Studies on anthracenes. 1. Human telomerase inhibition and lipid peroxidation of 9-acyloxy 1,5-dichloroanthracene derivatives

Article abstract of DOI:10.1248/cpb.49.969

The synthetically useful approaches to 9-acyloxy 1,5-dichloroanthracene derivatives are reported. The system selectively reduces the carbonyl group flanked by the peri substituents of the anthracenediones to give the corresponding 1,5-dichloro-9(10H)-anth

Full text of DOI:10.1248/cpb.49.969

August 2001
Chem. Pharm. Bull. 49(8) 969—973 (2001)
969
Studies on Anthracenes. 1. Human Telomerase Inhibition and Lipid
Peroxidation of 9-Acyloxy 1,5-Dichloroanthracene Derivatives
Hsu-Shan HUANG,*,a Jing-Min HWANG,b Yee-Min JEN,b Jing-Jer LIN,c Kung-Yuan LEE,a
a
Chang-Hsin SHI,a and Hsien-Chin HSU
School of Pharmacya and Department of Radiation Oncology,b National Defense Medical Center, Neihu, 114, Taipei,
Taiwan, R.O.C. and Institute of Bio-Pharmaceutical Science, National Yang-Ming University,c ShihPai, 112, Taipei,
Taiwan, R.O.C. Received February 13, 2001; accepted March 19, 2001
The synthetically useful approaches to 9-acyloxy 1,5-dichloroanthracene derivatives are reported. The sys-
tem selectively reduces the carbonyl group flanked by the peri substituents of the anthracenediones to give the
corresponding 1,5-dichloro-9(10H)-anthracenone. Simple regioselective acylation of anthracenone is applied
with appropriate acyl chlorides in CH2Cl2 with catalytic amount of pyridine to give the novel 9-acyloxy 1,5-
dichloroanthracene derivatives. Considerable interest has developed in the mechanism of how anthracenone
achieves this desirable selectivity. In an attempt to understand the mechanism of this reaction, solid-state struc-
tures of anthracene derivatives have been obtained. In addition, the inhibition of lipid peroxidation in model
membranes was determined as was their ability to inhibit the telomere-addition function of the human telom-
erase enzyme together with their inhibition of the Taq polymerase enzyme. In contrast to (؉)-a-tocopherol, 3b,
3c, 3d, 3g, and 3i do not enhance lipid peroxidation in model membranes. Implications for 9-acyloxy 1,5-
dichloroanthracene analogues as potential anticancer agents are discussed.
Key words peri substituent; anthracenedione; anthracenone; anthracene; acylation; telomerase; lipid peroxidation
A series of novel 9-acyloxy 1,5-dichloroanthracene deriva- substitution with an electron-withdrawing acyl group at the
tives were synthesized by a general synthetic route. An- 9-position impairs the reduction ability of anthracenones. We
thraquinones have proved to be important in the development report here a simple and general methodology for the synthe-
of electrochemically switchable ligands in studies conducted sis of 9-acyloxy 1,5-dichloroanthracene derivatives.
in many laboratories,1—6) and are increasingly popular in a
variety of related applications.7—12) Anthracene derivatives Chemistry
have been the subject of extensive research mainly due to
The synthesis of the desired anthracenes is shown in Chart
their well-recognized biological importance and the signifi- 1. 1,5-Dichloro-9(10H)-anthracenone 2, prepared from 1 and
cant biological applications of their derivatives. Introduction SnCl2 in boiling HCl and acetic acid according to the litera-
of side chains onto the anthracenone skeleton is usually ac- ture,15) was treated with appropriate acyl chloride in the pres-
complished by a stepwise procedure via the anthracenedione ence of pyridine to afford corresponding 9-acyloxy 1,5-
1
because of the chemical instability of many anthracenones.13) dichloroanthracenes in 75—90% yield (Table 1). In the H-
There is continuing interest in the development of new NMR spectrum the 10-H aromatic proton signal was identi-
agents that modify the chromophore anthracenone moiety yet fied while that at d 8.80 was one single proton signal, explic-
exhibit different potent spectra, together with reduced overall able downfield chemical shift of acyloxy anthracene 10-H
toxicity. Our present research work has been hampered by the proton signal (d 8.80) is due to the deshielding effect of aro-
lack of synthetic methods for the preparation of substituted matic anthracene ring not to C-9 acyloxy group effect. The
anthracene derivatives and more suitable antitumor agents. 1H-NMR spectrum of 2 in CDCl3 lends credence to the struc-
Reduction of 1,5-dichloroanthraquinone is required as a first ture as it shows evidence for the methylene protons (4.08
step in the synthesis of 9-acyloxy 1,5-dichloroanthracenes. ppm) at C-10. Of particular importance are the one 10-H
The reaction of anthracenone with an acyl chloride is ex- proton chemical shifts between d 8.69 and 9.23, which are
pected to lead to anthracene. Introduction of the substituted different from the range of the 1,5-dichloro-9(10H)-anthra-
functionality onto the anthrone skeleton was achieved by re- cenone possessing two 10-H protons and the IR stretching
action of the appropriate acyl chloride with anthracenone frequency 1735 cmϪ1, which indicate the normal ester CϭO
under weakly basic conditions, where acylation took place at stretch. Furthermore, the 13C-NMR spectra of these com-
the carbonyl oxygen via the isomerization. C-9-Acylated an-
thracenes were afforded in good yields under base-catalyzed
(pyridine). Halide ions from acyl chloride are good leaving
groups that elimination from the complex, and take place al-
most synchronously with addition to the carbonyl group.14)
Analysis of the reaction mixture from the reaction showed,
after hydrolysis, the presence of anthracene and the absence
of high molecular weight by-products, even after the reaction
mixture had been allowed to high temperature. In each exper-
iment the acyl chloride was added directly to the anthra-
cenone, which it may well be that such material is less nucle-
ophilic and hence more selective in its reaction. In particular,
Chart 1
To whom correspondence should be addressed. e-mail: huanghs@ndmctsgh.edu.tw
© 2001 Pharmaceutical Society of Japan

970
Vol. 49, No. 8
Table 1. Chemical and Physical Data of 9-Acyloxy 1,5-Dichloroan- Table 2. Taq Inhibition and Inhibition of Lipid Peroxidation of 9-Acyloxy
thracene Derivatives
Compd.
