O-Benzoyl pyridine aldoxime and amidoxime derivatives: Novel efficient DNA photo-cleavage agents

Article abstract of DOI:10.1039/c4md00548a

O-Benzoyl derivatives of meta-, ortho- and para-pyridine aldoximes and amidoximes are novel efficient DNA photo-cleavage agents. In particular O-p-nitro-benzoyl derivatives 4, 8 and 15 were effective at concentrations as low as 1 μM. Both aldoximes and amidoximes were active under aerobic and anaerobic conditions, with a double-stranded to single-stranded DNA cleavage ratio of up to 1. These results give prospects for multiple applications, including phototherapeutic treatment of solid tumors. This journal is

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O-Benzoyl pyridine aldoxime and amidoxime
derivatives: novel efficient DNA photo-cleavage
Cite this: DOI: 10.1039/c4md00548a
agents†
Paraskevi Karamtzioti,‡a Asterios Papastergiou,‡a John G. Stefanakis,b
Alexandros E. Koumbis,b Ioanna Anastasiou,a Maria Koffaa
a
*
and Konstantina C. Fylaktakidou
Received 8th December 2014,
Accepted 14th January 2015
O-Benzoyl derivatives of meta-, ortho- and para-pyridine aldoximes and amidoximes are novel efficient
DNA photo-cleavage agents. In particular O-p-nitro-benzoyl derivatives 4, 8 and 15 were effective at
concentrations as low as 1 μM. Both aldoximes and amidoximes were active under aerobic and anaerobic
conditions, with a double-stranded to single-stranded DNA cleavage ratio of up to 1. These results give
prospects for multiple applications, including phototherapeutic treatment of solid tumors.
DOI: 10.1039/c4md00548a
www.rsc.org/medchemcomm
Aldoximes, ketoximes and amidoximes (Fig. 1, I, II and III,
respectively) retain a complementary position in drug design
1. Introduction
Organic compounds with DNA cleaving activity are involved
in biological diagnostic, therapeutic and mechanistic aspects
such as gene and cancer therapies, DNA electron and energy
transfers and design of DNA targeted drugs.1–6 Photo-
activated “chemical nucleases”, known also as “photo-cleavers”,
interact with DNA and cause its cleavage using light. As a
result, the need for external chemical initiators is eliminated
due to the fact that the chemical reaction is initiated only
when the mixture of the organic compound and DNA is irra-
diated. Light causes selective excitation of the photo-
cleavaging agents,6,7 which via various mechanistic pathways
lead to single-stranded (ss) DNA damage, repairable by enzy-
matic processes,8 and/or double-stranded (ds) DNA photo-
cleavage.9–11 The latter, which is more difficult to repair, may
trigger self-programmed cell death, featuring this approach as
an efficient tool for cancer therapy.
and
discovery
being
individually
considered
as
pharmacophores and participating as parent compounds in
multiple transformations.22–25 However, the existence of
hydroxy-imino/amino tautomerization in amidoximes differ-
entiates them from their other close relatives (Fig. 1). Thus,
amidoximes are bi-functional molecules which exhibit a rich
chemistry, providing among others one of the shortest ways
to reach various heterocycles.26–29 Amidoximes possess
numerous and diverse biological activities, which make them
important and attractive pharmacophores in medicinal
chemistry. Furthermore, their ability to complex with metals
allows them to act as radiopharmaceuticals and anti-
pollutants.29
The pyridine oxime moiety is rarely found in natural prod-
ucts;30 however several synthetic derivatives possess various
biological activities including cytotoxic, antiviral, analgesic,
cardiovascular, anti-inflammatory, antidiabetic and antispas-
modic activities.24c Additionally, charged31,32 as well as
uncharged derivatives33–35 are efficient reactivators of nerve
agent inhibited acetylcholinesterases aiming treatment for
organophosphorous compound poisoning. Inspired by acetyl-
cholinesterase reactivation, Terenzi et al. has examined the
ability of oximes to cleave phosphate bonds in nucleic acids
and act as metal-free artificial nucleases.36 Compounds
Several organic compounds including certain enediynes,10–12
pyrrolecarboxamide conjugated 4′-bromo-acetophenones,13
riboflavins,14 naphthalimides,15 pyrenes,16 anthraquinone based
derivatives,17,18 quinolines19,20 and benzo[b][1,8]naphthyridines21
were demonstrated as DNA photo-cleavaging agents.
a Laboratory of Organic, Bioorganic and Natural Product Chemistry, Molecular Biology
and Genetics Department, Democritus University of Thrace, University Campus,
Dragana, 68100, Alexandroupolis, Greece. E-mail: kfylakta@mbg.duth.gr;
Fax: +30 25510 30613
b Laboratory of Organic Chemistry, Chemistry Department, Aristotle University of
Thessaloniki, 54124, Thessaloniki, Greece
† Electronic supplementary information (ESI) available: 1H and 13C-NMR spectra
of all compounds, photochemistry and pH experiments, and UV spectra are
available. See DOI: 10.1039/c4md00548a
‡ Equal contribution.
