Structure-based design of a new bisintercalating anthracycline antibiotic

Article abstract of DOI:10.1021/jm9607414

A new bisintercalating anthracycline antibiotic, WP631, has been designed and synthesized. The rational design of the new compound was based upon the geometry of monomeric anthracyclines bound to DNA oligonucleotides observed in high-resolution crystal structures. Monomeric units of daunorubicin have been linked through their reactive 3' NH₂ substituents on the daunosamine moieties to form the new bisanthracycline WP631. Viscosity studies confirmed that WP631 binds to DNA by bisintercalation. Differential scanning calorimetry and UV melting experiments were used to measure the ultratight binding of WP631 to DNA. The binding constant for the interaction of WP631 with herring sperm DNA was determined to be 2.7 x 10¹¹ M^-1 at 20 °C. The large, favorable binding free energy of -15.3 kcal mol^-1 was found to result from a large, negative enthalpic contribution of -30.2 kcal mol^{- 1}. A molecular model was generated that shows the favorable stereochemical fit of the linker in the DNA minor groove. The cytotoxicity of WP631 was compared to that of doxorubicin using MCF-7-sensitive and MCF-7/VP-16 MRP- mediated multidrug-resistant cell lines. These initial studies showed that while WP631 is slightly less cytotoxic than doxorubicin in the sensitive cell line, it appears to overcome MRP-mediated multidrug resistance and was much more cytotoxic against the MCF-7/VP-16 cell line than was doxorubicin. The design of new potential anticancer agents based on known structural principles was found to produce a compound with significantly increased DNA binding affinity and with interesting biological activity.

Full text of DOI:10.1021/jm9607414

© Copyright 1997 by the American Chemical Society
Volume 40, Number 3
J anuary 31, 1997
Expedited Articles
Str u ctu r e-Ba sed Design of a New Bisin ter ca la tin g An th r a cyclin e An tibiotic
J onathan B. Chaires,*,† Fenfei Leng,† Teresa Przewloka,‡ Izabela Fokt,‡ Yi-He Ling,‡ Roman Perez-Soler,‡ and
Waldemar Priebe‡
Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street,
J ackson, Mississippi 39216-4505, and M. D. Anderson Cancer Center, The University of Texas,
1515 Holcombe Boulevard, Houston, Texas 77030
Received October 24, 1996X
A new bisintercalating anthracycline antibiotic, WP631, has been designed and synthesized.
The rational design of the new compound was based upon the geometry of monomeric
anthracyclines bound to DNA oligonucleotides observed in high-resolution crystal structures.
Monomeric units of daunorubicin have been linked through their reactive 3′ NH2 substituents
on the daunosamine moieties to form the new bisanthracycline WP631. Viscosity studies
confirmed that WP631 binds to DNA by bisintercalation. Differential scanning calorimetry
and UV melting experiments were used to measure the ultratight binding of WP631 to DNA.
The binding constant for the interaction of WP631 with herring sperm DNA was determined
to be 2.7 × 1011 M-1 at 20 °C. The large, favorable binding free energy of -15.3 kcal mol-1
was found to result from a large, negative enthalpic contribution of -30.2 kcal mol-1. A
molecular model was generated that shows the favorable stereochemical fit of the linker in
the DNA minor groove. The cytotoxicity of WP631 was compared to that of doxorubicin using
MCF-7-sensitive and MCF-7/VP-16 MRP-mediated multidrug-resistant cell lines. These initial
studies showed that while WP631 is slightly less cytotoxic than doxorubicin in the sensitive
cell line, it appears to overcome MRP-mediated multidrug resistance and was much more
cytotoxic against the MCF-7/VP-16 cell line than was doxorubicin. The design of new potential
anticancer agents based on known structural principles was found to produce a compound
with significantly increased DNA binding affinity and with interesting biological activity.
