Synthesis, antitumor and DNA photocleaving activities of novel naphthalene carboxamides: Effects of different thio-heterocyclic rings and aminoalkyl side chains

Article abstract of DOI:10.1016/j.tet.2005.06.097

Two kinds of thio-heterocyclic fused naphthalene carboxamides, 3a-b, 4a-b, were designed, synthesized and quantitatively evaluated as efficient antitumor and DNA photocleaving agents. Compound 3a or 3b, having the thiophene ring, intercalated into DNA mor

Full text of DOI:10.1016/j.tet.2005.06.097

Tetrahedron 61 (2005) 8711–8717
Synthesis, antitumor and DNA photocleaving activities of novel
naphthalene carboxamides: effects of different thio-heterocyclic
rings and aminoalkyl side chains
Zhigang Li,a Qing Yangb and Xuhong Qiana,c,
*
aState Key Lab. of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
bDepartment of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China
cShanghai Key Lab. of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Received 28 March 2005; revised 21 June 2005; accepted 24 June 2005
Available online 19 July 2005
Abstract—Two kinds of thio-heterocyclic fused naphthalene carboxamides, 3a–b, 4a–b, were designed, synthesized and quantitatively
evaluated as efficient antitumor and DNA photocleaving agents. Compound 3a or 3b, having the thiophene ring, intercalated into DNA more
strongly than compound 4a or 4b, having the thioxanthene ring. Compound 4a or 4b, photocleaved DNA more efficiently than 3a or 3b via
superoxide anion. Compound 4a was the strongest inhibitor for P388 (murine leukemia cell), while 3a was the most cytotoxic one against
A549 (human lung cancer cell). Each new compound showed stronger DNA photocleaving activity than corresponding naphthalimide.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
continued efforts for simple but efficient antitumor and/or
DNA photocleaving agents, we report here, the molecular
design and chemical synthesis of a novel heterocyclic
family of 3a–b and 4a–b (Fig. 1) by modification of the
naphthalimide skeletons via mercuric oxide mediated
decarbonylation. Compounds 3a–b or 4a–b, which could
be considered as ring opened models of 1a–b or 2a–b, were
expected to intercalate into DNA more strongly to shown
DNA intercalating agents form an important class of drugs
in anticancer therapy.1 To study the interaction between
such agents and DNA, in particular, effects of structural
characters of them on the interaction, could offer novel
insights into the design of new DNA targeting antitumor
drugs.2 Naphthalimides are significant examples of
DNA-intercalating agents,1,3–5 many of, which have
shown efficient antitumor activities upon a variety of
murine and human tumor cells,4 and/or DNA photocleaving
activities.5 They are generally characterized by the presence
of a planar tri- or tetracylic aromatic chromophore and one
or two flexible basic side chains.
To promote the antitumor and/or DNA photocleaving
activities, previous attempts were focused on incorporating
substituents or fusing aromatic rings to the naphthalimide
skeletons.4,5 The presence of a larger aromatic chromophore
was proved to improve the affinity of the intercalator for the
DNA molecule, consequently to a greater cytotoxic and/or
DNA photocleaving activity.4,5 We have ever reported a
series of thio-heterocyclic fused naphthalimides, 1a–d5f and
2a–d,5e as efficient DNA photocleavers (Fig. 1). In our
Keywords: DNA photocleavage; Antitumor; Intercalating.
*
Corresponding author. Address: State Key Lab. of Fine Chemicals,
Dalian University of Technology, Dalian 116024, China. Tel.: C86 411
83673466; fax: C86 411 83673488; e-mail: xhqian@ecust.edu.cn
Figure 1. Structures of the reported naphthalimides (1, ,2) and novel
designed naphthalene carboxamides (3, 4).
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.097

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Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
Scheme 1. (a) PhSH, EtOH, reflux, 4 h, 59% yield; (b) SnCl2/concentrated HCl, 65% yirld; (c) Pochorr cyclization: NaNO2, H2O–HCl–HOAc, 0–5 8C, 2 h;
CuSO4, HOAc, reflux, 2 h; (d) HgO (yellow), NaOH, AcOH, H2O, reflux, 4 days, 95% yield; (e) concentrated HCl, reflux, 2 h, 75% yield; (f) SOCl2, CHCl3,
Et3N, reflux, 20 h; (g) propitiate amine, CH2Cl2, room temperature, 24 h; (h) careful column chromatography.
efficient medical and biological use due to the reduction of
the steric effect of carbonyl anchor and the flexibility of the
aminoalkyl side chains.
reported.6 In our efforts to synthesize target compounds
3a–b, we found that only yellow mercuric oxide success-
fully mediated the decarbonylation of benzothiopheno-
naphthalic anhydride 5, synthesized from 4-bromo-3-
nitro anhydride through Pochorr intramolecular cycliza-
tion.5f,7 Two benzothiophenonaphthoic acid isomers, 8
and 9, were obtained after the reaction, which were then
converted to a pair of isomers, 3a, 3a0 or 3b, 3b0.
