Terbenzimidazoles: Influence of 2''-, 4-, and 5-substituents on cytotoxicity and relative potency as topoisomerase I poisons

Article abstract of DOI:10.1021/jm960658g

Terbenzimidazoles poison the nuclear enzyme topoisomerase I and possess significant cytotoxic activity against several human tumor cell lines. The relative pharmacological activity of 4,5- and 5,6-benzoterbenzimidazoles was compared to that of 5-phenylterbenzimidazole (3). 5,6-Benzoterbenzimidazole is inactive as a topoisomerase I poison and did not exhibit significant cytotoxic activity. In contrast, 4,5-benzoterbenzimidazole retained activity as a topoisomerase I poison but exhibited weak cytotoxic activity relative to 3. While 5-(1-naphthyl)-terbenzimidazole is less potent than 3 as a topoisomerase I poison and cytotoxic agent, 5-(2-naphthyl)terbenzimidazole has comparable activity to 3. The presence of a p-methoxy or p-chloro substituent on the phenyl moiety did not dramatically alter the pharmacological activity of 3. Several analogs of 3 were synthesized wherein the 2''-substituent varied from methyl, ethyl, propyl, isopropyl, phenyl to p-methoxyphenyl. Evaluation of the intrinsic activity of these analogs as topoisomerase I poisons indicates that topoisomerase I poisoning was not diminished by the presence of a methyl, ethyl, propyl, and isopropyl substituent at the 2''-position. Among the various 2''-substituted analogs evaluated, only in the case of 2''-(p-methoxyphenyl)-5-phenylterbenzimidazole was a significant decrease in cytotoxicity observed.

Full text of DOI:10.1021/jm960658g

2818
J . Med. Chem. 1997, 40, 2818-2824
Ter ben zim id a zoles: In flu en ce of 2′′-, 4-, a n d 5-Su bstitu en ts on Cytotoxicity a n d
Rela tive P oten cy a s Top oisom er a se I P oison s
J ung Sun Kim,† Chiang Yu,‡ Angela Liu,‡ Leroy F. Liu,‡ and Edmond J . LaVoie*,†
Department of Pharmaceutical Chemistry, Rutgers, The State University of New J ersey, Piscataway, New J ersey 08855, and
Department of Pharmacology, The University of Medicine and Dentistry of New J ersey, Robert Wood J ohnson Medical School,
Piscataway, New J ersey 08855
Received September 20, 1996X
Terbenzimidazoles poison the nuclear enzyme topoisomerase I and possess significant cytotoxic
activity against several human tumor cell lines. The relative pharmacological activity of 4,5-
and 5,6-benzoterbenzimidazoles was compared to that of 5-phenylterbenzimidazole (3). 5,6-
Benzoterbenzimidazole is inactive as a topoisomerase I poison and did not exhibit significant
cytotoxic activity. In contrast, 4,5-benzoterbenzimidazole retained activity as a topoisomerase
I poison but exhibited weak cytotoxic activity relative to 3. While 5-(1-naphthyl)terbenzimi-
dazole is less potent than 3 as a topoisomerase I poison and cytotoxic agent, 5-(2-naphthyl)-
terbenzimidazole has comparable activity to 3. The presence of a p-methoxy or p-chloro
substituent on the phenyl moiety did not dramatically alter the pharmacological activity of 3.
Several analogs of 3 were synthesized wherein the 2′′-substituent varied from methyl, ethyl,
propyl, isopropyl, phenyl to p-methoxyphenyl. Evaluation of the intrinsic activity of these
analogs as topoisomerase I poisons indicates that topoisomerase I poisoning was not diminished
by the presence of a methyl, ethyl, propyl, and isopropyl substituent at the 2′′-position. Among
the various 2′′-substituted analogs evaluated, only in the case of 2′′-(p-methoxyphenyl)-5-
phenylterbenzimidazole was a significant decrease in cytotoxicity observed.
Ch a r t 1
In tr od u ction
DNA topoisomerases are nuclear enzymes that are
involved in generating the necessary topological and
conformational changes in DNA critical to many cellular
processes such as replication and transcription.1-3
Poisoning of mammalian topoisomerase has been rec-
ognized as an effective approach for the development
of cancer chemotherapeutics.4-7 Recently, several biben-
zimidazoles and terbenzimidazoles have been identified
as topoisomerase I poisons.8-13 Among the terbenzimi-
dazoles evaluated, 1 and 2 (Chart 1) were active as
topoisomerase poisons but did not exhibit significant
cytotoxicity. The basis for this lack of cytotoxicity was
ascribed to their poor penetration into cells. In contrast,
the presence of a 5-phenyl or 5-pyridyl substituent on
these terbenzimidazoles, as in 3 and 4, resulted in
retention of activity as topoisomerase I poisons, as well
as significant cytotoxicity against several tumor cell
lines.12
Ch em istr y
The method used for the preparation of 4,5-benzo-
terbenzimidazole (9) and 5,6-benzoterbenzimidazole (10)
is outlined in Scheme 1. Condensation of either 1,2-
diaminonaphthalene (6) (prepared from 1-nitro-2-(acetyl-
amino)naphthalene14) or 2,3-diaminonaphthalene (7)
with 5-formyl-2-(benzimidazol-5′-yl)benzimidazole12 (5)
at 145 °C in nitrobenzene provided 9 and 10 in yields
of 67% and 53%, respectively. The preparation of the
4-phenylterbenzimidazole, 11, was accomplished under
similar reaction conditions by reaction of 3-phenyl-1,2-
phenylenediamine15 (8) with 5 as outlined in Scheme
1.
The 5-naphthylterbenzimidazoles 17 and 18, as well
as 5-(p-methoxyphenyl)- and 5-(p-chlorophenyl)terben-
zimidazoles 19 and 20, were similarly prepared by
condensation of the appropriate 1,2-phenylenediamine
with 5. The variously substituted 1,2-phenylenedi-
amine intermediates required for the formation of these
terbenzimidazoles were prepared from their respective
The cytotoxic activity observed for 3 prompted further
studies on the effect of various aryl substituents at the
4- and 5-positions of these terbenzimidazoles on their
pharmacological activity. In the present study, the
relative activity of 4,5- and 5,6-benzo-fused terbenzimi-
dazoles and 4-phenyl- and 5-naphthylbenzimidazoles as
topoisomerase I poisons and cytotoxic agents was com-
pared to the 5-phenylterbenzimidazole, 3. Addition of
a 2′′-(p-methoxyphenyl) substituent to 2 resulted in a
loss of topoisomerase I poisoning activity.12 The influ-
ence of steric factors associated with various substitu-
ents at the 2′′-position of 3 was also assessed with
regard to their influence on intrinsic activity as topo-
isomerase I poisons and on cytotoxicity.
