Synthesis of thiazo- or thiadiazo- naphthalene carboxamides via mercuric intermediates and their antitumor and DNA photocleavage activities

Article abstract of DOI:10.1016/j.tet.2005.03.135

Two kinds of thiazo- or thiadiazo- naphthalene carboxamides with aminoalkyl side chains at 5- or 6-position modified from naphthalimides were designed, synthesized and quantitatively evaluated as antitumor and DNA photocleaving agents. The compound with a

Full text of DOI:10.1016/j.tet.2005.03.135

Tetrahedron 61 (2005) 6634–6641
Synthesis of thiazo- or thiadiazo- naphthalene carboxamides
via mercuric intermediates and their antitumor and DNA
photocleavage activities
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 21 January 2005; revised 28 March 2005; accepted 30 March 2005
Available online 23 May 2005
Abstract—Two kinds of thiazo- or thiadiazo- naphthalene carboxamides with aminoalkyl side chains at 5- or 6-position modified from
naphthalimides were designed, synthesized and quantitatively evaluated as antitumor and DNA photocleaving agents. The compound with
aminoalkyl side chain at 6-position showed stronger antitumor (A549, P388) and DNA photocleaving abilities than its isomer with that at 5-
position. B2, the most efficient DNA photocleaver, also exhibited the highest cytotoxicity with the IC50 of 2.53 and 0.11 mM against cell lines
of A549 and P388, respectively. These compounds also photocleaved DNA more efficiently than their corresponding naphthalimides.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
proven actively2c,d in DNA photocleavage were remained.
The aminoalkyl side chains serving as a DNA groove binder
and/or external electrostatic binder were introduced into two
different regio-positions (5-, 6-) of the chromophores to
enhance the affinity to DNA and the effect of substitution
position on their bioactivities was also studied (Fig. 1).
The naphthalimide skeletons incorporated with substituents
or fused with (substituted) phenyl or heterocyclic rings form
an important class of compounds which is structurally
characterized by the presence of a planar chromophore
portion, possibly capable of intercalation into base pairs of
DNA, which resulted in high antitumor activity upon a
variety of murine and human tumor cells.1 Many of them
have also shown the capability of generating various
reactive intermediates that resulted in DNA photocleavage.2
Two kinds of phenyl thiazole (1, 2) or thiadiazole (3, 4)
conjugated naphthalimides have been reported to bind with
DNA and lead to DNA cleavage under photo-irradiation in
our previous work.2c,d These results promoted us to design
novel heterocyclic families by further modification of the
naphthalimide skeletons. Herein, we first proposed the
molecular modification of the reported heterocycle families,
1, 2 and 3, 4 through mercuric intermediates to their
corresponding ring opened models, phenyl thiazo- (A1–2
B
,
1–2) or thiadiazo- naphthalene carboxamides (C1–2, D1–2),
whose planar tri-cyclic ring systems may also potentially
intercalate into DNA to show medical or biological use.
Furthermore, the functional thiazo- or thiadiazo- groups
Keywords: Photocleavage; Antitumor; Intercalating.
*
Corresponding author. Tel.: C86 411 83673466; fax: C86 411
83673488; e-mail: xhqian@ecust.edu.cn
Figure 1. Structures of the reported and new photonucleases.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.135

Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
6635
2. Results and discussion
2.1. Synthesis and spectra
Structures of all the final products were well confirmed by
1H NMR, HRMS and IR spectra. Furthermore, the above
experiments provided two ways to synthesize different
region-isomers of heterocyclic conjugated naphthalene
carboxamides which may have potentials in other fields.
The replacement of one of the carbonyl groups by mercury
in (substituted) naphthalic anhydride has been known,3 but
the mercuric oxide mediated decarbonylation of 4-bromo-3-
nitro-1,8-naphthalic anhydride4 has not been reported.
When 4-bromo-3-nitro-1,8-naphthalic anhydride was
reacted with mercuric oxide (yellow or red) and acetic
acid according to the reported methods,3 two isomers, 4-
bromo-3-nitro-1-naphthoic acid and 4-bromo-3-nitro-8-
The UV–vis and fluorescent data for these compounds were
shown in Table 1. Slight difference was found between the
data of An and Bn, or Cn and Dn (nZ1, 2) of either
absorption wavelength or emission wavelength indicating
that the positions of aminoalkyl side chains had no obvious
effect on the spectra data.
