Design, synthesis, and evaluation of N-aroyloxy-2-thiopyridones as DNA photocleaving reagents

Article abstract of DOI:10.1016/S0968-0896(98)00211-9

N-Benzoyloxy-2-thiopyridone (12) was shown to induce single-strand nicks in duplex DNA upon irradiation with visible light (λ~350nm). This finding led to the design of a series of compounds, in which an acridinyl nucleus was covalently linked to the N-benzoyloxy-2-thiopyridone unit. These conjugates (15, 16, 17 and 18) were synthesized and evaluated as novel DNA photocleaving reagents. Optimal photocleaving activity was observed for conjugate 16, in which a flexible polymethylene spacer of 4 carbons was used to connect the aminoacridine entity to the thiopyridone. This compound was shown to cleave DNA at low μM concentrations and was approximately two-orders of magnitude more efficient than the parent N-benzoyloxy-2-thiopyridone (12). Furthermore, the DNA cleavage ladders induced by 16 and 12 were found to be identical and of no significant sequence selectivity. These data suggest that the N-aroyloxy-2-thiopyridones can be used for the design of new DNA photocleaving reagents with potential use as 'photofootprinting agents' or as 'site-directed photonucleases'. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00211-9

Bioorganic & Medicinal Chemistry 7 (1999) 727±736
Design, Synthesis, and Evaluation of N-Aroyloxy-2-thiopyridones
as DNA Photocleaving Reagents
Petra Blom, Alan X. Xiang, David Kao and Emmanuel A. Theodorakis*
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Received 2 July 1998; accepted 10 September 1998
AbstractÐN-Benzoyloxy-2-thiopyridone (12) was shown to induce single-strand nicks in duplex DNA upon irradiation with visible
light (l&350 nm). This ®nding led to the design of a series of compounds, in which an acridinyl nucleus was covalently linked to the
N-benzoyloxy-2-thiopyridone unit. These conjugates (15, 16, 17 and 18) were synthesized and evaluated as novel DNA photo-
cleaving reagents. Optimal photocleaving activity was observed for conjugate 16, in which a ¯exible polymethylene spacer of 4
carbons was used to connect the aminoacridine entity to the thiopyridone. This compound was shown to cleave DNA at low mM
concentrations and was approximately two-orders of magnitude more ecient than the parent N-benzoyloxy-2-thiopyridone (12).
Furthermore, the DNA cleavage ladders induced by 16 and 12 were found to be identical and of no signi®cant sequence selectivity.
These data suggest that the N-aroyloxy-2-thiopyridones can be used for the design of new DNA photocleaving reagents with
potential use as `photofootprinting agents' or as `site-directed photonucleases'. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
as chemical nucleases. In the initial reports, generation
of such radicals was accomplished by redox chemistry
of organometallic reagents, a concept pioneered by Sig-
man,6 Dervan,7 and Tullius8 using Cu(II) and Fe(II)
complexes. More recently, the repertoire of organome-
tallic reagents has been expanded by the work of Hecht9
and Burrows10 to include Co(II) and Ni(II) complexes.
Although all these compounds have been extensively
used over the years, they su€er from practical limitations,
since they are sensitive to the presence of various biolo-
gical species, including glycerol, thiols or metal ions,
and require high concentrations of chemical initiators.
The design of functional molecules has been a major
goal of research in chemistry, biology, and medicine,
encompassing topics such as catalysis, drug design,
host±guest interactions, catalytic antibodies, and syn-
thetic enzymes. This last topic includes, among others,
research directed toward the development of nucleolytic
reagents that react with DNA and/or RNA, ultimately
leading to cleavage of the phosphodiester backbone.1
These reagents, commonly referred to as `arti®cial
nucleases', hold great promise as tools in molecular
biology2 and as potential chemotherapeutic agents for
cancer or antiviral treatment.3 In addition, attachment
of these reagents to selective carriers could produce
`arti®cial restriction enzymes' and speci®c gene-targeted
drugs.4
The search for alternative, perhaps more ecient, che-
mical nucleases led several groups to examine light-
induced activation techniques.11 These methods are
advantageous since the cleavage proceeds using light,
eliminating the need of external chemical initiators.
These photoactivated reagents, commonly referred to as
`photonucleases', include complexes of Ru and Rh,12
uranyl salts13 and even simply UV light14 and have been
pioneered by Barton, Nielsen and Wang, respectively.
However all the above methods have several drawbacks,
particularly as related to the reagent concentration, the
wavelength and intensity of the light source and the low
quantum yield of the photoinitiation step. Owing to
these limitations, despite the inherent advantages of
light-activation, these reagents have not been widely
applied.
The challenge in the design of arti®cial nucleases lies in
choosing a suitable chemical moiety, which upon acti-
vation will generate reactive species, capable of dama-
ging and cleaving nucleic acid biopolymers. Within this
context, oxygen-centered radicals appear to be the most
promising candidates, since they are characterized by an
inherent reactivity pro®le and are of great biological
importance.5 Indeed, literature reports dated more than
15 years ago demonstrate the potential of these radicals
Key words: DNA cleavage; photochemistry; thiopyridone; amino-
acridine.
The ability of organic (non-metal containing) molecules
to cleave nucleic acids upon visible light activation has
*Corresponding author: Tel.: +1-619-822-0456; fax: +1-619-822-
0386; e-mail: etheodor@ucsd.edu
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00211-9

728
P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
not yet been well explored.15 Undoubtedly, such mole-
cules could display a di€erent, perhaps unique reaction
pro®le and, thereby, expand and complement the
repertoire of the already known DNA- and RNA-
cleaving agents. Our own studies in this domain are
directed toward the design and evaluation of N-
hydroxy-2-thiopyridone and its acyloxyl derivatives as
photoactivated DNA-cleaving species.16±18
in biological systems. Indeed, early studies were focused
on the employment of the N-hydroxy-2-thiopyridone (2)
as a progenitor of hydroxyl radicals (11) in a biological
environment (Scheme 2, eq (1)).20 Although the repor-
ted results, by us16 and others,21 indicated that 2 could
produce hydroxyl radicals, the photoexcitation process
proved to be slow and rather inecient. This drawback
was attributed to the tendency of N-hydroxy-2-thiopyr-
idone (2) to exist in equilibrium of di€erent forms
(Scheme 2, eq (2)).22 This equilibrium decreases the
concentration of the thiocarbonyl containing species
and thereby hampers the homolytic cleavage of the N±O
bond.
Results and Discussion
Photochemistry of N-hydroxy-2-thiopyridone (2) and
derivatives
The undesirable e€ects of the above equilibrium can be
minimized if one uses the O-acyl derivatives of N-
hydroxy-2-thiopyridone (such as 3) as sources of radi-
cals. Among them, the N-aroyloxy-2-thiopyridones are
particularly attractive, since they do not undergo rapid
decarboxylation23 and they could therefore be used as
precursors of oxygen-centered radicals. With this in
mind, we set out to examine the N-benzoyloxyl-2-thio-
pyridone (12) as a potential photoactivated DNA-
cleaving agent. Photoexcitation of 12 was expected to
produce benzoyloxyl radicals (13), which are more per-
sistent than the acyloxyl species 4 and do not undergo
ecient decarboxylation below 120 ꢀC.23 We anticipated
that radicals 13 would exhibit similar reactivity with
hydroxyl counterparts (11) and react with DNA, ulti-
mately resulting in strand cleavage. Indeed, generation
and trapping of benzoyloxyl radicals (13) was veri®ed
by photolysis of 12 in the presence of tert-BuSH. This
resulted in the isolation of benzoic acid (14) and dis-
ul®de 10 in 92% and 89% yields, respectively, in accor-
dance to a radical chain process (Scheme 3, eq (2)).16
Following the pioneering work of Barton and co-work-
ers, the O-acyl derivatives of N-hydroxy-2-thiopyridone
(such as 3), commonly referred to as Barton's esters,
have emerged as highly valuable sources of carbon-,
nitrogen-, and oxygen-centered radicals.19 The generally
accepted mechanism of this process is shown in Scheme
1. Thus, photoexcitation of the thiocarbonyl group of 3
(l&350 nm) results in homolytic cleavage of the N±O
bond and generation of the acyloxyl radical 4 and the
thiyl radical 5. Rapid decarboxylation of 4 subsequently
provides the carbon-centered radical 6, which in the
speci®c example of Scheme 1 is shown to abstract
hydrogen from tert-butylthiol (7) thereby generating the
hydrocarbon 8 and the propagating radical 9. The syn-
thetic eciency of this process has been clearly demon-
strated by the trapping of the carbon-centered radical 6
in a variety of ways, allowing a facile and high yielding
entry into a variety of functionalized molecules.19 Fur-
thermore, this strategy permits the generation of radi-
cals under mild conditions, using only visible light
irradiation and without temperature restrictions.
