Design, synthesis, DNA binding, and biological evaluation of water-soluble hybrid molecules containing two pyrazole analogues of the alkylating cyclopropylpyrroloindole (CPI) subunit of the antitumor agent CC-1065 and polypyrrole minor groove binders

Article abstract of DOI:10.1021/jm0108404

We have synthesized and evaluated a series of hybrids, denoted 22-27, for in vitro cytotoxic activity against a variety of cancer cell lines. These hybrids represent a molecular combination of polypyrrole minor groove binders structurally related to the n

Full text of DOI:10.1021/jm0108404

2536
J . Med. Chem. 2001, 44, 2536-2543
Design , Syn th esis, DNA Bin d in g, a n d Biologica l Eva lu a tion of Wa ter -Solu ble
Hybr id Molecu les Con ta in in g Tw o P yr a zole An a logu es of th e Alk yla tin g
Cyclop r op ylp yr r oloin d ole (CP I) Su bu n it of th e An titu m or Agen t CC-1065 a n d
P olyp yr r ole Min or Gr oove Bin d er s
Pier Giovanni Baraldi,*,† Gianfrano Balboni,† Maria Giovanna Pavani,† Giampiero Spalluto,†
Mojgan Aghazadeh Tabrizi,† Erik De Clercq,‡ J an Balzarini,‡ Toshikazu Bando,§ Hiroshi Sugiyama,§ and
Romeo Romagnoli†
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Rega Institute for Medical Research,
Laboratory of Virologyand Chemotherapy, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and Institute of Biomaterials
and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda, Tokyo 101-0062, J apan
Received February 7, 2001
We have synthesized and evaluated a series of hybrids, denoted 22-27, for in vitro cytotoxic
activity against a variety of cancer cell lines. These hybrids represent a molecular combination
of polypyrrole minor groove binders structurally related to the natural antitumor agent
distamycin A and two pyrazole analogues of the left-hand segment called cyclopropylpyrro-
loindole (CPI) of the potent antitumor antibiotic (+)-CC-1065. These novel water-soluble hybrids
have been designed to enhance the minor groove binding ability of alkylating units 20 and 21,
which should increase their clinical appeal by overcoming the administration problems of (+)-
CC-1065 derivatives. The DNA alkylating and cytotoxic activities against several tumor cell
lines are reported and discussed in terms of their structural differences in relation to both the
number of N-methyl pyrrole rings and the type of the alkylating unit tethered to the
oligopeptidic frame. It may be noted that, in general, and especially for 22-24, the cytotoxicity
of the hybrids was much greater than that of the alkylating units alone. In only one case,
compound 27, did the hybrid have cytotoxic activity comparable to that of the alkylating unit
alone against FM3A/0 cells. The broadest spectrum of activity and greatest potency was shown
by the hybrid 24, in which the alkylating unit 20 and the deformyl distamycin A are tethered
by 1-methyl 2,5-dicarbonyl pyrazole, with IC50 values for the different tumor cell lines ranging
from 7 to 71 nM. For compounds 22-24, the increase of the length of the pseudopeptidic moiety
from one to three N-methylpyrrole residues led to an increased cytotoxicity. Among the hybrids
tested for their inhibitory effects on the proliferation of murine L1210 leukemia cell line,
compound 24 proved to be the most active (IC50 ) 7.4 nM), and in the sequencing gel
experiments, it showed the strongest and most highly sequence-specific DNA alkylation activity.
For compounds 22-24, the sequence specificity of DNA alkylation appears to be affected by
the modification of the number of pyrrole rings, and the correlation between cytotoxicity and
alkylation pattern suggests that 24 exerts its cytotoxicity through DNA sequence-specific
alkylation of the third adenine located in the sequence 5′-ACAAAAATCG-3′. The two other
hybrids 22 and 23 were slightly less active for tumor cell proliferation, with IC50 values of 58
and 19 nM, respectively. With only one exception, none of the compounds was endowed with
antiviral activity at subtoxic concentrations. Compound 24 inhibited the effect of vaccinia virus
at a concentration that was significantly lower than its minimum cytotoxic concentration for
the E6SM host cells. These compounds gave distinct patterns of alkylation in AT-rich sequences,
indicating that minor structural changes produced marked alterations in sequence selectivity.
In tr od u ction
manner and modify the function of nucleic acids ir-
reversibly. Distamycin A is a naturally occurring anti-
biotic characterized by the presence of N-methylpyrrole-
2-carboxamide units ending with an amidino moiety,1,2
which binds to the DNA minor groove, preferentially to
AT-rich sequence, and in a reversible manner.3-5 Dis-
tamycin A (1) can be used as a DNA sequence-selective
vector for both alkylating and nonalkylating agents
leading to a substantial increase of cytotoxicity against
cancer cell lines in comparison to that of distamycin
alone.6-8
Many natural and synthetic anticancer agents with
the ability to interact with DNA have been discovered,
but most have little sequence specificity and often
exhibit severe toxicity to normal tissues. For these
reasons, there has been considerable interest in molec-
ular biology and human medicine to find small mol-
ecules able to alkylate the DNA in a sequence-specific
* Corresponding author. Tel: +39-(0)532-291293. FAX: +39-(0)-
532-291296. E-mail: pgb@ifeuniv.unife.it.
† Universita` di Ferrara.
(+)-CC-1065 (2) is a member of the class of cyclopro-
pylindole antitumor antibiotics first isolated from Strep-
‡ Rega Institute for Medical Research.
§ Tokyo Medical and Dental University.