† Rutgers, The State University of New J ersey.
‡ The University of Medicine and Dentistry of New J ersey.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(96)00658-9 CCC: $14.00 © 1997 American Chemical Society

Terbenzimidazoles as Topoisomerase I Poisons
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2819
Sch em e 1
Sch em e 2
Sch em e 3
crude diamine with 22 provided 5-phenyl-2-[2′-(3,4-
dinitrophenyl)benzimidazol-5′-yl]benzimidazole (24) in
94% yield. Compound 24 was used as the general
intermediate for the preparation of a series of 2′′-
substituted 5-phenylterbenzimidazoles. The crude di-
amine formed from the catalytic hydrogenation of 24
was reacted with acetaldehyde, propionaldehyde, bu-
tyraldehyde, isobutyraldehyde, benzaldehyde, and p-
methoxybenzaldehyde in yields which ranged from 28%
to 89%.
2-nitroaniline precursors, which were synthesized as
outlined in Scheme 2. Reaction of the tributyltin
derivatives formed from 1-bromonaphthalene, 2-bro-
monaphthalene, 4-bromoanisole, or 4-chlorobromoben-
zene with 12 provided the 2-nitroaniline derivatives 13-
16. Compounds 13-16 were hydrogenated in the
presence of Pd/C to generate their 1,2-phenylenediamine
derivatives which were used without further purifica-
tion.
Resu lts a n d Discu ssion
Condensation of the various 1,2-phenylenediamines
generated from 13-16 with 5 as outlined in Scheme 3
provided the 5-naphthyl- and phenyl-substituted ter-
benzimidazoles 17-20.
The synthetic approach used for the preparation of
2′′-substituted derivatives of 5-phenylterbenzimidazole
is outlined in Scheme 4. Reaction of 4-phenyl-1,2-
phenylenediamine12 (21) with 3,4-dinitrobenzaldehyde16
(22) resulted in the formation of 2-(3,4-dinitrophenyl)-
5-phenylbenzimidazole (23) in 66% yield. Catalytic
hydrogenation of 23 followed by reaction of the resulting
The cytotoxicity of the naphthylterbenzimidazoles 17
and 18, 5-(p-methoxyphenyl)terbenzimidazole (19), and
5-(p-chlorophenyl)terbenzimidazole (20) against the hu-
man lymphoblastoma cell line, RPMI 8402, and their
relative activity as topoisomerase I poisons are listed
in Table 1. While the 5-(2-naphthyl)terbenzimidazole,
18, had similar activity to 5-phenylterbenzimidazole, 3,
5-(1-naphthyl)terbenzimidazole, 17, was less cytotoxic.
While 19 was slightly more potent as a topoisomerase
I poison than either 3 or 20 (Table 1), all of these
5-phenylterbenzimidazoles had similar cytotoxic activ-

2820 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Kim et al.
Sch em e 4
Ta ble 1. Topoisomerase I-Mediated DNA Cleavage and
Cytotoxicity of 4,5- and 5,6-Benzoterbenzimidazoles, 4- and
5-Phenylterbenzimidazoles, and 2′′-Substituted
5-Phenylterbenzimidazoles
ity. These data indicate that para-substituents on the
5-phenyl ring retain activity. The pharmacological
activity observed for 18, in which there is a 2-naphthyl
substituent at the 5-position of these terbenzimidazoles,
suggests that meta-substituents on a 5-phenyl moiety
could also be well tolerated. The loss of antitumor
activity in the case of the 1-naphthyl analog 17 suggests
that 5-(ortho-substituted phenyl)terbenzimidazoles may
exhibit diminished activity.
Topo I-mediated
DNA cleavageb
cytotoxicitya
IC50 (µM)
compd
3
9
1
0.09
25
1c
10
11
17
18
19
20
25
26
27
28
29
30
>1000
5c
>100d
7.0
The relative activity of 4-phenylterbenzimidazole, 11,
and the 4,5-benzo-fused and 5,6-benzo-fused terbenz-
imidazoles 9 and 10 as topoisomerase I poisons is also
listed in Table 1. In contrast to 3 and 17, 10 was not a
topoisomerase I poison (Figures 1 and 2) and did not
possess cytotoxic activity. Its lack of activity relative
to 3 as a topoisomerase I poison provides a useful probe
for further comparison and examination of the biophysi-
cal interactions associated with DNA and topoisomerase
which are critically linked to the topoisomerase I
poisoning activity of terbenzimidazoles. Studies have
demonstrated that the principal interaction of 3 with
DNA occurs by binding to its minor groove.17 Recently,
the X-ray structure of the Hoechst 33258-related tris-
benzimidazole-oligonucleotide complex has been identi-
fied by Clark et al.18 Comparative studies are in
progress to assess the biophysical basis for the differ-
ences in pharmacological activity between 3 and this
benzo-fused terbenzimidazole. An altered DNA frag-
mentation pattern was obtained for 9 and 11 when
assayed as topoisomerase I poisons (Figures 1 and 2).
2
1
0.5
2
2
2.1
0.08
0.09
0.09
0.11
0.11
0.06
0.06
0.06
0.38
1
0.5
0.5
20
20
a
IC50 was calculated after 4 days of continuous exposure of
b
RPMI 8402 cells to drug. Topoisomerase I cleavage values are
reported as REC, relative effective concentration, i.e., concentra-
tions relative to 3, whose value is arbitrarily assumed as 1, that
produce the same extent of cleavage on the plasmid DNA in the
presence of recombinant human topoisomerase I. Cleavage is
calculated from the intensity of the strongest specific band. c The
altered DNA fragmentation pattern associated with these substi-
tuted terbenzimidazoles limits the accuracy of comparative data
d
on relative RECs as compared to 3. No indication of cytotoxicity
was considered indicative of IC50 values substantially greater than
the highest doses assayed.

