Studies of Pyrrolo[1,2-α]benzimidazolequinone DT-diaphorase substrate activity, topoisomerase II inhibition activity, and DNA reductive alkylation

Article abstract of DOI:10.1021/jm960546p

The influence of structure on DT-diaphorase substrate activity, topoisomerase II inhibition activity, and DNA reductive alkylation was studied for the 6-aziridinylpyrrolo[1,2-α]benzimidazolequinones (PBIs) and the 6-acetamidopyrrolo[1,2-α]benzimidazolequi

Full text of DOI:10.1021/jm960546p

J . Med. Chem. 1997, 40, 1327-1339
1327
Stu d ies of P yr r olo[1,2-a ]ben zim id a zolequ in on e DT-Dia p h or a se Su bstr a te
Activity, Top oisom er a se II In h ibition Activity, a n d DNA Red u ctive Alk yla tion
Edward B. Skibo,* Samantha Gordon, Laura Bess, Romesh Boruah, and Matthew J . Heileman
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604
Received J uly 26, 1996X
The influence of structure on DT-diaphorase substrate activity, topoisomerase II inhibition
activity, and DNA reductive alkylation was studied for the 6-aziridinylpyrrolo[1,2-a]benzimi-
dazolequinones (PBIs) and the 6-acetamidopyrrolo[1,2-a]benzimidazolequinones (APBIs). The
PBIs are reductively activated by DT-diaphorase and alkylate the phosphate backbone of DNA
via major groove interactions, while the APBIs are reductively inactivated by this enzyme since
only the quinone form inhibits topoisomerase II. Bulk at the 7-position (butyl instead of methyl)
significantly decreases kcat/Km for DT-diaphorase reductase activity for both PBIs and APBIs.
As a result, a 7-butyl PBI has little cytotoxicity while the 7-butyl APBI has enhanced
cytotoxicity. The type of 3-substituent and the configuration of the 3-position of the PBIs and
APBIs influence DT-diaphorase substrate activity to a lesser degree. Bulk at the 7-position
(butyl instead of methyl) had an adverse effect on APBI inhibition of topoisomerase II while
the configuration of the 3-position had either an adverse or positive effect on inhibition of this
enzyme. The configuration of the 3-position, when substituted with a hydrogen bond donor,
influences the PBI reductive alkylation of DNA homopolymers. The rationale for this
observation is that the R or S stereoisomers will determine if the 3-substituent points in the
3′ or 5′ direction and thereby influence the hydrogen-bonding interactions. The above findings
were used to rationalize the relative cytotoxicity of various PBI and APBI derivatives.
Ch a r t 1
The pyrrolo[1,2-a]benzimidazolequinone-based anti-
tumor agents have been investigated in this laboratory
for some time.1-11 These agents possess a 7-methyl
substituent and either a 6-acetamido group (APBIs) or
a 6-aziridinyl group (PBIs), inset of Chart 1. The PBIs
require two-electron reductive activation by DT-diapho-
rase as evident by the strong correlation between
cytotoxicity and cellular levels of this enzyme.9 The
reduced PBI then alkylates DNA at the phosphate
backbone.11 In contrast, the APBIs are inactivated by
two-electron reduction and therefore show a strong
inverse correlation with DT-diaphorase levels (the high-
est inverse correlation of 20 000 compounds in the
National Cancer Institute’s archives).9 The unchanged
(oxidized) APBI, rather than the reduced form, acts as
an inhibitor of the first step of topoisomerase IIs
mediated relaxation of supercoiled DNA.4 The findings
presented above prompted a study of the structural
requirements for pyrrolo[1,2-a]benzimidazolequinone-
DT-diaphorase substrate activity and topoisomerase II
inhibition as well as a study of how structural changes
influence the level of cytotoxicity.
Presented herein is such a study of the compounds
1-3 shown in Chart 1 along with a comparison of
results with those obtained with previously reported
PBIs and APBIs, inset of Chart 1. These previously
reported PBIs and APBIs are designated as found in
previous publications (i.e. APBI-A and PBI-A for the
3-acetate derivatives). The effects of the following three
parameters were addressed in the study: steric bulk
at the 7-position, the bulk of the 3-substituent, and the
configuration of the 3-stereocenter. In addition, the
ester in the 3-position was substituted with an amide
group. It was anticipated that the bulk of the 7-sub-
stituent would have the greatest effect on the specificity
(kcat/Km) for DT-diaphorase reductase activity. Indeed,
the 7-butyl APBI derivatives 1a ,b were slowly reduced
by the enzyme, resulting in enhanced cytotoxicity
compared to the 7-methyl analogues. The 7-butyl PBI
2 was likewise slowly reduced by the enzyme, resulting
in a complete loss of cytotoxicity. The bulk of the
3-substituent and the configuration of the 3-stereocenter
has a less dramatic effect on DT-diaphorase reductase
activity. An inverse correlation between the kcat/Km for
DT-diaphorase reductase activity and the level of cyto-
toxicity of the APBIs is consistent with reductive
inactivation by the enzyme. In contrast, the cytotoxicity
X Abstract published in Advance ACS Abstracts, March 15, 1997.
S0022-2623(96)00546-8 CCC: $14.00 © 1997 American Chemical Society

1328 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
Sch em e 1
The racemic and S-(-) forms of 3 were prepared from
the racemic and S-(-) forms of 18, which were previ-
ously prepared in this laboratory,1 Scheme 3. The
synthetic steps leading to 3 did not result in racemiza-
tion, as verified by chiral HPLC; see the Experimental
Section.
The R-(+) and S-(-) forms of APBI-A, inset of Chart
1, have been prepared from the R and S forms of the
amino acid 26 (Scheme 4), employing an internal
Phillips reaction.12 Similarly, an internal Phillips reac-
tion was employed in the unambiguous preparation of
R-(+)- and S-(-)-PBI-A, also shown in the inset of Chart
1. Illustrated in Scheme 4 is the preparation of the
enantiomers of 23, which were readily converted to
PBI-A by employing the synthetic sequence2 illustrated
in Scheme 3, starting with 19. A nucleophilic aromatic
substitution reaction of 25 with either enantiomer of 26
afforded R- or S-27. Catalytic reduction of the nitro
group of 27 followed by acid-catalyzed condensation of
the carboxylic acid group with the ortho diamine result-
ing from reduction (the Phillips reaction12) afforded
R-(+)-24 or S-(-)-24. Acetylation of the enantiomers
of 24 afforded the enantiomers of intermediate 23.
Unfortunately, the yields of 23 from the amino acid
incorporation method were too low, and therefore the
enzymatic resolution of the enantiomers of 23 had to
be employed for bulk synthesis. With the pure enan-
tiomers of 23 and 24 in hand, optical rotations and
retention times on chiral HPLC were employed to follow
the course of enzymatic reduction and measure enan-
tiomeric excesses (ee %). The acyl transfer enzyme
Candida antarctica “B” lipase was observed to hydrolyse
the R-(+) form of 23 much more rapidly than the S-(-)
form. Treatment of racemic 23 with the lipase for 2 h
afforded R-(+)-24 (98% ee) along with an enantiomeric
mixture of 23. A 24 h reaction time afforded S-(-)-23
(98% ee) along with an enantiomeric mixture of 24.
of the PBIs directly correlates with the kcat/Km for DT-
diaphorase reductase activity, consistent with the re-
ductive alkylation mechanism. The level of topoi-
somerase II inhibition was affected by the configuration
of the 3-stereocenter of the APBI and the 7-substituent.
As a result of this study, conclusions have been drawn
with respect to the design of new APBI-based topoi-
somerase II inhibitors.
DT-Dia p h or a se Str u ctu r e-Activity Stu d ies
Ch em istr y
The goal of these studies was to determine the
specificity (kcat/Km) of the APBI and PBI quinones for
two-electron reduction by rat liver DT-diaphorase. The
relative specificity of the quinones would provide in-
sights into both the structure-activity relationship for
quinone reduction and the mechanism for cytotoxicity.
Since cytotoxicity also pertains to topoisomerase II
inhibition (APBIs) or DNA alkylation (PBIs), the influ-
ences of structural changes in the APBIs and PBIs on
these processes are discussed in the following sections.
Rat liver DT-diaphorase generally reduces quinones
much more rapidly than the human enzyme.13-15 In
spite of high activity of the rat liver enzyme, the APBI
The n-butyl derivatives 1 and 2 were prepared start-
ing with the known compound 4, Schemes 1 and 2. The
synthetic steps shown in Scheme 1 are identical to steps
previously carried out in this laboratory for the prepa-
ration of 7-methyl analogues.2 The synthetic steps in
Scheme 2 were likewise uneventful except for the
preparation of 13 from 4. Nitration of 4 afforded a
mixture of 12 and two other mononitro isomers which
could not be separated. The mixture was then treated
with pyrrolidine to afford a mixture from which 13 was
readily isolated. Verification of structure was made by
the conversion of 13 to 14, a transformation which
requires ortho nitro and pyrrolidino substituents.
Sch em e 2

