Synthesis and antitumor evaluation of symmetrical 1,5-diamidoanthraquinone derivatives as compared to their disubstituted homologues

Article abstract of DOI:10.1248/cpb.54.458

A series of symmetrical 1,5-diamidoanthraquinone derivatives with potentially bioreducible groups has been synthesized and their cytostatic activity against the panel of various cancer cell lines in vitro has been studied. Preliminary structure-activity relationships were established. The results indicated that compounds 5 and 18 exhibited significant potent cytotoxicity at 1.24-1.75 μM for Hepa G2 cell line; compounds 5, 16, and 18 exhibited cytotoxicity at 0.14-1.82 μM for 2.2.15 cell line as determined by XTT colorimetric assay. Two structurally related compounds, mitoxantrone and adriamycin, were tested in parallel as positive controls. In addition, it was found that compounds 5 and 18 were a more potent and specific human hepatoma cell line than mitoxantrone and showed comparable activity to adriamycin. Among them, compound 18 was the most potent for 2.2.15 cells. We have demonstrated that the anthraquinone moiety is essential for activity and that less sterically hindered substituents contribute to enhanced in vitro efficacy. Implications for amidoanthraquinone cytotoxicity as potential anticancer agents are discussed. We further delineate the nature of the pharmacophore for this class of compounds, which provides a rational basis for the structure-activity relationships.

Full text of DOI:10.1248/cpb.54.458

458
Chem. Pharm. Bull. 54(4) 458—464 (2006)
Vol. 54, No. 4
Synthesis and Antitumor Evaluation of Symmetrical
1,5-Diamidoanthraquinone Derivatives as Compared to
Their Disubstituted Homologues
a
Hsu-Shan HUANG,*,a Hui-Fen CHIU,b Chi-Wei TAO,c and In-Been CHEN
a School of Pharmacy, National Defense Medical Center; No. 161, Minquan E. Rd., Taipei 11490, Taiwan, R.O.C.:
b Department of Pharmacology, Kaohsiung Medical University; Kaohsiung, 80708, Taiwan, R.O.C.: and c Cheng-Hsin
Medical Center; Taipei, 11258, Taiwan, R.O.C. Received September 9, 2005; accepted November 30, 2005
A series of symmetrical 1,5-diamidoanthraquinone derivatives with potentially bioreducible groups has
been synthesized and their cytostatic activity against the panel of various cancer cell lines in vitro has been stud-
ied. Preliminary structure–activity relationships were established. The results indicated that compounds 5 and
18 exhibited significant potent cytotoxicity at 1.24—1.75 mM for Hepa G2 cell line; compounds 5, 16, and 18 ex-
hibited cytotoxicity at 0.14—1.82 mM for 2.2.15 cell line as determined by XTT colorimetric assay. Two struc-
turally related compounds, mitoxantrone and adriamycin, were tested in parallel as positive controls. In addi-
tion, it was found that compounds 5 and 18 were a more potent and specific human hepatoma cell line than mi-
toxantrone and showed comparable activity to adriamycin. Among them, compound 18 was the most potent for
2.2.15 cells. We have demonstrated that the anthraquinone moiety is essential for activity and that less sterically
hindered substituents contribute to enhanced in vitro efficacy. Implications for amidoanthraquinone cytotoxicity
as potential anticancer agents are discussed. We further delineate the nature of the pharmacophore for this class
of compounds, which provides a rational basis for the structure–activity relationships.
Key words amidoanthraquinone; mitoxantrone; adriamycin; cytotoxicity; XTT colorimetric assay; structure–activity relation-
ship
In a continuation of our work on anthraquinones that vitro cytotoxicity in C6, Hepa G2 and 2.2.15 cell lines; the
widely occur in the plant kingdom and that may have biologi- results are presented in Table 1.
cal activities, we have developed and synthesized a series of
We recently described the human telomerase inhibition
small-molecule tricyclic anthraquinone structural motifs and cytotoxicity properties of a series of regioisomeric di-
which represents an attractive target for the rational design of functionalized anthraquinones at the 1,4-, 1,5- and 1,8-posi-
new anticancer agents.1—8) Antineoplastic drugs generally tions, respectively.3—6,14) Their in vitro cytotoxicity was re-
have a narrow therapeutic index and are delivered at doses ported and compared with those of their isomers, and it was
close to toxicity. Despite extensive and long-standing clinical proposed that their activity may be due to their ability to bind
utilization, the mechanisms responsible for the antiprolifera- to and stabilize G-quadruplex structures.8,15) The positional
tive and cytotoxic effects of anthracycline antibiotic doxoru- attachment and character of the diacyloxy-, dithio-, diamino-,
bicin are still uncertain and have been the subject of consid- and diamido side chains have been shown to profoundly in-
erable controversy.9) Several naturally occurring compounds fluence their mode of DNA binding and cytotoxicity. For ex-
having substituted anthraquinone moieties are widely used in ample, 1,4-diamidoanthraquinones have been shown to bind
cancer chemotherapy and constitute some of the most power- to duplex DNA by classical intercalation,16) whereas their cy-
ful cytostatics.10—12) In previous papers,1—6,13,14) we reported totoxicity and human telomerase inhibition,5,14) in which the
a series of anthraquinone derivatives (Chart 1) that showed in different functionalized side chains may simultaneously oc-
vitro anticancer activity, together with some of human telom- cupy both the DNA major and minor grooves, with intercala-
erase evaluation. The structure–activity relationships indi- tion of the planar chromophore.17) Biological results support-
cated amido substitution may lead to a different mechanism ing these structural motif and side arms have shown struc-
of cytotoxicity.5) Although an explanation for the mechanism ture–activity relationships. However, 1,4-diamido-isomers
has not yet emerged, the cytotoxic activity in cultured tumor were, in general (approximately 6 of 38 test compounds),
cell lines is well understood. We still envisioned a new ap- more cytotoxic than 1,5-diamido-, 1,5-diamino-, 1,5-diacy-
proach to the anthraquinone, taking advantage of the amido loxy-, or 1,5-dithio-substituted regioisomers in these three
side arms at different positions and pharmacophore moiety to cultured tumor cell lines. This has enabled us to address the
the pharmacophore that, if it is not active in the anticancer, issue of how, and to what extent, the position and their longer
can develop activity to produce a synergistic effect. The pri- side chains and better intercalation with DNA of side chain
mary aim for developing anticancer drugs has been for their substituents affects cytotoxic and telomerase activity. Their
cytotoxic and cytostatic properties and for their binding ca- in vitro cytotoxicity and some human telomerase inhibition
pacity to a recombinant fragment of the multi-drug-resist- properties are reported and compared with those of their 1,4-
ance transporter.12) These hypotheses suggest that the three- and 1,5-regioisomers in Table 2.
ring aromatic moiety gives DNA-intercalating ability to
In the present paper we describe the study of a further se-
cross-linkable side arms of substituents, and the an- ries of regioisomeric 1,5-diamidoanthraquinones. Hence,
thraquinone analog is considered as a possible antitumor lead amido substitution at anthraquinone may lead to a different
pharmacophore. We established a novel strategy to obtain mechanism of cytotoxicity, suppressing tumor growth mainly
new derivatives of anthraquinone and screened against the in by intercalation into DNA and inhibition of topoisomerase II,
∗ To whom correspondence should be addressed. e-mail: huanghs@ndmctsgh.edu.tw
© 2006 Pharmaceutical Society of Japan

April 2006
459
Chart 1
for the synthesis of target compounds. Compounds 2—27
were synthesized using a novel synthetic strategy (Chart 2).
