Synthesis of benzo-annulated tryptanthrins and their biological properties

Article abstract of DOI:10.1016/j.bmc.2012.06.034

A series of benzo-annulated derivatives of tryptanthrin were prepared and their optical and redox properties were studied. Tryptanthrin and its benzo-annulated derivatives showed selective inhibitory activity on topo I with an increase of activity on topo II by benzo-annulation on quinazolin-4(3H)-one moiety. Although the benzo-annulation on quinazolin-4(3H)-one ring did not affect significantly on the inhibitory activities against topo I and II, the benzoannulation on indolin-3-one ring affected the inhibitory activity very much especially by linear annulation. Cytotoxicities were not significantly changed upon benzoannulation, which were not directly related either to the inhibitory activities against topo I and II or to the reduction potentials.

Full text of DOI:10.1016/j.bmc.2012.06.034

Bioorganic & Medicinal Chemistry 20 (2012) 4962–4967
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of benzo-annulated tryptanthrins and their biological properties
a
b
b
a,
⇑
Jing Lu Liang , So-Eun Park , Youngjoo Kwon , Yurngdong Jahng
a
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
College of Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzo-annulated derivatives of tryptanthrin were prepared and their optical and redox prop-
erties were studied. Tryptanthrin and its benzo-annulated derivatives showed selective inhibitory activ-
ity on topo I with an increase of activity on topo II by benzo-annulation on quinazolin-4(3H)-one moiety.
Although the benzo-annulation on quinazolin-4(3H)-one ring did not affect significantly on the inhibitory
activities against topo I and II, the benzoannulation on indolin-3-one ring affected the inhibitory activity
very much especially by linear annulation. Cytotoxicities were not significantly changed upon benzoann-
ulation, which were not directly related either to the inhibitory activities against topo I and II or to the
reduction potentials.
Received 30 May 2012
Accepted 19 June 2012
Available online 2 July 2012
Keywords:
Tryptanthrin
Benzotryptanthrin
Topoisomerase I
Topoisomerase II
Isatin
Ó 2012 Elsevier Ltd. All rights reserved.
3
-Amino-2-naphthoic acid
Isatoic anhydride
Cytotoxicity
1
. Introduction
tryptanthrin.14 Electron accepting ability also plays an important
role in the antitumor activity for mitomycin C and adriamycin.
1
6
17
Tryptanthrin (1a) was first reported as a sublimation product of
indigo under reduced pressure and later was isolated as an indo-
Such a variety of intriguing physicochemical and biological
properties have led continuous efforts not only to find other plant
1
1
8
loquinazoline alkaloid from the culture of the yeast Candida lipoly-
sources but also to trigger the development of new methods for
2
19
20
tica and continuously from petroleum extract of dried and
the total synthesis as well as cultivation methods.
3
powdered fruit of Couroupita guaianensis Abul., and other higher
Although a series of tryptanthrin derivatives were reported, the
introduction of substituents was limited only to the two external
4
plant sources. Higher plants include Chinese traditional medicine
2
1
‘
Qing Dai’ (Isatis indigotica) which has long been used as an anti-
inflammatory agent. Isolated tryptanthrin showed various biologi-
benzene rings. Our interest in derivatization of simple alkaloids
2
2
as well as in the effect of benzoannulation on the chemical
g/mL),5 antimicrobial
and/or biological properties spurred us to prepare a series of
benzotryptanthrins and examine their physicochemical as well as
biological properties focusing on the relationships between the
reduction potentials, inhibitory activities on topoisomerases I and
II and cytotoxicities against selected human cancer cell lines.
