Inhibitors of topoisomerase II based on the benzodiimidazole and dipyrroloimidazobenzimidazole ring systems: Controlling DT-diaphorase reductive inactivation with steric bulk

Article abstract of DOI:10.1021/jm990210q

Described herein are the synthesis, cytotoxic properties, and topoisomerase II inhibition assays of benzodiimidazole and dipyrroloimidazobenzimidazole structural variants of the pyrrolo[1,2- a]benzimidazole or APBI ring system. These ring variants were designed to inhibit topoisomerase II, much as the APBIs are able to do. Since only the quinone form of the APBIs can intercalate DNA, two-electron reduction to the hydroquinone by DT-diaphorase is known to deactivate these compounds. Indeed, the APBIs possess a high inverse correlation with the cellular concentration of DT-diaphorase. Therefore one feature of the ABPI structural variants is the excessive bulk about the quinone ring, which was predicted to diminish DT-diaphorase substrate activity. Another feature is the presence of one or two alkylating centers, which would permit alkylation of DNA and/or topoisomerase II. Inhibition assays for topoisomerase II-mediated relaxation of supercoiled DNA indicate that the benzodiimidazole and dipyrrolo- imidazobenzimidazole quinone ring systems are catalytic inhibitors of topoisomerase II. Both quinone systems exhibit cytotoxicity perhaps due to the lack of inactivation by DT-diaphorase as well as topoisomerase II inhibition. One quinone displayed the novel feature of cytotoxicity selectively against melanoma cell lines. In conclusion, the benzodiimidazole and dipyrroloimid-azobenzimidazole quinone ring systems will be subjected to future analogue development and structure-activity studies.

Full text of DOI:10.1021/jm990210q

J . Med. Chem. 2000, 43, 629-638
629
In h ibitor s of Top oisom er a se II Ba sed on th e Ben zod iim id a zole a n d
Dip yr r oloim id a zoben zim id a zole Rin g System s: Con tr ollin g DT-Dia p h or a se
Red u ctive In a ctiva tion w ith Ster ic Bu lk
William G. Schulz and Edward B. Skibo*
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604
Received April 26, 1999
Described herein are the synthesis, cytotoxic properties, and topoisomerase II inhibition assays
of benzodiimidazole and dipyrroloimidazobenzimidazole structural variants of the pyrrolo[1,2-
a]benzimidazole or APBI ring system. These ring variants were designed to inhibit topo-
isomerase II, much as the APBIs are able to do. Since only the quinone form of the APBIs can
intercalate DNA, two-electron reduction to the hydroquinone by DT-diaphorase is known to
deactivate these compounds. Indeed, the APBIs possess a high inverse correlation with the
cellular concentration of DT-diaphorase. Therefore one feature of the ABPI structural variants
is the excessive bulk about the quinone ring, which was predicted to diminish DT-diaphorase
substrate activity. Another feature is the presence of one or two alkylating centers, which would
permit alkylation of DNA and/or topoisomerase II. Inhibition assays for topoisomerase
II-mediated relaxation of supercoiled DNA indicate that the benzodiimidazole and dipyrrolo-
imidazobenzimidazole quinone ring systems are catalytic inhibitors of topoisomerase II. Both
quinone systems exhibit cytotoxicity perhaps due to the lack of inactivation by DT-diaphorase
as well as topoisomerase II inhibition. One quinone displayed the novel feature of cytotoxicity
selectively against melanoma cell lines. In conclusion, the benzodiimidazole and dipyrroloimid-
azobenzimidazole quinone ring systems will be subjected to future analogue development and
structure-activity studies.
Ch a r t 1
In tr od u ction
The design of antitumor agents utilizing the two-
electron reducing enzyme DT-diaphorase is envisioned
either by reductive activation or by the absence of
reductive inactivation. The reductive activation of anti-
tumor agents by DT-diaphorase specifically in cancer
cell lines is well-known in the literature.1,2 The reductive
activation process usually involves reduction of a qui-
none species to the hydroquinone by DT-diaphorase
followed by leaving group elimination to afford an
alkylating quinone methide species.3,4 DT-diaphorase is
also known to play a role in the detoxification of toxins,1
and therefore cancers low in DT-diaphorase could be
sensitive to some agents based on the absence of
reductive inactivation
The 6-acetamidopyrrolo[1,2-a]benzimidazole-based an-
titumor agents, the APBIs in Chart 1, have been
investigated in this laboratory for some time.5-8 The
APBIs are inactivated by two-electron reduction and
therefore show a strong inverse correlation with DT-
diaphorase levels (the highest inverse correlation of
20 000 compounds in the National Cancer Institute’s
archives).6 The unchanged (oxidized) APBI, rather than
the reduced form, appears to act as an inhibitor of the
first step of topoisomerase II-mediated relaxation of
supercoiled DNA.5,7 These findings prompted a study
of structural variants of the pyrrolo[1,2-a]benzimidazole
ring system: the benzodiimidazole and dipyrroloimid-
azobenzimidazole quinone systems shown in Chart 1,
along with their complete ring names.
The rationale for studying these ring variants is that
the increased steric bulk about the quinone ring, com-
pared to the APBIs, would decrease the DT-diaphorase
substrate activity. Since the 7-butyl APBI derivatives
were slowly reduced by DT-diaphorase,7 the bulkier
benzodiimidazoles and the dipyrroloimidazobenzimid-
azoles should be even more resistant to reduction
resulting in enhanced cytotoxicity. Previous studies of
the APBIs revealed the importance of a substituent at
the 3-position, which is usually an acetoxy derivative,6
* To whom correspondence should be addressed. Tel: 480-965-3581.
Fax: 480-965-2747. E-mail: ESkibo@ASU.EDU.
10.1021/jm990210q CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/26/2000

630 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schulz and Skibo
Sch em e 1
second annelation gives rise to cis/trans isomers, which
are not readily separated until converted to the quinone
products. Quinone elaboration9-11 afforded three prod-
ucts: the cis and trans isomers of 19 and the deacety-
lated product 20. The latter product very likely arose
from reductive removal of one of the acetates during
nitro group reduction. Identification of the cis and trans
forms of 19 was based on the independent synthesis
outlined in Scheme 4. Nucleophilic substitution of 1,5-
dibromo-2,4-dinitrobenzene with (R)- 4-amino-2-hydroxy-
butanoic acid15 afforded 22, which was converted to
trans-17 by an “ internal Phillips reaction”.10 Conversion
of authentic trans-17 to the quinone species resulted in
a product spectrally identical to 19b.
Differ en tia l Cytotoxicity. This section presents a
discussion of the relative cytotoxicities of 5, 19a ,b, and
11-13 against seven histological types of cancer: leu-
kemia, non-small-cell lung, colon, CNS, melanoma,
ovarian, and renal. The measure of cyotoxicity employed
is the LC50 value, which is the concentration of drug
lethal to 50% of cancer cells. The LC50 data were
obtained by screening the compound against up to 60
human cancer cell lines in the National Cancer Insti-
tute’s in vitro screen.16,17 The LC50 data in Figures 1
and 2 are the average values for each type of cancer
expressed as the -log(LC50) on the y-axis. The x-axis
shows the histological cancer type, and the z-axis shows
the compound. Lower LC50 values represent higher
potency, and therefore higher -log(LC50) values repre-
sent higher potency.