Compounds 5—10 were synthesized by a two-stage reaction.
The first stage involved acylation of the 1,5-diaminoan-
thraquinone with chloroacyl chloride in DMF as a solvent,
with a catalytic quantity of pyridine, producing the 1,5-bis-
(w-chloroalkaneamido) side chain compounds 2—4 in es-
sentially quantitative yield. Further amination of the interme-
diate 2 with the appropriate primary and secondary amines in
DMSO gave the desired disubstituted anthraquinones 5—10.
Depending on the structure of the amine used further reac-
tions occurred, broadening the range of structures. Reaction
of the starting material with 2-chloropropionyl chloride
under similar conditions afforded compound 11 which was
used for the synthesis of compounds 12—18. Compounds
19—27 were synthesized by direct one-stage acylation reac-
tion. Structures of these compounds were obtained in good
yield and determined to be pure by 1H- and 13C-NMR. We re-
port here a simple and general methodology for the synthe-
sis; all compounds were used for the cytotoxicity studies and
telomerase inhibition which the results of which will be re-
ported elsewhere.
Biological Activity and Discussion
In previous papers, we reported a series of 1,4-, 1,5- and
1,8-difunctionalized substituted anthraquinone derivatives
that inhibit human telomerase and their cytotoxicity.1—6,14)
Chart 2
and is suspected of generating free radicals leading to DNA The present study details the preparation of 1,5-diamidoan-
strand scission.8,18—20) In addition, biophysical studies on a thraquinones and their in vitro cytotoxicity compared with
range of side arms and structure–activity relationships have those of other types of regioisomeric difunctionalized an-
been conducted in order to examine binding to the proposed thraquinones substituted at the 1,4-, 1,5- and 1,8-positions.
target structure formed by the cytotoxicity and telomerase in- These compounds with diamido side arms are assumed to
hibition.
provide better intercalation with DNA but the mechanisms
are still uncertain. Synthesized 1,5-diamidoanthraquinones
were tested against a panel of cancer cells (G2, 2.2.15. and
Chemistry
There has been continuing interest in the synthesis of natu- C6 cells) using XTT assay as described previously.5) Two
rally occurring anthraquinones largely on account of their bi- structurally related compounds, anthraquinone-based antitu-
ological activities. In this paper we report a very convenient mor agents mitoxantrone and adriamycin, were tested in par-
methodology for the synthesis of 1,5-difunctionalized ami- allel as positive controls. Both the control agents are highly
doanthraquinones with side chains having basic nitrogen cytotoxic in the three cell lines used in this study. The results
atoms. We have prepared two intermediates which are useful showed that some tested compounds inhibited the growth of

460
Vol. 54, No. 4
cancer cells at micromolar concentrations, and presented in played remarkable results; this is the 2-isopropylaminopropi-
Table 1 are the concentrations required to inhibit cell growth onamido-substituted derivative 18 which had an IC50 of
by 50% (IC50 values). The 1,5-diamidoanthraquinones de- 0.14 mM. Compounds 5 and 18 also showed good cytotoxicity
scribed here exhibit no cytotoxicity except for 5, 16, and 18, (with IC50 values of 1.24 and 1.75 mM, respectively). Of note,
which have IC50 values in the low-micromolar range com- by locating the disubstituents at the anthraquinone skeleton,
pared with mitoxantrone and adriamycin, against a panel of the activities of the 1,5-diamino isomers with disubstituted of
three cell lines (Murine C6, human Hepa G2, and 2.2.15). (CH2)2N(CH3)2 are significantly lower than other isomers,
However, the difference between the 1,4-isomer,5) 1,5- with IC50 values of 0.09 mM in Hepa G2 cell lines, 0.12 mM in
isomer1,2,4) and 1,8-isomers6) has a significant effect on the C6 cell lines and 0.13 mM in 2.2.15 cell lines, respectively.
shape of chromophore. Keppler et al.21) have indicated that Comparison of Tables 1 and 2 shows that the most two active
1,5-disubstituted anthraquinones interacted with duplex compounds in Hepa G cell lines, with an IC50 of 0.02 and
DNA in molecular modeling studies, which provide a ra- 0.04 mM, are two of the 1,5-diacyloxy-isomers.1) This sug-
tional basis for the SARs. By comparison, the amidoan- gests that for these derivatives, simple butyryloxy and 2-
thraquinones reported here are, in general, less effective as chlorobenzoyl side chains were essentially found to be the
cytotoxic agents, although two of the most active com- most active, with 1,5-bis(butyryloxy)anthraquinone indicat-
pounds, 5, and 18, are better than mitoxantrone in the Hepa ing about 100 times the potency of the positive control of mi-
G2 cell line; compound 18 is better than mitoxantrone and toxantrone and 45 times that for adriamycin. If such com-
adriamycin in the 2.2.15 cell line. Compounds 18 and 1,5-di- pounds are to have application as antitumor agents, with cy-
aminoanthraquinone with (CH2)2N(CH3)2 substitutents were totoxicity in tumor cell lines leading to the attrition of tumor
found to be the most potent of the compounds studied in the cell growth and consequent apoptosis, then conventional cy-
2.2.15 cell line (Table 2). Members of the 1,5-difunctional- totoxicity should be required. By contrast, the established an-
ized series (compounds 2—4, 6—15, 17, 19—27) are gener- thraquinone-based anticancer drugs mitoxantrone and adri-
ally less cytotoxic than their 1,4-isomers depending on the amycin, even though they show cytotoxicity at levels similar
character of side chains. Prior to the evaluation of types of to those exhibited by some compounds presented here (Table
test compounds in the XTT assay, the derivatives were tested 1). These represent some of the most potent non-nucleoside
for their ability to inhibit telomerase and cytotoxicity to ex- small molecule anthraquinones for Hepa G2 cell lines re-
amine the selectivity of cytotoxic versus telomerase inhibi- ported to date further investigation is worthwhile. Further-
tion. In addition, two of the most active compounds of 1,5- more, there does not appear to be any correlation between
diacyloxy isomers (compounds with CH2CH2CH3 disubsti- telomerase inhibition and cytotoxicity.3) The precise role of
tuted and 2-ClC6H4 disubstituted) are better than mitox- cytotoxicity has yet to be fully established, and thus its rele-
antrone and adriamycin in the Hepa G2 cell line. We were vance as a selective target for chemotherapy remains to be
unable to determine mechanisms from IC50 values for any proven. However, apart from the well-documented large dif-
type of 1,4-diamido-isomers, 1,5-diamido-isomers, 1,5-di- ferences in length between murine and human cytotoxicity, it
amino-isomers, 1,5-diacyloxy-isomers, 1,5-dithio-isomers, or is apparent there are a minority of mechanisms, and it is con-
1,8-diamido-isomers due to inhibition of tumor cell prolifer- ceivable that some of the selective murine and human tumor
ation at concentrations above those of mitoxantrone and cell lines operate by an analogous control process. In addi-
adriamycin. However, both drugs of 1,5-diacyloxy-isomers tion to this favorable feature, some compounds revealed in-
with CH2CH2CH3 disubstituted and 2-ClC6H4 disubstituted teresting binding properties and detail SARs, and reversed
showed cytotoxic activity against Hepa G2 cell lines at the the anthraquinone-based phenotype of cells, thus making
lowest concentrations.