In tr od u ction
might be attributed, in part, to flaws in the design
process. Early efforts in anthracycline synthesis were
not guided by any known structural or mechanistic
principles. In the last decade, however, the anthra-
cylines have become perhaps the best understood of the
DNA intercalators.2-4 Over 22 high-resolution struc-
tures have now been reported for a variety of anthra-
cyclines intercalated into DNA oligonucleotides of vari-
ous sequences (see Table 1 in ref 2). The DNA sequence
preference of daunorubicin binding to DNA has been
characterized by high-resolution footprinting,5 and the
origin of its preferential binding to certain triplet
sequences has been explored by computational chem-
istry.6,7 The thermodynamics and kinetics of daunoru-
The anthracycline antibiotics daunorubicin (dauno-
mycin) and doxorubicin (Adriamycin) are among the
most potent and clinically useful agents currently used
in cancer chemotherapy. Although there have been
intense efforts to synthesize more efficacious anthracy-
clines with fewer toxic side effects, these efforts have
not succeeded,1 and the parent compounds remain the
best chemotherapeutic agents of the chemical class. The
failure to obtain new, improved anthracycline antibiotics
* To whom correspondence should be addressed.
† University of Mississippi Medical Center.
‡ The University of Texas.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00741-8 CCC: $14.00 © 1997 American Chemical Society

262 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Chaires et al.
F igu r e 1. A schematic of the structure of monomeric anthra-
cycline antibiotic molecules bound to a DNA hexanucleotide
as revealed by high-resolution crystallographic structures. The
daunosamine moieties of the drug monomers point toward the
center of the hexanucleotide, placing the reactive 3′ NH2
substituents within <7 Å of one another.
bicin binding to DNA have been thoroughly studied.8-10
This wealth of detailed information provides a founda-
tion for the rational design of new anthracyclines
targeted toward DNA. The design, synthesis, and initial
characterization of one such compound is described here.
F igu r e 2. Structure of WP631, a newly synthesized bisan-
thracycline in which two daunorubicin molecules are linked
through their 3′ NH2 substituents by a p-xylene linker.
site size of WP631 could impart a binding specificity on
par with such enzymes.
The logic of the design of WP631, a bisanthracycline
composed of two daunorubicin monomers, is based on
the high-resolution crystallographic structures of an-
thracyclines bound to DNA hexanucleotides. In all of
the structures reported for noncovalent complexes of
anthracyclines bound to DNA, a 2:1 stoichiometry of
drug to DNA duplex was observed. In all such com-
plexes, each drug was observed to be intercalated at
either end of the hexanucleotide, with the daunosamine
moieties of the two drug molecules pointing toward one
another, lining the minor groove. Figure 1 shows a
schematic of the observed geometry. This arrangement
brings the reactive NH2 substituents of each drug
molecule to within <7 Å of one another. The simple
and obvious design strategy to emerge from these
considerations is to link the two amine groups to form
a bisanthracycline with the potential of bisintercalating
into DNA. The linker must be designed to be of the
appropriate length (approximately 7 Å) and must fit into
the minor groove of DNA without steric hindrance. A
compound designed and synthesized according to these
specifications, WP631, is shown in Figure 2.
Bisanthracyclines have been synthesized before.14-16
In these cases, monomers were linked through either
the C13 or C14 positions, a strategy we believe to have
led to a design flaw. The C13 and C14 positions are
labeled in one of the monomeric units in Figure 2. High-
resolution structures show that substituents near these
positions may participate in specific molecular interac-
tions within the drug-DNA complex. Attachment of a
bulky linker at those positions might sterically hinder
the binding of the monomeric units. Linkage through
the NH2 groups is likely to be less invasive and should
be stereochemically preferred. Indeed, the linker might
provide additional favorable interactions within the
minor groove to further enhance DNA binding and
specificity. Daunorubicin alone is a polyfunctional
molecule, with both an intercalating domain and a
groove binding domain.2 An appropriately designed
linker might serve to expand and enhance the groove
binding domain, in contrast to linkers attached at C13
or C14, which would likely not fit properly into the
minor groove.