23
cal properties such as antifungal (MIC = 5
l
6
7
(
MIC’s of 3.1–6.3 lg/mL), tuberculostatic (MIC = 10 lg/mL),
COX-2 inhibitory (IC50 = 64 nM),8 5-LOX inhibitory (IC50 = 0.15
9
10
11
l
M), NO synthase inhibitory, cytotoxic (10
l
M for HT-1376),
and antimalarial activities, and apoptosis by downregulation of
glutathione S-transferase(GST)
-expression.13
1
2
p
In addition, tryptanthrin has two characteristic reduction
2
2
. Results and discussion
potentials (À0.75 and À1.40 V vs SCE) for the reduction of two car-
1
4
bonyl groups, thus having potentials for photoelectronic photo-
.1. Chemistry
1
5
receptor. The more facile electron transfer to the oxygen atom
of the five-membered ring could be a plausible path in the mech-
anism of action indicating that electron accepting ability of the car-
bonyl atoms is crucial for the antileishmanial activity of
A series of benzotryptanthrins were prepared by employing
1
9j
previously reported method shown below. One-pot reaction of
isatin (2a) and its benzo-analogues (2b–d)
2
4–26
with anthranilic
acid (3a) and 3-amino-2-naphthoic acid (3b) in the presence of
SOCl2, respectively, afforded the corresponding tryptanthrin (1a)
and benzotryptanthrins (1b–h) in 51–84% yields.
⇑
Corresponding author.
E-mail addresses: ydjahng@yumail.ac.kr, ydjahng@yu.ac.kr (Y. Jahng).
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.034

J. L. Liang et al. / Bioorg. Med. Chem. 20 (2012) 4962–4967
4963
O
H
N
N
O
N
O
O
2a
1
a,b
O
N
NH
O
N
O
O
O
O
SOCl2
1c,d
2
b
OH
+
O
pyridine
NH2
O
3
O
N
N
H
N
2
c
1
e,f
O
O
N
HN
O
N
O
2
d
1
g,h
Alternatively, anions generated from isatin (2a) and its benzo-
annalogues (2b–d) were condensed with isatoic anhydride (4a)
and its benzo-annalogue (4b),27 respectively, to afford the corre-
sponding tryptanthrin (1a) and benzotryptanthrins (1b–h) in
somewhat lower yields. The prerequisite 4b were readily prepared
by reaction of 3-amino-2-napthoic acid with bis(trichloromethyl)
carbonate (triphosgene).
the more conjugated and linear systems. The electronic spectrum
⁄
of the most linear 1f showed a
p–p
absorptions band at 293 nm
(e 41,000) which is red-shifted from the parent tryptanthrin by
42 nm with little effect on the intensity of the absorption from
the parent 1a. It should be noted that benzoannulation led an addi-
tional absorption band in the region of 320–400 nm, of which 1d
showed the most intense and bathochromic shifted absorption
due to the additional benzene ring which is comparable to those
of 1f and 1h although 1f is the most linear one.
O
O
2
.2.2. Cyclic voltammetry
Cyclic voltammetry has been one of the efficient and powerful
N
H
O
NaH
DMF
electrochemical methods to provide reliable quantitative assess-
ment of electron transfer ability of the molecules. Reduction
4
2
a-d
1a-h
28
potentials of the compounds prepared were measured using
CV-50 W Voltammetric Analyzer by previously reported methods
and are summarized in Table 2. In every case, two reversible reduc-
tion waves in the region of À0.64 to À0.76 V and À1.29 to À1.46 V
versus SCE were observed. Camptothecin (CPT) also showed two
cathodic reduction peaks at À0.73 and À0.93 V for the ester car-
bonyl and lactam carbonyl, respectively, which are well matched
to those in the literature, while etoposide (ETO) did not show
any reduction potentials. The first reduction potentials covering
the carbonyl in the five-membered ring were less negative by
2
2
.2. Properties
.2.1. UV absorption
UV absorption spectra (Fig. 1) were obtained from EtOH
À5
(
2.5 Â 10 M) and the absorption maxima and extinction coeffi-
29
cients are summarized in Table 1. Four distinct absorption bands
are observed in the region of 225–230, 245–255, 280–375, and
75–420 nm which correspond quite closely to the
tions. The intensity of this band increases considerably for the
more linear compound where only a weak shoulder can be ob-
served at 500 nm. These data are consistent with an electronic
transition state in which the energy of the receptor
lowered by increasing delocalization which would be found for
⁄
3
p
–
p
absorp-
0
.07–0.11 V compared to that of tryptanthrin while the second
for the carbonyl in the six-membered ring by 0.04–120 mV. Such
a shift indicates that annulation of the benzene ring results in eas-
ier reduction compared to the parent tryptanthrin. The most planar
linear 1f showed the lowest reduction potentials À0.64 and
À1.18 V, respectively.