them promising potential anti-tumor drugs. The activity of
In conclusion, we previously developed a series of iso- telomerase in human cells has not been clarified and the re-
meric symmetrical substituted anthraquinone derivatives as sults of such studies will be reported elsewhere.
duplex DNA-interactive agents, inhibiting telomerase as well
Experimental
as being known for their antitumor activities.1—6,13,14) The an-
titumor activity of anthraquinone derivatives on a panel of
Melting points were determined with a Büchi B-545 melting point appa-
ratus and are uncorrected. All reactions were monitored by TLC (silica gel
1
cancer cell lines during the last 30 years led investigators to
synthesize thousands of anthracycline analogs and test their
cytotoxicity to identify compounds superior to the parent
drugs in terms of increased therapeutic effectiveness, re-
duced toxicity or both. Perry and Neidle8) have shown that
amidoanthraquinones favoring duplex binding are related to
the inhibition of telomerase activity and assumed to be nons-
elective. This may be the necessary initial event that results
in telomerase inhibition and cytotoxicity. From a structure-
activity relationship point of view cytotoxicity is evident
from the remarkable results presented here and previously re-
ported. Compounds containing –(CH2)n– side chains termi-
nating in basic groups such as aminoalkyl-substituted,
showed cytotoxic activity in several cell lines.5) For example,
the most active compound from the XTT assay in hepatitis B
60 F254). H-NMR: Varian GEMINI-300 (300 MHz) and Brucker AM-500
(500 MHz); d values are in ppm relative to TMS as an internal standard.
Fourier-transform IR spectra (KBr): Perkin–Elmer 983G spectrometer. The
UV spectra were recorded on a Shimadzu UV-160A. Mass spectra (EI,
70 eV, unless otherwise stated): Finnigan MAT TSQ-46, Finnigan MAT
TSQ-700 (Universität Regensburg, Germany) and Finnigan MAT LCQ-MS
(National Research Institute of Chinese Medicine, Taipei, Taiwan). Typical
experiments illustrating the general procedures for the preparation of the an-
thraquinones are described below.
General Procedure for the Preparation of 1,5-Diamidoanthra-
quinones Method A (Compounds 2—10): Chloroacylchloride (12 mmol)
was added dropwise at 0 °C under N2 to a solution of 1,5-diaminoan-
thraquinone (1 mmol) and pyridine (0.5 ml) in N,N-dimethylformamide
(20 ml). The reaction mixture was stirred for 24 h at room temperature. The
resulting precipitate was collected by filtration, washed with diethyl ether
and purified by crystallization from ethyl acetate to afford desired compound
2—4. A solution of appropriate amine (20 mmol) in DMF was added drop-
wise under N2 to a suspended solution of 2 (1 mmol) in 40 ml of DMF and
virus transfected hepatoma cell lines (HepG 2.2.15) dis- triethylamine (0.5 ml). The reaction mixture was heated and refluxed at

April 2006
461
Table 1. Structures and Cytotoxic Activities of 1,5-Diamidoanthraquinones (2—27) in Suspended Murine and Human Tumor Cell Lines
IC50 (mM)a)
Compd.
No.
R
C6b)
Hepa G2c)
2.2.15d)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
CH2Cl
(CH2)2Cl
(CH2)3Cl
143.4ꢀ10.8
37.1ꢀ2.3
208.6ꢀ10.7
13.4ꢀ1.5
11.5ꢀ1.2
17.7ꢀ0.5
167.3ꢀ5.2
ꢁ200
208.6ꢀ7.8
86.5ꢀ5.3
52.5ꢀ1.8
36.6ꢀ2.1
63.6ꢀ1.9
32.9ꢀ4.7
ꢁ200
2.51ꢀ0.23
86.5ꢀ1.2
351.8ꢀ10.7
163.4ꢀ9.7
273.3ꢀ7.6
124.6ꢀ10.3
54.9ꢀ4.2
88.1ꢀ1.5
132.6ꢀ9.2
41.4ꢀ2.1
191.4ꢀ6.2
0.07ꢀ0.01
1.00ꢀ0.16
134.3ꢀ11.3
50.0ꢀ5.7
131.3ꢀ10.4
1.24ꢀ0.24
11.9ꢀ1.1
54.4ꢀ2.3
17.3ꢀ0.5
39.0ꢀ4.1
27.2ꢀ3.2
119.0ꢀ7.1
15.1ꢀ1.7
14.4ꢀ1.7
110.8ꢀ9.6
27.7ꢀ2.3
6.8ꢀ1.3
7.46ꢀ0.32
1.75ꢀ0.17
222.6ꢀ11.7
135.6ꢀ5.7
354.3ꢀ12.7
139.4ꢀ7.2
54.9ꢀ1.7
98.5ꢀ3.2
123.9ꢀ8.4
125.3ꢀ6.2
182.1ꢀ3.1
2.00ꢀ0.50
0.90ꢀ0.01
229.2ꢀ21.2
243.0ꢀ10.7
190.8ꢀ14.2
1.75ꢀ0.21
12.1ꢀ1.4
132.7ꢀ7.6
15.1ꢀ0.7
65.0ꢀ5.9
17.7ꢀ3.4
ꢁ200
CH2NHCH2CH3
CH2NH(CH2)2CH3
CH2NH(CH2)3CH3
CH2NH(CH2)4CH3
CH2NH(CH2)5CH3
CH2N(CH2CH3)2
(CH)CH3Cl
(CH)CH3NHCH2CH3
(CH)CH3NH(CH2)2CH3
(CH)CH3NH(CH2)3CH3
(CH)CH3NH(CH2)4CH3
(CH)CH3NH(CH2)5CH3
(CH)CH3NH(CH2)3N(CH3)2
(CH)CH3NHCH(CH3)2
CH3
21.2ꢀ1.1
8.4ꢀ1.2
109.9ꢀ2.4
185.4ꢀ9.5
1.82ꢀ0.17
11.00ꢀ0.26
0.14ꢀ0.02
350.6ꢀ6.7
56.3ꢀ4.1
278.4ꢀ9.7
201.0ꢀ6.1
212.6ꢀ3.6
231.0ꢀ8.4
213.9ꢀ9.2
207.4ꢀ10.7
219.1ꢀ9.1
0.40ꢀ0.02
1.60ꢀ0.04
CH2CH3
3-C6H4CH3
C6H10 (cyclohexane)
(CH2)2C5H9
C5H9
C3H5
trans-CH(CH2)CHC6H5
CH2SC6H5
Mitoxantrone
Adriamycin
a) IC50, drug concentration inhibiting 50% of cellular growth following 48 h of drug exposure. Values are in mM and represent an average of three experiments. The variance
for the IC50 values was less than ꢀ20%. Inhibition of cell growth was significantly different with respect to that of the control; nꢂ3 or more, pꢃ0.01. Inhibition was compared with
that of the control and standard errors. b) Hepa G2, human hepatoma G2 cells. c) C6 cells, rat glioma C6 cells. d) 2.2.15 cells, hepatitis B virus transfected hepatoma cell
lines, Hepa G 2.2.15 cells.