Inspection of Figure 1 reveals that unsubstituted
benzodiimidazole quinone 5 possesses a high specificity
toward melanoma cell lines. In contrast the agent APBI-
A, which shows in vivo activity against melanoma,6
displays high specificity toward both melanoma and
renal cancers. The combination of the structural fea-
tures of 5 and APBI-A, represented by 19a ,b, results
in either complete loss of cytotoxicity (19b) or retained
cytotoxicity but with complete loss of specificity (19a in
Figure 1). Compound 19b was tested as the racemate,
and therefore neither enantiomer (RR or SS) is active.
In conclusion, the meso form (19a ) appears to be the
most suitable stereoisomer for further analogue devel-
opment.
as well as the importance of the 3-center configuration
on DT-diaphorase reductase activity.7 Therefore, both
the cis- and trans-diacetoxy derivatives of the dipyrrolo-
imidazobenzimidazole system were investigated. The
benzodiimidazoles had to be substituted with chloride,
rather than acetoxy groups, to observe cytotoxicity. The
present report provides a description of the synthesis,
cytotoxicity, DT-diaphorase substrate activity, and topo-
isomerase II activity of the ring variants shown in Chart
1.
Resu lts a n d Discu ssion
Syn th esis. The preparation of the unsubstituted
dipyrroloimidazobenzimidazole quinone 5 was carried
out as outlined in Scheme 1. Treatment of 1,5-dibromo-
2,4-dinitrobenzene with pyrrolidine afforded 1, which
was converted to 2 by reduction and then acetylation.
Cyclization of 2 to 3 was carried out as previously
described for the synthesis of the ABPIs.9,10 Quinone
elaboration involved nitration, nitro group reduction to
the amine, and finally Fremy oxidation of the amine.9-11
Preparation of the benzodiimidazoles 11-13 was
carried out starting with 6 as outlined in Scheme 2.
Reduction and tetrachloroacetylation of 6 afforded 8,
which was ring-closed to the benzodiimidazole system
9 by a Phillips type reaction.12 Quinone elaboration by
the usual method9-11 afforded a mixture of 11-13, all
of which were important to the present study.
The data in Figure 2 reveal that the unsubstituted
benzodiimidazole quinone (13) possesses no cytotoxicity
while analogues bearing leaving groups (11 and 12)
possess cytotoxicity, but with little or no specificity. In
order for the benzodiimidazole quinones to show ap-
preciable cytotoxicity, substitution with an excellent
leaving group (chloride as opposed to acetate) appears
to be required. The nearly equivalent cytotoxicity of 11
and 12 suggests that alkylation, but not necessarily
cross-linking, is occurring. In contrast to the benzodi-
imidazoles, the dipyrroloimidazobenzimidazoles and the
APBIs are still cytotoxic when substituted with acetate
or even when unsubstituted.
DT-Dia p h or a se Su b st r a t e Act ivit y. The com-
pounds evaluated for rat liver DT-diaphorase substrate
activity include 5, 11-13, 19a ,b, and 20. The substrate
specificity was compared with that of the known anti-
tumor agent APBI-A, Chart 1. We had proposed that
increasing the steric bulk about the quinone ring of
APBI-A would slow DT-diaphorase-mediated reduction.
The preparation of the dipyrroloimidazobenzimidazole
derivatives 19a ,b and 20 was carried out as outlined
in Schemes 3 and 4. Annelation of both of the pyrrolo-
imidazo rings was carried out stepwise, 14 f 15 and
16 f 17, employing the “tert-amino effect”.10,13,14 The

Inhibitors of Topoisomerase II
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 631
Sch em e 2
Sch em e 3
However, the dipyrroloimidazobenzimidazole quinones
5 and 20 were substantially better substrates for the
enzyme than ABPI-A, Figure 3. Both 5 and 20 had
cytotoxicity against all cancer panels, while the less
bulky APBIs (represented by APBI-A) possess cytotox-
icity toward melanoma and renal cancers. The lack of
cancer cell specificity we observe with 19a , 11, and 12
may originate with the absence of DT-diaphorase inac-
tivation in all the cancer cell lines. In contrast compound
20 was inactive against all cancer cell lines, perhaps
due to the efficient reductive inactivation by DT-
diaphorase.
The quinones 5 and 19b do not fit into the relation-
ship between DT-diaphorase substrate activity and
specific cytotoxicity discussed above. Quinone 5 is an
excellent substrate for DT-diaphorase and also has a
high specificity toward melanoma cell lines. Quinone
19b, on the other hand, is not reduced (inactivated) by
DT-diaphorase, is an inhibitor of topoisomerase II, and
is noncytotoxic. Clearly other factors beside DT-dia-
phorase and topoisomerase II must be involved in the
biological activity of these quinones.
similar Vmax/Km values (6.24 and 7.1 × 10-4 s-1
)
compared to that of 2.6 × 10-4 s-1 for APBI-A. These
results suggest that the presence of one or more fused
pyrrolo rings is important for the DT-diaphorase sub-
strate activity of these systems. Indeed, none of the
benzodiimidazole quinones 11-13 were reduced by rat
liver DT-diaphorase. The presence of the fused pyrrolo
ring in mitomycin C may be in part responsible for its
efficient reductve activation in tumor cells. When the
dipyrroloimidazobenzimidazole ring possesses two ace-
tate substituents, 19a ,b, DT-diaphorase substrate activ-
ity was lost perhaps for steric reasons.
Some of the cytotoxicity results presented in Figures
1 and 2 show a relationship between the absence of DT-
diaphorase substrate activity and the lack of specific
cancer cell cytotoxicity. Thus 19a , 11, and 12 exhibit

632 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schulz and Skibo
F igu r e 1. Plots of -log(LC50) versus cancer type for 5, APBI-
A, and 19a . Each cancer type represents the average of six to
eight different cancer cell lines. Compound 5 has high specific-
ity for melanoma, while APBI-A has specificity for melanoma,
ovarian, and renal cancers. In contrast, 19a is completely
nonspecific.
F igu r e 2. Plots of -log(LC50) versus cancer type for 11-13.
Each cancer type represents the average of six to eight
different cancer cell lines. Compound 13 is considered to be
devoid of activity; i.e., -log(LC50) is 3-4. Both 11 and 12 are
cytotoxic but with little or no selectivity for cancer type.
Sch em e 4
F igu r e 3. Reciprocal plots of velocity (nM/s) versus concen-
tration (10-5 M) for substrate activity of APBI-A, 5, and 20
with purified rat liver DT-diaphorase. Both 5 and 20 fall on
the same plot due to similar Vmax/Km values. The relative
specificity or kcat/Km of these substrates was calculated from
the Vmax/Km value and the concentration of enzyme.
COMPARE correlation of 0.9 was observed between 19a
and tamoxifen, which is also active against all cell lines.
Top oisom er a se II In h ibition . Previous studies with
the APBIs indicated that they are catalytic inhibitors
of topoisomerase II rather than poisons.7,19 Topo-
isomerase II poisons are usually clinically active and
act at the religation step by stabilizing the enzyme-
DNA complex formed upon double-strand cleavage.
Examples of agents which stabilize the cleavable com-
plex include m-AMSA, etoposide, doxorubicin,20-22 natu-
rally occurring naphthoquinones,23-25 and many other
structurally diverse compounds.26,27 However, catalytic
inhibitors of topoisomerase II are being reported more
frequently in the literature and may also be clinically
useful.28-31 Of particular note are the pyrroloimino-
quinones,32,33 which are mechanistic and structurally
related to the APBIs and imino-APBIs. Both the pyr-
roloiminoquinones and the ABPIs are known to inhibit
the topoisomerase II-mediated relaxation of supercoiled
DNA by intercalation.5 The data shown in Figures 4 and
To gain insight into other possible cytotoxicity mech-
anisms, we employed the National Cancer Institute’s
COMPARE program.16 This program along with the
DISCOVERY program18 were developed to compare the
patterns of cytotoxicity in 60-cell line cancer screens
with those of other compounds. Antitumor agents with
identical mechanisms of action possess identical or
nearly identical cytotoxicity patterns (correlation coef-
ficient > 0.8). For example anthracycline analogues
(doxorubicin, rubidazone, daunamycin) have a high
correlation (>0.9) with each other as do the DNA
alkylating agents (chlorambucil, thiotepa, triethylene-
melamine). Quinone 5 does not correlate well with any
known compound in the National Cancer Institute’s
archives suggesting a unique mechanism of cytotoxicity.