DNA cleaving studies using N-benzoyloxy-2-thiopyridone
(12)
Due to its eciency and simplicity, the above method
could be used for the generation and study of free radicals
Can the N-benzoyloxy-2-thiopyridone (12) induce DNA
cleavage upon photoactivation? A comparison of con-
centration-dependent photocleavage of DNA by 2 and
12 is shown in Figure 1.24 The results revealed an
increased eciency of strand scission using 12, indicat-
ing that the generation of radicals happens before any
saponi®cation (that could a€ord 2 from 12). The
enhanced reactivity of 12 could be explained if we con-
sider that: (a) compound 12 exists entirely in the thio-
carbonyl containing form (shown in Scheme 3) and not
Scheme 1.
Scheme 2.

P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
729
Figure 2. Concentration-dependent photocleavage of DNA using 12
and 3b. The irradiation (all samples except those in lanes 2 and 7) was
performed for 1 h at 4 ꢀC with two lamps (GE, 300 W), placed at about
20 cm from the samples. Lane 1: fX174 DNA (control); lane 2: DNA
and 1.0 mM of 12 (no hn); lanes 3±6: DNA and 12 at concentrations of
12: 0.1, 0.3, 0.6, and 1.0 mM repectively. Lane 7: DNA and 1.0 mM of
3b (no hn); lanes 8±11: DNA and 3b at concentrations of 3b: 0.1, 0.3,
0.6, and 1.0 mM respectively; lane 12: fX174 DNA (control).
Scheme 3. Reagents and conditions: (a) 1.1 equiv of PhCOCl,
1.3 equiv of pyridine, CH2Cl2, 0 ꢀC, 1 h, 92%; (b) 5.0 equiv of tBuSH,
CH2Cl2, hn (GE, 300 W, >350 nm), 0 ꢀC, 1 h, 92% (for 14) and 89%
(for 10).
in an equilibrium of forms, as it was proposed for 2; and
(b) the thiocarbonyl group of 12 is excited at higher
wavelength, leading to faster N±O bond cleavage.22 In
fact, this di€erence is re¯ected in the color of 12, which
is bright yellow, while 2 is colorless and the UV-Vis
absorption of the thiocarbonyl moieties of 12
(lmax=368) and 2 (lmax=349). Subsequent studies
using 12 demonstrated that the observed DNA scission
proceeds at a range of temperatures (0±25 ꢀC) and is pH
independent (pH range: 6±8.5).
Attachment of the N-benzoyloxy-2-thiopyridone to 9-
aminoacridine; design, synthesis, and DNA-photocleaving
studies
The above experiments con®rmed that the N-benzoyl-
oxy-2-thiopyridone (12) could be used as a photo-
activated DNA-cleaving agent. In order to increase the
eciency of the cleavage and test the versatility of our
method, we synthesized the acridine±thiopyridone con-
jugates 15±18 (Fig. 3). The rationale for this design is
based on covalently attaching the photocleaving entity
to a `recognition element'. The acridinyl group could
assure high anity for duplex DNA via intercalation,25
while the thiopyridone entity could account for the
strand scission.
Comparison of lanes 6 and 7 of Figure 1 indicate a
strong inhibition of DNA-scission when the same
experiment was conducted in the presence of glu-
tathione (radical scavenger). This result further supports
the notion that radical species, probably the benzoyl-
oxyl radicals (13), are responsible for the cleavage.
Subsequent time-controlled DNA cleavage studies
demonstrated that these radicals are generated from 12
at a relatively linear rate.18 This anticipated feature
The synthesis of compounds 15±17 is depicted in
Scheme 4 and began with condensation of commercially
available o-bromocarbonyl chlorides 20 with p-amino
methyl benzoate (19), to produce bromides 21. Com-
pounds 21 were then converted to azides 22, which upon
saponi®cation of the methylester moiety and reduction
of the azido functionality gave rise to amino acid 25,
through intermediates 23 and 24. Coupling of 25 with 9-
chloroacridine (phenol, 110 ꢀC), followed by esteri®ca-
tion with 2-mercaptopyridine-N-oxide (2) (EDC, DMF)
a€orded conjugate 15±17 in good overall yields (indivi-
dual yields are given in the experimental section). Con-
jugate 18 was synthesized from the commercially
available acridine-9-carboxylic acid (26) as illustrated in
Scheme 5, in 79% yield.
constitutes
a fundamental di€erence between our
method and those involving metal complexes, where
radicals are usually produced in a rapid burst.20
We also compared the cleaving eciency of 12 and 3b
(Fig. 2). Under the conditions tried, only 12 was shown
to cleave DNA, while the acyloxyl adduct 3b (and simi-
larly 3a) did not produce any measurable strand scis-
sion. The lack of reactivity of the acyloxyl derivatives
could be attributed to the facile radical decarboxylation
of such species, which generates less reactive carbon-
centered radicals.
Figure 1. Concentration-dependent photocleavage of DNA using 12
and 2. The irradiation (all samples except those in lanes 2 and 8) was
performed for 1 h at 4 ꢀC with two lamps (GE, 300 W), placed at about
20 cm from the samples. Lane 1: fX174 DNA (control); lane 2: DNA
and 1.0 mM of 12 (no hn); lanes 3±6: DNA and 12 at concentrations of
12: 0.5, 1.0, 1.5, and 2.0 mM respectively; lane 7: DNA, 2.0 mM of 12
and 3.0 mM of glutathione. Lane 8: DNA and 1.0 mM of 2 (no hn);
lanes 9±12: DNA and 2 at concentrations of 2: 0.5, 1.0, 1.5, and
2.0 mM respectively.
Figure 3. Molecular structure of acridine±thiopyridone ester con-
jugates 15±18.

730
P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
Table 1 presents the extent of DNA photocleavage
induced by the above compounds in two di€erent con-
centrations (10 and 60 mM). Compounds 15, 16, and 17
were found to induce ecient strand cleavage at low
mM concentrations. Among them, conjugate 16 (n=4
carbons) was the most ecient and was approximately
two orders of magnitude more reactive than the parent
N-benzoyloxy-2-thiopyridone (12).17 This substantial
increase in eciency of cleavage was attributed to the
intercalating properties of 9-aminoacridine.25 Interest-
ingly, much less cleavage was observed with 18,
(equivalent to 12), testifying to the importance of a
¯exible polymethylene linker for our design. Again, the
least reactive derivatives were the N-acyloxy-2-thiopyr-
idones 3a and 3b presumably due to the rapid radical
decarboxylation event, which produces relatively stable
carbon-centered radicals.
To verify that conjugate 16 intercalates onto duplex
DNA we conducted UV±vis and ¯uorescence spectro-
scopy studies. The UV±vis studies were performed by
titrating a solution of 16 (in 50 mM Tris±HCl) with sal-
mon testes DNA and indicated a hypochromic and
bathochromic shift of the UV spectrum of 16, in accor-
dance with the intercalation models.26 For the ¯uores-
cence experiments the aminoacridine moiety of 16 was
selectively excited at 412 nm and its ¯uorescence emis-
sion was monitored at 420±540 nm (Figs 4 and 5).
Measurements of the emission spectra of 16 upon titra-
tion with salmon testes DNA (Fig. 5), in comparison to
Scheme 4. Reagents and conditions: (a) 1.0 equiv of 19, 1.0 equiv of
20, 1.2 equiv of Et3N, CH2Cl2, 0 ꢀC, 1 h; (b) 2.0 equiv of NaN3,
0.05 equiv of [18-c-6], DMF, 25 ꢀC, 3 h; (c) 2.0 equiv of KOH, THF/
H2O: 1/1, 25 ꢀC, 24 h; (d) 0.1 equiv of 10% Pd/C, H2, MeOH, 24 h; (e)
1.0 equiv of 9-chloroacridine, PhOH, 110 ꢀC, 1 h; (f) 1.0 equiv of 2,
1.0 equiv of EDC, DMF, 25 ꢀC, 1 h.
Figure 4. Fluorescence quenching studies of 16 with DNA; titration
with bu€er.
Scheme 5. Synthesis pathway to 1-methyl-4-(3,4-dimethylpyrrol-2-yl)-
1,2,3,6-tetrahydropyridine (23). Reagents and conditions: (i) n-BuLi,
THF; (ii) ZnCl2, THF; (iii) 4-bromopyridine, Pd(PPh3)4; (v) Ch3I,
acetone; (v) NaBH4, MeOH; (vi) H2C2O4, Et2O.
