Design, synthesis and biological evaluation of a novel class of anticancer agents: Anthracenylisoxazole lexitropsin conjugates

Article abstract of DOI:10.1016/j.bmc.2008.12.056

The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.) (22-33) are described. The molec

Full text of DOI:10.1016/j.bmc.2008.12.056

Bioorganic & Medicinal Chemistry 17 (2009) 1671–1680
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of a novel class of anticancer agents:
Anthracenylisoxazole lexitropsin conjugates
Xiaochun Han b, Chun Li b, Michael D. Mosher a,b, Kevin C. Rider b, , Peiwen Zhou b,d
,
Ronald L. Crawford c, William Fusco c, Andrzej Paszczynski c, Nicholas R. Natale b, ,
*
a Department of Chemistry, University of Nebraska—Kearney, Kearney, NE 68849-1150, United States
b Department of Chemistry, University of Idaho, Moscow, ID 83844, United States
c Environmental Biotechnology Institute, University of Idaho, Moscow, ID 83844, United States
d AK Scientific Inc., Mountain View CA 94043, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides
(AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I.
Meyers.) (22–33) are described. The molecules consist of an isoxazole that pre-organizes a planar aro-
matic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were
prepared via doubly activated amidation modification of Weinreb’s amide formation technique, using
SmCl3 as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazole-
carboxylic esters. The results of the National Cancer Institute’s (NCI) 60 cell line screening assay show a
distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with
one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these
compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of
the AIMs for G4 over B-DNA is presented.
Received 16 October 2008
Revised 19 December 2008
Accepted 22 December 2008
Available online 31 December 2008
Keywords:
Anthracene
Anti-tumor
G-quadruplex
Isoxazole
Pyrrole
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
cancer efficacy.11,12 While our initial foray into this arena met with
encouraging anti-tumor activity in screens provided by the Na-
Small molecules that bind DNA have found application in med-
icine, notably as cancer chemotherapeutic agents.1,2 That these
agents are also highly cytotoxic is attributed to their tendency to
bind rather indiscriminately to DNA, and any beneficial effect
arises from the more rapid death rates for faster replicating cancer
cells.3 Thus, it seems a reasonable proposition that agents which
are able to selectively recognize specific sequences of DNA could
potentially have higher therapeutic indices.4 In fact, considerable
effort has been devoted toward the conjugation of DNA binding
pharmacophores in an attempt to recognize and selectively bind
specific DNA sequences.5,6 The bulk of the focus of this work has,
until recently, been on B-DNA.7–10 While this has produced
a greatly increased understanding of the principles relating to
the binding of small molecules to DNA, only limited progress in
the development of new chemotherapeutic agents has been
achieved.
tional Cancer Institute’s Developmental Therapeutics Program
(NCI-DTP)13,14 (vide infra), we will present evidence herein that
our lead molecules did not appear to exert their bioactivity by bind-
ing B-DNA. Concurrent with our studies at this time, numerous
workers recognized that non-B-DNA conformations are potentially
feasible targets for anti-tumor drug development. Two prominent
theories recognize the G-quadruplex (G4) conformation of DNA as
a potential molecular target. The first line of reasoning involves
the inhibition of telomere maintenance via stabilization of the G4
conformer.7–10,15–18 The second theory postulates that G4 may rep-
resent an ancient off-switch for gene expression in specific onco-
genes, such as c-myc.19 It has been argued that molecules that
selectively target G4 could plausibly have unprecedented selectiv-
ity. Proof-of-concept has emerged in the form of a G4 binder which
advanced to clinical trials as an anti-cancer agent.20 Based on this
information, we have examined systematic structural changes in
our initial lead compound to test this revised hypothesis, and de-
scribe in this work the synthesis and anti-tumor activity of these
compounds in the NCI-DTP 60 cell line screening protocol. The
structure activity relationship (SAR) that emerges is consistent
with the G4 binding working hypothesis, and is supported by evi-
dence of G4 interaction from spectroscopy, telomerase inhibition
assays, and electrospray mass spectrometry.
The initial rationale for our work involved the use of an isoxazole
to pre-organize B-DNA binding groups and thereby increase anti-
* Corresponding author. Tel.: +1 406 243 4132; fax: +1 406 243 5228.