In contrast, compound 19a is active against all cell lines
and there is no pattern to utilize in correlations. A

Inhibitors of Topoisomerase II
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 633
F igu r e 4. Agarose gels of topoisomerase II-catalyzed relaxations of pRYG supercoiled DNA (form I along with supercoiled dimer
and trimer) run in the presence or absence of ethidium bromide. In both Figures 4 and 5, lanes E-H are controls: lane G is
unrelaxed pRYG supercoiled DNA, lane H is the linear form of pRYG supercoiled DNA, lanes E and F are relaxation reactions
carried out with and without DMSO. Lanes A and B are relaxation reactions carried out in the presence of 0.19 and 0.39 mM 19a
and lanes C and D likewise carried out in the presence of 0.13 and 0.26 mM 19b. The higher concentrations of both compounds
show inhibition with 19b causing complete inhibition.
5 indicate that the benzodiimidazole and dipyrrolo-
imidazobenzimidazole quinone systems likewise inhibit
topoisomerase II.
reveals significant amounts of form I DNA in lanes B-D
indicating that both 19a ,b can inhibit topoisomerase II-
mediated relaxation. Consistent with the results ob-
tained with the non-EB gel, 0.38 mM 19b completely
inhibited relaxation, lane D.
The non-EB-containing gel in Figure 5 shows that
both 5 and 11 can inhibit topoisomerase II by virtue of
the presence of significant amounts of form I DNA in
lanes A-D, with both concentrations of 11 (0.25 and 0.5
mM) causing complete inhibition.
Relaxation assays were carried out with recombinant
human topoisomerase II, and completed reactions were
assayed on agarose gels run with or without ethidium
bromide (EB). The EB-containing gels will readily detect
the presence of linear DNA, but these gels bearly resolve
form I and form II DNAs (form II traveled slightly faster
than form I). If linear DNA is present, then the agent
acts as a topoisomerase II poison; i.e., the agent
stabilizes the cleavable complex. In contrast, non-EB-
containing gels will readily resolve the form II topomers
arising from the supercoiled or form I DNA, and
catalytic inhibition could then be documented by noting
the decrease in relaxation with increaasing inhibitor
concentration.
Shown in Figure 4 are the assays for the relaxation
of pRYG supercoiled DNA by p170 human topo-
isomerase II in the presence of 19a (lanes A and B) and
19b (lanes C and D). The other lanes are either controls
or references (see legend of Figure 4). Inspection of the
non-EB gel of Figure 4 reveals the absence of linear
DNA indicating the absence of topoisomerase II poison-
ing. Inspection of the non-EB gel shown in Figure 4
Con clu sion s
The goal at the outset of this research was to develop
more effective cytotoxic analogues of the pyrrolo[1,2-a]-
benzimidazole (APBI) system previously developed in
this laboratory. The strategy employed was to add fused
pyrrolo and fused imidazo rings to the APBI system in
the hope of increasing the bulk about the quinone ring
so as to prevent DT-diaphorase reductive inactivation.
The resulting dipyrroloimidazobenzimidazole quinones
5 and 20 were found to be even better substrates for
DT-diaphorase than the APBIs. These results suggest
that the presence of one or more fused pyrrolo rings is
important for the DT-diaphorase substrate activity of
these systems. Indeed, none of the benzodiimidazole

634 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schulz and Skibo
Exp er im en ta l Section
All solutions and buffers for DT-diaphorase and topo-
isomerase II assays used doubly distilled water. Human
recombinant p170 topoisomerase II and pRYG plasmid were
purchased from TopoGEN Inc. All analytically pure compounds
were dried under high vacuum in a drying pistol over refluxing
toluene. Elemental analyses were run at Atlantic Microlab,
Inc., Norcross, GA. All TLCs were performed on silica gel
plates using a variety of solvents and a fluorescent indicator
for visualization. IR spectra were taken as thin films and the
strongest absorbances reported. 1H NMR spectra were ob-
tained from a 300-MHz spectrometer. All chemical shifts are
reported relative to TMS.
DT-Dia p h or a se Red u ction Kin etics Stu d ies. Rat liver
DT-diaphorase was isolated as previously described.7,34 Kinetic
studies were carried out in 0.05 M pH 7.4 Tris‚HCl buffer,
under anaerobic conditions, employing Thunberg cuvettes. A
2 mM stock solution of the appropriate quinone was prepared
in dimethyl sulfoxide (DMSO). To the top port was added the
quinone stock and to the bottom port was added DT-diaphorase
and NADH in the Tris buffer. The top and bottom ports were
purged with argon for 20 min and equilibrated to 30 °C. The
ports were then mixed and the reaction was followed at 296
nm for 25 min to obtain initial rates. The concentrations upon
after mixing were 0.3 mM NADH, 1-20 × 10-5 M quinone,
and 14.5 nM (based on flavin) enzyme active sites. The value
of ∆ꢀ was calculated from the initial and final absorbance
values for complete quinone reduction; usual value for ꢀ is
6000-8000 M-1 cm-1. The value of ∆ꢀ was used to calculate
F igu r e 5. Agarose gels of topoisomerase II-catalyzed relax-
ations of pRYG supercoiled DNA (form I along with supercoiled
dimer and trimer) run in the absence of ethidium bromide.
The control lanes E-H are the same as described in the Figure
4 legend. Lanes A and B are relaxation reactions carried out
in the presence of 0.1 and 0.2 mM 5 and lanes C and D likewise
carried out in the presence of 0.25 and 0.5 mM 11. Compound
11 shows complete inhibition at both concentrations.
V
max in M s-1. The results were fitted to a Lineweaver-Burke
quinones 11-13 were reduced by rat liver DT-dia-
phorase. When 5 was substituted with acetates to afford
19a ,b, neither quinone was reduced by DT-diaphorase
presumably for steric reasons.
plot from which kcat/Km values were calculated based on 14.5
nM active sites.
Cytotoxicity Stu d ies. Cytotoxicity studies were carried out
at the National Cancer Institute.16,35 The submitted sample
(∼5 mg) was subjected to the 60-cell line in vitro assay, which
resulted in mean graph data including LC50 (lethal concentra-
tion, 50%), GI50 (growth inhibition concentration, 50%), and
TGI (total growth inhibition concentration). Average LC50
values off each cancer type are plotted in Figures 1 and 2.
Top oisom er a se II Assa ys. The topoisomerase relaxation
reactions were carried out with 0.25 µg of pRYG supercoiled
DNA (Form I), 4 units of topoisomerase II in 20 µL of 50 mM
Tris‚Cl, pH 8, containing 120 mM KCl, 10 mM MgCl2, 0.5 mM
dithiothreitol, and 0.5 mM ATP. Varying amounts of the
quinone-based inhibitors were added to some reactions. The
reactions were run for 45 min at 37 °C and then mixed with
2 µL of a stop solution consisting of 10% SDS. After the
reaction was stopped, 1 µL of a solution of 1 mg of proteinase
K in 1 mL of water was added and the resulting mixture
incubated at 37 °C for 15 min. The reaction mixture was
extracted once with a 20-µL solution of chloroform:isoamyl
alcohol (24:1) and then combined with 2 µL of 10X loading
solution (0.25% bromophenol blue and 50% aqueous glycerol).