Table 2. In Vitro Cytotoxicity Data for Isomeric Anthraquinone Derivatives
IC50 (mM)a)
Isomers
R
C6b)
Hepa G2c)
2.2.15d)
1,5-diamido
CH2NHCH2CH3 (5)
(CH)CH3NH(CH2)5CH3 (16)
(CH)CH3NHCH(CH3)2 (18)
CH2CH2CH3
2-ClC6H4
CH2CH2C6H5
13.43ꢀ0.10
ꢁ200
86.48ꢀ1.21
38.5ꢀ2.8
40.7ꢀ4.7
40.1ꢀ5.5
1.24ꢀ0.24
6.76ꢀ1.36
1.75ꢀ2.17
0.02ꢀ0.01
0.04ꢀ0.01
0.4ꢀ0.1
1.75ꢀ0.01
1.82ꢀ0.17
0.14ꢀ1.92
ND
ND
ND
1,5-Diamido
1,5-Diamido
1,5-Diacyloxy1
1,5-Diacyloxy1
1,5-Diacyloxy1
1,5-Dithio2
CH2CH3
4-NH2C6H4
0.02ꢀ0.01
0.05ꢀ0.01
0.12ꢀ0.01
1.17ꢀ0.03
16.03ꢀ0.68
0.60ꢀ0.10
1.06ꢀ0.12
0.40ꢀ0.09
13.25ꢀ0.03
0.49ꢀ0.05
0.61ꢀ0.01
0.02ꢀ0.01
0.15ꢀ0.04
0.11ꢀ0.01
0.07ꢀ0.01
1.00ꢀ0.16
12.2ꢀ1.1
17.4ꢀ1.5
ND
ND
1,5-Dithio2
1,5-Diamino4
1,5-Diamino4
1,5-Diamino4
1,4-Diamido5
1,4-Diamido5
1,4-Diamido5
1,4-Diamido5
1,4-Diamido5
1,8-Diamino6
1,8-Diamino6
1,8-Diamino6
1,8-Diamino6
CH2CH2N(CH3)2
CH2CH2NH(CH2)2OH
CH2CH2CH3
CH2N(CH2CH3)2
CH2CH2N(CH2CH3)2
CH2CH2NHCH2CH(CH2)2
Cyclopropane
2,5-(CF3)2C6H3
CH2CH2CH3
CH2CH2OH
CH2CH2N(CH3)2
CH2CH2CH2CH2CH2NH2
0.09ꢀ0.01
1.20ꢀ0.02
1.74ꢀ0.14
1.43ꢀ0.24
0.89ꢀ0.02
1.01ꢀ0.01
0.53ꢀ0.01
6.51ꢀ0.07
0.19ꢀ0.01
16.0ꢀ0.1
0.13ꢀ0.01
7.01ꢀ0.11
1.94ꢀ0.02
1.98ꢀ0.01
1.15ꢀ0.04
6.04ꢀ0.59
2.61ꢀ0.03
6.57ꢀ0.06
1.06ꢀ0.03
91.25ꢀ0.8
8.55ꢀ0.09
1.29ꢀ0.06
0.40ꢀ0.02
1.60ꢀ0.04
0.16ꢀ0.04
0.09ꢀ0.01
2.00ꢀ0.50
0.90ꢀ0.01
Mitoxantrone
Adriamycin
For significant selective cytotoxicity against Hepa G2, human hepatoma G2 cells; C6 cells, rat glioma C6 cells; 2.2.15 cells, hepatitis B virus transfected hepatoma cell lines,
Hepa G 2.2.15 cells. ND: not determined. a) IC50, drug concentration inhibiting 50% of cellular growth following 48 h of drug exposure. Values are in mM and represent an aver-
age of three experiments. The variance for the IC50 values was less than ꢀ20%. Inhibition of cell growth was significantly different with respect to that of the control; nꢂ3 or more,
pꢃ0.01. Inhibition was compared with that of the control and standard errors. b) Hepa G2, human hepatoma G2 cells. c) C6 cells, rat glioma C6 cells. d) 2.2.15 cells, hepa-
titis B virus transfected hepatoma cell lines, Hepa G 2.2.15 cells.

462
Vol. 54, No. 4
130 °C in a miniclave (Büchi®) for 1 h. After cooling the reaction mixture
was treated with crushed ice and the resulting precipitate was collected by
filtration, washed and purified by crystallization from ethyl acetate to afford
desired compounds 5—10.
185.51 (C-9,10), 172.47 (NCO), 140.62 (C-1,5), 135.05 (C-4a,8a), 134.46
(C-3,7), 125.72 (C-5a,9a), 122.20 (C-4,8), 117.50 (C-2,6), 53.79 (CH2),
50.05 (CH2), 29.33 (CH2), 28.97 (CH2), 22.20 (CH2), 13.62 (CH3). IR
(KBr): 1495, 1647, 2918, 3339 cmꢄ1. PI-EI-MS m/z: 492 (Mꢅ), 393, 365,
266.
1,5-Bis(2-hexylaminoethylamido)anthraquinone (9) 76% yield. mp
204 °C (EA). 1H-NMR (CDCl3) d: 0.86 (6H, t, Jꢂ7.2 Hz, CH3), 1.23—1.41
(12H, m, CH2), 1.63—1.68 (4H, m, CH2), 2.72 (4H, t, Jꢂ7.2 Hz, CH2), 3.52
(4H, s, CH2), 7.76 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.08 (2H, dd, Jꢂ7.8, 1.2 Hz, Ar-
H), 9.21 (2H, dd, Jꢂ7.2, 1.2 Hz, Ar-H), 13.06 (2H, s, NH). 13C-NMR
(CDCl3) d: 185.52 (C-9,10), 172.50 (NCO), 140.67 (C-1,5), 135.03 (C-
4a,8a), 134.48 (C-3,7), 125.75 (C-5a,9a), 122.22 (C-4,8), 117.52 (C-2,6),
53.80 (CH2), 50.10 (CH2), 31.38 (CH2), 29.64 (CH2), 26.46 (CH2), 22.23
(CH2), 13.61 (CH3). IR (KBr): 1695, 2937, 3186 cmꢄ1. PI-EI-MS m/z: 520
(Mꢅ), 407, 292, 266.
Method B (Compounds 11—18): 2-Chloropropionyl chloride (12 mmol)
was added dropwise at 0 °C under N2 to a solution of 1,5-diaminoan-
thraquinone (1 mmol) and pyridine (0.5 ml) in N,N-dimethylformamide
(20 ml). The reaction mixture was stirred for 24 h at room temperature. The
resulting precipitate was collected by filtration, washed with diethyl ether
and purified by crystallization from ethyl acetate to afford desired compound
11. A solution of an appropriate amine (20 mmol) in DMF was added drop-
wise under N2 to a suspended solution of 11 (1 mmol) in 40 ml of DMF and
triethylamine (0.5 ml). The reaction mixture was heated and refluxed at
130 °C in a miniclave (Büchi®) for 1 h. After cooling the reaction mixture
was treated with crushed ice and the resulting precipitate was collected by
filtration, washed and purified by crystallization from ethyl acetate to afford
desired compounds 12—18.
1,5-Bis(N,N-diethylaminoethylamido)anthraquinone (10) 68% yield.