Fig. 1 Structures of aldoximes (I), ketoximes (II) and amidoximes (III).
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containing N–O bonds (aldoximes and ketoximes included)
have been found to be efficient metal-free artificial DNA
photo-cleavers16,17,19,37–39 due to the homolysis of the weak
N–O bonds.40
adopted in order to obtain four new compounds, 14–17
(Scheme 2).
All reactions (Schemes 1 and 2) furnished a single product
in good yield, without the need of chromatographic purifica-
tion. The structures of all derivatives were fully assigned
based on their spectral data. Additionally, all compounds
were found to absorb UV light at least partially over 300 nm,
as it was deduced from their UV-vis spectra (see the ESI†).
The stereochemistry of synthesized aldoxime esters was
found to be syn (to the H atom). This was evidenced by a
standard procedure, which involves the ester cleavage in alka-
line methanolic solution, leading to the formation of the par-
ent syn-oxime.47 Additionally, all amidoxime derivatives have
the Z-conformation in accordance with what was previously
reported.27
To the best of our knowledge amidoximes have never been
tested as metal or metal-free artificial photo-cleavers. Our
interest in the chemistry and biology of oximes29,41–43 has
prompted us to synthesize and study several pyridine amido-
xime ester conjugates and compare them with their corre-
sponding aldoximes. The fact that the number of aldoximes
acting as efficient photo-cleavers is limited19 prompted us to
study novel scaffolds and provide a structure–activity relation-
ship based on electronic phenomena on the ester conjugates
(electron donor or acceptor) as well as steric factors and
hydrogen bonding capacities (H vs. NH2).
Photolysis causes homolytic cleavage of the N–O bond of
O-aryl-oximes and production of iminyl and acyloxyl radicals
(Scheme 3). The latter react with DNA, via several mechanis-
tic pathways, to give single- and double-stranded cleavages.
The reactive species are, according to the work of
Theodorakis et al.,37,38 the oxygen centred radicals. Irradia-
tion of compounds in the absence of DNA under various con-
ditions initially gives iminyl radicals IJR1R2CN˙). This is
eventually transformed to an imine which is further hydro-
lyzed to the corresponding ketone. The aryloxyl radical
(ArCOO˙) gives the corresponding acid, whereas in some
cases it is subsequently decarboxylated to the aryl radical.
Other researchers observed photo-Beckman transformations,
along with the expected ketone and acid products.16
In the case of amidoximes we have observed the same
homolysis of the amidoxime N–O bond, which gave pyridine
amidinyl radicals (R1 = pyridine, R2 = NH2) and p-nitro-
benzoyl radicals (Ar = PNP), respectively, upon irradiation at
312 nm for 5 h in a MeOH–H2O solution or benzene
containing 1,4-cyclohexadiene (see the ESI†).
2. Results and discussion
2.1. Chemistry
meta-Pyridine aldoxime 1 (ref. 44) (Scheme 1) reacted with
acyl anhydrides or acid chlorides in various solvents to give
O-acyl derivatives 2–4. Under similar reaction conditions,
meta-pyridine amidoxime 5 (ref. 45) (Scheme 1) delivered
O-acyl derivatives 6–9. It is known that the aliphatic acyl-oxy
derivatives do not give radicals with the same efficiency as
the aromatic ones probably due to the facile radical decarbox-
ylation of the former which generate less reactive carbon
centred radicals.37 Since, however, amidoximes are tested for
the first time, we needed to verify that the same applies to
this class of oximes as well. Thus, amidoxime methyl-ester 9
was also synthesized and tested.
Interestingly, although several meta-pyridine O-acyl aldoximes
and amidoximes are known,46 derivatives 3, 4, 7 and 8 are
new. Nevertheless, none of the rest had been fully character-
ized, either because their synthesis was very old or because
these particular amidoxime derivatives had been solely used
as intermediates.
2.2. Biological assays
The photocleaving ability of the above compounds
(vide infra) suggested that the p-nitro-benzoyl substituent is
the most efficient at low concentrations. Based on this obser-
vation, the p-nitrobenzoyl group is further linked to the o-
and p-pyridine aldoxime and amidoxime scaffolds, in an
effort to examine the effect of the position of the pyridine
ring nitrogen atom. A similar experimental protocol was
Compounds 1–9 and 14–17 were irradiated with UV light
(312 nm, 90 W) at room temperature for 15 min, under aero-
bic conditions, at various concentrations in a DMF solution
(1–20%) and in a Tris buffer solution (25 μM, pH = 6.8)
containing the supercoiled circular pBluescript KS II DNA
(form I, 500 ng). Plasmid DNA was then analyzed by gel
electrophoresis on 1% agarose stained with ethidium bro-
mide. Plasmid DNA can exist in three conformations:
Scheme 1 Synthesis of meta-pyridine derivatives 2–4 and 6–9. Reac-
tion conditions: acylating agent, Et3N, solvent (CHCl3 or THF or DMF),
0 °C to r.t., 74–89% yield for 2–4, 50–72% yield for 6–9 [PMP =
p-methoxy-phenyl, PNP = p-nitro-phenyl].