1
naphthoic acid were obtained with the ratio of 4:1 via H
NMR. The acids were refluxed in water with sodium
disulfide for 8 h. The reaction mixture was then dropped
into glacial acetic acid containing benzaldehyde or 4-
methoxybenzaldehyde immediately and refluxed for further
4 h to give two phenylthiazole or 4-methoxy phenylthiazole
conjugated naphthoic acid isomers. Finally, the correspond-
ing naphthoyl chlorides were prepared from the acids by
refluxing with thionyl chloride in chloroform for 20 h and
then condensed with N,N-dimethylethylenediamine in
dichloromethane to form a pair of isomers, A1, B1 or A2,
B2. The isomers could be separated by careful column
chromatography (Scheme 1).
2.2. DNA-intercalating property
The Scatchard binding constants for compounds, A1, A2, B1,
B2, individually and calf thymus (CT) DNA (dissolved in
20 mM Tris–HCl, pH 7.5) were determined to be 6.39!104,
6.73!104, 1.22!105 and 1.29!105 MK1, respectively, by
the fluorescence quenching method5 (Fig. 2). The slight
difference in DNA binding constants between An and Bn
(nZ1, 2) suggested that positions of the carboxalimide side
chain in the chromophores almost had no effect on their
corresponding intercalating activities. However, the con-
stant of A2 or B2 was as twice higher as that of A1 or B1
suggesting the A2–DNA or B2–DNA complex was more
stable with the aid of hydrogen bond possibly formed
between the oxygen atom on the phenyl ring of A2 or B2 and
hydrogen atoms in base pairs of DNA molecule.
We noted that the decarbonylation of larger heterocyclic
conjugated naphthalic anhydride had not been reported yet.
So we tried an alternative strategy to synthesize the desired
products of C1–2 and D1–2. Thiadiazole conjugated
naphthalic anhydride was firstly synthesized,2d however,
the decarbonylation was only reacted with yellow mercuric
oxide in the way we used in the above experiment. Two
thiadiazole conjugated naphthoic acid isomers 18 and 19
The intercalation experiments of C1–2 and D1–2 were carried
out by using an electronic absorption spectra technique (Fig.
3) instead of fluorescence quenching method due to their
weak fluorescence. In the process of adding calf thymus
DNA into the compound solution, their absorption
intensities decreased with the increase of DNA
concentrations.
1
were obtained with the ratio of 1:1 by H NMR. The pure
desired compounds C1–2 and D1–2 were then synthesized
and purified by following the procedures described above
(Scheme 2).
Scheme 1. Synthesis of new compounds, A1–2 and B1–2. (a) HgO (yellow or red), NaOH, AcOH, H2O, reflux 96 h, 98% yield; (b) concentrated HCl, reflux 3 h,
88% yield; (c) Na2S2, H2O, 4 h; (d) benzaldehyde or 4-methoxybenzaldehyde, AcOH, N2, reflux 4 h; (e) SOCl2, CHCl3, Et3N; (f) N,N-
dimethylethylenediamine, CH2Cl2, 20 h.

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Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
Scheme 2. Synthesis of new compounds, C1–2 and D1–2. (a) Benzyl mercaptan, K2CO3, DMF, N2, 8 h, 93% yield; (b) SnCl2, HCl, 2 h, 80% yield; (c) NaNO2,
HCl, 0–5 8C, 3.5 h, 86% yield; (d) HgO (yellow), NaOH, AcOH, H2O, reflux 96 h, 95% yield; (e) concentrated HCl, reflux 2 h, 75% yield; (f) SOCl2, CHCl3,
Et3N; (g) corresponding RNH2, CH2Cl2, 20 h.
2.3. Antitumor evaluation
electrophoresis. The reaction mixture containing plasmid
DNA and each compound was put under a transluminator
(360 nm) at a distance of 20 cm at 0 8C for 3 h under aerobic
conditions, and no cleavages were observed without the
light irradiation. The photocleavage efficiency was defined
by the ratio of obtained nicked DNA (form II) converted
from supercoiled pBR322 DNA (form I). The different
cleavage efficiency orders of these compounds were
revealed as B2OB1OA2OA1 (Fig. 4a) and D2OC2O
D1OC1 (Fig. 4b). The effect of the position of carboxamide
side chain was obviously exhibited by the order of BnOAn,
DnOCn (nZ1 or 2) indicating that the compound with the
carboxalimide side chain at 6-position photocleaved DNA
more efficiently than its isomer with the side chain at 5-
position. Compounds B2 and D2, being more efficient than
their counterparts under the identical conditions, exhibited
detectable cleavage (21 and 27% form II) at concentration
of 0.5 and 5 mM, respectively. While the total cleavage of
supercolied plasmid DNA from form I to 100% form II by
B2 was found at the concentration of 50 mM. The
photocleavage abilities of B2 (Fig. 4e) and D2 (Fig. 4f)
increased remarkably with the prolongation of photo-
irradiation time, showing the cleavage a time-dependent
process. No damage was observed in the absence of the light
(lane 3), proving the function of UV light as a trigging factor
to initiate the DNA strand scission.