1
mp 224 °C (EA). H-NMR (CDCl3) d: 1.16 (6H, t, Jꢂ6.9 Hz, CH3), 2.72
(8H, q, Jꢂ7.2 Hz, CH2), 3.26 (4H, s, CH2), 7.74 (2H, t, Jꢂ8.7 Hz, Ar-H),
8.07 (2H, d, Jꢂ7.8 Hz, Ar-H), 9.20 (2H, d, Jꢂ8.7 Hz, Ar-H), 13.13 (2H, s,
NH). 13C-NMR (CDCl3) d: 185.55 (C-9,10), 173.48 (NCO), 140.87 (C-1,5),
135.21 (C-4a,8a), 135.02 (C-3,7), 125.97 (C-5a,9a), 122.49 (C-4,8), 118.06
(C-2,6), 59.16 (CH2), 48.70 (CH2), 12.17 (CH3). IR (KBr): 1695 cmꢄ1. PI-
EI-MS m/z: 464 (Mꢅ), 393, 86.
1,5-Bis(2-chloropropionylamido)anthraquinone (11) 70% yield. mp
288 °C (EA). 1H-NMR (CDCl3) d: 1.87 (3H, d, CH3), 4.60 (1H, q,
Jꢂ6.9 Hz, CH), 7.81 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.14 (2H, d, Jꢂ6.9 Hz, Ar-H),
9.12 (2H, d, Jꢂ8.7 Hz, Ar-H), 12.94 (2H, s, NH). 13C-NMR (CDCl3) d:
185.90 (C-9,10), 169.40 (NCO-1ꢆ,5ꢆ), 140.70 (C-1,5), 135.45 (C-4a,8a),
134.21 (C-3,7), 125.86 (C-5a,9a), 123.08 (C-4,8), 114.66 (C-2,6), 55.54
(CH), 22.05 (CH3). IR (KBr): 1680, 1705, 3192 cmꢄ1. EI-MS m/z: 418
(Mꢅ), 355, 264.7.
Method C (Compounds 19—27): Acyl chloride (12 mmol) was added
dropwise at 0 °C under N2 into a solution of 1,5-diaminoanthraquinone
(1 mmol) and pyridine (0.5 ml) in N,N-dimethylformamide (20 ml). The re-
action mixture was stirred for 24 h at room temperature. The resulting pre-
cipitate was collected by filtration, washed with diethyl ether and purified by
crystallization from ethyl acetate to afford desired compounds 19—27.
1,5-Bis(2-chloroacetamido)anthraquinone (2) 93% yield. mp 370 °C
(EA). 1H-NMR (CDCl3) d: 4.33 (4H, s, CH2), 7.80 (2H, t, Jꢂ8.1 Hz, Ar-H),
8.15 (2H, d, Jꢂ6.9 Hz, Ar-H), 9.12 (2H, d, Jꢂ8.7 Hz, Ar-H), 11.70 (2H, s,
NH). IR (KBr): 1684, 3200 cmꢄ1. EI-MS m/z: 392 (Mꢅ), 341, 264.9.
1,5-Bis(2-chloropropionamido)anthraquinone (3) 48% yield. mp
275 °C (EA). 1H-NMR (CDCl3) d: 3.01 (4H, t, Jꢂ6.3 Hz, CH2), 3.92 (4H, t,
Jꢂ6.6 Hz, CH2), 7.80 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.06 (2H, d, Jꢂ7.5 Hz, Ar-
H), 9.15 (2H, d, Jꢂ8.4 Hz, Ar-H), 12.39 (2H, s, NH). 13C-NMR (CDCl3) d
186.64 (C-9,10), 169.24 (NCO-1ꢆ,5ꢆ), 141.62 (C-1,5), 136.05 (C-4a,8a),
134.54 (C-3,7), 126.40 (C-5a,9a), 122.96 (C-4,8), 117.19 (C-2,6), 41.53
(CH2), 39.29 (CH2). IR (KBr): 1681, 3205 cmꢄ1. EI-MS m/z: 418 (Mꢅ), 382,
355, 328, 237.9.
1,5-Bis(4-chlorobutylamido)anthraquinone (4) 90% yield. mp 206 °C
(EA). 1H-NMR (CDCl3) d: 2.28—2.37 (4H, m, CH2), 2.82 (4H, t, Jꢂ7.2 Hz,
CH2), 3.75 (4H, t, Jꢂ6.3 Hz, CH2), 7.84 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.11 (2H,
dd, Jꢂ7.6, 1.1 Hz, Ar-H), 9.18 (2H, dd, Jꢂ8.7, 1.1 Hz, Ar-H), 12.39 (2H, s,
NH). 13C-NMR (CDCl3) d: 185.62 (C-9,10), 171.65 (NCO-1ꢆ,5ꢆ), 141,90
(C-1,5), 135.95 (C-4a,8a), 134.59 (C-3,7), 126.24 (C-5a,9a), 122.66 (C-4,8),
117.06 (C-2,6), 44.13 (CH2), 35.40 (CH2), 27.93 (CH2). IR (KBr): 1680,
3215 cmꢄ1. EI-MS m/z: 446 (Mꢅ), 342, 265, 238.
1,5-Bis(2-ethylaminoethylamido)anthraquinone (5) 77% yield, mp
163 °C (EA). 1H-NMR (CDCl3) d: 1.28 (6H, t, Jꢂ7.2 Hz, CH3), 2.79 (4H, q,
Jꢂ7.2 Hz, CH2), 3.53 (4H, s, CH2), 7.76 (2H, t, Jꢂ8.4 Hz, Ar-H), 8.09 (2H,
d, Jꢂ6.3 Hz, Ar-H), 9.21 (2H, d, Jꢂ7.2 Hz, Ar-H), 13.08 (2H, s, NH). 13C-
NMR (CDCl3) d: 185.96 (C-9,10), 172.87 (NCO), 141.09 (C-1,5), 135.46
(C-4a,8a), 134.90 (C-3,7), 126.14 (C-5a,9a), 122.66 (C-4,8), 117.93 (C-2,6),
53.97 (CH2), 44.73 (CH2), 15.25 (CH3). IR (KBr): 1685, 3330 cmꢄ1. EI-MS
m/z: 408 (Mꢅ), 351, 266.
1,5-Bis(2-propylaminoethylamido)anthraquinone (6) 80% yield. mp
172 °C (EA). 1H-NMR (CDCl3) d: 0.99 (6H, t, Jꢂ7.5 Hz, CH3), 1.64—1.74
(4H, m, CH2), 2.71 (4H, t, Jꢂ6.9 Hz, CH2), 3.52 (4H, s, CH2), 7.75 (2H, t,
Jꢂ8.1 Hz, Ar-H), 8.06 (2H, dd, Jꢂ7.8, 1.2 Hz, Ar-H), 9.20 (2H, d,
Jꢂ7.5 Hz, Ar-H), 13.04 (2H, s, NH). 13C-NMR (CDCl3) d: 185.92 (C-9,10),
172.91 (NCO), 141.04 (C-1,5), 135.46 (C-4a,8a), 134.86 (C-3,7), 126.13 (C-
5a,9a), 122.64 (C-4,8), 117.90 (C-2,6), 53.13 (CH2), 52.32 (CH2), 23.19
(CH2), 11.65 (CH2). IR (KBr): 1647, 3339 cmꢄ1. PI-EI-MS m/z: 437 (Mꢅ),
436, 365, 337, 266, 238, 72.