E-mail address: Nicholas.natale@umontana.edu (N.R. Natale).
NIH-COBRE Center for Structural and Functional Neuroscience, Department of
Biomedical and Pharmaceutical Sciences, University of Montana, Skaggs Building 480,
Missoula, MT 59812-1522, United States.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.056

1672
X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
amidation.12 This amidation is actually a modification of Weinreb’s
2. Chemistry
amidation, which has been an effective method for direct conver-
sion from ester to amide for many years. In the classical Weinreb’s
amidation,24 trialkyl aluminum is mixed with free primary or sec-
ondary amine to generate dialkylaluminum amide in situ, which
not only increases the nucleophilicity of the amine but also makes
carboxylic ester group susceptible to attack.
The central strategy for the preparation of the target molecules
necessitated improved synthetic methodology which could over-
come formidable steric encumbrance, hindrance which was
required by the premise of our working hypothesis. The route to
these compounds (Scheme 1) involves the initial acetylation and
nitration of N-methylpyrrole (1). Amide bond formation between
3 and appropriate primary and secondary amines gave rise to the
expected amidopyrroles (6 and 12). Reduction of the nitro group
allows step-wise extension of the pyrrole chain to the desired
chain length.
Linkage via an amide bond of the relatively unreactive heteroar-
omatic amines (7, 9, 11, 13, and 15) with rigid anthracenyl-isoxaz-
ole carboxylates was accomplished using a modification of our
previously reported double activation (Scheme 2). Based on this
previously developed methodology,21,22 we prepared a set of pyr-
role-type lexitropsin oligopeptides possessing at least one moiety
which would be protonated at physiological pH.
Double activated amidation was applied after we tried typical
Weinreb’s amidation on the amide formation between an amino-
lexitropsin and 3-(90-antracenyl) 5-methyl 4-isoxazocarboxylic es-
ter and obtained products in only modest yield. We had previously
observed by NMR that the ester group of 3-(90-anthracenyl)
5-methyl 4-isoxazolcarboxylate is located proximal to the tricyclic
aromatic system as evidenced by significant magnetic anisot-
ropy.23 This has also been supported by subsequent X-ray stud-
ies.25 It is a reasonable expectation that the low reactivity arises
from considerable steric hindrance preventing ester group interac-
tion with the aluminum center of Weinreb’s amide. In our modified
methodology, the carboxylic ester was pre-mixed with a mild
Lewis acid (SmCl3) to avoid a coordinative interaction with the
aluminum activated amine. In order for the Lewis acid to be com-
patible to the existence of basic tertiary amino group of lexitropsin
as well as the utilization of alkyl aluminum, mild Lewis acid
lanthanide chloride was applied. No or minimum additional coor-
dination interaction between the lanthanide center and aluminum
center is required. Two decades ago, in the study of the character-
istics of lanthanide coordination catalysts for polymerization, it
was suggested that SmCl3 and EuCl3 gave the lowest coordination
interaction with alkylaluminum.26 This finding indicates that
The series containing two tertiary amine groups was explored
by molecular modeling studies and target molecules (23–26) were
docked with the solid state coordinates of the G4 DNA reported by
Neidle. Such studies indicated that the two ‘tails’ could plausibly
increase the interaction of a lexitropsin-type molecule with the
DNA phosphate backbones.
The amide formation between lexitropsin and 3-(90-anthrace-
nyl) 5-methyl 4-isoxazolecarboxylate 17 (prepared via the 1,3-
cycloaddition of 9-anthracenyl nitrile oxide and the enamine of
ethyl acetoacetate)23 was achieved through doubly activated
N
O2N
R
a
b
5, c
CCl3
N
CCl3
N
N
N
N
N
O
O
O
CH3
3
CH3
CH3
1
CH3
2
6 R=NO2
7 R=NH2
d
4, c
R
3, e
H
N
R
N
N
H
N
N
O
CH3
N
N
O
CH3
12 R=NO2
13 R=NH2
N
N
i
O
CH3
3, e
8 R=NO2
9 R=NH2
f
R
H
N
3, g
N
H
N
R
N
O
CH3
H
N
N
O
CH3
N
CH3
H
N
N
O
N
14 R=NO2
15 R=NH2
j
N
O
h
CH3
N
N
O
CH3
10 R=NO2
11 R=NH2
Scheme 1. Reagents and conditions: (a) trichloroacetyl chloride, ether, 0 °C to room temperature; (b) HNO3 (fuming), acetyl anhydride, ꢀ40 °C to room temperature; (c) 5
NH[(CH2)3N(CH3)2]2 or 4 NH2(CH2)3N(CH3)2, THF, room temperature; (d) Pd/C, H2 (37 psi), MeOH, room temperature; (e) 3, THF, room temperature; (f) Pd/C, H2 (37 psi),
MeOH, room temperature; (g) 3, THF, room temperature; (h) Pd/C, H2 (37 psi), MeOH, room temperature; (i) Pd/C, H2 (37 psi), MeOH, room temperature; (j) Pd/C, H2 (37 psi),
MeOH, room temperature.