The antitumor activities of these new compounds against
human lung cancer cell (A549) and murine leukemia cell
(P388) were evaluated in vitro (under scattered light) as
shown in Table 2. The IC50 represents the drug concen-
tration (mM) required to inhibit cell growth by 50%. All
these compounds were found to be more cytotoxic against
P388 than against A549. The compound with the ami-
noalkyl side chain at 6-position inhibited the growth of
tumor cells more efficiently than its 5-position isomer which
was in agreement with the comparison of their DNA
photocleaving abilities (shown as follows). B2 exhibited the
highest cytotoxicity with the IC50 of 2.53 and 0.11 mM
against cell lines of A549 and P388, respectively. The
phenylthiazole conjugates showed stronger cytotoxicity
than thiadiazole conjugates. The modification of such
agents for potent antitumor drugs seemed to be not very
successful because some other reported efficient naphtha-
limides inhibited the tumor cells efficiently in nM or even
lower orders.1 However, these data also proved the
important role of the six-membered piperidinedione ring
in naphthalimides played on their antitumor activities.
2.4. DNA photocleavage
The cleavage abilities to supercoiled plasmid pBR322 DNA
by these compounds were evaluated by 1% agarose gel
Compared with that of B2, the concentration of the
Table 1. Spectra data of the synthesized compoundsa
Compound
R
X
PSCb
UV lmax (lg 3)
FL lmax
A1
A2
B1
B2
C1
C2
D1
D2
H
5
5
6
6
5
5
6
6
358 (3.43)
360 (4.11)
358 (3.46)
360 (4.15)
340 (3.21)
340 (3.39)
342 (2.96)
342 (3.25)
394
396
394
396
408
408
410
410
OCH3
H
OCH3
(CH2)2
(CH2)3
(CH2)2
(CH2)3
a In absolute DMSO.
b PSC indicates the position of the side chain.

Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
6637
Figure 2. Fluorescence spectra before and after interaction of compound B2
and calf thymus DNA. Curves F and F-CT correspond to compound B2
before and after being mixed with DNA. Numbers 1–4 indicated the
concentration of B2, 5, 10, 20, 40 mM, respectively. DNA applied was
50 mM (bp).
corresponding naphthalimide 1 required for detectable (19%
form II) or total cleavage of supercoiled plasmid DNA, was
5 or 100 mM reported in our previous work,2c while B2 was
0.5, 50 mM. Only 67% form II DNA was obtained by 1 at
50 mM. under identical conditions, 100 mM of D2 could
convert supercoiled DNA (form I) into 90% nicked DNA
(form II) while its corresponding naphthalimide 3 could
only convert 58%.2d Thus, we concluded that B2 and D2
exhibited stronger cleavage abilities than their correspond-
ing naphthalimide derivatives. Additionally, B2 (4-methyl-
oxyl phenyl thizaole) could obtain higher form II DNA ratio
(100%) even at lower concentration (50 mM) than that of D2
(thiadiazole) (90%, 100 mM) indicating the phenyl thizaole
group might be a better choice than thiadiazole group in
photocleavage area.
Figure 4. Photocleavage of closed supercoiled pBR322 DNA in the buffer
of Tris–HCl (20 mM, pH 7.5). (a, b) Photocleavage of plasmid pBR322
DNA by different compounds for 3 h. lane 1, DNA alone (no hn); lane 2,
DNA alone (hn 3 h); lanes 3–6, (a) compounds, A1, B1, A2, B2, (50 mM)
individually and DNA, (b) C1, D1, C2, D2 (100 mM) individually and DNA.
(c, d) Photocleavage of plasmid pBR322 DNA by B2 and D2 at various
concentrations for 3 h. Lane 1, DNA alone (no hn); lane 2, DNA alone (hn
3 h); lanes 3–8, (c) DNA and B2 at concentration of 0.5, 1, 5, 10, 20, 50 mM,
individually. (d) DNA and D2 at concentration of 1, 5, 10, 20, 50, 100 mM,
respectively. (e, f) Photocleavage of plasmid pBR322 DNA B2 and D2 at
various time intervals. Lane 1, DNA alone (no hn); lane 2, DNA alone (hn
3 h); lanes 3–7, (e) B2 (50 mM), (f) D2 (100 mM) and DNA at time intervals
of 0, 30, 60, 90, 120, 180 min, respectively.