1,5-Dichloroanthracene Derivatives
Ra)
log P mp (°C)
Yield (%) Anal.b)
Inhibition of Lipid
Peroxidation (%)b)
Taq Inhibitiona)
Compd.
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3
4.53 164—166
4.69 180—181
4.50 181—182
4.80 134—135
4.29 116—118
4.99 120—122
4.96 138—140
5.14 132—134
5.27 118—120
5.12 130—132
5.98 120—121
5.24 166—168
5.69 162—164
5.60 172—173
5.56 204—206
5.84 172—174
5.67 169—171
5.16 198—200
5.38 216—218
89
64
66
88
76
86
70
93
70
69
84
61
81
78
77
76
90
75
83
70
73
77
71
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H, N
C, H, N
C, H
C, H
C, H
C, H
CH2Br
CH2Cl
CH2CH3
CH(CH3)2
CH3CH(Cl)
CHCl2
(CH2)2CH3
(CH2)3Br
(CH2)3Cl
1 mM
0.1 mM
0.01 mM
10 mM
1 mM
3a
3b
3c
NDc)
ϩ
NDc)
ϩ
NDc)
ϩ
Ϫ
ϩ
NDc)
NDc)
ϩ
ϩ
Ϫ
Ϫ
ϩ
NDc)
Ϫ
34Ϯ0.1
100
26Ϯ0.1
100
Ϫ
Ϫ
100
100
3d
3e
3f
ϩ
ϩ
100
100
NDc)
NDc)
ϩ
NDc)
NDc)
ϩ
29Ϯ0.1
61Ϯ0.2
100
18Ϯ0.4
100
2Ϯ0.1
10Ϯ0.2
62Ϯ0.4
12Ϯ0.2
38Ϯ0.2
10Ϯ0.1
12Ϯ0.3
5Ϯ0.2
0
3j
3g
3k
3l
(CH2)4CH3
C6H5
3h
3i
ϩ
Ϫ
ϩ
Ϫ
3m
3n
3o
3p
3q
3r
3s
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
3-ClC6H4
4-ClC6H4
3-NO2C6H4
4-NO2C6H4
3j
ϩ
ϩ
29Ϯ0.2
17Ϯ0.5
14Ϯ0.3
23Ϯ0.2
22Ϯ0.1
26Ϯ0.2
31Ϯ0.1
NDc)
3k
3l
3m
3n
3o
3p
3q
3r
ϩ
ϩ
NDc)
ϩ
NDc)
Ϫ
ϩ
ϩ
Ϫ
11Ϯ0.1
3Ϯ0.1
9Ϯ0.1
NDc)
NDc)
ϩ
NDc)
Ϫ
NDc)
Ϫ
3t
4-Cl, 2-CH3O-C6H3 3.97 195—196
ϩ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
NDc)
Ϫ
Ϫ
ϩ
ϩ
3u
3v
3w
2,4-Cl2C6H3
CH2C6H5
CH2CH2C6H5
6.06 190—192
5.31 152—154
5.63 126—128
NDc)
NDc)
3s
3t
3u
3v
Ϫ
Ϫ
36Ϯ0.2
10Ϯ0.3
16Ϯ0.1
33Ϯ0.2
37Ϯ0.2
100
6Ϯ0.1
0
2Ϯ0.1
14Ϯ0.1
30Ϯ0.4
62
NDc)
ϩ
NDc)
ϩ
a) All new compounds displayed 1H-NMR, FTIR, UV and MS spectra consistent
with the assigned structure. b) Carbon, hydrogen, and nitrogen elemental analyses
were obtained for all new targets and were within Ϯ0.4% of the theoretical values ex-
cept where stated otherwise.
ϩ
ϩ
3w
ϩ
ϩ
ϩ
ϩ
Controld)
a) Key: (ϩ) inhibition, (Ϫ) no inhibition. b) Relative percentage of inhibition. In-
hibition was significantly different with respect to that of the control [(ϩ)-a-toco-
pherol], pϽ0.01, meanϮS.E., nϭ4. Values in parentheses are percent inhibition at the
indicated concentration (mM), and standard errors average 10% of the indicated values.
c) NDϭnot determined. d) Control: Taq Inhibition: doxorubicin, mitomycin-C, and
methotrexate; Inhibition of lipid peroxidation: (ϩ)-a-tocopherol.
