Benzo[f]azino[2,1-a]phthalazinium Cations: Novel DNA Intercalating Chromophores with Antiproliferative Activity

Article abstract of DOI:10.1021/jm0310434

New azaquinolizinium-type cations have been obtained from isochromane. The synthesis was completed over seven steps and included as the key feature an intramolecular Westphal condensation. This first example of the intramolecular process allowed the preparation of benzo[f]pyrido[2,1-a]phthalazinium and benzo[f]quino[2,1-a]phthalazinium salts, which were evaluated as DNA intercalators, DNA topoisomerase I inhibitors, and antiproliferative compounds. Both cationic systems behave as DNA intercalators and exhibit antiproliferative activity. The pentacyclic benzo[f]quino[2,1-a]phthalazinium cations also have an inhibitory effect on the catalytic activity of DNA topoisomerase I, without trapping of cleavage complexes. Structural characterization using density functional theory indicates that the fused ring systems are slightly nonplanar, and additional molecular modeling studies suggest a preferred orientation for the intercalating chromophores within a typical CpG or TpG intercalation site.

Full text of DOI:10.1021/jm0310434

1136
J . Med. Chem. 2004, 47, 1136-1148
Ben zo[f]a zin o[2,1-a ]p h th a la zin iu m Ca tion s: Novel DNA In ter ca la tin g
Ch r om op h or es w ith An tip r olifer a tive Activity
Valent´ın Mart´ınez,† Carolina Burgos,† J ulio Alvarez-Builla,† Gero´nimo Ferna´ndez,‡ Alberto Domingo,*,‡
Raquel Garc´ıa-Nieto,§ Federico Gago,§ Ignacio Manzanares,|| Carmen Cuevas,|| and J uan J . Vaquero*,†
Departamento de Qu´ımica Orga´nica, Departamento de Bioqu´ımica y Biolog´ıa Molecular, and Departamento de Farmacolog´ıa,
Universidad de Alcala´, 28871-Alcala´ de Henares, Madrid, Spain, and Lab. PharmaMar, Avda. de los Reyes, 1, Pol´ıgono
Industrial La Mina-Norte, 28770-Colmenar Viejo, Madrid, Spain
Received September 23, 2003
New azaquinolizinium-type cations have been obtained from isochromane. The synthesis was
completed over seven steps and included as the key feature an intramolecular Westphal
condensation. This first example of the intramolecular process allowed the preparation of benzo-
[f]pyrido[2,1-a]phthalazinium and benzo[f]quino[2,1-a]phthalazinium salts, which were evalu-
ated as DNA intercalators, DNA topoisomerase I inhibitors, and antiproliferative compounds.
Both cationic systems behave as DNA intercalators and exhibit antiproliferative activity. The
pentacyclic benzo[f]quino[2,1-a]phthalazinium cations also have an inhibitory effect on the
catalytic activity of DNA topoisomerase I, without trapping of cleavage complexes. Structural
characterization using density functional theory indicates that the fused ring systems are
slightly nonplanar, and additional molecular modeling studies suggest a preferred orientation
for the intercalating chromophores within a typical CpG or TpG intercalation site.
In tr od u ction
Many effective anticancer drugs in clinical use inter-
act with DNA,1-4 and some of them do so through
intercalation.5,6 The intercalation process reflects the
ability of a planar aromatic or heteroaromatic system
to become inserted between adjacent base pairs of a
DNA molecule without disturbing the overall stacking
pattern.7 This class of DNA-binding ligands can affect
many biological properties of DNA, including replication
and transcription, and several antitumor antibiotics are
recognized as the most important class of DNA inter-
calating reagents.8,9
The intercalative reaction is envisaged as a complex
process that depends on multiple variables, with micro-
heterogeneity of the DNA molecule being one of the
factors that more strongly contributes to hamper our
F igu r e 1. General structure and representative examples
(1, 2) of azaquinolizinium-type DNA intercalators.
understanding of the rules that govern high-affinity
binding and selection of a particular sequence.10 Thus,
in this field there is still room for the design and
uisite for intercalation in this class of hetearoaromatic
exploration of new structural classes of chromophores
cations bearing a bridgehead quaternary nitrogen is the
with intercalating properties11-16 or for rational struc-
existence of at least four fused rings, preferably non-
tural modifications on well-established chromophores,
linearly fused. Moreover, while affinity seems to be little
even though we must assume that understanding the
affected by simple substituents, the antiproliferative
mechanisms that ultimately lead to cell death is still
activity is strongly dependent on chromophore substi-
tution.17-21
an elusive goal.
We recently reported the promising antiproliferative
activity associated with a new class of DNA intercala-
tors based on an azaquinolizinium core, such as γ-car-
bolinium (1)17,18 and benzimidazolium (2)19,20 systems
(Figure 1). Results from our lab are demonstrating that,
contrary to other charged DNA intercalators, the req-
These considerations prompted us to explore a new
series of azaquinolizinium-type chromophores with
potential DNA intercalating properties. Herein we
report the synthesis and DNA binding affinity of one of
these fused heteroaromatic cationic systems by using
the intramolecular Westphal reaction, a version of the
intermolecular process22 widely used in the synthesis
of quinolizinium and azaquinolizinium cations.
* To whom correspondence should be addressed. FAX: 34-1-
918854660. E-mail: juanjose.vaquero@uah.es.
† Departamento de Qu´ımica Orga´nica, Universidad de Alcala´.
‡ Departamento de Bioqu´ımica y Biolog´ıa Molecular, Universidad
de Alcala´.
Ch em istr y
In earlier reports we established the potential of the
Westphal condensation reaction (Scheme 1) to prepare
§ Departamento de Farmacolog´ıa, Universidad de Alcala´.
| Lab. PharmaMar.
10.1021/jm0310434 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/24/2004

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1137
Sch em e 1
with 4-picolinmethylide. Carrying out the nucleophilic
substitution under different conditions did not improve
the regioselectivity.
Completion of the synthesis of 3 would require the
following synthetic operations: (1) the preparation of
the diketones 9, (2) formation of the salts 10, and finally
(3) intramolecular Westphal reaction cyclization of the
N-aminopyridinium salts under basic conditions.
Sch em e 2
The most successful methods for conversion of alkenes
into diketones are based on the use of DMSO. More
specifically, good yields in diketone are described by
reaction of arylalkenes and I2/DMSO or HBr/DMSO. In
our case, compounds 8a -e (R1 ) Ar) were oxidized to
the corresponding diketones in moderate yields by using
either iodine25,26 or HBr27 in DMSO at reflux temper-
ature, although in a comparative study with compounds
8a ,b the latter procedure gave better yields (9a , 77%
vs 60%; 9b, 71% vs 56%). All our attempts to transform
the alkene with R1 ) Me into the corresponding dike-
tones failed with oxidative degradation of the double
bond and concomitant formation of aldehydes being
observed.
Compounds 9 were easily transformed into the cor-
responding N-aminopyridinium salts 10 by reaction
with (O-mesitylenesulfonyl)hydroxylamine (MSH) in
CH2Cl2 at reflux temperature in good yields. Initial
experiments on the intramolecular Westphal condensa-
tion focused on the selection of the most appropriate
base and solvent for the success of the reaction. On the
basis of our previous work on the intermolecular pro-
cess,22 the condensation was achieved using triethyl-
amine in ethanol and sodium acetate in acetone. In most
of the cases, the reaction afforded a complex mixture
from which the expected condensation product and the
required fully oxidized compound 3 were identified.
Although both compounds were difficult to separate, it
was found that prolonged reaction times gave 3 as the
only compound without appreciable loss of yield.
To prove the DNA intercalating properties of com-
pounds 3, both electrophoresis and UV determination
(for details, see below) were carried out. It was shown
that compound 3c does not interact with DNA and that
compounds 3a , 3b, and 3e intercalate into DNA only
weakly. The solubility of compound 3d was too low to
allow an accurate measurement of its intercalating
behavior. In the light of these findings, we decided to
modify the size of the chromophore, thereby extending
the fused system with an additional aromatic ring.
To use the same approach for the preparation of the
new chromophore, 2-methylquinoline, 3-methylisoquin-
oline, and 2-methylbenzothiazole were chosen as het-
erocycles for the reaction with 7. However, only the use
of 2-methylquinoline allowed us to complete the syn-
thetic route leading to the pentacyclic benzo[f]quino[2,1-
a]phthalazinium system 4 (Scheme 4). The reaction with
3-methylisoquinoline failed under different conditions
with the heterocycle being recovered unaltered. On the
contrary, 3-methylbenzothiazole reacted with 7a , af-
fording the substitution compound. However, the sub-
sequent oxidation step was also troublesome and com-
plex reaction mixtures were formed under the tested
conditions.
a great variety of heteroaromatic cations bearing a
quaternary bridgehead nitrogen, some of which behave
as DNA intercalators.17-22
The search in our lab for new azaquinolizinium-type
cations having the requisites previously established for
intercalation led us to focus on different possibilities for
incorporating the azaquinolizinium moiety into a non-
linearly fused tetracyclic cation. The retrosynthetic
analysis of one of the desired systems (Scheme 2)
indicated that the intramolecular Westphal reaction23
could be the choice for a straightforward synthesis of
benzo[f]pyrido[2,1-a]phthalazinium cations 3 from the
salt 10, which was envisioned to originate from the
alkene 8. Thus, our approach was to use isocromane 5
as the key raw material, convert it to a 2-haloethyl
benzaldehyde 6, and prepare 8 through a Wittig reac-
tion.
Formation of the 2-bromoethylbenzaldehyde 6 (X )
Br) from 5 was carried out following the procedure
described by Rieche.24 The method rests on the treat-
ment of 5 with bromine in CH2Cl2, at reflux temperature
under sunlight. The Wittig reaction of 6 with alkyl and
arylmethylphosphines in THF gave rise to a 1:1 Z/E
mixture of the required alkenes 7 in moderate to good
yields. The success of the reaction required slow addition
of the bromo derivative to avoid undesirable competitive
dehydrohalogenation.
Reaction of 7 with methylides generated from pico-
line and lutidine in the presence of nBuLi in THF
afforded the corresponding derivatives 8 in good yield,
except when 7a reacted with the 2,4-dimethylpyridine,
where the desired derivative 8c was obtained in only
37% yield because of the formation of the undesired
regioisomer (34% yield) coming from the reaction of 7a
In this case an alternative method for the formation
of diketones 13 with substituent R1 being an alkyl group

1138 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mart´ınez et al.
Sch em e 3
Sch em e 4
was also attempted using the Katritzky benzotriazole
methodology,28 but up to now all our attempts have been
unsuccessful.
Table 1 shows the heteroaromatic cations 3 and 4
prepared along with the best conditions for the intramo-
lecular Westphal reaction and the yields obtained in this
last key step of the synthesis.
ium salts 4. Supporting evidence for an intercalative
mode of binding to DNA of compounds 3a ,b,e and 4a -d
was first obtained by electrophoretic titration of a
bacterial plasmid, as shown in Figure 2. This assay
exploits the conformational changes induced by inter-
calating ligands on covalently closed, double-stranded
DNA molecules, such as bacterial plasmids, together
with their differential electrophoretic behavior in aga-
rose gels.
Resu lts a n d Discu ssion
DNA Bin d in g Stu d ies. As indicated above, two
different studies were carried out to investigate the
DNA-binding behavior of the benzo[f]pyrido[2,1-a]ph-
thalazinium salts 3 and benzo[f]quino[2,1-a]phthalazin-
Bacterial plasmids are dynamically maintained in
vivo in an underwound topology that induces a nega-
tively supercoiled conformation. Upon binding of a
number of intercalating molecules of any kind, the