Terbenzimidazoles as Topoisomerase I Poisons
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2821
F igu r e 1. Stimulation of enzyme-mediated DNA cleavage by
4- or 5-substituted terbenzimidazoles using recombinant hu-
man topoisomerase I. Enzyme-mediated DNA cleavage using
human topoisomerase I was performed as described in the
Experimental Section. The left-most lane is the DNA control
without topoisomerase I. The second lane from the left is the
DNA control with topoisomerase I. The rest of the lanes are
DNA with topoisomerase I and serially (10-fold each) diluted
compounds from 0.01 to 10 µg/mL for 3, 9, 17, and 20.
F igu r e 3. Stimulation of enzyme-mediated DNA cleavage by
3 and 2′′-substituted terbenzimidazoles using recombinant
human topoisomerase I. The assays were performed as de-
scribed for Figure 1. The left-most lane is the DNA control
with topoisomerase I. The relative doses employed ranged from
0.01 to 10 µg/mL for 3, 26, 27, and 30.
phenyl substituent did result in a significant loss of
activity as a topoisomerase I poison for 29 and 30
(Figure 3). While 29 did retain significant cytotoxic
activity, 30 was less active than the other 2′′-substituted
5-phenylterbenzimidazoles. These data indicate that in
the case of human topoisomerase I there exists greater
structural tolerance at the 2′′-position of these 5-phe-
nylterbenzimidazoles than previously anticipated. Stud-
ies are in progress to evaluate these various analogs for
their relative activity as poisons toward various camp-
tothecin-resistant mutant forms of topoisomerase I.
Exp er im en ta l Section
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column chroma-
tography refers to flash chromatography conducted on Sil-
iTech, 32-63 µm (ICN Biomedicals, Eschwegge, Germany),
using the solvent systems indicated. Radial chromatography
refers to the use of a Model 8924 chromatotron (Harrison
Research, CA). Infrared spectral data (IR) were obtained on
a Perkin-Elmer 1600 Fourier transform spectrophotometer and
are reported in cm-1. Proton (1H NMR) and carbon (13C NMR)
nuclear magnetic resonance were recorded on a Varian Gemini-
200 Fourier transform spectrometer. NMR spectra (200 MHz
for 1H and 50 MHz for 13C) were recorded in the deuterated
solvent indicated with chemical shifts reported in δ units
downfield from tetramethylsilane (TMS). Coupling constants
are reported in hertz (Hz). A few drops of CF3COOH improved
13C NMR spectra by allowing for increased solubility and
formation of the protonated form of the terbenzimidazoles,
thereby eliminating tautomeric differences among carbon
atoms. Mass spectra were obtained from Washington Uni-
versity Resource for Biomedical and Bio-organic Mass Spec-
trometry within the Department of Chemistry at Washington
University, St. Louis, MO. The purity of all compounds for
which HRMS data are provided was determined by analytical
reverse-phase HPLC. Compounds were analyzed using both
of the following conditions: method A, a Vydac C-18 column
(The Separations Group) using methanol:H2O (87:13) with a
flow rate of 1 mL/min; method B, a Microsorb C-8 column
(Rainin Instrument Co., Inc.) using methanol:0.1 M potassium
phosphate buffer (pH 7.0) (95:5) with a flow rate of 1 mL/min.
HPLC analyses were performed with a Hewlett-Packard 1090
liquid chromatograph equipped with a diode array UV detector
monitoring at 254 and 335 nm. The percent purity of these
F igu r e 2. Stimulation of enzyme-mediated DNA cleavage by
4- or 5-substituted terbenzimidazoles using recombinant hu-
man topoisomerase I. The assays were performed as described
for Figure 1. The left-most lane is the DNA control without
topoisomerase I. The relative doses employed ranged from 0.01
to 10 µg/mL for 3, 11, 10, and 19.
This altered DNA fragmentation pattern hampers ef-
forts to provide an accurate assessment of their potency
relative to 3. Both 9 and 11 clearly exhibited less
cytotoxic activity. These data indicate that the presence
of substituents at the 4-position appear to be associated
with reduced cytotoxic activity.
Table 1 lists the relative activity of the 2′′-substituted
5-phenylterbenzimidazoles 25-30. Previous studies on
the relative activity of terbenzimidazoles suggested that
the presence of a 2′′-(p-methoxyphenyl) substituent, as
in the case of 5-cyanoterbenzimidazole,12 would result
in a loss of topoisomerase I poisoning activity. The
results in Table 1 indicate that alkyl analogs 25-28 of
5-phenylterbenzimidazole, which range from methyl,
ethyl, propyl, to isopropyl, retain activity as topo-
isomerase I poisons and possess similar cytotoxic activ-
ity. Only modest differences in their activity as either
topoisomerase I poisons or cytotoxic agents were ob-
served relative to 3 (Figure 3). Introduction of a 2′′-

2822 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Kim et al.
compounds was calculated from the peak area assuming that
the extinction coefficient of the compound of interest and the
impurity are the same. On the basis of these analyses, all
the compounds were found to be 98.0-99% pure in these
systems. Combustion analyses were performed by Atlantic
Microlabs, Inc., Norcross, GA, and were within (0.4% of the
theoretical values.
Recrystallization in CH2Cl2 and hexanes provided orange
crystals: mp 189 °C; IR (KBr) 3467, 3351, 3172, 1629, 1524,
1408, 1344, 1244; H NMR (CDCl3) δ 6.29 (brs, 2H), 6.94 (d,
1H, J ) 9.0), 7.39-7.59 (m, 6H), 7.88 (d, 2H, J ) 9.0), 8.29 (d,
1H, J ) 2.0); 13C NMR (DMSO-d6) δ 120.6, 122.8, 124.2, 124.8,
126.4, 126.9, 127.5, 128.0, 128.9, 132.5, 134.1, 134.3, 135.5,
136.1, 140.7. Anal. (C16H12N2O2) C,H,N.