Reported here is the synthesis and initial character-
ization of the DNA binding properties and biological
activity of the novel bisanthracycline WP631. WP631
binds to DNA by bisintercalation, as judged by viscosity
studies and molecular models, and has greatly enhanced
DNA binding affinity. The biological activity of WP631
shows outstanding promise. WP631 appears to over-
come MRP-mediated multidrug resistance in cultured
cells, while retaining activity near that observed for
doxorubicin in sensitive cells.
The potential advantages of a bisintercalator over its
monomeric counterpart are many.11 First, DNA binding
affinity should be greatly enhanced. The binding con-
stant for the bisintercalator should be roughly the
square of the binding constant for the monomer. The
biological activity of the anthracyclines has been cor-
related with their DNA binding affinity,12 so the poten-
tial of WP631 as an improved chemotherapeutic agent
is great. Second, the site size of a bisintercalator is
increased relative to the monomer. Increased site size
can potentially lead to increased sequence selectivity.13
Daunorubicin binds preferentially to triplets of the type
5′(A/T)CG and 5′(A/T)GC, where the notation (A/T)
means that either A or T can occupy the position.5 The
bisanthracycline WP631 potentially will occupy 6 bp
instead of 3, with a concomitant increase in potential
sequence selectivity. Six base pairs define the specific
cleavage sites of many restriction enzymes, so the larger
Resu lts a n d Discu ssion
WP631 (Figure 2) is a rationally designed bisanthra-
cycline that is intended to bisintercalate into DNA, with
the concomitant advantages of enhanced DNA binding
affinity and sequence selectivity over its monomeric
counterpart, daunorubicin. These advantages will po-
tentially make WP631 an improved anticancer agent,

New Bisintercalating Anthracycline Antibiotic
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 263
steric hindrance in the minor groove and, most impor-
tantly, to be of the appropriate length to connect to the
two amine without any radical change in the structure
of the intercalated monomeric units. The exact orienta-
tion of the aromatic ring of the linker was found to be
flexible and might be free to rotate within the minor
groove.
Ultr a tigh t Bin d in g of WP 631 to DNA. The bind-
ing affinity of a bisintercalator for DNA ought to
approximately equal the square of the binding constant
of the corresponding monomer. Since daunorubicin
binds to DNA with a binding constant of 107 M-1 under
the ionic conditions used here, a binding constant of 1014
M-1 is expected for WP631. Traditional spectrophoto-
metric methods for measuring affinity fail for such
ultratight binding, but optical melting studies or dif-
ferential scanning calorimetry provides reliable alterna-
tives for the accurate determination of binding
constants.21-23 UV melting studies were used to deter-
mine the binding constant for the interaction of WP631
with herring sperm DNA in BPE buffer (16 mM total
Na+). In the absence of WP631, the Tm of herring sperm
DNA was found to be 67.2 °C. In the presence of 10
µM WP631, a concentration sufficient to saturate the
DNA lattice, the Tm was elevated to 93.5 °C. McGhee22
has shown that the shift in Tm is a function of the ligand
binding constant and site size. Assuming no interaction
of ligand with single-stranded DNA, McGhee derived
the equation
F igu r e 3. Viscosity studies of WP631-DNA interaction. The
cubed root of the relative viscosity (η/η0) is shown as a function
of the ratio of bound drug per DNA base pair: solid circles,
ethidium bromide; open diamonds, daunorubicin; solid dia-
monds, WP631. Linear least squares fits to these data yielded
the following slopes: ethidium, 0.84 ( 0.02; daunorubicin, 0.89
( 0.04; WP631, 1.58 ( 0.08.
since the anthracycline antibiotics upon which it is
based have been successfully used in cancer chemo-
therapy for over three decades. The initial character-
ization of the DNA binding and biological activity of
WP631 will be described.