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1139
Ta ble 1. Synthesis and DNA Binding Properties of
Benzo[f]pyrido[2,1-a]phthalazinium Salts 3 and
Benzo[f]quino[2,1-a]phthalazinium Salts 4a
in the case of intercalating compounds that form highly
stable complexes with DNA, that is, when the kinetic
dissociation constant (koff) of the binding reaction is very
low. Such slow dissociation behavior is usually found
in the case of bis-intercalating compounds. Ditercalin-
ium29 was included in Figure 1 to show an example of
the typical results obtained with a bisintercalating
molecule, for which this assay was initially designed.
In the case of monointercalating molecules, the koff is
usually much higher, so that dissociation occurs very
rapidly when the DNA:ligand complexes enter the
agarose gel. In this case, the relaxed conformation
occurs only as a transient state. All of the plasmid DNA
can be considered free of the intercalating ligand after
a few minutes of electrophoresis and, in this situation,
the plasmids recover the fast migrating, negatively
supercoiled conformation. Nevertheless, in some lanes
the plasmids start from, or pass through, the relaxed
state inside the gel, and this conformation has a much
slower electrophoretic migration than the negative or
positive supercoiled states. The resulting effect is a
transient reduction in the migration speed of the DNA
band that is revealed by its slight delay when compared
to a control lane, or by a smearing of the DNA band.
reaction yield
compd
R1
C6H5
C6H5
C6H5
4-EtO-C6H4
4-EtO-C6H4 Me
C6H5
C6H5
R2 R3 conditions (%) ∆λ
H
K
n
3a
3b
3c
3d
3e
4a
4b
4c
4d
EtBr
H
Me
H
H
H
Me
H
H
A
B
B
A
B
A
B
B
B
65
50
28
22
28
75
50
28
50
10
0
0
-
0
6 ND ND
0
0
-
0
-
-
-
-
H
NM NM
-
-
2.3
H
Me
H
-
-
-
-
12 33 2.3
10 11 1.03 1.8
12 52 7.20 2.3
4-EtO-C6H4
4-EtO-C6H4 Me
14
6
1.12 2.2
2.06 2.1
a
Yields refer to the intramolecular Westphal reaction step. A:
NaOAc/acetone. B: Et3N/EtOH. ∆λ ) (λbound - λfree ), where λfree
and λbound are the wavelengths of maximum absorption for free
and DNA bound compounds. H ) percent hypochromicity [%H )
(1 - ꢀbound/ꢀfree) × 100], where ꢀfree and ꢀbound are the extinction
coefficients for free and DNA bound compounds. K ) the affinity
constant for DNA (105 M-1). n ) the number of base pairs per
bound molecule. EtBr )ethidium bromide. ND ) not determined.
NM ) not measurable due to the insolubility of the compound
above the micromolar range.
Compounds with high kinetic dissociation constants
(koff) or with an overall reduced intercalative binding
capacity may produce negative or somewhat unclear
results in this preliminary, but semiquantitative, screen-
ing assay. Ethidium is a noteworthy example of a well-
known DNA-intercalating compound that produces a
negative result in this assay, due to the high dissociation
rate of its complex with DNA. For this reason, a further
indication of the intercalative binding capacity can be
obtained by a different electrophoretic assay, which is
much more sensitive as a DNA intercalation test, but
only qualitative and also more demanding in terms of
labor, reagents, and expense of analyte. In this type of
experiment, the compound under study is included in
the mass of the agarose gel, as well as in the samples,
to maintain the plasmids in a saturating environment
during the electrophoresis. The complete assay involves
four lanes. In two of them the plasmid is previously
treated with DNA topoisomerase I in order to obtain a
covalently closed double-stranded DNA in a relaxed
topology. The other two lanes are loaded with the
original plasmids, as purified from bacterial culture in
a natural negatively supercoiled topology. If intercala-
tion occurs, all the plasmids in the four lanes should
adopt a positively supercoiled conformation, character-
ized by a likewise fast electrophoretic migration. If the
compound under study is not able to intercalate at all,
the lanes loaded with the topoisomerase I-relaxed
plasmids show the characteristic slow migrating band,
clearly distinguishable from the fast migrating band
that appears in the lanes loaded with the negatively
supercoiled plasmids. In two of the four lanes, the
putative ligand is not included in the sample. These two
lanes serve as an internal control to ascertain that
conditions inside the gel are saturating as expected. As
an additional control, the same set of samples is also
applied to a second agarose gel without the compound
under study. This assay complements the previous one,
mainly to resolve some unclear or conspicuous situations
as in the noteworthy case of compound 3c, for which a
F igu r e 2. Electrophoretic titration of supercoiled bacterial
plasmid DNA. Each lane was loaded with a 6 µL sample
containing a preincubated mixture of 200 ng of plasmid DNA
plus the compound under study at the final molar concentra-
tion indicated in the top row. Pictures were taken after
postelectrophoretic staining with ethidium bromide. The ar-
rowhead in each gel indicates the lane where a complete
relaxation of the plasmid DNA has been reached or exceeded.
The effect was confirmed in all cases by a second assay
exploring a narrower range of concentrations. The left panel
shows the results obtained with compounds 3 (3d could not
be analyzed because of its very low solubility in aqueous
media). The same assay applied to ditercalinium (D) is also
shown for comparison. The right panel shows the results
obtained with compounds 4.
plasmids reach a relaxed conformation. The binding of
more intercalating molecules then drives the plasmids
to adopt a positively supercoiled form. Such complex
conformational changes can be detected by viscometry
as well as by agarose gel electrophoresis. Nevertheless,
the electrophoretic assay, described in the Experimental
Section, is much faster and requires very small amounts
of both plasmid DNA and ligand. We use this electro-
phoretic assay as a first screening test to determine the
intercalative capacity of every new molecule. Both
positive and negative supercoiled conformations behave
as compact structures, with a fast electrophoretic mi-
gration in agarose gel, whereas the more extended and
flexible relaxed conformation has a much slower elec-
trophoretic migration. The results obtained by this
assay with compounds 3 and 4 are shown in Figure 2.
The complete conformational transition is clearly visible

1140 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mart´ınez et al.
F igu r e 3. Electrophoretic assay of supercoiled and relaxed bacterial plasmid DNA. Each lane was loaded with 6 µL samples
containing 200 ng of bacterial plasmid DNA that was untreated, negatively supercoiled in the case of odd numbered lanes, or
relaxed by treatment with DNA topoisomerase I in the case of even numbered lanes. The samples applied to lanes 3, 4, 7, and 8
contain the compound indicated on the left at a final concentration of 0.1 mM. The agarose gel on the right-hand side of each row
contains the same compound in the mass of the gel at a final concentration of 0.01 mM. Pictures were taken after postelectrophoretic
staining with ethidium bromide. The left panel shows the results obtained with compounds 3 (3d could not be analyzed because
of its complete insolubility in aqueous media). The right panel shows the results obtained with compounds 4a and 4b. The results
of the same assay when applied to ethidium bromide (EtBr) and DAPI are also shown to compare the behavior of a well-known
intercalator and a groove binder, respectively.
simple substituent completely abolishes the intercalat-
ing capacity of the molecule. The results obtained from
this assay are shown in Figure 3.
Compounds 4a -d show a similar intercalative bind-
ing behavior in these two electrophoretic assays. In the
titration assay the fully relaxed conformation occurs at
concentrations around 10-5 M in the four cases (inter-
calator:base-pair molar ratio of 0.195). Compounds 3a ,
3b, and 3e also display intercalating properties. In these
cases the fully relaxed conformation is reached at 10-3
M (intercalator:base-pair molar ratio of 19.53). Interest-
ingly, when a methyl substituent is introduced on the
parent chromophore, the resulting compound 3c does
F igu r e 4. Binding curves for 4a to calf thymus DNA showing
the isosbestic point. Spectrophotometric titration was per-
not intercalate at all (see Figures 2 and 3).
UV-visible spectrophotometric determinations of se-
formed by serial addition of 100 µL aliquots of a DNA solution
(47 × 10-6 M) into a 10 cm path length quartz cell containing
ries 3 and 4 showed that compounds 3b, 3c, and 3e
exhibit neither hypochromic nor bathochromic effects
on their UV spectra in the presence of DNA, thus
confirming the very weak interaction of these com-
pounds with DNA. Compound 3a and the pentacyclic
salts 4a -d showed characteristic changes in their
visible absorption spectra as a result of formation of
more stable complexes. Both a hypochromic effect
(percent of hypochromicity between 6 and 52%) and
bathochromic shifts between 10 and 14 nm relative to
the spectra of the free compounds30,31 were observed
(Table 1).
a solution of 4a (1.5 × 10-6 M) and scanning the UV-visible
spectrum after each addition.
Composite spectra exhibit well-defined isosbestic
points for this series (one single binding mode) (Figure
4). From the spectral changes, nonlinear Scatchard
binding isotherms (Figure 5) were generated for the
interaction of these compounds with DNA using the
noncooperative McGhee-Von Hippel equation.32
The binding parameters (K, n) determined in this
manner are shown in Table 1, with K being the apparent
binding constant and n the number of base pairs per
bound molecule. For compound 3a the numerical data
generated in different spectrophotometric titrations
could not be adequately fitted to the Scatchard equation
because of its dispersion. All derivatives 4a -d have
DNA affinity constant values around 105 M-1, which are
essentially on the same order of magnitude as that of
the well-known intercalator ethidium bromide,33-35 used
here as a reference and measured under the same
conditions (Table 1).
F igu r e 5. Scatchard plot for the binding of 4a to calf thymus
DNA. Values for binding ratio υ and free compound concentra-
tion c were determined from data taken from spectrophoto-
metric titrations of the compound by DNA. Points in the figure
are the data, and solid lines are the best fits from the
noncooperative McGhee-von Hippel model.32
Th eor et ica l Ca lcu la t ion s. Representative com-
pounds 3a and 4a were studied using density functional
theory with the well-established B3LYP/6-31G* method.
The presence of the phenyl substituent induces some
distortions that cause the fused ring system to which it
is attached to be slightly nonplanar (Figure 6). This
phenyl ring is locked into position as rotation is ham-
pered by neighboring atoms. Similar distortions due to
steric congestion have been recently reported for related
phthalocyanines.36

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1141
F igu r e 6. Front and side views of representative compounds 3a (left) and 4a (right) optimized using the density functional
B3LYP/6-31G* method.
F igu r e 7. Stereoview from the minor groove of compound 4a docked into a TpG intercalation step (for clarity, only non-H atoms
are displayed).
Ta ble 2. Antiproliferative Activity (GI50, µM)a on Human
When the complexes between 3a and 4a -d with
Cancer Cells Lines of Compounds 3 and 4
prototypical CpG and TpG intercalation steps were
cell line
prostate
3a 3b 3c 3d 3e 4a 4b 4c 4d Adb
simulated using molecular dynamics, only the com-
plexes in which the phenyl ring substituent protruded
from the minor groove led to stable trajectories (Figure
7). The alternative orientation of the chromophore with
the phenyl pointing out from the major groove resulted,
in all cases, in extrusion of the ligand out of the
intercalation step. We take these results as an indica-
tion that the intercalation mode of these molecules is
similar to that of ethidium bromide.10,33-35
DNA Top oisom er a se I in h ibition . This enzyme is
a well-known biological target for some potent antican-
cer drugs, which exert their actions due a particular
mechanism of inhibition that involves trapping the
covalent enzyme-DNA “cleavage” complex.37-40 The
inhibitory effects on the catalytic activity of Topo1 were
semiquantitatively determined41 for all synthesized
compounds, and the results (not shown) indicate that
compounds 4a , 4b, and 4d have inhibitory effects on
Topo1. Compound 4d inhibits this enzyme completely
at a concentration of 10 µM, and partially at 5 µM,
whereas compounds 4a and 4b show only partial
inhibition at 10 µM. In all cases the mechanism of the
inhibition does not involve trapping of the cleavage
complex. This fact very likely rules out this particular
enzyme as the molecular target responsible for the
cytotoxic effects of these compounds, but the inhibition
does not seem to result simply from nonspecific steric
interference, since the inhibitory effect is detectable at
ligand concentrations well below saturation of the DNA
substrate. Compounds 3a -c, 3e, and 4c do not show
any effect on the enzymatic activity of Topo1.
DU-145
LN-caP
IGROV
NS 3.1 NS 4.8 1.8 1.0 0.5 5.8 0.5 0.02
5.2 0.4 4.0 0.5 0.4 0.3 0.2 2.6 0.07 0.02
0.1 1.7 6.5 3.8 2.4 0.5 0.5 2.6 0.4 4.4
ovarian
breast
melanoma MEL-28
NSCL A-549
pancreas PANC-1
IGROV-ET NS 2.9 NS 8.6 4.5 2.0 1.8 NS 1.7 2.4
SK-BR-3
9.7 0.5 4.6 1.2 0.5 0.5 0.5 3.9 0.5 1.1
NS 2.5 8.4 2.6 1.2 1.0 0.5 5.3 0.4 3.1
NS 4.2 NS 3.0 0.8 0.8 0.6 6.0 0.5 0.07
NS 3.4 NS 2.1 1.0 1.3 0.7 4.9 0.4 3.5
NS 1.2 NS 1.1 0.6 0.4 0.4 5.4 0.2 3.1
NS 3.0 NS 2.9 1.0 0.6 0.4 6.3 0.3 0.01
colon
HT-29
LOVO
LOVO-DOX NS 5.4 NS 8.0 7.0 3.5 3.6 NS NS 2.4
HELA NS 3.8 NS 3.1 1.3 0.7 0.5 4.6 0.3 0.01
HELA-APL NS 4.1 NS 5.0 1.4 0.1 NS 7.4 0.6 0.01
cervix
leukemia K-562
7.6 1.5 NS 1.2 0.6 0.4 0.2 6.7 0.1 0.03
a
GI50 ) concentration that causes 50% growth inhibition. GI50
values higher than 10 µM are considered not significant (NS). All
b
assays were performed in triplicate. Ad ) adriamycin.
shown in Table 2. According to these values some
generalizations can be deduced: (i) the results show that
although both series have antiproliferative activity in
the micromolar range, compounds in series 4 are clearly
more potent than compounds in series 3. (ii) The role of
intercalative binding in the cytotoxicity is presently
unclear. Thus, the lack of intercalating capacity of 3c
seems to result in an appreciable decrease in cytotoxicity
when compared with the DNA intercalator 3b, but the
capacity is similar to that found for 3a , which also
exhibits intercalative binding behavior. (iii) The cyto-
toxicity of DNA intercalators 4a -d seems to correlate
to some extent with their Topo1 inhibitory activity.
Thus, the most potent compound (4d ) completely inhib-
its the topoisomerase reaction, whereas 4a and 4b are
more potent than 4c, which does not inhibit this enzyme
at all. Nevertheless, the mechanism of inhibition, as
stated before, does not seem to point to this enzyme as
the molecular target responsible for the cytotoxic effects.
(iv) Although the substitution pattern with simple
An tip r olifer a tive Activity. In vitro activity poten-
cies (GI50) of compounds 3 and 4 against different
human cancer cells including two multidrug-resistant
(MDR) cell lines (IGROV-ET and LOVO-DOX) are

