. The nucleotide sequence of the labeled
strand of the duplex is shown at the left.
3. Discussion
We carried out experiments of DNA cleavage by aminyl
(R2NÅ) and iminyl (R2C@NÅ) radicals, which were gen-
erated by photolytic fission of the N–N single bond in
arylhydrazones under aerobic conditions. Our results in
Table 2 show that the efficiency, indicated by the ratio of
(form II)/(form I), depended upon the substituents
attached to both aryl units. In comparison with the
parent hydrazone 1, hydrazone 6 exhibited much greater
cleaving ability (1.3 vs 16 for the ratio). Thus we con-
clude that an electron-donating (+M) group, such as
OMe, attached to the aminyl nucleus can enhance its
DNA-cleaving ability. An electron-withdrawing ()M)
group NO2 in 7 disabled its DNA-cleaving ability. The
similar trend resulting from substituents on the iminyl
Figure 2. Dose measurements of arylhydrazone 10 for its DNA-
cleaving ability in a sodium phosphate buffer (pH 6.0) upon irradiation
with 350 nm UV light.

J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
2513
nucleus is reflected by the ratios of 5.4 and 0.42 for
hydrazones 2 (OMe, +M) and 4 (NO2, )M), respectively.
aerobic conditions.45 Results from our control experi-
ment involving the addition of sodium azide as a singlet
oxygen scavenger indicate that the oxygen-centered
radicals contributed up to ꢁ18% to the DNA scission.
We believe that an arylaminyl or an aryliminyl group can
be stabilized by bearing an electron-donating group.
After photolytic fission occurs to the N–N single bond in
arylhydrazones, the lifetime of the resultant nitrogen-
centered radicals bearing a methoxy group could be long
enough to allow themselves to attack DNA. Accordingly,
the DNA scission takes place. Having a methoxy group
on each of the aryl rings enabled hydrazone 10 to exhibit
great efficiency for DNA cleavage; its ratio of (form II)/
(form I) reached 24. This rationale is further sup-
ported by the results of DNA cleavage resulting from
arylhydrazones with various substitutions on both aryl
rings.
4. Conclusion
Ability of arylhydrazones to cleave DNA was studied;
these compounds could be also developed as photo-in-
duced DNA-cleaving agents. Upon irradiation with
350 nm UV light, hydrazones bearing the electron-
donating OMe group showed great cleaving activity.
Our results from systematic studies provide evidence to
show that the nitrogen-centered aminyl and iminyl
radicals, generated by photolysis of arylhydrazones, are
active species for DNA scission.
On the other hand, hydrazones 4 and 7–9 possess an
NO2 moiety, which is a strong electron-withdrawing
group. Their ratios of (form II)/(form I) were between
0.31 and 0.42 at the concentration of 250 lM. Thus, the
presence of an NO2 group in hydrazones decreases their
DNA cleaving ability significantly regardless the pres-
ence of an OMe group. Results from our systematic
studies clearly indicate that the substituents on each of
the aryl rings exerted influence on the DNA scission.
Therefore we conclude that both the aminyl and the
iminyl radicals are responsible for DNA cleavage.
5. Experimental
5.1. General procedure
All reactions were carried out in oven-dried (120 ꢁC) or
autoclaved glassware and eppendorf tubes. Absolute
ethyl alcohol was obtained from RDH Chemical Co.
Hydrazine hydrate from Merck Inc. was dried and dis-
tilled from NaOH. Ammonium persulfate and boric
acid were purchased from Serva Chemical Co. Brom-
ophenol blue, salmon testing DNA, t-RNA, agarose,
tetramethylethylenediamine, tris(hydroxymethyl)amino-
methane, and xylene cyanol were purchased from Sigma
Chemical Co. and used as received. Supercoiled circular
/X174 RFI DNA, pBR322 DNA, calf intestinal alka-
line phosphatase (CIP),T4 polynucleotide kinase as well
as SalI and SphI restriction endonucleases were pur-
chased from New England Biolabs. Maxam–Gilbert
sequencing kit was purchased from NEN-DuPont
Chemical Co. and deoxyadenosine 50-[c-32P]-triphos-
phate (>5000 Ci/mmol) from Amersham Co. Acrylam-
ide and N,N0-methylenebisacrylamide were purchased
from Amersco Chemical Co.
The research teams of Wender,35 Sortino,16 Kawani-
shi,36 and Davis37 reported individually that aminyl
radicals can damage DNA. An iminyl radical generated
from oxime ester of anthraquinone by photolysis may
also be responsible for DNA scissions.38 Two possible
mechanisms can be envisaged for DNA lesion by these
nitrogen-centered radicals. The first is the hydrogen
abstraction from the deoxyribose moieties.39 The second
is radical attack on the nucleobases to generate hot
piperidine sensitive sites.40 At the current stage, our data
shown in Figure 1 (without piperidine treatment) and
Figure 2 (with piperidine treatment) cannot differentiate
these two possibilities. The cleaving mechanism in detail
will be studied in due course.