WP 631 Bisin ter ca la tes in to DNA. In the absence
of high-resolution structural data, hydrodynamic stud-
ies, especially viscosity, provide the most reliable means
of inferring the binding mode of agents that interact
with DNA.17 Figure 3 shows the results of viscosity
experiments that verify that WP631 binds to DNA by
bisintercalation. The theory of Cohen and Eisenberg18
predicts that, for monointercalation, plots of the cubed
root of the relative viscosity ((η/η0)1/3) versus the binding
ratio (bound drug/DNA bp) ought to have a slope of 1.0.
Figure 3 shows that the proven monointercalators
ethidium and daunorubicin, run as controls in the
experiment, both show viscosity changes in excellent
agreement with the theory. For bisintercalators, the
slope is expected to be twice that observed for monoin-
tercalators, an expectation that has been verified for a
variety of bisintercalating compounds.11 The slope
observed for WP631 in Figure 3 is nearly double that
observed for daunorubicin, consistent with a bisinter-
calative binding mode. Such a binding mode is stere-
ochemically reasonable, as was determined by construct-
ing a molecular model of WP631 bound to a DNA
hexanucleotide (figure not shown at the suggestion of a
reviewer). The model was constructed using Hyper-
Chem software (v. 4.5, Hypercube, Inc.) by using the
coordinates of the high-resolution structure reported by
Wang et al.19 for a 2:1 daunorubicin-DNA complex. The
sequence of the hexanucleotide in that study was d(G‚
CTAG‚C)2, where the two equivalent intercalation sites
are indicated. WP631 was built in the complex by
attaching the p-xylene linker to the daunorubicin amines
within the minor groove. The geometry of the drug was
then optimized using the MM+ force field in Hyper-
Chem while constraining the DNA conformation to that
observed in the crystal structure. In the resultant
complex, the p-xylene linker was found to fit without
0
(1/Tm - 1/Tm) ) (∆Hm/R) ln(1 + KL)1/n
where Tm0 is the melting temperature of the DNA alone,
Tm is the melting temperature in the presence of
saturating amounts of ligand, ∆Hm is the enthalpy of
DNA melting (per bp), R is the gas constant, K is the
ligand binding constant at Tm, L is the free ligand
concentration (approximated at the Tm by the total
ligand concentration), and n is the ligand site size. A
value of ∆Hm ) 7.0 ( 0.3 kcal mol-1 for herring sperm
DNA, determined by separate differential scanning
calorimetry experiments, was used. From the experi-
mentally determined increase in Tm observed for WP631,
a value of K ) 8.8 × 106 M-1 (at 93.5 °C) was computed,
assuming n ) 6 bp. Correction of this value to lower
temperatures requires knowledge of the binding en-
thalpy, which was determined by differential scanning
calorimetry.
DSC Deter m in a tion of th e Bin d in g En th a lp y.
Figure 4 shows the results of differential scanning
calorimetry experiments using herring sperm DNA in
the presence and absence of saturating amounts of
WP631. The area under the curves shown in Figure 4
provides a model-free estimate of the enthalpy of
melting of the DNA alone and the DNA-WP631 com-
plex. By Hess’s law, these data may be used to
determine the enthalpy of WP631 binding to DNA. The
equilibria to be considered, along with the experimen-
tally determined enthalpy values, are as follows:
duplex a 2(single strand)
∆H1 ) 7.0 ( 0.3 kcal mol-1
WP631-duplex a 2(single strand) + WP631
∆H2 ) 11.4 ( 1.0 kcal mol-1

264 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Chaires et al.
Ta ble 1. In Vitro Evaluation of the Cytotoxicity of WP631 and
Doxorubicin
ID50 (µM)
cell line
MCF-7
doxorubicin
WP631
0.9 ( 0.5
14.2 ( 0.8
16
4.8 ( 2.5
2.5 ( 3.4
0.5
MCF-7/VP-16
RIa
a
RI is the ratio ID50(MCF-7/VP-16)/ID50(MCF-7).