However, only pure 3a or 3b was obtained after careful
column chromatography due to the slight amount of 3a0 or
3b0 (Scheme 1a).
2. Results and discussion
2.1. Synthesis and spectra
The mercuric oxide (yellow or red) mediated decarbonyl-
ation of (substituted) naphthalic anhydrides has been
Scheme 2. (a) HgO (yellow or red), NaOH, AcOH, H2O, reflux, 4 days, 94% yield; (b) concentrated HCl, reflux, 2 h, 70% yield; (c) recrystalized with AcOH,
40% yield; (d) SOCl2, reflux, 5 h; methanol, reflux, 2 h; (e) 2-aminobenzenethiol, K2CO3, DMF, reflux, 80 h; (f) Pochorr cyclization: NaNO2, HOAc H2SO4,
0–5 8C, 12 h; CuSO4, HOAc, reflux, 2 h; (g) NaOH, Methanol, reflux, 2 h; HCl; (h) SOCl2, CHCl3, Et3N, reflux 20 h; (i) propitiate amine, CH2Cl2, room
temperature, 24 h; (j) careful column chromatography.

Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
8713
The decarbonylation of benzothioxanthenenaphthalic
anhydride 105e was also only mediated by yellow mercuric
oxide. The pair of isomer mixtures, 4a, 4a0 or 4b, 4b0 was
obtained. However, neither pure product could be separated
after careful column chromatography due to the similar
molecular polarities between 4a, 4a0 or 4b, 4b0 (Scheme
1b). Then we tried the other way outlined in Scheme 2.
Either yellow or red mercuric oxide was found to mediate
the decarbonylation of 4-bromo-1,8-naphthalic anhydride to
obtain a pair of isomers of 15 and 16 with the ratio of 2:3 via
1H NMR. Pure 5-bromo-1-naphthaloic acid 16 was obtained
after recrystallization from acetic acid. It was transformed
to methyl-5-bromo-1-naphthoate 17, which was then
condensed with 2-aminobenzenthiol to obtain 18. Followed
by the Pochorr intramolecular cyclization, methyl benzo-
thioxanthenenaphthoate 19 was obtained, which was
converted to corresponding acid 20, then to target product
4a or 4b, which was purified by careful column
chromatography.
the intercalation orders of 3aO4a, 3bO4b, it is obvious that
the chromophore of 3a or 3b, having the thiophene ring
could intercalated more strongly than that of 4a or 4b,
having the thioxanthene ring, in agreement with the
comparison of 1a (2.8!105 MK1 5f
and 2a (8.27!
)
103 MK1).5e As expected, 3a or 4a, exhibited greater
affinity for DNA than 1a or 2a, indicating that the reduction
of the steric effect of carbonyl anchor and/or the higher
flexibility of the side chain facilitated its intercalation with
DNA (Fig. 2).
Structures of all the final products were well confirmed by
1H NMR, HRMS and IR. Furthermore, the above
experiments provided two different ways to synthesize
side-armed heterocyclic fused naphthalene derivatives,
which may have potentials in other fields.
Figure 2. Fluorescence spectra before and after interaction of compound 3b
and CT-DNA. Curves F and F-CT corresponded to compound 3b before
and after mixed with DNA. Numbers 1–4 indicated the concentration of 3b,
5, 10, 20, 40 mM, respectively. DNA applied was 50 mM (bp).
Spectra data of these new compounds were measured and
summarized in Table 1. Compared with those of their
corresponding naphthalimides,5e–f values of both the
emission wavelength and the absorption wavelength of
3a–b, 4a–b, were blue-shifted due to the reduction of their
conjugation areas and electronic pushing–pulling ICT
(intramolecular charge transfer) effects caused by the
decarbonylation reaction. Slight effect of the side chains
was found on spectra data of these compounds.