10.1021/jm0108404 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/28/2001

Antitumor Agent CC-1065 and Polypyrrole Minor Groove Binders J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2537
tomyces zelensis by scientists at the Upjohn Company,9
and it is a highly potent natural agent with activity both
in vitro and in experimental animals.10-12 This activity
has generated great interest in this agent as a potential
anticancer drug.13,14 Studies on the mechanism of cy-
totoxic action have shown that CC-1065 affords its
biological activity through binding to double-stranded
B-DNA within the minor groove to AT-rich sequences
and selectively alkylating at the N3 position of the 3′-
adenine by its cyclopropylindole (CPI) subunit.15-17 It
was also demonstrated that synthetic enantiomer (-)-
CC-1065 alkylates the adenine 5′end of the AT-rich
sequence.18 Despite its high potency and broad spectrum
of antitumor activity, CC-1065 cannot be used in
humans because it causes delayed death in experimen-
tal models.19
Lown and colleagues have synthesized a series of
hybrid compounds belonging to the general structures
3-5, which contain the CPI moiety (in racemic form)
coupled with various lexitropsins constituted by one or
two N-methyl pyrroles.20,21 Among the synthesized
compounds, the two most potent CPI-lexitropsin hy-
brids were the compounds possessing structure 3, where
the CPI unit was linked to one or two N-methyl pyrroles
via a trans double bond. The reason for this enhanced
cytotoxic activity due to the presence of a trans double
bond linker on CPI compounds 3 is not well understood.
In all derivatives 3-5, the alkylating units were syn-
thesized and linked in racemic form.
Here we wish to report the synthesis and biological
evaluation of a novel series of conjugates comprising of
either of these two CPI pyrazole analogues 6 and 7 and
three mixed pyrazole-pyrrole compounds 8-10, called
lexitropsins (or information-reading oligopeptides), con-
sisting of a varying number of pyrrole amide units (from
one to three) tethered on the N-terminus to a 3,5-
pyrazole dicarboxylic acid moiety and structurally re-
lated to the DNA minor groove binder distamycin A. The
DNA binding domains chosen are structurally related
to distamycin A, which is known to bind to the minor
groove of DNA in AT-rich sequences. In addition, the
amidino moiety would also enhance the DNA-binding
affinity by providing noncovalent electrostatic DNA
binding. In synthesizing these novel water-soluble
hybrids, we wanted to increase the potency of pyrazole
CPI analogues 20 and 21 by increasing their affinity
for DNA and to determine the structure-activity rela-
tionship among the length of the oligopyrrolic frame,
antitumor activity, and sequence specificity.
Ch em istr y
The synthesis of the alkylating units 2022,23 and 2124
has been previously reported. The key intermediate for
the synthesis of lexitropsins 8-10 was the partially
masked pyrazole 13, which acts as a rigid linker. In fact,
by the carbonyl functions present in the 3 and 5 position
on the pyrazolic moiety, it was possible to join the
alkylating units 20-21 and the polypyrrolic aminoam-
idines 14-16. The known compound 1125 was trans-
formed into the corresponding benzyl ester 12 by
treatment with benzyl bromide and potassium carbon-
ate. The latter compound was selectively deprotected
by selective alkaline hydrolysis with 1 equiv of sodium
hydroxide, affording the acid 13 after acidification with
diluted hydrochloric acid (5% v/v). Coupling of carboxylic
acid-benzyl ester 13 with the well-known amines 14,26
15,27 and 16 (deformyl distamycin A)1 was readily
achieved with 1-ethyl-3-[3-(dimethylamino)propyl]car-
bodiimide hydrochloride (EDCI) and 1H-hydroxy-1,2,3-
benzotriazole (HOBt) to give 17, 18, and 19, respec-
tively. The protecting benzyl group in 17-19 was
cleaved off by catalytic hydrogenation (10% Pd on C) in
methanol, in the presence of a few drops of diluted
hydrochloric acid (5% v/v) to give compounds 8-10,
respectively. The final incorporation of the alkylating
units 20 and 21 into the DNA-binding moieties, consti-
tuted by one, two, and three pyrrole residues (com-
Recently, we have reported the synthesis and biologi-
cal evaluation of two CPI pyrazole analogues, (()-N-
Boc-CPzI 622,23 and (()-N-Boc-N-BnCPzI 7,24 which
demonstrate a cytotoxicity against L1210 leukemia cells
that was comparable to and 10-fold lower than that of
the reference compound N-Boc-CPI [IC50 ) 330 nM for
(+)-N-Boc-CPI vs IC50 ) 370 nM for (()-6 and IC50
)
3064 nM for (()-7, respectively]. Because of their
limited sequence specificity, low affinity for DNA, and
poor water solubility, it was reasoned that it may be
beneficial to tether these alkylating compounds to a
DNA binding vector, such as polypyrrole pseudopep-
tides, which can permeate cell membranes and has the
potential to control specific gene expression. The vector
could therefore deliver the reactive group more ef-
ficiently and in a sequence-specific manner to the DNA.
Moreover, water solubility made these hybrid com-
pounds attractive to overcome the administration prob-
lem of CC-1065 derivatives.

2538 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Baraldi et al.
Sch em e 1
in improved cytotoxic potency against most of the tumor
cell lines as compared to that of the alkylating units
alone. This is likely due to the more efficient delivery
of 20 and 21 to the DNA, followed by a covalent reaction
with the DNA at selected sites. The results show that
the hybrids 22-24 were about 8- to 70-fold more potent
than the alkylating unit 20. Among these, the hybrid
24 demonstrated the highest potency across the panel
of tumor cell lines, especially against T- and B-lympho-
blast cells, with IC50 values between 7.4 and 71 nM.
Against L1210 cells, the tripyrrole analogue 24 was 2-
to 8-fold more active than the bis and monopyrrole
counterparts (compounds 23 and 22, respectively). This
is presumably due to the increased DNA binding of the
“longer” compounds. The IC50 for 23 ranged between 19
and 45 nM with respect to the tumor cell lines L1210,
Molt4, CEM, and Daudi, but the same compound was
somewhat less active against FM3A (IC50 ) 190 nM).
Compound 24 was more active against L1210 cells than
against the other tumor cell lines. For this series of
hybrids, it is possible to correlate structure with biologi-
cal activity, as previously reported,6-8 increasing the
number of pyrrole rings from one to three results in
increased cytotoxic activity.
The hybrids 25-27 demonstrated potent cytotoxic
activity against Daudi cells (IC50 values ranging from
11 to 100 nM). While being somewhat less toxic to the
other tumor cells (IC50 values ranging between 70 and
19 300 nM), they are always more cytotoxic than the
alkylating unit 21 alone (with only few exceptions). The
FM3A cell line was substantially less sensitive to the
hybrids 25-27, which exhibited cytotoxic activities
comparable to or 3-fold higher (IC50 values ranging from
4 to 19 µM) than that of the alkylating unit 21 alone.