1142 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mart´ınez et al.
quenched with NaHCO3 solution, extracted with CH2Cl2, and
dried (MgSO4). Elimination of the solvent under reduced
pressure and purification by flash chromatography [silica gel,
hexane:ethyl acetate (9:1)] afforded a Z/E 1:1 mixture of
alkenes 7.
electrodonating substituents seems to be critical for
intercalation and Topo1 inhibition, it appears to be less
relevant for activity in these series in contrast to what
was observed with benzimidazolium and γ-carbolinium
cations.17-20 A methyl substituent does not significantly
vary the activity in series 4 (4a vs 4b, both DNA
intercalators and Topo1 inhibitors) and moderately
improves the cytotoxicity in series 3 (3a vs 3b, both
DNA intercalators). A comparison between 3b/3e (both
DNA intercalators) and 4b/4d (both DNA intercalators
and Topo1 inhibitors) to evaluate the role of the ethoxy
substitution indicates that this group moderately in-
creases the activity of 3e and 4d when compared to the
unsubstituted derivatives 3b and 4b, respectively.
(Z/E)-2-(2′-Br om oeth yl)stilben e (7a ): A 1:1 mixture of
alkenes; colorless oil (75%); IR (neat) νmax 3061, 1724, 1599,
1
1494, 1447 cm-1; H NMR (300 MHz, CDCl3) δ 7.63 (d, 1H, J
) 7.3 Hz), 7.53 (d, 2H, J ) 7.6 Hz), 7.41-7.35 (m, 3H), 7.32-
7.08 (m, 13H), 7.02 (d, 1H, J ) 15.7 Hz), 6.70 (d, 2H, J ) 2,0.9
Hz), 3.55 (t, 2H, J ) 7.3 Hz), 3.54 (t, 2H, J ) 7.3 Hz), 3.32 (t,
2H, J ) 7.3 Hz), 3.20 (t, 2H, J ) 7.3 Hz) ppm; 13C NMR (75
MHz, CDCl3) δ 137.2, 137.0, 136.8, 136.5, 136.4, 136.3, 131.5,
131.3, 130.0, 129.8, 129.6, 128.9, 128.7, 128.3, 128.1, 127.8,
127.7, 127.5, 127.4, 127.2, 127.0, 126.6, 126.2, 125.3, 37.2, 37.0,
31.9, 31.8 ppm; MS (EI, 70 eV) m/z (relative intensity) 286,
288 (M+, 36, 38), 178 (41), 90 (100); HRMS (EI) calcd for C16H15
Br (M+) 286.0353, found 286.0376.
-
Con clu sion s
(Z/E)-2-(2′-Br om oeth yl)-4′′-eth oxystilben e (7b): A 1:1
mixture of alkenes; colorless oil (82%); IR (neat) νmax 3060,
1606, 1511, 1476, cm-1; 1H NMR (300 MHz, CDCl3) δ 8.26 (d,
1H, J ) 7.8 Hz), 8.12 (d, 2H, J ) 10.0 Hz), 7.97-7.80 (m, 10H),
7.68-7.55 (m, 5H), 7.34 (d, 2H, J ) 8.8 Hz), 4.73 (q, 2H, J )
7.2 Hz), 4.63 (q, 2H, J ) 7.0 Hz), 4.24-4.14 (m, 4H), 3.97 (t,
2H, J ) 7.4 Hz), 3.83 (t, 2H, J ) 7.4 Hz), 2.10 (t, 3H, J ) 7.2
Hz), 2.03 (t, 3H, J ) 7.0 Hz) ppm; 13C NMR (75 MHz, CDCl3)
δ 158.1, 137.4, 136.8, 131.0, 130.2, 129.8, 129.6, 128.9, 127.3,
127.1, 126.2, 114.1, 63.3, 37.3, 32.0, 14.7 ppm; MS (EI, 70 eV)
m/z (relative intensity) 332, 330 (M+, 26, 26), 107 (100), 93
(30), 77 (14); HRMS (EI) calcd for C18H19BrO (M+) 330.0638,
found 330.0653.
Isochromane was used as the starting material for the
preparation of new azaquinolizinium-type cations. The
synthesis was completed over seven steps and included
as the key feature an intramolecular Westphal conden-
sation. The resulting benzo[f]pyrido[2,1-a]phthalazini-
um and benzo[f]quino[2,1-a]phthalazinium salts dis-
played a broad spectrum of antiproliferative activity
against human cancer cell lines and were evaluated for
their ability to bind DNA through intercalation and
their capacity to inhibit Topoisomerase I activity. Topo1
inhibition was associated only with the pentacyclic
benzo[f]quino[2,1-a]phthalazinium cations, even though
both cationic systems behaved as DNA intercalators.
Theoretical calculations using density functional theory
showed fused ring system 3 and 4 to be slightly
nonplanar due to distortions induced by the phenyl
substituent. Additional molecular modeling studies sug-
gest a preferred orientation for the intercalating cho-
mophores within CpG and TpG intercalation sites that
resembles that found for the prototypical intercalator
ethidium bromide.
(Z/E)-2-(2-Br om oeth yl)-2-p r op en yl ben zen e (7c): A 1:1
mixture of alkenes; colorless oil (45%); IR (neat) νmax 3064,
1603, 1511 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.35-7.04 (m,
8H), 6.51 (d, 2H, J ) 12.0 Hz), 5.91-5.85 (m, 2H), 7.68-7.55
(m, 5H), 3.48 (t, 4H, J ) 7.5 Hz), 3.15 (t, 4H, J ) 7.5 Hz), 1.91
(d, 3H, J ) 7.0 Hz), 1.71 (t, 3H, J ) 7.0 Hz) ppm; 13C NMR
(75 MHz, CDCl3) δ 137.0, 136.4, 135.5, 129.8, 129.7, 129.4,
128.4, 128.1, 127.8, 127.2, 127.0, 126.6, 126.3, 37.2, 37.0, 31.8,
31.7, 14.2 ppm; MS (EI, 70 eV) m/z (relative intensity) 224,
226 (M+, 30, 30), 93 (30), 77 (20); HRMS (EI) calcd for C11H13
Br (M+) 224.0197, found 224.0221.
-
Gen er a l P r oced u r e for P r ep a r a tion of 8 a n d 12. To a
solution of the corresponding methylazine (2 mmol) in dry THF
(5 mL) was added n-BuLi (1 mmol, solution 1.6 M in hexanes)
dropwise at -20 °C under an atmosphere of dry argon. The
reaction mixture was allowed to warm to room temperature
and stirred for 1 h. Then, a solution of 7 (1 mmol) in dry THF
(5 mL) was added and stirring was continued at room tem-
perature for 2 h more. The reaction mixture was quenched
with NaHCO3 solution, extracted with CH2Cl2, dried (MgSO4),
and evaporated under reduced pressure. The crude product
was purified by flash chromatography [silica gel, hexane:ethyl
acetate (8:2)], affording a Z/E 1:1 mixture of alkenes 8 and
12, respectively.
(Z/E)-2-[3-(2-Styr ilp h en yl)p r op yl]p yr id in e (8a ): A 1:1
mixture of alkenes; colorless oil (90%); IR (neat) νmax 3064,
1738, 1594, 1462 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.55 (dd,
2H, J ) 10.6, 5.8 Hz), 7.65-6.99 (m, 26H), 6.71 (d, 1H, J )
11.1 Hz), 6.60 (d, 1H, J ) 11.1 Hz), 2.94-2.82 (m, 6H), 2.74
(t, 2H, J ) 7.69 Hz), 2.12-2.04 (m, 4H) ppm; 13C NMR (75
MHz, CDCl3) δ 161.8, 149.2, 149.1, 140.9, 139.9, 136.8, 136.6,
136.1, 130.5, 130.1, 129.7, 129.3, 128.9, 128.5, 127.9, 127.5,
127.4, 127.3, 126.9, 126.5, 126.3, 125.8, 125.7, 122.7, 122.6,
120.8, 38.1, 37.9, 33.2, 32.8, 30.9, 30.4 ppm; MS (EI, 70 eV)
m/z (relative intensity) 299 (M+, 21), 106 (100), 93 (58), 91 (65);
HRMS (EI) calcd for C22H21N (M+) 299.1668, found 299.1666.
Exp er im en ta l Section
Gen er a l Meth od s. 1H and 13C NMR spectra were recorded
on a 300 MHz spectrometer. General experimental conditions,
such as absorbance measurements in the UV-visible region,
were carried out as described previously.18,20 2-(2′-Bromoethyl)-
benzaldehyde (6) was prepared according to the literature
procedure.24
Th eor etica l Ca lcu la tion s. Ring systems 3 and 4 were
model-built in Insight II42 using standard bond lengths and
angles. The initial geometries were first refined by means of
the semiempirical quantum mechanics program MOPAC43 and
further optimized using hybrid density functional theory
(B3LYP) and the 6-31G* basis set as implemented in the
Gaussian program.44 The AMBER all-atom force-field param-
eters for the chromophores were derived, by analogy or through
interpolation, from those already present in the AMBER
database,45 and point charges that reproduced the molecular
electrostatic potential were calculated with MOPAC (ESP
keyword). Model complexes of the intercalators sandwiched
at a d(CG)2 and a d(TG)2 dinucleotide step in all different
orientations were built and simulated as described before.20
Solvent polarization effects were implicitly included by use of
a generalized Born model.46
Gen er a l P r oced u r e for P r ep a r a tion of 7. To a suspen-
sion of the phosphonium salt (1 mmol) in dry THF (5 mL) at
-78 °C in an atmosphere of dry argon was added n-BuLi (1
mmol, solution 1.6 M in hexanes), and the mixture was stirred
for 1 h at the same temperature. A solution of 6 (X ) Br, 1
mmol) in dry THF (5 mL) was added dropwise via cannula
and the mixture was stirred for 2 h more. The reaction was
(Z/E)-2-Met h yl-6-[3-(2-st yr ylp h en yl)p r op yl]p yr id in e
(8b): A 1:1 mixture of alkenes; colorless oil (62%); IR (neat)
ν
max 3024, 1602, 1514, 1492. cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.65-6.83 (m, 26H), 6.70 (d, 1H, J ) 8.0 Hz), 6.57 (d, 1H, J
) 10 Hz), 2.92-2.65 (m, 8H), 2.50 (s, 6H), 2.15-1.95 (m, 4H)
ppm; 13C NMR (75 MHz, CDCl3) δ 164.3, 164.0, 161.6, 149.0,
148.9, 147.1, 144.5, 140.2, 140.0, 137.6, 137.1, 136.9, 136.8,