Scheme 2 Synthesis of ortho- and para-pyridine derivatives 14–17.
Reaction conditions: PNP-COCl, Et3N, THF, 0 °C to r.t, 1.5 h, 75–88%
yield.
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Fig. 3 Comparative dose measurement results from DNA cleavage by
the meta-pyridine amidoxime O-acyl derivatives 6–9. Top: the gel
electrophoresis data: lane 1: DNA without UV irradiation; lane 2: DNA
with UV irradiation; lane 3: DNA + 6 (100 μM); lane 4: DNA + 7
(100 μM); lane 5: DNA + 8 (100 μM); lane 6: DNA + 9 (100 μM); lane 7:
DNA + 6 (500 μM); lane 8: DNA + 7 (500 μM); lane 9: DNA +
8 (500 μM); lane 10: DNA + 9 (500 μM); lane 11: DNA + 6 (1000 μM);
lane 12: DNA + 7 (1000 μM); lane 13: DNA + 8 (1000 μM); lane 14:
DNA + 9 (1000 μM). Bottom: % conversion of pBluescript KS II with
derivatives 6–9. For the calculation of ss and ds %, see ref. 48.
Scheme 3 Photo-cleavage of the N–O bond of oxime containing ester
conjugates.
supercoiled (form I), relaxed or open-circular (form II), and
linear (form III). For the same size, supercoiled DNA runs
faster than relaxed circular DNA. Linear DNA sustains less
friction than relaxed circular DNA, but more than supercoiled
DNA. We found that in the presence of the compounds the
supercoiled plasmid DNA sustained single-stranded nicks
of the double helix, generating the relaxed circular DNA
(form II) found in many experiments, whereas in some cases
the linear DNA (form III) was formed as well, generated by
double-stranded nicks. None of the tested compounds
showed any activity towards DNA in the absence of UV irradi-
ation. Blank experiments with DMF up to 20% did not show
any effect on DNA, and pH did not affect the activity of the
cleavage (see the ESI†). Neither aldoxime 1 nor amidoxime 5
has shown any DNA photocleavage activity, a result that is in
accordance with the lack of reactivity of other N–OH
derivatives.37a All experiments were performed at least three
times.
For the structure–activity relationships, we initially used
concentrations of 100, 500 and 1000 μM for all aldoxime and
amidoxime derivatives (Fig. 2 and 3, respectively). Aromatic
derivatives 2–4 and 6–8 have shown conversion mainly at
high concentrations, with derivatives 4 (Fig. 2, lanes 5, 8, and 11)
and 8 (Fig. 3, lanes 5, 9, and 13), bearing an electron with-
drawing substituent, to give the best results. The latter com-
pounds also promoted the formation of linear DNA (form III)
at levels of up to 36% (at a concentration of 1 mM). In con-
trast, aliphatic O-acetyl derivative 9 showed very low conver-
sion (Fig. 3, lanes 6, 10, and 14) in accordance with the
literature.37
electron withdrawing p-nitro-benzoyl conjugate strongly pro-
motes DNA photo-cleavage in both classes of compounds. No
significant difference is observed in the other end for the less
reactive compounds that bear an electron donating group
(i.e. p-methoxy-derivatives). However, benzoyl substituted
aldoximes clearly showed an enhanced nicking effect relative
to the corresponding amidoximes. Since it was shown that
the S0 → S1 transition and the triplet state are localized on
the oxime moiety, the mechanism of dissociation is most
probably affected by the substituent effects.40b
All four o- and p-pyridine derivatives, 14–17, interacted
strongly with DNA under UV irradiation at the tested concen-
trations (Fig. 4) and exhibited similar behaviour to that of
the m-pyridine p-nitro-benzoyl derivatives 4 and 8. Most
importantly, all PNP derivatives regardless of the N position
of the pyridine moiety caused not only single-stranded nicks
but also double-stranded DNA scission (50% for derivative 17
at a concentration of 1 mM, Fig. 4, lane 9).
The above results have prompted us to examine the lowest
concentrations, which are sufficient for DNA photo-cleavage
for the PNP ester conjugates 4, 8 and 14–17. This concentra-
tion was determined as the amount of compound capable of
cleaving at least 50% of the supercoiled plasmid DNA, with
the following calculation: (form II + form III)/form I ≥ 1.
Three compounds, one aldoxime (4) and two amidoximes (8
and 15), were active even at concentrations as low as 1 μM
(Fig. 5, lanes 2, 5, and 6, and Fig. 6).