In the CEM cell line, compounds 25-27 demonstrated
IC50 values between 70 and 400 nM, which were 30- to
100-fold higher than that reported for 21, with com-
pound 25 exhibiting potent cytotoxicity (IC50 ) 70 nM).
A fairly marked dependence on the number of pyrrolic
rings for the antiproliferative activity has been observed
in the 22-24 series, with compound 24 comprising three
pyrrolic rings proving to be the most active. The
relationship between the number of pyrrole groups in
the 25-27 series and their corresponding cytotoxicity
did not seem to follow this pattern. In fact, the cytotox-
icity was higher for compound 25, which possesses only
one pyrrole ring. It is interesting to note that the L1210
cell line was 50-fold more susceptible to the cytotoxic
action of compound 27 than FM3A cells. For all cell
lines, taken together, compounds possessing the same
number of pyrrole rings and the alkylating unit 20
appeared to be more cytotoxic than those containing 21
as the alkylating agent.
pounds 8, 9 and 10, respectively), was achieved using
the classical coupling procedure mediated by EDCI after
deprotection of the BOC group in (()-seco-N-Boc-CPzI
2022,23 and (()-seco-N-Boc-N-BnCPzI 2124 by acid hy-
drolysis with dry HCl in ethyl acetate. The final hybrid
compounds 22-27 possessed improved water solubility
with respect to the alkylating units 20 and 21 (which
are poorly soluble in aqueous solution) and can be easily
stored as the stable chlorohydrate salt after purification
by preparative HPLC and lyophilization.
Resu lts a n d Discu ssion
An titu m or Activity. In Table 1, we have reported
the in vitro cytotoxic activity of compounds 22-27
against a panel of tumor cell lines using the alkylating
units 20 and 21 as reference compounds. By previous
in vitro assay on L1210 cell line,22-24 we have observed
that there was not any detectable difference between
the cytotoxic activies of the seco-derivatives 20 and 21
(IC50 ) 520 ( 6 and 2710 ( 490 nM, respectively) and
that of the corresponding spiro derivatives 6 and 7 (IC50
) 370 ( 4 and 3064 ( 220 nM, respectively). All
compounds 20-27 demonstrated profound inhibitory
effects on the proliferation of murine leukemia (L1210),
murine mammary carcinoma (FM3A), human T-lym-
phoblastoid (Molt/4 and CEM), and human B-lympho-
blastoid (Daudi) cells.
An t ivir a l Act ivit y. The most active compound,
hybrid 24, has been also tested for its antiviral activity
against herpes simplex virus (HSV)-1 and -2, vaccinia
virus (VV), vesicular stomatitis virus (VSV), herpes
simplex virus-1 TK- KOS ACV, and herpes simplex
virus-1 TK- VMW 1837 in E6SM (embryonic skin
muscle) cell cultures. Results were compared both with
alkylating unit 20 alone and with the antiviral agents
BVDU, ribavirin, ACG, and DHPG (Table 2). Compound
24 proved to be rather toxic to the host cells (morphology
and growth), and no specific antiviral activity could be
As evident from Table 1, tethering the pyrazole CPI
analogues 20 and 21 to the DNA binders 8-10 (to afford
the hybrids 22-27) resulted, with only a few exceptions,

Antitumor Agent CC-1065 and Polypyrrole Minor Groove Binders J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2539
Ta ble 1. In Vitro Activity of Hybrids 22-27 and Alkylating Units 20 and 21 against the Proliferation of Murine Leukemia (L1210),
Murine Mammary Carcinoma (FM3A), Human T-Lymphoblast (Molt/4 and CEM) and Human B-Lymphoblast (Daudi) Cellsa
IC50 (nM ( SE)
compound
L1210
FM3A
Molt/4
CEM
Daudi
20
21
22
23
24
25
26
27
520 ( 6.6
2710 ( 490
58 ( 17
1400 ( 40
18300 ( 200
1600 ( 50
190 ( 6
1740 ( 50
8550 ( 280
340 ( 20
45 ( 1
1260 ( 30
6720 ( 1040
230 ( 10
39 ( 1
680 ( 150
7520 ( 30
150 ( 40
22 ( 10
8.8 ( 0.1
11 ( 6.0
38 ( 7.0
100 ( 10
19 ( 2
7.4 ( 0.4
240 ( 30
600 ( 90
400 ( 16
31 ( 11
17 ( 4
71 ( 9
4000 ( 1000
5600 ( 1400
19300 ( 3400
130 ( 20
160 ( 60
310 ( 70
70 ( 21
210 ( 110
400 ( 50
a
IC50 ) compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean (SE from the
dose-response curves of at least three indipendent experiments.
SM
Ta ble 2. Cytotoxicity and Antiviral Activity of Compounds 20 and 24 against Several Viruses in E6 Cell Cultures
MICb (µg/mL)
HSV-1
TK-KOS
ACV
HSV-1
TK-VMW
1837
MCCa
(µg/mL)
HSV-1
(KOS)
HSV-2
(G)
compound
VV
0.019
>1.6
0.077
16
>400
>100
VSV
24
20
BVDU
Ribavirin
ACG
0.8
8
>400
>400
>400
>100
>0.16
>1.6
0.015
240
0.077
0.004
0.16
>1.6
240
48
0.077
0.004
>0.16
>1.6
>400
48
>400
>100
>0.16
>1.6
240
240
48
>0.16
>1.6
0.128
240
0.384
0.019
DHPG
1.92
a
Minimum cytotoxic concentration (MCC) required to cause a microscopically detectable alteration of normal cell morphology when
b
incubated with cells for the same duration as that required to measure antiviral activity. Minimum inhibitory concentration (MIC)
required to reduce virus-induced cytopathogenicity by 50%. Viral cytopathogenicity was recorded as soon as it reached completation in
the control virus-infected cell cultures.
discerned at subtoxic concentrations with respect to
HSV-1 and -2, VSV, HSV-1 TK- KOS ACV, and HSV-1
TK- VMW 1837. Furthermore, compound 24 showed a
minimum inhibitory concentration (MIC) of 0.0192 µg/
mL against vaccinia virus replication, this concentration
being about 40-fold below the toxicity threshold of the
compound for the E6SM host cells (0.8 µg/mL).