1
1,2-Diaminonaphthalene (6) was prepared from 1-nitro-2-
(acetylamino)naphthalene14 which was hydrolyzed by refluxing
with 1.2 N HCl to 1-amino-2-nitronaphthalene. This reaction
mixture was made basic and extracted with EtOAc, and the
resulting crude 1-amino-2-nitronaphthalene was hydrogenated
using 10% Pd/C in EtOAc. 2,3-Diaminonaphthalene (7) was
commercially available (Aldrich Chemical Co., Milwaukee, WI).
The syntheses of 5,12 8,15 12,12 21,12 and 2216 have been detailed
in the literature.
Gen er a l P r oced u r e for P r ep a r in g 4,5- a n d 5,6-Ben zo-
F u sed Ter b en zim id a zoles a n d 4-P h en ylt er b en zim id a -
zoles: 2-[2′-(Ben zim id a zol-5′′-yl)b en zim id a zol-5′-yl]-
n a p h th [1,2-d ]im id a zole (9). A stirred solution of 5 (119 mg,
0.45 mmol) and 1,2-naphthalenediamine14 (144 mg, 0.90 mmol)
in 10 mL of nitrobenzene was heated at 145 °C under N2
overnight. The cooled reaction mixture was then purified
directly by column chromatography. Elution with 0-20%
MeOH/EtOAc provided 110 mg (61%) of a brownish white
solid: mp >280 °C; IR (KBr) 3058, 1435, 1383; 1H NMR
(DMSO-d6) δ 7.46-7.54 (m, 1H), 7.61-7.87 (m, 5H), 8.03 (d,
1H, J ) 8.1), 8.12-8.23 (m, 2H), 8.38-8.44 (m, 2H), 8.52-
8.60 (m, 2H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ
113.3, 113.8, 115.7, 115.8, 115.9, 116.3, 118.4, 121.4, 122.1,
123.0, 124.9, 126.0, 126.8, 127.3 128.1, 128.5, 129.5, 129.9,
131.0, 132.4, 133.5, 139.2, 141.3, 148.5, 153.7; HRMS (FAB)
calcd for C25H17N6 (MH+) 401.1515, found 401.1508.
4-(2-Naph th yl)-2-n itr oan ilin e (14): prepared from 2-(tribu-
tylstannyl)naphthalene (5.17 g, 12.4 mmol), which was pre-
pared from 2-bromonaphthalene (5 g, 24 mmol), 1.6 M n-BuLi
(16.6 mL), and tributyltin chloride (6.51 mL, 24 mmol), with
4-bromo-2-nitroaniline (3.3 g, 15 mmol) in DMF (70 mL) using
Pd(PPh3)2Cl2 (528 mg, 0.75 mmol) and PPh3 (1.97 g, 7.5 mmol)
to provide 766 mg (35%) of orange crystals; mp 187 °C; IR
(KBr) 3467, 3351, 3172, 1629, 1524, 1408, 1344, 1244; 1H NMR
(DMSO-d6) δ 7.20 (d, 1H, J ) 9.0), 7.50-7.59 (m, 2H), 7.62
(brs, 2H), 7.84 (dd, 1H, J ) 8.5, 2.0), 7.91-8.02 (m, 4H), 8.21
(s, 1H), 8.40 (d, 1H, J ) 2.0); 13C NMR (DMSO-d6) δ 120.4,
122.9, 124.2, 124.5, 126.2, 126.7, 127.4, 127.7, 128.3, 128.8,
130.3, 132.3, 133.7, 134.7, 135.8, 145.9. Anal. (C16H12N2O2)
C,H,N.
4-(p-Meth oxyp h en yl)-2-n itr oa n ilin e (15): prepared from
(p-methoxyphenyl)tributyltin (1.92 g, 4.83 mmol), which was
prepared from p-bromoanisole (1.28 g, 6.86 mmol), 1.6 M
n-BuLi (4.75 mL), and tributyltin chloride (2.83 mL, 10 mmol),
with 4-bromo-2-nitroaniline (700 mg, 3.22 mmol) in DMF (18
mL) using Pd(PPh3)2Cl2 (111 mg, 0.16 mmol) and PPh3 (414
mg, 1.6 mmol) to provide 300 mg (38%) of orange crystals; mp
170-171 °C; IR (KBr) 3479, 3367, 1648, 1502, 1350, 1235; 1H
NMR (DMSO-d6) δ 3.80 (s, 3H), 7.01 (d, 2H, J ) 9.0), 7.12 (d,
1H, J ) 9.0), 7.51 (brs, 2H), 7.58 (d, 2H, J ) 9.0), 7.75 (dd,
1H, J ) 9.0, 2.0), 8.15 (d, 1H, J ) 2.0); 13C NMR (DMSO-d6)
δ 55.5, 114.7, 120.2, 121.8, 127.2, 127.5, 130.7, 131.9, 134.4,
145.4, 158.8. Anal. (C13H12N2O3) C,H,N.
2-[2′-(Ben zim id a zol-5′′-yl)b en zim id a zol-5′-yl]n a p h t h -
[2,3-d ]im id a zole (10): prepared from 5 (102 mg, 0.39 mmol)
and 2,3-diaminonaphthalene (124 mg, 0.78 mmol) in 10 mL
of nitrobenzene; elution with 10% MeOH/EtOAc provided 125
mg (80%) of a brownish white solid; mp >280 °C; IR (KBr)
3043, 1626, 1441, 1389, 1271; 1H NMR (DMSO-d6) δ 7.37-
7.42 (m, 2H), 7.80 (d, 2H, J ) 8.5), 8.01-8.06 (m, 2H), 8.11-
4-(p-Ch lor op h en yl)-2-n itr oa n ilin e (16): prepared from
(p-chlorophenyl)tributyltin (2.02 g, 5.04 mmol), which was
prepared from p-chlorobromobenzene (1.0 g, 5.16 mmol), 1.6
M n-BuLi (3.54 mL), and tributyltin chloride (2.10 mL, 7.76
mmol), with 4-bromo-2-nitroaniline (730 mg, 3.36 mmol) in
DMF (18 mL) using Pd(PPh3)2Cl2 (118 mg, 0.17 mmol) and
PPh3 (440 mg, 1.70 mmol) to provide 270 mg (32%) of scarlet
needles; mp 170 °C; IR (KBr) 3467, 3351, 1634, 1509, 1487,
8.24 (m, 4H), 8.40 (s, 1H), 8.51 (s, 2H); 13C NMR (DMSO-d6
+
3 drops of CF3COOH) δ 110.6, 110.7, 113.7, 115.7, 116.3, 116.9,
117.3, 123.4, 125.9, 126.4, 128.4, 131.3, 131.9, 132.1, 133.2,
139.8, 142.5, 154.0, 154.2; HRMS (FAB) calcd for C25H17N6
(MH+) 401.1515, found 401.1499.