Results and Discussion
9-Acyloxy 1,5-dichloroanthracene derivatives (3a—w) were
synthesized and Taq inhibition and lipid peroxidation were
evaluated. Prior to the evaluation of compounds in the PCR-
based telomerase assay, the agents were tested for their abil-
ity to inhibit Taq polymerase in order to address the selectiv-
ity of polymerase/telomerase inhibition.10,16—18) Approxi-
mately most of the compounds examined completely inhib-
ited Taq polymerase activity at concentration of 1 mM, 0.1
mM, and 0.01 mM, respectively, whereas 3b, 3d, 3g, 3h, 3k,
and 3w, showed significant inhibitory effect at the three dif-
ferent concentrations. Further, there does not appear to be
any correlation between Taq polymerase inhibition and cyto-
toxicity. The anthraquinone-based control drugs doxorubicin,
Fig. 1. X-Ray Crystal Structure of Compound 3o
pounds lack the usual carbonyl chemical shift in the d 180— mitomycin-C, and methotrexate showed total inhibition of
190 region that the carbonyl resonance of anthrone, itself oc- Taq polymerase activity at 1 mM, 0.1 mM, and 0.01 mM, re-
curs at d 183.7.2) The new method provides a simple and spectively.
convenient route to 9-acyloxy 1,5-dichloroanthracene deriva-
The inhibitory effect on lipid peroxidation of these com-
tives. Pyridine in CH2Cl2 at room temperature for several pounds was evaluated with rat brain phospholipid liposomes
hours to yield high yield of desired products. The data for all which provide an ideal model system for lipid peroxidation
of the direct reactions are presented in Table 1. The structure studies.19) With respect to the (ϩ)-a-tocopherol, we found
of 3o including the side chain was confirmed by X-ray analy- better inhibitory effect in 10 mM by compounds 3b, 3c, 3d,
sis. The ORTEP plot of 3o is shown in Fig. 1. In the crystal, 3g, and 3i. Furthermore, compounds 3b, 3c, and 3d were sig-
the toluoyl ring is twisted from the plane of the anthracene nificantly more efficient than (ϩ)-a-tocopherol in 1 mM. Al-
ring. However, upon consideration of the intermolecular in- though not a potent inhibitor of lipid peroxidation in itself,
teraction present in the crystal the reason for this geometry in can provide a useful template for the design of potential anti-
the crystal became apparent.
cancer agents. Moreover, the results support our hypothesis
that structural modification of 1,5-dichloroanthracenone may

August 2001
971
C, 63.97; H, 3.79. Found: C, 63.99; H, 3.88.
lead to control of the release of active oxygen species. Taq
inhibitory action of 9-substituted 1,5-dichloroanthracenes
was not proportionally related to the lipid peroxidation; com-
pounds which had potent human telomerase inhibition were
9-Isobutyryloxy 1,5-Dichloroanthracene (3e) 1H-NMR (CDCl3) d:
8.77 (H, s), 7.98 (H, d, Jϭ8.4 Hz), 7.87 (H, d, Jϭ8.8 Hz), 7.60—7.55 (2H,
m), 7.40—7.33 (2H, m), 3.21—3.13 (H, m), 1.60—1.39 (6H, m). 13C-NMR
(CDCl3) d: 176.45, 141.64, 134.53, 132.62, 130.47, 129.90, 129.62, 128.51,
also effective in lipid peroxidation (3b, 3c, 3d, 3g, 3i). In ad- 127.18, 126.85, 125.93, 123.83, 123.66, 121.45, 35.30, 20.16, 19.07. MS
m/z: 332 (Mϩ), 262. IR (KBr) cmϪ1: 1761. Anal. Calcd for C18H14O2Cl2: C,
dition to the redox properties, other factors such as an appro-
priate geometry of the molecules when bound to the active
site of the substrate may be responsible for the human telom-
65.08; H, 3.94. Found: C, 64.86; H, 4.18.
9-(2-Chloropropionyloxy) 1,5-Dichloroanthracene (3f) 1H-NMR
(CDCl3) d: 8.80 (H, s), 8.11 (H, d, Jϭ11.4 Hz), 7.99 (H, d, Jϭ10.6 Hz),
erase inhibition activities of the novel anthracene analogs.
This is supported by the fact that for these compounds, a def-
inite length of the chain linking the anthracene nucleus and
the phenyl rest was optimal for activity. In conclusion, we
have described 9-acyloxy 1,5-dichloroanthracene derivatives
which show potent Taq inhibition and antioxidant activity. To
understand whether or not these compounds have potent anti-
tumor and antiproliferative activities, we examined their ef-
fects in other cell lines. The results will be reported else-
where.
7.63—7.55 (2H, m), 7.43—7.33 (2H, m), 4.98—4.93 (H, q), 1.86 (3H, d,
Jϭ8.5 Hz). 13C-NMR (CDCl3) d: 166.90, 142.76, 133.55, 133.25, 131.58,
130.76, 129.89, 129.74, 128.61, 128.37, 127.36, 127.15, 127.04, 126.02,
124.32, 121.36, 52.53, 31.87, 21.17. MS m/z: 352 (Mϩ), 262. IR (KBr)
cmϪ1: 1766. Anal. Calcd for C17H11O2Cl3: C, 57.74; H, 3.14. Found: C,
57.85; H, 2.99.