The relaxation reactions were assayed in 1X TAE buffer (50X
is 242 g of Tris base, 57.1 mL of acetic acid, and 100 mL of 0.5
M EDTA in 1 L of dd water) using a 1% agarose gel. The gel
and running buffer either did or did not have 0.5 µg/mL
ethidium bromide. The gel was loaded with 20 µL of reaction
mixture/gel loading solution and run at 2 V/cm for 6-8 h. The
gels without ethidium were soaked in a solution of 0.5 µg/mL
ethidium bromide in the TAE buffer followed by washing for
30 min in the same buffer without ethidium. Gels with
ethidium bromide were washed for 30 min in the TAE buffer.
1,5-Dia ceta m id o-2,4-d ip yr r olid in oben zen e (2) was pre-
pared by the two-step procedure described as follows. To 1.0 g
(3.26 mmol) of 1,3-dibromo-4,6-dinitrobenzene in a round-
bottom flask was added 1.5 mL of pyrrolidine dropwise. After
addition of the pyrrolidine the solution was heated at 90 °C
until starting material had disappeared by TLC, about a 12-h
reaction time. The completed reaction was poured over 100 g
of ice resulting in precipitation of the product. The product
was filtered off and dried under vacuum. An analytical sample
was prepared by flash chromatography using silica gel with
chloroform as the eluent. The product 1 was recrystallized from
The lack of cancer cell specificity we observe with 19a ,
11, and 12 may originate with the absence of DT-
diaphorase inactivation in all the cancer cell lines. Thus
the absence of DT-diaphorase inactivation in all cells
results in cytotoxicity in all panels. In contrast com-
pound 20 was inactive against all cancer cell lines,
perhaps due to the efficient reductive inactivation by
DT-diaphorase.
The quinones 5 and 19b do not fit into the relation-
ship between DT-diaphorase substrate activity and
specific cytotoxicity discussed above. Quinone 5 is an
excellent substrate for DT-diaphorase and also has a
high specificity toward melanoma cell lines. Quinone
19b, on the other hand, is not reduced by DT-diaphorase
and is noncytotoxic. There are no others compounds in
the National Cancer Institute’s archives with the LC50
mean graph profile of 5 suggesting the presence of a
novel cytotoxicity profile.
The benzodiimidazole and dipyrroloimidazobenz-
imidazole quinones 19a ,b, 5, and 11 were found to
inhibit topoisomerase II suggesting a possible cytotox-
icity mechanism. The absence of linear DNA formation
in the topoisomerase II assays indicates the absence of
cleavable complex stabilization as seen in the clinically
used topoisomerase II poisons.
This study has succeded in identifying novel lead
compounds for further development. Quinone 5 is a
highly selective cytotoxic agent whose mechanism of
action must be investigated. The rational design of
selective anticancer compounds would be possible based
on these mechanistic studies. On the other hand,
quinones 19a and 11 possess broad-spectrum cytotox-
icity and could also be effective cancer chemotherapeutic
agents. These systems will also be subjected to mecha-
nistic and structure-activity studies.

Inhibitors of Topoisomerase II
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 635
chloroform/hexane: 892 mg (89%) yield; TLC (CHCl3) Rf )
0.37; mp >190 °C dec; IR (KBr pellet) 2874, 1599, 1554, 1489,
1354, 1323, 1309, 1259 cm-1; 1H NMR (CDCl3) δ 8.55 and 5.90
(2H, 2s, aromatic protons), 3.28 (8H, t, J ) 6.6 Hz, pyrrolo
protons), 2.00 (8H, quintet, J ) 3.3 Hz, pyrrolo protons); MS
(EI mode) m/ z 306 (M+), 289 (M+ - OH).
chloroform. The chloroform extracts were dried (Na2SO4),
filtered, and concentrated in vacuo to afford crude product.
The product was further purified by column chromatography
using silica gel and chloroform/methanol (99:1) as eluent. The
purified product was recrystallized from chloroform/hexane:
95 mg (40%) yield; mp >260 °C; TLC (chloroform/methanol
[80:20]) Rf ) 0.47; IR (KBr pellet) 1676, 1656, 1521, 1485, 1438,
1408, 1307, 740 cm-1; 1H NMR (CDC13) δ 4.25 (4H, t, J ) 7.13
Hz, C(1) and C(9) methylene), 2.96 (4H, t, J ) 6.1 Hz, C(3)
and C(7) methylene), 2.72 (4H, quintet, J ) 7.77 Hz, C(2) and
C(8) methylene); 13C NMR (CDC13) δ 175, 168, 160, 147, 129,
45, 26, 22; MS (EI mode) m/z 268 (M+). Anal. (C14H12N4O2) C,
H, N.
1,5-Bis(m eth yla m in o)-2,5-d in itr oben zen e (6). To 2.48 g
(7.6 mmol) of 1,5-dibromo-2,4-dinitrobenzene was added 25 mL
of dimethylformamide followed by 15 mL of 17% methylamine
in dimethylformamide. The reaction was stirred at room
temperature for 15 min and then 30 mL of ice water was added
to precipitate the product. The yellow product was filtered off,
washed with ice water, and dried under vacuum: 1.62 g (95%)
yield; mp 260 °C dec; TLC (CH2Cl2) Rf ) 0.62; IR (KBr pellet)
3385, 1629, 1593, 1541, 1425, 1410, 1342, 1294, 1195, 1010,
800 cm-1; 1H NMR (dimethyl sulfoxide-d6) δ 8.97 and 5.69 (2H,
2s, C(5) and C(2) protons), 8.5 (2H, s, amine protons), 3.0 (6H,
s, N-methyl’s); MS (EI mode) m/z 226. Anal. (C8H10N4O4) C,
H, N.
1,5-Dia m in o-2,4-bis(m eth yla m in o)ben zen e‚HC1 (7). A
suspension consisting of 1.05 g (4.66 mmol) of 6 and 200 mg
of 5% Pd on carbon in 150 mL of methanol was shaken under
50 pi H2 for 2 h. The reaction mixture was filtered through
Celite into 5 mL of concentrated hydrochloric acid and the
methanol was removed in vacuo to afford the HCl salt as a
greenish solid: 1.36 g (93%) yield; mp 130-145 °C dec; IR (KBr
pellet) 3346, 3219, 3022, 2858, 1531, 1460, 1288, 1130, 887,
468 cm-1; MS (EI mode) m/z 166 (M+), 155 (M+ - methyl),
124 (M+ - methyl, NH2).
1,5-Bis(ch lor oa ce t a m id o)-2,4-b is(N -m e t h ylch lor o-
a ceta m id o)ben zen e (8). To a solution consisting of 100 mL
of dry dimethylformamide and 2.5 mL of pyridine was added
815 mg (2.6 mmol) of 7 followed by the addition of 1.0 mL (13
mmol) of chloroacetyl chloride. The reaction was stirred for 2
h and then the dimethylformamide was removed in vacuo to
afford a solid residue. To the residue was added 75 mL of water
and the solution was neutralized with aqueous sodium bicar-
bonate. The resulting precipitate was filtered off and dried in
vacuo. The supernatant was extracted thrice with 50-mL
portions of chloroform. The chloroform extracts were dried
(Na2SO4), filtered, and evaporated to a solid residue. The
precipitate and extracted solids were combined and recrystal-
lized from chloroform/hexane: 848 mg (69%) yield; mp 193-
200 °C dec; TLC (chloroform/methanol [90:10]) Rf ) 0.42; IR
(KBr pellet) 1670, 1597, 1534, 1534, 1507, 1428, 1385, 1344,
1255, 1252, 1125, cm-1. Anal. (C16 H18Cl4N4O4) H; N: found
11.31; found 11.86. This intermediate has some triacetylated
impurity; all acetylated species were converted to pure 9.