) -9.9 kcal mol-1, ∆H ) -10.8 kcal mol-1, and ∆S )
-3 cal mol-1 K-1 (Leng and Chaires, unpublished data).
Comparison shows that while the DNA binding of both
daunorubicin and WP631 is driven by the enthalpy
contribution, WP631 shows a comparatively larger
unfavorable entropic contribution. Two plausible con-
tributions to this behavior could be WP631 effects on
DNA structure and losses of conformational freedom in
WP631. The bisintercalation of WP631 could result in
a proportionally larger increase of the stiffness of the
DNA helix relative to daunorubicin, and thus a greater
unfavorable entropic cost for the loss of DNA confor-
mational freedom. In addition, preliminary molecular
modeling studies have indicated that there is consider-
able conformational freedom in WP631, with free rota-
tion about many of the bonds in the linker. An
unfavorable entropic contribution might come from the
restriction of this conformational freedom upon bisin-
tercalation, an effect in addition to the general entropic
cost for bimolecular complex formation resulting from
the loss of translational and rotational freedom of the
reacting partners.
F igu r e 4. Results of differential scanning calorimetry studies
of the melting of herring sperm DNA alone (A) or in the
presence of near saturating amounts of WP631 (B). Excess
heat capacity (cal mol-1 °C-1) is plotted as a function of
temperature.
Combining these two reactions, the binding reaction and
its enthalpy may be obtained:
WP631-duplex a duplex + WP631
∆H3 ) ∆H2 - ∆H1
The enthalpy ∆H3 needs to be corrected for the amount
of WP631 bound to the DNA, and the sign changed, to
get the binding enthalpy,
∆Hb ) -∆H3/(mol of WP631/mol of bp)
Cytotoxicity of WP 631. The biological activity of
WP631 was studied by its in vitro cytotoxicity against
MCF-7 and MCF-7/VP-16 cell lines. The former is a
sensitive human breast carcinoma cell line, while the
latter is its multidrug-resistant counterpart. The re-
sults are shown in Table 1. In comparison with doxo-
rubicin, WP631 is slightly less toxic toward the sensitive
MCF-7 cells, by a factor of 4-5. Against the multidrug-
resistant MCF-7/VP-16 cell line, however, WP631 is
considerably more cytotoxic than is doxorubicin, by a
factor of 6. The ratio of the cytotoxicities for the two
cell lines (RI ) ID50(MCF-7/VP-16)/ID50(MCF-7)) was
found to be 16 and 0.5 for doxorubicin and WP631,
respectively. The exciting implication of this finding is
that WP631 can overcome multidrug resistance in these
cell lines. These studies indicate promising biological
activity for WP631, and a potentially improved anti-
cancer activity as was intended in the design.
Com p a r ison w ith P r eviou sly Syn th esized Bisa n -
th r a cyclin es. WP631 appears to offer several advan-
tages over previously synthesized bisintercalating
anthracyclines.11,14-16 In these earlier attempts, mono-
mers were linked through the C13 and C14 positions.
The primary advantage of WP631 over these earlier
designs is that it expands the inherent groove binding
domain of daunorubicin, creating a molecule with
distinctive intercalating and groove binding function-
alities. Molecular modeling studies have shown that the
linkers attached to the C13 and C14 positions do not
fit into the DNA minor groove and consequently do not
themselves add any favorable interactions with the
DNA. In contrast, WP631 was designed and built to
include intercalating and groove binding moieties that
will both contribute to DNA binding affinity and speci-
From five determinations, a value of ∆Hb ) -30.2 (
2.6 kcal mol-1 was obtained for the association of WP631
with DNA.
Com p lete Th er m od yn a m ic P r ofile for th e Bin d -
in g of WP 631 to DNA. ∆Hb may be used, assuming
that it is constant with temperature, to calculate the
binding constant at 20 °C by application of the standard
van’t Hoff equation, yielding a value of 2.7 × 1011 M-1
.