The other derivatives 22, 23, and 25-27 were also
evaluated for their antiviral activity against cytomega-
lovirus (CMV), varicella-zoster virus (VZV), herpes
simplex virus type 1 (HSV-1) and type 2 (HSV-2), and
vaccinia virus (VV). The compounds proved to be very
toxic to human embryonic lung cells (estimated by
altered morphology and inhibition of growth), and no
specific antiviral activity could be discerned at subtoxic
concentrations. All five hybrids were not selectively
active against CMV, VZV, HSV-1 and-2, and VVsthat
is, they did not inhibit the cytopathic effect of the viruses
at a concentration that was lower than their minimum
cytotoxic concentration for the host cells.
DNA Alk yla tion a n d Molecu la r Mod elin g Stu d -
ies. The sequence-selective alkylation by these hybrids
22-27 was assayed using the 5′-Texas Red-end-labeled
450 base pair DNA fragments. Alkylated DNA frag-
ments were cleaved by heating at 90 °C, and the
fragments analyzed using a DNA sequencer were cleaved
as described previously28 (Figure 1).
When compounds 25-27 were incubated at different
concentrations (ranging from 0.1 to 100 µM), no signifi-
cant cleavage was observed by thermally induced strand
cleavage of DNA fragment. For compounds 25-27, the
presence of a benzyl group at the azaindole moiety 21
significantly disrupted the alkylation reaction, and the
effect of this substitution has presumably altered the
positioning of the compound on the DNA. In the
orientation that is suitable for DNA alkylation, the
benzyl group needs to protrude from the minor groove,
and it is assumed that this causes large energy loss.
Therefore, the introduction of a benzyl group decreased
the alkylating activity and also the corresponding
cytotoxicity.
In contrast to the case of hybrids 25-27, hybrids 22-
24 show unique DNA sequence-selective alkylation in
AT-rich sequences. High-resolution denaturating gel
electrophoresis indicated that 24 selectively alkylates
the third adenine of the 5′-ACAAAAATCG-3′ motif
within a 400 bp DNA fragment (Figure 1). This com-
pound elicited the strongest and most highly sequence-
specific DNA alkylation activity. For compound 24, DNA
alkylation was observed even at 50 nM. Interestingly,
DNA alkylation sites by 22-24 in AT-rich sequence
were slightly different, as shown in inset of Figure 1a.
The results clearly suggest the different orientation of
the binding of these agents, as shown in Figure 1b, in
which pyrrole moiety of 24 and 23 is oriented N f C
with respect to the 3′ f 5′ direction of the reacting DNA
strand, whereas compound 22 pyrrole moiety is oriented
N f C with respect to the 5′ f 3′ direction of the
reacting DNA strand. Differences in binding orientation
might derived from the balance of binding preference
of polypyrrole moiety and the alkylating site of 20. The
results also suggest the interesting possibility that only
the 4S enantiomer of 24 and 23 and 4R isomer of 22
were used for DNA alkylation. When the Texas Red-
labeled DNA fragment was incubated with increased
concentrations of the hybrids 22-24, the intensity of
the DNA alkylation also increased without altering the
selectivity. These results clearly indicate the importance
of the hybrid structure 24 constructed from 20 and the
lexitropsin 10 for the DNA binding.
On the basis of the results of the sequencing gel
experiment for compound 24, we constructed the bind-

2540 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Baraldi et al.
F igu r e 2. Energy-minimized structure of the 24-5′-
ACAAAAATCG-3′/5′-CGATTTTTGT-3′ complex. Minimization
was performed in the presence of 18 sodium cations and a 10
Å layer of H2O by CFF force field. For simplicity, hydrogen
atom, sodium ions, and H2O are not presented. DNA is drawn
in purple. Compound 24 is drawn in blue and red.
nunced cytotoxicity toward five different tumor cell
lines. Results from this investigation suggest a promis-
ing approach for developing a new generation of DNA-
alkylating agents based on CPI analogues and lexitrop-
sin hybrid system that can alkylate purine bases in a
sequence-selective fashion. Because of the high ef-
ficiency of alkylation, results from the present investi-
gation suggest that these molecules should be useful in
the design of compounds that target a single gene.
Further studies on the generality and the optimization
of this new class of DNA alkylation systems are cur-
rently in progress.
F igu r e 1. Thermally induced strand cleavage of 5′-Texas Red-
labeled pUC 18F 780-1229 DNA fragment incubated with
hybrids 22-24 (a) and the schematic prepresentation of
binding mode of hybrids 22-24 (b). Lane 1: DNA control.
Lanes 2-6: 1000, 500, 250, 100, and 50 nM 22, respectively.
Lanes 7-11: 1000, 500, 250, 100, and 50 nM 23, respectively.
Lanes 12-16: 1000, 500, 250, 100, and 50 nM 24, respectively.
The pyrrole rings and CPI are represented by circles and
triangles, respectively.
Exp er im en ta l Section
Ch em istr y. Ma ter ia ls a n d Meth od s. 1H NMR spectra
were recorded on a Bruker AC 200 spectrometer. Chemical
shifts (δ) are given in parts per million upfield from tetra-
methylsilane as internal standard, and the spectra were
recorded in appropriate deuterated solvents indicated in the
procedure. Melting points (mp) were determined on a Buchi-
Tottoli apparatus and are uncorrected. All products reported
showed 1H NMR spectra in agreement with the assigned
structures. Elemental analyses were conducted by the Mi-
croanalytical Laboratory of the Chemistry Department of the
University of Ferrara. All reactions were carried out under
an inert atmosphere of dry nitrogen, unless otherwise de-
scribed. Standard syringe techniques were applied for trans-
ferring dry solvents. Reaction courses and product mixtures
were routinely monitored by TLC on silica gel (precoated F254
Merk plates) and visualized with aqueous KMnO4. Flash
chromatography was performed using 230-400 mesh silica gel
and the solvent system indicated in the procedure. All com-
mercially available compounds were used without further
purification. Organic solutions were dried over anhydrous
MgSO4. Dioxane was distilled from calcium hydride, and dry
DMF was distilled from calcium chloride and stored over
molecular sieves (3 Å). In high-pressure hydrogenation experi-
ments, a Parr shaker on a high-pressure autoclave was used.