⁄
p
orbital is
.
O
:O :
OH
N
e-, H+
redox 1
+ e , H+
-
+
N
N
N
N
N
redox 2
O
HO
HO

4
964
J. L. Liang et al. / Bioorg. Med. Chem. 20 (2012) 4962–4967
Figure 1.
Table 1
UV absorption data for tryptanthrin and its benzo-annulated derivatives
À1
À1) (2.5 Â 10À5 mol in EtOH)
Compd
k
max
(e, cm
M
1
1
1
1
1
1
1
1
a
b
c
d
e
f
225 (31,900), 251 (47900), 279 (7,900), 310 (13,900), 330 (8900), 398 (7200)
220 (28,200), 259 (40,100), 284 (15,400), 294 (16,600), 353 (5900), 425 (7100)
222 (31,200), 277 (18,000), 465 (1300)
239 (34,200), 271 (21,600), 327 (5600), 389 (13,600)
231 (40,100), 270 (34,500), 290 (29,500), 346 (7900), 469 (1500)
232 (20,300), 232 (19,200), 274 (35,700), 293 (41,000), 339 (6300), 485 (2600)
227 (32,800), 274 (23,200), 291 (17,300), 346 (9700), 440 (2700)
230 (31,100), 243 (33,500), 276 (22,600), 286 (22,900), 302 (18,200), 328 (15,000), 412 (8000)
g
h
Table 2
Reduction potentials for tryptanthrin and its benzoannulated-derivatives in CH
3
CN (0.1 M TBAP) at 25 °C
Compd
E
1/2 V (vs SSCE)
Compound
E1/2 V (vs SSCE)
Reduction 1
Reduction 2
Reduction 1
Reduction 2
a
b
1
1
1
1
1
a
b
c
d
e
À0.75 (75)
À1.40 (73)
À1.36 (75)
À1.40 (76)
À1.35 (73)
À1.21 (74)
1f
1g
1h
À0.64 (74)
À0.64 (76)
À0.64 (76)
À1.18 (75)
À0.74 (73)
À0.68 (75)
À0.67 (73)
À0.64 (74)
À1.42 (73)
À1.34 (75)
b
b
Camptothecin
Etoposide
À0.73
À0.93
Not observed
a
Numbers in parentheses are the differences between the cathodic and anodic potentials and reported in mV.
Potentials for cathodic peak, anodic peak was not observed in CV.
b
Although redox properties of tryptanthrin and its benzo-annu-
selectivity on topo I of which the activity is comparable to that of
CPT at the 100 M level. Although the benzo-annulation did not
change the inhibitory activities on topo I, the activities on topo II
were significantly increased especially benzo-annulation on the
indolin-3-one ring (1d, e, and h).
lated derivatives were not directly related to the biological proper-
ties, the more readily reducible 1e and 1f showed somewhat
stronger cytotoxicity against selected cancer cell lines.