Pyrrolo[1,2-a]benzimidazolequinone Activities
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9 1329
Sch em e 3
Sch em e 4
the specificity for reduction. Bulk about the quinone
may interfere with the stacking interaction between the
quinone and the reduced flavin as well as with the
hydrogen bonding of tyrosine residues with one of the
quinone oxygens. Both of these interactions have been
documented to occur when duroquinone binds to the
active site.16
The (()-7-n-butyl APBI 1b therefore has a specificity
3-fold less than the 7-methyl analogue (()-APBI-A,
Table 1, while the 7-n-butyl PBI (()-2 has a specificity
8.7-fold less than (()-PBI-A, Table 2. The presence of
the 7-n-butyl group decreases the inhibition of topoi-
somerase II by 1b, but this group does not affect DNA
reductive alkylation by 3; see the following sections.
Therefore the cytotoxicity differences noted (()-2 in
Table 2 on changing 7-methyl to 7-n-butyl are largely
attributed to differences in DT-diaphorase substrate
specificity. Since (()-2 is reductively activated more
slowly than (() PBI-A, (()-2 possesses substantially less
cytotoxicity than (()-PBI-A in both cell lines shown in
Table 2. In contrast, (()-1b is reductively inactivated
more slowly than (()-APBI-A but has less inhibitory
activity against topoisomerase II. The net effect of the
n-butyl group appears to increase the cytotoxicity of (()-
1b compared to that of (()-APBI-A, Table 2.
The low substrate specificity of APBIs and PBIs,
compared to benzoquinone and naphthoquinone deriva-
tives, is attributed to the steric hindrance of the fused
pyrrolo ring.
and PBI quinones were found to possess a specificity
(kcat/Km) up to 2 orders of magnitude less than those of
commonly studied quinones (menadione and DZQ). In
order to compensate for the slow rates of reduction, a
relatively large concentration of enzyme was present in
assay reactions (14.5 nM compared to 0.4-0.8 nM14),
and the reactions were followed under strict anaerobic
conditions in Thunberg cuvettes. Under these condi-
tions, in the presence of excess NADH (0.3 mM), the
quinone substrates were completely converted to hyd-
roquinones, permitting the calculation of ∆ꢀ values at
296 nm for the two-electron reduction. Initial rate
measurements and reciprocal (Lineweaver-Burk) plots
provided values of Km, Vmax, and kcat/Km values found
inTables 1 and 2. The kcat/Km values (M-1 s-1) were
calculated from reciprocal plot data and the 14.5 mM
of enzyme present in the reactions.
The next structural modification to be discussed is the
3-substituent. Even though this substituent is far
removed from the site of reduction, it can have a
substantial effect on the specificity for DT-diaphorase.
Previous studies with mitosenes,17 which are structur-
ally similar to the APBIs and PBIs, revealed that the
configuration of the 1-substituent (equivalent to the
pyrrolo[1,2-a]benzimidazole 3-substituent) influences
the specificity for DT-diaphorase. Comparison of the
specificities of 1a and 1b reveals that the presence of a
3-substituent results in a 4.8-fold decrease in specificity
for DT-diaphorase. When the size of the 3-substituent
is increased in APBIs, from acetate to propionate to
benzoate, the specificity does not change ((()-APBI-A:
(()-APBI-F:(()-APBI-G possess kcat/Km values of 1.73:
1.8:1.98, respectively (see Table 1 for structures of these
analogues)). The presence of a more polar 3-substituent,
the methoxyacetate of (()-APBI-I, results in a decrease
The first structural modification to be discussed is the
conversion of 7-methyl to 7-butyl in the APBI (1 and 2)
and PBI (3) quinones. Increasing the bulk at the
quinone ring, where hydride transfer occurs from the
reduced flavin cofactor, would be expected to decrease
in kcat/Km to 1.09 M-1 s-1
.
This influence of the
3-substituent polarity on kcat/Km is also apparent in the
PBI series; when 3-acetate is changed to 3-acetamido
there is a 4.4-fold decrease in specificity. Apparently,

1330 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
Ta ble 1. Parameters for DT-Diaphorase Substrate Activity and Melanoma and Ovarian Cancer log10 LC50 (mol/L)Values for APBI
Analogues
Ta ble 2. Parameters for DT-Diaphorase Substrate Activity
and Melanoma and Ovarian Cancer log10 LC50 (mol/L) Values
for PBI Analogues
enzyme resulted in high cytotoxicity for other APBI
analogues (see beginning of this section).
The 3-substituent of the PBIs is known to influence
Km
×
Vmax
kcat
/
melanoma ovarian
the degree and base-pair specificity of DNA bioreductive
alkylation.9,11 The 3-acetate and 3-acetamido PBI sub-
stituents have identical influence on DNA bioreductive
alkylation (see following sections). Therefore differences
in cytotoxicity must pertain to differences in lipophilicity
and the specificity for DT-diaphorase. A 16-fold de-
crease in cytotoxicity against the melanoma cell lines
was observed when the racemic 3-acetate (PBI-A) was
compared to the less lipophilic carbamate analogue.9
The observed 48-fold decrease in cytotoxicity against the
melanoma cell lines when the racemic 3-acetate (PBI-
A) is changed to the racemic 3-acetamido ((()-3) ana-
logue must in part be due to the decreased specificity
for DT-diaphorase.
structure
105 (nM s-1
)
Km × 10-4 log10 LC50 log10 LC50
(()-PBI-A
4.5
66.5
83.4
62.6
8.5
8.3
2.2
10.2
14.7
7.8
1.17
2.3
3.4
<-7.36
-6.96
-6.08
-4.77
-5.67
-5.00
-6.56
-6.12
-5.59
-4.56
-5.65
-4.94
R-(+)-PBI-A 3.9
S-(-)-PBI-A 5.5
(()-2
5.0
2.5
4.5
(()-3
S-(-)-3
a more polar 3-substituent interferes somewhat with
DT-diaphorase substrate specificity.
The 3-substituent of the APBIs interacts with the
active site of topoisomerase II as well as with the active
site of DT-diaphorase. Therefore correlations between
kcat/Km for DT-diaphorase substrate activity and cyto-
toxicity are not clear-cut. For example, the analogue
(()-APBI-I has a low specificity for the enzyme and low
cytotoxicity. Recall that low substrate specificity for the
The influence of the configuration of the 3-stereo-
center on the substrate specificity for DT-diaphorase
will now be considered. It was expected that one

Pyrrolo[1,2-a]benzimidazolequinone Activities
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9 1331
Sch em e 5
stereoisomer would have higher specificity for the
enzyme than the other while the specificity of the
racemate would be in between those of the stereoiso-
mers. Both APBI-I and PBI-A fit this expectation with
the S enantiomer possessing a lower specificity for DT-
diaphorase than the R enantiomer. The S enantiomer
of 3 possesses a higher specificity for the enzyme than
the racemate (the R enantiomer of 3 has not yet been
synthesized).
In conclusion, the greatest effect of structural changes
on the specificity for DT-diaphorase are substitution of
a large group (n-butyl) on the quinone ring and the
presence of a polar (methoxyacetate or acetamido)
substituent in the 3-position. The configuration of the
3-position and the size of 3-substituents have small
effects on specificity for the enzyme.
Top oisom er a se In h ibition Stu d ies
An unusual finding was noted for the APBI-A stereo-
isomers: The R-(+) and S-(-) enantiomers have nearly
equal specificity for the enzyme while the racemate has
a higher specificity for the enzyme than either enanti-
omer. An explanation for this observation is the pres-
ence of two substrates bound to the DT-diaphorase
dimer resulting in either RR, SS, or RS forms of
substrate-enzyme complex. The active sites of the DT-
diaphorase dimer are located next to each other,18
resulting in the stereocenters of the substrates “seeing
each other”. Therefore the use of pure enantiomers as
substrates will result in an RR or SS complex, while
the racemate will afford a diastereomeric RS complex
which must be associated with a higher kcat/Km than the
enantiomeric forms.
Topoisomerases I and II cause relaxation of super-
coiled DNA by single or double strand cleavage respec-
tively to afford an enzyme-DNA complex followed by
religation of the cleaved strands19 after relaxation,
Scheme 5. The activity of the topoisomerases is impor-
tant in cell division, and therefore many types of cancer
cells possess elevated levels of these enzymes.20 Most
topoisomerase II inhibitors of clinical value act at the
religation step by stabilizing the enzyme-DNA complex
formed upon double strand cleavage, the cleavable
complex, Scheme 5. Examples of clinically used agents
which stabilize the cleavable complex include m-AMSA,
etoposide, and doxorubicin,21-24 Scheme 6. Both m-
AMSA and doxorubicin are intercalative inhibitors while
etopside operates by a nonintercalative mechanism. In
Sch em e 6