Table 1 Comparison ofDNAcleavage(%)with2, 3a, 3b, 12and15±18a
Conc.
Agent
12 15
2
3a
3b
16
17
18
10 mM
60 mM
4
8
0.5
0.6
1
0.9
9
19
40
81
47
98
45
96
10
18
aThe reported data refer to % yield of DNA photocleavage and were
obtained by densitometry reading of the scission event.
Figure 5. Fluorescence quenching studies of 16 with DNA; titration
with salmon testes DNA.

P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
731
Figure 7. Relative intensity of strand scission induced by compounds
16, 12 and MPE-Fe(II) in a 18 base pairs region of the Sal I/Sph I
fragment of pBR322 duplex DNA (93 mer). Quanti®cation was per-
formed using ImageQuant software.
the conformation of the DNA was ruled out, since less
cleavage was detected when 9-aminoacridine was added
as an external intercalator (compare lines 12,13). Fur-
thermore, the cleavage is more enhanced upon sub-
sequent treatment with piperidine at 90 ꢀC for 30 min
without any change in sequence or base speci®city (lines
7,8). Based on the above data we believe that in the case
of 16 the DNA cleavage is performed by the intercalation
complex and is probably mediated by aroyloxyl radicals.
Conclusion
It is evident from the above studies that the N-aroyloxy-
2-thiopyridones (such as 12 or 16) can induce single
strand nicks in duplex DNA in a light-dependent reac-
tion. These compounds are potentially interesting
nucleic acid-cleaving agents since they possess the fol-
lowing characteristics: a purely organic structure, facile
preparation and prolonged stability in the absence of
light. Furthermore, they undergo an ecient photo-
excitation, with very good quantum yields (&0.5)22
using visible light (l&350 nm). The DNA cleavage
occurs simply by irradiation, without the need of exter-
nal additives and is concentration- and time-dependent.
In addition, the strand scission is neither base- nor
sequence-speci®c and is probably mediated by aroyloxyl
radicals. The eciency and/or selectivity of the cleavage
could be tuned by the proper attachment of the DNA-
recognition element. In addition, the light intensity that
is responsible for the photoactivation could be tuned by
structurally modifying the thiopyridone core. Thus, the
N-aroyloxy-2-thiopyridones can be used for the design
of new DNA photocleaving reagents with potential
use as `photofootprinting reagents' or as `site-directed
photonucleases'.
Figure 6. Autoradiogram of a 10% denaturing polyacrylamide gel
showing photocleavage of 50-32P end-labeled Sal I/Sph I restriction
fragment of pBR322 duplex DNA (93 mer), induced by 16 and 12.
DNA was incubated for 1 h at 25 ꢀC with compounds 16 or 12 in
bu€ered solution (30 mM Tris±HCl, 20 mM NaCl) and then irradiated
(with two GE 300 W lamps placed at 20 cm from the samples) for
another 2 h at 4 ꢀC (lanes 3, 4 and 7±13). The resulting solution was
treated with piperidine (1 M) at 90 ꢀC for 30 min, followed by ethanol
precipitation (lanes 8±13). Lane 1: DNA cut by PIe I (14 base pairs)
and EcoN I (28 base pairs); lane 2: DNase footprinting; lane 3: DNA
(control); lane 4: DNA irradiated and piperidine treated without 12
nor 16; lane 5: DNA and 200 mM of 12 (no hn); lane 6: DNA and
60 mM of 16 (no hn); lane 7: DNA and 5 mM of 16 (no piperidine
treatment); lanes 8±10: DNA and 16 at concentrations of 16: 5, 10,
20 mM respectively; lanes 11, 12: DNA and 12 at concentrations of 40,
60 mM respectively; lane 13: DNA, 12 (60 mM) and 9-aminoacridine
(30 mM).
those of the control experiments (Fig. 4) indicated a
signi®cant quenching of ¯uorescence, supporting the
intercalation event.27
The base-selectivity of the DNA cleavage induced upon
irradiation of 12 and 16 was evaluated using `single-hit'
conditions (Fig. 6).17 These experiments were performed
with the 50-32P labeled Sal I/Sph I restriction fragment of
pBR322 duplex DNA (93-mer). Our data (Figs 6 and 7)
show that both 12 and 16 generate identical DNA ladders
and the photocleavage is indisputably neither base- nor
sequence-speci®c. In addition, comparison of lanes 8±12
of Figure 6 indicate that 16 is more ecient than 12 in
cleaving DNA and can accurately cut the duplex at
concentrations as low as 5 mM. The possibility that the
9-aminoacridinyl group enhances cleavage by altering
Experimental
General techniques
All reactions were carried out under an argon atmo-
sphere in dry, freshly distilled solvents under anhydrous
conditions, unless otherwise noted. Tetrahydrofuran

732
P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
1
(THF) and diethyl ether (Et2O) were distilled from
sodium/benzophenone; dichloromethane (CH2Cl2) from
calcium hydride; DMF from calcium chloride. Yields
refer to chromatographically and spectroscopically (1H
NMR) homogeneous materials, unless otherwise stated.
Reagents were purchased at highest commercial quality
and used without further puri®cation unless otherwise
stated. Reactions were monitored by thin-layer chro-
matography carried out on 0.25 mm E. Merck silica gel
plates (60F±254) using UV light as visualizing agent and
7% ethanolic phosphomolybdic acid, or p-anisaldehyde
solution and heat as developing agents. E. Merck silica
gel (60, particle size 0.040±0.063 mm) was used for ¯ash
chromatography. Preparative thin-layer chromato-
graphy separations were carried out on 0.25 or 0.50 mm
E. Merck silica gel plates (60F±254). NMR spectra were
recorded on a Varian 500 instrument and calibrated
using residual undeuterated solvent as an internal refer-
ence. The following abbreviations were used to explain
the multiplicities: s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet, b=broad. IR spectra were
recorded on a Perkin±Elmer Model 781 spectrometer.
UV and ¯uorescence spectra were recorded on a Hew-
lett±Packard 8452A Diode Array Spectrophotometer
and a Perkin±Elmer LS50B luminescence spectrometer
respectively. High resolution mass spectra (HRMS)
were recorded on a VG 7070 HS mass spectrometer
under chemical ionization (CI) conditions or on a VG
ZAB-ZSE mass spectrometer under fast atom bom-
bardment (FAB) conditions.
89%). 10: colorless liquid; H NMR (500 MHz, CDCl3)
d 8.48±8.31 (m, 1H), 7.65±7.50 (m, 2H), 6.91±7.02 (m,
1H), 1.35 (s, 9H, tert Bu).
Bromide 21b. To a solution of methyl 4-aminobenzoate
(19) (14.02 g, 92.74 mmol) and triethylamine (13.9 mL,
99.7 mmol) in methylene chloride (100 mL) at 0 ꢀC was
added 5-bromovaleryl chloride (20b) (14.61 mL,
99.7 mmol). The reaction mixture was stirred at 0 ꢀC for
30 min and at 25 ꢀC for an additional 1 h. The mixture
was then diluted with methylene chloride and washed
with three portions of aqueous saturated sodium bicar-
bonate (3Â200 mL). The organic layer was dried
(MgSO4), ®ltered, and concentrated to give compound
21b (28.56 g, 90.89 mmol, 98%). 21b: white solid;
Rf=0.20 (silica, 50% ethyl acetate in hexanes); IR (KBr
plate) nmax 3319 (s), 3199 (m), 2951 (m), 1721 (s), 1676
(s), 1602 (s), 1537 (s), 1509 (m), 1407 (m), 1279 (s), 1169
(s), 1099 (m), 771 (m) cm
;
1H NMR (500 MHz,
1
CDCl3) d 7.99 (d, 2H, J=9.0 Hz), 7.60 (d, 2H,
J=9.0 Hz), 7.56 (s, 1H), 3.89 (s, 3H), 3.42 (t, 2H,
J=6.5 Hz), 2.42 (t, 2H, J=6.5 Hz), 1.91 (m, 4H); 13C
NMR (125 MHz, CDCl3) d 171.3, 166.8, 142.2, 130.8,
125.5, 119.0, 52.0, 36.4, 33.0, 31.8, 23.7; HRMS calcd
for C13H16NO3Br, (M+Cs+) 445.9368, found 445.9387.