1
1339, 1238; H NMR (DMSO-d6) δ 7.14 (d, 1H, J ) 9.0), 7.49
(d, 2H, J ) 8.5), 7.61 (brs, 2H), 7.68 (d, 2H, J ) 8.5), 7.80 (dd,
1H, J ) 9.0, 2.0), 8.23 (d, 1H, J ) 2.0); 13C NMR (DMSO-d6)
δ 120.4, 122.0, 126.2, 127.7, 128.2, 130.7, 132.0, 134.3, 137.4,
146.0. Anal. (C12H9N2O2Cl), C,H,N.
4-P h en yl-2-[2′-(ben zim id a zol-5′′-yl)ben zim id a zol-5′-yl]-
ben zim id a zole (11): prepared from 5 (128 mg, 0.49 mmol)
and 3-phenyl-1,2-phenylenediamine15 (100 mg, 0.49 mmol) in
6 mL of nitrobenzene to provide 107 mg (51%) of a brownish
white solid; mp >280 °C; IR (KBr) 3043, 1626, 1441, 1389,
1271; 1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 7.55-
7.68 (m, 5H), 7.84-7.90 (m, 3H), 8.00 (d, 1H, J ) 8.5), 8.12 (d,
1H, J ) 8.5), 8.23 (dd, 1H, J ) 8.5, 1.5), 8.46 (d, 1H, J ) 9.0),
Gen er a l P r oced u r e for P r ep a r in g 5-Su bstitu ted Ter -
ben zim id a zoles: 5-(1-Na p h th yl)-2-[2′-(ben zim id a zol-5′′-
yl)ben zim id a zol-5′-yl]ben zim id a zole (17). Hydrogenation
of 13 (166 mg, 0.63 mmol) was accomplished at 45 psi of H2 at
room temperature for 32 h using 10% Pd/C (33 mg) in EtOAc
(100 mL). The crude diamine (132 mg, 0.56 mmol), obtained
after passage through a bed of Celite and removal of EtOAc
in vacuo, was dissolved in 10 mL of nitrobenzene containing
5 (74 mg, 0.28 mmol). This reaction mixture was heated at
145 °C under N2 overnight. The cooled reaction mixture was
then purified directly by column chromatography. Elution
with 10-20% MeOH/EtOAc provided a 67% yield of a brownish
white solid; mp >280 °C; IR (KBr) 3048, 2925, 1624, 1436,
1385, 1290; 1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ
7.59-7.73 (m, 4H), 7.78-7.83 (m, 2H), 7.95 (s, 1H), 8.02-8.21
(m, 6H), 8.50 (d, 1H, J ) 9.0), 8.68 (s, 1H), 8.75 (s, 1H), 9.69
(s, 1H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ 113.9,
114.1, 114.9, 115.8, 116.1, 116.4, 117.8, 123.2, 124.0, 125.3,
125.8, 125.9, 126.4, 127.0, 127.5, 127.7, 128.3, 128.4, 129.4,
131.2, 131.7, 131.8, 132.5, 133.0, 133.7, 138.1, 138.8, 141.5,
150.6, 153.8, 158.1; HRMS (FAB) calcd for C31H21N6 (MH+)
477.1828, found 477.1813.
8.66 (s, 1H), 8.72 (s, 1H), 9.63 (s, 1H); 13C NMR (DMSO-d6
+
3 drops of CF3COOH) δ 112.9, 114.0, 115.7, 115.8, 116.5, 116.6,
118.9, 124.1, 125.1, 125.3, 125.6, 125.7, 126.1, 128.6, 129.0,
129.2, 131.6, 132.1, 133.3, 133.4, 136.5, 138.4, 140.6, 151.4,
153.3; HRMS (EI) calcd for C27H18N6 (M+) 426.1593, found
426.1605.
Gen er a l P r oced u r e for th e Syn th esis of 4-Su bstitu ted
2-Nit r oa n ilin e Der iva t ives:
4-(1-Na p h t h yl)-2-n it r o-
a n ilin e (13). 1-Bromonaphthalene (5 g, 24 mmol) was dis-
solved in freshly distilled THF (40 mL); 1.1 equiv of 1.6 M
n-BuLi (16.6 mL) was added dropwise at -78 °C. After 30
min of stirring, tributyltin chloride (6.51 mL, 24 mmol) was
added and the reaction mixture was brought to room temper-
ature overnight. The reaction was quenched by stirring in
open air. THF was removed in vacuo, and the mixture was
quickly passed through a short silica gel column eluting with
100% hexanes. Without further purification, the crude 1-(tribu-
tylstannyl)naphthalene (9.4 g, 23 mmol), together with 4-bromo-
2-nitroaniline (3.3 g, 15 mmol), Pd(PPh3)2Cl2 (528 mg, 0.75
mmol), and PPh3 (1.97 g, 7.5 mmol) in DMF (70 mL), was
heated under N2 at 120 °C overnight. After the reaction
mixture had cooled, DMF was removed in vacuo and the crude
mixture purified on a silica gel column eluting with 5-20%
EtOAc/hexanes to provide 395 mg (10%) of a scarlet solid.