In BPE buffer at 20 °C, the binding constant for the
interaction of daunorubicin with herring sperm DNA
is 1.6 × 107 M-1 (Leng and Chaires, unpublished data),
so the binding constant of WP631 indeed approaches
the value expected for a bisintercalator. The magnitude
of the WP631 binding constant approaches that ob-
served for the binding of many regulatory proteins for
their specific DNA binding sites. Knowledge of the
binding constant and the binding enthalpy allows us to
construct the complete thermodynamic profile for the
binding of WP631 to DNA. The free energy is obtained
from the standard relation ∆G0 ) -RT ln K, yielding a
value of -15.3 kcal mol-1 at 20 °C. The entropy may
be evaluated from the equation -T∆S ) ∆G - ∆H,
yielding ∆S ) -51 cal mol-1 K-1 at 20 °C. The
thermodynamic profile indicates that the large, favor-
able binding free energy of -15.3 kcal mol-1 is derived
from the large negative enthalpic contribution of -30.2
kcal mol-1
. Binding is opposed by an unfavorable
entropic contribution of T∆S ) -14.9 kcal mol-1 at 20
°C.
Com p a r ison w ith Da u n or u bicin Bin d in g. Under
comparable ionic conditions, the thermodynamic profile
for daunorubicin binding to herring sperm DNA is ∆G0

New Bisintercalating Anthracycline Antibiotic
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 265
(100 mg) and R,R-dibromo-p-xylene (68.7 mg, 0.26 mmol) were
added. The reaction mixture was stirred at room temperature
for 20-28 h. The progress of the reaction was monitored by
TLC (chloroform:methanol:NH4OH[aq], 86:13:1). After the
reaction was complete, the reaction mixture was diluted with
CH2Cl2 (100 mL) and poured into water (100 mL). The organic
layer was separated and washed with water until neutral pH,
dried over anhydrous Na2SO4, and evaporated under dimin-
ished pressure. The crude product was purified by column
chromatography (Silicagel 60, 230-400 mesh; Merck), eluted
with CHCl3, and then eluted with CHCl3:MeOH at v/v ratios
of 98:2 and 95:5. The final product was isolated as the free
amine (WP630) and precipitated from CH2Cl2:hexane to give
a red solid (178 mg, 0.153 mmol) in 61.5% yield. Anal.
(C62H64N2O20‚H2O) C, H; N: calcd, 2.38; found, 2.28. 1H-NMR
(CDCl3): δ 13.95 (s, 2H, OH), 8.01 (d, 2H, J 3,2 ) 7.7 Hz, H-3),
7.77 (app t, 2H, J 2,3 ) 7.7 Hz, H-2), 7.38 (d, 2H, J 1,2 ) 8.5 Hz,
H-1), 7.18 (s, 4H, p-xylene), 5.50 (d, 2H, J 1′,2′a ) 3.4 Hz, H-1′),
5.28 (bs, 2H, H-7), 4.66 (bs, 2H, 9-OH), 4.07 (s, 6H, OCH3),
4.05 (q, 2H, J 5′,6′ ) 6.25 Hz, H-5′), 3.77 (d, 2H, J ) 12.78 Hz,
CH2 from p-xylene), 3.63 (m, 4H, CH2 from p-xylene and H-4′),
ficity. The biological activity of previously synthesized
bisanthracyclines was judged to be disappointing,16 in
spite of increases in the DNA affinity and of the
lifetimes of the complexes. In contrast, WP631 shows
exciting biological activity, with an unexpected ability
to apparently overcome multidrug resistance in the
initial studies.