X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
1673
R
N
or
or
N
N
N
O
O
C
O
O
O
O
H3C
O
H3C
O
O
O
O
21
16
17 R=H
18 R=Cl
19 R=Br
20 R=Ph
b
a
7, 9, 11, 13, or 15
R
N
N
O
N
N
O
O
O
or
or
N
NH
NH
NH
H3C
H3C
H3C
N
O
O
H
N
H
N
n
N
N
H3C
N
H3C
N
N
H3C
N
O
O
O
n
2
22
23 n=0
24 n=1
25 n=2
26 n=3
27 R=H, n=0
28 R=H, n=1
29 R=H, n=2
30 R=Cl, n=1
31 R=Br, n=1
32 R=Ph, n=1
N
O
O
H
N
H
N
or
H
N
N
O
HN
N
O
N
CH3
O
O
CH3
H
N
N
H
O
H3C
N
H3C
N
N
33
Scheme 2. Reagents and conditions: (a) SmCl3 (10%), THF, room temperature, 2 h; (b) Al(CH3)3 (1 equiv), THF, 0 °C to room temperature, 1 h; (c) THF, reflux, 36 h.
SmCl3 or EuCl3 may serve as ideal lanthanide Lewis acid for the
doubly activated amidation.
The modified Weinreb’s amidation afforded up to 80–90% yield
(Scheme 2), with a variety of hindered substrates.27,28 This method
had also been previously used to prepare the bis-lexitropsin 33.29
anti-HIV test showed both 22 and 29 were inactive to HIV. But sur-
prisingly, compound 29 showed slight activity against certain hu-
man tumor cells in NCI’s 60 cell line screen.13,14 Subsequently
COMPARE31,32 analysis with the NCI Standard Agent Database
was performed. However, it did not give a significant correlation
with agents of known mechanism of action (all Pairwise Correla-
tion Coefficients were <0.5); in contrast to the good correlations
usually observed for intercalating agents.
3. Results and discussion
A plethora of G-quadruplex ligands have been developed as
potential molecular targets for cancer chemotherapy, such as
PIPER, TMPyP, 2,6-diamidoanthraquinones, and bisamido acri-
dines.15–19,33–35 Almost all of these G-quadruplex ligands have
Our work was initiated to find a compound that meets the
needs of both DNA-intercalation and B-DNA’s minor-groove bind-
ing and thus targets HIV.23 It was reported that DNA minor groove
binders (such as netropsin or distamycin analogues) linked with
acridine showed greater binding affinity for DNA than either acri-
dine or minor groove binders, and optimum linker length should
consist of a chain of 5 atoms.30 An isoxazole ring was then designed
to tether two biologically active portions, acridine (22) or anthra-
cene (29) and lexitropsin peptide containing polyamidopyrroles,
which exhibit preference for binding to poly-AT DNA and target
HIV’s tat gene. The results from National Cancer Institute (NCI)’s
extended planar chromophores, and
end(s) is an important factor in their binding.19,33–36 The anthra-
cene moiety in 29 may serve as an analogous -electron rich planar
p–p stacking on the G-quartet
p
chromophore, and provide binding to the top stack of G-quadru-
plex.36 The lexitropsin peptide moiety was presumed to bind the
TTA loop of G-quadruplex.4,5,37,38 This hypothesis inspired us to
design a series of analogues (23–28, 30–32) based on 29.39

1674
X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
The isoxazole moiety plays a pivotal role in our working
potent than 27 and 29, which inhibited 1 and 12 cell lines, respec-
tively. This suggests the interaction of the conjugate molecule oc-
curs with folded DNA structures possessing n + 1 (two) ATT-rich
bases instead of with duplex DNA’s minor groove, which requires
long-chained base pair recognition structure for binding. On the
other hand, compound 23, which has no pyrrole ring in the struc-
hypothesis.40 It is a rigid linker, pre-organizing the anthracene
and peptide in three dimensions, and potentially can also function
to deliver the bioconjugate molecule to the cellular DNA. Lexitrop-
sin analogues were in different lengths, to test the hypothesis that
given the binding site for ‘n’ pyrrole rings is ‘n + 1’ base pairs long
in terms of contacted base pairs,37,38 the relationship for the n = 0–3
series should be linear and increasing for B-DNA, but in contrast for
G4-DNA would be expected to be maximum at n = 1 based on our
working model (vide infra). Furthermore, introduction of an elec-
tron-pair bearing element (Cl) or group (phenyl) may provide
interaction to cations in the G-quadruplex cavity, thus preferen-
tially enhancing binding of these compounds to G4 DNA.