Bromide 21a. Preparation of this compound was
accomplished following the procedure described for
bromide 21b. 21a: (92%); white solid; Rf=0.25 (silica,
50% EtOAc in hexanes); IR (®lm) nmax 3306 (s), 1716
(s), 1602 (s), 1539 (s), 1411 (s), 1279 (s), 1173 (m), 1102
1
N-Benzoyloxy-2-thiopyridone 12. To a solution of N-
hydroxy-2-thiopyridone (2) (12.7 g, 100 mmol) in
methylene chloride (200 mL) was added, at 0 ꢀC and in
the dark, pyridine (10.5 mL, 130 mmol) followed by
benzoyl chloride (12.8 mL, 110 mmol). The mixture was
stirred for 3 h, and then diluted with methylene chloride
(300 mL) and washed twice with aqueous saturated
sodium bicarbonate (2Â300 mL). The organic layer was
dried (MgSO4), ®ltered, and concentrated under
reduced pressure at 20 ꢀC in the dark. Rapid ®ltration
over silica gel (60% ethyl ether in hexane) followed by
recrystallization from methylene chloride±hexane a€or-
ded the benzoyloxyl derivative 12 (21.25 g, 92 mmol,
(m), 953 (m), 857 (m), 771 (s), 697 (m), 512 (m) cm
;
1H NMR (500 MHz, CDCl3) d 8.01 (d, 2H, J=9.0 Hz),
7.86 (d, 2H, J=8.5 Hz), 7.32 (s, 1H), 3.80 (s, 3H), 3.54
(t, 2H, J=6.5 Hz), 2.60 (t, 2H, J=7.0 Hz), 2.28 (t, 2H,
J=6. 5 Hz); 13C NMR (500 MHz, CDCl3) d 171.3,
166.8, 142.5, 130.8, 125.5, 118.9, 51.9, 35.2, 33.1, 27.7;
HRMS, calcd for C12H14NO3Br (M+H+) 300.0235,
found 300.0245.
Bromide 21c. Preparation of this compound was
accomplished following the procedure described for
bromide 21b. 21c: (95%); white solid; Rf=0.25 (silica,
50% EtOAc in hexanes); IR (®lm) nmax 3340 (s), 2948
(m), 1691 (s), 1596 (s), 1532 (s), 1432 (s), 1258 (s), 1161
(s), 861 (m), 772 (s), 702 (m) cm 1; 1H NMR (500 MHz,
CDCl3) d 7.99 (d, 2H, J=9.0 Hz), 7.60 (d, 2H,
J=9.0 Hz), 7.56 (s, 1H), 3.88 (s, 3H), 3.40 (t, 2H,
J=7.0 Hz), 2.39 (t, 2H, J=7.0 Hz), 1.88 (m, 2H), 1.75
(m, 2H), 1.51 (m, 2H); 13C NMR (500 MHz, CDCl3) d
171.5, 166.8, 142.2, 130.8, 125.5, 118.9, 51.9, 37.3, 33.4,
32.3, 27.6, 24.3; HRMS, calcd for C14H18NO3Br
(M+H+) 328.0548, found 328.0540.
1
92%). 12: yellow solid; H NMR (500 MHz, CDCl3) d
8.25±8.15 (d, 2H, J=8 Hz), 7.72±7.62 (m, 3H), 7.58±
7.48 (m, 2H), 7.26±7.18 (m, 1H), 6.72±6.65 (dt, 1H,
J=1.8, 6.9 Hz); 13C NMR (125 MHz, CDCl3) d 175.5,
162.4, 137.8, 137.0, 134.8, 133.6, 130.4, 128.7, 125.4,
112.7.
Photolysis of 12 in the presence of tert-butylthiol. The
photolysis was performed under argon, using degassed,
dry methylene chloride as solvent. A solution of 12
(0.462 g, 2.0 mmol) and tert-butylthiol (1.1 mL,
10 mmol) in methylene chloride (4 mL) at 0 ꢀC was irra-
diated with one tungsten lamp (GE, 300 W) in a Pyrex
¯ask from a distance of about 20 cm. The consumption
of 12 was followed by TLC and completed after 20 min.
The solvent was then removed under reduced pressure
and the residue subjected to ¯ash chromatography
(silica, 5!60% ethyl ether in hexane) to give 14
(0.217 g, 1.84 mmol, 92%) and 10 (0.350 g, 1.78 mmol,
Azide 22b.
A solution of bromide 21b (28.43 g,
90.5 mmol), sodium azide (11.76 g, 181 mmol) and 18-
crown-6 (100 mg) in dry DMF (180 mL) was stirred at
25 ꢀC for 3 h. The reaction mixture was then diluted
with ethyl ether and washed with three portions of
aqueous saturated sodium bicarbonate (3Â200 mL).
The organic layer was dried (MgSO4), ®ltered, con-
centrated and chromatographed (silica, 20!50% ethyl
acetate in hexanes) to give 22b (22.84 g, 82.67 mmol,

P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
733
91%). 22b: white solid; Rf=0.45 (silica, 50% ethyl
acetate in hexanes); IR( KBr plate) nmax 3312 (s), 3192
(m), 3123 (m), 2950 (s), 2872 (m), 2091 (s), 1721 (s),
1674 (m), 1602 (s), 1543 (s), 1431 (s), 1410 (s), 1251 (s),
23a: (85%); white solid; Rf=0.15 (silica, 50% ethyl
acetate in hexane); IR (KBr plate) nmax 3311 (m), 2944
(m), 2094 (s), 1655 (s), 1524 (s), 1427 (m), 1302 (m),
1
1182 (m), 942 (m), 861 (m) cm 1; H NMR (500 MHz,
1
1174 (s), 1100 (s), 867 (m), 770 (s), 697 (m) cm 1; H
DMSO-d6) d 10.34 (s, 1H), 7.87 (d, 2H, J=9.0 Hz), 7.70
(d, 2H, J=9.0 Hz), 3.38 (t, 2H, J=7.0 Hz), 2.44 (t, 2H,
J=7.0 Hz), 1.82 (m, 2H); 13C NMR (125 MHz, DMSO-
d6) d 171.3, 167.2, 143.5, 130.5, 125.1, 118.5, 50.3, 33.4,
24.2; HRMS, calcd for C11H12N4O3 (M+Na+)
271.0807, found 271.0809.
NMR (500 MHz, CDCl3) d 7.99 (d, 2H, J=9.0 Hz), 7.61
(s, 1H), 7.60 (d, 2H, J=9.0 Hz), 3.89 (s, 3H), 3.31 (t,
2H, J=7.0 Hz), 2.42 (t, 2H, J=7.0 Hz), 1.81 (m, 2H),
1.66 (m, 2H); 13C NMR (125 MHz, CDCl3) d 171.5,
166.9, 142.4, 130.8, 125.4, 119.1, 52.0, 51.0, 36.7, 28.2,
22.5; HRMS, calcd for C13H16N4O3 (M+H+)
277.1301, found 277.1304.
Acid 23c. Preparation of this compound was accom-
plished following the procedure described for acid 23b.
23c: (93%); white solid; Rf=0.15 (silica, 50% ethyl
acetate in hexane); IR (KBr plate) nmax 3315 (s), 2950
(m), 2094 (s), 1662 (s), 1520 (s), 1300 (m), 1181 (m), 769
Azide 22a. Preparation of this compound was accom-
plished following the procedure described for azide 22b.
22a: (93%); white solid; Rf=0.3 (silica, 50% ethyl acet-
ate in hexanes); IR (KBr plate) nmax 3312 (m), 2957 (m),
2098 (s), 1720 (s), 1674 (s), 1596 (s), 1432 (s), 1279 (s),
1
(m) cm 1; H NMR (500 MHz, DMSO-d6) d 10.19 (s,
1H), 7.86 (d, 2H, J=8.5 Hz), 7.69 (d, 2H, J=8.5 Hz),
3.32 (t, 2H, J=7.0 Hz), 2.34 (t, 2H, J=7.5 Hz), 1.60 (m,
2H), 1.55 (m, 2H), 1.34 (m, 2H); 13C NMR (125 MHz,
DMSO-d6) d 172.2, 167.5, 143.9, 130.8, 125.4, 118.7,
50.9, 36.7, 28.4, 26.1, 24.8; HRMS, calcd for
C13H16N4O3 (M+Na+) 299.1120, found 299.1124.
1
1172 (s), 1101 (m), 770 (m), 697 (m) cm 1; H NMR
(500 MHz, CDCl3) d 8.01 (d, 2H, J=7.0 Hz), 7.59 (d,
2H, J=7.5 Hz), 7.35 (s, 1H), 3.90 (s, 3H), 3.43 (t, 2H,
J=7.0 Hz), 2.50 (t, 2H, J=7.5 Hz), 2.02 (t, 2H,
J=7.0 Hz); 13C NMR (125 MHz, CDCl3) d 170.8, 166.8,
142.2, 130.8, 125.5, 118.9, 51.9, 50.5, 34.0, 24.3; HRMS,
calcd for C12H14N4O3 (M+H+) 263.1144, found
263.1150.