From the comparison of aldoxime and amidoxime deriva-
tives with the same O-substituent, we conclude that the
Fig. 2 Comparative dose measurement results from DNA cleavage by
the meta-pyridine oxime O-acyl derivatives 2–4. Top: the gel electro-
phoresis data: lane 1: DNA without UV irradiation; lane 2: DNA with UV
irradiation; lane 3: DNA + 2 (100 μM); lane 4: DNA + 3 (100 μM); lane
5: DNA + 4 (100 μM); lane 6: DNA + 2 (500 μM); lane 7: DNA + 3
(500 μM); lane 8: DNA + 4 (500 μM); lane 9: DNA + 2 (1000 μM); lane
10: DNA + 3 (1000 μM); lane 11: DNA + 4 (1000 μM). Bottom: % con-
version of pBluescript KS II with derivatives 2–4. For the calculation of
ss and ds %, see ref. 48.
Fig. 4 Comparative dose measurement results from DNA cleavage by
the ortho- and para-pyridine derivatives 14–17. Top: the gel electro-
phoresis data: lane 1: DNA with UV irradiation; lane 2: DNA + 14
(100 μM); lane 3: DNA + 16 (100 μM); lane 4: DNA + 14 (1000 μM); lane
5: DNA + 16 (1000 μM); lane 6: DNA + 15 (100 μM); lane 7: DNA + 17
(100 μM); lane 8: DNA + 15 (1000 μM); lane 9: DNA + 17 (1000 μM).
Bottom: % conversion of pBluescript KS II with derivatives 14–17. For
the calculation of ss and ds %, see ref. 48.
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Fig. 5 Comparative dose measurement results from DNA cleavage by
the pyridine oxime 4, 14, and 16 and amidoxime derivatives 8, 15, and
17 at a concentration of 1 μM. Top: the gel electrophoresis data: lane
1: DNA with UV irradiation; lane 2: DNA + 4; lane 3: DNA + 14; lane 4:
DNA + 16; lane 5: DNA + 8; lane 6: DNA + 15; lane 7: DNA + 17.
Fig. 7 Mechanistic studies involved at the DNA cleavage by derivative
4 (100 μM). Top: the gel electrophoresis data: lane 1: DNA with UV
irradiation; lane 2: DNA + 4; lane 3: DNA + 4 + argon; lane 4: DNA + 4 +
DMSO (20%); lane 5: DNA + 4 + NaN3 (20 mM); lane 6: DNA + 4 + D2O.
Bottom: % conversion of pBluescript KS II with compound 4. For the
calculation of ss and ds %, see ref. 47.
Bottom:
% conversion of pBluescript KS II with derivatives 4, 8,
and 14–17. For the calculation of ss and ds %, see ref. 48.
Fig. 8 Mechanistic studies involved at the DNA cleavage by derivative
8 (100 μM). Top: the gel electrophoresis data: lane 1: DNA without UV
irradiation; lane 2: DNA with UV irradiation; lane 3: DNA + 8; lane 4:
DNA + argon; lane 5: DNA + 8 + argon; lane 6: DNA + DMSO (20%);
lane 7: DNA + 8 + DMSO (20%); lane 8: DNA + NaN3 (20 mM); lane 9:
DNA + 8 + NaN3 (20 mM); lane 10: DNA + D2O; lane 11: DNA + 8 +
D2O. Bottom: % conversion of pBluescript KS II with compound 8. For
the calculation of ss and ds %, see ref. 47.
Fig. 6 (ss + ds) % DNA cleavage caused by aldoximes 4, 14, and 16 and
amidoximes 8, 15, and 17 at concentrations of 1, 10, 100 and 1000 μM.
The results presented in the figure show the mean values
least three runs.
SD of at
Nevertheless, the cleavage of plasmid DNA is not enhanced
when D2O was used as a solvent. It is known that D2O
increases the lifetime of singlet oxygen and thus leads to a
more effective cleavage when this radical is involved in the
mechanism.49
All three pyridine amidoximes exhibited better photo-
cleaving ability at a concentration of 10 μM compared to
their related aldoximes (4 vs. 8, 14 vs. 15 and 16 vs. 17,
Fig. 6). At higher concentrations both classes of oximes are
highly efficient with their differences to stay within the statis-
tical error. Nevertheless, the percentage of the double-
stranded photo-cleavage is much more enhanced in the case
of amidoximes (4 vs. 8, Fig. 2, lane 11, and Fig. 3, lane 13,
respectively; 14 vs. 15, Fig. 4, lanes 4 and 8, respectively; 16
vs. 17, Fig. 4, lanes 5 and 9, respectively). It seems possible
that NH2 in amidoximes positively affects the affinity with
DNA providing extra points for hydrogen bonding.
In an effort to understand the mechanistic pathways
involved in the DNA cleavage we have also performed experi-
ments under anaerobic (argon atmosphere) and aerobic con-
ditions in the presence of molecular oxygen with hydroxyl
radical scavengers, like DMSO, and singlet oxygen quenchers,
such as sodium azide, for representative aldoxime (4) and
amidoxime (8) derivatives. Results for these compounds are
presented in Fig. 7 and 8, respectively.