1,5-Bis(2-ethylaminopropionamido)anthraquinone (12) 86% yield.
1
mp 217 °C (EA). H-NMR (CDCl3) d: 1.26 (6H, t, Jꢂ6.9, CH3), 1.47 (6H,
d, Jꢂ5.7, CH3), 2.74 (4H, q, Jꢂ5.7, 1.5 Hz, CH2), 3.40 (2H, q, Jꢂ6.9 Hz,
CH), 7.76 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.10 (2H, dd, Jꢂ6.3, 1.2 Hz, Ar-H), 9.21
(2H, dd, Jꢂ7.2, 1.2 Hz, Ar-H), 13.08 (2H, s, NH). 13C-NMR (CDCl3) d:
186.01 (C-9,10), 176.35 (NCO), 141.36 (C-1,5), 135.44 (C-4a,8a), 134.98
(C-3,7), 126.14 (C-5a,9a), 122.60 (C-4,8), 118.06 (C-2,6), 59.92 (CH),
43.32 (CH2), 20.12 (CH3), 15.31 (CH3). IR (KBr): 1666, 2947, 3339 cmꢄ1
.
PI-EI-MS m/z: 436 (Mꢅ), 365, 266.
1,5-Bis(2-propylaminopropionamido)anthraquinone (13) 70% yield.
mp 203 °C (EA). 1H-NMR (CDCl3) d: 0.91 (6H, t, Jꢂ7.8, CH3), 1.33—1.40
(4H, m, CH2), 1.44 (4H, d, Jꢂ6.9 Hz, CH2), 1.58—1.69 (4H, m, CH2),
2.66—2.71 (4H, t, Jꢂ6.9 Hz, CH2), 3.38 (2H, q, Jꢂ6.9 Hz, CH), 7.76 (2H, t,
Jꢂ8.1 Hz, Ar-H), 8.10 (2H, dd, Jꢂ6.3, 1.2 Hz, Ar-H), 9.21 (2H, dd, Jꢂ7.5,
1.2 Hz, Ar-H), 13.05 (2H, s, NH). 13C-NMR (CDCl3) d: 185.97 (C-9,10),
176.38 (NCO), 141.35 (C-1,5), 135.42 (C-4a,8a), 134.97 (C-3,7), 126.14 (C-
5a,9a), 122.56 (C-4,8), 118.04 (C-2,6), 60.02 (CH), 50.95 (CH2), 23.29
(CH2), 20.09 (CH3), 11.71 (CH3). IR (KBr): 1671, 2851, 3157, 3320 cmꢄ1
.
PI-EI-MS m/z: 464 (Mꢅ), 463, 435, 379, 266.
1,5-Bis(2-butylaminopropionamido)anthraquinone (14) 70% yield.
1
mp 196 °C (EA). H-NMR (CDCl3) d: 0.95 (6H, t, Jꢂ7.8 Hz, CH3), 1.44
(6H, d, Jꢂ6.9 Hz, CH3), 1.54—1.69 (6H, m, CH2), 1.63—1.68 (4H, m,
CH2), 2.66 (4H, t, Jꢂ6.9 Hz, CH2), 3.38 (2H, q, Jꢂ6.9 Hz, CH), 7.76 (2H, t,
Jꢂ8.1 Hz, Ar-H), 8.10 (2H, dd, Jꢂ6.3, 1.2 Hz, Ar-H), 9.21 (2H, dd, Jꢂ7.5,
1.2 Hz, Ar-H), 13.05 (2H, s, NH). 13C-NMR (CDCl3) d: 186.42 (C-9,10),
176.78 (NCO), 141.80 (C-1,5), 135.88 (C-4a,8a), 135.42 (C-3,7), 126.59 (C-
5a,9a), 122.99 (C-4,8), 118.50 (C-2,6), 60.53 (CH), 49.18 (CH2), 32.72
(CH2), 20.82 (CH2), 20.53 (CH3), 14.42 (CH3). IR (KBr): 1642, 3444 cmꢄ1
.
EI-MS (APCI) m/z: 493.1 (Mꢅ), 491.4, 267.
1,5-Bis(2-pentylaminopropionamido)anthraquinone (15) 55% yield.
1,5-Bis(2-butylaminoethylamido)anthraquinone (7) 77% yield. mp
170 °C (EA). 1H-NMR (CDCl3) d: 0.93 (6H, t, Jꢂ7.5 Hz, CH3), 1.39—1.46
(4H, m, CH2), 1.60—1.69 (4H, m, CH2), 2.74 (4H, t, Jꢂ7.2 Hz, CH2), 3.53
(4H, s, CH2), 7.75 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.07 (2H, d, Jꢂ7.5 Hz, Ar-H),
9.20 (2H, d, Jꢂ8.7 Hz, Ar-H), 13.02 (2H, s, NH). 13C-NMR (CDCl3) d:
185.51 (C-9,10), 172.47 (NCO), 140.62 (C-1,5), 135.05 (C-4a,8a), 134.46
(C-3,7), 125.72 (C-5a,9a), 122.20 (C-4,8), 117.50 (C-2,6), 53.77 (CH2),
49.73 (CH2), 31.76 (CH2), 19.90 (CH2), 13.60 (CH3). IR (KBr): 1647, 2354,
2928, 3339 cmꢄ1. PI-EI-MS m/z: 464 (Mꢅ), 379, 351, 266.
1,5-Bis(2-pentylaminoethylamido)anthraquinone (8) 78% yield. mp
190 °C (EA). 1H-NMR (CDCl3) d: 0.89 (6H, t, Jꢂ7.2 Hz, CH3), 1.23—1.36
(8H, m, CH2), 1.64—1.68 (4H, m, CH2), 2.72 (4H, t, Jꢂ6.9 Hz, CH2), 3.52
(4H, s, CH2), 7.75 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.08 (2H, d, Jꢂ7.8 Hz, Ar-H),
9.21 (2H, d, Jꢂ8.7 Hz, Ar-H), 13.05 (2H, s, NH). 13C-NMR (CDCl3) d:
1
mp 208 °C (EA). H-NMR (CDCl3) d: 0.87 (6H, t, Jꢂ7.2 Hz, CH3), 1.24—
1.52 (18H, m, CH2, CH3), 2.67 (4H, t, Jꢂ7.2 Hz, CH2), 3.38 (2H, q,
Jꢂ6.9 Hz, CH), 7.76 (2H, t, Jꢂ8.4 Hz, Ar-H), 8.09 (2H, d, Jꢂ7.8 Hz, Ar-H),
9.22 (2H, d, Jꢂ8.4 Hz, Ar-H), 13.07 (2H, s, NH). 13C-NMR (CDCl3) d:
186.96 (C-9,10), 176.40 (NCO), 141.36 (C-1,5), 135.44 (C-4a,8a), 135.44
(C-3,7), 126.14 (C-5a,9a), 122.56 (C-4,8), 118.41 (C-2,6), 60.11 (CH),
49.08 (CH2), 29.85 (CH2), 29.47 (CH2), 22.62 (CH2), 20.14 (CH3), 14.03
(CH3). IR (KBr): 1647, 1680, 2918, 3425 cmꢄ1. PI-EI-MS m/z: 520 (Mꢅ),
407, 266.
1,5-Bis(2-hexylaminopropionamido)anthraquinone (16) 70% yield.