Amino acid 24b. To a solution of the acid 23b (18.98 g,
72.36 mmol) in methanol (500 mL) was added 10% Pd/C
(200 mg) and the mixture was stirred under H2 atmo-
sphere at 25 ꢀC for 24 h. The Pd/C was then removed by
®ltration, and the methanol was concentrated to a€ord
crude amino acid 24b, which was used in the next step
without any further puri®cation (15.2 g, 64.41 mmol,
89%). 24b: white solid; Rf=0.10 (silica, 40% MeOH in
CH2Cl2); IR (KBr plate) nmax 3313 (m), 3040 (m), 2951
(m), 1668 (s), 1609 (m), 1528 (m), 1379 (s), 1311 (m),
Azide 22c. Preparation of this compound was accom-
plished following the procedure described for azide 22b.
22c: (89%); white solid; Rf=0.5 (silica, 50% ethyl acet-
ate in hexanes); IR (KBr plate) nmax 3306 (m), 2947 (m),
2094 (s), 1720 (s), 1674 (s), 1538 (s), 1407 (m), 1280 (m),
1
1175 (m), 1103 (m), 852 (m), 770 (m) cm 1; H NMR
(500 MHz, CDCl3) d 8.00 (d, 2H, J=8.5 Hz), 7.60 (d,
2H, J=8.5 Hz), 7.37 (s, 1H), 3.89 (s, 3H), 3.28 (t, 2H,
J=7.0 Hz), 2.40 (t, 2H, J=7.0 Hz), 1.76 (m, 2H), 1.63
(m, 2H), 1.46 (m, 2H); 13C NMR (125 MHz, CDCl3) d
171.7, 166.8, 142.4, 130.7, 125.3, 118.9, 51.9, 51.0, 37.2,
28.4, 26.1, 24.7; HRMS, calcd for C14H18N4O3
(M+Na+) 313.1277, found 313.1286.
1
1173 (m), 786 (m) cm 1; H NMR (500 MHz, DMSO-
d6) d 10.25 (s, 1H), 7.80 (d, 2H, J=8.0 Hz), 7.60 (d, 2H,
J=8.5 Hz), 2.79 (t, 2H, J=7.0 Hz), 2.34 (t, 2H,
J=6.5 Hz), 1.62 (m, 4H); 13C NMR (125 MHz, DMSO-
d6) d 171.4, 169.1, 141.7, 130.0, 127.0, 118.1, 38.4, 35.8,
26.8, 22.1; HRMS, calcd for C12H16N2O3 (M+Na+)
259.1059, found 259.1078.
Acid 23b. To a solution of the azide 22b (20.94 g,
75.78 mmol) in tetrahydrofuran (200 mL) was added a
solution of potassium hydroxide (10 g, 178 mmol) in
H2O (200 mL). The resulting milky mixture was stirred
vigorously at 25 ꢀC for 24 h until it turned clear. Acid-
i®cation with concentrated hydrochloric acid to pH=1
and ®ltration a€orded compound 23b (19.49 g,
74.30 mmol, 98%). 23b: white solid; Rf=0.12 (silica,
50% ethyl acetate in hexane); IR (KBr plate) nmax 3313
(s), 2947 (m), 2862 (m), 2669 (m), 2550 (m), 2093 (s),
Amino acid 24a. Preparation of this compound was
accomplished following the procedure described for
amino acid 24b. 24a: (81%); white solid; Rf=0.10
(silica, 40% MeOH in CH2Cl2); IR (KBr plate) nmax
3151 (m), 3040 (m), 1691 (s), 1596 (s), 1531 (s), 1408
1
(m), 1314 (s), 1171 (s), 965 (m), 870 (m), 774 (m) cm
;
1H NMR (500 MHz, DMSO-d6) d 10.49 (s, 1H), 7.96 (s,
1H), 7.87 (d, 2H, J=9.0 Hz), 7.73 (d, 2H, J=8.5 Hz),
2.82 (m, 2H), 2.47 (m, 2H), 1.86 (m, 2H); 13C NMR
(125 MHz, DMSO-d6) d 171.1, 167.1, 143.4, 130.4,
125.1, 118.4, 38.2, 33.1, 22.9; HRMS, calcd for
C11H14N2O3 (M+H+) 223.1083, found 223.1085.
1674 (s), 1609 (s), 1525 (s), 1427 (s), 1299 (s), 1182 (s),
1
938 (m), 868 (m), 769 (s), 693 (m) cm
;
1H NMR
(500 MHz, DMSO-d6) d 10.27 (s, 1H), 7.88 (d, 2H,
J=8.0 Hz), 7.72 (d, 2H, J=8.0 Hz), 3.33 (t, 2H
J=6.5 Hz), 2.38 (t, 2H, J=6.5 Hz), 1.62 (m, 2H), 1.55
(m, 2H); 13C NMR (125 MHz, DMSO-d6) d 171.7,
167.2, 143.5, 130.5, 125.1, 118.4, 50.5, 35.9, 27.9, 22.2;
HRMS, calcd for C12H14N4O3 (M+Na+)285.0984,
found 285.0989.
Amino acid 24c. Preparation of this compound was
accomplished following the procedure described for
amino acid 24b. 24c: (88%); white solid; Rf=0.15
(silica, 40% MeOH in CH2Cl2); IR (KBr plate) nmax
3331 (m), 2953 (m), 1686 (s), 1596 (s), 1531 (s), 1416
(m), 1292 (s), 1163 (s), 864 (m), 772 (m), 700 (m), 546
1
Acid 23a. Preparation of this compound was accom-
plished following the procedure described for acid 23b.
(m) cm 1; H NMR (500 MHz, DMSO-d6) d 10.22 (s,
1H), 7.87 (d, 2H, J=9.0 Hz), 7.68 (d, 2H, J=7.0 Hz),

734
P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
2.52 (t, 2H, J=6.5 Hz), 2.49 (m, 2H), 2.38 (t, 2H,
J=7.5 Hz), 1.66 (m, 2H), 1.59 (m, 2H); 13C NMR
(125 MHz, DMSO-d6) d 171.4, 167.3, 143.2, 130.5,
120.7, 118.4, 39.5, 35.5, 24.4, 24.0, 16.0; HRMS, calcd
for C13H18N2O3 (M+H+) 251.1396, found 251.1403.
(m), 1595 (s), 1528 (s), 1411 (m), 1249 (m), 1171 (m), 978
1
(m), 748 (m) cm 1; H NMR (500 MHz, DMSO-d6) d
10.61 (s, 1H), 9.85 (m, 1H), 8.61 (m, 2H), 8.48 (d, 1H,
J=6.5 Hz), 8.08 (d, 2H, J=9.0 Hz), 7.96 (t, 2H,
J=8.0 Hz), 7.90 (d, 2H, J=9.0 Hz), 7.84 (d, 2H,
J=8.5 Hz), 7.58 (d, 1H, J=9.0 Hz), 7.50 (m, 3H), 6.92
(t, 1H, J=6.5 Hz), 4.12 (m, 2H), 2.44 (m, 2H), 1.95 (m,
2H), 1.74 (m, 2H); 13C NMR (125 MHz, DMSO-d6) d
174.8, 171.8, 167.1, 161.8, 157.4, 147.5, 145.5, 143.5,
140.1, 138.7, 135.9, 134.9, 131.6, 130.4, 127.2, 118.7,
118.5, 113.4, 48.3, 35.8, 28.4, 22.2; HRMS, calcd for
C30H26N4O3S (M+H+) 523.1804, found 523.1786; UV
(DMF, c=1.9Â10 4 M) lmax=250, 271, 347, 390,
428 nm.
Acid 25b. To a solution of the amino acid 24b (2.43 g,
10.3 mmol) in phenol (15 g) at 40 ꢀC was added the 9-
chloroacridine (2.2 g, 10.3 mmol) and the mixture was
stirred at 110 ꢀC for 1 h. The phenol was then removed
under reduced pressure and the residue puri®ed by
chromatography on silica (0!30% methanol in methy-
lene chloride) to a€ord acid 25b (3.80 g, 9.07 mmol,
90%). 25b: yellow solid; Rf=0.15 (silica, 20% MeOH in
CH2Cl2); IR (KBr plate) nmax 3416 (s), 1655 (m), 1603
1
(m), 1527 (m), 1408 (m), 863 (m), 790 (m) cm 1; H
Aminoacridine 15. Preparation of this compound was
accomplished following the procedure described for
aminoacridine 16. 15: (61%); yellow solid; Rf=0.60
(silica, 20% MeOH in CH2Cl2); IR (KBr plate) nmax
NMR (500 MHz, DMSO-d6) d 10.15 (s, 1H), 8.28 (m,
2H), 7.85 (d, 2H, J=8.5 Hz), 7.64 (m, 6H), 7.25 (m,
2H), 3.83 (t, 2H, J=6.5 Hz), 2.35 (t, 2H, J=6.5 Hz),
1.76 (m, 2H), 1.69 (m, 2H); HRMS, calcd for
C25H23N3O3 (M+Na+) 436.1637, found 436.1671; UV
(DMF, c=2.0Â10 4 M) lmax=253, 270, 379 nm.