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1143
136.6, 135.5, 130.5, 130.0, 129.7, 129.9, 128.9, 128.6, 128.0,
127.5, 127.4, 127.3, 126.9, 126.5, 126.2, 125.8, 125.6, 123.7,
123.5, 122.0, 121.9, 38.0, 37.8, 33.3, 32.9, 31.0, 30.5, 20.9 ppm;
MS (EI, 70 eV) m/z (relative intensity) 313 (M+, 27), 222 (100),
194 (24); HRMS (EI) calcd for C23H23N (M+) 313.1824, found
313.1830.
6H), 7.28-7.04 (m, 20H), 6.72 (d, 1H, J ) 12.1 Hz), 6.57 (d,
1H, J ) 12.1 Hz), 3.03 (m, 4H), 2.82 (t, 2H, J ) 7.6 Hz), 2.78
(t, 2H, J ) 7.69 Hz), 2.52 (s, 6H), 2.19-2.09 (m, 4H) ppm; 13
C
NMR (75 MHz, CDCl3) δ 164.0, 159.5, 147.8, 146.6, 146.4,
145.5, 145.4, 143.9, 143.8, 142.7, 140.3, 140.1, 139.8, 139.6,
136.8, 136.6, 135.4, 135.3, 131.4, 130.5, 129.4, 129.3, 128.9,
128.4, 127.9, 127.3, 126.9, 126.6, 126.2, 125.8, 121.1, 38.8, 33.3,
30.5, 21.3, 14.1 ppm; MS (EI, 70 eV) m/z (relative intensity)
363 (M+, 17), 157 (100), 91 (10), 78 (7); HRMS (EI) calcd for
(Z/E)-4-Meth yl-2-[3-(2-styr ylph en yl)pr opyl]pyr idin e (8c).
Following the general procedure, from 2,4-lutidine was ob-
tained a mixture of regioisomers that were separated by flash
chromatography (37%): a 1:1 mixture of alkenes; IR (neat) νma´x
C
27H25N (M+) 363.1980, found 363.1982.
3024, 1602, 1514, 1492 cm-1
;
1H NMR (300 MHz, CDCl3) δ
(Z/E)-2-(3-{2-[2-(4-Eth oxyp h en yl)vin yl]p h en yl}p r op yl)-
8.38 (d, 1H, J ) 6.2 Hz), 8.35 (d, 1H, J ) 6.6 Hz), 7.60 (m 1H),
7.46 (d, 2H, J ) 8.0 Hz), 7.38-6.90 (m, 21H), 6.68 (d, 1H, J )
12.1 Hz), 6.56 (d, 1H, J ) 12.1 Hz), 2.85-2.76 (m, 6H), 2.69
qu in olin e (12c): A 1:1 mixture of alkenes; colorless oil (88%);
IR (neat) νmax 3058, 1606, 1510, 1476, cm-1 1H NMR (300
;
MHz, CDCl3) δ 8.03 (dd, 4H, J ) 8.4, 5.1 Hz), 7.75 (dd, 2H, J
) 8.4, 1.5 Hz), 7.67 (ddd, 2H, J ) 8.4, 6.6, 1.5 Hz), 7.47 (ddd,
2H, J ) 8.1, 1.5 Hz), 7.25-7.16 (m, 10H), 7.10-6.98 (m 6H),
6.70-6.63 (m, 4H), 6.57 (d, 1H, J ) 12.4 Hz), 6.47 (d, 1H, J )
12.4 Hz), 3.94 (q, 2H, J ) 6.9 Hz), 3.91 (q, 2H, J ) 6.9 Hz),
3.01 (m, 4H), 2.82 (t, 2H, J ) 7.6 Hz), 2.74 (t, 2H, J ) 7.6 Hz),
2.16-2.06 (m, 4H), 1.35 (t, 3H, J ) 6.9 Hz), 1.31 (t, 3H, J )
6.9 Hz) ppm; 13C NMR (75 MHz, CDCl3) δ 162.4, 157.9, 156.9,
148.8, 148.7, 148.3, 147.8, 146.9, 146.5, 145.4, 144.3, 140.1,
137.1, 136.1, 131.9, 130.8, 130.1, 130.0, 129.7, 129.5, 129.3,
129.2, 128.8, 127.4, 127.2, 127.1, 126.7, 125.9, 125.6, 121.2,
113.9, 63.2, 59.9, 39.0, 33.4, 30.5, 30.1, 14.7 ppm; MS (EI, 70
eV) m/z (relative intensity) 393 (M+, 10), 143 (100), 115 (20),
77 (10); HRMS (EI) calcd for C28H27NO (M+) 393.2085, found
393.2090.
(t, 2H, J ) 7.32 Hz), 2.27 (s, 6H), 2.11-1.96 (m, 4H) ppm; 13
C
NMR (75 MHz, CDCl3) δ 164.3, 164.0, 161.6, 149.0, 148.9,
147.1, 144.5, 140.2, 140.0, 137.6, 137.1, 136.9, 136.8, 136.6,
135.5, 130.5, 130.0, 129.7, 129.9, 128.9, 128.6, 128.0, 127.5,
127.4, 127.3, 126.9, 126.5, 126.2, 125.8, 125.6, 123.7, 123.5,
122.0, 121.9, 38.0, 37.8, 33.3, 32.9, 31.0, 30.5, 20.9 ppm; MS
(EI, 70 eV) m/z (relative intensity) 313 (M+,27), 222 (100), 120
(98), 91 (60); HRMS (EI) calcd for C23H23N (M+) 313.1824,
found 313.1831.
(Z/E)-2-(3-{2-[2-(4-Eth oxyp h en yl)vin yl]p h en yl}p r op yl)-
p yr id in e (8d ): A 1:1 mixture of alkenes; colorless oil ( 97%);
IR (neat) νmax 3009, 1606, 1590, 1510, 1254 cm-1; 1H NMR (300
MHz, CDCl3) δ 8.51 (dd, 2H, J ) 5.5, 1.8 Hz), 8.53 (ddd, 1H,
J ) 7.6, 1.8 Hz), 7.25-7.15 (m, 8H), 7.09-6.99 (m, 9H), 6.69-
6.64 (m, 6H), 6.54 (d, 1H, J ) 12.1 Hz), 6.50 (d, 1H, J ) 12.1
Hz), 3.97 (q, 2H, J ) 6.9 Hz), 3.95 (q, 2, J ) 6.9 Hz), 2.83 (m,
4H), 2.70 (t, 2H, J ) 7.7 Hz), 2.08-1.97 (m, 4H), 1.36 (t, 3H,
J ) 6.96 Hz), 1.30 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz,
CDCl3) δ 163.9, 161.9, 157.9, 157.6, 155.5, 149.1, 141.3, 141.2,
140.1, 137.0, 136.1, 130.9, 130.1, 129.9,129.7, 129.3, 128.8,
128.7, 127.5, 127.2, 127.1, 126.3, 125.8, 122.6, 120.8, 113.9,
63.2, 61.2, 38.1, 33.2, 30.3, 30.4, 22.0, 14.7 ppm; MS (EI, 70
eV) m/z (relative intensity) 343 (M+, 5), 93 (100), 78 (10);
HRMS (EI) calcd for C24H25NO (M+) 343.1929, found 343.1934.
(Z/E)-2-(3-{2-[2-(4-Eth oxyp h en yl)vin yl]p h en yl}p r op yl)-
6-m eth ylqu in olin e (12d ): A 1:1 mixture of alkenes; colorless
oil (74%); IR (neat) νmax 3060, 1604, 1510, 1476, 1448 cm-1
;
1H NMR (75 MHz, CDCl3) δ 7.92 (d, 4H, J ) 8.4 Hz), 7.52-
7.48 (m, 4H), 7.25-7.16 (m, 8H), 7.08-6.99 (m, 8H), 6.6-6.64
(m, 4H), 6.56 (d, 1H, J ) 12.1 Hz), 6.48 (d, 1H, J ) 12.1 Hz),
3.96 (q, 2H, J ) 6.9 Hz), 3.90 (q, 2H, J ) 6.9 Hz), 2.99 (m,
4H), 2.82 (t, 2H, J ) 7.6), 2.74 (t, 2H, J ) 7.6 Hz), 2.51 (s,
6H), 2.17-2.07 (m, 4H), 1.35 (t, 3H, J ) 6.9 Hz), 1.30 (t, 3H,
J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CDCl3) δ 161.4, 157.9,
157.8, 148.9, 148.1, 147.3, 146.9, 146.3,141.2, 140.1, 137.0,
135.5, 135.3, 131.5, 130.1, 130.0, 129.8, 129.7, 129.6, 129.3,
128.4, 127.6, 127.2, 127.1, 126.7, 126.3, 125.9, 121.2, 114.6,
113.9, 63.2, 59.8, 38.8, 33.4, 30.5, 30.2, 21.4, 14.7 ppm; MS
(EI, 70 eV) m/z (relative intensity) 407 (M+, 10), 170 (50), 157
(100), 107 (25); HRMS (EI) calcd for C29H29NO (M+) 407.2241,
found 407.2264.
(Z/E)-2-(3-{2-[2-(4-Eth oxyp h en yl)vin yl]p h en yl}p r op yl)-
6-m eth ylp yr id in e (8e): A 1:1 mixture of alkenes; colorless
oil (98%); IR (neat) νmax 3061, 1606, 1591, 1511, 1476, 1258
1
cm-1; H NMR (300 MHz, CDCl3) δ 7.42 (t, 2H, J ) 7.6 Hz),
7.26-7.16 (m, 4H), 7.09-7.01 (m 4H), 6.93 (d, 2H, J ) 7.6 Hz),
6.88 (d, 2H, J ) 7.6 Hz), 6.69-6.65 (m, 10H), 6.58 (d, 1H, J )
12.4 Hz), 6.51 (d, 1H, J ) 12.4 Hz), 3.93 (q, 2H, J ) 6.9 Hz),
3.90 (q, 2H, J ) 6.9 Hz), 2.82 (m, 4H), 2.75 (t, 2H, J ) 7.6
Hz), 2.72 (t, 2H, J ) 7.6 Hz), 2.53 (s, 6H), 2.08-1.97 (m, 4H),
1.36 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CDCl3) δ
161.1, 157.8, 157.4, 145.5, 145.0, 144.9, 144.8, 147.1, 140.1,
140.0, 136.9, 136.2, 135.5, 135.4, 134.2, 131.9, 130.0, 129.9,
129.8, 129.2, 127.1, 127.0, 125.7, 120.2, 119.3, 113.9, 63.0, 61.1,
38.1, 33.2, 30.9, 30.6, 24.3, 14.6 ppm; MS (EI, 70 eV) m/z
(relative intensity) 357 (M+, 10), 107 (100), 92 (99); HRMS (EI)
calcd for C25H27NO (M+) 357.2085, found 357.2097.
Gen er a l P r oced u r e for P r ep a r a tion of Dik eton es 9
a n d 13. To a solution of 8 or 12 (1 mmol) in DMSO (5 mL)
was added HBr (1.2 mmol) and the mixture was heated for 12
h at 100 °C. Then, a NaHCO3 solution was added until the
pH was basic, the mixture was extracted with CH2Cl2 and
dried (MgSO4), and the solvent was evaporated under reduced
pressure. The crude products were purified by flash chroma-
tography [silica gel, hexane:ethyl acetate (8:2)], yielding a pale
yellow oil.
(Z/E)-2-[3-(2-Styr ylp h en yl)p r op yl]qu in olin e (12a ):
1:1 mixture of alkenes; colorless oil (75%); IR (neat) νmax 3060,
1620, 1600, 1510, 694 cm-1 1H NMR (300 MHz, CDCl3) δ
A
1-P h en yl-2-[2-(3-p yr id in -2-yl-p r op yl)p h en yl]et h a n e-
1,2-d ion e (9a ): 77%; IR (neat) νmax 3065, 1719, 1678, 1596,
1
;
1448 cm-1; H NMR (300 MHz, CDCl3) δ 8.52 (d, 1H, J ) 5.1
8.08-8.01 (m, 2H), 7.77 (dd, 4H, J ) 8.0, 2.5 Hz), 7.69 (tq,
2H, J ) 8.8, 6.9, 1.8 Hz), 7.62-7.59 (m, 2H), 7.52-7.43 (m,
3H), 7.40 (dd, 2H, J ) 8.4, 1.5), 7.34-7.20 (m, 9H), 7.17-6.96
(m, 8H), 6.70 (d, 1H, J ) 12.4 Hz), 6.56 (d, 1H, J ) 12.4 Hz),
3.11-3.01 (m, 4H), 2.89 (t, 2H, J ) 7.6 Hz), 2.77 (t, 2H, J )
7.6 Hz), 2,20-2.11 (m, 4H) ppm; 13C NMR (75 MHz, CDCl3) δ
162.3, 162.2, 147.8, 140.1, 139.9, 137.6, 136.8, 136.7, 136.2,
136.1, 135.9, 130.5, 130.2, 129.8, 129.3, 129.0, 128.9, 128.8,
128.5, 128.0, 127.6, 127.4, 127.3, 126.9, 126.7, 126.5, 126.3,
126.2, 125.9, 125.7, 121.3, 38.9, 38.7, 33.4, 33.0, 31.0, 30.5 ppm;
MS (EI, 70 eV) m/z (relative intensity) 349 (M+, 27), 143 (100),
91 (30), 77 (20); HRMS (EI) calcd for C26H23N (M+) 349.1824,
found 349.1831.
Hz), 7.96 (d, 2H, J ) 7.7 Hz), 7.67-7.47 (m, 6H), 7.37 (d, 1H,
J ) 7.7 Hz), 7.26 (t, 1H, J ) 7.7 Hz), 7.18 (d, 1H, J ) 7.7 Hz),
7.09 (dd, 1H, J ) 6.6, 5.1 Hz), 3.13 (t, 2H, J ) 7.7 Hz), 2.91 (t,
2H, J ) 7.7 Hz), 2.17-2.07 (m, 2H) ppm; 13C NMR (75 MHz,
CDCl3) δ 196.5, 194.4, 161.5, 148.7, 145.2, 136.3, 134.4, 133.5,
133.0, 131.7, 130.0, 129.9, 129.7, 128.8, 126.0, 122.7, 120.9,
37.8, 37.6, 31.1 ppm; MS (EI, 70 eV) m/z (relative intensity)
329 (M+, 100), 242 (30), 224 (20), 123 (60); HRMS (EI) calcd
for C22H19NO2 (M+) 329.1410, found 329.1434.
1-{2-[3-(6-Meth ylp yr id in -2-yl)p r op yl]p h en yl}2-p h en yl-
eth a n e-1,2-d ion e (9b): 71%; IR (neat) νmax 3030, 1762, 1720,
1598, 1450 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.98-7.95 (m,
2H), 7.65 (tt, 1H, J ) 6.6, 1.1 Hz), 7.60 (dd, 1H, J ) 7.7, 1.5
Hz), 7.54-7.44 (m, 4H), 7.38 (dd, 1H, J ) 7.7, 1.1 Hz), 7.25
(ddd, 1H, J ) 7.7, 1.5 Hz), 6.96 (t, 2H, J ) 8.1 Hz), 3.14 (t,
2H, J ) 7.7 Hz), 2.87 (t, 2H, J ) 7.7 Hz), 2.52 (s, 3H), 2.14-
2.03 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3) δ 196.6, 194.6,
(Z/E)-6-Met h yl-2-[3-(2-st yr ylp h en yl)p r op yl]q u in olin e
(12b): A 1:1 mixture of alkenes; colorless oil (80%); IR (neat)
1
νmax 3058, 3022, 2943, 1599, 1495, 1481 cm-1; H NMR (300
MHz, CDCl3) δ 7.95 (dd, 4H, J ) 9.1, 6.9 Hz), 7.53-7.50 (m,