In order to reveal the reactive species responsible for the
plasmid DNA cleavage, B2 and D2 were chosen as example
compounds to perform mechanistic experiments by addition
of histidine (singlet oxygen quencher), dithiothreitol (DTT,
superoxide radical scavenger), ethanol (radical scavenger),
respectively (Fig. 5a and b). It was clear that histidine had
no obvious effects on the cleavage reaction (lanes 4 and 7),
indicating singlet oxygen (1O2) was not involved in the
cleavage. However, DTT and ethanol obviously retarded
Figure 5. Effects of additives on the photocleavage of closed supercoiled
pBR322 DNA by (a) compound B2 (30 mM), (b) D2 (80 mM) in the buffer of
Tris–HCl (20 mM, pH 7.5) for 3 h. lane 1, DNA alone (no hn); lane 2, DNA
alone (hn 3 h); lanes 3 DNA and (a) compound B2, (b) D2, lanes 4–6, DNA
and (a) compound B2, (b) D2, in the presence of histidine (12 mM),
dithiothreitol (DTT, 60 mM), ethanol (3.4 M), respectively.
Figure 3. Interaction of D2 (50 mM) and calf thymus DNA. Absorption
changes of D2 during addition of calf thymus DNA (0, 50, 100, 200 mM) in
20 mM Tris–HCl (pH 7.5) solution.

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Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
Table 2. Cytotoxicity of compounds against the A-549, P388 cell lines
Compound
Cytotoxicity (IC50, mM)
A549a
P388b
A1
A2
B1
B2
C1
C2
D1
D2
206
20
12
2.53
517
320
15,000
1170
2.36
6.03
2.16
0.11
129
51.5
493
275
a Cytotoxicity (CTX) against human lung cancer cell (A549) was measured by sulforhodamine B dye-staining method.6
b CTX against murine leukemia cells (P388) was measured by microculture tetrazolium–formazan method.7
Figure 6. The simulated electron cloud densities of these compounds at excited triplet state by AM1 semi-empirical calculation.
photocleaving process as for both B2 and D2 (lanes 5 and 6),
suggesting that superoxide anion (OK2 ) and radical played
roles in the DNA cleavage. In our cases, the damage to DNA
by these compounds might be mainly by superoxide anions
generated through electron transfer from the chromophores
to oxygen, and partly by radicals produced by the phenyl
thiazole, thiadiazole group or/and the C]O bond via photo-
excited (n–p*) state under photo-irradiation.
3
The mechanism of B2 was different from that of its
corresponding naphthalimides which photocleaved the
plasmid only via radical produced in excited triplet state.2c
The electron densities of thiazo-naphthalene chromophores
were relatively higher than that of their corresponding
naphthalimides due to the reduction of one strong electron-
withdrawing group, carbonyl group. The higher electron
densities were inferred to grant them the abilities to transfer
electron from the chromophores to oxygen to form the
superoxide anion responsible for cleavage of the plasmid
under photo-activation. It must be the emergence of new
reactive species (superoxide anion) that accounted for the
mechanism change. The photocleavage ability of B2 also
increased with the emergence of active superoxide anion.
With AM1 semi-empirical quantum calculations
(Hyperchem7.0), the electron densities of these compounds
were obtained. The electron densities of one specific
compound at either excited singlet state or at triplet state
Figure 7. The simulated electron cloud densities of A2 and B2 at groud state
by AM1 semi-empirical calculation.

Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
6639
had slight difference. However, there seems to exist some
correlation between electron densities on the carbonyl
groups of these compounds at excited state, for example,
excited triplet state (Fig. 6) and their photocleavage
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.
abilities. The carbonyl groups of compounds, A1–2, C1–2
,
with the carboxalimide side chains at 5-position, had thicker
electron cloud, while that of compounds, B1–2, D1–2, with
the carboxalimide side chains at 6-position, had almost
‘naked’ electron cloud. The electron densities of these
compounds at ground state were also calculated which
showed no obvious difference between each pair of isomers
(Fig. 7, isomers of A2, B2 as examples). Comparing the
electron cloud at ground and excited states of A2 and B2, we
can see that the electron on the carbonyl group of B2 at
ground state was transferred to the thiazo-naphthalene
chromophore at excited state under activation, for example,
UV light. However, such electron transfer was not found at
the course of A2 which possibly exhibited the electron
transfer ability difference of the pair of isomers. The higher
electron transfer ability of B2, with the side chain at 6-
position, was believed to grant it stronger ability to produce
superoxide anion responsible for DNA cleavage than its
corresponding isomer, A2, with that at 5-position, which
accounted for the cleavage ability difference between the
pair of regio-isomers in the end.
4.2. Synthesis
4.2.1. Anhydro-8-hydroxylmercuri-1-naphthoic acids 5
and 6. 4-Bromo-3-nitro-1,8-naphthalic anhydride (9.66 g,
30 mmol) was suspended in 100 mL aqueous sodium
hydroxide (3.75 g, 94 mmol) and refluxed until the solid
material dissolved, a solution of HgO (yellow or red) (6.6 g)
in a mixture of H2O (10 mL) and AcOH (6 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 overnight to give
the mixture of 5 and 6 (15 g, 98% yield). Attempts to purify
and separate the anhydro compounds were unsuccessful. It
is insoluble in organic solvents.