Im p lica tion s for Ra tion a l Dr u g Design . The
rational, structure-based design of new DNA binding
agents is an area of great current interest. Small
synthetic molecules that could bind to DNA with high
affinity and sequence specificity would be valuable tools
for molecular biology and of potential medical use as
chemotherapeutic agents against a variety of infections
and diseases. WP631 represents a promising new
compound resulting from a novel approach to the
structure-based design of DNA binding agents. Previ-
ous design efforts have focused largely on groove binding
agents, whereas WP631 represents an attempt to com-
bine the intercalation and groove binding motifs to yield
a molecule that exploits the advantages of each binding
mode. Groove binding agents, as exemplified by ne-
tropsin and distamycin, in general bind selectively to
runs of AT base pairs, in part because of the steric
hindrance resulting from the protrusion of the N2 amino
group of guanine into the minor groove. The “lexitrop-
sins” were perhaps the first structure-based DNA bind-
ing agents to be synthesized and were designed to
incorporate the reading of GC base pairs into the groove
binding motif.24-26 More recently, impressive progress
for the recognition of DNA by groove binding pyrrole-
imidazole polyamides has been achieved,27,28 an ap-
proach that exploits the side-by-side groove binding
structural motif first described by Pelton and Wem-
mer.29 Such agents bind to DNA with sequence-specific
3.22 (d, 2H, J 10e,10a ) 18.8 Hz, H-10e), 2.96 (d, 2H, J 10a,10e
)
18.8 Hz, H-10a), 2.98-2.93 (m, 2H, H-3′), 2.41 (s, 6H, H-14),
2.37 (d, 2H, J 8a,8e ) 14.7 Hz, H-8e), 2.09 (dd, 2H, J 8a,8e ) 14.9
Hz, J 7,8a ) 4.1 Hz, H-8a), 1.76 (td, 2H, J 2′a,3′ ) 13.0 Hz, J 1′,2′a
) 3.8 Hz, H-2′a), 1.68 (dd, 2H, J 2′e,3′ ) 4.7 Hz, J 2′a,2′e ) 13.2
Hz, H-2′e), 1.37 (d, 6H, J 5′,6′ ) 6.65 Hz, H-6′).
The free amine WP630 was suspended in methanol (2 mL).
Then 1 N dry HCl in MeOH was added (to pH 4), followed by
an excess of diethyl ether to precipitate the hydrochloride of
WP630. The red solid was washed with ether until neutral
pH and then dried to give analytically pure WP631 (170 mg,
0.138 mmol, 55.3% yield calculated from daunorubicin). Mp:
160-170 °C dec. [R]D29: 184.7° (c 0.05, CHCl3:CH3OH, 1:1).
Anal. (C62H65N2O20‚2HCl‚4H2O) C, H, Cl, N.
Viscosity Stu d ies. Relative viscosity studies were con-
ducted exactly as described previously.17 Data were cast into
a plot of (η/η0)1/3 versus ratio of bound drug per DNA base pair,
in accord with the theory of Cohen and Eisenberg.18 Ethidium
bromide and daunorubicin were run as controls for these
experiments.
binding constants of 108-1010 M-1 28
The strategy used
.
UV Meltin g Stu d ies. Ultraviolet DNA melting curves
were determined using a Cary 3E UV/visible spectrophotom-
eter (Varian, Inc., Palo Alto, CA), equipped with a thermo-
electric temperature controller. Sonicated herring sperm DNA
at a concentration near 20 µM bp in BPE buffer (2 mM NaH2-
PO4, 6 mM Na2HPO4, 1 mM Na2EDTA, pH 7.0) was used for
to construct WP631 is complementary to these designs
based on the groove binding mode. Intercalators in
general preferentially bind to CpG or GpC sites,30
a
property that might be exploited in the design of new
agents. Linking GC specific intercalators by tethers
that bind in the minor groove can expand the repertoire
of available design elements. WP631 shows the promise
of such an approach. While the sequence specificity of
WP631 is not yet known, its affinity for DNA surpasses
that of the polyamides28 and its biological activity shows
outstanding promise in the initial studies described
here.
melting studies. Samples were heated at a rate of 1 °C min-1
,
while the absorbance at 260 nm was continuously monitored.