Summarizing these results, we believe that not a single
mechanistic pathway is implicated in the cleavage and, cer-
tainly, the one involving singlet oxygen is not the dominant
one. Nitro substituted compounds have been studied for
their ability to cleave DNA; thus, the p-nitro-phenyl moiety
may also contribute to the DNA photo-cleavage. It has been
reported that radicals are formed upon irradiation of nitro
compounds; nevertheless, their triplet state rapid deactiva-
tion may cause insufficient photochemistry. The mechanism
of action of nitro-phenyl containing compounds is not fully
clarified, since researchers have suggested hydrogen abstrac-
tion from the deoxyribose DNA backbone and/or oxygen
donation, electron transfer, or hydrogen abstraction from the
thymine methyl group. Nevertheless, other factors such as
affinity of the compound with DNA are also a require-
ment.6,50 The role of the substituent of the oxime or amido-
xime moieties, as well as of the NO2 group, is currently inves-
tigated with the synthesis and evaluation of more derivatives.
3. Conclusions
It seems that both compounds can react under anaerobic
conditions and the mode of action most probably does not
involve hydroxyl radicals (no reaction with DMSO). Their
activity is notably reduced in the presence of sodium azide
(Fig. 7, lane 5, for compound 4, Fig. 8, lane 9, for compound
8), which indicates that singlet oxygen is possibly involved.
We have found that O-benzoyl-pyridine aldoxime and amido-
xime derivatives are DNA photo-cleavers. In particular, meta-,
ortho- and para-pyridine O-p-nitrobenzoyl aldoximes and the
corresponding amidoximes are highly effective at concentra-
tions as low as 1 μM, with amidoxime 15 being the most
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efficient. The fact that these compounds are able a) to react
under anaerobic conditions and b) to cause relatively high
ratios of ds/ss DNA cleavage may potentially lead to multiple
applications, including phototherapeutic treatment of solid
tumors and cancer therapy in general. Finally, we believe that
amidoximes in particular may be recognized as a novel class
of “photo-cleavers” along with aldoximes and ketoximes.
filtered off. The solid residue was recrystallized to give
228 mg (89%) of oxime 3. Off-white crystals, m.p. 133–135 °C
(ethanol), IR (KBr): 1736, 1606 cm−1; 1H NMR (500 MHz,
CDCl3) δ 3.87 (s, 3H), 6.96 (d, J = 5.4 Hz, 2H), 7.39 (dd, J =
4.8, 2.9 Hz, 1H), 8.07 (d, J = 5.4 Hz, 2H), 8.26 (dt, J = 4.8,
1.0 Hz, 1H), 8.57 (s, 1H), 8.70 (dd, J = 2.9, 1.0 Hz, 1H), 8.86
(d, J = 0.9 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 55.5, 113.9,
120.3, 123.8, 126.6, 131.9, 134.5, 150.0, 152.4, 153.5, 163.4,
163.9; HRMS (ESI) calc C14H13N2O3 [M + H]+ 257.0921; found
257.0917.
4.3. Synthesis of nicotinaldehyde O-4-nitrobenzoyl oxime
(4). Aldoxime 1 (ref. 44) (244 mg, 2 mmol) was dissolved in
tetrahydrofuran (0.15 M) and triethylamine (0.30 mL,
2.2 mmol) was added at 0 °C under an Ar atmosphere,
followed by 4-nitro-benzoyl chloride (408 mg, 2.2 mmol). The
mixture was stirred for 1.5 h. Water (100 mL) was added and
the mixture was extracted with ethyl acetate (2 × 100 mL).
The organic layers were further washed with water (100 mL)
and dried with Na2SO4, and the solvents were evaporated to
dryness. The crude residue was recrystallized to give 445 mg
(83%) of oxime 4. Off-white crystals, m.p. 161.2 °C (ethyl ace-
tate), IR (KBr): 1739, 1614 cm−1; 1H NMR (500 MHz, DMSO-
d6) δ 7.58 (dd, J = 7.9, 4.9 Hz, 1H), 8.24 (dt, J = 8.0, 1.8 Hz,
1H), 8.31 (dd, J = 8.9, 1.8 Hz, 2H), 8.42 (dd, J = 8.9, 1.8 Hz,
2H), 8.76 (dd, J = 4.8, 1.6 Hz, 1H), 8.95 (d, J = 1.6 Hz, 1H),
9.07 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 124.1, 124.3,
126.0, 130.8, 133.4, 134.8, 149.7, 150.5, 152.7, 156.8, 161.6;
HRMS (ESI) calc C13H10N3O4 [M + H]+ 272.0666; found
272.0656.
4. Experimental
Mps were measured using a Kofler hot-stage apparatus or a
melting point meter M5000 KRÜSS, and were uncorrected.