HPLC separations were conducted with a Waters Delta Prep
3000 A reversed-phase column (30 × 3 cm; 15 mm). The
ingmodelof24 and 5′-ACAAAAATCG-3′/5′-CGATTTTTGT-
3′. The initial structure of a 1:1complex was constructed
by manual docking of the 4S isomer of 24 in the
observed binding orientation to the minor groove of
duplex decanucleotides in ideal B-form DNA. This was
generated using the builder module of the Discover/
InsightII program (MSI). The assembled structure in
the presence of 18 sodium cations and 10 Å layer of
water was minimized with a CFF force field (MSI) with
convergence criteria of less than 0.001 kcal/Å. The
energy minimized binding model is shown in Figure 2.
Inspection of the minimized structure suggested that
only 4R stereoisomer of 24 can akylate 5′ side of adenine
of AT-rich sequence. Further study to evaluate the
contribution of the individual enantiomers is in progress.
Con clu sion s
The present work demonstrated not only the molec-
ular design and the chemical synthesis of novel CPzI-
lexitropsin hybrids, but also their DNA-binding profiles
and cytotoxic activities. Many of the compounds pre-
pared in this study were shown to demonstrate pro-

Antitumor Agent CC-1065 and Polypyrrole Minor Groove Binders J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2541
compounds were eluted with a gradient of 0-60% B in 25 min
at a flow rate of 30 mL/min, and the mobile phases were
solvent A (10%, v/v, acetonitrile in 0.1% TFA) and solvent B
(60%, v/v, acetonitrile in 0.1% TFA). The pure products were
converted into the corresponding hydrochloride forms by the
addition of 0.1 N HCl aqueous solution to the mobile phase
containing the pure product. Analytical HPLC analyses were
performed on a Bruker liquid chromatography LC 21-C instru-
ment using a Vydac 218 TP 5415 C18 column (250 × 4 mm, 5
mm particle size) and equipped with a Bruker LC 313 UV
variable-wavelength detector. Recording and quantification
were accomplished with a chromatographic data processor
coupled to an Epson computer system (QX-10).
1-Methyl-3-benzyloxycar bon yl-5-m eth oxycar bonylP yr a-
zole (12). A stirred solution of acid-ester 11 (860 mg, 4.67
mmol), DBU (0.8 mL, 5.14 mmol), and benzyl bromide (1.4 mL,
10.3 mmol) in dry DMF (5 mL) was stirred at room temper-
ature for 18 h, and the reaction mixture was concentrated in
vacuo. The residue was dissolved in EtOAc (10 mL), washed
with 5% HCl (3 mL), and a saturated solution of NaHCO3 (3
mL). The organic phase was separated and dried, and after
concentration in vacuo, the crude product was purified by flash
chromatography (EtOAc/petroleum ether 1:1), yielding com-
pound 12 as a white solid: yield 1.14 g (89%); mp 60-63 °C;
1H NMR (CDCl3) δ 3.89 (s, 3H), 4.26 (s, 3 H), 5.38 (s, 2H),
7.37 (m, 5 H); IR (KBr) cm-1 3441, 1705, 1557, 1443, 1264,
1231, 1187, 1123, 757. Anal. (C14H14N2O4) C, H, N.
3-[1-Meth yl-4-[[1-m eth yl-4-[1-m eth yl-4-(1-m eth yl-3-ben -
zyloxyca r b on ylp yr a zole-5-ca r b oxa m id o)p yr r ole-2-ca r -
boxam ido]pyr r ole-2-car boxam ido]pyr r ole-2-car boxam ido]-
p r op ion a m id in e Hyd r och lor id e (19). Yield 46%; brown
solid; mp 195-200 °C; 1H NMR (DMSO-d6) δ 2.72 (m, 2H),
3.47 (m, 2H), 3.76-3.88 (m, 9H), 4.19 (s, 3H), 5.34 (s, 2H), 6.96
(s, 1H), 7.11 (s, 1H), 7.21 (s, 1H), 7.22 (s, 1H), 7.26 (s, 1H),
7.31 (s, 1H), 7.45 (m, 5H), 7.61 (s, 1H), 8.27 (t, 1H, J ) 7.4
Hz), 8.70 (bs, 2H), 9.02 (bs, 2H), 9.96 (s, 1H),10.0 (s, 1H), 10.58
(s, 1H). Anal. (C34H38ClN11O6) C, H, Cl, N.
Gen er a l P r oced u r e for th e Deben zyla tion of Lex-
itr op sin s (17-19). A solution of benzyluretane 17-19 (1
mmol) in 10 mL of a MeOH containing 0.5 mL of 5% HCl was
hydrogenated over 50 mg of 10% Pd/C at 50 p.s.i for 4 h. The
catalyst was removed by filtration, and the filtrate was
concentrated to give a foam oil which was used without
purification for the next step.
3-[1-Meth yl-4-(1-m eth yl-3-ca r boxylp yr a zole-5-ca r boxa -
m id o)-p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r o-
ch lor id e (8). Yield 78%; green solid; mp 205-207 °C; 1H NMR
(DMSO-d6) δ 2.68 (t, 2H, J ) 5.8 Hz), 3.52 (m, 2H), 3.82 (s,
3H), 4.23 (s, 3H), 6.98 (s, 1H), 7.18 (s, 1H), 7.56 (s, 1H), 8.28
(t, 1H, J ) 7.2 Hz), 8.62 (bs, 2H), 9.00 (bs, 2H), 10.58 (s, 1H).