Primary data were transferred to the graphics program Origin
(Microcal, Inc., Northampton, MA) for plotting and analysis.
Differ en tia l Sca n n in g Ca lor im etr y. Differential scan-
ning calorimetry (DSC) experiments utilized a Microcal MC2
instrument (Microcal, Inc., Northampton, MA) along with its
DA2 software (J uly 1986 version) for data acquisition and
analysis. Sonicated herring sperm DNA at a concentration of
1 mM bp in BPE buffer was used for all experiments. A scan
rate of 1 °C min-1 was used. Primary data were corrected by
substraction of a buffer-buffer baseline, normalized to the
concentration of DNA base pairs, and further baseline-cor-
rected using the Cp(0) software option. Baseline-corrected,
normalized data were transferred to Origin graphics software
for integration and plotting. Samples for DSC of DNA +
WP631 were prepared by weighing appropriate amounts of
solid WP631 and dissolving the solid directly into 2 mL of 1
mM DNA solution. Any undissolved drug was removed by low-
speed centrifugation. The exact amount of WP631 bound to
the DNA was determined spectrophotometrically.
Su m m a r y
The design, synthesis, and initial characterization of
WP631, a novel bisintercalating bisanthracycline, is
described. The structure of WP631 is based on the
proximity and orientation of daunorubicin molecules
bound to DNA hexanucleotides observed in high-resolu-
tion crystal structures. As intended in the design,
WP631 was found to bisintercalate into DNA with
ultratight binding affinity. WP631 appears to overcome
a clinically relevant MRP-mediated multidrug resis-
tance in a human breast carcinoma cell line, a promising
biological activity showing the potential of this ratio-
nally designed compound.
In Vitr o Cytotoxicity a ga in st MCF -7 a n d MCF -7/VP -
16 Cell Lin es. In vitro drug cytotoxicities against human
breast carcinoma wild-type MCF-7 and MRP-resistant MCF-
7/VP-16 cells were assessed by using the MTT reduction assay,
as previously reported.20 The MTT dye was obtained from
Sigma Chemical Co. (St. Louis, MO). Cells were plated in 96-
well microassay culture plates (104 cells/well) and grown
overnight at 37 °C in a 5% CO2 incubator. Drugs were then
Exp er im en ta l Section
Syn th esis of WP 631. Daunorubicin hydrochloride (282
mg, 0.5 mmol) was dissolved in a mixture of N,N-dimethyl-
formamide (DMF) and CH2Cl2 (1:1 v/v; 6 mL). Then, Na2CO3

266 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
Chaires et al.
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added to the wells to achieve a final drug concentration
ranging from 0.1 to 50 µg/mL. Four wells were used for each
concentration. Control wells were prepared by adding ap-
propriate volumes of calcium- and magnesium-free PBS (pH
7.4). Wells containing culture medium without cells were used
as blanks. The plates were incubated at 37 °C in a 5% CO2
incubator for 72 h. Upon completion of the incubation, 20 µL
of stock MTT dye solution (5 mg/mL) was added to each well.
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dimethylformamide and 20% SDS was added to solubilize the
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placing the plate in a mechanical shaker for 30 min at room
temperature. The optical density of each well was then
measured with a microplate spectrophotometer at a wave-
length of 570 nm. The percent cell viability was calculated
by the following equation:
% cell viability ) (OD treated wells/OD control wells) ×
100
where OD is the mean optical density from four determina-
tions. The percent cell viability values were plotted against
the drug concentrations used, and the ID50 was calculated from
the curve. Cytotoxicity experiments were repeated at least
three times.
Ack n ow led gm en t. Supported by grants from the
Elsa U. Pardee Foundation (J .B.C.) and from the
National Cancer Institute (CA35635 to J .B.C. and
CA55320 to W.P.). We thank Loren Williams and
Susan Wellman for helpful comments on the manu-
script. We thank one reviewer for an especially thor-
ough and helpful review.
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