2.3. Antitumor evaluation
The antitumor activities of these compounds were evaluated
in vitro (under scattered light) against A549 (human lung
cancer cell) and P388 (murine leukemia cell) cell lines,
respectively. As shown in Table 2, all these compounds
exhibited efficient antitumor activities with IC50 ranging
from 0.176 to 29 mM. Also, all of them were more cytotoxic
against P388 than against A549, reflecting an excellent
selectivity for a special murine or leukemia cell type.
Cytotoxic potencies of these compounds against two tumor
cells were highly dependent on the length of side chains.
The compound with two methylene units in the side chain
between two nitrogen atoms was more cytotoxic than
corresponding homologue with one more methylene unit, as
indicated by the cytotoxicity orders of 3aO3b, 4aO4b.
Compound 4a was found to be the strongest inhibitor for
P388 with IC50 of 0.176 mM, while 3a was the most
cytotoxic one against A549 with IC50 of 1.16 mM.
Table 1. Spectra data,a,b and Scatchard binding constants of compounds 1–4
Compd
UV lmax/nm
(log 3)
FL lmax/nm
(F)
Scatchard binding
constants (MK1
)
3a
3b
4a
350 (3.32)
350 (3.42)
389 (3.73)
388 (3.91)
384 (4.23)
462 (4.42)
423 (0.009)
421 (0.011)
455 (0.013)
454 (0.014)
437 (0.0215)
521 (0.51)
4.46!105
6.86!105
1.95!104
4.02!104
2.8!105
4b
1a4f
2a4e
8.27!103
a In absolute ethanol.
b With quinine sulfate in sulfuric acid as quantum yield standard (fZ0.55).
Table 2. Cytotoxicity of these compounds against A549 a and P388 b cells
Compounds
Cytotoxicity (IC50, mM)
P388b
2.2. DNA intercalation
A549a
The fluorescences of these compounds were quenched upon
addition of Calf thymus DNA. The Scatchard binding
constants8 were calculated and summarized in Table 1 with
the orders of 3bO3a, 4bO4a, clearly indicating the
importance of the aminoalkyl side chains serving as DNA
groove binders and/or external electrostatic binders. The
chain length is important in placing the protonated side
chain nitrogen in proximity to functional groups suitable for
hydrogen bonding on the DNA double helix after the thio-
heterocyclic fused naphthalene chromophore has inter-
calated. In our cases, the side chain with three methylene
units between two nitrogen atoms can significantly enhance
the intercalating abilities of the thiazonaphthalimides. As to
3a
3b
4a
4b
1.16
19.1
20.5
29
0.428
0.89
0.176
0.295
a Cytotoxicity (CTX) against human lung cancer cell (A549) was measured
by sulforhodamine B dye-staining method.9
b CTX against murine leukemia cells (P388) was measured by microculture
tetrazolium–formazan method.10
2.4. DNA photocleavage
The photocleavage of these compounds to supercoiled
plasmid pBR322 DNA were evaluated by 1% agarose gel
electrophoresis. The reaction mixture containing each

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Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
compound and plasmid DNA was put under photo-
irradiation (2300 W/cm2) through a transluminator
(360 nm) at a distance of 20 cm at 0 8C for 3 h under
aerobic conditions. The photocleavage efficiency was
defined by the conversion ratio from supercoiled pBR322
DNA (form I) to relaxed circular DNA (form II) and linear
DNA (form III).
addition of histidine (singlet oxygen quencher), dithio-
threitol (DTT, superoxide anion scavenger) and ethanol
(hydroxyl radical scavenger), respectively. As shown in
Figure 3c or d, with 3b, 4b as examples, histidine, and
ethanol clearly had no obvious effects on the photocleavage,
indicating that singlet oxygen and hydroxyl radical were not
likely to be cleavage contributors. However, the DNA
cleaving activities both decreased greatly in the presence of
DTT (lane 6), indicating superoxide anion was most likely
to be the reactive specie responsible for plasmid cleavage.
Under identical conditions, DTT inhibited the photo-
cleavage activity of 4b (form II: 68% to 22%) more
efficiently than that of 3b (form II: 47% to 29%), indicating
superoxide anion was more easily produced in the
photocleaving reaction of 4b.
As shown in Figure 3a, all these compounds efficiently
cleaved DNA from form I to form II and form III at the
concentration of 100 mM with an order of 4bO4aO3bO3a.