1332 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
should influence topoisomerase II inhibition. The re-
sults provided below indicate that configuration does
have a small effect on inhibition. The results for R-(+)-
and S-(-)-APBI-I indicate that the R-(+) enantiomer
inhibits relaxation at 4-fold lower concentrations than
the S-(-) enantiomer. In contrast, the S-(-) form of
APBI-A inhibits at 5-fold lower concentrations than the
R-(+) enantiomer. The differences between APBI-A and
APBI-I cited above may result from the hydrophobic/
hydrophilic interactions between acetate and methoxy-
acetate and the enzyme.
The results of the DT-diaphorase and topoisomerase
II studies suggest that S-(-)-APBI-A should be a good
antitumor agent. This enantiomer has a specificity for
DT-diaphorase lower than that of the racemate and is
therefore more slowly inactivated by reduction. This
feature and the higher activity of the S-(-) enantiomer
against topoisomerase II likely account for its higher
cytotoxicity than either the R-(+) enantiomer or the
racemate against melanoma and ovarian cancer cell
lines, Table 1. Currently S-(-)-APBI-A is undergoing
in vivo screening at the National Cancer Institute.
F igu r e 1. Effect of APBI-A and APBI-G on the relaxation of
SV-40 supercoiled DNA by Drosophila topoisomerase II. A
1.3% agarose gel run in 0.045 M Tris‚borate buffer with 1 mM
EDTA was employed to assay the relaxation reactions. The
topoisomerase relaxation reactions were carried out with 0.25
µg of SV-40 supercoiled (form I) DNA and10 units of topoi-
somerase II as described in the Experimental Section: lane
A, control without enzyme; lane B, control with enzyme; lane
C, 0.23 mM APBI-A; lane D, 0.48 mM APBI-A; lane E, 0.69
mM APBI-A; lane F, 0.954 mM APBI-A; lane G, 1.15 mM
APBI-A; lane H, 0.15 mM APBI-G; lane I, 0.31 mM APBI-G;
lane J , 0.45 mM APBI-G; lane K, 0.63 mM APBI-G; lane L,
0.75 mM APBI-G.
addition, Scheme 6 shows relatively new compounds
which also stabilize this complex: the pyrimido[1,6-a]-
benzimidazole,25 the pyrrolo imino quinone,26 and the
naphthoquinone.27 In contrast to the other agents
above, the quinobenoxazine28 acts at the noncleavable
complex and inhibits the double-strand cleavage step
leading to the cleavable complex.
DNA Alk yla tion Stu d ies
The PBIs are reductive alkylating agents of the DNA
phosphate backbone.5 Extensive structure-activity
studies of the PBIs have been carried in this labora-
tory,6,7,9-11 leading to the conclusions that the 4-nitrogen
is required for the alkylation reaction7 and that the
3-position influences the base-pair specificity.9 The
fused pyrrolo ring is not important for the alkylation
reaction, and this ring can either be removed or ho-
mologated to a tetrahydropyrido ring.6 These findings
are consistent with the major groove binding model4,5
wherein the reduced PBI hydrogen bonds to the major
groove by utilizing the hydroquinone hydroxyl, the
4-nitrogen, and the 3-substituent. The fused pyrrolo
ring points away from the major groove, and therefore
this group does not influence the alkylation reaction.
In the model, the 7-n-butyl substituent would also point
away from the major groove and not influence the
alkylation of DNA. In order to compare the 7-n-butyl
(2) and the 7-methyl (PBI-A) analogues with respect to
DNA alkylation, both compounds in their reduced forms
were reacted with 600 bp calf thymus DNA; see the
Experimental Section. The percent incorporation of PBI
into DNA, based on one alkylation per base pair
representing 100% alkylation, was calculated from
The APBI topoisomerase II inhibitors have structural
similarities to some of the compounds in Scheme 6,
particularly the pyrimidobenzimidazole, pyrrolo imino
quinone, and naphthoquinone analogues. Previous
work showed that the APBIs intercalate DNA and
prevent the noncleavable complex from proceeding to
the cleavable complex and eventual formation of relaxed
(form I) DNA, Scheme 5.4,10 Shown in Figure 1 are
inhibition assays of APBI derivatives bearing an acetate
(APBI-A) and a benzoate (APBI-G) in the 3-position.
These assays show that the supercoiled DNA (form I)
builds up without apparent formation of linear DNA
(form III) in the presence of 20 units of topoisomerase
II. The use of such high concentrations of enzyme is
required to see the linear DNA since this DNA is formed
by proteinase digestion of the cleavable complex consist-
ing of supercoiled DNA/APBI/topoisomerase II. The
current view of APBI inhibition is that the APBI can
slow the formation of the cleavable complex. Even the
clinically used topoisomerase II inhibitors are thought
to slow the formation of the cleavable complex as well
as stabilize this complex; see discussion in ref 28.
To follow is a discussion of the structural features
which influence the APBI mediated formation of the
cleavable complex. The results shown in Figure 1 reveal
that the size of the 3-substituent (acetate vs benzoate)
does not influence the degree of inhibition. Even the
absence of a 3-substituent in the APBI still resulted in
inhibition of cleavable complex formation. In the pres-
ence of 10-20 units of topoisomerase II, the APBIs
studied so far inhibit formation of cleavable complex at
or above 0.3-0.5 mM concentrations. However, the
n-butyl analogue (1b) is much less active than APBI-A,
and only concentrations of 1b greater than 1 mM caused
inhibition of topoisomerase II. The n-butyl group very
likely interferes with the intercalation of DNA, which
is important in the inhibition of topoisomerase II. The
substituent at the 3-position could interact with topoi-
somerase II while the benzimidazolequinone interca-
lates DNA. Therefore the configuration of the 3-position
absorbance measurements at 550 nm (ꢀ ) 785 m-1 cm-1
)
for the PBI chromophore; see ref 11 for details. As
expected from the major groove binding model, 2 and
PBI-A reductively alkylated DNA to nearly the same
degree, 10% and 13%, respectively. This observation
suggests that the low cytotoxicity of 2 is largely due to
the slow reductive activation by DT-diaphorase; see DT-
Diaphorase Structure-Activity Studies.
Previous studies showed that the 3-carbamate PBI
(PBI-B) reductively alkylated the DNA phosphate to a
greater degree than any PBI bearing a 3-ester substitu-
ent.4,9 This property of the 3-carbamate is attributed
to a hydrogen-bonding interaction in the major groove
not present with esters. Similarly, the 3-acetamido PBI
(()-3 alkylates calf thymus DNA to a greater degree
than PBI-A, 24% compared to 10%. If the 3-substituent
interacts in the major groove of DNA, the configuration

Pyrrolo[1,2-a]benzimidazolequinone Activities
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9 1333
Ta ble 3. Percentages of PBI Incorporation into Various DNAs
the 7-butyl substituent is an increase in cytotoxicity for
1b compared to the 7-methyl analogue APBI-A. The
n-butyl substituent does not influence the reductive
alkylation of DNA by 2. Therefore the loss of cytotox-
icity exhibited by 2, compared to the 7-methyl analogue
PBI-A, is due to the slow reductive activation. The
conclusion is that the 7-butyl substituent confers some
cytotoxic advantage in the APBIs.
DNA
(()-3
S-(-)-3
R-(+)-PBI-A
S-(-)-PBI-A
poly(dG)
8.2
12.1
poly(dC)
poly(dA)
poly(dT)
poly(dA-dT)
poly(dT-dA)
41.5
28.0
32.7
38.0
5.9
8.5
The bulk of the APBI 3-substituent does not affect
the specificity for DT-diaphorase or topoisomerase II
inhibition. Previous studies showed that the lipophi-
licity of the 3-substituent is important in APBI cyto-
toxicity.9 Given the absence of a significant effect of the
3-substituent on the enzyme systems studied, future
APBI analogues will bear 3-substituents of appropriate
lipophilicity. Previous studies showed that a hydrogen-
bonding donor (carbamate) at the 3-position of a PBI
resulted in reductive alkylation predominately at AT
base pairs.9,11 Consistent with this finding, the 3-ac-
etamido PBI derivative 3 exhibited up to a 5-fold
preference for AT base pairs over the 3-acetate deriva-
tive (PBI-A). Furthermore, 3 and the carbamate de-
rivative reductively alkylate DNA to a greater degree
than a 3-ester derivative. It is concluded that PBIs
bearing a 3-substituent with hydrogen bond donor
capability will be efficient reductive alkylating agents
of DNA and show high A-T base pair specificity.
The configuration of the 3-position is important in the
cytotoxicity of both the PBIs and APBIs because the
configuration influences the interaction with both DT-
diaphorase and topoisomerase II. The high cytotoxicity
of S-(-)-APBI-A, compared to the R-(+) enantiomer and
racemate, is due to the low specificity of the S-(-)
enantiomer for reductive inactivation by DT-diaphorase
as well as the more potent inhibition of topoisomerase
II by this enantiomner. The high cytotoxicity of R-(+)-
PBI-A, compared to its enantiomer, is due to the high
specificity of the R-(+) enantiomer for reductive activa-
tion by DT-diaphorase.
It is concluded that the configuration of the 3-position
is important in PBI and APBI cytotoxicity, and therefore
the resolution of enantiomers will be a part of future
analogue development.
The configuration of the 3-position does not influence
the reductive alkylation of DNA by PBIs if an ester
substituent is present at this position. However, the
studies with (()-3 and its S-(-) enantiomer suggest that
configuration at the 3-position becomes important in
DNA reductive alkylation if a hydrogen bond donor is
present. The results of this study suggests that R-(+)-3
has a high selectivity for A-T base pairs and therefore
has greater cytotoxicity than the S-(-) enantiomer.
of the 3-position should influence base-pair specificity
because the R- or S stereoisomers will determine if the
3-substituent points in the 3′ or 5′ direction. This
possibility is discussed below in conjunction with Table
3.
Shown in Table 3 are DNA incorporation percentages
for racemic and S-(-)-3 as well as for R-(+)- and S-(-
)-PBI-A. Racemic 3 and its S-(-) enantiomer show up
to a 5-fold preference for A-T base pairs over G-C base
pairs. The explanation for this preference is hydrogen
bonding of the amide N-H with a thymine carbonyl.
Preferences for A-T are also seen in the 3-carbamate4,9
as well as the 3-ureido (unpublished result) derivative.
When only an ester is present in the 3-position, as in
PBI-A, the amount of DNA reductive alkylation de-
creases and there is equal preference for A-T and G-C
base pairs [see PAGEs (polyacrylamide gels) on page
3052 of ref 5]. The configuration of the 3-stereocenter
influences base-pair specificity very little for PBI-A and
3 (at G-C base pairs). In these cases there are only
small differences in small percentages for incorporation
into DNA, Table 3. In the case of 3, reductive alkylation
of poly(dA)‚poly(dT) and the alternating polymer poly-
(dA-dT)‚poly(dT-dA) show a definite enantiomeric se-
lectivity. Racemic 3 reductively alkylates poly(dA)‚-
poly(dT) to a greater degree than the S-(-) enantiomer,
which suggests that the R-(+) enantiomer shows a
preference for this DNA. In contrast, the S-(-) enan-
tiomer of 3 alkylates an alternating polymer to a greater
degree than the R-(+) enantiomer. The higher cytotox-
icity of (()-3 against melanoma and ovarian cancer cell
lines (Table 2) compared to the S-(-) enantiomer could
be due to the greater selectivity of the R-(+) enantiomer
for a run of A-T base pairs. A-T rich DNA is known to
be the target of other antitumor agents such as CC-
106529 and the distamyin and netropsin30 based sys-
tems.
Con clu sion s
The influence of APBI and PBI structure on DT-
diaphorase substrate specificity, topoisomerase II inhi-
bition, and DNA reductive alkylation was investigated
to determine the structural requirements for cytotox-
icity. The particular structural elements of interest
include the 7-substituent, the 3-substituent, and the
configuration of the 3-position. The conclusions of this
investigation are of value in the design of new APBI
and PBI antitumor agents.
The change from 7-methyl to 7-butyl has a profound
effect on both DT-diaphorase substrate specificity and
topoisomerase II inhibition. The bulk of the 7-butyl
substituent significantly slows the enzymatic reduction
of both APBI and PBI systems. The slow reduction
should enhance the cytotoxicity of APBIs which are
inactivated by reduction. However, the 7-butyl sub-
stituent decreases the ability of APBIs to inhibit topoi-
somerase II. The net result of these opposing effects of
Exp er im en ta l Section
All solutions and buffers for kinetic, DNA, and electrophore-
sis studies used doubly distilled water. 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 per-
formed 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 obtained from a 300 MHz spectrometer. All
chemical shifts are reported relative to TMS. Optical rotations
were obtained from a Perkin-Elmer polarimeter employing a
10-cm quartz cell, 0.2 g/dL enantiomer in methanol. Enam-
tiomeric purity was determined using optical rotations and