3240 (m), 1752 (m), 1697 (m), 1636 (s), 1595 (s), 1528
1
(s), 1445 (m), 1251 (s), 1173 (m), 987 (m), 746 (m) cm
;
1H NMR (500 MHz, DMSO-d6) d 10.59 (s, 1H), 9.86 (s,
1H), 8.62 (m, 2H), 8.49 (d, 1H, J=6.5 Hz), 8.05 (d, 2H,
J=8.5 Hz), 7.95 (t, 2H, J=7.5 Hz), 7.87 (d, 2H,
J=8.5 Hz), 7.76 (d, 2H, J=8.0 Hz), 7.59 (d, 1H,
J=8.5 Hz), 7.53 (m, 3H), 6.93 (t, 1H, J=6.5 Hz), 4.19
(t, 2H, J=6.5 Hz), 2.60 (t, 2H, J=6.0 Hz), 2.25 (m, 2H);
13C NMR (125 MHz, DMSO-d6) d 190.0, 175.2, 172.3,
170.2, 162.2, 158.2, 156.4, 145.5, 140.5, 136.3, 135.4,
135.4, 132.0, 130.1, 127.4, 119.7, 119.0, 113.8, 48.9,3 4.0,
24.7; HRMS calcd for C29H24N4O3S (M+H+)
509.1647, found 509.1633; UV (DMF, c=4.0Â10 4 M)
Acid 25a. Preparation of this compound was accom-
plished following the procedure described for acid 25b.
25a: (87%); yellow solid; Rf=0.15 (silica, 20% MeOH
in CH2Cl2); IR (KBr plate) nmax 3397 (m), 1698 (m),
1648 (s), 1603 (s), 1533 (s), 1373 (s), 1309 (m), 1164 (s),
1
789 (m) cm 1; H NMR (500 MHz, DMSO-d6) d 10.06
(s, 1H), 8.31 (d, 2H, J=7.5 Hz), 7.79 (d, 2H, J=8.0 Hz),
7.55 (m, 6H), 7.23 (m, 2H), 3.86 (t, 2H, J=7.0 Hz), 2.45
(t, 2H, J=7.0 Hz), 1.22 (m, 2H); HRMS calcd for
C24H21N3O3 (M+Na+) 422.1481, found 422.1488; UV
(DMF, c=8.0Â10 5 M) lmax=254, 270, 348, 360, 390,
419 nm.
l
max=270, 286, 349, 390 nm.
Aminoacridine 17. Preparation of this compound was
accomplished following the procedure described above,
for the synthesis of aminoacridine 16. 17: (55%); yellow
solid; Rf=0.60 (silica, 20% MeOH in CH2Cl2); IR (KBr
plate) nmax 3386 (m), 1772 (s), 1591 (s), 1530 (s), 1408
Acid 25c. Preparation of this compound was accom-
plished following the procedure described above, for the
synthesis of acid 25b. 25c: (76%); yellow solid; Rf=0.10
(silica, 20% MeOH in CH2Cl2); IR (KBr plate) nmax
3402 (s), 1685 (m), 1596 (m), 1527 (m), 1376 (m), 1254
1
(m), 1253 (m), 1169 (m), 987 (m), 750 (m) cm 1; H
NMR (500 MHz, DMSO-d6) d 10.74 (s, 1H), 10.05 (m,
1H), 8.65 (m, 2H), 8.49 (d, 1H, J=7.0 Hz), 8.06 (d, 2H,
J=8.5 Hz), 8.01 (d, 2H, J=8.0 Hz), 7.95 (t, 2H,
J=7.5 Hz), 7.87 (d, 2H, J=9.0 Hz), 7.58 (d, 1H,
J=9.0 Hz), 7.49 (m, 3H), 6.92 (t, 1H, J=6.5 Hz), 4.10
(m, 2H), 2.42 (t, 2H, J=7.5 Hz), 1.95 (m, 2H), 1.66 (m,
2H), 1.43 (m, 2H); 13C NMR (125 MHz, DMSO-d6) d
190.0, 161.8, 157.4, 145.6, 140.2, 135.8, 134.5, 134.2,
131.5, 127.0, 118.7, 118.6, 118.5, 115.8, 113.4, 111.1,
99.2, 94.4, 48.3, 36.2, 28.5, 25.7, 24.4; HRMS calcd for
C31H28N4O3S (M+H+) 537.1961, found 537.1992; UV
(DMF, c=1.9Â10 4 M) lmax=253, 271, 295, 340, 392,
410, 434 nm.
1
(m), 1165 (m) cm 1; H NMR (500 MHz, DMSO-d6) d
10.31 (s, 1H), 8.55 (m, 2H), 7.86 (m, 6H), 7.68 (d, 2H,
J=8.5 Hz), 7.47 (m, 2H), 4.04 (t, 2H, J=7.0 Hz), 2.35
(t, 2H, J=7.0 Hz), 1.9 (m, 2H), 1.63 (m, 2H), 1.41 (m,
2H); 13C NMR (125 MHz, DMSO-d6) d 172.3, 157.4,
143.7, 140.6, 134.9, 130.6, 126.5, 126.3, 123.5, 118.7,
118.7, 48.9, 48.9, 36.5, 29.0, 26.1, 24.9; HRMS, calcd for
C26H25N3O3 (M+H+) 428.1974, found 428.1991; UV
(DMF, c=9.75Â10 5 M) lmax=269, 358, 395, 430 nm.
Aminoacridine 16. To a solution of the acid 25b (2.0 g,
4.84 mmol) in dry DMF (20 mL) was added EDC
(927 mg, 4.84 mmol) and the mixture was stirred at
25ꢀC for 30min. To this solution was added 2-mercapto-
pyridine-N-oxide (2) (615 mg, 4.84 mmol), the ¯ask was
covered with alumina foil and the mixture was stirred
at 25 ꢀC for 12 h. The DMF was then removed under
reduced pressure and the residue chromatographed
(silica, 0!20% methanol in methylene chloride) to give
compound 22 (1.09 g, 2.08 mmol, 43%). 22: yellow
solid; Rf=0.60 (silica, 20% MeOH in CH2Cl2); IR
(KBr plate) nmax 3255 (m), 3098 (m), 2940 (m), 1772
Acridine 18. A suspension of the 9-aminoacridine car-
boxylic acid (26) (780 mg, 3.5 mmol) in thionyl chloride
(20 mL) was heated under stirring at 50 ꢀC for 3 h. The
excess thionyl chloride was then removed under reduced
pressure, the residue was dissolved in dry methylene
chloride (5 mL) and transferred dropwise via syringe to
a solution of 2-mercaptopyridine-N-oxide (2) (490 mg,
3.8 mmol) and pyridine (0.62 mL, 7.0 mmol) in methy-
lene chloride (20 mL). The reaction ¯ask was covered

P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
735
with alumina foil and the mixture was stirred at 25 ꢀC
for 2 h. The reaction mixture was then diluted with
methylene chloride (30 mL) and washed with saturated
aqueous sodium bicarbonate (3Â30 mL). The organic
layer was extracted, dried (MgSO4), ®ltered, con-
centrated and the residue was crystallized (methylene
chloride/hexanes) to give 18 (920 mg, 2.76 mmol, 79%).
18: yellow solid; Rf=0.75 (silica, 10% MeOH in
CH2Cl2); IR (KBr plate) nmax 3412 (m), 1792 (s), 1609
(m), 1525 (s), 1449 (s), 1405 (m), 1226 (m), 1103 (s), 933
(s), 755 (s) cm 1; 1H NMR (500 MHz, DMSO-d6) d 8.71
(m, 2H), 8.34 (d, 2H, J=8.0 Hz), 7.88 (t, 4H,
J=7.0 Hz), 7.73 (t, 2H, J=7.0 Hz), 7.33 (t, 1H,
J=7.5 Hz), 6.79 (m, 1H); 13C NMR (125 MHz, DMSO-
d6) d 148.5, 138.0, 137.5, 133.6, 130.6, 130.1, 128.6,
125.3, 123.1, 113.1, 113.0, 105.1, 84.4; HRMS calcd for
C19H12N2O2S (M+Cs+) 464.9674, found 464.9659; UV
(DMF, c=2.5Â10 4 M) lmax=217, 254, 292, 350, 364,
388 nm.
electrophores is in a nondenaturing 10% polyacryl-
amide gel and subsequent column chromatography on
Sephadex G-25.