Compound 4b was more active than its analogues in that it
could produce more percentage of form III (36%), generally,
the result from double-strand cuts or proximal single-strand
cuts on opposite strands. The concentration-dependent
experiment showed that 4b exhibited detectable cleavage
(21% form II) even at 0.5 mM (Fig. 3b). No damage was
observed in the absence of either compound (Fig. 3c, lane 2)
or light (lane 3), indicating that they were obligate factors
for DNA strand scission. In this case, UV light actually
functioned as a trigger to initiate the strand scission.
The effect of side chains on the order of photocleavage
abilities was parallel to that of their DNA intercalating
abilities (3bO3a, 4bO4a), but anti-parallel to that of their
antitumor activities (3aO3b, 4aO4b). The reason account-
ing for it probably lies on that: the cytotoxicity of one
compound is determined by two conflicting factors
including cell membrane crossing ability and DNA binding
ability. The side chains could be protonated to different
extent under physiological pH due to their different basicity
of corresponding nitrogen atoms. The protonation extent
significantly affected the ability of the molecule to pass
through lipophilic memebranes to bind to DNA. Lower
degree of protonation is desirable for cell penetration, but
higher degree of protonation favored DNA binding. Once
these two factors effectively compromise, the higher
antitumor potency is possible, such as 3a, 4a. Also, the
selective cytotoxicities of these compounds were possibly
determined by their different memebrane crossing abilities
for different cell lines.
The effect of heterocyclic rings on the order of photo-
cleavage abilities (4aO3a, 4bO3b) was anti-parallel to that
of their DNA intercalating abilities (3aO4a, 3bO4b). In
our case, the five-membered thiophene ring was better
conjugated to the naphthalene skeleton than the six-
membered thioxanthene ring, resulting in the better planar
chromophore of 3a or 3b. The better planar chromophore of
3 in turn accounted for the more efficient intercalating
activity of 3. As to 4a or 4b, its worse conjugating structure
made it less stable under photo-irradiation. Then electrons
were more easily transferred from the chromophore of 4 to
oxygen to generate superoxide anions responsible for DNA
cleavage to lead to its stronger photocleaving activity.
Figure 3. Photocleavage of closed supercoiled pBR322 DNA (200 mM/bp)
in the buffer of Tris–HCl (20 mM, pH 7.5). (a) Photocleavage of plasmid
DNA by different compounds (100 mM) for 3 h. Lane 1, DNA alone; lane 2,
DNA with UV; lane 3–6, each compound of 3a, 3b, 4a, 4b, and DNA,
respectively. (b) Photocleavage of plasmid DNA by 4b at various
concentrations for 3 h. Lane 1, DNA alone; lane 2, DNA with UV; lane
3, DNA with 4b (100 mM) without UV; Lane 4–9, DNA with 4b at
concentration of 0.5, 5, 10, 25, 50, 100 mM, respectively, following UV
irradiation. (c) and (d) Effect of additives on the photocleavage by
compound 3b or 4b (50 mM) for 3 h. Lane 1, DNA alone; lane 2, DNA with
UV; lanes 3 DNA and compound 3b (c), 4b (d) with UV; lane 4–6, DNA
and compound in the presence of histidine (6 mM), ethanol (1.7 M),
dithiothreitol (DTT, 30 mM), respectively, following UV irradiation.
Furthermore, 3a–b or 4a–b photocleaved DNA more
efficiently than corresponding napthalimides 1a–b or
2a–b.5e–f Electron densities of the thio-heterocyclic fused
naphthalene chromophores were relatively higher than those
of their corresponding naphthalimides due to the reduction
of one strong electron-withdrawing carbonyl group. The
higher chromophores’ electron densities were inferred to
grant 3a–b or 4a–b stronger abilities of transferring
electrons, which were from their chromophores to oxygen
to form more superoxide anions for photocleavage under
photo-activation, consequently to higher photocleavage
abilities than 1a–b or 2a–b.
Photocleavage can be via a variety of mechanisms involving
free radical, electron transfer and singlet oxygen.11 In order
to establish the reactive species responsible for cleavage of
the plasmid, mechanistic experiments were performed by

Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
8715
3. Conclusion
1.0 Hz, J2Z7.4 Hz, 1H, 11-H), 8.30–8.26 (m, 1H, 2-H),
8.04 (d, J1Z8.2 Hz, J2Z7.4 Hz, 1H, 8-H), 7.70–7.66 (m,
2H, 9-H, 10-H). HRMS (ESI): Calcd for C18H9O3S (MC
H)C: 305.0272. Found: 305.0281.