4.2.2. 4-Bromo-3-nitro-1-naphthoic acid (7) and 4-
bromo-3-nitro-8-naphthoic acid (8). The obtained pre-
cipitates were suspended in 80 mL concentrated HCl,
stirred, heated under reflux for 3 h. Hot filtration gave the
mixture of 4-bromo-3-nitro-1-naphthoic acid 7 and 4-
bromo-3-nitro-8-naphthoic acid 8 (7.1 g, 88% yield) with
As to the cleavage efficiency order of A2OA1, B2OB1, the
methyloxyl group on phenyl ring not only have stronger
ability to push electron, then facilitate the electron transfer
from the chromophore to oxygen to form the reactive
superoxide anion, but also contribute to A2 or B02 stronger
intercalation ability, which both accounted for the above
orders.
1
ratio of 4:1 via H NMR. APCI-MS (negative) m/z: 293.9
(MKH)C.
4.2.3. 2-Phenyl naphtha[2,1-d]thiazole-5-acid (9) and 2-
phenyl naphtha[2,1-d]thiazole-6-acid (10). Na2S$9H2O
(4.32 g, 18 mmol) and S (1.152 g, 36 mmol) were refluxed
in 50 mL H2O for 0.5 h till S was all dissolved. The acids, 7
and 8 (1.18 g, 4 mmol) was added within 0.5 h and refluxed
for 8 h, the reaction mixture cooled and filtered to get the
dark red solution. Then it was dropped into glacial acetic
acid containing benzaldehyde (0.448 mL) immediately
under the protection of N2, and refluxed for 4 h, cooled
and poured into 1000 mL ice-water, filtered, washed with
water and dried under vacuum at 40 8C overnight to obtain
the brown solid mixture of phenyl thiazole conjugated
naphthalic acids 9 and 10 (0.75 g, 65% yield). APCI-MS
(negative) m/z: 306.0 (MKH)C.
3. Conclusion
The present work demonstrated the design, synthesis and
quantitative evaluation of two kinds of thiazo- or thiadiazo-
naphthalene carboxamides with the aminoalkyl side chains
at 5- or 6-position as antitumor and photocleaving agents.
All these compounds were found to be more active against
P388 than against A549. Compounds with aminoalkyl side
chains at 6-position showed stronger DNA photocleaving
and antitumor abilities than the corresponding isomers with
that at 5-position. Thiazole conjugates showed stronger
cleavage abilities than thiadiazole conjugates. B2, the most
efficient cleaver, showed detectable DNA cleavage at
0.5 mM and total cleavage from form I to form 100% II at
50 mM which was more efficient than its corresponding
naphthalimide. Mechanism experiment showed that elec-
tron transfer involving superoxide anion and radical were
involved.
4.2.4.
N-(2-(Dimethylamino)ethyl)-2-phenyl
naphtha[2,1-d] thiazole-5-carboxamide (A1) and N-(2-
(dimethylamino) ethyl)-2-phenyl naphtha[2,1-d]thia-
zole-6-carboxamide (B1). The acids 9 and 10 (0.75 g)
were treated with thionyl chloride (15 mL) and DMF (1
drop) in CHCl3 (15 mL) at reflux temperature for 20 h. After
removal of the solvent and excess thionyl chloride, the crude
solid and N,N-dimethyl ethyl diamine (0.5 mL) were
combined in 25 mL CH2Cl2. The mixture cooled in an ice
bath while Et3N (0.55 mL) was added dropwise with
stirring. The stirring continued for 20 h at room tempera-
ture. Removal of solvent and separation on silica gel
chromatography afforded the pure products. Separated on
silica gel chromatography (CH2Cl2/MeOHZ6:1, v/v) to get
pure A1 (0.45 g, 51% yield) and B1 (0.11 g, 12% yield).
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
1
Compound A1. Mp 186–187 8C. H NMR (d6-CDCl3) d
(ppm): 2.33 (s, 6H, NCH3), 2.66 (t, J1Z6 Hz, J2Z5.2 Hz,

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Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
2H, NCH2), 3.69 (d, J1Z4 Hz, 2H, CONHCH2), 7.02 (s,
1H, CONH), 7.52 (d, JZ5.2 Hz, 3H, 30-H, 40-H, 50-H), 7.62
(m, J1Z7.6 Hz, J2Z8 Hz, J3Z7.6 Hz, J4Z7.6 Hz, 2H,
7-H, 8-H), 8.05 (d, J1Z7.6 Hz, 9-H), 8.13 (t, J1Z2 Hz, J2Z
3.2 Hz, 2H, 10-H, 60-H), 8.26 (s, 1H, 4-H), 8.50 (d, JZ8 Hz,
1H, 6-H). HRMS (ESI): calcd for C22H22SN3O (MCH)C:
376.1484. Found: 376.1475. IR (KBr): 3285, 2921, 2850,
of 14 (1.81 g, 80%yield), mp 177–181 8C. APCI-MS
(positive) m/z: 336.0 (MCH)C.