ture, has similar GI50 (4.57
lines) to that of compound 24. Both 30 and 32 had less than
M GI50 values, and inhibited almost all the tumor cell lines
lM) and inhibition spectrum (42 cell
2
l
tested, indicating that introduction of electron-rich substituents
at C10’ of anthracene (30 and 32) increased anti-tumor potency.
An interesting result is that compounds 23, 27, 30, and 32
showed higher activity against some colon cancer cell lines and
non-small cell lung cancer cell lines, in which the c-myc oncogene
is implicated as a potential contributing factor (Table 1).43 C-myc
expression has been reported to be downregulated by G-quadru-
plex stabilization.19 This suggests that compounds 23, 27, 30, and
32 inhibit cells, in which c-myc oncogene is over expressed, by
stabilizing G-quadruplex.
3.1. Testing of biological activities
3.1.1. In vitro anti-tumor activity
Structure–activity relationship (SAR) data for the anthracenyl
isoxazole amides synthesized (23–32) were acquired by evaluating
their in vitro anti-tumor activity against NCI’s 60 human tumor cell
lines.13,14,41,42 These cell lines have been derived from nine cancer
types that adequately meet minimal quality assurance criteria rep-
resenting leukemia, melanoma and cancers of the lung, colon,
brain, ovary, breast, prostate, and kidney. The results are shown
in Table 1. Several compounds were selected for re-screening by
the Biological Evaluation Committee of NCI, and for those ranges
are given (AIMs 23, 29, 30, and 32). Since single digit micromolar
is a practical measure of encouraging anti-tumor activity, we use
the total number of cell lines inhibited at single digit micromolar
(N in Table 1) as an additional benchmark for overall activity. We
also note those cell lines for which the anti-tumor efficacy was
within the nanomolar regime. We started the SAR by conjugating
different lengths of lexitropsin peptide derivatives (23–32), and
anticipating higher anti-tumor activity as peptide length increased.
Even though varying the length of peptide had a dramatic effect on
anti-tumor potency, the anti-tumor activity of this series of mole-
cules had no linear relationship with the length of lexitropsin
peptide. Inside each series, either double tail (23–26) or single tail
(27–32), compounds which had one pyrrole (n = 1) moiety gave the
strongest anti-tumor activity and the broadest spectrum of inhibi-
3.1.2. In vivo activity and acute toxicity
In light of the observation that the in vitro activity lies outside
the category of adequately studied anti-tumor agents, the lead
compound 29 was selected for the hollow fiber in vivo screen.44
The total Score of 10 reflected some in vivo effect, however, the
score usually required for further development is 20. The mouse
toxicity assay indicated no acute toxicity for 29, and the animals
showed weight gain at day 14 for all three dose regimens (IP, single
dose at day 1, as a homogeneous smooth suspension of saline plus
Tween 80 at 100, 200, and 400 mg/kg). Therefore, further studies
into SAR development to increase the efficacy (op cit) and under-
stand the mechanism of action (vide infra) appeared warranted
(Fig. 1).