9-Dichloroacetoxy 1,5-Dichloroanthracene (3g) 1H-NMR (CDCl3) d:
8.84 (H, s), 8.05—8.00 (2H, m), 7.62—7.59 (2H, m), 7.47—7.36 (2H, m),
6.51 (H, s). 13C-NMR (CDCl3) d: 163.31, 140.68, 133.73, 132.12, 130.49,
129.27, 129.15, 127.21, 127.15, 126.67, 126.19, 125.62, 124.39, 120.96,
120.19, 64.59. MS m/z: 374 (Mϩ), 262. IR (KBr) cmϪ1: 1769. Anal. Calcd
for C16H8O2Cl4: C, 51.38; H, 2.16. Found: C, 51.16; H, 1.99.
9-Butyryloxy 1,5-Dichloroanthracene (3h) 1H-NMR (CDCl3) d: 8.76
(H, s), 7.97 (H, d, Jϭ8.4 Hz), 7.86 (H, d, Jϭ8.8 Hz), 7.59—7.55 (2H, m),
7.40—7.32 (2H, m), 2.90—2.84 (2H, m). 13C-NMR (CDCl3) d: 173.14,
142.77, 134.51, 132.60, 130.47, 129.61, 129.21, 128.44, 127.12, 126.92,
126.85, 125.94, 123.67, 122.13, 121.68, 37.21, 18.73, 14.53. MS m/z: 332
(Mϩ), 262. IR (KBr) cmϪ1: 1754. Anal. Calcd for C18H14O2Cl2: C, 64.88; H,
4.23. Found: C, 64.82; H, 4.23.
Experimental
Melting points were determined with a Büchi 530 melting point apparatus
and are uncorrected. All reactions were monitored by TLC, which was per-
formed on precoated sheets on silica gel 60 F254, and flash column chro-
matography was done in silica gel (E. Merck, 70—230 mesh) with CH2Cl2
as eluant. 1H-NMR spectra were recorded with a Varian GEMINI-300
(300 MHz); d values are in ppm relative to a tetramethylsilane internal stan-
dard. Fourier-transform-IR spectra (KBr) were recorded on a Perkin-Elmer
983G spectrometer. Mass spectra (EI, 70 eV, unless otherwise stated) were
obtained on a Finnigan MAT TSQ-46 and Finnigan MAT TSQ-700. The X-
ray Crystallographic Laboratory of the University of Taiwan performed X-
ray structural analysis of compound 3o.
1,5-Dichloro-9(10H)-anthracenone (2) To a solution of 1,5-dichloro
anthraquinone (5.56 g, 20 mmol) in glacial acetic acid (200 ml) heated to re-
flux was added dropwise over 3 h, a solution of SnCl2 (50 g, 220 mmol) in
37% HCl (100 ml). The solution was then cooled, and the resulting crystals
were collected by filtration, washed with water, purified by crystallization
(acetic acid) and subjected to column chromatography.
General Procedure for the Preparation of 9-Acyloxy 1,5-Dichloro An-
thracenes To a solution of 2 (1 mmol) and pyridine (0.1 ml) in dry CH2Cl2
(20 ml) was added dropwise a solution of appropriate acyl chlorides
(3 mmol) in dry CH2Cl2 (10 ml) under N2. The reaction mixture was stirred
at room temperature or refluxed for several hours. The solvent was removed
and the residue purified by recrystallization (EtOH) and chromatography.
9-Acetoxy 1,5-Dichloroanthracene (3a) 1H-NMR (CDCl3) d: 8.79 (H,
s), 8.01 (H, d, Jϭ8.4 Hz), 7.90 (H, d, Jϭ8.8 Hz), 7.62—7.58 (2H, m),
7.43—7.34 (2H, m), 2.60 (3H, s). 13C-NMR (CDCl3) d: 170.58, 142.00,
134.51, 132.31, 130.54, 129.87, 129.61, 128.37, 126.97, 126.93, 126.80,
125.97, 123.81, 121.79, 121.67, 22.12. MS m/z: 304 (Mϩ), 262, 227. IR
(KBr) cmϪ1: 1753. Anal. Calcd for C16H10O2Cl2: C, 62.98; H, 3.30. Found:
C, 62.98; H, 3.21.
9-(4-Bromobutyryloxy) 1,5-Dichloroanthracene (3i) 1H-NMR (CDCl3)
d: 8.78 (H, s), 7.99 (H, d, Jϭ8.4 Hz), 7.86 (H, d, Jϭ8.8 Hz), 7.61—7.57
(2H, m), 7.42—7.33 (2H, m), 3.63—3.60 (2H, m), 3.15—3.11 (2H, m),
2.41—2.36 (2H, m). 13C-NMR (CDCl3) d: 172.25, 142.42, 134.48, 132.67,
130.60, 129.87, 129.66, 128.28, 127.06, 127.00, 126.01, 123.91, 122.02,
121.53, 33.33, 33.18, 27.85. MS m/z: 412 (Mϩ), 262. IR (KBr) cmϪ1: 1747.
Anal. Calcd for C18H13O2Cl2Br: C, 52.46; H, 3.18. Found: C, 52.49; H, 3.16.