1144 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mart´ınez et al.
164.0, 161.1, 157.6, 145.5, 136.4, 134.6, 133.7, 133.2, 131.8,
131.6, 129.9, 128.9, 126.1, 120.4, 119.9, 38.3, 34.0, 31.4, 24.5
ppm; MS (EI) m/z (rel int) 343 (M+, 2), 121 (100), 107 (99), 77
(30); HRMS (EI) calcd for C23H21NO2 (M+) 343.1575, found
343.1584.
1-(4-Eth oxyph en yl)-2-[2-(3-qu in olin -2-ylpr opyl)ph en yl]-
eth a n e-1,2-d ion e (13c): 41%; IR (neat) νmax 3060, 1734, 1658,
1
1506, 1428 cm-1; H NMR (300 MHz, CDCl3) δ 8.02 (d, 1H, J
) 8.4 Hz), 7.87 (d, 1H, J ) 2.2 Hz), 7.80 (dd, 1H, J ) 8.8, 2.2
Hz), 7.75-7.71 (m, 1H), 7.67-7.59 (m, 2H), 7.48-7.41 (m, 3H),
7.36 (d, 1H, J ) 7.7 Hz), 7.31 (d, 1H, J ) 8.4 Hz), 7.25-7,19
(m, 2H), 6.84 (d, 1H, J ) 8.8 Hz), 4.09 (q, 2H, J ) 6.9 Hz),
3.18 (t, 2H, J ) 7.7 Hz), 3.08 (t, 2H, J ) 7.7 Hz), 2.21-2.12
(m, 2H), 1.42 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz,
CDCl3) δ 197.0, 193.1, 162.2, 161.9, 147.8, 145.0, 136.0, 133.4,
133.0, 131.7, 131.6, 131.4, 129.1, 128.6, 128.4, 127.9, 127.3,
126.6, 125.5, 121.5, 111.0, 64.2, 38.9, 33.9, 31.1, 14.4 ppm; MS
(EI, 70 eV) m/z (relative intensity) 423 (M+, 2), 274 (100), 156
(30); HRMS (EI) calcd for C28H25NO3 (M+) 423.1827, found
423.1844.
1-{2-[3-(4-Meth ylp yr id in -2-yl)p r op yl]p h en yl}2-p h en yl-
eth a n e-1,2-d ion e (9c): 43%; IR (neat) νmax 3060, 1718, 1606,
1
1444 cm-1; H NMR (300 MHz, CDCl3) δ 8.37 (d, 1H, J ) 8.1
Hz), 7.98-7.95 (m, 2H), 7.65 (tt, 1H, J ) 6.6, 1.1 Hz), 7.69
(dd, 1H, J ) 7.7, 1.5 Hz), 7.54-7.44 (m, 3H), 7.38 (dd, 1H, J
) 7.7, 1.1 Hz), 7.25 (ddd, 1H, J ) 7.7, 1.5 Hz), 7.00 (s, 1H),
6.91 (d, 1H, J ) 8.1 Hz), 3.13 (t, 2H, J ) 7.7 Hz), 2.86 (t, 2H,
J ) 7.7 Hz), 2.30 (s, 3H), 2.15-2.05 (m, 2H) ppm; 13C NMR
(75 MHz, CDCl3) δ 195.1, 193.0, 160.1, 154.6, 147.5, 135.4,
127.9, 127.8, 127.7, 127.1, 125.5, 124.8, 124.7, 124.5, 123.9,
122.4, 35.0, 33.7, 30.8, 20.9 ppm; MS (EI, 70 eV) m/z (relative
intensity) 343 (M+, 2), 121 (100), 107 (99), 77 (39); HRMS (EI)
calcd for C23H21NO2 (M+) 343.1575, found 343.1572.
1-(4-E t h oxyp h e n yl)-2-{2-[3-(6-m e t h ylq u in olin -2-yl)-
p r op yl]p h en yl}eth a n e-1,2-d ion e (13d ): 41%; IR (neat) νmax
3020, 1702, 1662, 1598 cm-1 1H NMR (300 MHz, CDCl3) δ
;
7.97 (d, 2H, J ) 8.4 Hz), 7.92 (d, 2H, J ) 8.8 Hz), 7.61 (d, 1H,
J ) 7.7 Hz), 7.53-7.45 (m, 3H), 7.37 (d, 1H, J ) 6.6 Hz), 7.30
(d, 1H, J ) 8.4 Hz), 7.25 (ddd, 1H, J ) 7.7, 1.5 Hz), 6.94 (d,
2H, J ) 8.8 Hz), 4.10 (q, 2H, J ) 6.9 Hz), 3.17 (t, 2H, J ) 7.7
Hz), 3.06 (t, 2H, J ) 7.7 Hz), 2.51 (s, 3H), 2.22-2.17 (m, 2H),
1.44 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CDCl3) δ
197.1, 193.3, 164.2, 161.4, 146.4, 145.3, 135.5, 135.3, 133.5,
133.2, 132.3, 131.9, 131.8, 131.5, 128.5, 126.4, 126.1, 126.0,
121.4, 114.7, 63.9, 39.0, 34.1, 31.3, 14.5 ppm; MS (EI, 70 eV)
m/z (relative intensity) 437 (M+, 2), 288 (100), 149 (30), 121
(20), 93 (72); HRMS (EI) calcd for C29H27NO3 (M+) 437.1977,
found 437.1991.
Gen er a l P r oced u r e for P r ep a r a tion of Sa lts 10 a n d 14.
A solution of the dicarbonyl compound 9 or 13 (1 mmol) in
CH2Cl2 (5 mL) was added dropwise to a solution of (O-
mesitylensulfonyl)hydroxylamine (MSH, 1.5 mmol) in dichlo-
romethane (5 mL) and the mixture was stirred at room
temperature for 1 h. Diethyl ether was added (25 mL) and the
precipitated solid was filtrated and recrystallized from absolute
ethanol.
1-Am in o-2-{3-[2-(2-oxo-2-p h en yla cetyl)p h en yl]p r op yl}-
p yr id in iu m m esitylen esu lfon a te (10a ): yellow solid (94%);
mp 100-102 °C; IR (KBr) νmax 3140, 2938, 2917, 1660, 1598,
1450, 1204 cm-1; 1H NMR (300 MHz, CD3OD) δ 8.74 (d, 1H, J
) 7.3 Hz), 8.23 (t, 1H, J ) 8.4 Hz), 7.98 (d, 1H, J ) 6.6 Hz),
7.92 (d, 2H, J ) 8.4 Hz), 7.80 (t, 1H, J ) 7.7 Hz), 7.72 (t, 1H,
J ) 7.7 Hz), 7.63-7.50 (m, 5H), 7.35 (t, 1H, J ) 8.1 Hz), 6.85
(s, 2H), 3.29 (t, 2H, J ) 7.7 Hz), 3.20 (t, 2H, J ) 7.7 Hz), 2.60
(s, 6H), 2.22 (s, 3H), 2.20-2.14 (m, 2H) ppm; 13C NMR (75
MHz, CD3OD) δ 195.5, 195.0, 164.3, 163.9, 163.2, 145.5, 142.9,
142.5, 140.1, 138.2, 136.2, 135.5, 134.7, 133.3, 131.6, 130.8,
130.3, 129.6, 127.9, 126.7, 34.5, 32.3, 29.1, 23.2, 20.8 ppm.
Anal. Calcd for C31H32N2O5S: C, 68.36; H, 5.92; N, 5.14.
Found: C, 68.07; H, 5.87; N, 5.05.
1-(4-Eth oxyp h en yl)-2-[2-(3-p yr id in -2-yl-p r op yl)p h en yl]-
eth a n e-1,2-d ion e (9d ): 61%; IR (neat) νmax 3050, 1672, 1652,
1
1596, 1570 cm-1; H NMR (300 MHz, CDCl3) δ 8.51 (d, 1H, J
) 5.1 Hz), 7.93 (d, 2H, J ) 8.8 Hz),7.61-7.55 (m, 2H), 7.47
(ddd, 1H, J ) 7.7, 1.5 Hz), 7.35 (d, 1H, J ) 7.7 Hz), 7.23 (ddd,
1H, J ) 7.7, 2.2 Hz), 7.17 (d, 1H, J ) 8.1 Hz), 7.09 (dd, 1H, J
) 7.7, 5.1 Hz), 6.95 (d, 2H, J ) 8.8 Hz), 4.11 (q, 2H, J ) 6.9
Hz), 3.12 (t, 2H, J ) 7.7 Hz), 2.90 (t, 2H, J ) 7.7 Hz), 2.16-
2.06 (m, 2H), 1.44 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz,
CDCl3) δ 197.0, 193.3, 164.2, 164.0, 161.8, 149.1, 145.3, 136.2,
133.5, 133.2, 132.3, 131.9, 131.7, 126.0, 122.8, 120.9, 114.7,
63.9, 38.2, 33.9, 31.2, 14.5 ppm; MS (EI, 70 eV) m/z (relative
intensity) 373 (M+, 2), 224 (100), 149 (99), 121 (40), 93 (32);
HRMS (EI) calcd for C24H23NO3 (M+) 373.1671, found 373.1684.
1-(4-Eth oxyph en yl)-2-{2-[3-(6-m eth ylpyr idin -2-yl)pr opyl]-
p h en yl}eth a n e-1,2-d ion e (9e): 61%; IR (neat) νmax 3030,
1
1666, 1595, 1454 cm-1; H NMR (300 MHz, CDCl3) δ 7.86 (d,
1H, J ) 2.2 Hz), 7.81 (dd, 1H, J ) 8.4, 2.2 Hz), 7.59 (dd, 1H,
J ) 8.1, 1.5 Hz), 7.49-7.42 (m, 3H), 7.34 (dd, 1H, J ) 6.2, 1.5
Hz), 7.22 (ddd, 1H, J ) 6.7, 1.1 Hz), 6.98-6.88 (m, 3H), 4.13
(q, 2H, J ) 6.9 Hz), 3.12 (t, 2H, J ) 7.7 Hz), 2.85 (t, 2H, J )
7.7 Hz), 2.57 (s, 3H), 2.12-2.02 (m, 2H), 1.45 (t, 3H, J ) 6.9
Hz) ppm; 13C NMR (75 MHz, CDCl3) δ 196.9, 193.2, 164.0,
163.9, 161.8, 161.1, 157.5, 145.3, 136.4, 133.4, 133.0, 131.7,
131.4, 127.9, 125.9, 125.4, 120.3, 119.5, 111.1, 64.2, 38.2, 33.9,
31.4, 14.3, 14.5 ppm; MS (EI, 70 eV) m/z (relative intensity)
387 (M+, 10), 238 (90), 209 (100), 133 (50), 107 (22), 77 (12);
HRMS (EI) calcd for C25H25NO3 (M+) 387.1827, found 387.1844.
1-P h en yl-2-[2-(3-q u in olin -2-ylp r op yl)p h en yl]et h a n e-
1,2-d ion e (13a ): 75%; IR (neat) νmax 3062, 1718, 1662 cm-1
;
1H NMR (300 MHz, CDCl3) δ 8.05 (t, 2H, J ) 8.2 Hz), 7.98-
7.95 (m, 2H), 7.77 (d, 1H, J ) 8.2), 7.70-7.59 (m, 3H), 7.52-
7.54 (m, 4H), 7.39 (d, 1H, J ) 6.8 Hz), 7.34 (d, 1H, J ) 8.4
Hz), 7.25 (t, 1H, J ) 7.3 Hz), 3.20 (t, 2H, J ) 7.7 Hz), 3.10 (t,
2H, J ) 7.7 Hz), 2.27-2.19 (m, 2H) ppm; 13C NMR (75 MHz,
CDCl3) δ 196.7, 194.6, 162.3, 145.3, 136.1, 134.6, 133.7, 133.2,
133.1, 131.9, 131.6, 130.1, 129.8, 129.2, 128.9, 128.8, 127.4,
126.7, 126.1, 125.6, 121.4, 39.0, 34.1, 31.2 ppm; MS (EI, 70
eV) m/z (relative intensity) 379 (M+, 3), 274 (100), 256 (10),
1-Am in o-2-m eth yl-6-{3-[2-(2-oxo-2-ph en ylacetyl)ph en yl]-
pr opyl}pyr idin iu m m esitylen esu lfon ate (10b): yellow solid
(83%); mp 102-104 °C; IR (KBr) νmax 3758, 3238, 3114, 2934,
1
1724, 1672, 1622, 1600 cm-1; H NMR (300 MHz, CD3OD) δ
8.10 (ddd, 1H, J ) 10.2, 7.7, 2.5 Hz), 8.00 (d, 1H, J ) 7.7 Hz),
7.93-7.89 (m, 2H), 7.77-7.69 (m, 3H), 7.62-7.24 (m, 5H), 6.83
(s, 2H), 3.20 (t, 2H, J ) 7.7 Hz), 2.89 (t, 2H, J ) 7.7 Hz), 2.81
143 (15), 128 (15), 105 (15), 77 (17); HRMS (EI) calcd for C26H21
-
NO2 (M+) 379.1566, found 379.1584.
(s, 3H), 2.57 (s, 6H), 2.20 (s, 3H), 2.19-2.09 (m, 2H) ppm; 13
C
NMR (75 MHz, CD3OD) δ 198.6, 196.3, 157.1, 145.5, 143.1,
140.1, 138.9, 136.2, 135.5, 134.7, 133.4, 132.5, 131.7, 130.7,
129.9, 129.4, 128.4, 127.9, 127.3, 127.2, 126.8, 34.5, 32.3, 29.4,
23.2, 20.8, 17.6 ppm. Anal. Calcd for C32H34N2O5S: C, 68.79;
H, 6.13; N, 5.81. Found: C, 68.93; H, 6.32; N, 5.89.
1-{2-[3-(6-Met h ylq u in olin -2-yl)p r op yl]p h en yl}2-p h e-
n yleth a n e-1,2-d ion e (13b): 46%; IR (neat) νmax 3060, 3024,
1
1718, 1672, 1446 cm-1; H NMR (300 MHz, CDCl3) δ 7.98-
7.92 (m, 4H), 7.64 (tt, 1H, J ) 6.2, 1.1 Hz), 7.61 (dd, 1H, J )
7.7, 1.1 Hz); 7.52-7.47 (m, 4H), 7.38 (d, 1H, J ) 6.6 Hz), 7.31
(s, 1H), 7.27 (d, 1H, J ) 7.7 Hz), 7.25 (ddd, 1H, J ) 7.7, 1.5
Hz), 3.19 (t, 2H, J ) 7.7 Hz), 3.08 (2H, J ) 7.7 Hz), 2.51 (s,
3H), 2.26-2.15 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3) δ
196.6, 194.6, 161.3, 146.4, 145.4, 135.6, 135.3, 134.7, 134.6,
133.2, 131.9, 131.6, 131.5, 130.0, 129.8, 128.9, 128.4, 126.7,
126.3, 126.1, 121.3, 38.9, 34.1, 31.2, 21.4 ppm; MS (EI, 70 eV)
m/z (relative intensity) 393 (M+, 2), 374 (5), 288 (100), 105 (22),
77 (19); HRMS (EI) calcd for C27H23NO2 (M+) 393.1722, found
393.1728.
1-Am in o-4-m eth yl-2-{3-[2-(2-oxo-2-ph en ylacetyl)ph en yl]-
pr opyl}pyr idin iu m m esitylen esu lfon ate (10c): yellow solid
(76%); mp 118-120 °C; IR (KBr) νmax 3082, 2974, 2934, 1664,
1
1634, 1598, 1450, 1208 cm-1; H NMR (300 MHz, CD3OD) δ
8.56 (d, 1H, J ) 6.9 Hz), 7.91 (t, 2H, J ) 7.7 Hz), 7.80 (s, 1H),
7.70 (t, 1H, J ) 8.0 Hz), 7.69-7.51 (m, 5H), 6.85 (s, 2H), 3.26
(t, 2H, J ) 7.7 Hz), 3.20 (t, 2H, J ) 7.7 Hz), 2.60 (s, 6H), 2.22
(s, 3H), 2.21-2.14 (m, 2H), 1.28 (s, 3H) ppm; 13C NMR (75
MHz, CD3OD) δ 198.6, 196.3, 157.1, 145.6, 143.1, 140.1, 138.2,