In summary, the present work demonstrated the design,
synthesis and quantitative evaluation of two kinds of thio-
heterocyclic fused naphthalene carboxamides as efficient
antitumor and DNA photocleaving agents. All these
compounds were found to be more cytotoxic to P388 than
to A549. Compounds with the five-membered thiophene
rings were proved to be more efficient DNA intercalators
while those with the six-membered thioxanthene rings were
more efficient DNA photocleavers. The side chains played
different roles in intercalating/photocleaving and antitumor
activities.
4.2.2. Anhydro-8-hydroxylmercuri-1-benzothiopheno-
naphthoic acids 8 and 9. The obtained precipitate 5
(2.63 g) was suspend in 60 mL aqueous sodium hydroxide
(1 g) and refluxed until the solid material dissolved, a
solution of HgO (yellow) (2.34 g) in a mixture of H2O
(10 mL) and AcOH (7 mL) was added with stirring to result
in slow evolution of carbon dioxide. The reaction mixture
refluxed for 96 h, then cooled and filtered. The highly
insoluble yellow solid was washed with water and dried
under vacuum at 50 8C over night to give the mixture of 6
and 7 (4.11 g, 95% yield). Attempts to purify and separate
the anhydro compounds were unsuccessful. It is insoluble in
organic solvents. The above obtained solid (4.11 g) were
suspended in 30 mL concentrated HCl, stirred, heated under
reflux for 3 h. Hot filtration gave the mixture thio-
heterocyclic fused naphthoic acids 8 and 9 (1.86 g, 76%
yield). HRMS (ESI): Calcd for C17H12O2S (MCH)C:
279.0480. Found: 279.0472.
4. Experimental
4.1. Materials
1
All the solvents were of analytic grade. H NMR were
measured on a Bruker AV-400 spectrometer with chemical
shifts reported as ppm (in DMSO/CDCl3-d6, TMS as
internal standard). Mass spectra were measured on a HP
1100 LC–MS spectrometer. Melting points were determined
by an X-6 micro-melting point apparatus and uncorrected.
Absorption spectra were determined on PGENERAL
TU-1901 UV–VIS Spectrophotometer.
4.2.3. N-(2-(Dimethylamino)ethyl)benzothiopheno-
naphalene-4-carboxamide 3a. The acids 9 and 10 (0.6 g)
were treated with thionyl chloride (10 mL) and DMF (1
drop) in CHCl3 (10 mL) under reflux for 20 h. After removal
of the solvent and excess thionyl chloride, the crude solid
and N,N-dimethyl ethyl diamine (0.4 mL) were combined in
25 mL CH2Cl2. The mixture cooled in an ice bath while
Et3N (0.45 mL) was added dropwise with stirring. The
stirring continued for 20 h at room temperature. Removal
of solvent and separated on silica gel chromatography
(CH2Cl2:MeOHZ6:1, v/v) to get pure 3a (0.32 g, 42%
yield). Mp: 194–195 8C. 1H NMR (d6-DMSO) d (ppm): 2.42
(s, 6H, NCH3), 2.75 (t, J1ZJ2Z6.4 Hz, 2H, NCH2), 3.55 (s,
2H, J1Z4 Hz, J2Z6.8 Hz, CONHCH2), 7.58 (t, J1Z6.8 Hz,
J2Z6.4 Hz, 2H, 8-H, 9-H), 7.72 (t, J1Z6.0 Hz, J2Z7.2 Hz,
2H, 2-H, 3-H), 8.13 (t, J1Z5.2 Hz, J2Z3.6 Hz, 1H, 10-H),
8.22 (t, J1Z4 Hz, J2Z5.2 Hz, 1H, 1-H), 8.30 (d, JZ8 Hz,
1H, 6-H), 8.42 (s, 1H, 7-H), 8.44 (s, 1H, 5-H). HRMS (ESI):
Calcd for C21H21N2OS (MCH)C: 349.1375. Found:
349.1368. IR (KBr): 3268, 2921, 2815, 1634, 1555,
4.2. Synthesis
4.2.1. Benzothiophenonaphthalic anhydride 5. 4-Bromo-
3-nitro-1,8-naphthalic anhydride (6.44 g) was stirred under
reflux in ethanol (60 mL) with thiophenol (2.6 mL) for 5 h.