4.2.8. Thiadiazole conjugated naphthalic anhydride (15).
The above solid (1.46 g), AcOH (57 mL), H2O (7 mL) and
concentrated HCl (8.5 mL) were stirred for 30 min at room
temperature, cooled with ice bath, 10 mL aqueous solution
of NaNO2 (0.31 g) was added within 20 min, and continued
to react for 3.5 h, filtered, washed with 1% aqueous solution
of NaHCO3 and dried to get yellow-green solid of 15
(0.97 g, 86% yield), mp 165–167 8C. APCI-MS (negative)
m/z: 255.2 (MKH)C.
1635, 1536, 761 cmK1
.
Compound B1. Mp 142–143 8C. 1H NMR (d6-CDCl3) d
(ppm): 2.33 (s, 6H, NCH3), 2.65 (t, J1Z6 Hz, J2Z5.6 Hz,
2H, NCH2), 3.68 (d, 2H, JZ5.2 Hz, CONHCH2), 6.93 (s,
1H, CONH), 7.51 (d, JZ5.2 Hz, 3H, 30-H, 40-H, 50-H), 7.59
(t, J1Z7.6 Hz, J2Z7.6 Hz, 1H, 8-H), 7.67 (d, JZ6.8 Hz,
1H, 9-H), 8.09 (d, JZ8 Hz, 7-H), 8.14 (d, JZ8.4 Hz, 3H,
20-H, 60-H, 4-H), 8.37 (d, JZ9.2 Hz, 1H, 5-H). HRMS
(ESI): calcd for C22H22SN3O (MCH)C: 376.1484. Found:
376.1487. IR (KBr): 3285, 2921, 2850, 1635, 1536,
4.2.9. Naphtha[2,1-d][1,2,3]thiadiazole-5-carboxylic acid
(18) and naphtha[2,1-d][1,2,3]thiadiazole-6-carboxylic
acid (19). Compound 15 (0.97 g) was suspended in 15 mL
aqueous sodium hydroxide (0.38 g, 9.5 mmol) and refluxed
until the solid material dissolved, a solution of HgO
(yellow) (0.67 g) in a mixture of H2O (5 mL) and AcOH
(1 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 then dried under vacuum at 50 8C
overnight to give the mixture of 16 and 17 (1.71 g, 95%
yield). The obtained precipitates were suspended in 15 mL
concentrated HCl, stirred, heated under reflux for 3 h. Hot
filtration gave the mixture acids of 18 and 19 (0.66 g, 75%
yield) with the ratio of 1:1 via 1H NMR. APCI-MS
(negative) m/z: 229.1 (MKH)C.
761 cmK1
.
4.2.5. N-(2-(Dimethylamino)ethyl)-2-(4-methoxyphenyl)
naphtha[2,1-d]thiazole-5-carboxamide (A2) and N-(2-
(dimethylamino)ethyl)-2-(4-methoxyphenyl) naphtha[2,
1-d]thiazole-6-carboxamide (B2). Prepared and purified in
a similar manner as that in A1 and B1, 4-methoxybenzalde-
hyde was used here instead of benzaldehyde and separated
on silica gel chromatography (CH2Cl2/MeOHZ4:1, v/v).
1
Compound A2. Mp 194–195 8C. H NMR (d6-CDCl3) d
(ppm): 2.92 (s, 6H, NCH3), 3.44 (s, 2H, NCH2), 3.86 (s, 3H,
OCH3), 3.98 (s, 2H, CONHCH2), 6.92 (d, JZ8.8 Hz, 2H,
30-H, 50-H), 7.46 (m, J1Z7.6 Hz, J2Z8.8 Hz, J3Z8.8 Hz,
J4Z6.4 Hz, 2H, 7-H, 8-H), 7.82 (d, JZ7.6 Hz, 6-H), 7.89
(d, JZ8.4 Hz, 2H, 20-H, 60-H), 8.30 (s, 1H, 4-H), 8.33 (d,
JZ8 Hz, 1H, 9-H), 8.57 (s, 1H, CONH). HRMS (ESI):
calcd for C23H24SN3O2 (MCH)C: 406.1545. Found:
406.1554. IR (KBr): 3285, 2921, 2850, 1635, 1606,
4.2.10. N-(2-(Dimethylamino)ethyl) naphtha[2,1-
d][1,2,3] thiadiazole-5-carboxamide (C1) and N-(2-
(dimethylamino)ethyl) naphtha[2,1-d][1,2,3] thiadia-
zole-6-carboxamide (D1). The acids 18 and 19 (0.56 g)
were treated with thionyl chloride (10 mL) and DMF (1
drop) in CHCl3 (10 mL) at reflux temperature for 20 h. After
removal of the solvent and excess thionyl chloride, the
obtained crude solid and propitiate amine (0.4 mL) were
combined in CH2Cl2 (25 mL). 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 tempera-
ture. Removal of solvent and separation on silica gel
chromatography afforded the pure products. Separated on
silica gel chromatography (CHCl3/MeOHZ1:1, v/v) to get
pure C1 (0.22 g, 31% yield) and D1 (0.29 g, 40% yield).