To confirm the DNA cleavages by radicals from benz-
aldehyde phenylhydrazones, we performed control
experiments by adding ascorbic acid1a and DMSO,1a
respectively, as radical quenchers in the DNA media.
Ascorbic acid was found to obstruct the scission process
completely; DMSO degraded the scissions to a minimal
extent. Ethanol can also function as a moderate radical
quencher.1a;41 Our experimental results however indicate
that its addition to the samples of benzaldehyde phen-
ylhydrazones 1–15 did not exert significant influence on
their cleaving efficiency as displayed by gel electropho-
resis. Thus we chose 10% ethanol as the co-solvent42 for
dissolving compounds 1–15 in the DNA cleaving
experiments.
Ultraviolet (UV) spectra were recorded on a Hitachi
U-3300 spectrophotometer and kmax was obtained in nm
(e) units. Infrared (IR) spectra were recorded on a Per-
kin–Elmer Paragon 1000 FTIR spectrophotometer. The
wavenumbers reported are referenced to the 1601 cmꢀ1
absorption of polystyrene. Proton NMR spectra were
obtained on Bruker AC-200 (200 MHz) and AC-300
(300 MHz) spectrometers and 13C NMR spectra were
recorded on a Bruker AC-200 (50 MHz) or AC-300
(75 MHz) spectrometer. Chloroform-d or dimethylsulf-
oxide-d6 was used as solvent; Me4Si (d 0.00 ppm) and the
center of the CDCl3 triplet (d 77.00 ppm) were used as
an internal standard for 1H and 13C NMR spectra,
respectively. All NMR chemical shifts are reported as d
values in parts per million (ppm) and coupling constants
(J) are given in hertz (Hz). The splitting pattern abbre-
viations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; br, broad; m, unresolved multiplet; dd, doublet
of doublets.
Autoxidation of benzaldehyde phenylhydrazones pro-
duces peroxy intermediates and free-radical species.43;44
Thus phenylhydrazones 1–15 oxidized by molecular
oxygen may generate radicals during photolysis under

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J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
Photolytic experiments were carried out at room tem-
perature by use of a 16-Watt Rayonet 350 nm lamp, and
equipped with Merry-go-round apparatus. The NIH
1.60 image program, provided by Dr. R. Wayne of
National Institutes of Health, U.S.A., was used for the
quantitative analysis of DNA cleavage.
Calcd for C11H9N4Cl: C, 56.78; H, 3.90; N, 24.08.
Found: C, 56.80; H, 3.89; N, 24.09.
5.2. Cleavage of supercoiled circular /X174 RFI DNA by
arylhydrazones
The reaction mixtures (10 lL) containing supercoiled
circular /X174 RFI DNA stock solution (form I,
50 lM/base pair), an arylhydrazone (250 lM), phos-
phate buffers (0.10 M, pH 5.0–8.0), and 10% EtOH in a
Pyrex vial were irradiated with UV light (350 nm, 16 W)
under aerobic conditions at room temperature for 2.0 h.
After addition of the gel-loading buffer (0.25% bromo-
phenol blue, 0.25% xylene cyanol, and 30% glycerol), the
reaction mixtures were loaded on a 1% agarose gel with
ethidium bromide staining. The electrophoresis tank
was attached to a power supply at a constant current
(ꢁ100 mA). The gel was visualized by 312 nm UV
transilluminator and photographed by an FB-PBC-34
camera. Quantitation of DNA-cleaving activities was
performed by integration of the optical density as a
function of the band area by use of a Microtek scanner
and an NIH 1.60 image program.
5.1.1. Standard procedure for preparation of substituted
N-aryl arylhydrazones. Arylaldehyde (1.1–1.2 equiv) was
added into aqueous ethanol (20%) containing aryl-
hydrazine (1.0 equiv) and a trace amount of glacial
acetic acid. After the reaction mixture was stirred at
room temperature in the dark for 3.0 h, the crystals of
€
hydrazones were collected on a Buchner funnel. Their
structures were determined by spectroscopic methods.
Hydrazones 1–13 have been reported before;46–58 their
physical properties and spectroscopic characteristics are
consistent with those in the literature.