FT-IR spectra were obtained with
a Perkin-Elmer 1310
spectrometer using potassium bromide pellets. For the UV
spectra a reader TECAN M1000 was used. NMR spectra were
1
recorded on an Agilent 500/54 (500 MHz and 125 MHz for H
and 13C, respectively) spectrometer using CDCl3 and/or
DMSO-d6 as solvent. J values are reported in hertz. High reso-
lution mass spectra (HRMS) were recorded on a micrOTOF
GC-MS QP 5050 Shimadzu single-quadrupole mass spectro-
meter. Mass spectra were determined using a Shimadzu
LCMS-2010 EV system under electrospray ionization (ESI)
conditions. All reactions were monitored on commercially
available pre-coated TLC plates (layer thickness, 0.25 mm) of
Kieselgel 60 F254. Yields were calculated after recrystalliza-
tion. Samples containing plasmid DNA were irradiated with a
Macrovue 2011 transilluminator LKB BROMMA at 312 nm,
90 W, 0.225 W cm−2, 10 cm distance, whereas samples with-
out the plasmid were irradiated with Philips 2 × 9W/12/2P
UV-B broadband lamps at 312 nm.
4.4. Synthesis of picolinaldehyde O-4-nitrobenzoyl oxime
(14). From aldoxime 10 (ref. 44) following the procedure used
for 4, the crude residue was recrystallized to give 452 mg
(84%) of oxime 14. Light yellow crystals, m.p. 178–180 °C
Synthesis of pyridine aldoxime derivatives
4.1. Synthesis of nicotinaldehyde O-benzoyl oxime (2)46a
.
1
Aldoxime 1 (ref. 44) (122 mg, 1 mmol) was dissolved in tetra-
hydrofuran (0.2 M) and triethylamine (0.15 mL, 1.1 mmol)
was added at 0 °C under an Ar atmosphere, followed by
benzoic anhydride (249 mg, 1.1 mmol). The mixture was
stirred for 12 h, then water (30 mL) was added and the mix-
ture was extracted with ethyl acetate (2 × 30 mL). After drying
with Na2SO4 the organic solvents were removed using a rotary
evaporator and the crude residue was recrystallized to give
170 mg (75%) of oxime 2. White crystals, m.p. 151–153 °C
(ethyl acetate), IR (KBr): 1736, 1603; 1H NMR (500 MHz,
DMSO-d6) δ 7.53–7.62 (m, 3H), 7.74 (tt, J = 7.5, 1.2 Hz, 1H),
8.08 (br dd, J = 8.4, 1.3 Hz, 2H), 8.23 (br dt, J = 8.0, 1.7 Hz,
1H), 8.74 (bd, J = 4.7, 1.6 Hz, 1H), 8.95 (br d, J = 1.2 Hz, 1H),
8.99 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 124.3, 126.3,
127.9, 129.0, 129.3, 134.0, 134.8, 149.6, 152.5, 156.0, 163.0;
HRMS (ESI) calc C13H11N2O2 [M + H]+ 227.0815; found
227.0817.
(ethyl acetate), IR (KBr): 1755, 1696, 1607 cm−1; H NMR (500
MHz, CDCl3) δ 7.42 (dd, J = 6.6, 4.9 Hz, 1H), 7.82 (dt, J = 7.8,
1.3 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 8.9 Hz, 2H),
8.35 (d, J = 8.9 Hz, 2H), 8.67 (s, 1H), 8.71 (d, J = 4.3 Hz, 1H);
13C NMR (125 MHz, CDCl3) δ 122.3, 123.8, 125.8, 130.9,
133.8, 136.8, 149.5, 150.1, 150.9, 158.2, 161.9; HRMS (ESI)
calc C13H10N3O4 [M + H]+ 272.0666; found 272.0662.
4.5. Synthesis of isonicotinaldehyde O-4-nitrobenzoyl
oxime (16). From aldoxime 12 (ref. 44) following the proce-
dure used for 4, the crude residue was recrystallized to give
475 mg (88%) of oxime 16. Pale yellow crystals, m.p. 194–196 °C
1
(ethyl acetate), IR (KBr): 1760, 1608 cm−1; H NMR (500 MHz,
DMSO-d6) δ 7.75 (d, J = 5.1 Hz, 2H), 8.29 (d, J = 8.5 Hz, 2H),
8.41 (d, J = 8.5 Hz, 2H), 8.76 (d, J = 5.1 Hz, 2H), 9.03 (s, 1H);
13C NMR (125 MHz, DMSO-d6) δ 122.0, 124.1, 130.9, 133.3,
137.1, 150.6, 150.7, 157.4, 161.5; HRMS (ESI) calc C13H10N3O4
[M + H]+ 272.0666; found 272.0661.
4.2. Synthesis of nicotinaldehyde O-4-methoxybenzoyl
oxime (3). Aldoxime 1 (ref. 44) (122 mg, 1 mmol) was
dissolved in dry CHCl3 (0.2 M) and triethylamine (0.15 mL,
1.1 mmol) was added at 0 °C under an Ar atmosphere,
followed by 4-methoxy-benzoyl chloride (188 mg, 1.1 mmol)
and DMAP (0.05%). The mixture was stirred for 2 h and
Synthesis of pyridine amidoxime derivatives
4.6. Synthesis of N′-IJbenzoyloxy)nicotinimidamide (6)51
Amidoxime 5 (ref. 45) (274 mg, 2 mmol) was dissolved in a
.
mixture of chloroform and DMF (ratio, 30/1; 0.15 M) and
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cooled to 0 °C under an Ar atmosphere. Triethylamine
(0.28 mL, 2 mmol) was added, followed by benzoic anhydride
(452 mg, 2 mmol), and the mixture was stirred for 6 h. Water
(70 mL) was added and the mixture was extracted with
dichloromethane (3 × 70 mL) and ethyl acetate (3 × 70 mL).