1
mp 184 °C (EA). H-NMR (CDCl3) d: 0.84 (6H, t, Jꢂ6.6 Hz, CH3), 1.27—
1.68 (22H, m, CH2, CH3), 2.67 (t, 4H, Jꢂ7.2 Hz, CH2), 3.38 (q, 2H, Jꢂ6.9,
CH), 7.76 (t, 2H, Jꢂ8.1 Hz, Ar-H), 8.09 (d, 2H, Jꢂ7.5 Hz, Ar-H), 9.22 (d,

April 2006
463
m/z: 430.9 (Mꢅ), 335, 317.
2H, Jꢂ8.4 Hz, Ar-H), 13.06 (s, 2H, NH). 13C-NMR (CDCl3) d: 186.36 (C-
9,10), 176.83 (NCO), 141.75 (C-1,5), 135.86 (C-4a,8a), 135.34 (C-3,7),
126.55 (C-5a,9a), 122.99 (C-4,8), 118.41 (C-2,6), 60.52 (CH), 49.54 (CH2),
32.20 (CH2), 30.53 (CH2), 27.36 (CH2), 23.04 (CH2), 20.57 (CH3), 14.45
(CH3). IR (KBr): 1680, 2918, 3435 cmꢄ1. APCI-EI-MS m/z: 549 (Mꢅ), 281,
266.
1,5-Bis(cyclopropylamido)anthraquinone (25) 75% yield. mp 328 °C
1
(EA). H-NMR (CDCl3) d: 0.92—0.98 (4H, m, CH2), 1.12—1.17 (4H, m,
CH2), 1.75—1.79 (2H, m, CH), 7.75 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.02 (2H, dd,
Jꢂ6.6, 1.2 Hz, Ar-H), 9.11 (2H, d, Jꢂ9 Hz, Ar-H), 12.52 (2H, s, NH). 13C-
NMR (CDCl3) d: 186.28 (C-9,10), 173.24 (NCO), 141.81 (C-1,5), 135.44
(C-4a,8a), 134.23 (C-3,7), 125.82 (C-5a,9a), 121.85 (C-4,8), 116.40 (C-2,6),
16.64 (CH), 8.29 (CH2). IR (KBr): 1680, 2342, 2368, 3454 cmꢄ1. EI-MS
m/z: 374.2 (Mꢅ), 306.3, 238.4.
1,5-Bis(N,N-dimethylaminopropylpropionamido)anthraquinone (17)
1
40% yield. mp 190 °C (EA). H-NMR (CDCl3) d: 0.91 (12H, t, Jꢂ7.8 Hz,
CH3), 1.33—1.40 (4H, m, CH2), 1.44 (4H, d, Jꢂ6.9, CH2), 1.58—1.69 (4H,
m, CH2), 2.74 (4H, t, Jꢂ6.9 Hz, CH2), 3.38 (2H, q, Jꢂ6.9 Hz, CH), 7.75
(2H, t, Jꢂ8.4 Hz, Ar-H), 8.07 (2H, dd, Jꢂ7.8, 1.2 Hz, Ar-H), 9.21 (2H, d,
Jꢂ7.5 Hz, Ar-H), 13.08 (2H, s, NH). 13C-NMR (CDCl3) d: 185.91 (C-9,10),
176.36 (NCO), 141.35 (C-1,5), 135.43 (C-4a,8a), 134.89 (C-3,7), 126.11 (C-
5a,9a), 122.51 (C-4,8), 117.96 (C-2,6), 60.09 (CH), 58.09 (CH2), 47.65
(CH2), 45.56 (CH3), 28.00 (CH3), 20.05 (CH3). IR (KBr): 1661, 2938,
3430 cmꢄ1. PI-EI-MS m/z: 550 (Mꢅ), 505, 492, 450, 129.
1,5-Bis(trans-2-pheny-1-cyclopropanecarboxamido)anthraquinone
(26) 75% yield. mp 249 °C (EA). 1H-NMR (CDCl3) d: 1.43—1.49 (4H, m,
CH2), 1.75—1.78 (2H, m, CH), 1.99—2.02 (2H, m, CH), 7.16—7.33 (10H,
m, CH) 7.75 (2H, t, Jꢂ8.4 Hz, Ar-H), 8.01 (2H, d, Jꢂ7.8 Hz, Ar-H), 9.15
(2H, d, Jꢂ8.4 Hz, Ar-H), 12.55 (2H, s, NH). 13C-NMR (CDCl3) d: 186.64
(C-9,10), 171.97 (NCO), 142.09 (C-1,5), 140.17 (C), 135.44 (C-4a,8a),
134.60 (C-3,7), 128.60 (CH), 126.59 (CH), 126.24 (CH), 126.32 (C-5a,9a),
122.44 (C-4,8), 116.58 (C-2,6), 28.56 (CH), 26.74 (CH), 17.19 (CH2). IR
(KBr): 1633, 1676, 3224, 3435 cmꢄ1. APCI-EI-MS m/z: 527.1 (Mꢅ), 509.5,
383.2, 365.4.
1,5-Bis(phenylthioacetamido)anthraquinone (27) 70% yield. mp
197 °C (EA). 1H-NMR (CDCl3) d: 3.87 (4H, s, CH2), 7.17 (2H, t, Jꢂ6.9 Hz,
Ar-H), 7.26 (2H, t, Jꢂ7.2 Hz, Ar-H), 7.45 (2H, d, Jꢂ7.8 Hz, Ar-H), 7.75
(2H, t, Jꢂ5.1 Hz, Ar-H), 8.06 (2H, d, Jꢂ7.8 Hz, Ar-H), 9.09 (2H, d,
Jꢂ8.4 Hz, Ar-H), 12.92 (2H, s, NH). 13C-NMR (CDCl3) d: 186.16 (C-9,10),
168.80 (NCO), 141.27 (C-1,5), 135.68 (C-4a,8a, SC), 134.66 (C-3,7),
129.81 (C-5a,9a), 129.26 (C-4,8), 127.11 (CH), 126.25 (CH), 123.09
(CH,C-2,6), 40.27 (CH2). IR (KBr): 1614, 3301, 3416 cmꢄ1. EI-MS m/z: 538
(Mꢅ), 428.2, 415.2, 388.2, 305.4, 238.4.
Cell Culture Various cancer cell lines (G2, 2.2.15. cells and C6 cells)
were cultured in minimum essential medium (MEM), supplemented with
10% fetal calf serum, 100 units/ml penicillin and 100 mg/ml streptomycin in
a humidified atmosphere in 5% CO2 at 37 °C. Cell culture media were re-
newed every three days, up to the confluence of the monolayer. The cell cul-
ture was passaged when they had formed confluent cultures, using trypsin-
EDTA to detach the cells from their culture flasks or dishes. Test compounds
were stored at ꢄ70 °C and solublized in 100% DMSO. All the drug solu-
tions were prepared immediately before the experiments and were diluted in
complete medium before addition to cell cultures. All data presented in this
report are from at least three independent experiments showing the same
pattern of expression.