3.2. Approaches toward understanding the mechanism of
action (MoA)
3.2.1. Spectroscopy with oligonucleotides
An oligonucleotide microarray experiment was conducted
wherein the effect of 29 on Calf thymus DNA and an oligonucleo-
tide designed by Hurley, (50-CATGGTGGTTT(GGGTTA)4CCAC-30)
known to form a quadruplex in solutions of KCl and NaCl,45 was
examined. The microarray fluorescence experiment for the mixture
of Calf Thymus (CT) DNA with 29 indicates a quenching of the fluo-
rescence but shows no bathochromic shift which would be consis-
tion at single digit micro-molar (
had GI50 of 3.89 M and inhibited 44 tumor cell lines, was 3.98
and 6.30-fold more potent than 25 and 26, which inhibited 7 and
cell lines, respectively. Compound 28, which had GI50 as
7.94 M and inhibited 23 cell lines, was 4.06 and 1.32-fold more
lV) GI50. Compound 24, which
l
0
l
Table 1
Synopsis of in vitro anti-tumor activities of compounds (22–33) against the NCI-DTP 60 cell line screen
a
AIM compound
ꢀLog (mean GI50
)
Mean GI50
(lM)
Nb
Efficacyc
16
22
23
24
25
26
27
28
29
30
31
32
33
4.69
4.68
5.31–5.34
5.41
4.81
4.61
4.49
5.10
4.91–4.98
5.72–5.75
5.06
5.71–6.04
4.69
20.4
20.9
4.90–4.57
3.89
15.5
24.5
32.4
7.94
12.3–10.5
1.91–1.78
8.71
1.95–0.91
20.4
2
0
37–42
44
7
0
1
23
12–14
53–57
18
Non-small cell lung cancer EKVX: 6.18, colon cancer HCC-2998: 6.11, CNS cancer SF-295: 7.52
Non-small cell lung cancer NCI-H226: <8.00
Non-small cell lung cancer HOP-62: 6.39
Non-small cell lung cancer HOP-92: 7.03
CNS cancer SNB-75: 6.80
CNS cancer SF-539: <8.00, Non-small cell long cancer HOP-62: 7.98, HOP-92: 7.2
60
2
The full 60 cell line data is available free of charge on the internet at the NCI-DTP site [http://dtp.nci.nih.gov/index.html]. NSC numbers are provided for each compound in
this table in the Supplementary data.
a
Range given for those compounds selected for a second screening by NCI’s Biological Evaluation Committee.
The number of cell lines inhibited at single digit micromolar.
b
c
Cell lines inhibited in the nanomolar range, with ꢀLog GI50
.

X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
1675
Figure 1. Fluorescence spectra of oligonucleotide microarray experiment. Left: Normalized Fluorescence spectra of AIM-29 alone (solid line), CT-DNA alone (long-dashed
line) and the Hurley Oligonucleotide alone (small-dashed line). Right: Normalized Fluorescence spectra of AIM-29 alone (solid line), with CT-DNA (long-dashed line) and the
Hurley-sequence Oligonucleotide (small-dashed line).
tent with an anthracene intercalation mechanism (Fig. 2).46,47 The
Hurley-Oligonucleotide experiment with 29 shows considerable
quenching of the fluorescence which is consistent with a p-stack-
were an 11 nm hypsochromic shift in the kmax which is inconsis-
tent with an intercalative mechanism47 and is likely due to hydro-
gen bonding interactions and/or solvation of 29 in the presence of
the DNA. Further fluorescence-DNA-titration experiments are
warranted in light of these results.
ing interaction with the G-tetrad of the quadruplex structure that
forms in situ under the experimental conditions.48,49
The oligonucleotide and CT-DNA solutions were prepared in a
10 mM KCl solution containing a TE buffer at pH = 7.0 with a final
3.2.2. COMPARE analysis31,32,50,51
concentration of 400
hybridized using a cyanine dye (Cy3-AP3-dCTP) utilizing a 96-well
plate. AIM-2 was in an aqueous 25% DMSO solution (10 M). All
experiments were carried out at 37 °C with oligonucleotide solu-
tions being incubated for 5 min prior to printing on the slide for
analysis. All spectra were taken at four excitation wavelengths
(310 nm, 380 nm, 410 nm, and 485 nm) with 530 nm emission.