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1145
136.2, 135.5, 134.7, 133.4, 132.5, 131.7, 130.7, 129.9, 129.4,
128.4, 127.9, 127.3, 127.2, 126.8, 34.5, 32.3, 29.4, 23.2, 21.4,
20.8 ppm. Anal. Calcd for C32H34N2O5S: C, 68.79; H, 6.13; N,
5.01. Found: C, 68.60; H, 6.20; N, 5.28.
143.2, 140.0, 138.9, 138.3, 136.2, 134.4, 134.0, 133.6, 133.1,
133.0, 132.8, 131.5, 129.2, 129.1, 128.7, 126.1, 123.4, 117.6,
111.8, 63.9, 33.1, 32.9, 28.6, 21.5, 19.8, 19.0 ppm. Anal. Calcd
for C37H38N2O6S: C, 69.57; H, 6.00; N, 4.39. Found: C, 69.32;
H, 6.13; N, 4.57.
1-Am in o -2-(3-{2-[2-(4-e t h o x y p h e n yl)-2-o xo a c e t yl]-
p h en yl}p r op yl)p yr id in iu m m esitylen esu lfon a te (10d ):
yellow solid (68%); mp 108-110 °C; IR (KBr) νmax 3452, 3106,
1-Am in o-2-(3-{2-[2-(4-et h oxyp h en yl)-2-oxo-2-a cet yl]-
ph en yl}pr opyl)-6-m eth ylqu in olin iu m m esitylen esu lfon ate
(14d ): yellow solid (84%); mp 110-112 °C; IR (KBr) νmax 3424,
1
2980, 1664, 1600, 1568, 1264 cm-1; H NMR (300 MHz, CD3-
1
2984, 1654, 1600, 1512, 1266 cm-1; H NMR (300 MHz, CD3-
OD) δ 8.73 (d, 1H, J ) 5.5 Hz), 8.19 (t, 1H, J ) 8.0 Hz), 7.92
(d, 1H, J ) 6.6 Hz), 7.87 (d, 2H, J ) 8.8 Hz), 7.76 (t, 1H, J )
6.6 Hz), 7.61-7.54 (m, 2H), 7.48 (d, 1H, J ) 7.0 Hz), 7.33 (t,
1H, J ) 8.0 Hz), 7.04 (d, 2H, J ) 8.8 Hz), 6.83 (s, 2H), 4.13 (q,
2H, J ) 6.9 Hz), 3.25 (t, 2H, J ) 7.7 Hz), 3.16 (t, 2H, J ) 7.7
Hz), 2.59 (s, 6H), 2.20 (s, 3H), 2.19-2.10 (m, 2H), 1.40 (t, 3H,
J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CD3OD) δ 198.3, 192.2,
166.1, 163.9, 142.9, 142.5, 140.8, 140.1, 138.1, 135.3, 134.5,
133.4, 133.2, 131.7, 129.6, 127.8, 126.9, 126.7, 123.2, 116.1,
115.7, 65.3, 34.4, 32.3, 29.1, 23.2, 20.8, 14.8 ppm. Anal. Calcd
for C33H36N2O3S: C, 67.33; H, 6.16; N, 4.76. Found: C, 67.51;
H, 6.27; N, 4.82.
OD) δ 8.77 (d, 1H, J ) 8.7 Hz), 8.52 (d, 1H, J ) 8.7 Hz), 8.01-
7.98 (m, 2H), 7.92 (d, 1H, J ) 8.4 Hz), 7.84 (d, 2H, J ) 9.1
Hz), 7.59-7.48 (m, 3H), 7.31 (t, 1H, J ) 7.3 Hz), 7.02 (d, 2H,
J ) 9.1 Hz), 6.79 (s, 2H), 4.13 (q, 2H, J ) 6.9 Hz), 3.55 (t, 2H,
J ) 7.7 Hz), 3.21 (t, 2H, J ) 7.7 Hz), 2.60 (s, 3H), 2.55 (s, 6H),
2.30-2.18 (m, 2H), 2.19 (s, 3H), 1.40 (t, 3H, J ) 6.9 Hz) ppm;
13C NMR (75 MHz, CD3OD) 196.6, 195.0, 164.0, 163.9, 163.3,
145.3, 144.8, 141.8, 140.0, 138.3, 138.1, 135.2, 134.5, 133.4,
133.2, 131.6, 130.4, 129.7, 127.9, 124.6, 119.1, 116.0, 63.2, 34.7,
30.5, 24.0, 23.2, 21.1, 20.8, 14.8 ppm. Anal. Calcd for
C
38H40N2O6S: C, 69.92; H, 6.18; N, 4.29. Found: C, 69.80; H,
6.13; N, 4.50.
1-Am in o-2-(3-{2-[2-(4-e t h o x y p h e n y l)-2-ox o a c e t yl]-
ph en yl}pr opyl)-6-m eth ylpyr idin iu m m esitylen esu lfon ate
(10e): yellow solid (92%); mp 99-101 °C; IR (KBr) νmax 3189,
Gen er a l P r oced u r e for P r ep a r a tion of Ben zo[f]p yr id o-
[2,1-a ]p h th a la zin iu m Mesitylen esu lfon a tes 3 a n d Ben zo-
[f]qu in o[2,1-a ]p h th a la zin iu m Mesitylen esu lfon a tes 4.
Sodium acetate (method A) or Et3N (method B) (1.2 mmol) was
added to a suspension of the pyridinium (10) or quinolinium
(14) salts (1 mmol) in acetone (method A) or EtOH (method
B) (20 mL) and the mixture was heated at reflux for 6 h. Then,
the solvent was concentrated under vacuum, and the solid
residue was washed with EtOAc and Et2O and crystallized
from EtOH/Et2O.
1
2102, 1664, 1597, 1568, 1191 cm-1; H NMR (300 MHz, CD3-
OD) δ 8.12 (t, 1H, J ) 8.1 Hz), 7.96-7.88 (m, 3H), 7.78-7.72
(m, 2H), 7.62-7.55 (m, 2H), 7.48 (d, 1H, J ) 8.8 Hz), 7.33 (t,
1H, J ) 7.7 Hz), 7.19 (d, 1H, J ) 8.8 Hz), 6.84 (s, 2H), 4.22 (q,
2H, J ) 6.9 Hz), 3.17-3.08 (m, 4H), 2.59 (s, 6H), 2.21 (s, 3H),
2.15-2.10 (m, 2H), 1.44 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75
MHz, CD3OD) δ 195.4, 193.5, 171.1, 168.1, 166.2, 163.3, 163.1,
162.7, 162.2, 161.2, 159.7, 156.0, 155.8, 155.4, 155.3, 154.8,
154.4, 154.3, 153.4, 141.1, 138.3, 63.2, 34.8, 30.5, 24.1, 23.3,
21.1, 20.8, 14.8 ppm. Anal. Calcd for C34H38N2O6S: C, 67.75;
H, 6.35; N, 4.65. Found: C, 67.32; H, 6.34; N, 4.44.
5-P h en ylben zo[f]p yr id o[2,1-a ]p h th a la zin -7-iu m m esi-
tylen esu lfon a te (3a ): method A; yellow solid (65%); mp 199-
201 °C; IR (KBr) νmax 3419, 2946, 1602, 1563, 1451, 1203 cm-1
;
1H NMR (300 MHz, CD3OD) δ 9.59 (d, 1H, J ) 6.9 Hz), 9.51
(d, 1H, J ) 9.5 Hz), 9.04 (d, 1H, J ) 9.1 Hz), 8.78 (t, 2H, J )
9.1 Hz), 8.30 (t, 1H, J ) 7.3 Hz), 8.26 (d, 1H, J ) 9.1 Hz),
7.85-7.63 (m, 7H), 7.44 (t, 1H, J ) 6.9 Hz), 6.79 (s, 2H), 2.60
(s, 6H), 2.23 (s, 3H) ppm; 13C NMR (75 MHz, CD3OD) δ 142.9,
142.4, 140.8, 140.3, 140.1, 139.0, 138.9, 138.3, 138.2, 137.6,
132.0, 131.7, 131.4, 130.7, 130.6, 130.1, 129.4, 129.3, 129.0,
126.9, 124.6, 120.9, 23.3, 20.8 ppm. Anal. Calcd for
1-Am in o-2-{3-[2-(2-oxo-2-p h en yla cetyl)p h en yl]p r op yl}-
qu in olin iu m m esitylen esu lfon a te (14a ): yellow solid (92%);
mp 97-99 °C; IR (KBr) νmax 3428, 3064, 1672, 1602, 1248 cm-1
;
1H NMR (75 MHz, CD3OD) δ 9.33 (d, 1H, J ) 8.8 Hz), 8.91 (d,
1H, J ) 9.5 Hz), 8.59 (d, 1H, J ) 9.5 Hz), 8.38 (dd, 1H, J )
8.1, 1.5 Hz), 8.24 (ddd, 1H, J ) 8.8, 7.0, 1.5 Hz), 8.09 (t, 1H,
J ) 8.1 Hz), 7.87-7.80 (m, 2H), 7.67-7.54 (m, 3H), 7.49 (d,
1H, J ) 7.0 Hz), 7.41 (ddd, 1H, J ) 8.8, 7.0, 1.5 Hz), 7.09-
7.02 (m 2H), 6.77 (s, 2H), 3.46 (t, 2H, J ) 7.7 Hz), 3.24 (t, 2H,
J ) 7.7 Hz), 2.54 (s, 6H), 2.18 (s, 3H), 2.17-2.09 (m, 2H) ppm;
13C NMR (75 MHz, CD3OD) δ 195.5, 194.6, 140.9, 139.9, 138.1,
137.1, 137.0, 135.5, 132.9, 132.3, 132.1, 131.6, 130.9, 130.8,
130.7, 130.5, 130.2, 129.8, 129.5, 129.4, 127.6, 120.4, 119.2,
27.6, 25.2, 24.2, 23.2, 20.8 ppm. Anal. Calcd for C35H34N2O5S:
C, 70.69; H, 5.76; N, 4.71. Found: C, 70.72; H, 5.61; N, 4.51.
C
31H26N2O3S: C, 73.49; H, 5.17; N, 5.53. Found: C, 73.20; H,
4.98; N, 5.78.
5-P h en yl-8-m eth ylben zo[f]p yr id o[2,1-a ]p h th a la zin -7-
iu m m esitylen esu lfon a te (3b): method B; yellow solid (50%);
mp 221-223 °C; IR (KBr) νmax 2930; 1625, 1471, 1369, 1190,
1015 cm-1; 1H NMR (300 MHz, CD3OD) δ 9.39 (d, 1H, J ) 9.3
Hz), 9.00 (d, 1H, J ) 8.7 Hz), 8.73 (d, 1H, J ) 8.7 Hz), 8.63 (t,
1H, J ) 8.1 Hz), 8.24-8.28 (m, 2H), 7.84-7.65 (m, 7H), 7.44
(t, 1H, J ) 7.7 Hz), 6.79 (s, 2H), 3.17 (s, 3H), 2.54 (s, 6H), 2.18
(s, 3H) ppm; 13C NMR (75 MHz, CD3OD) δ 143.1, 141.4, 140.8,
140.2, 140.0, 138.7, 138.2, 132.1, 131.6, 131.2, 130.9, 130.6,
130.3, 130.1, 129.5, 129.4, 129.1, 128.4, 128.0, 127.9, 127.7,
127.5, 127.4, 127.3, 23.2, 20.8, 18.3 ppm. Anal. Calcd for
1-Am in o-6-m eth yl-2-{3-[2-(2-oxo-2-ph en ylacetyl]ph en yl]-
p r op yl}qu in olin iu m m esitylen esu lfon a te (14b): yellow
solid (98%); mp 86-88 °C; IR (KBr) νmax 3252, 1679, 1674,
1600, 1450, 1210 cm-1 1H NMR (300 MHz, CD3OD) δ 8.73
;
(dd, 1H, J ) 8.8, 5.1 Hz), 8.51 (d, 1H, J ) 9.1 Hz), 7.99-7.83
(m, 5H), 7.70 (t, 1H, J ) 7.3 Hz), 7.61-7.24 (m, 6H), 6.77 (s,
2H), 3.22 (t, 2H, J ) 7.7 Hz), 2.96 (t, 2H, J ) 7.7 Hz), 2.60 (s,
3H), 2.54 (s, 6H), 2.23-2.20 (m, 2H), 2.18 (s, 3H) ppm; 13C
NMR (75 MHz, CD3OD) δ 197.5, 194.3, 144.8, 141.8, 140.8,
140.1, 138.3, 138:2, 138.1, 133.3, 132.8, 131.8, 131.6, 131.2,
130.8, 130.5, 130.4, 129.9, 129.5, 127.6, 127.4, 124.5, 120.2,
119.1, 110.3, 34.6, 33.9, 30.7, 23.2, 20.8, 18.3 ppm. Anal. Calcd
for C36H36N2O5S: C, 71.03; H, 5.96; N, 4.60. Found: C, 70.95;
H, 5.98; N, 4.59.
C
32H28N2O3S: C, 73.82; H, 5.42; N, 5.38. Found: C, 74.11; H,
5.19; N, 5.40.
5-P h en yl-10-m eth ylben zo[f]p yr id o[2,1-a ]p h th a la zin -7-
iu m m esitylen esu lfon a te (3c): method B; yellow solid (28%);
mp 222-224 °C; IR (KBr) νmax 3028, 1632, 1405, 1194 cm-1
;
1H NMR (300 MHz, CD3OD) δ 9.33 (d, 1H, J ) 6.6 Hz), 8.61
(s, 1H), 8.06 (d, 1H, J ) 6.6 Hz), 7.80-7.32 (m, 9H), 7.00-
6.91 (m, 2H), 6.83 (s, 2H), 2.82 (s, 3H), 2.59 (s, 6H), 2.21 (s,
3H) ppm; 13C NMR (75 MHz, CD3OD) δ 143.7, 142.4, 140.6,
134.6, 134.4, 134.3, 134.0, 133.7, 133.6, 133.5, 133.1, 133.0,
132.7, 132.4, 132.3, 132.2, 132.1, 131.8, 131.7, 130.8, 129.8,
126.4, 27.2, 25.6, 23.2 ppm. Anal. Calcd for C32H28N2O3S: C,
73.82; H, 5.42; N, 5.38. Found: C, 73.98; H, 5.45; N, 5.23.
1-Am in o-2-(3-{2-[2-(4-et h oxyp h en yl)-2-oxo-2-a cet yl]-
p h en yl}p r op yl)qu in olin iu m m esitylen esu lfon a te (14c):
yellow solid (75%); mp 106-108 °C; IR (KBr) νmax 3156, 1663,
1599, 1446, 1189 cm-1; 1H NMR (300 MHz, CD3OD) δ 8.82 (d,
1H, J ) 8.4 Hz), 8.70 (d, 1H, J ) 8.4 Hz), 8.23 (t, 2H, J ) 7.7
Hz), 8.14 (t, 1H, J ) 7.3 Hz), 8.05-7.86 (m, 5H), 7.60-7.56
(m, 1H), 7.51 (t, 1H, J ) 5.5 Hz), 7.32 (t, 1H, J ) 7.0 Hz), 7.23
(d, 1H, J ) 8.0 Hz), 6.79 (s, 2H), 4.25 (q, 2H, J ) 6.9 Hz), 3.55
(t, 2H, J ) 7.7 Hz), 3.21 (t, 2H, J ) 7.7 Hz), 2.55 (s, 6H), 2.19
(s, 3H), 2.25-2.11 (m, 2H), 1.46 (t, 3H, J ) 6.9 Hz) ppm; 13C
NMR (75 MHz, CD3OD) δ 196.7, 193.4, 143.9, 143.7, 143.6,
5-(4-Eth oxyp h en yl)ben zo[f]p yr id o[2,1-a ]p h th a la zin -7-
iu m m esitylen esu lfon a te (3d ): method A; yellow solid (22%);
mp 225-227 °C; IR (KBr) νmax 2932, 1604, 1458, 1298 cm-1
;
1H NMR (300 MHz, CD3OD) δ 9.55 (d, 1H, J ) 6.5 Hz), 9.43
(d, 1H, J ) 8.0 Hz), 8.97 (d, 1H, J ) 8.8 Hz), 8.73 (dd, 2H, J
) 8.4, 6.9 Hz), 8.30-8.24 (m, 2H), 7.97 (d, 1H, J ) 8.8 Hz),