1334 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
chiral HPLC, see Chiral High-Pressure Liquid Chromatogra-
phy in this section. Purified topoisomerase II (Drosophila
melanogaster) was purchased from United States Biochemical;
activity: 20 units/mL. SV-40 DNA was purchased from Gibco
BRL (Life Technologies). NADH and reagents employed in
affinity column preparation were purchased from Sigma.
Candida antarctica “B” lipase (Altus 13) was purchased from
Altus Biologics, Inc.
(dimethyl sulfoxide-d6) δ 9.27 (1H, s, amide proton), 7.38 and
7.24 (2H, 2s, aromatic protons), 4.06 (2H, t, J ) 6.94 Hz, C(3)
methylene), 2.91 (2H, t, J ) 7.23 Hz, C(1) methylene), 2.62
(4H, m, C(2) and C (1′) methylene protons), 2.03 (3H, s, acetate
methyl), 1.51 (2H, quintet, J ) 7.74 Hz, C(2′)), 1.33 (2H, sextet,
J ) 7.37 Hz, C(3′)), 0.90 (3H, t, J ) 7.29 Hz C(4′)); mass
spectrum (EI mode), m/ z 271 (M+), 228 (M+ - acetyl). Anal.
(C16H21N3O‚0.5H2O) C, H, N.
5-Bu tyl-2,4-d in itr o-1-N-p yr r olid in oben zen e (6). The
synthesis of 6 was carried out in the two-step process described
below.
7-Bu tyl-6-n itr o-2,3-d ih yd r o-1H-p yr r olo[1,2-a ]ben zim i-
d a zol-3-yl Aceta te (8). A mixture containing 0.5 (1.7 mmol)
of 6, 0.45 g (3.30 mmol) of ZnCl2, and 5 mL of acetic anhydride
was refluxed at 150 °C for 6 h. The reaction mixture was
cooled and combined with 50 mL of water. The mixture was
then extracted with 3 × 50 mL portions of chloroform. The
extracts were combined, dried (Na2SO4), and the solvent
removed in vacuo. The residue was placed on a silica gel flash
column, and the product was eluted off with ethyl acetate.
Recrystallization with chloroform/hexane afforded pure 8:
0.119 g (22% yield); mp 98-99 °C; TLC (ethyl acetate) Rf )
0.35; IR (KBr pellet) 2974, 2862, 1745, 1629, 1525, 1458, 1334,
1217, 1084, 977 cm-1; 1H NMR (dimethyl sulfoxide-d6) δ 8.23
(1H, s, C(5)), 7.63 (1H, s, C(8)), 6.13 (1H, dd, J ) 7.50 Hz, J )
3.75 Hz, C(3)), 4.28 and 4.17 (2H, 2m, C(1) diastereomeric
methylene), 3.11 and 2.57 (2H, 2m, C(2) diastereomeric
methylene), 2.89 (2H, t, J ) 7.77 Hz, C(1′)), 2.04 (3H, s, acetate
methyl), 1.55 (2H, quintet, J ) 7.47 Hz, C(2′), 1.32 (2H, sextet,
J ) 7.23 Hz, C(3′)), 0.87 (3H, t, J ) 7.32 Hz, C(4′)); mass
spectrum (EI mode), m/ z 317 (M+), 300 (M+ - OH), 274 (M+
- acetyl), 258 (M+- acetate). Anal. (C16H19N3O4) C, H, N.
6-Acetam ido-7-bu tyl-2,3-dih ydr o-1H-pyr r olo[1,2-a ]ben -
zim id a zol-3-yl Aceta te (9). A suspension of 0.394 g (1.24
mmol) of 8 and 79 mg of 5% Pd on charcoal in 60 mL of
methanol was shaken under 50 psi of H2 for 3 h. The catalyst
was removed by filtering the solution through Celite, and the
filtrate was added to a flask containing 20 mL of acetic acid
and 4.0 mL of acetic anhydride. The resulting solution was
stirred at room temperature for 2 h. The solvent was removed
in vacuo, and the residue was triturated with ether. Recrys-
tallization with ethyl acetate afforded analytically pure 9: mp
191 °C; TLC (chloroform/methanol, 80:20) Rf ) 0.66; IR (KBr
pellet) 3244, 2960, 2874, 1743, 1647, 1529, 1371, 1234, 1030
To 10 mL of fuming nitric acid, chilled in an ice bath, was
added 1.99 g (9.33 mmol) of 1-bromo-5-butylbenzene (4) over
a period of 20 min. The mixture was stirred at room temper-
ature for 14 h and then poured over ice. The resulting mixture
was extracted five times with 10 mL portions of chloroform.
The extracts were combined and dried (Na2SO4), and the
solvent was removed by evaporation. The product (5) was
isolated as an orange oil, which was carried on to the next
step.
A mixture of 3.30 mL (39.4 mmol) of pyrrolidine, 20 mL of
ethanol, and crude 5 obtained above were refluxed for 3 h. The
ethanol was then removed by evaporation. The residue was
dissolved in chloroform and washed five times with 25 mL
portions of 1 N HCl. The organic layer was dried (Na2SO4)
and the chloroform removed by evaporation. The residue was
placed on a silica gel flash column, and the product was eluted
with hexane/chloroform (80:20). Evaporation of the eluant
afforded a yellow residue. Recrystallization from hexane
produced fine yellow crystals of 6: 1.51 g (55%) yield from 4;
mp 93.5-94 °C; TLC (chloroform/hexane, 70:30) Rf ) 0.54; IR
(KBr pellet) 2986, 2957, 2928, 2872, 2859, 1607, 1566, 1503,
1464, 1370, 1315, 1285 cm-1; 1H NMR (dimethyl sulfoxide-d6)
δ 8.54 and 6.99 (2H, 2s, C(3) and C(6) aromatic protons), 3.28
(4 H, m, pyrrolidine protons next to nitrogen), 2.97 (2H, dd, J
≈ 7.6 Hz, C(1′)), 1.94 (4H, m, other pyrrolidine protons), 1.54
(2 H, quintet, J ) 7.3 Hz, C(2′), 1.38 (2H, sextet, J ) 7.5 Hz,
C(3′)), 0.92 (3 H, t, J ) 7.3 Hz C(4′)); mass spectrum (EI mode),
m/ z 293 (M+). Anal. (C14H19N3O4) C, H, N.
6-Acetam ido-7-bu tyl-2,3-dih ydr o-1H-pyr r olo[1,2-a ]ben -
zim id a zole (7) was prepared from 6 according to the following
two-step procedure: A suspension of 1.0 g (3.41 m mol) of 6
and 0.24 g of 5% Pd on charcoal in 100 mL of methanol was
shaken under 50 psi of H2 for 3.5 h. The mixture was then
filtered through Celite. The filtrate was added to a solution
of 10 mL of acetic anhydride and 5 mL of acetic acid. This
solution was stirred at room temperature for 2.5 h, and then
the solvent was evaporated in vacuo. The oily residue was
dried under high vacuum and triturated with ether to afford
2,4-diacetamido-5-butyl-2-N-pyrrolidinobenzene. Yield of
crude product, suitable for the next step, was 0.973 g (90%).
An analytical sample was prepared by recrystallization from
chloroform: mp 179 °C dec; TLC (chloroform/methanol, 85:
15) Rf ) 0.60; IR (KBr pellet) 3250, 2957, 2932, 2870, 2816,
cm-1 1H NMR (dimethyl sulfoxide-d6) δ 9.28 (1H, s, amide
;
proton), 7.45 and 7.32 (2H, 2s, aromatic protons), 6.06 (1H,
dd, J ) 7.62 Hz, J ) 3.27 Hz, C(3)), 4.14 (2H, m, C(1)), 3.08
and 2.53 (2H, 2m, C(2)-diastereomeric methylene), 2.61 (2H,
t, J ) 7.65 Hz, C(1′)), 2.03 and 2.00 (6H, 2s, acetamido and
acetate methyl), 1.48 (2H, J ) 7.53 Hz, C(2′)), 1.29 (2H, sextet,
J ) 7.35 Hz, C(3′)), 0.865 (3H, t, J ) 7.29 Hz, C(4′)); mass
spectrum (EI mode), m/ z 329 (M+), 286 (M+ - acetyl), 270
(M+ - acetate), 244. Anal. (C18H23N3O3) C, H, N.
6-Acet a m id o-7-b u t yl-5-n it r o-2,3-d ih yd r o-1H -p yr r olo-
[1,2-a ]ben zim id a zole (10). To a mixture of 4.5 mL of fuming
nitric acid and 0.5 mL of concentrated surfuric acid, chilled at
0 °C, was added 0.143 g (0.527 mmol) of 7. The resulting
solution was stirred for 6 min and then poured over 50 mL of
ice. The solution was neutralized with sodium bicarbonate and
then extracted with 5 × 50 mL portions of chloroform. The
extracts were combined and dried (sodium sulfate), and the
solvent was evaporated in vacuo. Recrystallization was carried
out from chloroform/hexane. Purified product was obtained
by silica gel flash chromatography employing chloroform/
methanol (95:5) as the eluant: 0.110 g (66%) yield; mp 210-
211 °C; TLC (chloroform/methanol, 80:20) Rf ) 0.65; IR (KBr
pellet) 3229, 3175, 3007, 2957, 2932, 2870, 1684, 1520, 1450,
1358, 1267 cm-1; 1H NMR (dimethyl sulfoxide-d6) δ 9.68 (1H,
br s, amide proton), 7.62 (1H, s, aromatic proton), 4.16 (2H, t,
J ) 7.04 Hz, C(1) methylene), 2.98 (2H, t, J ) 7.62 Hz, C(3)
methylene), 2.65 (4H, m, C(2) and C(1′) methylene), 1.52 (2H,
quintet, J ) 7.78, Hz, C(2′) methylenes), 1.32 (2H, sextet, J )
7.37 Hz, C(3′) methylene), 0.90 (3H, t, J ) 7.28 Hz, C(4′)
methylene); mass spectrum (EI mode), m/ z 316 (M+), 270 (M+
- NO2), 227 (M+ - NO2 - acetyl). Anal. (C16H20N4O3) C, H,
N.
1651, 1527, 1420, 1370, 1281 cm-1 1H NMR (dimethyl sul-
;
foxide-d6) δ 9.11 and 9.07 (2H, 2s, amide protons), 7.14 and
6.63 (2H, 2s, aromatic), 3.13 (4H, t, J ) 6.12 Hz pyrrolidine
protons next to nitrogen), 2.45 (2H, t, J ) 7.63 Hz, C(1′)), 2.00
and 1.97 (6H, 2s, amide methyls), 1.85 (4H, m, other pyrroli-
dine methylenes), 1.45 (2H, quintet, J ) 7.71 Hz, C(2′), 1.29
(2H, sextet, J ) 7.37 Hz, C(3′)), 0.88 (3H, t, J ) 7.22 Hz C(4′));
mass spectrum (EI mode), m/ z 317 (M+), 274 (M+ - acetyl),
259 (M+ - acetamido).
A mixture of 0.30 g (0.95 mmol) of the diacetamido deriva-
tive obtained above, 1.6 mL formic acid, and 0.8 mL of 30%
hydrogen peroxide was stirred at 70-75 °C for 1 h. The cooled
reaction mixture was then neutralized with sodium bicarbon-
ate. The neutralized solution was extracted with 4 × 50 mL
portions of chloroform. The extracts were combined and dried
(sodium sulfate), and the solvent was removed in vacuo.
Trituration of the residue in chloroform with a small volume
of hexane afforded crude 7 in 0.16 g (65%) yield. An analyti-
cally pure sample was prepared by silica gel flash chromatog-
raphy eluting with chloroform: mp 177-179 °C; TLC (chlo-
roform/methanol, 80:20) Rf ) 0.62; IR (KBr pellet) 3250, 2957,
2931, 2870, 1651, 1613, 1528, 1420, 1370, 1281 cm-1; 1H NMR
6-Acet a m id o-7-b u t yl-5-n it r o-2,3-d ih yd r o-1H -p yr r olo-
[1,2-a ]ben zim id a zol-3-yl Aceta te (11). To a mixture of 9
mL of fuming nitric acid and 1 mL of concentrated surfuric