3-[1-Meth yl-4-[1-m eth yl-4-(1-m eth yl-3-car boxylpyr azole-
5-ca r boxa m id o) p yr r ole-2-ca r b oxa m id o]p yr r ole-2-ca r -
boxa m id o]p r op ion a m id in e Hyd r och lor id e (9). Yield 72%;
green-yellow solid; mp 187-190 °C; 1H NMR (DMSO-d6) δ 2.72
(m, 2H), 3.36 (m, 2H), 3.81 (s, 3H), 3.86 (m, 3H), 4.12 (s, 3H),
6.95 (s, 1H), 7.10 (s, 1H), 7.21 (s, 1H), 7.33 (s, 1H), 7.54 (s,
1H), 8.28 (t, 1H, J ) 7.2 Hz), 8.78 (bs, 2H), 9.08 (bs, 2H), 10.0
(s, 1H), 10.60 (s, 1H).
3-Ben zyloxyca r b on yl-1-m et h ylp yr a zole-5-ca r b oxylic
Acid (13). To a solution of ester 12 (548 mg, 2 mmol) in
dioxane (10 mL) was added 2 N KOH (1 mL), and the mixture
was stirred at r.t. for 24 h. At the end of this time, the solvent
was evaporated, the crude residue was dissolved in water (8
mL), and the resulting solution was extracted with EtOAc (5
mL). The water phase was acidified to pH 1 with concentrated
HCl and then extracted with EtOAc (2 × 10 mL). The
recombined organic phases were dried on Na2SO4 and evapo-
rated, and the residue was purified by flash chromatography
(EtOAc), yielding compound 13 as a white solid: yield 458 mg
3-[1-Meth yl-4-[[1-m eth yl-4-[1-m eth yl-4-(1-m eth yl-3-ca r -
b oxyp yr a zole-5-ca r b oxa m id o)p yr r ole-2-ca r b oxa m id o]-
p yr r ole-2-ca r b oxa m id o]p yr r ole-2-ca r b oxa m id o]p r op i-
on a m id in e Hyd r och lor id e (10). Yield 68%; brown solid; mp
257-260 °C; 1H NMR (DMSO-d6) δ 2.74 (m, 2H), 3.52 (m, 2H),
3.72 (s, 3H), 3.76 (s, 3H), 3.87 (s, 3H), 4.23 (s, 3H), 6.98 (s,
1H), 7.11 (s, 1H), 7.21 (s, 1H), 7.24 (s, 1H), 7.29 (s, 1H), 7.34
(s, 1H), 7.61 (s, 1H), 8.29 (t, 1H, J ) 7.4 Hz), 8.70 (bs, 2H),
9.08 (bs, 2H), 9.96 (s, 1H),10.06 (s, 1H), 11.04 (s, 1H).
1
(88%); mp 128-130 °C; H NMR (DMSO-d6) δ 4.13 (s, 3 H),
5.31 (s, 2H), 7.40 (m, 5 H), 13.6 (bs, 1H); IR (KBr) cm-1 3392,
1738, 1707, 1455, 1284, 1204, 1009, 710. Anal. (C13H12N2O4)
C, H, N.
Gen er a l P r oced u r e for Cou p lin g Rea ction betw een
Ca r boxylic Acid Lexitr op sin s (8-10) a n d Alk yla tin g
Un its 20 a n d 21. A solution of (20-21) (0.1 mmol) in 3M HCl-
EtOAc (5 mL) at 0 °C was stirred for 30 min before the solvent
was removed under reduced pressure. The resulting unstable
crude amine hydrochloride was dissolved in dry DMF (3 mL)
and treated sequentially with the appropriate lexitropsin acid
(8-10) (0.1 mmol) and EDCI (0.5 mmol, 86 mg). After 18 h of
stirring, the solution was evaporated, and the crude residue
was purified by semipreparative HPLC to give compounds 22-
27.
Gen er a l P r oced u r e for th e Cou p lin g of 3-Ben zyloxy-
ca r bon yl-1-m eth ylp yr a zole-5-ca r boxylic Acid (13) w ith
P yr r ole-P yr a zole Oligom er s (14-16). To a stirred 0.4 M
solution of the pyrrole oligomer 14-16 in anhydrous DMF
under argon atmosphere was added the Hunig’s base (1 equiv)
at 0 °C. After 5 min, acid 13 (1.1 equiv) was added, followed
by EDCI (2 equiv) and then HOBT (1.1 equiv). The resulting
mixture was stirred for 18 h as it warmed to room tempera-
ture, acidified with 10% HCl to pH 3, and then evaporated to
dryness in a vacuum. The residue was dissolved into a small
volume of MeOH, and then ethyl ether was added to precipi-
tate the crude product as a brown solid: this procedure was
repeated 5 times. The solid residue was purified by column
chromatography (CH2Cl2/CH3OH 1.5:8.5 and then 2:8) and
recrystallized (CH3OH/diethyl ether) to give 17-19.
3-[1-Meth yl-4-[1-m eth yl-3-[6-(3-m eth yl-4-ch lor om eth yl-
8-h yd r oxy-4,5-d ih yd r o-6H -p yr r ole-[3,2-e]-1-H -in d a zole]
ca r boxylp yr a zole-5-ca r boxa m id o]-p yr r ole-2-ca r boxa m i-
d o]p r op ion a m id in e Hyd r och lor id e (22). Yield 78%; green
solid; mp 192-195 °C; 1H NMR (DMSO-d6) δ 2.59 (s, 3H), 2.64
(t, 2H, J ) 6.4 Hz), 3.56 (m, 2H), 3.83 (s, 3H), 3.89-3.97 (m,
3H), 4.20 (s, 3H), 4.48-4.76 (m, 2H), 6.99 (s, 1H), 7.27 (s, 1H),
7.54 (s, 1H), 7.87 (s, 1H), 8.32 (t, 1H, J ) 7.2 Hz), 8.56 (bs,
2H), 8.96 (bs, 2H), 10.29 (s, 1H), 10.57 (s, 1H), 12.21 (bs, 1H).
Anal. (C26H30Cl2N10O4) C, H, Cl, N.