5-(2-Na p h th yl)-2-[2′-(ben zim id a zol-5′′-yl)ben zim id a zol-
5′-yl]ben zim id a zole (18). The crude diamine (107 mg, 0.45
mmol), which was obtained by hydrogenation of 14 (123 mg,
0.45 mmol) in EtOAc (100 mL) using 10% Pd/C (33 mg) for 9
h, with 5 (119 mg, 0.45 mmol) provided 114 mg (53%) of a
brownish white solid; mp >280 °C; IR (KBr) 3046, 2954, 2923,
1
2862, 1441, 1287; H NMR (DMSO-d6) δ 7.52-7.59 (m, 2H),
7.66-7.81 (m, 4H), 7.93-8.07 (m, 5H), 8.12-8.19 (m, 2H), 8.29

Terbenzimidazoles as Topoisomerase I Poisons
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2823
(s, 1H), 8.40 (s, 1H), 8.45 (s, 1H), 8.49 (s, 1H); 13C NMR
(DMSO-d6 + 3 drops of CF3COOH) δ 112.0, 113.9, 114.7, 115.8,
116.1, 116.3, 117.8, 123.2, 125.3, 125.7, 125.8, 126.3, 126.7,
126.8, 126.9, 127.8, 128.6, 129.0, 131.0, 132.7, 133.0, 133.1,
133.6, 134.5, 135.5, 137.0, 138.6, 139.1, 141.4, 150.7, 153.7;
HRMS (FAB) calcd for C31H21N6 (MH+) 477.1828, found
477.1828.
(5.7 mg, 0.13 mmol) in 3 mL of nitrobenzene were heated at
145 °C in a sealed tube overnight. The cooled reaction mixture
was then purified directly by column chromatography. Elution
with 10% MeOH/EtOAc provided 26 mg (45%) of a brownish
white solid; mp >280 °C; IR (KBr) 3033, 2968, 2932, 1551,
1
1443; H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 2.88 (s,
3H), 7.47-7.61 (m, 3H), 7.80 (d, 2H, J ) 8.5), 7.88-8.01 (m,
2H), 8.04-8.29 (m, 4H), 8.44 (d, 1H, J ) 8.5), 8.65 (s, 2H); 13
C
5-(4-Met h oxyp h en yl)-2-[2′-(b en zim id a zol-5′′-yl)b en z-
im id a zol-5′-yl]ben zim id a zole (19). The crude diamine (148
mg, 0.69 mmol), which was obtained by hydrogenation of 15
(205 mg, 0.84 mmol) in EtOAc (100 mL) using 10% Pd/C (40
mg) for 4 h, with 5 (165 mg, 0.63 mmol) provided a 54% yield
of a brownish white solid; mp 275 °C; IR (KBr) 3395, 3064,
NMR (DMSO-d6 + 3 drops of CF3COOH) δ 14.3, 111.7, 114.6,
114.9, 116.2, 116.4, 117.5, 117.6, 123.0, 123.1, 124.8, 125.5,
126.0, 126.2, 127.5, 128.2, 129.5, 131.8, 133.0, 133.1, 134.9,
138.7, 139.7, 150.7, 153.8, 153.9; HRMS (FAB) calcd for
C28H21N6 (MH+) 441.1828, found 441.1846.
1
2821, 1610, 1517, 1437, 1243; H NMR (DMSO-d6) δ 3.83 (s,
5-P h en yl-2-[2′-(2′′-eth ylben zim id a zol-5′′-yl)ben zim id a -
zol-5′-yl]ben zim id a zole (26): prepared using the crude
diamine derived from 24 (75 mg, 0.16 mmol) and propional-
dehyde (7.3 mg, 0.13 mmol) using 3 mL of nitrobenzene in a
sealed tube overnight to provide a 28% yield of a brownish
white solid; mp >280 °C; IR (KBr) 3019, 2961, 1624, 1436,
3H), 7.07 (d, 2H, J ) 8.5), 7.48 (dd, 1H, J ) 8.5, 1.5), 7.64-
7.81 (m, 6H), 8.10-8.28 (m, 2H), 8.40 (s, 1H), 8.42 (s, 1H), 8.50
(s, 1H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ 55.5,
107.4, 110.9, 113.8, 114.5, 114.7, 114.8, 115.8, 116.1, 116.3,
117.6, 123.1, 125.2, 126.2, 128.6, 131.2, 131.9, 132.0, 133.0,
138.5, 139.2, 141.5, 150.3, 154.0, 159.8; HRMS (FAB) calcd
for C28H21N6O (MH+) 457.1777, found 457.1784.
1
1261; H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 1.48 (t,
3H), 3.23 (q, 2H), 7.46-7.61 (m, 3H), 7.79-8.01 (m, 4H), 8.06-
8.28 (m, 4H), 8.45 (dd, 1H, J ) 9.0, 1.0), 8.66 (s, 2H); 13C NMR
(DMSO-d6 + 3 drops of CF3COOH) δ 11.6, 20.3, 111.7, 113.3,
114.6, 115.1, 116.2, 123.6, 125.1, 125.6, 127.5, 128.2, 129.3,
129.4, 131.7, 132.9, 133.3, 133.5, 138.9, 139.7, 140.4, 150.4,
153.5, 158.9; HRMS (FAB) calcd for C29H23N6 (MH+) 455.1985,
found 455.1974.
5-(4-Ch lor op h en yl)-2-[2′-(ben zim id a zol-5′′-yl)ben zim i-
d a zol-5′-yl]ben zim id a zole (20). The crude diamine, which
was obtained by hydrogenation of 16 (177 mg, 0.66 mmol) in
EtOAc (100 mL) using 10% Pd/C (40 mg) for 4 h, with 5 (99
mg, 0.38 mmol) in 5 mL of nitrobenzene provided a 49% yield
of a brownish white solid; mp >280 °C; IR (KBr) 3046, 2820,
1621, 1549, 1426, 1282; 1H NMR (DMSO-d6 + 3 drops of CF3-
COOH) δ 7.56 (d, 2H, J ) 8.5), 7.82 (d, 2H, J ) 8.5), 7.88-
8.12 (m, 6H), 8.48 (d, 1H, J ) 8.5), 8.63 (s, 1H), 8.72 (s, 1H),
9.69 (s, 1H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ
111.8, 113.8, 114.7, 115.8, 116.1, 117.7, 123.0, 124.1, 125.1,
125.2, 125.3, 125.4, 129.2, 129.3, 129.4, 131.9, 132.1, 133.0,
133.1, 137.2, 138.5, 139.3, 141.6, 150.8, 153.8; HRMS (FAB)
calcd for C27H18N6Cl (MH+) 461.1281, found 461.1273.
2-(3,4-Din itr op h en yl)-5-p h en ylben zim id a zole (23). A
stirred solution of 4-phenyl-1,2-phenylenediamine12 (340 mg,
1.9 mmol) and 22 (374 mg, 1.9 mmol) in 10 mL of nitrobenzene
was heated at 145 °C under N2 overnight. The cooled reaction
mixture was then purified directly by column chromatography.