All microarray experiments were run on a GenePix 4000 micro-
array fluorescence scanner with 10 lm resolution and a dynamic
detection range of four orders of magnitude which is linear over
three orders of magnitude.
An additional fluorescence experiment involving 29 with hu-
man DNA (1:1 M ratio), sans the histones, was performed (data
not shown). The results of the second fluorescence experiment
l
g/
l
L. The oligonucleotide solution was
We used 29 as a probe or seed compound in the NCI’s COMPARE
Algorithm to rank the similarity of responses of the 60 cell lines to
the standard agent database.13,14,41,42 Similarity of pattern to that
of the seed is expressed quantitatively as a Pearson correlation
coefficient (PCC). The results obtained with the COMPARE algo-
rithm indicate that compounds high in this ranking may possess
a mechanism of action (MoA) similar to that of the seed com-
pound.31,32 COMPARE works quite well for intercalating/topoiso-
merase II inhibitors. The top ten matches for adriamycin, with a
PCC range of 0.758–0.950, were all topoisomerase II inhibitors. In
contrast, 29—originally thought by our group to be a potential
intercalating/minor groove binder—indicted no strong correlation
to any consistent MoA, with the highest PCC being a relatively
weak 0.517 (Table 2). Similar COMPARE analysis of 23–26
l
Figure 2. Plausible Docking modes of compounds 29 and 30. (a) Left, the Intercalative mode, where 29 is shown displacing a guanine after minimization. (b) Right, the
stacking mode with ligand 30, after minimization the chlorine nestles within a reasonable association distance with the potassium in the G-tetrad.

1676
X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
Table 3
indicated no significant correlation with any MoA in the Standard
Agent Data Base.
Energy calculation of rotation toward isoxazole/anthracene coplanarity
3.2.3. Computation
The dihedral angle between the tricyclic planar aromatic moiety
and the isoxazole from our crystallographic studies of intermedi-
ates and analogs, is in the range of 74–80°.25,27–29,52,53 The ideal-
ized helical pitch angle for B-DNA was estimated to be ca. 47.1°
by Goodsell and Dickerson in their isohelical analysis of groove-
binding drugs.54,55 Therefore, we calculated the energy associated
with conformational changes to this dihedral angle in order to
determine whether B-DNA binding seemed plausible. The rota-
tional barrier calculations were performed using the torsion force
constraint in the Discover module.56 The bond in question was ro-
tated through 360 intervals (1° increments) using a force constant
of 10. After each rotation, the structure was subjected to 1000
steps of minimization, or until an rms value of 0.01 was reached,
using the VA09A algorithm. The results of the conformational
searches were examined with the Analysis module by constructing
a table of total energy verses dihedral angle. Calculations from the
INSIGHT II program suggests that this energetic cost is high, precisely
in the vicinity of the helical pitch angle requisite for binding of a
C-3 aromatic isoxazole to B-DNA (Table 3).
within 3 Å of the NH2 of the guanine of the lower, intact G-tetrad.
Alternatively, we considered an external stacking (Fig. 2b), and in
the case of analog 30 which contained a group bearing lone pairs
at C(10) of the anthracenyl group, C(10) tended to orient—again
after minimization—within 2.62 Å over the potassium in the cavity
of the G-tetrad.
In combination with the COMPARE analysis, we felt it was
prudent to consider alternatives to B-DNA as a molecular target.
3.2.4. Revision of the working hypothesis
Similar features which emerged from both of these minimized
structures were (1) the isoxazole nitrogen was within hydrogen
bonding distance of the guanine 2-amino group of the G-tetrad,
and (2) the amine of the dimethylamino tail would move slightly
to associate proximal to a group peripheral to the intact G-tetrad,
as seen in the case of 30, with the phosphate in the sugar-phos-
phate TTA loop.