The liquor was reduced in volume to 30 mL and filtered,
washed with some ethanol, dried, giving 6.83 g (97.3%) of
3-nitro-4-phenylthio-1,8-naphthalic anhydride. The recrys-
tallization from glycol monomethyl ether gave long golden
needles. Mp: 177–178 8C. The above nitro compound
(2.7 g) was stirred into a mixture of stannous chloride
(8.81 g) and concentrated hydrochloric acid (12 mL). After
warming to 40 8C, the temperature rose spontaneously to
85 8C and was maintained at 85 8C for 1 h (color change
from orange to olive-green). The suspension was cooled and
filtered to give 2.4 g crude product. Recrystallization from
pyridine gave greenish-yellow needles of 3-amino-4-
768 cmK1
.
1
phenylthio-1,8-naphthalic anhydride. Mp: 224–225 8C H
NMR (d6-CDCl3) d (ppm): 8.69 (d, JZ8.5 Hz, 1H, 7-H),
8.37 (d, JZ7.6 Hz, 1H, 5-H), 8.15 (s, 1H, 2-H), 8.15 (t, J1Z
7.6 Hz, J2Z8.5 Hz, 1H, 6-H), 7.25–7.20 (m, 2H, 30-H,
50-H), 7.17–7.13 (m, 1H, 40-H), 7.06–7.02 (m, 2H, 20-H,
60-H), 5.08 (s, 2H, NH2). EI-MS: (m/z): 321.3 (MC).
Sodium nitrite (0.7 g) and glacial acid (2 mL) were added
dropwise to concentrated sulfuric acid (10 mL). The
mixture was cooled to 0–5 8C and 3-amino-4-phenylthio-
1,8-naphthalic anhydride (3.2 g) was slowly added over 1 h.
After stirring for 1 h, the dark red viscous liquor was added
over 90 min to a boiling solution of copper sulfate (70 g) in
water (1000 mL) and glacial acetic acid (120 mL). After the
addition was complete, the liquor was refluxed for 30 min,
cooled, filtered, dried and an orange solid (2.63 g, 85%) was
collected. Recrystallization from DMF gave deep red
4.2.4. N-(2-(Dimethylamino)propyl)benzothiopheno-
naphthalene-4-carboxamide 3b. Prepared and purified in
a similar manner as that in 3a, N,N-dimethylpropyl diamine
was used here, instead of N,N-dimethylethyl diamine and
separated on silica gel chromatography (CH2Cl2:MeOHZ
1
5:1, v/v) to get pure 3b (39% yield). Mp: 183–184 8C. H
NMR (d6-DMSO) d (ppm): 1.82 (t, J1Z6.4 Hz, J2Z7.2 Hz,
2H, CH2), 2.42 (s, 6H, NCH3), 3.16 (s, 2H, NCH2),
4.06 (t, J1Z7.2 Hz, J2Z7.2 Hz, 2H, CONCH2), 7.56 (t,
J1Z4.4 Hz, J2Z4 Hz, 2H, 8-H, 9-H), 7.69 (d, JZ
7.8 Hz, 1H, 3-H), 7.74 (t, J1ZJ2Z8 Hz, 1H, 2-H), 8.17
(t, J1Z4 Hz, J2Z4.8 Hz, 1H, 1-H), 8.23 (d, JZ8.8 Hz,
2H, 7-H, 10-H), 8.47 (t, J1Z8.8 Hz, J2Z7.8 Hz, 2H,
5-H, 6-H). HRMS (ESI): calcd for C22H23N2OS (MC
H)C: 363.1531, Found: 363.1523. IR (KBr): 3257,
1
needles. Mp: 284–286 8C. H NMR (d6-DMSO) d (ppm):
9.4 (s, 1H, 7-H), 8.77–8.73 (m, 2H, 3-H, 1-H), 8.59 (d, J1Z
2922, 2853, 1632, 1544, 767 cmK1
.

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Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
4.2.5. 5-Bromo-1-naphthoic acid 16. 4-Bromo-1,8-
naphthalic anhydride (2.77 g) was suspend in 50 mL
aqueous sodium hydroxide (1.25 g) and refluxed until the
solid material dissolved, a solution of HgO (red or yellow)
(2.2 g) in a mixture of H2O (2 mL) and AcOH (2 mL) was
added with stirring to result in slow evolution of carbon
dioxide. The reaction mixture refluxed for 96 h, then cooled
and filtered. The highly insoluble yellow solid was washed
with water and dried under vacuum at 100 8C over night to
give the mixture of 13 and 14 (4.9 g, 96% yield). The
obtained precipitates were suspended in 25 mL concen-
trated HCl, stirred, heated under reflux for 3 h. Hot filtration
gave the mixture of 4-bromo-1-naphthoic acid 15 and
5-bromo-1-naphthoic acid 16 with ratio of 2:3 via 1H NMR.