Pyrrolo[1,2-a]benzimidazolequinone Activities
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9 1335
acid, chilled at -8 °C, was added 250 mg (0.761 mmol) of 9.
The reaction mixture was stirred for 20 min at ice bath
temperature and then poured into a mixture of 50 g of ice and
50 mL of chloroform. The chloroform layer was removed, and
the aqueous layer was extracted with 5 × 30 mL portions of
chloroform. The extracts were combined and dried (Na2SO4),
and the solvent was removed in vacuo. The residue was
recrystallized from chloroform and hexane to afford yellow
crystals of 11: 191 mg (67%) yield; mp 199-200 °C; TLC
(chloroform/methanol, 95:5) Rf ) 0.35; IR (KBr pellet) 3167,
(EI mode), m/ z 359 (M+), 317 (M+ - ketene), 257 (M+
acetate - ketene). Anal. (C18H21N3O5) C, H, N.
-
4-Bu tyl-1-n itr o-2-N-p yr r olid in oben zen e (13). The con-
version of 4 to 13 was carried out by a two-step process
described below. A total of 9.98 g (0.0468 mol) of 4 was divided
in half and added to two 20 mL portions of chilled (-8 °C)
fuming nitric acid over a 20 min period. The resulting reaction
mixtures were stirred at -5 °C for an additional 10 min, and
then they were poured over ice. Both mixtures were extracted
with 5 × 50 mL portions of chloroform. All extracts were
combined and dried (sodium sulfate), and the solvent was
removed in vacuo. The mixture of mononitrated isomers of
12 was carried on to the next step without further purification.
A solution of 15 mL of pyrrolidine, 60 mL of ethanol, and
the isomers of 12 was refluxed for 3.5 h. The reaction mixture
was cooled, and the solvent was removed in vacuo. The
residue was dissolved in 100 mL of chloroform and extracted
with 3 × 40 mL portions of 1 N HCl. The extracts were
combined, dried (Na2SO4), and concentrated to a residue, which
was chromatographed on silica gel, employing hexane/chloro-
form (80:20) as the eluant. Evaporation of the solvent afforded
13 as an oil: 3.03 g (26%) overall yield. An analytically pure
sample was obtained by sublimation: TLC (chloroform) Rf )
0.66; IR (KBr pellet) 2957, 2872, 2361, 1616, 1568, 1506, 1361,
1282, 1010, 829 cm-1; 1H NMR (CDCl3) δ 7.66, 6.66, and 6.52
(3H, ABX, J ortho ) 8.46 Hz, J meta ) 1.68 Hz, J para ≈ 0 Hz, C(6),
C(3), and C(5) aromatic protons, respectively), 3.18 (4H,
quintet, J ) 3.93 Hz, pyrrolidine methylenes adjacent to N),
2.56 (2H, t, J ) 7.7 Hz, C(1′)), 1.93 (4H, quintet, J ) 3.51 Hz,
other pyrrolidine methylenes), 1.56 (2H, m, C(2′)), 1.33 (2H,
sextet, J ) 7.2 Hz, C(3′)), 0.898 3H, t, J ) Hz, C(4′)); mass
spectrum (EI mode), m/ z 248 (M+), 231 (M+ - OH), 201 (M+
- H - NO2). Anal. (C14H20N2O2) C, H, N.
2955, 1749, 1678, 1525, 1460, 1363, 1234, 1084, 1058 cm-1
;
1H NMR (dimethyl sulfoxide-d6) δ 9.73 (1H, s, amide proton),
7.72 (1H, s, aromatic proton), 6.11 (1H, dd, J ) 7.7 Hz, J )
3.6 Hz, C(3)), 4.29 and 4.18 (2H, 2m, C(1)-diastereomeric
methylene), 3.10 and 2.55 (2H, 2m, C(2)-diastereomeric me-
thylene), 2.64 (2H, t, J ) 7.4 Hz, C(1′)) 2.05 and 1.97 (6H, 2s,
acetamido and acetate protons), 1.49 (2H, quintet, J ) 7.29
Hz, C(2′)), 1.29 (2H, sextet, J ) 7.35 Hz, C(3′)), 0.866 (3H, t,
J ) 7.29 Hz, C(4′)); mass spectrum (EI mode), m/ z 374 (M+),
356 (M+ - H2O), 328 (M+ - NO2), 254 (M+ - H2O - ketene -
acetic acid). Anal. (C18H22N4O5) C, H, N.
6-Acetam ido-7-bu tyl-2,3-dih ydr o-1H-pyr r olo[1,2-a ]ben -
zim id a zole-5,8-d ion e (1a ). The synthesis of 1a was carried
out in a two-step process described below.
A suspension of 0.109 g (0.344 mmol) of 10 in 50 mL of
methanol with 0.023 g of Pd on carbon was shaken under 50
psi of H2 for 3 h. The solution was filtered through Celite and
the solvent evaporated in vacuo. After being washed through
the Celite, a solution of 0.085 g of potassium phosphate
monobasic in 10 mL of water was added to the residue. To
this mixture was added another solution consisting of 0.2 g of
potassium phosphate monobasic and 0.5 g of Fremy salt in 45
mL of water. The resulting purple solution was stirred at room
temperature for 4 h and then extracted with 6 × 50 mL
portions of chloroform. The extracts were combined and dried
(sodium sulfate), and the solvent was evaporated in vacuo. The
concentrated oil was then flash chromatographed, employing
silica gel with ethyl acetate/methanol (90:10) as eluant. Pure
product was obtained by recrystallization from chloroform/
hexane: 0.0462 g (44%) yield; mp 176 °C dec; TLC (chloroform/
methanol, 80:20) Rf ) 0.67; IR (KBr pellet) 3237, 3173, 3136,
7-Bu tyl-2,3-d ih yd r o-1H-p yr r olo[1,2-a ]ben zim id a zol-3-
yl Aceta te (14). A mixture consisting of 4.4 g (17.7 mmol) of
14, 2.5 g (18.3 mmol) of ZnCl2, and 70 mL of acetic anhydride
was refluxed at 100-110 °C for 5 h. The reaction mixture was
cooled and then combined with 150 mL of water. The resulting
solution was extracted with 5 × 100 mL portions of chloroform.
The extracts were combined, dried (Na2SO4), and concentrated
in vacuo. Chromatography of the residue on a silica gel flash
column employing chloroform/methanol (95:5) as the eluant
afforded crude 14 in 2.0 g (41%) yield. Recrystallization from
chloroform and hexane afforded analytically pure 14: mp 141-
141.5 °C; TLC (chloroform/methanol, 95:5) Rf ) 0.38; IR (KBr
pellet) 2955, 2931, 2858, 1747, 1622, 1537, 1373, 1249, 1051,
2996, 2957, 2932, 2870, 1653, 1518, 1267 cm-1 1H NMR
;
(dimethyl sulfoxide-d6) δ 9.46 (1H, br s, amide proton), 4.17
(2H, t, J ) 7.12 Hz, C(1) methylene), 2.86 (2H, t, J ) 7.50 Hz,
C(3) methylene), 2.61 (2H, m, C(2) methylene), 2.36 (2H, t, J
) 7.4 Hz, C(1′) methylene), 1.3 (4H, m, C(2′) and C(3′)
methylenes), 0.856 (3H, t, J ) 7.1 Hz, C(4′) methylene); mass
spectrum (EI mode), m/ z 301 (M+). Anal. (C16H19N3O3) C,
H, N.
814 cm-1 1H NMR (dimethyl sulfoxide-d6) δ 7.49, 7.31 and
;
7.03 (3H, ABX, J ortho ) 8.34 Hz, J meta ) 1.65 Hz, J para ≈ 0 Hz,
C(5), C(8) and C(6) aromatic protons, respectively), 6.07 (1H,
dd, J ) 7.56 Hz, J ) 3.27 Hz, C(3)), 4.14 (2H, m, C(1)), 3.06
and 2.54 (2H, 2m, C(2) diasteriomeric methylene), 2.03 (3H,
s, acetate methyl), 1.57 (2H, quintet, J ) 7.59 Hz, C(2′)), 1.28
(2H, sextet, J ) 1.53 Hz, C(3′)), 0.863 (3H, t, J ) 7.29 Hz,
6-Aceta m id o-7-bu tyl-2,3-d ih yd r o-5,8-d ioxo-1H-p yr r olo-
[1,2-a ]ben zim id a zol-3-yl Aceta te (1b). A suspension of
0.102 g (0.272 mmol) of 11 and 20 mg of 5% Pd on charcoal in
80 mL of methanol was shaken at 50 psi of H2 for 3 h. The
solution was then filtered through Celite and concentrated to
a residue. A solution containing 20 mL of water and 85 mg of
potassium phosphate monobasic was washed through the
Celite and then added to the residue. To this solution was
added a solution consisting of 45 mL of water, 200 mg of
potassium phosphate monobasic, and 500 mg of Fremy salt.
The resulting mixture was stirred at room temperature for 5
h, and then it was extracted with 8 × 25 mL portions of
chloroform. The combined extracts were dried (Na2SO4), and
the solvent was removed in vacuo. Pure product was obtained
by silica gel chromatography, employing ethyl acetate/
chloroform (80:20) as the eluant. Recrystallization from
chloroform and hexane afforded yellow crystals of 1b: 25 mg
(26%) yield; mp 178-179 °C; TLC (chloroform/methanol, 95:
5) Rf ) 0.55; IR (KBr pellet) 2960, 1745, 1658, 1608, 1521,
C(4′)); mass spectrum (EI mode), m/ z 272 (M+), 229 (M+
acetyl). Anal. (C16H20N2O2‚0.75H2O) C, H, N.
-
6-Br om o-7-b u t yl-2,3-d ih yd r o-1H -p yr r olo[1,2-a ]b en z-
im id a zol-3-yl Aceta te (15). A mixture containing 0.972 g
(3.57 mmol) of 14, 13 mL of dioxane, 1.14 g of K2CO3, and 2.7
mL of 1 M bromine in dioxane was stirred at room temperature
for 30 min. At this time, another 2.6 mL of the 1 M bromine
solution was added to the mixture followed by stirring for 50
min at room temperature. The reaction mixture was then
combined with 150 mL of water and then extracted with 5 ×
100 mL portions of chloroform. The extracts were combined
and dried (Na2SO4), and the solvent was removed in vacuo.
Pure product was obtained by flash column chromatography
on silica gel, employing ethyl acetate/chloroform (50:50) as the
eluant. Trituration with hexane produced white crystals of
15: 1.02 g (64% yield); mp 111-112 °C, TLC (chloroform/ethyl
acetate, 50:50) Rf ) 0.32; IR (KBr pellet) 2960, 2926, 2858,
1741, 1520, 1454, 1373, 1228, 1037, 858 cm-1; 1H NMR (CDCl3)
δ 7.94 (1H, s, C(8)), 7.19 (1H, s, C(5)), 6.14 (1H, dd, J ) 7.62
Hz, J ) 3.33 Hz, C(3)), 4.22 and 4.08 (2H, 2m, C(1) diastere-
omeric methylene), 3.16 and 2.62 (2H, 2m, C(2) diastereomeric
methylene), 2.81 (2H, t, J ) 7.71 Hz, C(1′)), 2.09 (3H, s, acetate
methyl), 1.61 (2H, m, C(2′)), 1.41 (2H, sextet, J ) 7.29 Hz,
1
1438, 1371, 1319, 1236, 1035 cm-1; H NMR (CDCl3) δ 7.53
(1H, s, amide proton), 6.06 (1H, dd, J ) 7.55 Hz, J ) 3.0 Hz,
C(3)), 4.24 (2H, m, C(1)), 3.14 and 2.63 (2H, 2m, C(2)-
diastereomeric methylene), 2.51 (2H, t, J ) 6.93 Hz, C(1′)),
2.21 and 2.08 (6H, 2s, acetamido and acetate methyls), 1.39
(2H, quintet, J ) 7.08 Hz, C(2′)), 1.28 (2H, sextet, J ) 7.26
Hz, C(3′)), 0.862 (3H, t, J ) 7.23 Hz, C(4′)); mass spectrum