Acknowledgements
The authors gratefully acknowledge the ®nancial sup-
port provided by the Cancer Research Coordinating
Committee, the UCSD Academic Senate, the Hellman
Foundation, and the donors of the Petroleum Research
Funds administered by the American Chemical Society.
We also thank Professors M. Goodman and Y. Tor of
this department for allowing us access to their IR and
UV/Fluorescence instruments and for useful discus-
sions. This paper is dedicated with respect and appre-
ciation to the memory of our mentor, Professor Sir
Derek H. R. Barton.
References and Notes
Photocleavage of supercoiled DNA using N-aroyloxy-2-
thiopyridones. fX174 (50 mM per base pair) was incu-
bated for 1 h at 25 ꢀC with the appropriate N-aroyloxy-
2-thiopyridones and then irradiated for 1 h at 4 ꢀC. The
irradiation was performed with two GE 300W lamps
placed at approximately 20 cm from the samples. Intact
fX174 DNA runs at the position noted as I, relaxed
circular DNA (single-stranded cleavage) at position II,
and linear DNA (double-stranded cleavage) at position
III. The results were analyzed on 1% agarose gel (Tris-
acetate bu€er) stained with ethidium bromide and are
displayed in Figures 1±3, 7 and Table 1.
1. For related reviews see: (a) Pratviel, G.; Bernadou, J.;
Meunier, B. Angew. Chem., Int. Ed. Engl. 1995, 34, 746. (b)
Sigman, D. S.; Bruice, T. W.; Mazumder, A.; Sutton, C. L.
Acc. Chem. Res. 1993, 26, 98. (c) Sigman, D. S.; Mazumder,
A.; Perrin, D. M. Chem. Rev. 1993, 93, 2295. (d) Sigman, D. S.
Biochemistry 1990, 29, 9097.
2. (a) Sigman, D. S.; Chen, C.-H. Annu. Rev. Biochem. 1990,
59, 207. (b) Dervan, P. B. Nature 1992, 359, 87. (c) Huber, P.
W. Genes Dev. 1993, 7, 1367. (d) Papavassiliou A. G. Biochem.
J. 1995, 305, 345. (e) Revzin, A. Footprinting of Nucleic Acid-
Protein Complexes; Academic Press: San Diego, 1993.
3. (a) Remers, W. A. In Textbook of Organic, Medicinal and
Pharmaceutical Chemistry; Delgrado, J. W.; Remers, W. A.;
Eds.; Lippincott: Philadelphia, PA, 1991; 315±358. (b) Kane,
S. A.; Hecht, S. M. Prog. Nucleic Acid Res. Mol. Biol. 1994,
49, 313. (c) Henderson, D.; Hurley, L. H. Nature 1995, 1, 525.
(d) Boger, D. L.; Zhou, J. J. Org. Chem. 1993, 58, 3018.
4. (a) Nicolaou, K. C.; Dai, W. M. Angew. Chem., Int. Ed.
Engl. 1991, 30, 1387. (b) Nicolaou, K. C.; Dai, W. M.; Tsay, S.
C.; Estevez, V. A.; Wrasidlo, W. Science 1992, 256, 1172. (c)
Nicolaou, K. C.; Smith, A. L.; Yue, E. W. Proc. Natl. Acad.
Sci. USA 1993, 90, 5881. (d) Nicolaou, K. C.; Pitsinos, E. N.;
Theodorakis, E. A.; Saimoto, H.; Wrasidlo, W. Chem. Biol.
1994, 1, 57. (e) Murphy, J. A.; Grith, J. Nat. Prod. Rep.
1993, 551. (f) Ikemoto, N.; Kumar, R. A.; Ling, T. T.; Elles-
tad, G. A.; Danishefsky, S. J.; Patel, D. J. Proc. Natl. Acad.
Sci. USA 1995, 92, 10506. (g) Danishefsky, S. J.; Shair, M. D.
J. Org. Chem. 1996, 61, 16. (h) Kahne, D. Chem. Biol. 1995, 2,
7. (i) Kabanov, A. V.; Kabanov, V. A. Bioconjugate Chem.
1995, 6, 7.
5. (a) Guyton, K. Z.; Kensler, T. W. Br. Med. Bull. 1993, 49,
523. (b) Halliwell, B.; Gutteridge, M. C. Biochem. J. 1984, 219,
1. (c) Dizdaroglu, M. Mutat. Res. 1992, 275, 331. (d) Sara®an,
T. A.; Bredesen, D. E. Free Radical Res. 1994, 21, 1. (e) Khan,
A. U.; Wilson, T. Chem. Biol. 1995, 2, 437. (f) Breen, A. P.;
Murphy, J. A. Free Radical Biol. Med. 1995, 18, 1033.
6. (a) D'Aurora, V.; Stern, A. M.; Sigman, D. S. Biochem.
Biophys. Res. Commun. 1978, 80, 1025. (b) Sigman, D. S.;
Graham, D. R.; D'Aurora, V.; Stern, A. M. J. Biol. Chem.
1979, 254, 12269. (c) Pope, L. M.; Reich, K. A.; Graham, D.
R.; Sigman, D. S. J. Biol. Chem. 1982, 257, 12121. (d) Pope, L.
E.; Sigman, D. S. Proc. Natl. Acad. Sci. USA 1984, 81, 3.
7. (a) Hertzberg, R. P.; Dervan, P. B. J. Am. Chem. Soc. 1982,
104, 313. (b) VanDyke, M. W.; Dervan, P. B. Nucleic Acids
Res. 1983, 11, 5555. (c) Hertzberg, R. P.; Dervan, P. B. Bio-
chemistry 1984, 23, 3934.
UV±vis and ¯uorescence studies of conjugate 16. For the
UV±vis experiments compound 16 (9.1 mg) was dis-
solved in 1 mL of DMSO and diluted 60 times with
ꢁ
50 mmol Tris HCl bu€er to give a ®nal concentration of
0.29 mmol. A 2.4 mL of this solution was titrated with
50 mL aliquots of salmon testes DNA (2.25 mmol) dis-
ꢁ
solved in 50 mmol Tris HCl bu€er and the spectra was
recorded after each titration. Control tests were per-
formed in the same fashion by replacing the DNA
solution with plain bu€er. For the ¯uorescence
quenching studies, conjugate 16 was selectively excited
at 412 nm and its ¯uorescence was recorded at 420±
580 nm. Compound 16 (10.2 mg) was dissolved in 10 mL
ꢁ
of DMSO and diluted 100 times with 50 mmol Tris HCl
bu€er to give a ®nal concentration of 19 mmol. A 3 mL
of this solution was titrated with 200 mL aliquots of sal-
mon testes DNA (2.25 mmol) dissolved in 50 mmol
ꢁ
Tris HCl bu€er and the spectra was recorded after each
titration. Control tests were performed in the same
fashion by replacing the DNA solution with plain
bu€er.
Isolation of the 50 32P end-labeled Sal I/Sph I restriction
fragment of the pBR322 duplex DNA. BR322 DNA was
cleaved at the unique Sal I si0te, dephosphorylated using
calf intestine phosphatase, 5 -32P-phosphorylated using
T4 polynucleotide kinase and [g-32P] ATP (4.5 mCi/
pmol) and cleaved again at the unique Sph I site. This
generated a 93-base-pair double-stranded fragment,
50-32P-labeled at the Sal I end, which was puri®ed by gel

736
P. Blom et al. / Bioorg. Med. Chem. 7 (1999) 727±736
8. (a) Dixon W. J.; Hayes, J. J.; Levin, J. R.; Weidner, M. F.;
Dombroski, B. A.; Tullius, T. D. Methods Enzymol. 1991, 208,
380. (b) Tullius, T. D. Nature 1988, 332, 663. (c) Tullius, T. D.
Trends Biochem. Sci. 1987, 12, 297. (d) Pogozelski, W. K.;
McNeese, T. J.; Tullius, T. D. J. Am. Chem. Soc. 1995, 117,
6428.
1992, 48, 2529. (e) Newcomb, M.; Deeb, T. M.; Marquardt, D.
J. Tetrahedron 1990, 46, 2317. (f) Newcomb, M.; Marquardt,
D. J.; Deeb, T. M. Tetrahedron 1990, 46, 2329.
20. (a) Hess, K. M.; Dix, T. A. Anal. Biochem. 1992, 206, 309.