9-(4-Chlorobutyryloxy) 1,5-Dichloroanthracene (3j) 1H-NMR (CDCl3)
d: 8.74 (H, s), 7.95 (H, d, Jϭ8.4 Hz), 7.84 (H, d, Jϭ8.8 Hz), 7.59—7.55
(2H, m), 7.40—7.30 (2H, m), 3.75 (2H, t, Jϭ6.1 Hz), 2.31 (2H, m), 1.56
(2H, m). 13C-NMR (CDCl3) d: 172.36, 142.41, 134.46, 132.65, 130.59,
129.84, 129.65, 128.27, 127.03, 126.98, 125.99, 123.88, 121.99, 121.51,
44.58, 31.95, 27.79. MS m/z: 366 (Mϩ), 262. IR (KBr) cmϪ1: 1747. Anal.
Calcd for C18H13O2Cl3: C, 58.80; H, 3.56. Found: C, 58.55; H, 3.49.
9-Hexanoyloxy 1,5-Dichloroanthracene (3k) 1H-NMR (CDCl3) d:
8.69 (H, s), 7.90 (H, d, Jϭ8.4 Hz), 7.79 (H, d, Jϭ8.8 Hz), 7.52—7.48 (2H,
m), 7.33—7.26 (2H, m), 2.84—2.79 (2H, m), 1.85—1.79 (2H, m), 1.46—
1.32 (4H, m), 0.89 (3H, t, Jϭ7.2 Hz). 13C-NMR (CDCl3) d: 173.34, 142.44,
134.52, 132.61, 130.48, 129.87, 129.62, 128.44, 126.93, 126.85, 125.95,
123.67, 122.13, 121.67, 35.29, 32.03, 24.87, 23.01, 14.55. MS m/z: 360
(Mϩ), 262. IR (KBr) cmϪ1: 1753. Anal. Calcd for C20H18O2Cl2: C, 66.49; H,
5.02. Found: C, 66.44; H, 5.02.
9-Benzoyloxy 1,5-Dichloroanthracene (3l) 1H-NMR (CDCl3) d: 8.82
(H, s), 8.40 (2H, d, Jϭ8.1 Hz) 8.01 (H, d, Jϭ8.4 Hz), 7.91 (H, d, Jϭ8.8 Hz),
7.71 (H, t, Jϭ7.4 Hz), 7.61—7.58 (2H, m), 7.56—7.52 (2H, d, Jϭ7.2 Hz),
7.37—7.33 (2H, m). 13C-NMR: (CDCl3) d: 166.62, 142.41, 134.49, 132.64,
131.38, 130.43, 130.11, 129.98, 129.53, 129.40, 128.65, 127.24, 126.98,
126.00, 125.84, 122.45, 121.81. MS m/z: 366 (Mϩ), 262, 105. IR (KBr)
cmϪ1: 1737. Anal. Calcd for C21H12O2Cl2: C, 68.68; H, 3.29. Found: C,
68.61; H, 3.25.
9-Bromoacetoxy 1,5-Dichloroanthracene (3b) 1H-NMR (CDCl3) d:
8.80 (H, s), 8.03 (H, d, Jϭ8.8 Hz), 7.99 (H, d, Jϭ8.4 Hz), 7.62—7.57 (2H,
m), 7.45—7.34 (2H, m), 4.32 (2H, m). 13C-NMR (CDCl3) d: 166.80,
156.93, 134.39, 132.61, 130.83, 129.88, 129.74, 127.96, 127.39, 127.16,
126.99, 126.09, 125.75, 123.44, 121.21, 26.31. MS m/z: 384 (Mϩ), 262. IR
(KBr) cmϪ1: 1759. Anal. Calcd for C16H9O2Cl2Br: C, 50.04; H, 2.36. Found:
C, 50.00; H, 2.36.
9-(o-Toluoyloxy) 1,5-Dichloroanthracene (3m) 1H-NMR (CDCl3) d:
8.82 (H, s), 8.59 (H, d, Jϭ7.8 Hz), 8.02 (H, d, Jϭ8.4 Hz), 7.97 (H, d,
Jϭ8.8 Hz), 7.61—7.56 (4H, m), 7.45 (H, t, Jϭ7.6 Hz), 7.40—7.35 (2H, m),
2.72 (3H, s). 13C-NMR (CDCl3) d: 166.37, 143.14, 142.98, 134.58, 133.78,
132.83, 132.67, 130.44, 129.95, 129.59, 128.70, 128.52, 127.30, 126.97,
126.80, 126.01, 123.71, 122.49, 121.85, 31.52, 22.78. MS m/z: 380 (Mϩ),
262, 119. IR (KBr) cmϪ1: 1731. Anal. Calcd for C22H14O2Cl2: C, 69.31; H,
3.70. Found: C, 69.11; H, 3.64.
9-Chloroacetoxy 1,5-Dichloroanthracene (3c) 1H-NMR: (CDCl3) d:
8.81 (H, s), 8.00 (H, d, Jϭ8.4 Hz), 7.92 (H, d, Jϭ8.8 Hz), 7.62—7.58 (2H,
m), 7.45—7.35 (2H, m), 4.57 (2H, m). 13C-NMR (CDCl3) d: 166.93,
142.53, 134.39, 132.67, 130.87, 129.84, 129.73, 127.92, 127.41, 127.14,
126.81, 126.11, 126.01, 124.47, 121.69, 121.18, 44.88. MS m/z: 388 (Mϩ),
262. IR (KBr) cmϪ1: 1756. Anal. Calcd for C16H9O2Cl3: C, 56.59; H, 2.67.
Found: C, 56.46; H, 2.67.