827 cmK1
.
Compound B2. Mp 154–155 8C. 1H NMR (d6-CDCl3) d
(ppm): 2.58 (s, 6H, NCH3), 2.84 (t, J1Z6 Hz, J2Z5.6 Hz,
2H, NCH2), 3.81 (d, JZ5.2 Hz, 2H, CONHCH2), 3.90 (s,
3H, OCH3), 7.01 (d, JZ8.4 Hz, 2H, 30-H, 50-H), 7.58 (t, 2H,
8-H, 9-H), 7.78 (s, 1H, 7-H), 8.07 (d, JZ8.4 Hz, 2H, 20-H,
60-H), 8.12 (d, JZ8.8 Hz, 4-H), 8.39 (d, JZ8.4 Hz, 1H,
5-H). HRMS (ESI): calcd for C23H24SN3O2 (MCH)C:
406.1545. Found: 406.1554. IR (KBr): 3285, 2921, 2850,
1
Compound C1. Mp 149–150 8C. H NMR (d6-CDCl3) d
1635, 1606, 827 cmK1
.
(ppm): 2.32 (s, 6H, NCH3), 2.65 (t, J1Z4.2 Hz, J2Z4.5 Hz,
2H, NCH2), 3.68 (d, JZ4.2 Hz, 2H, CONHCH2), 7.02 (s,
1H, CONH), 7.75 (m, J1Z6.8 Hz, J2Z7.2 Hz, J3Z6.0 Hz,
J4Z8 Hz, J5Z6.8 Hz, 2H, 7-H, 8-H,), 8.10 (d, JZ8 Hz, 1H,
9-H), 8.51 (d, JZ8 Hz, 6-H), 8.64 (s, 1H, 4-H). HRMS
(ESI): calcd for C15H17SN4O (MCH)C: 301.1123. Found:
301.1127. IR (KBr): 3266, 2918, 2849, 1638, 1548,
4.2.6. 4-Benzylmercapto-3-nitro-1,8-naphthalic anhy-
dride (13). 4-Bromo-3-nitro-1,8-naphthalic anhydride
(2.32 g, mmol), K2CO3 (0.52 g), benzylmercaptan
(0.88 mL) was dissolved in 45 mL DMF under the
protection of N2, the mixture reacted for 8 h at 80 8C,
cooled and poured into 1000 mL water, filtered and dried to
get yellow solid of 13 (2.47 g, 93% yield), mp 188–191 8C.
APCI-MS (negative) m/z: 400.0 (MCClK)C.
762 cmK1
.
1
Compound D1. Mp 129–130 8C. H NMR (d6-CDCl3) d
(ppm): 2.31 (s, 6H, NCH3), 2.63 (t, J1Z5.6 Hz, J2Z6 Hz,
2H, NCH2), 3.67 (d, 2H, JZ5.6 Hz, CONHCH2), 6.81 (s,
1H, CONH), 7.68 (t, J1Z6 Hz, J2Z7.8 Hz, 1H, 8-H), 7.84
(d, JZ7.2 Hz, 1H, 9-H), 8.16 (d, JZ8.4 Hz, 1H, 7-H), 8.48
(d, JZ9.6 Hz,1H, 4-H), 8.54 (d, JZ9.2 Hz,1H, 5-H).
HRMS (ESI): calcd for C15H17SN4O (MCH)C: 301.1123.
4.2.7. 4-Benzylmercapto-3-amino-1,8-naphthalic anhy-
dride (14). The above solid and SnCl2$2H2O (7.6 g) were
suspended in 30 mL concentrated HCl, raised the reaction
temperature to 90 8C and stirred for 2 h, cooled and poured
into 500 mL ice water, filtered and dried to get yellow solid

Z. Li et al. / Tetrahedron 61 (2005) 6634–6641
6641
Found: 301.1133. IR (KBr): 3285, 2921, 2850, 1635, 1536,
808 cmK1
4.5. Photocleavage of supercoiled pBR322 DNA
.
pBR322DNA (250 ng, form I), 1 mL solution of chemical in
DMSO 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. The samples were
analyzed by gel electrophoresis in 1% Agarose and gel was
stained with ethidium bromide.