In light of the increasingly numerous reports postulating G4
binding as a mechanism for anti-tumor activity, and given an over-
all similarity of some of the salient structural features of our com-
pounds, we conducted a number of computational studies with the
G4 coordinates available from Neidle’s elegant crystallographic
studies.57,58
In a typical example, the minimum energy structure of NSCD
694332 and quadrulex DNA57 was calculated in a SGI INSIGHT
2000 docking study, using the cvff forcefield, and all the solution
molecules (H2O) were removed. The DNA molecule was con-
strained and the distance between first potassium and C-10 hydro-
gen was initially set to 4.00 Å. After 3000 iterations using steepest
descent minimization, in most cases the drug-receptor interaction
had converged to a final energy, which the program reported as
consisting of separate electrostatic and VdW components. If the
minimization did not reach convergence at a set number of itera-
tions, reasonable and slight adjustments were made to the ligand
structure, and minimization was repeated until a convergent struc-
ture solution was obtained.27
Thus, there is a specific structure-based reason for the expecta-
tion that the AIMs should be selective for G4 DNA at the expense of
B. The energy cost of B-DNA binding is increased by the mismatch with
the helical pitch angle.
3.2.5. Telomeric repeat amplification protocol (TRAP) assay
Stabilizing G-quadruplex inhibits telomerase activity, and this
has been correlated with inhibition of cancer growth.59 Thus, until
recently the TRAP assay has been widely used to assess G-quadru-
plex interaction. We reported in 2004 that the fluorescence analy-
sis TRAPese assay was marred by the inhibition of taq polymerase
by 32,60 and this recently has been verified by Mergny’s group.61 In
the more reliable gel electrophoresis TRAP analysis, AIM 30 did
indeed appear to inhibit telomere elongation rather than taq poly-
merase (data for both TRAPese and TRAP is shown in the Supple-
mentary data), however, we sought a more direct method of
determining G4 interaction with our compounds, and therefore
also examined electrospray mass spectrometry (vide infra).
Two of the possible docking modes we considered are illus-
trated in Figure 2. In the first we initiated the docking with the
anthracenyl moiety intercalated between the G-tetrads. During
minimization, the anthracene of 29 displaced two of the guanines
(in red). In the minimized structure (Fig. 2a), the isoxazole ring N is
3.2.6. Electrospray ionization mass spectrometry (ESI-MS) study
of G-quadruplex DNA stabilization by 30 or 3262
Table 2
COMPARE analysis using 29, NSC D-694332, as ‘seed’
Total ions of G-quadruplex DNA, i-motif DNA, and duplex DNA
were measured under the same conditions (Fig. 3). In the absence
of G4 stabilizing ligands (negative control), ion intensities of sin-
gle-stranded DNA dropped dramatically, while duplex formation
is rather fast. Rehybridization in the absence of G4 stabilizing
ligands proceeded to 50% completion in 10 min and was essentially
complete in 45 min (negative control, shown in Supplementary
data).
When incubated with either TMPyP4 (positive control), 30 or
32, the decrease of single-stranded DNA signals and increase of du-
plex signals were dramatically slowed. Although no DNA-ligand
complex ion was detected, the inhibition of duplex formation
Anti-tumor agent
PCC
Mechanism
Tetrandrine
Macbecin II
Didemnin B
Tetrocarcin A
Spirogermanium
Pibenzimol
Thalicarpine
Neocarzinostatin
Fostriecin
0.517
0.488
0.468
0.462
0.459
0.453
0.450
0.436
0.421
0.415
0.414
Calcium channel blocker
DNA anti-metabolite
Inhibits ribosomal protein synthesis
Modulates mitochondrial apoptosis
Alkylating agent
Minor groove binder
Unknown
Enediyne strand breaker
Topo II catalytic inhibitor
DHFR inhibitor
Emofolin sodium
Vinblastin sulfate
Antimitotic agent

X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
1677
Figure 3. ESI-MS spectra comparison between 32 and TMPyP4. Left: ESI-MS spectra of a mixture of 5
spectra of a mixture of 5 M G-DNA, 5 M TMPyP4, and 5 M C-DNA at the times indicated.
lM G-DNA, 5
l
M 32, and 5
lM C-DNA at different time. Right: ESI-MS
l
l
l
indicated interference from both TMPyP, 30, and 32 in DNA anneal-
ing, which suggests the ability for G-quadruplex stabilization. The
slopes of ion intensity trend lines give approximate inhibition abil-
ities of TMPyP4 and 32. For example, slopes in total duplex ion
intensity trends showed that TMPyP4 inhibited duplex formation
at a comparable rate to 32, where both took about 80 min to
proceed to 50% hybridization, and significant ions corresponding
to single stranded oligonuclotides were still present even at 2 h.
the MoA and the effects of substitution at C5 of the isoxazole,
and on the anthracene appears warranted. Those results will be
reported in due course.