1,5-Bis(2-isopropylaminopropionamido)anthraquinone
(18) 65%
1
yield. mp 271—273 °C (EA). H-NMR (CDCl3) d: 1.17 (6H, d, Jꢂ8.7 Hz),
1.34 (6H, d, Jꢂ6.6 Hz, CH3), 1.43 (6H, d, Jꢂ7.2 Hz, CH3), 2.85—2.93 (2H,
m, CH), 3.46 (2H, q, Jꢂ7.2 Hz, CH), 7.75 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.08
(2H, d, Jꢂ7.8 Hz, Ar-H), 9.21 (2H, d, Jꢂ8.4 Hz, Ar-H), 13.13 (2H, s, NH).
13C-NMR (CDCl3) d: 185.81 (C-9,10), 177.12 (NCO), 141.22 (C-1,5),
135.38 (C-4a,8a), 134.92 (C-3,7), 125.99 (C-5a,9a), 122.50 (C-4,8), 117.97
(C-2,6), 57.41 (CH), 48.40 (CH), 23.26 (CH3), 22.87 (CH3), 20.69 (CH3). IR
(KBr): 1662, 2940, 3420 cmꢄ1. PI-EI-MS m/z: 464 (Mꢅ), 266.
1,5-Bis(methylacetamido)anthraquinone (19) 45% yield. mp 321 °C
(EA). 1H-NMR (CDCl3) d: 2.33 (6H, s, CH3), 7.76 (2H, t, Jꢂ8.7 Hz, Ar-H),
8.03 (2H, d, Jꢂ7.8 Hz, Ar-H), 9.12 (2H, d, Jꢂ8.7 Hz, Ar-H), 12.25 (2H, s,
NH). 13C-NMR (CDCl3) d: 186.61 (C-9,10), 169.89 (NCO-1ꢆ,5ꢆ), 142.04
(C-1,5), 135.90 (C-4a,8a), 134.57 (C-3,7), 126.15 (C-5a,9a), 122.48 (C-4,8),
116.96 (C-2,6), 25.70 (CH3). IR (KBr): 1637, 1702, 3209 cmꢄ1. APCI-EI-
MS m/z: 323 (Mꢅ), 281, 238.
1,5-Bis(ethylacetamido)anthraquinone (20) 70% yield. mp 265 °C
(EA). 1H-NMR (CDCl3) d: 1.33 (6H, t, Jꢂ7.5 Hz, CH3), 2.57 (4H, q,
Jꢂ7.5 Hz, CH2), 7.75 (2H, t, Jꢂ8.1 Hz, Ar-H), 8.01 (2H, dd, Jꢂ6.6, 1.2 Hz,
Ar-H), 9.14 (2H, dd, Jꢂ7.2, 1.2 Hz, Ar-H), 12.26 (2H, s, NH). 13C-NMR
(CDCl3) d: 186.62 (C-9,10), 173.79 (NCO-1ꢆ,5ꢆ), 142.15 (C-1,5), 135.87
(C-4a,8a), 134.57 (C-3,7), 126.17 (C-5a,9a), 122.39 (C-4,8), 116.96 (C-2,6),
31.94 (CH2), 9.52 (CH3). IR (KBr): 1656 cmꢄ1. EI-MS m/z: 350 (Mꢅ), 294,
265, 238.
XTT Method The tetrazolium reagent (XTT) was designed to yield a
suitably colored, aqueous-soluble, non-toxic formazan upon metabolic re-
duction by viable cells. Approximately 2ꢇ103 cells, suspended in MEM
medium, were plated in each well of a 96-well plate and incubated in 5%
CO2 at 37 °C for 24 h. Test compounds were then added to the culture
medium at designated various concentrations. After 72 h, fresh XTT 50 ml
and electron coupling reagent (PMS) 1 ml were mixed together, and 50 ml of
this mixture was added to each well. After an appropriate incubation at
37 °C for 6 h, the absorbency at 490 nm was measured with the ELISA
reader.
1,5-Bis(3-methylbenzamido)anthraquinone (21) 63% yield. mp
290 °C (EA). H-NMR (CDCl3) d: 2.55 (6H, s, CH3), 7.48—7.52 (6H, m,
1
Ar-H), 7.89 (2H, t, Jꢂ7.5 Hz, Ar-H), 7.99 (2H, d, Jꢂ6 Hz, Ar-H), 8.19 (2H,
d, Jꢂ6.3 Hz, Ar-H), 9.40 (2H, d, Jꢂ8.7 Hz, Ar-H), 13.24 (2H, s, NH). 13C-
NMR (CDCl3) d: 186.91 (C-9,10), 166.83 (NCO-1ꢆ,5ꢆ), 138.86 (C), 136.02
(C), 134.51 (C), 133.19 (C), 128.84 (C), 128.53 (CH), 126.44 (CH), 124.59
(CH), 122.84 (CH), 21.51 (CH3). IR (KBr): 1637, 3245 cmꢄ1. EI-MS m/z:
474 (Mꢅ), 356, 119.
1,5-Bis(cyclohexylamido)anthraquinone (22) 68% yield. mp 296 °C
1
(EA). H-NMR (CDCl3) d: 1.24—1.76 (12H, m, CH2), 1.86—1.90 (4H, m,
CH2), 2.06—2.10 (4H, m, CH2), 2.43 (2H, m, CH), 7.76 (2H, t, Jꢂ8.4 Hz,
Ar-H), 8.04 (2H, d, Jꢂ7.8 Hz, Ar-H), 9.17 (2H, d, Jꢂ8.4 Hz, Ar-H), 12.29
(2H, s, NH). 13C-NMR (CDCl3) d: 186.73 (C-9,10), 176.28 (NCO), 142.40
(C-1,5), 135.82 (C-4a,8a), 134.67 (C-3,7), 126.34 (C-5a,9a), 122.39 (C-4,8),
117.14 (C-2,6), 47.47 (CH), 29.69 (CH2), 25.82 (CH2), 25.78 (CH2). IR
(KBr): 1640, 1690, 2856, 2931 cmꢄ1. APCI-EI-MS m/z: 459 (Mꢅ), 441,
439, 252, 238.
Acknowledgments This research was supported by National Science
Council Grant NSC94-2113-M-016-003. The authors are indebted to Dr.
Klaus K. Mayer (Universität Regensburg, Germany) for the mass spectrome-
try and analytical determinations.
References
1,5-Bis(cyclopentanepropylamido)anthraquinone (23) 72% yield. mp
221 °C (EA). 1H-NMR (CDCl3) d: 1.18—1.23 (8H, m, CH2), 1.54—1.63
(8H, m, CH2), 1.54—1.63 (2H, m, CH), 1.81 (4H, q, Jꢂ5.7 Hz, CH2), 2.55
(4H, t, Jꢂ8.1 Hz, CH2), 7.76 (2H, t, Jꢂ8.4 Hz, Ar-H), 8.03 (2H, dd, Jꢂ1.2,
7.8 Hz, Ar-H), 9.15 (2H, d, Jꢂ8.1 Hz, Ar-H), 12.27 (s, 2H, NH). 13C-NMR
(CDCl3) d: 187.10 (C-9,10), 173.79 (NCO), 142.60 (C-1,5), 136.29 (C-
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40.18 (CH2), 38.69 (CH2), 32.99 (CH2), 32.15 (CH), 25.65 (CH2). IR (KBr):
1698, 3190 cmꢄ1. APCI-EI-MS m/z: 487 (Mꢅ), 453, 431, 363, 285.
1,5-Bis(cyclopentylamido)anthraquinone (24) 77% yield. mp 258 °C
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