After drying with Na2SO4 the organic solvents were removed
using a rotary evaporator and the residue was recrystallized
to give 343 mg (69%) of amidoxime 6. White crystals, m.p.
200–202 °C (ethanol); IR (KBr): 3425, 3331, 1727, 1637, 1605;
1H NMR (500 MHz, DMSO-d6) δ 7.17 (br s, 2H), 7.58–7.48 (m,
3H), 7.67 (tt, J = 7.4, 1.3 Hz, 1H), 8.15 (dt, J = 8.3, 1.8 Hz, 1H),
8.21 (dd, J = 8.2, 1.8 Hz, 2H), 8.71 (dd, J = 4.8, 1.7 Hz, 1H),
8.95 (dd, J = 2.3, 0.8 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
123.5, 127.8, 128.6, 129.3, 129.5, 133.1, 134.7, 147.8, 151.4,
155.2, 163.5. HRMS (ESI) calc C13H12N3O2 [M + H]+, 242.0924;
found 242.0920.
4.7. Synthesis of N′-IJ(4-methoxybenzoyl)oxy)nicotinimidamide
(7). Amidoxime 5 (ref. 45) (137 mg, 1 mmol) was dissolved in
a mixture of dry chloroform and DMF (ratio, 10/1; 0.2 M) and
cooled to 0 °C under an Ar atmosphere. Triethylamine (0.15 mL,
1.1 mmol) was added followed by 4-methoxy-benzoyl chloride
(188 mg, 1.1 mmol) and DMAP (0.05%). The mixture was stirred
for 12 h and filtered off. The crude solid was recrystallized to give
196 mg (72%) of amidoxime 7. White crystals, m.p. 201–203 °C
(ethanol); IR (KBr): 3410, 3327, 1716, 1638, 1604 cm−1; 1H NMR
(500 MHz, DMSO-d6) δ 3.85 (s, 3H), 7.05 (dd, J = 8.9, 1.9 Hz, 2H),
7.11 (br s, 2H), 7.51 (dd, J = 7.9, 4.8 Hz, 1H), 8.12 (dt, J = 8.0,
1.8 Hz, 1H), 8.16 (dd, J = 8.9, 2.0 Hz, 2H), 8.70 (dd, J = 4.8, 1.5 Hz,
1H), 8.93 (dd, J = 2.2, 0.6 Hz, 1H); 13C NMR (125 MHz, DMSO-d6)
δ 55.6, 113.9, 121.4, 123.5, 127.9, 131.7, 134.7, 147.8, 151.3,
154.9, 163.1, 163.2; HRMS (ESI) calc C14H14N3O3 [M + H]+,
272.1035; found 272.1027.
(500 MHz, DMSO-d6) δ 2.14 (s, 3H), 6.99 (br s, 2H), 7.49 (ddd,
J = 8.0, 4.9, 0.9 Hz, 1H), 8.06 (ddd, J = 8.0, 2.3, 1.8 Hz, 1H),
8.68 (dd, J = 4.8, 1.7 Hz, 1H), 8.87 (dd, J = 2.3, 0.8 Hz 1H);
13C NMR (125 MHz, DMSO-d6) δ 19.8, 123.5, 127.6, 134.4, 147.6,
151.3, 154.7, 168.4; HRMS (ESI) calc C8H10N3O2 [M + H]+,
180.0768; found 180.0764.
4.10. Synthesis of N′-IJ(4-nitrobenzoyl)oxy)picolinimidamide
(15). Amidoxime 11 (ref. 52) (274 mg, 2 mmol) was dissolved
in tetrahydrofuran (0.15 M) and triethylamine (0.30 mL,
2.2 mmol) was added at 0 °C under an argon atmosphere,
followed by 4-nitro-benzoyl chloride (408 mg, 2.2 mmol). The
mixture was stirred for 1.5 h, and the mixture was filtered off
to give part of the crude product. The filtrate was extracted
with ethyl acetate (2 × 70 mL) and water (70 mL). The organic
phases were collected, dried with Na2SO4, and evaporated to
dryness. The crude residues were recrystallized to give 450 mg
(79%) of amidoxime 15. Yellow crystals, m.p. 205–207 °C
(ethanol); IR (KBr): 3475, 3361, 1726, 1633 cm−1; 1H NMR
(500 MHz, DMSO-d6) δ 7.18 (br s, 1H), 7.36 (br s, 1H), 7.58 (t,
J = 5.3 Hz, 1H), 7.96 (t, J = 7.1 Hz, 1H), 8.02 (d, J = 7.9 Hz,
1H), 8.34 (d, J = 8.7 Hz, 2H), 8.47 (d, J = 8.7 Hz, 2H), 8.69 (d,
J = 4.4 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 121.3, 123.7,
126.0, 131.2, 134.7, 137.5, 148.1, 148.9, 150.3, 155.1, 162.1;
HRMS (ESI) calc C13H11N4O4 [M + H]+, 287.0775; found
287.0772.