1146 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mart´ınez et al.
7.82 (t, 1H, J ) 8.0 Hz), 7.66-7.61 (m, 2H), 7.49 (t, 1H, J )
8.8 Hz), 7.15 (d, 1H, J ) 8.8 Hz), 7.04 (t, 1H, J ) 8.8 Hz), 6.79
(s, 2H), 4.19 (q, 2H, J ) 6.9 Hz), 2.56 (s, 6H), 2.18 (s, 3H),
1.47 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CD3OD) δ
142.7, 142.0, 140.8, 138.7, 138.2, 132.6, 131.9, 131.6, 131.5,
130.7, 130.6, 130.0, 129.5, 129.4, 129.2, 129.1, 129.0, 127.5,
126.8, 126.7, 124.5, 116.4, 115.9, 30.4, 23.2, 20.8, 15.0 ppm.
Anal. Calcd for C33H30N2O4S: C, 71.98; H, 5.49; N, 5.09.
Found: C, 70.89; H, 5.30; N, 5.23.
7.49 (t, 1H, J ) 7.7 Hz), 7.17 (d, 2H, J ) 8.0), 6.69 (s, 2H),
4.20 (q, 2H, J ) 6.9), 2.58 (s, 3H), 2.50 (s, 6H), 2.13 (s, 3H),
1.49 (t, 3H, J ) 6.9 Hz) ppm; 13C NMR (75 MHz, CD3OD) δ
162.9, 160.6, 143.5, 141.6, 140.9, 139.8, 138.7, 138.2, 138.1,
137.7, 137.5, 132.1, 132.0, 131.5, 130.9, 130.6, 129.7, 129.6,
129.1, 124.5, 121.3, 120.4, 118.7, 116.6, 65.0, 23.2, 21.4, 20.8,
15.0 ppm. Anal. Calcd for C38H34N2O4S: C, 74.24; H, 5.57; N,
4.56. Found: C, 73, 91; H, 5.52; N, 4.37.
DNA Bin d in g Exp er im en ts.
5-(4-Eth oxyp h en yl)-8-m eth ylben zo[f]p yr id o[2,1-a ]p h -
th a la zin -7-iu m m esitylen esu lfon a te (3e): method B; yel-
low solid (28%); mp 180-182 °C; IR (KBr) νmax 3438, 1604,
1467, 1388, 1189 cm-1; 1H NMR (300 MHz, CD3OD) δ 9.31 (d,
1H, J ) 9.3 Hz), 8.96 (d, 1H, J ) 9.3 Hz), 8.71-8.59 (m, 1H),
8.42 (t, 1H, J ) 7.6 Hz), 8.23 (d, 1H, J ) 8.0 Hz), 8.14 (d, 1H,
J ) 7.6 Hz), 7.67 (d, 2H, J ) 8.5 Hz), 7.45 (d, 1H, J ) 8.0 Hz),
7.36 (t, 1H, J ) 7.6 Hz), 7.15 (d, 1H, J ) 8.3 Hz), 7.02 (d, 2H,
J ) 8.5 Hz), 6.79 (s, 2H), 4.11 (q, 2H, J ) 6.9 Hz), 3.17 (s, 3H),
2.55 (s, 6H), 2.18 (s, 3H), 1.42 (t, 3H, J ) 6.9 Hz) ppm; 13C
NMR (75 MHz, CD3OD) δ 143.3, 142.5, 140.6, 140.5, 134.7,
134.5, 134.2, 134.1, 133.8, 133.1, 133.0, 132.9, 132.6, 132.5,
132.4, 131.9, 131.5, 130.4, 130.3, 130.2, 130.1, 130.0, 125.6,
115.7, 30.2, 25.7, 23.2, 20.8, 11.6 ppm. Anal. Calcd for
Electr op h or etic Titr a tion of Su p er coiled Ba cter ia l
P la sm id DNA. Each reaction mixture, in a final volume of 6
µL, contained 200 ng of supercoiled bacterial plasmid DNA
(pBluescript SK+, Stratagene, 2961 pb, equivalent final
concentration 5.12 × 10-5 M base pairs), 5% glycerol, in 2×
TAE (TAE: 40 mM Tris-acetic acid, 1 mM EDTA, pH 8) plus
the compound under study at the final concentration indicated
in each case. The complete reaction mixture was incubated
for 1 h at 37 °C before electrophoresis, which was performed
in 1× TAE, 1% agarose gel at 60 V (4.7 V/cm, 100 mA) for 1 h.
Gels are stained in 5 µg/mL ethidium bromide in water and
visualized using a UV transilluminator. The plasmid DNA was
prepared using the Wizard Plus Midipreps DNA Purification
System (Promega, Cat.# A7640; http://www.promega.com),
following its recommended instructions, from 200 mL expo-
nential phase cultures of E. coli Dh5a carrying the artificial
cloning vector plasmid pBluescript SK+ (Stratagene). Bacte-
rial growth was carried out at 37 °C in Luria-Bertani (LB)
broth [10 g of Bacto-Tryptone (Difco 0123-01-1), 5 g of Bacto-
yeast extract (Difco 0127-05-3) 10 g of NaCl, doubly distilled
H2O to 1 L] supplemented with 100 mg/mL ampicillin.47-49
Electr op h or etic Assa y of Su p er coiled a n d Rela xed
Ba cter ia l P la sm id DNA. The complete assay involves four
sample mixtures (1-4) and two agarose gels. Each mixture,
in a final volume of 24 µL, contains 800 ng of bacterial plasmid
DNA (pBluescrips SK+, Stratagene, 2961 pb, equivalent final
concentration 5.12 × 10-5 M base pairs), 5% glycerol, and 2×
TAE. Mixtures 1 and 3 were prepared from untreated super-
coiled plasmid, whereas mixtures 2 and 4 were prepared from
a DNA topoisomerase I-relaxed, covalently closed plasmid.
Finally, mixtures 3 and 4 were supplemented with the
compound under study at a final concentration of 0.1 mM and
incubated for 1 h at 37 °C before electrophoresis. Samples of
6 µL from each mixture were subjected to electrophoresis in
two independent 1× TAE, 1% agarose gels at 60 V (4.7 V/cm,
100 mA) for 1 h. One of the two gels contained the compound
C
34H32N2O4S: C, 72.32; H, 5.71; N, 4.96. Found: C, 72.09; H,
5.99; N, 4.90.
15-P h en ylben zo[f]qu in o[2,1-a ]ph th alazin -13-in iu m m es-
itylen esu lfon a te (4a ): method A; yellow solid (75%); mp 280-
282 °C; IR (KBr) νmax 2930, 1600, 1507, 1398, 1353, 1205 cm-1
;
1H NMR (300 MHz, CD3OD) δ 9.40 (d, 1H, J ) 8.8 Hz), 9.36
(d, 1H, J ) 9.1 Hz), 9.15 (d, 2H, J ) 9.1 Hz), 8.81 (d, 1H, J )
9.1 Hz), 8.47 (d, 1H, J ) 8.0 Hz), 8.29 (t, 2H, J ) 9.1 Hz), 8.12
(t, 1H, J ) 8.0 Hz), 7.95 (d, 1H, J ) 8.8 Hz), 7.83-7.90 (m,
2H), 7.69-7.78 (m, 3H), 7.50 (t, 2H, J ) 7.3 Hz), 6.80 (s, 2H),
2.57 (s, 6H), 2.19 (s, 3H) ppm; 13C NMR (75 MHz, CD3OD) δ
142.2, 140.9, 139.9, 139.5, 139.2, 139.0, 138.2, 138.0, 135.9,
132.2, 132.1, 131.6, 130.8, 130.3, 129.5, 129.4, 129.3, 129.2,
125.0, 121.5, 120.7, 118.8, 23.2, 20.7 ppm. Anal. Calcd for
C
35H28N2O3S: C, 75.52; H, 5.07; N, 5.03. Found: C, 75.77; H,
5.30; N, 4.88.
15-P h en yl-10-m et h ylb en zo[f]q u in o[2,1-a ]p h t h a la zin -
13-iu m m esitylen esu lfon a te (4b): method B; yellow solid
(50%); mp 220-222 °C; IR (KBr) νmax 2927, 1601, 1448, 1188
cm-1; 1H NMR (300 MHz, CD3OD) δ 9.52 (d, 1H, J ) 8.8 Hz),
9.30 (d, 1H, J ) 9.1 Hz), 9.18 (t, 2H, J ) 9.1 Hz), 8.95 (d, 1H,
J ) 9.1 Hz), 8.38 (d, 1H, J ) 8.0 Hz), 8.32 (s, 1H), 8.13 (d, 1H,
J ) 8.0 Hz), 7.90 (t, 1H, J ) 8.8 Hz), 7.70-7.82 (m, 5H), 7.51-
7.62 (m, 2H), 6.69 (s, 2H), 2.66 (s, 3H), 2.45 (s, 6H), 2.13 (s,
3H) ppm; 13C NMR (75 MHz, CD3OD) δ 143.3, 141.7, 141.3,
140.6, 139.7, 138.9, 138.6, 137.9, 137.6, 137.4, 131.8, 131.3,
130.5, 130.1, 129.5, 129.4, 129.1, 129.0, 124.3, 121.2, 120.2,
118.5, 23.9, 21.1, 20.5 ppm. Anal. Calcd for C36H30N2O3S: C,
75.76; H, 5.30; N, 4.91. Found: C, 75.56; H, 5.23; N, 5.12.
15-(4-E t h oxyp h en yl)b en zo[f]q u in o[2,1-a ]p h t h a la zin -
13-iu m m esitylen esu lfon a te (4c): method B; yellow solid
(28%); mp 198-200 °C; IR (KBr) νmax 2933, 1604, 1190, 1088
cm-1; 1H NMR (300 MHz, CD3OD) δ 9.36 (d, 1H, J ) 8.8 Hz),
9.20 (t, 1H, J ) 8.0 Hz), 9.00 (d, 1H, J ) 8.8 Hz), 8.95 (t, 1H,
J ) 8.0 Hz), 8.32-8.22 (m, 4H), 8.02 (d, 2H, J ) 8.8 Hz), 7.87
(t, 1H, J ) 7.7 Hz), 7.70 (d, 2H, J ) 8.4 Hz), 7.50 (t, 1H, J )
7.7 Hz), 7.17 (d, 2H, J ) 8.0 Hz), 6.70 (s, 2H), 4.22 (q, 2H, J )
6.9 Hz), 2.60 (s, 6H), 2.20 (s, 3H), 1.60 (t, 3H, J ) 6.9 Hz) ppm;
13C NMR (75 MHz, CD3OD) δ 160.9, 160.0, 150.0, 144.5, 141.6,
140.9, 139.8, 138.7, 138.0, 137.7, 137.6, 135.4, 132.5, 132.2,
131.7, 131.0, 129.7, 129.1, 124.5, 120.9, 120.4, 119.0, 118.7,
116.2, 112.2, 110.1, 65.2, 23.2, 21.4, 20.8 ppm. Anal. Calcd for
under study in the mass of the agarose gel at
a final
concentration of 0.01 mM. Both gels were postelectrophoreti-
cally stained with 5 µg/mL ethidium bromide in water and
visualized using an UV transilluminator.
UV-Visible Sp ectr op h otom etr ic Deter m in a tion . Calf
thymus DNA for spectrophotometric binding analysis was
purchased from Sigma Chemical Co. DNA concentrations were
determined by absorbance measurements using an extinction
coefficient of 13 200 M-1 cm-1 at 260 nm and are expressed in
terms of base pairs equivalents per liter. The aqueous solution
used was TRIS buffer (50 mM Tris, 15 mM NaCl, pH 7.6).
All compounds examined obeyed Beer’s law over the range
of concentrations used (0-20 µM), and the molar extinction
coefficients were determined at their appropriate λmax values
by Beer’s law plots. Molar extinction coefficients of compounds
bound to DNA were determined at the same wavelength as
the molar extinction coefficient measurements of the free
compound, but a larger molar excess of DNA was present
[DNA nucleotides]/[compound] > 20-100). Spectrophotometric
titrations were performed by serial addition of 100 µL aliquots
of corresponding compound 3 or 4 (6-20 µM) into a 10 cm path
length quartz cell containing a DNA stock solution (0.1-0.5
mM DNA nucleotides in TRIS buffer) in the same buffer and
scanning the UV-visible spectrum after each addition. Titra-
tions were stopped when no shift to the lower energy range of
the maximum absorbance wavelength in the spectrum was
detected between additions. These absorbance values were
converted to υ (mol of bound compound/mol of DNA base pairs)
and c (free ligand concentrations) using the free and bound
C
37H32N2O4S: C, 73.98; H, 5.37; N, 4.66. Found: C, 74.30; H,
5.23; N, 4.77.
15-(4-Eth oxyph en yl)-10-m eth ylben zo[f]qu in o[2,1-a ]ph -
th a la zin -13-iu m m esitylen esu lfon a te (4d ): method B;
yellow solid (50%); mp 220-222 °C; IR (KBr) νmax 2947, 1603,
1
1507, 1452, 1372, 1249 cm-1; H NMR (300 MHz, CD3OD) δ
9.16 (d, 2H, J ) 8.8 Hz), 8.95 (t, 2H, J ) 8.0 Hz), 8.67 (d, 1H,
J ) 8.8 Hz), 8.21 (d, 1H, J ) 8.0 Hz), 8.15 (s, 1H), 8.02 (d, 2H,
J ) 8.8 Hz), 7.82 (t, 1H, J ) 7.7 Hz), 7.70 (d, 2H, J ) 8.4 Hz),