A solution consisting of 4.0 g (13.7 mmol) of 1 and 800 mg
of 5% Pd on carbon in 350 mL of acetic acid was shaken under
50 psi of H2 for 12 h and then filtered through Celite into a
flask containing 100 mL of acetic anhydride. An additional 50
mL of acetic anhydride was then added to the reaction mixture
followed by stirring at room temperature for 1.5 h. The reaction
was then concentrated in vacuo to an oil to which 350 mL of
diethyl ether was added. The product crystallized from solution
as an off-white solid pure enough for the next step. An
analytical sample was prepared by recrystallization from
chloroform/hexane: 4.0 g (88%) yield; mp 205 °C; TLC (chlo-
roform/methanol [90:10]) Rf ) 0.43; IR (KBr pellet) 3539, 3485,
1
1645, 1562, 1516, 1427, 1371, 1298, 972, 815 cm-1; H NMR
(dimethyl sulfoxide-d6) δ 6.94 and 6.15 (2H, 2s, aromatic
protons), 3.16 (4H, t, J ) 6.4 Hz, pyrrolidino protons), 2.5 (4H,
t, J ) 6.4 Hz, pyrrolodino protons); MS (EI mode) m/z 330 (M+),
287 (M+ - ketene). Anal. (C18H26N4O2‚0.5H2O) C, H, N.
3H ,7H -1,2,8,9-Tet r a h yd r op yr r olo[1,2-a ]p yr r olo[1′,2′:
1,2]im id a zo[4,5-f]ben zim id a zole (3). To a solution consist-
ing of 6 mL of 96% formic acid and 3 mL of 30% hydrogen
peroxide was added 1.5 g (4.5 mmol) of 2, and the reaction
stirred at 70 °C for 2 h. The reaction was then cooled and
diluted with 25 mL of water and the pH adjusted to ∼7 with
concentrated ammonium hydroxide. The resulting precipitate
as filtered off and the remaining solution extracted five times
with 25-mL portions of chloroform. The chloroform extracts
were dried (Na2SO4), filtered, and concentrated to afford the
crude product. An analytical sample was obtained by recrys-
tallization from chloroform/hexane: 165 mg (10%) yield; mp
240 °C dec; T1C (chloroform/methanol [80:20]) Rf ) 0.40; IR
(KBr pellet) 1545, 1491, 1433, 1354, 1302, 1228, 1118, 943,
848, 669 cm-1; 1H NMR (dimethyl sulfoxide-d6) δ 7.62 and 7.40
(2H, 2s, aromatic protons) 4.10 (4H, t, J ) 6.87 Hz, C(1) and
C(9) methylenes), 2.95 (4H, t, J ) 7.0 Hz, C(3) and C(7)
methylenes); MS (EI mode) m/z 238 (M+). Anal. (C14H14N4‚
0.2H2O) C, H, N.
5-Nitr o-3H,7H-1,2,8,9-tetr a h yd r op yr r olo[1,2-a ]p yr r olo-
[1′,2′:1,2]im id a zo[4,5-f]ben zim id a zole (4). To a solution
consisting of fuming nitric acid and concentrated sulfuric acid
(50:50), cooled in an ice/salt bath to 0 °C, was added 316 mg
(1.33 mmol) of 3 and the reaction was stirred for 15 min. The
reaction was then poured over crushed ice and pH adjusted
to 6.0-6.5 with concentrated ammonium hydroxide. The
resulting solution was then extracted five times with 25-mL
portions of chloroform. The chloroform extracts were combined,
dried (Na2SO4), filtered, and concentrated. Pure product was
obtained by recrystallization from chloroform/hexane. An
analytical sample was prepared by preparative TLC using
chloroform/methanol (9:1) as the eluent: 343 mg (90%) yield;
mp > 260 °C; TLC (chloroform/methanol [90:10]) Rf ) 0.23;
IR (KBr pellet) 3510, 3448, 3171, 1498, 1460, 1340, 1236, 1157,
2,6-Bis(ch lor om eth yl)-1,7-d im eth ylben zo[1,2-d :4,5-d ′]-
d iim id izole (9). To a 50-mL solution of 4 N HCl, held at 80
°C, was added 821 mg (1.74 mmol) of 8 and the reaction stirred
for 2 h at 80 °C. The completed reaction was cooled and
neutralized with aqueous sodium bicarbonate. The resulting
precipitate was filtered off and dried under high vacuum: 438
mg (88%) yield; mp 214-215 °C; TLC (1-butanol/acetic acid/
water [5:3:2]) Rf ) 0.55; IR (KBr pellet) 1528, 1470, 1424, 1370,
1
1033, 675 cm-1; H NMR (dimethyl sulfoxide-d6) δ 7.93 (1H,
s, aromatic proton), 4.20 (4H, t, J ) 7.08 Hz, C(1) and C(9)
methylenes), 3.04 (4H, t, J ) 7 Hz, C(3) and C(7) methylenes)
2.68 (4H, quintet, J ) 7.68 Hz, C(2) and C(8) methylenes); MS
(EI mode) m/z 238 (M+). Anal. (C14H13N5O2) C, H, N.
3H ,7H -1,2,8,9-Tet r a h yd r op yr r olo[1,2-a ]p yr r olo[1′,2′:
1,2]im id a zo[4,5-f]ben zim id a zole-5,10-d ion e (5). A solution
consisting of 250 mg (0.88 mmol) of 4, 62 mg of 5% palladium
on activated carbon, and 80 mL of methanol was shaken at
50 psi H2 for 4 h. The reaction was then filtered through Celite
and the methanol removed in vacuo to afford the amine as a
solid residue. To the amine was added a solution consisting of
1 g of monobasic potassium phosphate in 25 mL of water. To
this solution was added a second solution consisting of 1.8 g
of Fremy’s salt, 1.5 g of monobasic potassium phosphate, and
75 mL of water. The reaction was stirred at room temperature
for 2 h and then extracted thrice with 25-mL portions of
1257, 936, 909, 801 cm-1 1H NMR (CDCl3) δ 8.12 and 7.17
;
(2H, 2s, C(4) and C(8) protons), 4.88 (4H, s, C(2) and C(6),
chloromethyls), 3.93 (6H, s, N(1) and N(7) methyls); MS (EI
mode) m/z 282 (M+), 284 (M + 2). Anal. (C12H12Cl2N4) C, H,
N.