9-(m-Toluoyloxy) 1,5-Dichloroanthracene (3n) 1H-NMR (CDCl3) d:
8.74 (H, s), 8.15 (H, d, Jϭ7.3 Hz) 7.93 (H, d, Jϭ8.8 Hz), 7.85 (H, d,
Jϭ8.8 Hz), 7.53—7.39 (4H, m), 7.29—7.25 (2H, m), 2.42 (3H, s). 13C-NMR
(CDCl3) d: 166.41, 142.98, 139.25, 135.31, 134.55, 132.58, 131.83, 130.40,
129.94, 129.90, 129.52, 129.31, 128.72, 128.55, 127.19, 126.94, 126.00,
9-Propionyloxy 1,5-Dichloroanthracene (3d) 1H-NMR (CDCl3) d:
8.78 (H, s), 7.99 (H, d, Jϭ8.4 Hz), 7.87 (H, d, Jϭ8.8 Hz), 7.60—7.56 (2H,
m), 7.41—7.34 (2H, m), 2.95—2.90 (2H, q), 1.40 (3H, t, Jϭ7.5 Hz). 13C-
NMR (CDCl3) d: 173.99, 142.74, 134.51, 132.59, 130.49, 129.87, 129.61,
128.42, 127.09, 126.93, 126.88, 125.95, 123.68, 121.62, 58.96, 28.77, 18.94.
MS m/z: 318 (Mϩ), 262. IR (KBr) cmϪ1: 1757. Anal. Calcd for C17H12O2Cl2:
123.79, 122.38, 121.86, 21.97. MS m/z: 380 (Mϩ), 262, 119. IR (KBr) cmϪ1
1730. Anal. Calcd for C22H14O2Cl2: C, 69.31; H, 3.70. Found: C, 69.05; H,
:

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Vol. 49, No. 8
121.41, 42.59. MS m/z: 380 (Mϩ), 262. IR (KBr) cmϪ1: 1750. Anal. Calcd
for C22H14O2Cl2: C, 69.31; H, 3.70. Found: C, 69.34; H, 3.79.
3.71.
9-(p-Toluoyloxy) 1,5-Dichloroanthracene (3o) 1H-NMR (CDCl3) d:
9-(Phenylpropionyloxy) 1,5-Dichloroanthracene (3w) 1H-NMR (CDCl3)
d: 8.63 (H, s), 7.85 (H, d, Jϭ8.4 Hz), 7.45—7.43 (2H, m), 7.31 (H, d, Jϭ
8.8 Hz), 7.26—7.16 (6H, m), 7.13—7.10 (H, m), 3.15—3.07 (4H, m). 13C-
NMR (CDCl3) d: 172.52, 145.17, 134.46, 132.45, 130.51, 129.61, 129.31,
129.24, 127.14, 126.91, 126.86, 125.95, 123.76, 121.72, 37.03, 31.28. MS
m/z: 394 (Mϩ), 262. IR (KBr) cmϪ1: 1769. Anal. Calcd for C23H16O2Cl2: C,
69.89; H, 4.06. Found: C, 69.75; H, 4.08.
8.81 (H, s), 8.82 (H, d, Jϭ8.1 Hz) 8.01 (H, d, Jϭ8.4 Hz), 7.91 (H, d, Jϭ
8.8 Hz), 7.59—7.54 (2H, m), 7.39 (2H, d, Jϭ8.0 Hz), 7.36—7.32 (2H, m),
2.58 (3H, s). 13C-NMR (CDCl3) d: 166.28, 145.43, 143.03, 134.56, 132.56,
131.43, 130.38, 130.13, 129.91, 129.51, 128.76, 127.28, 127.24, 126.97,
126.91, 125.99, 123.74, 122.49, 121.89, 22.44. MS m/z: 380 (Mϩ), 262, 119.
IR (KBr) cmϪ1: 1735. Anal. Calcd for C22H14O2Cl2: C, 69.31; H, 3.70.
Found: C, 68.81; H, 3.65.
X-Ray Crystal Structure of 3o: C22H14O2Cl2, Mtϭ381.25. A needle with
approximate dimensions of 0.2ϫ0.25ϫ0.25 mm was mounted a glass fiber
along its longest axis. The crystal system was triclinic, space group P-1. Cell
constants aϭ8.6360(19), bϭ9.7774(21), and cϭ11.662(3) Å; aϭ85.178
(19)°; bϭ75.775(18)°; gϭ67.100(19)°; Vϭ879.2(3) Å3; Zϭ2; m(MoKa)ϭ
3.810 cmϪ1; F(000)ϭ393. Cell dimensions were determined using a Non-
ious CAD4 Kappa Axis XRD & Siemens Smart CCD XRD diffractometer
equipped with a graphite monochromator and molybdenum source (lϭ
7.1073 Å). Data were collected on the same instrument using w scans with
2q varied from 2—50°. A total of 3090 unique reflexions were determined
of which 1647 were Ͼ2.0s. The structure was solved using direct methods
and was refined using standard techniques. A total of 235 parameters were
varied in the final least-squares. The refinement converged at Rϭ0.040 and
Rwϭ0.040. Residual electron density varied from 0.20 to Ϫ0.20 e/Å3.