1336 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
C(3′)), 0.934 (3H, t, J ) 7.26 Hz, C(4′)); mass spectrum (EI
mode), m/ z 351 and 352 (M+, 79Br and M+, 81Br), 307 and 309
(M+ - acetyl). Anal. (C16H19BrN2O2‚0.125H2O) C, H, N.
(()- or (S)-(-)-3-Aceta m id o-2,3-d ih yd r o-7-m eth yl-1H-
p yr r olo[1,2-a ] ben zim id a zole (19). To a solution of 10 mL
of acetic acid and 10 mL of acetic anhydride was added 75 mg
(0.401 mmol) of 18‚HCl. The solution was stirred at room
temperature for 1.5 h, and the solvent was removed to afford
a solid residue, which was neutralized with aqueous sodium
bicarbonate. The product was extracted from the aqueous
layer with chloroform, and the extracts were dried (Na2SO4).
The dried extracts were concentrated, and the resulting solid
was recrystallized with chloroform/hexane to afford a white
solid: 78 mg (85%) yield; mp 218-220 °C; TLC (chloroform/
methane, 90:10) Rf ) 0.40; IR (KBr pellet) 3246, 3069, 2991,
6-Br om o-7-bu tyl-5-n itr o-2,3-d ih yd r o-1H-p yr r olo[1,2-a ]-
ben zim id a zol-3-yl Aceta te (16). To a mixture of 9 mL of
fuming nitric acid and 1 mL of concentrated sulfuric acid
chilled to -8 °C was added 256 mg (0.728 mmol) of 15. The
reaction mixture was stirred for 20 min at ice bath tempera-
ture. The mixture was then combined with 50 g of ice and 50
mL of chloroform. The resulting mixture was neutralized with
sodium bicarbonate and the chloroform layer separated. The
aqueous phase was then extracted with 4 × 50 mL portions of
chloroform. The extracts were combined and dried (Na2SO4),
and the solvent was removed in vacuo. The solid residue was
recrystallized from chloroform/hexane to afford pure 16: 186
mg (64%) yield; mp 101.5-103 °C; TLC (chloroform/methanol,
95:5) Rf ) 0.50; IR (KBr pellet) 2957, 2872, 1745, 1537, 1375,
1300, 1226, 1085, 881, 750 cm-1; 1H NMR (dimethyl sulfoxide-
d6) δ 7.84 (1H, s, C(8) aromatic proton), 6.11 (1H, dd, J ) 7.84
Hz, J ) 3.87 Hz, C(3)), 4.29 and 4.18 (2H, 2m, C(1) diastere-
omeric methylene), 3.09 and 2.57 (2H, 2m, C(2) diastereometric
methylene), 2.85 (2H, t, J ) 7.53 Hz, C(1′)), 2.06 (3H, s, acetate
methyl), 1.57 (2H, m, C(2′)), 1.35 (2H, sextet, J ) 7.32 Hz,
C(3′)), 0.901 (3H, t, J ) 7.23 Hz C(4′)); mass spectrum (EI
mode), m/ z 395 and 397 (M+, 79Br and M+, 81Br), 352 and 354
1
1637, 1562, 1421, 1500 cm-1; H NMR (CDCl3) 7.50 (1H, d, J
) 8.2 Hz, 5-aromatic proton), 7.04 (1H, d, J ) 8.3 Hz,
6-aromatic proton), 6.94 (1H, s, 8-aromatic proton), 5.44 (1H,
m, 3-methine proton), 4.03-3.93 (2H, m, 1-methylene protons),
2.56-2.50 (1H, m, 2-methylene protons), 2.45 (3H, s, methyl),
2.10 (3H, s, methyl); mass spectrum (EI), m/ z 229 (M+), 186
(M+ - COCH3), 171, 158, 145, 133, 116, 104. Rotation of S-(-
)-19 in methanol solvent, [R]25D ) -59.6°. Anal. (C13H15N3O)
C, H, N.
(()- or (S)-(-)-3-Acet a m id o-2,3-d ih yd r o-6-b r om o-7-
m eth yl-1H-p yr r olo[1,2-a ]ben zim id a zole (20). To a solu-
tion of 200 mg (0.87 mmol) of 19 in 10 mL of glacial acetic
acid was added 1 mL of 0.8 M bromine in acetic acid, and the
resulting mixture was stirred at room temperature for 20 min.
The reaction mixture was poured into 200 mL of 0.2 M
phosphate buffer (pH ) 7.0), and then the aqueous mixture
was extracted with 2 × 80 mL portions of ethyl acetate. The
extracts were washed with 2 × 30 mL portions of saturated
NaHCO3 and dried over Na2SO4. Removal of solvent gave 20
as a white solid, which was recrystallized from ethyl acetate:
188 mg (77%) yield; mp 249-250 °C; TLC (chloroform/
methanol, 90:10) Rf ) 0.30; IR (KBr pellet) 3265, 3065, 2982,
1641, 1560, 1523, 1423, 1296 cm-1; 1H NMR (CDCl3) 7.81 and
7.08 (2H, 2s, aromatic protons), 7.12 (1H, bs, NH), 5.43 (1H,
m, 3-methine proton), 4.17-3.96 (2H, m, 1-methylene protons),
3.32-4.09 (1H, m, 2-methylene proton), 2.60-2.51 (1H, m,
2-methylene proton), 2.46 (3H, s, methyl), 2.09 (3H, s, methyl);
mass spectrum (EI), m/ z 307, 309 (M+, Br81 and M+, Br79),
266, 264 (M+ - acetyl), 236, 211, 169, 131. Rotation of S-(-
(M+ - acetyl), 335 and 337 (M+ - acetic acid). Anal. (C16H18
BrN3O4) C, H, N.
-
7-Bu t yl-2,3-d ih yd r o-5,8-d ioxo-1H -p yr r olo[1,2-a ]b en z-
im id a zol-3-yl Aceta te (17). A suspension of 225 mg (0.568
mmol) of 16 and 45 mg of 5% Pd on charcoal in 100 mL of
methanol was shaken under 50 psi of H2 for 13 h. The solution
was then filtered through Celite and concentrated to a dry
residue consisting of the 5-amino derivative. A solution
consisting of 20 mL of water and 17 mg of potassium phosphate
monobasic was washed through Celite and then added to the
5-amino derivative. This solution was combined with
a
solution consisting of 90 mL of water, 100 mg of potassium
phosphate monobasic, and 1 g of Fremy salt. The resulting
mixture was stirred at room temperature for 5 h and then
extracted with 5 × 50 mL portions of chloroform. The extracts
were combined and dried (Na2SO4), and the solvent was
removed in vacuo. Pure product was obtained by flash column
chromatography on silica gel, employing chloroform as the
eluant. Recrystallization with acetone and hexane afforded
yellow crystals of 17: 58 mg (30%) yield; mp 105.5-106 °C;
TLC (ethyl acetate/chloroform, 80:20) Rf ) 0.37; IR (KBr pellet)
2960, 2931, 1747, 1655, 1525, 1371, 1224, 1153, 1084, 1043
)-20 in methanol solvent, [R]25 ) -57.7°. Anal. (C13H14
-
D
BrN3O) C, H, N.
(()- or (S)-(-)-3-Acet a m id o-2,3-d ih yd r o-6-b r om o-7-
m eth yl-5-n itr o-1H-p yr r olo[1,2-a ]ben zim id a zole (21). To
5 mL of ice-cooled fuming nitric acid was slowly added 220
mg (0.72 mmol) of 20 with stirring followed by 0.5 mL of acetic
anhydride. The reaction mixture was stirred at 10-25 °C for
1.5 h and then poured into 200 mL of ice-cold water. The
mixture was buffered to pH ) 7.0 using saturated NaHCO3
and then extracted with 2 × 30 mL portions of ethyl acetate.
The extracts were dried (Na2SO4), and the solvent was
removed to give an oil. Tritration of the oil with n-hexane
afforded a solid compound which was recrystallized from ethyl
acetate, resulting in light yellow crystals: 172 mg (68%) yield;
mp 248-250 °C (dec); TLC (chloroform/methanol, 90:10) Rf )
0.30; IR (KBr pellet) 3260, 3065, 2926, 1672, 1535, 1373, 1298
cm-1 1H NMR (CDCl3) δ 6.46 (1H, d, J ) 1.47 Hz, C(6)
;
aromatic proton), 6.06 (1H, dd, J ) 7.65 Hz, J ) 3.15 Hz, C(3)),
4.32 (2H, m, C(1) diastereomeric methylene), 3.14 and 2.63
(2H, 2m, C(2) diastereomeric methylene), 2.44 (2H, t, J ) 7.02
Hz, C(1′)), 2.07 (3H, s, acetate methyl), 1.48 (2H, m, C(2′)),
1.37 (2H, sextet, J ) 7.23 Hz, C(3′)), 0.907 (3H, t, J ) 7.29
Hz, C(4′)); mass spectrum (EI mode), m/ z 302 (M+), 259 (M+
- acetyl). Anal. (C16H18N2O4) C, H, N.
6-N-Azir idin yl-7-bu tyl-2,3-dih ydr o-5,8-dioxo-1H-pyr r olo-
[1,2-a ]ben zim id a zol-3-yl Aceta te (2). A solution containing
235 mg (0.777 mmol) of 17 and 16 mL of dry methanol was
chilled to 0 °C, at which time 0.85 mL of ethyleneamine was
added. The reaction mixture was stirred for 30 min at 0 °C
and then stirred at room temperature for 1.3 h. The methanol
was evaporated, and the residue was purified by flash chro-
matography on silica gel, employing chloroform as the eluant.
Pure 3 was recrystallized from ethyl acetate and hexane: 136
mg (51%) yield; mp 122-122.5 °C; TLC (chloroform/methanol,
99:1) Rf ) 0.21; IR (KBr pellet) 3448, 2958, 2872, 2362, 1749,
1
cm-1; H NMR (CDCl3) δ 8.08 (trace, s, 5-aromatic proton of
8-nitro isomer), 7.66 (1H, d, J ) 6 Hz, amide N-H), 7.36 (1H,
s, 8-aromatic proton), 5.45-5.39 (1H, m, 3-methine proton),
4.39-4.05 (2H, 2m, 1-methylene protons), 3.34-3.25 (1H, m,
2-methylene proton), 2.75-2.68 (1H, m, 2-methylene proton),
2.55 and 2.04 (6H, 2s, methyl); mass spectrum (E1), m/z 354
(M+, 81Br) 352 (M+, 79Br), 292 and 294 (M+ - NHCOCH3), 265,
236, 184, 156. Rotation of S-(-)-21 in methanol solvent, [R]25
) -42.2°. Anal. (C13H13BrN4O3) C, H, N.
D
(()- or (S)-(-)-3-Aceta m id o-2,3-d ih yd r o-7-m eth yl-1H-
p yr r olo[1,2-a ]ben zim id a zole-5,8-d ion e (22). A solution of
210 mg (0.60 mmol) of 21 in 100 mL of methanol containing
100 mg of 5% Pd on carbon was shaken under 50 psi of H2 for
4 h. The reaction mixture was filtered through Celite, and
the filter cake was washed with 2 × 20 mL methanol. The
combined filtrates were concentrated under reduced pressure
to afford a brown oily compound. The compound was dissolved
in a solution of 1 g of KH2PO4 in 50 mL water and treated
with a solution of 2.0 g (0.75 mmol) of Fremy salt dissolved in
1
1678, 1629, 1520, 1228, 1138 cm-1; H NMR (CDCl3) δ 6.02
(1H, dd, J ) 7.51 Hz, J ) 3.83 Hz, C(3)), 4.27 (2H, m, C(1)
diastereomeric methylene), 3.11 and 2.61 (2H, 2m, C(2)
diastereomeric methylene), 2.55 (2H, t, J ) 7.01 Hz, C(1′)),
2.34 (4H, s, aziridine protons), 2.06 (3H, s, acetate methyl),
1.37 (4H, m, C(2′) and C(3′)), 0.914 (3H, t, J ) 7.00 Hz, C(4′));
mass spectrum (EI mode), m/ z 343 (M+), 300 (M+ - acetyl)
283 (M+ - acetic acid), 240 (M+ - acetic acid - aziridine).
Anal. (C18H21N3O4‚0.125H2O) C, H, N.