4.11. Synthesis of N′-IJ(4-nitrobenzoyl)oxy)isonicotinimidamide
(17). From amidoxime 13 (ref. 53) following the procedure used
for product 15, the crude residue was recrystallized to give 428 mg
(75%) of amidoxime 17. Yellow crystals, m.p. 180.2 °C (etha-
nol); IR (KBr): 3470, 3372, 1726, 1630 cm−1; 1H NMR (500 MHz,
DMSO-d6) δ 7.36 (br s, 2H), 7.75 (dd, J = 6.0, 0.4 Hz, 2H), 8.35
(dd, J = 9.0, 2.1 Hz, 2H), 8.45 (dd, J = 9.0, 2.0 Hz, 2H), 8.72 (dd,
J = 6.0, 1.4 Hz, 2H); 13C NMR (1255 MHz, DMSO-d6) δ 121.3,
123.7, 131.2, 134.7, 139.1, 150.2, 150.3, 155.8, 162.1; HRMS
(ESI) calc C13H11N4O4 [M + H]+, 287.0775; found 287.0770.
4.8. Synthesis of N′-IJ(4-nitrobenzoyl)oxy)nicotinimidamide
(8). Amidoxime 5 (ref. 45) (137 mg, 1 mmol) was dissolved in
tetrahydrofuran (0.1 M) and triethylamine (0.15 mL, 1.1 mmol)
was added at 0 °C under an Ar atmosphere, followed by
4-nitro-benzoyl chloride (204 mg, 1.1 mmol). The mixture was
Molecular biology
stirred for
4
h
and filtered off. The crude solid was
Cleavage of supercoiled circular pBluescript KS II by acyl
aldoximes and amidoximes. The reaction mixtures (20 μL)
containing supercoiled circular pBluescript KS II DNA stock
solution (form I, 50 μM/base pair, ~500 ng), compounds, and
Tris buffer (25 μM, pH 6.8) in Pyrex vials were incubated for
30 min at 37 °C, centrifuged, and then irradiated with UV light
(312 nm, 90 W) under aerobic conditions at room temperature
for 15 min. After addition of the gel-loading buffer (6× orange
DNA loading dye 10 mM Tris–HCl (pH 7.6), 0.15% orange G,
0.03% xylene cyanol FF, 60% glycerol, and 60 mM EDTA, from
Fermentas), 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 (65 V for
1 h). The gel was visualized by using a 312 nm UV transillumi-
nator and photographed by using an FB-PBC-34 camera Vilber
Lourmat. Quantitation of DNA-cleaving activities was
performed by integration of the optical density as a function of
the band area using the program “Image J” available at the site
http://rsb.info.nih.gov/ij/download.html.
recrystallized to give 188 mg (65%) of amidoxime 8. Yellow
crystals, m.p. 226–228 ° C (ethanol); IR (KBr): 3398, 3323,
3198, 1741, 1640 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 7.33
(br s, 2H), 7.53 (dd, J = 7.8, 4.8 Hz, 1H), 8.14 (dt, J = 8.0,
1.8 Hz, 1H), 8.34 (d, J = 8.8 Hz, 2H), 8.45 (d, J = 8.8 Hz, 2H),
8.72 (dd, J = 4.8, 1.4 Hz, 1H), 8.94 (d, J = 1.7 Hz, 1H); 13C NMR
(125 MHz, DMSO-d6) δ 123.5, 123.6, 127.5, 131.1, 134.7, 134.8,
147.8, 150.1, 151.5, 155.8, 162.1; HRMS (ESI) calc C13H11N4O4
[M + H]+, 287.0775; found 287.0772.
4.9. Synthesis of N′-acetoxynicotinimidamide (9)46c. Amidoxime 5
(ref. 45) (137 mg, 1 mmol) was dissolved in tetrahydrofuran
(0.15 M) and triethylamine (0.15 mL, 1.1 mmol) was added at 0 °C
under an Ar atmosphere, followed by acetic anhydride (0.1 mL,
1.1 mmol). The mixture was stirred for 3 h, and since the product
was highly water soluble, the mixture was evaporated to dryness
and the crude residue was recrystallized to give 90 mg (50%) of
amidoxime 9. White crystals, m.p. 150–152 °C (ethyl acetate/eth-
anol); IR (KBr): 3399, 3341, 3223, 1751, 1647, 1605 cm−1; 1H NMR
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Acknowledgements
We would like to thank Assist. Prof. Dr. S. Boukouvala for
access to the TECAN instrument, Dr. H. Evgenidou for techni-
cal assistance with the LC-MS instrument, and Dr. A. Tsolou,
C. Karampelias, M.Sc., and E. Aggelou, M.Sc., for helpful
discussions.
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