l
2
.2.3. Topoisomerase-inhibitory activity
Inhibitory activities of the compounds 1 against topoisomerases
2.2.4. Cytotoxicities
I (topo I) and II (topo II) were evaluated as compared to topo I
selective inhibitor, camptothecin and topo II selective inhibitor,
etoposide by assessing the relaxation of supercoiled pBR 322 plas-
mid DNA employing previously described method,30 and results
are summarized in Table 3. The parent tryptanthrin showed strong
Cytotoxicities of the compounds prepared were screened by the
3
1
method previously described against selected human cancer cell
lines such as ductal breast epithelial tumor cell line (T47D), colo-
rectal adrenocarcinoma on tumor (HCT15), prostate tumor
(DU145), and embryonic kidney 293 cells (HEK293) as compared

J. L. Liang et al. / Bioorg. Med. Chem. 20 (2012) 4962–4967
4965
Table 3
Inhibitory activity of 1 against topoisomerases I and II and their cytotoxicities against selected human cancer cell lines
Compd
Inhibitory activity (%)
Cytotoxicity [IC50
HCT15
(lM)]
Topo I
Topo II
T47D
DU145
HEK293
100
lM
20
l
M
100
lM
20 lM
1
1
1
1
1
1
1
1
a
b
c
d
e
f
68.44
18.79
47.79
39.10
69.56
41.85
53.41
84.13
63.10
4.09
N/T
4.54
15.96
8.13
71.26
75.93
12.60
14.04
53.45
N/T
N/T
N/T
5.83
5.95
N/T
N/T
14.29 ± 0.17
23.08 ± 0.87
31.31 ± 0.52
25.22 ± 0.46
21.50 ± 0.50
25.68 ± 0.01
22.75 ± 0.12
34.93 ± 0.77
9.17 ± 1.12
1.21 ± 0.13
1.82 ± 0.68
8.49 ± 0.02
25.76 ± 0.03
24.69 ± 0.09
32.78 ± 0.14
6.15 ± 0.23
26.21 ± 0.01
12.63 ± 0.13
23.96 ± 0.17
9.92 ± 0.76
0.28 ± 0.03
1.76 ± 0.15
6.22 ± 0.04
26.02 ± 0.01
27.67 ± 0.09
34.03 ± 0.35
17.75 ± 0.21
26.09 ± 0.01
21.39 ± 0.27
32.28 ± 0.04
9.29 ± 0.71
1.42 ± 0.19
1.57 ± 0.22
4.11 ± 0.11
31.52 ± 0.66
25.73 ± 0.02
24.22 ± 0.88
10.69 ± 0.03
2.31 ± 0.24
22.14 ± 0.72
20.32 ± 0.61
7.94 ± 2.02
0.41 ± 0.11
1.00 ± 0.05
4.90
3.80
5.40
3.60
4.10
3.50
g
h
19.05
CPT
ETO
ADR
26.30
78.91
36.08
to those of adriamycin (ADR), and the results are summarized in
Table 3. The parent tryptanthrin (1a) showed strong cytotoxicities
against all the cancer cell lines while a benzo-annulation did not
lead any significant increase of activity except 1e and 1f against
especially HCT15 and HEK293, respectively.
evaporated in vacuo. The resulting residue was treated with H
gives the desired product (165 mg, 71%) as red cubes or orange
2
O,
o
26
o
leaflets: mp 299–300 C (lit. mp 299–300 C). Unreported spec-
1
tral data were as follows: H NMR (250 MHz, DMSO-d
(br s, 1H, NH), 8.28 (s, 1H, H4), 7.98 (d, J = 8.0 Hz, 1H, H5), 7.80
d, J = 8.0 Hz, 1H, H8), 7.53 (td, J = 8.0, 1.2 Hz, 1H, H6), 7.33 (td,
J = 8.0, 1.0 Hz, 1H, H7), 7.19 (s, 1H, H9), 2.32 (s, 3H). C NMR
62.5 MHz, DMSO-d ) d 183.93, 145.25, 145.03, 138.49, 130.96,
29.61, 128.07, 126.94, 126.12, 123.92, 122.14, 105.41.