3-[1-Meth yl-4-(1-m eth yl-3-ben zyloxyca r bon ylp yr a zole-
5-ca r b oxa m id o)-p yr r ole -2-ca r b oxa m id o]p r op ion a m i-
d in e Hyd r och lor id e (17). Yield 52%; brown solid; mp 112-
1
114 °C; H NMR (DMSO-d6) δ 2.62 (t, 2H, J ) 5.8 Hz), 3.46
(m, 2H), 3.81 (s, 3H), 3.82 (m, 3H), 4.18 (s, 3H), 5.33 (s, 2H),
6.98 (s, 1H), 7.26 (s, 1H), 7.42 (m, 5H), 7.60 (s, 1H), 8.34 (t,
1H, J ) 7.4 Hz), 8.68 (bs, 2H), 9.03 (bs, 2H), 10.55 (s, 1H).
Anal. (C22H26ClN7O4) C, H, Cl, N.
3-[1-Met h yl-4-[1-m et h yl-4-(1-m et h yl-3-[6-(3-m et h yl-4-
ch lor om eth yl-8-h yd r oxy-4,5-d ih yd r o-6H-p yr r ole-[3,2-e]-
1-H -in d a zole ]c a r b oxylp yr a zole -5-c a r b oxa m id o)p yr -
r ole -2-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]p r op ion -
a m id in e Hyd r och lor id e (23). Yield 72%; green-yellow solid;
mp 185-187 °C; 1H NMR (DMSO-d6) δ 2.58 (s, 3H), 2.62 (t, J
) 6.2 Hz, 2H), 3.28 (m, 2H), 3.82 (s, 3H), 3.89 (m, 3H), 3.92-
4.16 (m, 3H),4.23 (s, 3H), 4.42-4.64 (m, 2H), 6.98 (s, 1H), 7.12
(s, 1H), 7.22 (s, 1H), 7.38 (s, 1H), 7.60 (s, 1H), 7.92 (s, 1H),
8.18 (s, 1H), 8.22 (t, 1H, J ) 7.4 Hz), 8.58 (bs, 2H), 8.98 (bs,
3-[1-Meth yl-4-[1-m eth yl-4-(1-m eth yl-3-ben zyloxyca r bo-
n ylp yr a zole-5-ca r boxa m id o)p yr r ole-2-ca r boxa m id o]p y-
r r ole-2-car boxam ido]pr opion am idin e Hydr och lor ide (18).
Yield 52%; brown solid; mp 138-140 °C; 1H NMR (DMSO-d6)
δ 2.71 (m, 2H), 3.48 (m, 2H), 3.81 (s, 3H), 3.86 (m, 3H), 4.18
(s, 3H), 5.34 (s, 2H), 6.95 (s, 1H), 7.09 (s, 1H), 7.21 (s, 1H),
7.31 (s, 1H), 7.45 (m, 5H), 7.61 (s, 1H), 8.27 (t, 1H, J ) 7.4
Hz), 8.71 (bs, 2H), 9.03 (bs, 2H), 10.0 (s, 1H), 10.58 (s, 1H).
Anal. (C28H32ClN9O5) C, H, Cl, N.
2H), 10.00 (s, 1H), 10.58 (s, 1H), 10.59 (s, 1H). Anal. (C32H36
Cl2N12O5) C, H, Cl, N.
-

2542 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Baraldi et al.
3-[1-Meth yl-4-[[1-m eth yl-4-[1-m eth yl-4-(1-m eth yl-3-[6-
(3-m eth yl-4-ch lor om eth yl-8-h yd r oxy-4,5-d ih yd r o-6H-p yr -
r ole-[3,2-e]-1-H-in d a zole] ca r boxyp yr a zole-5-ca r boxa m -
id o)p yr r ole -2-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e
(24). Yield 64%; yellow solid; mp 275-278 °C; 1H NMR
(DMSO-d6) δ 2.54 (s, 3H), 2.59 (m, 2H), 3.64 (m, 2H), 3.69 (m,
1H), 3.81 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 3.94-4.6 (m, 2H),
4.22 (s, 3H), 4.55-4.72 (m, 2H), 6.97 (s, 1H), 7.09 (s, 1H), 7.14
(s, 1H), 7.18 (s, 1H), 7.25 (s, 1H), 7.34 (s, 1H), 7.55 (s, 1H),
7.87 (s, 1H), 8.22 (t, 1H, J ) 7.2 Hz), 8.50 (bs, 2H), 8.92 (bs,
2H), 9.94 (s, 1H), 10.03 (s, 1H), 10.29 (s, 1H), 10.59 (s, 1H),
12.75 (s, 1H). Anal. (C38H42Cl2N14O6) C, H, Cl, N.
Cytosta tic Assa ys. Murine leukemia L1210, murine mam-
mary carcinoma FM3A, human T-lymphocyte CEM and Molt
4/ clone 8, and human B-lymphocyte Daudi cells were seeded
in 96-well microtiter plates at 250 000-300 000 cells/mL and
were treated with different concentrations of the experimental
drugs. Cell cultures were incubated for 2-3 days at 37 °C. At
the indicated time, the cell number was determined using a
Coulter counter. The 50% cytostatic concentration (IC50) was
defined as the compound concentration required to reduce the
cell number by 50%.
P r ep a r a tion of 5′-Texa s Red -En d -Mod ified 450-Ba se
P a ir DNA F r a gm en ts. The 5′-Texas Red-end-modified 450-
base pair DNA fragments pUC18 F780*-1229 and pUC18
R1459-1908 (they are complementary) were prepared by the
PCR method using 5′-end Texas Red modified 18mers 5′-
AGAATCAGGGGATAACGCAG-3′ (pUC 18 forward 780-799)
as a primer28 and purified by filtration using Suprec-02. Their
concentration was determined by ethidium bromide staining.
The asterisk indicates Texas Red modification and the nucle-
otide numbering starts with the replication site.
High -Resolu tion Gel Eletr op h or esis. The 5′-Texas Red
labeled DNA fragment (75 nM) was alkylated by various
concentration of the agents in 10 mL of 10 mM Na phosphate
buffer (pH 7.0) containing 10% DMF at 23 °C for 16 h. The
reaction was quenched by adding 400 ng of calf thymus DNA
and heating for 5 min at 90 °C. The solvent was evaporated
in vacuo. The resulting residue was dissolved in 8 mL of
loading dye (formamide with fushin red), heated at 94 °C for
20 min, and then immediately cooled to 0 °C. A 2 mL of aliquot
was electrophoresed on 6% denaturing polyacrylamide gel
using a Hitachi 5500-S DNA sequencer.