Benzo[f]azino[2,1-a]phthalazinium Cations
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1147
molar extinction coefficients for the analyzed compound.
Experimental data that were in the fraction-bound range of
0.2-0.8 were plotted using the method of Scatchard (results
outside of this range are subject to large systematic errors as
a result of experimental errors in molar extinction coefficient
determinations50). The salts 3 and 4 were analyzed by the
extended neighbor-exclusion model of McGhee and von Hip-
pel32 using a nonlinear least-squares fitting procedure to derive
the binding parameters (K, n) with a computer program51 from
the following equation:
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DNA Top oisom er a se I In h ibition Assa y. Each reaction
mixture, in a final volume of 20 µL, contained 340 ng of
supercoiled bacterial plasmid DNA (pBluescrips SK+, Strat-
agene), 0.01% bovine serum albumin (BSA), in topoisomerase
I reaction buffer (35 mM Tris-HCl pH 8.0, 72 mM KCl, 5 mM
MgCl2, 5 mM DTT, 5 mM spermidine), plus 1 unit of bovine
DNA topoisomerase I (Amersham Pharmacia Biotech), and the
compound under study at the final concentration indicated in
each case. The complete reaction mixture was incubated for 1
h at 37 °C. The reaction was stopped by the addition of 20 µL
of 1% sodium dodecyl sulfate. Half of the resulting volume (20
µL) of each mixture was subjected to electrophoresis in 1× TAE
1% agarose gel at 60 V (4.7 V/cm, 100 mA) for 1 h, and the gel
was then stained postelectrophoretically with 5 µg/mL ethid-
ium bromide in water. The remaining 20 µL of each mixture
was subjected to electrophoresis under the same conditions
but in an agarose gel supplemented with ethidium bromide
at a final concentration of 0.1 mg/mL, without postelectro-
phoretic staining. The bands were visualized using an UV
transilluminator. The first two lanes of each gel, labeled as
C1 and C2, contained supercoiled and completely relaxed
plasmids, respectively, as controls. The following lanes con-
tained plasmid DNA treated with topoisomerase I in the
presence of different concentrations of the compound under
study (routinely 10, 5, and 1 µM).
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Ackn owledgm en t. Financial support from the Comi-
sio´n Interministerial de Ciencia y Tecnolog´ıa (CICYT,
projects SAF98-0093/BQU2002-0358) and a studentship
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National Foundation for Cancer Research for partial
support of this research.

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