6
) d 10.43
(
13
3
. Conclusions
(
6
1
4
6
In conclusion, a series of benzotryptanthrins were prepared by
eitherone-potreactionofisatinandbenzoisatinswithanthranilicacid
and 3-amino-2-naphthoic acid or condensation of isatin and benzois-
atins with isatoic anhydride and benzoisatoic anhydride in 34–84%
yields. Tryptanthrin and its benzo-annulated derivatives showed
somewhat selective inhibitory activity on topo I with an increase of
activity on topo II by benzo-annulation on quinazolin-4(3H)-one moi-
ety. Although the benzo-annulation on quinazolin-4(3H)-one ring did
not affect significantly on the inhibitory activities against topo I and II,
the benzoannulation on indolin-3-one ring affected the inhibitory
activityverymuchespeciallybylinearannulation. Cytotoxicitieswere
not significantly changed upon benzoannulation, which were not di-
rectly related either to the inhibitory activities against topo I and II
or to the reduction potentials.
.1.2. 2H-Naphtho[2,3-d][1,3]oxazine-2,4(1H)-dione (4b)
To a stirred solution of 3-amino-2-naphthoic acid (1.12 g,
o
.0 mmol) in CH
3
CN (100 mL) at 50–55 C were added dropwise
pyridine (0.95 g, 12.0 mmol) and
a solution of triphosgene
(
0.59 g, 2.0 mmol) in CH Cl (20 mL) at the same time. After com-
2
2
pletion of the addition, the temperature of the reaction mixture
o
was maintained at 50–55 C for an additional 4 h. The solvent
was removed under reduced pressure and water (50 mL) was
added to the residue. The precipitated solid collected by filtration
was washed with water followed by chilled dichloromethane,
and dried in a vacuum dryer to yield 1.19 g (93%) of 2 as orange
o
27
o
powder: mp 396 C. (lit. mp >290 C). Unreported spectral data
1
are as follows: H NMR (CDCl
.74 (s, 1H), 8.13 (d, 1H, J = 8.3 Hz), 7.95 (d, 1H, J = 8.3 Hz), 7.66
ddd, 1H, J = 8.3, 8.0, 1.3 Hz), 7.50 (ddd, 1H, J = 8.3, 8.0, 1.3 Hz),
.49 (s, 1H), C NMR (62.5 MHz, CDCl ) d 159.87, 146.84, 136.93,
3
35.90, 131.67, 130.11, 129.62, 128.81, 126.82, 125.41, 110.96,
10.37.
3
, 250 MHz) d 11.81 (s, 1H, NH),
8
4
. Experimental
(
1
3
7
1
1
Melting points were determined using a Fischer-Jones melting
points apparatus and are not corrected. UV absorption spectra
were recorded on a JASCO-V550 spectrophotometer. IR spectra
were obtained using a Perkin–Elmer 1330 spectrophotometer.
4
.1.3. Benzo[g]indolo[2,1-b]quinazoline-6,14-dione (1b, benzo
NMR spectra were obtained using a Bruker-250 spectrometer
50 MHz or 600 MHz for 1H NMR and 62.5 MHz for C NMR and
13
[k]tryptanthrin)
.1.3.1. Method A.
0.10 g, 0.53 mmol) and 2a (74 mg, 0.50 mmol) in dry pyridine
20 mL) was added SOCl (0.2 mL). The resulting mixture was re-
2
4
To a solution of 3-amino-2-naphthoic acid
are reported as parts per million (ppm) from the internal standard
tetramethylsilane (TMS). Chemicals and solvents were commercial
reagent grade and used without further purification. The solvent
(
(
2
fluxed for 1 h and then poured to ice-water. Reaction mixture
was made basic with saturated NH OH and the precipitate formed
was collected to give the desired compound (125 mg, 84%): mp
(
CH
3
CN) used for cyclic voltammetry was dried and distilled over
. Elemental analyses were taken on a Hewlett–Packard Model
85B elemental analyzer. Reduction potentials were measured
using on a CV-50 W Voltammetric Analyzer with a C2 cell stand
Bioanalytical Systems, West Lafayette, IN, USA). The starting 1H-
4
2 5
P O
1
o
32
o
3
17 C. (lit. mp >300 C). Unreported spectral data are as follows:
1
3
H NMR (CDCl , 250 MHz) d 9.00 (s, 1H), 8.65 (d, 1H, J = 8.3 Hz),
(
24
8.53 (s, 1H), 8.12–8.04 (m, 2H), 7.92 (dd, 1H, J = 7.5, 1.0 Hz), 7.80
benzo[e]indole-1,2(3H)-dione (2b), 1H-benzo[g]indole-2,3-dione
2
5
26
(td, 1H, J = 8.3, 1.3 Hz), 7.73–7.64 (m, 2H), 7.41 (td, 1H, J = 8.3,
(2c), and 1-acetylbenz[5,6]isatin 2-oxime were prepared by
+
0
.8 Hz). MS (ESI) calcd for C19
11
H N
2
O
2
[M+H] 299, found 299.