Pure 5-bromo-1-naphthoic acid 16 was obtained after
recrystallization from acetic acid (1.1 g, 42% yield), mp:
Et3N (0.45 mL) was added dropwise with stirring. The
stirring continued for 20 h at room temperature. Removal of
solvent and separated on silica gel chromatography to afford
the pure products. Separated on silica gel chromatography
(CH2Cl2:MeOHZ6:1, v/v) to get pure 4a (0.30 g, 42%
yield). Mp: 223–224 8C. 1H NMR (d6-CDCl3) d (ppm): 2.88
(s, 6H, NCH3), 3.05 (t, J1ZJ2Z3.6 Hz, 2H, NCH2), 3.67
(d, 2H, JZ5.6 Hz, CONHCH2), 7.40 (t, J1ZJ2Z3.6 Hz,
2H, 8-H, 9-H), 7.49 (d, JZ3.2 Hz, 1H, 1-H), 7.61 (t, J1Z
J2Z6.4 Hz, 1H, 5-H), 7.85 (s, 1H, 2-H), 8.17 (d, JZ8 Hz,
1H, 10-H), 8.23 (d, JZ6.8 Hz, 1H, 6-H). 8.30 (d, JZ8 Hz,
1H, 7-H), 8.94 (t, J1ZJ2Z3.6 Hz, 1H, 4-H). HRMS (ESI):
Calcd for C21H21SN2O (MCH)C: 349.1375. Found:
349.1378. IR (KBr): 3266, 2918, 2849, 1638, 1548,
762 cmK1
.
1
205–206 8C, H NMR (d6-DMSO) d (ppm): 7.57 (t, J1Z
4.3.3. N-(2-(Dimethylamino)propyl) benzothioxanthene-
naphthalene-3-carboxamide 4b. Prepared and purified in a
similar manner as that in 4a, N,N-dimethylpropyl diamine
was used here, instead of N,N-dimethylethyl diamine and
separated on silica gel chromatography (CH2Cl2: MeOHZ
8 Hz, J2Z7.6 Hz, 1H, 7-H), 7.77 (t, J1Z8 Hz, J2Z8.4 Hz,
1H, 3-H), 8.00 (d, JZ7.6 Hz, 1H, 6-H), 8.23 (d, JZ7.2 Hz,
1H, 4-H), 8.43 (d, JZ8.4 Hz, 1H, 2-H), 8.88 (d, JZ8.8 Hz,
1H, 8-H). HRMS (ESI): Calcd for C11H8BrO2 (MCH)C:
250.9708. Found: 250.9715.
1
4:1, v/v) to get pure 4b (36% yield). Mp: 217–218 8C. H
NMR (d6-CDCl3) d (ppm): 1.89 (t, J1Z6.4 Hz, J2Z7.2 Hz,
2H, CH2), 2.58 (s, 6H, NCH3), 2.62 (s, 2H, NCH2), 3.03
(t, J1Z7.2 Hz, J2Z7.2 Hz, 2H, CONCH2), 7.42 (t, J1ZJ2Z
6.4 Hz, 2H, 8-H, 9-H), 7.48 (d, JZ3.2 Hz, 1H, 1-H), 7.65
(t, J1ZJ2Z6.8 Hz, 1H, 5-H), 7.81 (s, 1H, 2-H), 8.16 (d, JZ
7.8 Hz, 2H, 7-H, 10-H), 8.247 (d, JZ6.4 Hz, 1H, 6-H). 8.29
(d, JZ6.4 Hz, 1H, 4-H). HRMS (ESI): Calcd for
C22H23N2OS (MCH)C: 363.1531. Found: 363.1535. IR
4.3. Methyl-5-bromo-1-naphthoate 17
The pure 5-bromo-1-naphthoic acid 16 (4.16 g) was
combined with thionyl chloride (30 mL) and DMF
(1 drop) under reflux for 20 h. After removal of the excess
thionyl chloride, 30 mL methanol was added, reacted for 1 h
under reflux. The crude methyl-5-bromo-1-naphthoate was
obtained after removal of the excess methanol. ESI-MS
(positive) m/z: 266.5 (MCH)C.