4.2.11. N-(3-(Dimethylamino) propyl) naphtha[2,1-
d][1,2,3] thiadiazole-5-carboxamide (C2) and N-(3-
(dimethyl amino) propyl) naphtha[2,1-d][1,2,3]thiadia-
zole-6-carboxamide (D2). Prepared and purified in a
similar manner as that in C1 and D1, N,N-dimethylpropyl
diamine was used here instead of N,N-dimethylethyl
diamine and separated on silica gel chromatography
(CHCl3/MeOHZ1:2, v/v) to get pure C2 (33% yield) and
D2 (35% yield).
Acknowledgements
1
Compound C2. Mp 167–168 8C. H NMR (d6-CDCl3) d
Financial support by the National Key Project for Basic
Research (2003CB114400) and under the auspices of
National Natural Science Foundation of China is greatly
appreciated.
(ppm): 1.93 (t, J1Z6 Hz, J2Z6.4 Hz, CH2), 2.33 (s, 6H,
NCH3), 2.65 (t, J1Z5.6 Hz, J2Z6 Hz, 2H, NCH2), 3.70 (d,
2H, JZ5.6 Hz, CONHCH2), 7.73 (m, J1Z6.8 Hz, J2Z
7.2 Hz, J3Z6.0 Hz, J4Z8 Hz, J5Z6.8 Hz, 2H, 7-H, 8-H),
8.07 (d, JZ8 Hz, 1H, 9-H), 8.39 (s, 1H, CONH), 8.52 (d,
JZ8.4 Hz, 6-H), 8.58 (s, 1H, 4-H). HRMS (ESI): calcd for
C16H19SN4O (MCH)C: 315.1280. Found: 315.1278. IR
(KBr): 3274, 2940, 2856, 1634, 1541, 763 cmK1
.
References and notes
1
Compound D2. Mp 141–142 8C. H NMR (d6-CDCl3) d
(ppm): 1.94 (t, J1Z6 Hz, J2Z6 Hz, CH2), 2.33 (s, 6H,
NCH3), 2.65 (t, J1Z5.6 Hz, J2Z6.4 Hz, 2H, NCH2), 3.68
(d, 2H, JZ5.6 Hz, CONHCH2), 7.66 (t, J1Z7.2 Hz, J2Z
8 Hz, 1H, 8-H), 7.83 (d, JZ7.2 Hz, 1H, 9-H), 8.13 (d, JZ
8.4 Hz, 2H, 7-H, CONH), 8.52 (d, J1Z9.6 Hz, J2Z9.2 Hz,
2H, 4-H, 5-H). HRMS (ESI): calcd for C16H19SN4O (MC
H)C: 315.1280. Found: 315.1270. IR (KBr): 3290, 2940,
1. (a) Bran˜a, M. F.; Cacho, M.; Gradillas, A.; Pascual-Teresa, B.;
Ramos, A. Curr. Pharm. Des. 2001, 7, 1745. (b) Bran˜a, M. F.;
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(c) Sami, S. M.; Dorr, R. T.; Alberts, D. S.; Remers, W. A.
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2856, 1629, 1537, 804 cmK1
.
2. (a) Qian, X.; Yao, W. Tetrahedron Lett. 2001, 42, 6175. (b)
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4.3. Intercalation studies of compounds to CT-DNA
(a) A compound (0.1 mL solution, A1–2, B1–2) 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.
(b) A compound (0.25 mL solution, C1–2, D1–2) in DMSO
(10K3 M) mixed with 20 mM Tris–HCl (pHZ7.5) to 5 mL.
Then, one group of samples were prepared in the
concentration of chemical at 50 mM, one did not contain
Calf-thymus DNA as control, the others contained DNA
with the concentration of. 50, 100, 200 mM, respectively.
All the above solution was shaken for 3 days at 25 8C in the
dark. Absorption spectra of samples were measured.
4. Yao, W.; Qian, X. Dyes Pigments 2001, 48, 43.
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Vistica, D.; Warren, J. T.; Bokesc, H.; Kenney, S.; Boyd, M. R.
J. Natl Cancer Inst. 1990, 82, 1107.
4.4. Cytocoxicity in vitro evaluation
The prepared compounds have been submitted to Shanghai
Institute of Materia Medica for testing their cytotoxicities.
7. Kuroda, M.; Mimaki, Y.; Sashida, Y.; Hirano, T.; Oka, K.;
Dobashi, A.; Li, H.; Harada, N. Tetrahedron 1997, 53, 11549.