employing previously reported methods.
4
.1.3.2. Method B.
A solution of isatin (2a, 0.10 g, 0.68 mmol)
4
4
.1. Chemistry
in dry DMF (5 mL) was slowly added to a mixture of NaH (20 mg,
.69 mmol) in dry DMF (7 mL). To the resulting purple solution
0
.1.1. 1H-Benzo[f]indole-2,3-dione (2d)
A mixture of 1-acetylbenz[5,6]isatin 2-oxime (300 mg.1.18 mmol)
was added compound 3b (0.15 g, 0.70 mmol). The reaction mixture
was stirred for 12 h and heated at 50 oC for 30 min. The yellow
in acetone (20 mL) and HCl (6 mL 1:1) was refluxed for 2 h, then

4
966
J. L. Liang et al. / Bioorg. Med. Chem. 20 (2012) 4962–4967
precipitate formed was collected and washed with CH
3
OH to give
[M+H]+ 439, found 439. Anal. calcd for C23
H N O C, 79.30; H,
12 2 2
the desired product (0.11 g). Concentration of the filtrate afforded
an additional product (0.05 g, 81% overall): mp 317 C. Spectral
3.47; N, 8.04. Found C, 79.36; H, 3.49; N, 8.02.
o
data are identical to those described above in Method A.
4.2. Cyclic voltammetry
4
.1.4. Benzo[4,5]indolo[2,1-b]quinazoline-8,14-dione (1c,
benzo[c]tryptanthrin)
.1.4.1. Method A.
CDCl , 250 MHz) d 8.96 (d, 1H, J = 8.8 Hz), 8.80 (d, 1H, J = 8.8 Hz),
.43 (dd, 1H, J = 8.0, 1.5 Hz), 8.27 (d, 1H, J = 8.8 Hz), 8.04 (d, 1H,
A glassy carbon working, a silver–silver chloride reference elec-
trode and a platinum auxiliary electrode were used in a 5 mL glass
cell. All samples were dissolved in dry CH CN (dried over P O )
3 2 5
o
1
4
(
8
Yellow needles (57%): mp 298 C. H NMR
3
with 0.1 M tetrabutylammonium hexafluorophosphate (Aldrich)
as the supporting electrolyte. The stock solution with each tryp-
tanthrin analogue was prepared at a concentration of 1 mM and
was degassed with nitrogen for 5 min prior to analysis. Samples
J = 7.5 Hz), 7.90 (d, 1H, J = 8.3 Hz), 7.83 (td, 1H, J = 8.0, 1.0 Hz),
7
1
1
1
1
.74 (td, 1H, J = 8.3, 1.3 Hz), 7.65 (td, 1H, J = 8.0, 1.3 Hz), 7.56 (td,
H, J = 8.0, 1.3 Hz). C NMR (CDCl
1
3
33
3
, 62.5 MHz) d 182.32, 157.94,
were run at several scans ranging from 20 to 1000 mV/s. Cyclic
48.90, 146.67, 144.05, 140.04, 135.15, 131.86, 131.19, 130.76,
voltammograms were recorded using A Hewlett–Packard X–Y re-
corder. Halfwave potentials (E1/2) were measured as the average
of the anodic and cathodic peak potentials.