Pyrrolo[1,2-a]benzimidazolequinone Activities
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9 1337
100 mL of water containing 3 g of KH2PO4. The reaction
mixture was stirred for 1 h during which the reaction turned
orange yellow. It was extracted with 4 × 25 mL portions of
chloroform and dried over Na2SO4. Removal of solvent gave
a yellow solid compound which was purified by flash column
chromatography, employing chloroform/methanol (95:5) as the
eluant. Recrystallization was carried out with ethyl acetate/
hexane: 110 mg (71%) yield; mp 186-88 °C; TLC (chloroform/
methanol, 9:1) Rf ) 0.30; IR (KBr pellet) 3289, 3041, 1664,
1541, 1481, 1273, 1157, 959 cm-1; 1H NMR (CDCl3) δ 7.41 (1H,
d, J ) 3.33 Hz, NH), 6.43 (1H, q, J ) 1.6 Hz, C-7 proton), 5.30
(1H, m, 3-methine proton), 4.36-4.35 (1H, m, 1-methylene
proton), 4.20-4.11 (1H, m, 1-methylene proton), 3.26-3.14
(1H, m, 2-methylene proton), 2.73-2.63 (1H, m, 2-methylene
proton), 2.05 and 2.04 (6H, 2s, methyls); mass spectrum (E1),
m/ z 259 (M+), 216 (M+ - acetyl). Anal. (C13H13N3O3) C, H,
N.
(()- or (S)-(-)-3-Aceta m id o-7-a zir id in yl-2,3-d ih yd r o-7-
m eth yl-1H-p yr r olo[1,2-a ]ben zim id a zole-5,8-d ion e (3). To
a solution of 60 mg (0.23 mmol) of 22 in 30 mL of dry methanol
was added 0.5 mL of ethyleneamine at 0 °C, and the mixture
was stirred for 30 min. The solvent was removed under
vacuum to afford a red mass. The product was purified by
flash column chromatography, employing chloroform/methanol
(95:5) as the eluant. Recrystallization was carried out with
chloroform/hexane: 38 mg (55%) yield; mp 238-240 °C dec;
TLC (chloroform/methanol, 9:1) Rf ) 0.25; IR (KBr pellet) 3294,
2995, 2928, 1686, 1638, 1520, 1309, 1138, 977 cm-1; 1H NMR
(CDCl3) δ 7.60 (1H, d, J ) 7.23 Hz, acetamido NH), 5.31 (1H,
m, 3-methine proton), 4.36-4.27 (1H, m, 1-methylene proton),
4.13-4.04 (1H, m, 1-methylene proton), 3.16-3.10 (1H, m,
2-methylene proton), 2.69-2.62 (1H, m, 2-methylene proton),
2.38 (4H, bs, aziridinyl protons), 2.06 and 2.00 (6H, 2s,
methyls); mass spectrum (EI), m/ z 300 (M+); CD for S-(-)-3
(c ) 0.08 in dioxane) at 25 °C [θ]600 ) -10, [θ]470 ) +2885,
[θ]400 ≈ 0. Anal. (C15H16N4O3‚0.25H2O) C, H, N.
methylene), 2.86 and 2.34 (2H, 2m, C(2) diastereomeric
methylene), 2.38 (3H, s, methyl). Rotation of 24 in methanol,
[R]25 ) -28° (S) and +28° (R).
D
(R)- or (S)-7-Meth yl-2,3-d ih yd r o-1H-p yr r olo[1,2-a ]ben -
zim id a zol-3-yl Aceta te (23). To a solution of 180 mg (0.96
mmol) of R- or S-24, dissolved in 10 mL of acetic anhydride,
was added 2 mL of glacial acetic acid and 1 mL of water. The
resulting mixture was stirred at room temperature for 2 h,
after which the solvents were evaporated in vacuo. The
remaining residue was flash chromatographed on silica gel
using chloroform to elute the first fraction and then chloroform/
methanol (99:1) to elute R- or S-23. The product was recrys-
tallized by dissolution in a small volume of chloroform followed
by addition of hexane: 146 mg (66%) yield; TLC (chloroform/
1
methanol, 80:20) Rf ) 0.88; H NMR (dimethyl sulfoxide-d6)
7.51, 7.34 and 7.05 (3H, ABX, J ortho ) 8.4 Hz, J meta ) 1.2 Hz,
J para ≈ 0 Hz, aromatic protons), 6.10 (1H, dd, J ) 7.5 Hz, J )
3.3 Hz, C(3) proton), 4.22 and 4.13 (2H, 2m, C(1) diastereo-
meric methylene), 3.12 and 2.57 (2H, 2m, C(2) diastereomeric
methylene), 2.43 (3H, s, 7-methyl), 2.07 (3H, s, acetate methyl).
Rotation of 23 in methanol, [R]25 ) +91° (R) and -90° (S).
D
En a n tioselective Hyd r olysis of (()-23. A solution of 500
mg of racemic 23 in 4 mL of dimethylformamide was added to
a suspension of 300 mg of Candida antarctica “B” lipase in
120 mL of pH 7 phosphate buffer. The resulting mixture was
stirred at room temperature for 2 h to obtain >97% ee of S-23
or for 24 h to obtain >98% ee of R-24. The solution was then
extracted with 3 × 100 mL of chloroform, and any emulsion
formed was filtered through a thin pad of Celite. The extract
was dried (sodium sulfate), concentrated to a small volume,
and then flash chromatographed on silica gel using chloroform/
methanol (95:5) as eluant. The products were recrystallized
by dissolution in chloroform/methanol (3:2), followed by addi-
tion of hexane. Enantiomeric excess was calculated from
optical rotations. [R]25
) -88° S-23 and +27.5° R-24.
D
Ch ir a l High -P r essu r e Liqu id Ch r om a togr a p h y. All
HPLC separations of enantiomers were done on Chirex
columns (50 × 3.2 mm) from Phenomenex. The detector was
set at 254 mm and sensitivity at 0.5. The flow rate was 0.5
mL/min with the mobile phase consisting of the indicated
solvent system. Chirex column no. 3014 was used for com-
pounds 23 and 24. Measured retention times in minutes were
as follows: 2.3, R-24; 3.0, S-24; 5.0, R-23; 6.5, S-23). The
mobile phase was hexane/1,2-dichloroethane/methanol, 99:11:
1. Chirex column no. 3017 was used for (R)- and (S)-APBI-A
(R)- a n d (S)-4-[(5′-Meth yl-2′-n itr op h en yl)a m in o]-2-h y-
d r oxybu ta n oic Acid (27). To a solution of 1.0 g (8.4 mmol)
of R- or S-26 in 10 mL of 0.84 mM dry sodium ethoxide/ethanol
was added 2.0 g (9.3mmol) of 25 in 4 mL of dry ethanol and
10 mL of dry dimethyl formamide. The resulting mixture was
refluxed for 16 h. After the mixture was cooled to room
temperature, 100 mL of saturated aqueous sodium bicarbonate
was added. Extraction of the aqueous solution with 3 × 50
mL portions of chloroform removed unreacted 25. The aqueous
solution was then acidified to pH 3 with 4 N hydrochloric acid,
and R- or S-27 was removed with 8 × 200 mL portions of
chloroform. The extract was dried (sodium sulfate) and
concentrated to an orange oil: 1.20 g (56%) yield; TLC
(butanol/water/acetic acid, 5:3:2) Rf ) 0.74; IR (NaCl thin film)
3366, 1742, 1628, 1576, 1408, 1225, 1161, 752 cm-1; 1H NMR
(CDCl3) δ 8.21 (1H, s, amine proton), 7.94, 6.60 and 6.38 (3H,
ABX, J ortho ) 9.0 Hz, J meta ) 1.5 Hz, J para < 0 Hz, aromatic
protons), 4.37 (1H, dd, J ) 7.2 Hz, J ) 4.2 Hz, C(2) methylene),
3.46 (2H, t, J ) 7.2 Hz, C(4) methylene), 2.29 (3H, s, methyl),
2.02 and 1.59 (2H, 2m, C(3) diastereomeric methylene).
which were previously prepared in this laboratory;1
measured
retention times in minutes were as follows: 11.2, (S)-APBI-A
and 12.5, (R)-APBI-A. The mobile phase was hexane/1,2-
dichloroethane/methanol, 85:10:5. Chirex column no. 3014
was also used for R- and S-3. Measured retention times in
minutes were as follows: 34, S-3; 44, R-3. The mobile phase
was hexane/1,2-dichloroethane, 60:40.
Alk yla tion of DNA by Red u ced P BIs. To a mixture of
1-5 mg of DNA (600 bp calf thymus, poly(dA)‚poly(dT), poly-
(dG)‚poly(dC), poly(dA-dT)‚poly(dT-dA)) in 2.0 mL of Tris
buffer, pH 7.4, and 0.1 mg of Pd on carbon was added a one-
to-one base-pair equivalent amount of the PBI dissolved in 0.5
mL of dimethyl sulfoxide. The resulting solution was degassed
under argon for 30 min, after which the mixture was purged
with H2 for 10 min. The solution was then purged with argon
for 10 min and placed in a 30 °C bath for 24 h. The reaction
was opened to the air, and the catalyst was filtered off. The
filtrate was combined with 0.385 mL of sodium acetate and
11 mL of ethanol. The mixture was chilled at -20 °C for 12
h and the DNA pellet collected by centrifuging at 12000g for
20 min. The pellet was redissolved in water and then
precipitated and centrifuged again. The resulting pellet was
suspended in ethanol, centrifuged, and dried.
Isola tion of DT-Dia p h or a se by Affin ity Ch r om a tog-
r a p h y. The azodicoumarol Sepharose 6B column material
was prepared according to the procedure by Ho¨jeberg et al.31
and equilibrated for 24 h in a 2.5 × 13 cm column with a
solution of 0.25 M sucrose in 50 mM pH 7.5 Tris‚HCl (buffer
A). The following steps were carried out in a cold room held
at 4 °C. Rat livers (200 g wet weight, Hooded rats) were added
to a 0.25 M sucrose solution and homogenized in a Waring
Rotation of 27 in methanol solvent, [R]25 ) +5.3° (S) and
D
-5.3° (R).
(R)- or (S)-3-Hydr oxy-7-m eth yl-2,3-dih ydr o-1H-pyr r olo-
[1,2-a ]ben zim id a zole (24). A solution of 1.0 g (3.93 mmol)
of R-27 in 20 mL of methanol was shaken under 50 psi of H2
in the presence of 700 mg of 5% Pd on activated carbon for 2
h. The completed reaction was treated with 4 N hydrochloric
acid and filtered through Celite. The filtrate was evaporated
in vacuo to an acid/amine mixture and then combined with
20 mL of 4 N hydrochloric acid. The mixture was neutralized
to pH 6 with saturated aqueous sodium bicarbonate, and R-
or S-27 was removed with 3 × 100 mL of chloroform. The
extract was dried (sodium sulfate) and concentrated to a tan
residue. The crude product was recrystallized by dissolution
in chloroform/methanol (3:2) followed by addition of hexane:
165 mg (19%) overall yield; TLC (chloroform/methanol, 80:20)
Rf ) 0.62; 1H NMR (dimethyl sulfoxide-d6) 7.43, 7.24, and 6.96
(3H, ABX, J ortho ) 8.4 Hz, J meta ) 1.2 Hz, J para ≈ 0 Hz, aromatic
protons), 5.77 (1H, d, J ) 6.0 Hz, hydroxyl proton), 5.03 (1H,
m, C(3) proton), 4.13 and 3.97 (2H, 2m, C(1) diastereomeric