(KBr): 3274, 2940, 2856, 1634, 1541, 763 cmK1
.
4.4. Intercalation studies of compounds to CT-DNA
4.3.1. Benzothioxanthenenaphthoic acid 20. Methyl-5-
bromo-1-naphthoate 17 (3.75 g) was stirred under reflux in
DMF (40 mL) with 2-aminobenzenethiol (2.12 g) and
K2CO3 (1.035 g) for 80 h. Cooled and poured into 100 mL
water, filtered and dried under vacuum to obtain the crude
solid of 18 (3.56 g, 74%). ESI-MS (positive) m/z: 310.1
(MCH)C. 11 mL aqueous solution of sodium nitrite
(8.35 g) was slowly added into mixture of 18 (3.56 g),
HCl (3 mL) glacial acetic acid (30 mL) and H2O (5 mL)
within 1 h at 0–5 8C. After stirring for 12 h, the dark red
viscous liquor was added over 90 min to a boiling solution
of copper sulfate (8.17 g) in water (120 mL) and glacial
acetic acid (7 mL). After the addition was complete, the
liquor was refluxed for 30 min, cooled, filtered, dried and an
dark-orange solid 19 (3.23 g, 95%) was collected. ESI-MS
(positive) m/z: 293.1 (MCH)C. The above obtained solid
19 was added into the mixture of 20%NaOH (50 mL) and
methanol (50 mL), then the mixture was refluxed for 1 h,
removal of excess methanol, cooled, filtered, dried under
vacuum to obtain 20 (1.95 g, 62%), mp: O300 8C. HRMS
(ESI): Calcd for C17H12O2S (MCH)C: 279.0480. Found:
279.0489.
0.1 mL solution of a compound in DMSO (10K3–10K4 M)
mixed with 20 mM Tris–HCl (pHZ7.5) to 5 mL. Then, two
groups of samples were prepared in the concentration of
chemical at 5, 10, 20, 40 mM, one contained Calf-thymus
DNA 50 mM, the other contained no DNA but had the same
concentration of chemical as control. All the above solution
was shaken for 3 days at 25 8C in the dark. Fluorescence
wavelength and intensity area of samples were measured.
4.5. Cytocoxicity in vitro evaluation
The prepared compounds have been submitted to Shanghai
Institute of Materia Medica for testing their cytotoxicities.
4.6. Photocleavage of supercoiled pBR322 DNA
250 mg pBR322DNA (form I), 1 mL solution of chemical
and 20 mM Tris–HCl (pHZ7.5) were mixed to 10 mL, then
irradiated for 3 h with light (360 nm) using lamp placed at
20 cm from sample. Supercoiled DNA runs at position I,
nicked DNA at position II, linear DNA at position III. The
samples were analyzed by gel electrophoresis in 1%
Agarose and gel was stained with ethidium bromide.
4.3.2. N-(2-(Dimethylamino)ethyl) benzothioxanthene-
naphthalene-3-carboxamide 4a. The acid 20 (0.6 g) was
treated with thionyl chloride (10 mL) and DMF (1 drop) in
CHCl3 (10 mL) under reflux for 20 h. After removal of
the solvent and excess thionyl chloride, the crude solid and
N,N-dimethyl ethyl diamine (0.4 mL) were combined in
25 mL CH2Cl2. The mixture cooled in an ice bath while
Acknowledgements
Financial support by the National Key Project for Basic
Research (2003CB114400) and under the auspices of

Z. Li et al. / Tetrahedron 61 (2005) 8711–8717
8717
5590–5591. (b) Saito, I.; Takayama, M.; Sugiyama, H.;
Nakatani, K. J. Am. Chem. Soc. 1995, 117, 6406–6408.
(c) Li, Y.; Xu, Y.; Qian, X. Bioorg. Med. Chem. Lett. 2003, 13,
3513–3515. (d) Li, Y.; Xu, Y.; Qian, X. Tetrahedron Lett.
2004, 45, 1247–1251. (e) Qian, X.; Li, Y.; Xu, Y. Bioorg. Med.
Chem. Lett. 2004, 14, 2665–2668. (f) Xu, Y.; Qu, B.; Qian, X.;
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National Natural Science Foundation of China is greatly
appreciated.
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