(b) Boivin, J.; Crepon, E.; Zard, S. Z. Bull. Soc. Chim. Fr.
1992, 129, 145. (c) Nocentini, G.; Castagnino, E.; Salvatori,
A.; Corsano, S.; Fioretti, M. C. Arzneim.-Forsch./Drug Res.
1995 (II), 10, 1127.
9. Kane, S. A.; Sasaki, H.; Hecht, S. M. J. Am. Chem. Soc.
1995, 117, 9107.
10. (a) Chen, X.; Woodson, S. A.; Burrows, C. J.; Rokita, S.
E. Biochemistry 1993, 32, 7610. (b) Muller, J. G.; Zheng, P.;
Rokita, S. E.; Burrows, C. J. J. Am. Chem. Soc. 1996, 118, 2320.
11. For a general review see: Bioorganic Photochemistry, Pho-
tochemistry and the Nucleic Acids; Morrison, H., Ed.; Wiley
and Sons: New York, 1990; Vol. 1.
12. (a) Uchida, K.; Pyle, A. M.; Morii, T.; Barton, J. K.
Nucleic Acids Res. 1989, 17, 10259. (b) Chow, C. S.; Behlen, L.
S.; Uhlenbeck, O. C.; Barton, J. K. Biochemistry 1992, 31, 972.
(c) Chow, C. S.; Hartmann, K. M.; Rawlings, S. L.; Huber, P.
W.; Barton, J. K. Biochemistry 1992, 31, 3534. (d) Chow, C.
S.; Barton, J. K. Biochemistry 1992, 31, 5423. (e) Stemp, E. D.
A; Arkin, M. R.; Barton, J. K. J. Am. Chem. Soc. 1997, 119,
2921.
21. (a) Adam, W.; Ballmaier, D.; Epe, B.; Grimm, G. N.;
Saha-Moller, C. H. Angew. Chem., Int. Ed. Engl. 1995, 34,
2156. (b) Epe, B.; Ballmaier, D.; Adam, W.; Grimm, G. N.;
Saha-Moller, C. R. Nucleic Acids Res. 1996, 24, 1625.
22. (a) Aveline, B. M.; Kochevar, I. E.; Redmond, R. W. J.
Am. Chem. Soc. 1996, 118, 289. (b) Aveline, B. M.; Koshevar,
I. E.; Redmond, R. W. J. Am. Chem. Soc. 1995, 117, 9699. (c)
Aveline, B. M.; Koshevar, I. E.; Redmond, R. W. J. Am.
Chem. Soc. 1996, 118, 10124. (d) Aveline, B. M.; Koshevar, I.
E.; Redmond, R. W. J. Am. Chem. Soc. 1996, 118, 289.
23. (a) Chateauneuf, J.; Lusztyk, J.; Ingold, K. U. J. Am.
Chem. Soc. 1988, 110, 2886. (b) Bottle, S. E.; Bus®eld, W. K.;
Jenkins, I. D. J. Chem. Soc., Perkin Trans 2 1992, 2145. (c)
Bevington, J. C. Angew. Makromol. Chem. 1991, 185/186, 1.
(d) Barson, C. A.; Bevington, J. C.; Huckerby, T. N. Polymer
1991, 32, 3415. (e) Barton, D. H. R.; Lacher, B.; Zard, S. Z.
Tetrahedron Lett. 1985, 26, 5939.
24. All DNA-cleaving tests were conducted in the presence of
supercoiled circular fX174 or pBR322 DNA and the photo-
cleavage eciency was determined as the degree of conversion
of supercoiled DNA (form I) to circular nicked (form II) and
linear (form III).
25. 9-Aminoacridine displays strong intercalating properties
onto DNA. For selected literature on this topic see: (a) Bailly,
C.; Henichart, J.-P. Perspectives in Bioconjugate Chem. 1991,
112. (b) Wang, A. H. J. Curr. Opin. Struct. Biol. 1992, 2, 361.
(c) Jeppesen, C.; Buchardt, O.; Henriksen, U.; Nielsen, P. E.
Nucl. Acids Res. 1988, 16, 5755. (d) Nielsen, P. E.; Jeppesen,
C.; Egholm, M.; Buchardt, O. Biochemistry 1988, 27, 6338. (e)
Lorente, A.; Espinosa, J. F.; Saiz, M. F.; Lehn, J.-M.; Wilson,
W. D.; Zhong, Y. Y. Tetrahedron Lett. 1996, 37, 4417. (f)
Chiu, F. C. K.; Brownlee, R. T. C.; Mitchell, K.; Phillips, D.
R. Bioorg. Med. Chem. Lett. 1995, 5, 1689.
13. (a) Nielsen, P. E. Nucleic Acids Res. 1992, 20, 2735. (b)
Jeppesen, C.; Nielsen, P. E. Nucleic Acids Res. 1989, 17, 4947.
(c) Nielsen, P. E.; Mollegaard, N. E.; Jeppesen, C. Nucleic
Acids Res. 1990, 18, 3847.
14. (a) Becker, M. M.; Wang, J. C. Nature 1984, 309, 682. (b)
Becker, M. M.; Lesser, D.; Kurpiewski, M.; Baranger, A.;
Jacobson, L. J. Proc. Natl. Acad. Sci. USA 1988, 85, 6247.
15. (a) Blacker, A. J.; Jazwinski, J.; Lehn, J.-M.; Wilhelm, F.
X. J. Chem. Soc., Chem. Commun. 1986, 1035. (b) Saito, I.;
Takayama, M.; Matsuura, T.; Matsugo, S.; Kawanishi, S. J.
Am. Chem. Soc. 1990, 112, 883. (c) Singh, U. S.; Scannell, R.
T.; An, H.; Carter, B. J.; Hecht, S. M. J. Am. Chem. Soc. 1995,
117, 12691. (d) Lytollis, W.; Scannell, R. T.; An, H.; Murty, V.
S.; Reddy, K. S.; Barr, J. R.; Hecht, S. M. J. Am. Chem. Soc.
1995, 117, 12683. (e) Adam, W.; Cadet, J.; Dall'Acqua, F.;
Epe, B.; Ramaiah, D.; Saha-Moller, C. R. Angew. Chem., Int.
Ed. Engl. 1995, 34, 91. (f) Adam, W.; Cadet, J.; Dall'Acqua,
F.; Epe, B.; Ramaiah, D.; Saha-Moller, C. R. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 107. (g) King, P. A.; Anderson, V. E.;
Edwards, J. O.; Gustafson, G.; Plumb, R. C.; Suggs, J. W. J.
Am. Chem. Soc. 1992, 114, 5430. (h) Bregant, T. M.; Groppe,
J.; Little, R. D. J. Am. Chem. Soc. 1994, 116, 3635.
16. Theodorakis, E. A.; Wilcoxen, K. M. J. Chem. Soc.,
Chem. Commun. 1996, 1927.
26. (a) Peacocke, A. R. In Heterocyclic Compounds: Acridines,
2nd ed.; 1973; 723. (b) Leupin, W. In Molecular Basis of Spe-
ci®city in Nucleic Acid-Drug Interactions; Pullman, B.; Jortner,
J., Eds.; Kluwer Academic Publishers: Netherlands, 1990;
579±603. (c) Intercalation Chemistry; Whittingham, M. S.;
Jacobson, A. J., Eds.; Academic Press: New York, 1982.
27. (a) Tubbs, R. K.; Ditmars, W. E., Jr.; van Winkle, Q. J.
Mol. Biol. 1964, 9, 545. (b) Oster, G. Trans. Faraday Soc.
1951, 47, 660. (c) Blake, A.; Peacocke, A. R. Biopolymers
1968, 6, 1225. (d) Peacocke, A. R.; Skerrett, J. N. H. Trans.
Faraday Soc. 1955, 52, 261. (e) Ellerton, N. F.; Isenberg, I.
Biopolymers 1969, 8, 767. (f) Strothcamp, K. G.; Strothcamp,
R. E. J. Chem. Educ. 1994, 71, 77.
17. Theodorakis, E. A.; Xiang, X.; Blom, P. Chem. Commun.
1997, 1463.
18. Theodorakis, E. A.; Xiang, X.; Lee, M.; Gibson, T. Tet-
rahedron Lett. 1998, 39, 3383.
19. For selected reviews on this topic see: (a) Barton, D. H.
R.; Zard, S. Z. Pure Appl. Chem. 1986, 58, 675. (b) Crich, D.
Aldrichimica Acta 1987, 20, 35. (c) Barton, D. H. R. Pure
Appl. Chem. 1994, 66, 1943. (d) Barton, D. H. R. Tetrahedron