30.20, 128.86 (two C’s), 127.66, 127.18, 124.29, 123.78, 116.00,
+
14.98. MS (ESI) calcd for C19
H
11
N
2
O
2
[M + H] 299, found 299.
10 2 2
Anal. calcd for C19H N O C, 76.50; H, 3.38; N, 9.39. Found C,
7
6.38; H, 3.41; N, 9.39.
4.3. Biology
4
.1.5. Benzo[g]benzo[4,5]indolo[2,1-b]quinazoline-8,16-dione
4.3.1. DNA relaxation assay of topoisomerase I
(
4
1d, dibenzo[c,k]tryptanthrin)
.1.5.1. Method A.
The test compounds were dissolved in DMSO at 10 mM as stock
solution. The activity of DNA topoisomerase I was determined by
assessing the relaxation of supercoiled DNA pBR322. The mixture
of 100 ng of plasmid pBR322 DNA and 0.2 units of calf thymus
DNA topoisomerase I (Fermentas, USA) was incubated without
and with the prepared compounds at 37 °C for 30 min in the relax-
Yellow powder (59%): mp >400 C. 1H
o
NMR (CDCl
1
3
, 250 MHz) d 9.06 (s, 1H), 8.87 (dd, 1H, J = 7.5,
.3 Hz), 8.78 (d, 1H, J = 7.5 Hz), 8.61 (s, 1H), 8.54 (d, 1H,
J = 7.5 Hz), 8.35 (dd, 1H, J = 7.5, 1.3 Hz), 8.24 (dd, 1H,
J = 7.5,1.3 Hz), 8.15 (d, 1H, J = 9.0 Hz), 7.86 (td, 1H, J = 7.5, 1.0 Hz),
7
.77 (td, 2H, J = 8.3, 1.3 Hz), 7.67 (td, 1H, J = 7.5, 1.3 Hz). MS (ESI)
ation buffer (35 mM Tris–HCl (pH 8.0), 72 mM KCl, 5 mM MgCl
5 mM dithiothreitol, 2 mM spermidine, 0.01% bovine serum albu-
min). The reaction in the final volume of 10 L was terminated
by adding 2.5 L of the stop solution containing 10% SDS, 0.2% bro-
2
,
+
calcd for C23
H
13
N
2
O
2
[M+H] 439, found 439. Anal. calcd for
C
7
23
H
12
N
2
O
2
C, 79.30; H, 3.47; N, 8.04. Found C, 79.48; H, 3.41; N,
l
.99.
l
mophenol blue, 0.2% xylene cyanol and 30% glycerol. DNA samples
were then electrophoresed on a 1% agarose gel at 15 V for 7 h with
a running buffer of TAE. Gels were stained for 30 min in an aqueous
4
.1.6. Benzo[5,6]indolo[2,1-b]quinazoline-6,14-dione (1e,
benzo[b]tryptanthrin)
.1.6.1. Method A.
CDCl , 250 MHz) d 9.00 (s, 1H), 8.65 (d, 1H, J = 7.8 Hz), 8.52 (s, 1H),
.12–8.03 (m, 2H), 7.79 (td, 1H, J = 7.8, 1.3 Hz), 7.69–7.64 (m, 2H),
o
1
4
(
8
7
Yellow powder (51%): mp 286 C. H NMR
solution of ethidium bromide (0.5 lg/mL). DNA bands were visual-
3
ized by transillumination with UV light and were quantitated using
TM
AlphaImager (Alpha Innotech Corporation).
+
11 2 2
.41 (t, 1H, J = 7.5 Hz). MS (ESI) calcd for C19H N O [M+H] 299,
found 299. Anal. calcd for C19
10
H N
2
O
2
C, 76.50; H, 3.38; N, 9.39.
4.3.2. DNA relaxation assay of topoisomerase II
Found C, 76.53; H, 3.35; N, 9.34.