9-(3-Chlorobenzoyloxy) 1,5-Dichloroanthracene (3p) 1H-NMR (CDCl3)
d: 8.83 (H, s), 8.40—8.39 (H, m), 8.30—8.28 (H, m), 8.02 (H, d, Jϭ8.4 Hz),
7.88 (H, d, Jϭ8.8 Hz), 7.71—7.68 (H, m), 7.62—7.57 (2H, m), 7.55 (H, t,
Jϭ8.0 Hz), 7.40—7.36 (2H, m). 13C-NMR (CDCl3) d: 165.06, 142.49,
135.61, 134.56, 134.52, 132.68, 131.76, 131.29, 130.75, 130.58, 129.89,
129.60, 129.46, 128.47, 127.14, 127.05, 126.08, 124.10, 122.39, 121.58,
112.02. MS m/z: 400 (Mϩ), 262, 139. IR (KBr) cmϪ1: 1740. Anal. Calcd for
C21H11O2Cl3: C, 62.79; H, 2.76. Found: C, 62.35; H, 2.76.
Log P Determination A standard reverse-phase HPLC procedure was
used. The method is based on the linear relationship between the capacity
factors log kЈ of the compound and their log P values. The log kЈ values of
five compounds (4-nitrophenol, 4-chlorophenol, benzophenone, naphtha-
lene, and anthracene) with known log P values (1.91, 2.39, 3.18, 3.44, and
4.49, respectively) were determined. A plot of log kЈ versus log P generated
from this calibration mixture was used for the calculation of log P values for
unknowns from the logarithms of their chromatographic capacity factors
(correlation coefficient Ͼ0.99; nϭ3). Methanol/water/acetic acid (77–23–
0.1), adjusted to pH 5.5 with concentrated NH3, was used as eluant. Calibra-
tion was performed as described.20) Log P values as a measure of lipophilic-
ity are given in Table 1.
Assay of Lipid Peroxidation19) Rat brain homogenate was prepared
from the brains of freshly killed Wistar rats and its peroxidation in the pres-
ence of iron ions was measured by the thiobarbituric acid (TBA) method as
described.19) Tetramethoxypropane was used as a standard, and the results
were expressed as nanomoles of malondialdehyde equivalents per milligram
of protein of rat brain homogenates. In brief, whole brain tissue, excluding
the cerebellum, was washed and homogenized in 10 volumes of ice-cold
Krebs buffer (10 mM N-2-hydroxyethyl piperazine-NЈ-2-ethanesulfonic acid
(Hepes), 10 mM glucose, 140 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 1.4 mM
KH2PO4, 0.7 mM MgSO4, pH 7.4) using a glass Dounce homogenizer. The
homogenate was centrifuged at low speed (1000ϫg) for 10 min, and the re-
sulting supernatant (adjusted to 2 mg/ml) was used immediately in lipid per-
oxidation assays. The reaction mixture with test compounds or vehicle was
incubated for 10 min, then stimulated by addition of ferrous ion (200 mM,
freshly prepared), and maintained at 37 °C for 30 min. The reactions were
terminated by adding 10 ml of ice-cold trichloroacetic acid solution (4%
(w/v) in 0.3 N HCl) and 200 ml of thiobarbituric acid-reactive substance
reagent (0.5% (w/v) thiobarbituric acid in 50% (v/v) acetic acid). After boil-
ing for 15 min, the samples were cooled and extracted with 1-butanol. The
extent of lipid peroxidation was estimated as thiobarbituric acid-reactive
substances and was read at 532 nm in a spectrophotometer (Shimadzu UV-
160). The results of this assay are provided in Table 2.
9-(4-Chlorobenzoyloxy) 1,5-Dichloroanthracene (3q) 1H-NMR (CDCl3)
d: 8.83 (H, s), 8.34—8.31 (2H, m), 8.02 (H, d, Jϭ8.4 Hz), 7.87 (H, d,
Jϭ8.8 Hz), 7.61—7.55 (4H, m), 7.38—7.34 (2H, m). 13C-NMR (CDCl3) d:
165.06, 148.41, 141.15, 134.56, 132.72, 132.44, 130.55, 129.93, 129.81,
129.60, 128.52, 128.44, 127.11, 127.04, 126.08, 124.04, 121.63, 112.01. MS
m/z: 400 (Mϩ), 262, 139. IR (KBr) cmϪ1
: 1732. Anal. Calcd for
C21H11O2Cl3: C, 62.79; H, 2.76. Found: C, 62.19; H, 2.76.
9-(3-Nitrobenzoyloxy) 1,5-Dichloroanthracene (3r) 1H-NMR (CDCl3)
d: 9.23 (H, s), 8.83 (H, s), 8.70—8.68 (H, m), 8.57—8.53 (H, m), 8.02 (H, d,
Jϭ8.4 Hz), 7.85—7.83 (H, m), 7.80 (H, t, Jϭ8.0 Hz), 7.62—7.56 (2H, m),
7.39—7.35 (2H, m). 13C-NMR (CDCl3) d: 164.18, 149.21, 136.87, 136.67,
134.51, 132.79, 131.83, 130.76, 129.90, 129.72, 128.21, 127.34, 127.13,
126.90, 126.25, 126.17, 125.97, 124.34, 121.33. MS m/z: 411 (Mϩ), 262,
120. IR (KBr) cmϪ1: 1744. Anal. Calcd for C21H11NO4Cl2: C, 61.19; H,
2.69; N, 3.40. Found: C, 60.30; H, 2.68; N, 3.69.