2,6-Bis(ch lor om eth yl)-1,7-d im eth yl-4-n itr oben zo[1,2-d :
4,5-d ′]d iim id izole (10). To 20 mL of dry acetonitrile was
added 50 mg (0.17 mmol) of 9 followed by 46 mg of (85%)
nitronium tetrafloroborate. The reaction was stirred for 1 h;
then the acetonitrile was removed in vacuo. The dry residue
was then dissolved in 25 mL of water and the pH adjusted to

636 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Schulz and Skibo
7.5 with aqueous sodium bicarbonate. It was then extracted
thrice with 20-mL portions of chloroform. The chloroform
extracts were dried (Na2SO4), filtered, and concentrated. The
resulting yellow solid was recrystallized from chloroform/
hexane: 36 mg (63%); mp 256 °C dec; TLC (chloroform/
methanol [90:10]) Rf ) 0.21; IR (KBr pellet) 1525, 1504, 1471,
ZnCl2 was heated at 120 °C for 20 h. (Note: Yield of products
decreases if anhydrous ZnCl2 is employed.) The reaction
mixture was allowed to cool and then poured into 25 mL of
H2O followed by extraction thrice with 25-mL portions of
chloroform. The chloroform extracts were dried (Na2SO4),
filtered, and concentrated to a solid residue. Pure products
were obtained by flash chromatography using silica gel with
chloroform/hexane [99:1] as the eluent followed by recrystal-
lization from ethyl acetate/hexane: 571.7 mg (56%) yield; mp
> 115 °C dec; TLC (chloroform/methanol [90:10]) Rf ) 0.60;
IR (KBr pellet) 1743, 1624, 1575, 1527, 1460, 1433, 1371, 1348,
1232, 1080 cm -1; 1H NMR (dimethyl sulfoxide-d6) δ 8.40 and
8.22 (2H, 2s, C(5)- and C(8)-aromatic protons), 6.18 (1H, dd, J
) 7.6 Hz, J ) 4 Hz, C(3)-proton), 4.34 (2H, m, C(1) methylene),
3.13 and 2.59 (2H, 2m, C(2) methylene), 2.09 (3H, s, acetate
methyl); MS (EI mode) m/z 339 and 341 (M+ 79Br and 81Br),
298 and 300 (M+ - acetyl), 234 and 236 (M+ - acetyl - nitro).
Anal. (C12H10BrN3O4‚0.4H2O) C, H, N.
1433, 1404, 1365, 1257, 1188, 1132, 1003 cm-1 1H NMR
;
(CDC13) δ 7.52 (1H, s, aromatic proton), 4.97 (4H, s, C(2)- and
C(6)-chloromethyls), 4.02 (6H, s, N(1)- and N(7)-methyls); MS
(EI mode) m/z 327 (M+), 329 (M + 2), 292 (M+ - C1), 246 (M+
- C1 - NO2). Anal. (C12H11C12N5O2) C, H, N.
2,6-Bis(ch lor om eth yl)-1,7-d im eth ylben zo[1,2-d :4,5-d ′]-
d iim id a zole-4,8-d ion e (11), 2-ch lor om eth yl-1,6,7-tr im eth -
ylben zo[1,2-d:4,5-d′]diim idizole-4,8-dion e (12), an d 1,2,6,7-
tetr a m eth ylben zo[1,2-d :4,5-d ′]d iim id izole-4,8-d ion e (13)
were synthesized by the following two-step procedure. A
suspension consisting of 207 mg (0.57 mmol) of 10 was
dissolved in 25 mL of chloroform and placed in a separatory
funnel. To the separatory funnel was added a sodium dithion-
ite solution, consisting of 800 mg sodium dithionite and 5 mL
of pH 7.0 phosphate buffer, and the funnel was vigorously
shaken. After addition of a second portion of sodium dithionite
followed by shaking, the chloroform layer was removed and
the aqueous dithionite layer was extracted thrice with 50-mL
portions of chloroform. The chloroform extracts were combined,
dried (Na2SO4), filtered, and concentrated to afford crude
amine product which was crystallized from chloroform/methanol/
hexane: 70 mg (40.4%) yield.
3-H yd r oxy-6-n it r o-7-p yr r olid in o-2,3-d ih yd r o-1H -p yr -
r olo[1,2-a ]ben zim id a zole (16). To 1.0 g (2.9 mmol) of 15 was
added 30 mL of pyrrolidine followed by heating at reflux for
12 h. The reaction mixture was concentrated in vacuo and the
residue diluted with 90 mL of chloroform. The resulting
solution was washed thrice with 30-mL portions of 20%
aqueous acetic acid and twice with 30-mL portions of water.
The chloroform solution was then dried (Na2SO4), filtered, and
concentrated to a solid residue. Recrystallization from chlo-
roform/hexane afforded pure product: 730 mg (87%) yield; mp
205-208 °C; TLC (chloroform/methanol [90:10]) Rf ) 0.42; IR
(KBr pellet) 3161, 1645, 1579, 1514, 1450, 1413, 1363, 1319,
To a suspension of 117 mg (0.39 mmol) of the crude amine
obtained above in 10 mL of water/dimethylformamide [9:1] was
added 20 mL of a solution consisting of 450 mg (1.56 mmol) of
Fremy’s salt and 35 mg of monobasic potassium phosphate.
The reaction was stirred for 8 h and then extracted thrice with
25-mL portions of chloroform. The chloroform extracts were
dried (Na2SO4), filtered, and concentrated to a solid residue.
The quinone products were purified by flash chromatography
using silica gel and chloroform/methanol [97:3] as the eluent.
11: 23 mg (17.5%) yield; mp 232 °C dec; TLC (chloroform/
methanol [90:10]) Rf ) 0.51; IR (KBr pellet) 3043, 1668, 1500,
1
1288, 825 cm-1; H NMR (dimethyl sulfoxide-d6) δ 8.00 and
7.02 (2H, 2s, aromatic protons), 6.05 (1H, d, J ) 5.7 Hz,
hydroxyl), 5.15 (1H, m, C(3)-proton), 4.29 and 4.12 (2H, 2m,
C(1)-diastereomeric methylene), 3.12 (4H, t, J ) 6.6 Hz, 2- and
5-pyrrolidine protons), 2.88 and 2.39 (2H, 2m, C(2) diastereo-
meric methylene), 1.92 (4H, q, J ) 3.3 Hz, 3- and 4-pyrrolidine
protons); MS (EI mode) m/z 288 (M+), 271 (M+ - OH), 241
(M+ - HNO2). Anal. (C14H16N4O3‚0.1H2O) C, H, N.
cis- a n d tr a n s-3,7-Dia cetoxy-3H,7H-1,2,8,9-tetr a h y-
dr opyr r olo[1,2-a ]pyr r olo[1′,2′:1,2]im idazo[4,5-f]ben zim id-
a zole (17). To 576 mg (2 mmol) of 16 in 20 mL of acetic
anhydride was added 272 mg of ZnCl2 and the reaction was
heated at reflux for 5 h. The reaction was concentrated in
vacuo to an oil followed by dilution with 100 mL of chloroform.
The chloroform solution was extracted twice with water and
dried over sodium sulfate. Chromatographic purification was
carried out employing a silica gel column utilizing chloroform/
methanol [9:1] as the eluent. The cis/trans mixture eluted off
just after a fast moving orange band corresponding to acety-
lated 16. The fractions corresponding to the cis/trans mixture
were combined and concentrated to a solid residue: 165 mg
(23%) yield; mp >300 °C dec; TLC (chloroform/methanol [90:
10]) Rf ) 0.36; IR (KBr pellet) 1743, 1639, 1548, 1437, 1373,
1404, 1344, 1085, 1006, 839, 754, 692, 636 cm-1 1H NMR
;
(CDC13) δ 4.74 (4H, s, C(2) and C(6) methylenes), 4.09 (6H, s,
N(1) and N(7) methyls); MS (EI mode) m/z 312 (M+), 314 (M
+ 2), 277 (M+ - Cl). Anal. (C12H10Cl2N4O2‚0.25H2O) C, H; N:
calcd, 17.64; found, 17.18. The low nitrogen percent found is
due to slow hydrolysis to the alcohol.