Elution with 10% EtOAc/hexanes provided 455 mg (66%) of a
yellow solid; mp 146 °C; IR (KBr) 3097, 2971, 1608, 1540, 1445,
1365, 1286; 1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ
7.37-7.55 (m, 3H), 7.67-8.79 (m, 3H), 7.85 (d, 1H, J ) 8.5),
7.97 (d, 1H, J ) 1.0), 8.50 (d, 1H, J ) 8.5), 8.71 (dd, 1H, J )
8.5, 2.0), 8.97 (d, 1H, J ) 2.0); 13C NMR (DMSO-d6 + 3 drops
of CF3COOH) δ 113.2, 116.3, 123.8, 124.4, 127.2, 127.2, 127.7,
129.4, 132.2, 133.7, 137.3, 137.4, 138.1, 140.5, 142.5, 142.8,
147.6; HRMS (FAB) calcd for C19H13N4O4 (MH+) 361.0937,
found 361.0935.
5-P h en yl-2-[2′-(2′′-p r op ylben zim id a zol-5′′-yl)ben zim i-
d a zol-5′-yl]ben zim id a zole (27). This was prepared from the
crude diamine derived from 24 (113 mg, 0.24 mmol) and
butyraldehyde (13.8 mg, 0.24 mmol) using 3 mL of nitroben-
zene in a sealed tube overnight. The cooled reaction mixture
was then purified directly by column chromatography. Elution
with 0-20% MeOH/EtOAc provided 45 mg (40%) of a brownish
white solid; mp >280 °C; IR (KBr) 3100, 2961, 2925, 2874,
1436, 1283; 1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 1.01
(t, 3H), 2.77 (q, 2H), 2.87 (t, 2H), 7.45-7.60 (m, 3H), 7.80 (d,
2H, J ) 7.0), 7.82-8.00 (m, 2H), 8.06-8.14 (m, 3H), 8.25 (d,
1H, J ) 7.0), 8.46 (d, 1H, J ) 9.0), 8.69 (s, 2H); 13C NMR
(DMSO-d6 + 3 drops of CF3COOH) δ 13.5, 20.3, 28.4, 111.7,
113.5, 115.1, 115.8, 115.9, 116.2, 116.6, 123.9, 124.5, 125.2,
125.6, 127.5, 128.2, 129.4, 131.7, 131.9, 132.9, 133.4, 137.6,
138.9, 139.6, 150.3, 153.4, 154.5, 157.1; HRMS (FAB) calcd
for C30H25N6 (MH+) 469.2141, found 469.2132.
5-P h en yl-2-[2′-(2′′-isop r op ylb en zim id a zol-5′′-yl)b en z-
im id a zol-5′-yl]ben zim id a zole (28): prepared from 24 (113
mg, 0.24 mmol) and isobutyraldehyde (13.6 mg, 0.19 mmol)
in 5 mL of nitrobenzene at 145 °C in a sealed tube overnight
in 51% yield, brownish white solid; mp >280 °C; IR (KBr) 3056,
1
2961, 2925, 2867, 1443, 1283; H NMR (DMSO-d6 + 3 drops
of CF3COOH) δ 1.53 (d, 6H, J ) 7.0), 3.54 (q, 1H, J ) 7.0),
7.46-7.61 (m, 3H), 7.79 (d, 2H, J ) 7.0), 7.87-8.00 (m, 2H),
8.04-8.08 (m, 3H), 8.21 (d, 1H, J ) 9.0), 8.46 (d, 1H, J ) 9.0),
8.66 (s, 2H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ
20.2, 27.6, 111.7, 113.1, 114.6, 115.1, 116.1, 116.2, 116.3, 123.2,
123.3, 125.0, 127.5, 128.2, 129.4, 131.7, 131.8, 133.0, 133.2,
138.8, 139.7, 141.2, 149.2, 150.7, 153.8, 161.2; HRMS (FAB)
calcd for C30H25N6 (MH+) 469.2141, found, 469.2154.
5-P h en yl-2-[2′-(2′′-p h en ylben zim id a zol-5′′-yl)ben zim i-
d a zol-5′-yl]ben zim id a zole (29): prepared from 24 (80 mg,
0.17 mmol) and benzaldehyde (15 mg, 0.14 mmol) using 3 mL
of nitrobenzene at 145 °C under N2 overnight to provide a 57%
yield of a brownish white solid; mp >280 °C; IR (KBr) 3056,
2925, 1624, 1436, 1283; 1H NMR (DMSO-d6 + 3 drops of CF3-
COOH) δ 7.44-7.61 (m, 4H), 7.72-8.13 (m, 9H), 8.20-8.41
(m, 4H), 8.66 (s, 2H); 13C NMR (DMSO-d6 + 3 drops of CF3-
COOH) δ 107.4, 111.8, 111.9, 114.1, 114.7, 115.6, 115.7, 116.1,
116.2, 123.5, 124.4, 127.5, 128.2, 129.5, 129.8, 130.2, 132.0,
133.2, 137.1, 138.7, 139.8, 140.4, 143.1, 150.5, 150.8, 152.5,
154.0, 160.7; HRMS (FAB) calcd for C33H23N6O (MH+) 503.1984,
found 503.1982.
5-P h en yl-2-[2′-(3,4-d in itr op h en yl)ben zim id a zol-5′-yl]-
ben zim id a zole (24). Compound 23 (370 mg, 1.03 mmol) was
dissolved in EtOAc (100 mL) and reduced by hydrogenation
using 10% Pd/C (80 mg) for 1.5 h. After passing through a
bed of Celite, the EtOAc was removed in vacuo. The crude
diamine (220 mg, 0.67 mmol) and 22 (131 mg, 0.67 mmol) in
10 mL of nitrobenzene were heated at 145 °C under N2
overnight. The cooled reaction mixture was then purified
directly by column chromatography. Elution with 0-10%
MeOH/EtOAc provided 300 mg (94%) of a yellow solid; mp
>280 °C; IR (KBr) 3065, 2960, 1603, 1540, 1439, 1360, 1280;
1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 7.46-7.60 (m,
3H), 7.80 (d, 2H, J ) 7.0), 7.87-8.00 (m, 2H), 8.05-8.10 (m,
2H), 8.19 (dd, 1H, J ) 8.5, 2.0), 8.50 (d, 1H, J ) 8.5), 8.66 (s,
1H), 8.77 (dd, 1H, J ) 8.5, 2.0), 9.00 (d, 1H, J ) 2.0); 13C NMR
(DMSO-d6 + 3 drops of CF3COOH) δ 111.6, 114.5, 116.0, 117.0,
117.3, 123.3, 125.6, 127.1, 127.5, 128.2, 129.4, 131.5, 132.3,
132.7, 134.8, 138.8, 139.6, 140.4, 142.3, 142.5, 142.8, 150.5,
150.9; HRMS (FAB) calcd for C26H17N6O4 (MH+) 477.1311,
found 477.1307.