5.1.1.1. Synthesis of benzaldehyde purin-6-yl-hydra-
zone (14). The standard procedure was followed by use
of benzaldehyde (325 mg, 3.06 mmol, 1.2 equiv), aque-
ous ethanol (20%, 10 mL), and purin-6-ylhydrazine
(383 mg, 2.55 mmol, 1.0 equiv). After the reaction mix-
ture was stirred at room temperature for 3.0 h, it was
worked up and the residue was recrystallized to give 14
as white crystals in 76% yield (462 mg, 1.94 mmol): mp
280–281 ꢁC; dH (200 MHz, CDCl3) 7.09–7.12 (m, 3H,
ArH), 7.50–7.55 (m, 2H, ArH), 7.78–7.93 (m, 2H,
N@CH + N@C(8)H), 8.14 (s, 1H, N@C(2)H); dC
(50 MHz, DMSO-d6) 127.24, 128.82, 129.41, 129.62,
134.34, 143.90, 142.24, 144.85, 150.03, 151.96; mmax
(KBr)/cmꢀ1 3410 (m), 3219 (m, NAH), 2796 (m, ArAH),
1640 (s), 1604 (s), 1490 (w), 1320 (s), 1251 (m), 1141 (m),
941 (w); kmax (EtOH)/nm (e) 211 (25,000), 268 (18,173);
m=z (EI) 238 (Mþ; 8%), 210 (1), 161 (12), 155 (2), 135
(100), 128 (2), 119 (6), 108 (48), 103 (13), 89 (13), 77
(27), 65 (15), 51 (35); exact mass calcd for C12H10N6
238.0967, found 238.0968; Anal. Calcd for C12H10N6: C,
60.50; H, 4.23; N, 35.27. Found: C, 60.48; H, 4.23; N,
35.28.
5.3. Cleavage of 32P-DNA by hydrazone 10
The reaction mixture containing 50-32P-end-labeled
SalI–SphI dsDNA fragment of pBR322, a phosphate
buffer (0.10 M, pH 6.0), and hydrazone 10 (250–
1000 lM) in a Pyrex vial were preincubated at room
temperature for 30 min. After irradiation with 312 or
350 nm UV light under aerobic conditions at room
temperature for 2.0 h, the reaction mixtures were quen-
ched with gel-loading buffer (80% formamide, 0.25%
bromophenol blue, and 0.25% xylene cyanol) or 95%
EtOH. The solutions were then subjected to piperidine
treatment and ethanol precipitation. The DNA pellet
was resuspended in aqueous piperidine solution (1.0 M,
60 lL) and maintained at 95 ꢁC for 30 min. Subse-
quently, the reaction mixtures were sequentially lyoph-
ilized, treated with water (30 lL), lyophilized, and
resuspended in the gel-loading buffer. The DNA solu-
tion and the Maxam–Gilbert markers were analyzed by
use of 20% polyacrylamide/8.0 M urea gel. The electro-
phoresis was performed at a voltage of 500 V for 30 min
and raised to 1000 V for another 2.0 h. The cleavage
positions were visualized by use of Kodak X-Omat Ar-5
film at )70 ꢁC for 24 h. Quantitation of the relative
intensity for the cleavage sites was integrated by the
optical density as a function of the band area by use of a
Microtek scanner and NIH 1.60 image program.
5.1.1.2. Synthesis of benzaldehyde 2-chloropyrimidin-4-
ylhydrazone (15). The standard procedure was followed
by use of benzaldehyde (558 mg, 5.26 mmol, 1.2 equiv),
aqueous ethanol (20%, 10 mL), and 2-chloropyrimidin-
4-ylhydrazine (482 mg, 4.38 mmol, 1.0 equiv). After the
reaction mixture was stirred at room temperature for
3.0 h, it was worked up and the residue was recrystal-
lized to give 15 as white crystals in 74% yield (754 mg,
3.24 mmol):
mp
248–249 ꢁC;
dH
(200 MHz,
Acknowledgements
CDCl3 + DMSO-d6) 6.65–6.71 (m, 1H, ArH), 6.89–6.94
(m, 2H, ArH), 7.17–7.24 (m, 4H, ArH), 7.59 (s, 1H,
N@CH), 7.66–7.69 (m, 1H, ArH); dC (50 MHz, DMSO-
d6) 102.37, 126.80, 128.78, 129.84, 134.03, 144.68,
158.41, 159.28, 162.93; mmax (KBr)/cmꢀ1 3461 (m), 3191
(m, NAH), 3048 (m), 2908 (m, ArAH), 1594 (s), 1422
(s), 1380 (s), 1228 (m), 1119 (s), 829 (m); exact mass
calcd for C11H9N4Cl 232.0516, found 232.0515; Anal.
For financial support, we thank Ministry of Education
(Grant 89-B-FA04-1-4) of the Republic of China. We
are also grateful to Dr. R. Wayne for providing the NIH
1.60 image program for the quantitative analysis of
DNA cleavage. Tzu-Rong Su is a graduate student from
Graduate Institute of Dental Sciences, Kaohsiung
Medical University.

J. Ru Hwu et al. / Bioorg. Med. Chem. 12 (2004) 2509–2515
2515
28. Sako, M.; Nagai, K.; Maki, Y. J. Chem. Soc. Chem.
Commun. 1993, 750–751.
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