12: 2.16 mg (2.3%) yield; mp 198 °C dec; TLC (chloroform/
methanol [90:10]) Rf ) 0.45; IR (KBr pellet) 3447, 3016, 2316,
1978, 1500, 1321, 1082, 1004, 748, 640 cm-1 1H NMR δ
;
(CDC13) δ 4.73 (2H, s, C(2)-methylene), 4.08 and 3.93 (6H, 2s,
N(1) and N(7) methyls), 2.51(3H, s, C(6)-methyl); MS (EI mode)
m/z 312 (M+), 314 (M+ + 2), 277 (M+ - Cl). Anal. (C12H11
ClN4O2) C, H, N.
-
13: 3.03 mg (3.16%) yield; mp > 300 °C dec; TLC (chloroform/
methanol [90:10]) Rf ) 0.34; IR (KBr pellet) 2924, 1670, 1643,
1498, 1369, 1315, 1080, 991, 742, 628 cm-1; 1H NMR (CDCl3)
δ 3.91 (6H, s, N(1)- and N(7)-methyls), 2.48 (6H, s, C(2)- and
C(6)-methyls); MS (EI mode) m/z 244 (M+), 229 (M+ - methyl),
216 (M+ - CO). Anal. (C12H12N4O2).
1
1300, 1232, 1006, 1232, 1080 cm-1; H NMR (dimethyl sulf-
oxide-d6) δ 7.92 and 7.70 (2H, 2s, aromatic protons), 6.17 (2H,
dd, J ) 7.2 Hz, J ) 3.9 Hz), 4.23 (4H, m, C(1) and C(9)
diastereomeric methylenes), 3.15 and 2.58 (4H, 2m, C(2) and
C(8) diastereomeric methylenes), 2.09 (6H, s, acetate methyl);
MS (EI mode) m/ z 354 (M+) 311 (M+ - acetyl), 295 (M+
-
1-Br om o-2,4-d in itr o-5-p yr r olid in oben zen e (14). To 4.0
g (0.012mol) of 1,5-dibromo-2,4-dinitrobenzene, suspended in
50 mLof ethanol and cooled in an ice bath, was added 2.2 mL
of pyrrolidine and the reaction was stirred for 1 h with
continued cooling. The resulting yellow precipitate was filtered
off, rinsed with ethanol, and vacuum-dried: 3.32 g (92%) yield;
TLC (CHCl3/hexane [80:20]) Rf ) 0.54; mp 165-171 °C; IR
acetate), 251 (M+ - acetic acid & acetyl). Anal. (C18H18N4O4)
C, H, N.
cis- a n d tr a n s-3,7-Dia cetoxy-5(a n d 10)-n itr o-3H,7H-
1,2,8,9-t et r a h yd r op yr r olo[1,2-a ]p yr r olo[1′,2′:1,2]im id -
a zo[4,5-f]ben zim id a zole (18). To a solution consisting of 81
mg (0.25 mmol) of 17 in 25 mL of acetonitrile was added 8
equiv of (85%) nitronium tetrafloroborate and the resulting
reaction mixture was stirred for 16 h at room temperature.
The solvent was removed in vacuo and the residue was
dissolved in 20 mL of H2O. The pH was adjusted to 6.0-6.5
with aqueous sodium bicarbonate and the solution was then
extracted thrice with 15-mL portions of chloroform. The
chloroform extracts were dried over sodium sulfate, filtered,
and concentrated to a solid residue. The product was purified
using silica gel preparative TLC plates employing chloroform/
(KBr pellet) 3113, 2876, 1579, 1554, 1510, 1369, 1321, 1273,
-1
1155, 991 cm
;
1H NMR (CDCl3) δ 8.60 (1 H, s, aromatic
proton), 7.18 (1 H, s, aromatic proton), 3.32 (4H, m, pyrrolidine
methylenes), 2.06 (4H, m, pyrrolidine methylenes); MS (EI
mode) m/z 315 and 317 (M+, 79Br and 81Br). Anal. (C10H10
-
BrN3O4) C, H, N.
3-Acetoxy-7-br om o-6-n itr o-2,3-d ih yd r o-1H-p yr r olo[1,2-
a ]ben zim id a zole (15). A mixture consisting of 906 mg (3
mmol) of 14, 10 mL of acetic anhydride, and 408 mg of 98%

Inhibitors of Topoisomerase II
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 637
methanol [90:10] as the developing solvent: 27 mg (30%)
combined yield of two products; TLC (chloroform/methanol [90:
10]) Rf ) 0.57 and 0.26; H NMR of high Rf product (DMSO-
3497, 3317, 1737, 1618, 1568, 1539, 1417, 1319, 1267, 1244,
1184, 1116 cm-1 1H NMR (DMSO-d6) δ 12.56 (1H, bs, acid
;
1
proton), 8.94 (1H, t, J ) 5.4 Hz, amine proton), 8.82 (1H, s,
aromatic proton), 7.49 (1H, s, aromatic proton), 5.61 (1H, bs,
hydroxy proton), 4.10 (1H, dd, J ) 3.3 Hz, J ) 7.8 Hz,
methine), 3.61 (2H, m, methylene), 2.00 & 1.88 (2H, 2m,
methylene); MS (EI mode) m/z 363 and 365 (M+, 79Br & 81Br),
274 (M+ - CH2CHOHCOOH). Anal. (C10H10BrN3O7) H, N; C:
calcd, 32.98; found, 33.74. This somewhat impure intermediate
was converted to the pure intermedeiate 22.
d6) δ 8.37 (1H, s, aromatic), 6.20 (2H, dd, J ) 2.7 Hz, J ) 5.8
Hz, C(3) and C(7) methines), 4.55 (4H, m, C(1)- and C(9)-
methylenes), 3.07 and 2.55 (4H, 2m, C(2) and C(8) methylenes),
2.11 (6H, s, acetate methyls); 1H NMR of low Rf product
(DMSO-d6) δ 8.19 (1H, s, aromatic), 6.21 (2H, m, C(3) and C(7)
methine), 4.39 and 4.28 (4H, 2m, C(1) and C(9) methylene),
3.21 and 2.65 (4H, 2m, C(2) and C(8) methylenes), 2.11 (6H,
s, acetate methyls).
(R,R)-1,5-Bis(3-ca r b oxy-3-h yd r oxyp r op yla m in o)-2,4-
d in itr oben zen e (22). To a solution consisting of 250 mg (0.68)
mmol) of 21 and 75 mL of H2O were added 200 mg of sodium
bicarbonate and 165 mg (1.38 mmol) of (R)-4-amino-â-hydroxy-
butyric acid. The reaction mixture was refluxed for 20 h and
then allowed to cool to room temperature. The solution was
then concentrated and placed on a 100-g phenyl Baker Bond
reverse-phase column and the product was eluted with H2O.