The mixture of 100 ng of supercoiled pBR322 plasmid DNA and
0
.2 units of human DNA topoisomerase IIa (Amersham, USA) was
4
.1.7. Benzo[g]benzo[5,6]indolo[2,1-b]quinazoline-7,15-dione
incubated without and with the prepared compounds in the assay
buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 5 mM
(
1f, dibenzo[b,k]tryptanthrin)
Yellow powder (54%): mp >400 C. 1H
o
MgCl
min) for 30 min at 37 °C. The reaction in a final volume of 10
was terminated by the addition of 3 L of 7 mM EDTA. Reaction
4
.1.7.1. Method A.
2
, 1 mM EDTA, 1 mM ATP, and 15 lg/mL bovine serum albu-
NMR (DMSO-d
6
, 600 MHz) d 9.09 (s, 1H), 8.94 (s, 1H), 8.66 (s,
lL
1
8
7
H), 8.61 (s, 1H), 8.36 (d, 1H, J = 7.8 Hz), 8.24 (d, 1H, J = 9.0 Hz),
.23 (d, 1H, J = 9.0 Hz), 8.19 (d, 1H, J = 8.4 Hz), 7.80–7.73 (m, 3H),
.63 (td, 1H, J = 7.8, 1.2 Hz). MS (ESI) calcd for C23
l
products are analyzed on a 1% agarose gel at 25 V for 4 h with a
running buffer of TAE. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 lg/mL). DNA bands were visual-
13 2 2
H N O
C, 79.30; H,
+
[
M+H] 439, found 439. Anal. calcd for C23
H
12
N
2
O
2
3
.47; N, 8.04. Found C, 79.34; H, 3.46; N, 8.09.
ized by transillumination with UV light and supercoiled DNA was
quantitated using AlphaImager (Alpha Innotech Corporation).
TM
4
.1.8. Benzo[6,7]indolo[2,1-b]quinazoline-7,13-dione (1g,
Benzo[a]tryptanthrin)
.1.8.1. Method A.
CDCl , 250 MHz) d 9.11 (d, 1H, J = 7.5 Hz), 8.43 (d, 1H, J = 7.5 Hz),
.99 (d, 1H, J = 7.5 Hz), 7.90–7.79 (m, 4H), 7.71–7.60 (m, 3H). MS
Acknowledgements
o
1
4
(
7
Yellow powder (54%): mp 297 C. H NMR
3
Financial support from Korean Science Foundation (KRF-2008-
521-E00189) and Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2010-0002646)
are gratefully acknowledged.
+
(
ESI) calcd for C19
H
11
N
2
O
2
[M+H] 299, found 299. Anal. calcd for
C
9
19 10 2 2
H N O C, 76.50; H, 3.38; N, 9.39. Found C, 76.47; H, 3.40; N,
.41.
.1.9. Benzo[g]benzo[6,7]indolo[2,1-b]quinazoline-7,15-dione
4
References and notes
(
4
1h, dibenzo[a,k]tryptanthrin)
.1.9.1. Method A.
Yellow powder (56%): mp >400 C. 1H
o
1.
Sommaruga, E. Annales 1879, 165, 302.
NMR (CDCl
3
, 250 MHz) d 9.07 (s, 1H), 8.98 (d, 1H, J = 7.5 Hz), 8.57
2. Schindler, W.; Zähner, H. Arch. Mikrobiol. 1971, 79, 187.
3.
Sen, A. K.; Mahato, S. B.; Dutta, N. L. Tetrahedron Lett. 1974, 609. in which authors
(
s, 1H), 8.32 (d, 1H, J = 7.5 Hz), 8.24 (d, 1H, J = 7.5 Hz), 8.13 (d,1H,
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proposed a different structure with a trivial name couroupitine A. However the
structure of couroupitine A was corrected later by Bergman et al.
structure 1a originally proposed.
34
as the
1
H
13
N
2
O
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