Taq Polymerase Assay10,16—18) Compounds were included at both 10
and 50 mM final concentrations in a PCR 50 ml master mix comprising 10 ng
of pCI-neo mammalian expression vector (Promega, Southampton, U.K.),
forward (GGAGTTCCGCGT-TACATAAC) and reverse (GTCTGCTC-
GAAGCATTAACC) primers (200 nmol), reaction buffer (75 mM Tris–HCl,
pH 8.8, 20 mM (NH4)2SO4, 0.01% v/v Tween 20), 2.5 mM MgCl2, 200 mM of
each deoxynucleotide triphosphate, and thermostable DNA polymerase (“red
hot”, Advanced Biotechnologies) 1.25 units. A reaction mix containing
water and no drug was used as a positive control, producing a product of ap-
proximately 1 kb. Amplification (30 cycles of 94 °C for 1 min, 55 °C for
1 min, and 72 °C for 2.5 min) was performed using a thermal cycler (Hybaid,
U.K.). PCR products were then separated by electrophoresis on a 2% w/w
agarose gel and visualized using ethidium bromide. The results of this assay
are provided in Table 2.
9-(4-Nitrobenzoyloxy) 1,5-Dichloroanthracene (3s) 1H-NMR (CDCl3)
d: 8.87 (H, s), 8.57 (2H, d, Jϭ8.8 Hz), 8.43 (2H, d, Jϭ8.8 Hz), 8.05 (H, d,
Jϭ8.4 Hz), 7.85 (H, d, Jϭ8.8 Hz), 7.63—7.57 (2H, m), 7.41—7.37 (2H, m).
13C-NMR (CDCl3) d: 165.04, 147.52, 135.35, 134.53, 132.83, 132.48,
130.79, 129.93, 129.74, 127.46, 127.38, 126.90, 126.19, 124.57, 124.47,
121.28, 111.04, 31.52. MS m/z: 411 (Mϩ), 262, 120. IR (KBr) cmϪ1: 1735.
Anal. Calcd for C21H11NO4Cl2: C, 61.19; H, 2.69; N, 3.40. Found: C, 60.57;
H, 2.71; N, 3.33.
9-(2-Methoxy-4-chlorobenzoyloxy) 1,5-Dichloroanthracene (3t) 1H-
NMR (CDCl3) d: 8.82 (H, s), 8.28 (H, d, Jϭ10.4 Hz), 8.06 (H, d,
Jϭ11.0 Hz), 8.01 (H, d, Jϭ10.6 Hz), 7.61—7.54 (2H, m), 7.40—7.33 (2H,
m), 7.14—7.09 (2H, m), 3.96 (3H, s). 13C-NMR (CDCl3) d: 161.66, 141.46,
134.89, 134.54, 132.52, 130.41, 129.56, 128.66, 126.96, 125.97, 123.79,
121.12, 121.44, 117.73, 113.68, 56.97. MS m/z: 430 (Mϩ), 262, 169. IR
(KBr) cmϪ1: 1738. Anal. Calcd for C22H13O3Cl3: C, 61.21; H, 3.04. Found:
C, 61.09; H, 3.02.
Acknowledgments This research was supported by grants from NSC of
the R.O.C. and assisted in part by the Globo Asia LLC. The authors are in-
debted to Dr. K. K. Mayer (University of Regensburg, Germany) for the
mass spectrometry analytical determination and to Prof. Dr. K. Müller (Uni-
versity of Münster, Germany) for his warm discussion.
9-(2,4-Dichlorobenzoyloxy) 1,5-Dichloroanthracene (3u) 1H-NMR
(CDCl3) d: 8.78 (H, s), 8.38 (H, d, Jϭ8.4 Hz), 7.96 (H, d, Jϭ8.4 Hz), 7.91—
7.89 (H, m), 7.57—7.52 (2H, m), 7.52—7.41 (2H, m), 7.35—7.29 (2H, m).
13C-NMR (CDCl3) d: 163.55, 140.40, 137.39, 134.79, 134.50, 132.69,
132.39, 130.07, 129.92, 129.66, 128.32, 128.05, 127.25, 127.05, 126.08,
124.22, 122.19, 121.63. MS m/z: 436 (Mϩ), 262, 173. IR (KBr) cmϪ1: 1741.
Anal. Calcd for C21H10O2Cl4: C, 57.84; H, 2.31. Found: C, 57.67; H, 2.32.
9-Phenylacetyloxy 1,5-Dichloroanthracene (3v) 1H-NMR (CDCl3) d:
8.68 (H, s), 7.90 (H, d, Jϭ8.4 Hz), 7.50—7.45 (4H, m), 7.42 (H, d,
Jϭ8.8 Hz), 7.36—7.33 (2H, m), 7.31—7.25 (2H, m), 7.17—7.13 (H, m),
4.15—4.03 (2H, m). 13C-NMR (CDCl3) d: 170.88, 145.32, 134.03, 133.29,
132.33, 130.54, 130.42, 129.63, 129.43, 128.18, 126.88, 125.97, 123.84,
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