The product fractions were combined and then acidified with
concentrated hydrochloric acid resulting in crystallization of
the product: 220 mg (80.6%) yield; TLC (butanol/acetic acid/
water [5:2:3]) Rf ) 0.43; mp 149-157 °C; IR (KBr pellet) 3333,
cis- a n d tr a n s-3,7-Dia cetoxy-3H,7H-1,2,8,9-tetr a h y-
dr opyr r olo[1,2-a ]pyr r olo[1′,2′:1,2]im idazo[4,5-f]ben zim id-
a zole-5,10-d ion e (19a ,b) was synthesized from crude 18 by
the following two-step procedure. A solution consisting of 138
mg (0.34 mmol) of 18, 75 mL of methanol, and 32 mg of 5%
palladium on activated carbon was shaken under 50 psi for 4
h. The reaction mixture was then filtered through a Celite cake
to remove the catalyst and concentrated to an off-white solid,
which was dissolved in 25 mL of H2O containing 200 mg of
monobasic potassium phosphate. To this solution was added
a second solution consisting of 50 mL of H2O, 1 g of monobasic
potassium phosphate, and 726 mg of Fremy’s salt. The
resulting mixture was stirred at room temperature. until it
turned from purple to orange. The solution was then extracted
thrice with 75-mL portions of chloroform. The extracts were
dried over sodium sulfate and concentrated to a residue, which
was purified by flash chromatography on silica gel employing
chloroform/methanol (95:5) as the eluent. The major yellow
band afforded 86 mg of cis/ trans-19 and the minor slower
moving yellow band afforded 20. Separation of the cis and
trans forms of 19 was accomplished by using a 34-cm silica
gel column employing chloroform/methanol (98:2) as the
eluent. The separated products were recrystallized from ethyl
acetate.
1761, 1720, 1626, 1577, 1404, 1329, 1302, 1219, 1107 cm-1
;
1H NMR (DMSO-d6) δ 8.97 and 5.94 (2H, 2s, aromatic protons),
8.63 (2H, t, J ) 4.8 Hz, amine protons), 4.12 (2H, dd, J ) 8.1
Hz, J ) 4.0 Hz, methines), 3.49, 2.06, &1.93 (8H, 3m,
methylenes). Anal. (C14H18N4O10‚0.25H2O), C, H, N.
(R,R)-3,7-Dia cetoxy-3H,7H-1,2,8,9-tetr a h yd r op yr r olo-
[1,2-a ]pyr r olo[1′,2′:1,2]im idazo[4,5-f]ben zim idazole (tr a n s-
17) was synthesized by the following sequence. A suspension
consisting of 200 mg (0.49) mmol) of 22 in 75 mL of 4 N HCl
and 40 mg of 5% Pd on activated carbon was shaken under 50
psi H2 for 4 h. The solution was then filtered through Celite
into a round-bottom flask and heated at 90 °C for 15 h. The
reaction mixture was then allowed to cool and the solvent was
removed under vacuum. The resulting solid was redissolved
in a minimum volume of water and the pH adjusted to 5.5-
6.0 with aqueous sodium hydroxide. The crystallized product
was filtered and dried under vacuum: 110 mg (82%) yield; mp
>260 °C; TLC (butanol/acetic acid/water [5:2:3]) Rf ) 0.14; IR
(KBr pellet) 3138, 2860, 1543, 1498, 1438, 1327, 1302, 1120,
tr a n s-19: 23.7 mg (18%) yield; TLC (chloroform/methanol
[90:10]) Rf ) 0.52; mp >260 °C; IR (KBr pellet) 1743, 1672,
1520, 1498, 1437, 1375, 1226, 1089, 1043 cm-1 1H NMR
;
(CDCl3) δ 6.09 (2H, dd, J ) 3.3 Hz, J ) 7.78 Hz, C(3) and C(7)
protons), 4.36 (4H, m, C4 and C(9) protons), 3.17 and 2.66 (4H,
2m, C(2) and C(8) protons), 2.12 (6H, s, acetate methyl); MS
(EI mode) m/z 384 (M+), 341 (M+ - acetyl), 325, 281. Anal.
(C18H16N4O6) C, H, N.
1
1105, 956 cm-1; H NMR (DMSO-d6) δ 7.76 and 7.50 (2H, 2s,
aromatic protons), 5.84 (2H, d, J ) 6 Hz, C(3) and C(7)
hydroxy), 5.10 (2H, m, C(3) and C(7) methines), 4.22-4.06 (4H,
m, C(1) and C(9) methylenes), 2.95 and 2.42 (4H, 2m, C(2) and
C(8) methylenes); MS (EI mode) m/z 270 (M+) 253 (M+ - OH).
cis-19: 27 mg (21%) yield; TLC (chloroform/methanol [90:
10]) Rf ) 0.46; mp 239-240 °C; IR (KBr pellet) 1739, 1670,
1521, 1498, 1437, 1373, 1228, 1080, 1047 cm-1 1H NMR
;
To a solution consisting of 48 mg (0.17 mmol) of the above
product in 15 mL of dry methylene chloride was added 100
µL of acetyl chloride followed by 400 µL of dry pyridine. The
reaction mixture was stirred for 4 h at room temperature and
then diluted with 10 mL of chloroform. The resulting mixture
was extracted five times with 25-mL portions of water and
the chloroform layer was dried over sodium sulfate, filtered,
and concentrated to a residue which was recrystallized from
ethyl acetate/hexane: 38 mg (62%) yield; 1H NMR (DMSO-
d6) δ 7.89 and 7.63 (2H, 2d, J ) 1 Hz, aromatic), 6.13 (2H, dd,
J ) 3.3 Hz, J ) 7.8 Hz, C(3) and C(7) protons), 4.24 (4H, m,
C(1) and C(9) methylene), 3.12 and 2.54 (4H, 2m, C(2) and
C(8) methylenes), 2.06 (6H, s, acetate methyls); MS (EI mode)
m/z 354 (M+).
(CDCl3) δ 6.08 (2H, dd, J ) 2.7 Hz, J ) 7.8 Hz, C(3) and C(7)
methine), 4.36 (4H, m, C(1) and C(9) methylenes), 3.20 and
2.65 (4H, 2m, C(2) and C(8) methylenes), 2.116 (6H, s, acetate
methyls), MS (EI mode) m/z 384 (M+), 341 (M+ - acetyl), 325,
281. Anal. (C18H16N4O6) C, H, N.
20: 11 mg (10%) yield; TLC (chloroform/methanol [90:10])
Rf ) 0.31; mp >240 °C; IR (KBr pellet) 1736, 1685, 1670, 1521,
1
1498, 1224, 1041 cm-1; H NMR (CDCl3) δ 6.09 (1H, dd, J )
3.3 Hz, J ) 8.5 Hz, C(3) methine), 4.34 (4H, m, C(1) and C(9)
methylenes), 3.18-2.64 (6H, 3m, C(2), C(7) and C(8) methyl-
enes), 2.11 (3H, s, acetate methyl); MS (EI mode) m/z 326 (M+),
383 (M+ - acetyl). Anal. (C16H14N4O4) C, H, N.
(R)-1-Br om o-5-(3-ca r boxy-3-h yd r oxyp r op yla m in o)-2,4-
d in itr oben zen e (21). To a solution consisting of 375 mg (3.15
mmol) of (R)-â-hydroxy-4-aminobutyric acid and 250 mg of
sodium bicarbonate in 75 mL of H2O was added a solution of
500 mg (1.5 mmol) of 1,5-dibromo-2,4-dinitrobenzene dissolved
in 50 mL of acetone. The reaction mixture was heated at reflux
for 22 h and then cooled to room temperature. The pH of the
reaction mixture was adjusted to 8 with sodium bicarbonate
and then extracted twice with 20-mL portions of chloroform
to remove unreacted 1,5-dibromo-2,4-dinitrobenzene. The aque-
ous layer was acidified with 4 N hydrochloric acid and then
extracted five times with 50-mL portions of chloroform. The
chloroform extracts were dried over sodium sulfate, filtered,
and concentrated to a solid residue, which was recrystallized
from chloroform/hexane: 347 mg (63%) yield; TLC (chloroform/
methanol [80:20]) Rf ) 0.63; mp 140-160 °C; IR (KBr pellet)
Ack n ow led gm en t. We thank the American Cancer
Society, the National Institutes of Health, and the
Arizona Disease Control Commission for their generous
support.
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