1338 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 9
Skibo et al.
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blender at high speed. The homogenate was centrifuged at
36 000 rpm (∼105000g) for 60 min and a supernatant filtered
through several layers of cheese cloth. The supernatant was
applied to the column at 90 mL/h, after which the column was
eluted with buffer A until no protein was detected by the
Bradford assay.32 To remove non-specifically-bound proteins,
a solution of 2 M potassium chloride and 0.25 M sucrose in 50
mM Tris‚HCl (pH 8.9) buffer was applied to the column. The
flow was then reversed with buffer A at 30 mL/h, and 11.2
mL of a solution of 20 mM NADH in buffer A was applied to
remove the bound DT-diaphorase. Fractions with the highest
activity were combined and concentrated to 10 mL by ultra-
filtration in a Diaflo cell (PM10 filter, 43 mm, N2 ) 20 psi).
The concentrated solution was applied to a Sephacryl S-200
column (3.2 × 55 cm) equilibrated with buffer A at a flow rate
of 30 mL/h. Fractions with a constant protein/flavin ratio (A270
/
A450 ) 6.7) were combined. The concentration of the flavin
was 8.7 µM in the combined fractions with a constant protein/
flavin ratio.
Kin etic Stu d ies. Kinetic studies were carried out in 0.05
M pH 7.4 Tris‚HCl buffer, under anaerobic conditions, employ-
ing Thunberg cuvettes. A 2 mM stock solution of the ap-
propriate PBI or APBI 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 followed at 296 nm for 25 min to
obtain initial rates. The concentrations after mixing were 0.3
mM NADH, (1-20) × 10-5 M quinone, and 14.5 nM (based on
flavin) of enzyme active sites. The value of ∆ꢀ was calculated
from the initial and final absorbance values for complete
quinone reduction; the usual value for ꢀ is 6000-8000 M-1
cm-1
. .
The value of ∆ꢀ was used to calculate Vmax in M s-1
The results were fitted to a Lineweaver-Burke plot from
which kcat/Km values were calculated based on 14.5 nM of active
sites.
Electr op h or esis Stu d ies. The relaxation reactions were
assayed in a 0.045 M Tris‚borate buffer using a 1.3% agarose
gel. The topoisomerase relaxation reactions were carried out
with 0.5 µg of SV-40 supercoiled DNA (form I), 1 µL of a
solution of 20 units of topoisomerase II in 20 µL of 100 mM
tris‚Cl, pH 7.9, containing 500 mM NaCl, 500 mM KCl, 50
mM MgCl2, 1 mM EDTA, 150 µg/mL BSA, and 10 mM ATP.
The reactions were run for 45 min at 37 °C and then mixed
with 1 µL of a stop solution consisting of 75 mg of SDS and
3.75 mg of proteinase K in 1.5 mL of water. After incubation
for an additional 45 min, the reactions were combined with 4
mL of gel loading solution. The gel was loaded with 5 µL of
gel-loading solution. The gel was loaded with 5 µL of reaction
solution per well and run at 40 V (12 milliamperes) for 13 h
after which the gel was washed with 100 µL of a 1 mg/mL
ethidium bromide solution in 700 mL of 0.045 M Tris‚borate
buffer.
Ack n ow led gm en t. The research was supported by
awards from the American Cancer Society and the
National Science Foundation.
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