Molecu la r Mod elin g Stu d ies. Minimizations were per-
formed with the Discover (MSI, San Diego, CA) program by
using CFF force-field parameters. The starting structure of
24-d(ACAAAAATCG)/d(CGATTTTTGT) complex was built us-
ing standard bond lengths and angles. The Du moiety of the
assembled initial structure was energy minimized using a
distance-dependent dielectric constant of e ) 4r (r stands for
the distance between atoms i and j) and the convergence
criteria with the rms gradient of less than 0.001 kcal/mol Å.
Twenty Na cations were placed at bifucating positions of
O-P-O angle at a distance of 2.51 Å form the phosphorus
atom. The resulting complex was soaked in 10 Å layer of water.
The whole system was minimized without any constraint to
the stage where the rms was less than 0.001 kcal/mol Å.
3-[1-Meth yl-4-(1-m eth yl-3-[6-(1-ben zyl-4-ch lor om eth yl-
8-h yd r oxy-4,5-d ih yd r o-6H -p yr r ole-[3,2-e]-1-H -in d a zole]
ca r boxylp yr a zole-5-ca r boxa m id o)-p yr r ole-2-ca r boxa m i-
d o]p r op ion a m id in e Hyd r och lor id e (25). Yield 62%; white
solid; mp 247-250 °C; 1H NMR (DMSO-d6) δ 2.51 (t, 2H, J )
6 Hz), 3.50 (m, 2H), 3.84 (s, 3H), 4.02-4.16 (m, 3H), 4.20 (s,
3H), 4.42-4.74 (m, 2H), 5.71 (s, 2H), 7.00 (s, 1H), 7.15-7.29
(m, 7H), 7.54 (s, 1H), 7.92 (s, 1H), 8.36 (t, 1H, J ) 7.2 Hz),
8.52 (bs, 2H), 8.94 (bs, 2H), 10.54 (s, 1H), 10.56 (s, 1H). Anal.
(C32H34Cl2N10O4) C, H, Cl, N.
3-[1-Met h yl-4-[1-m et h yl-4-(1-m et h yl-3-[6-(1-b en zyl-4-
ch lor om eth yl-8-h yd r oxy-4,5-d ih yd r o-6H-p yr r ole-[3,2-e]-
1-H -in d a zole ]c a r b oxylp yr a zole -5-c a r b oxa m id o)p yr -
r o l e -2 -c a r b o x a m i d o ]p y r r o l e -2 -c a r b o x a m i d o ]p r o -
p ion a m id in e Hyd r och lor id e (26). Yield 72%; green solid;
mp 262-265 °C; 1H NMR (DMSO-d6) δ 2.60 (t, J ) 6.4 Hz,
2H), 3.36 (m, 2H), 3.81 (s, 3H), 3.87 (m, 3H), 3.94-4.15 (m,
3H),4.22 (s, 3H), 4.51-4.76 (m, 2H), 5.79 (s, 2H), 6.96 (s, 1H),
7.10-7.33 (m, 8H), 7.55 (s, 1H), 7.96 (s, 1H), 8.18 (s, 1H), 8.21
(t, 1H, J ) 7.4 Hz), 8.52 (bs, 2H), 8.94 (bs, 2H), 10.00 (s, 1H),
10.55 (s, 1H), 10.59 (s, 1H). Anal. (C38H40Cl2N12O5) C, H, Cl,
N.
3-[1-Meth yl-4-[[1-m eth yl-4-[1-m eth yl-4-(1-m eth yl-3-[6-
(1-ben zyl-4-ch lor om eth yl-8-h yd r oxy-4,5-d ih yd r o-6H-p yr -
r ole-[3,2-e]-1-H-in d a zole] ca r boxyp yr a zole-5-ca r boxa m -
id o)p yr r ole -2-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e
(27). Yield 72%; yellow solid; mp 234-236 °C; 1H NMR
(DMSO-d6) δ 2.59 (m, 2H), 3.49 (m, 2H), 3.81 (s, 3H), 3.85 (s,
3H), 3.88 (s, 3H), 3.92-4.14 (m, 3H), 4.21 (s, 3H), 4.42-4.82
(m, 2H), 5.70 (s, 2H), 6.86 (s, 1H), 6.96 (s, 1H), 7.08 (s, 1H),
7.13-7.34 (s, 8H), 7.57 (s, 1H), 8.01 (s, 1H), 8.18 (s, 1H),8.20
(t, 1H, J ) 7.2 Hz), 8.64 (bs, 2H), 8.99 (bs, 2H), 9.96 (s, 1H),
10.05 (s, 1H), 10.61 (s, 2H). Anal. (C44H46Cl2N14O6) C, H, Cl,
N.
Ack n ow led gm en t. We wish to thank Pharmacia &
Upjohn for providing Distamycin A and Progetto Gio-
vani Ricercatori (1999) for financial support of this work.
Funding for this study was provided by the Fonds voor
Wetenschappelijk Onderzoek (FWO-Vlaanderen Krediet
G. 0/04.98) and the Geconcerteerde Onderzoeksacties
(Contract 2000/12) to J . Balzarini and E. De Clercq. We
thank Lizette van Berckelaer, Anita Van Lierde, and
Frieda De Meyer for excellent technical assistance.
Biologica l Assa ys. Cells. Human embryonic lung (HEL)
fibroblasts and E6SM cells were grown in minimum essential
medium (MEM) supplemented with 10% inactivated fetal calf
serum (FCS), 2 mM L-glutamine and 0.3% NaHCO3.
Vir u ses. The laboratory wild-type VZV strains Oka and YS,
the thymidine kinase-deficient VZV strains 07-1 and YS-R,
HSV-1 (KOS), HSV-2 (G), the thymidine kinase-deficient
HSV-1 strains B-2006 and VMW 1837, the CMV strains Davis
and AD-169, and vaccinia virus were used in the virus
inhibition assays.
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