Gen er a l P r oced u r e for P r ep a r in g 2′′-Su bstitu ted Ter -
ben zim id a zoles: 5-P h en yl-2-[2′-(2′′-m eth ylben zim id a zol-
5′′-yl)ben zim id a zol-5′-yl]ben zim id a zole (25). In EtOAc (50
mL) was dissolved 24 (75 mg, 0.16 mmol), which was reduced
by hydrogenation using 10% Pd/C (15 mg) for 1.5 h. After
passing through a bed of Celite and removal of the EtOAc in
vacuo, the crude diamine (63 mg, 0.13 mmol) and acetaldehyde
5-P h en yl-2-[2′-[2′′-(p-m eth oxyp h en yl)ben zim id a zol-5′′-
yl]ben zim id a zol-5′-yl]ben zim id a zole (30): prepared from
the crude diamine derived from 24 (80 mg, 0.17 mmol) and
p-anisaldehyde (22.9 mg, 0.17 mmol) using 3 mL of nitroben-
zene at 145 °C under N2 overnight to provide a 89% yield of a
brownish white solid; mp >280 °C; IR (KBr) 3056, 2933, 1611,

2824 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Kim et al.
1437, 1255; 1H NMR (DMSO-d6 + 3 drops of CF3COOH) δ 3.94
(s, 3H), 7.33-7.60 (m, 5H), 7.80 (d, 2H, J ) 8.0), 7.88-8.01
(m, 2H), 8.07-8.12 (m, 3H), 8.24-8.30 (m, 3H), 8.44 (d, 1H, J
) 9.0), 8.66 (d, 2H, J ) 2.0); 13C NMR (DMSO-d6 + 3 drops of
CF3COOH) δ 56.0, 111.7, 113.3, 114.6, 115.1, 115.6, 115.7,
116.2, 121.5, 122.8, 124.3, 125.2, 125.6, 127.5, 127.6, 129.4,
130.5, 130.8, 131.7, 132.9, 133.2, 134.9, 138.8, 139.7, 140.2,
150.3, 151.7, 153.6, 163.8; HRMS (FAB) calcd for C34H25N6O
(MH+) 533.2089, found 533.2072.
Top oisom er a se I-Med ia ted DNA Clea va ge Assa ys.
Human topoisomerase I was expressed in Escherichia coli and
purified as previously described.19 Plasmid YEpG was also
purified by the alkali lysis method followed by phenol depro-
teination and CsCl/ethidium isopycnic centrifugation as de-
scribed.20,21 The end-labeling of the plasmid was accomplished
as previously described.21 The cleavage assays were performed
as previously reported.8 The drug and DNA in the presence
of topoisomerase I was incubated for 30 min at 37 °C. After
development of the gels, 24 h exposure was typically used to
obtain autoradiographs outlining the extent of DNA fragmen-
tation.
Cytotoxicity Assa y. The cytotoxicity as listed for several
of the compounds which were prepared as part of this study
was determined using human lymphoblast RPMI 8402 cells
and the MTT microtiter plate tetrazolium cytotoxicity assay
(MTA).22-24 The cytotoxicity assay was performed using 2000
cells/well, in 200 µL of growth medium, which were grown in
suspension at 37 °C in 5% CO2 and maintained by regular
passage in RPMI medium supplemented with 10% heat-
inactivated fetal bovine serum, L-glutamine (2 mM), penicillin
(100 U/mL), and streptomycin (0.1 mg/mL). The cells were
exposed continuously for 4 days to different drug concentra-
tions and assayed at the end of the fourth day. Each assay
was performed with a control which did not contain any drug.
All assays were performed at least twice in six replica wells.
(6) Chen, A. Y.; Liu, L. F. DNA topoisomerases: Essential enzymes
and lethal targets. Annu. Rev. Pharmacol. Toxicol. 1994, 34,
191-218.
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totoxicity and drug resistance in human cell cultures. Cancer
Res. 1993, 53, 1332-1335.
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Synthesis and evaluation of terbenzimidazoles as topoisomerase
I inhibitors. J . Med. Chem. 1995, 38, 3638-3644.
(13) Kim, J . S.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J .
Substituted 2,5′-Bi-1H-benzimidazoles: Topoisomerase I inhibi-
tion and cytotoxicity. J . Med. Chem. 1996, 39, 992-998.
(14) 1-Nitro-2-(acetylamino)naphthalene:
Organic Syntheses;
Wiley: New York, 1943; Collect Vol. II, pp 438-439.
(15) Sako, S.-I. The formation of cyclic compounds from derivatives
of diphenyl. Part II. Bull. Chem. Soc. J pn. 1934, 9, 55-76.
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N. E.; Breslauer, K. J . Characterizing the DNA binding modes
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both intercalative and minor groove binding properties. Drug
Des. Discovery 1996, 13, 115-133.
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Isohelicity and phasing in drug-DNA sequence recognition:
crystal structure of a tris(benzimidazole)-oligonucleotide com-
plex. Biochemistry 1996, 35, 13745-13752.
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E. J .; Liu, L. F. Identification of topoisomerase I as the cytotoxic
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Ack n ow led gm en t. Mass spectrometry was provided
by the Washington University Mass Spectrometry Re-
source, an NIH Research Resource (Grant P41RR0954).
This study was supported by Grant CA 39662 from the
National Cancer Institute (L.F.L.) and a fellowship
grant from the J ohnson & J ohnson Discovery Research
Fund (E.J .L.).
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