5. Experimental
Mass spectra were obtained on a JEOL JMS-AX505 HA. The NMR
spectra (1H and 13C) were obtained on a Bruker AVANCE 300 and
500 Digital NMR (300 and 500 MHz, respectively) using SGI-IRIX
6.5. Elemental analysis was performed by Desert Analytics Labora-
tory, PO BOX 41838, Tucson, AZ 85717. All reactions were
performed under an inert atmosphere of nitrogen or argon. Tetra-
hydrofuran was distilled from sodium-benzophenone immediately
before use. Flash chromatography was performed on silica gel
(Merck 60 Å, 230–400 mesh) with freshly distilled solvents. Pyrrole
starting materials 2 and 3 were prepared according to Nishwaki.22
3-(90-Anthracenyl) 5-methyl 4-isoxazolecaboxylate 17, and the
corresponding acridine 16,23 anthracenyl ring analogs 18–2027,28
and final product 3329 were prepared according to methodology
previously reported by our lab.
4. Conclusion
The new compounds reported in this study showed in vitro
anti-tumor activity, and the mean GI50 against the NCI60 cell line
panel for the length of the oligopyrrole moiety (n = the number of
pyrroles) was observed as 1 > 0 ꢁ 2 > 3 for the bis-dimethylami-
nopropyl series 23–26) and 1 > 2 ꢁ 0 for the mono-dimethylami-
nopropyl series (27–29); and for the anthracenyl C(10) position
was Ph ꢂ Cl ꢁ H > Br (28, 30–32), with the most efficacious
examples having average activity comparable to agents currently
in general medical practice.13 The activity correlates inversely
with the length of lexitropsin oligopeptides within the conjugate
molecules, that is, conjugates containing a single pyrrole ring dem-
onstrated the strongest activity and broadest spectrum of inhibi-
tion against cancer cell lines. The SAR, spectroscopic and ESI-MS
assays are consistent with our current working hypothesis that
the compounds selectively target G4 sequences. In conclusion,
the results presented suggest that the synthesis of a new series
of anthracenyl isoxazole-lexitropsin conjugates, the AIMs, may
represent a potential useful addition to the arsenal of anti-cancer
molecules. In addition, we present structure-based evidence for
the contention that the AIMs are unique among G4 binding
agents in that they possess specific features which destabilize
their intercalative interaction with B-DNA, and therefore, the
expectation of selectivity is reasonable. Further clarification of
5.1. 2-[[[N,N-Bis[3-(N,N-dimethylamino)propyl]amino]
carbonyl]-1-methyl-4-nitropyrrole (6)
To a solution of 3 (9.24 g, 35.88 mmol) in THF (40 mL), was
added
a
solution of 3,30-iminobis(N,N-dimethylpropylamine)
(25.24 g, 134.74 mmol) at room temperature. The reaction mixture
was heated at reflux for 3 h. The reaction mixture was cooled to
room temperature and the solvent was evaporated. The residue
was then purified by chromatography on silica gel eluting with
MeOH/triethylamine (9/1) (Rf = 0.40) to give yellow oil 6 (4.82 g,
41.3%), which became a pale yellow solid after being triturated
with hexane (20 mL); mp: 60–61 °C; 1H NMR (CDCl3): d 7.69 (1H,
d, J = 1.7 Hz), 7.66 (1H, d, J = 1.7 Hz), 3.96 (3H, s), 3.67 (4H, t,

1678
X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
J = 7.5 Hz), 2.43–2.35 (16H, m), 1.94 (4H, m). 13C NMR (CDCl3): d
162.4, 135.4, 127.2, 125.5, 106.7, 57.0, 46.0, 45.7, 37.0, 26.2. MS
(CI): m/z (%) 340 (M+1, 100), 268 (8.62), 268 (8.62), 84 (12.83),
58 (18.52). Anal. Calcd for C16H29N5O3: C, 56.62; H, 8.61; N,
20.63. Found: C, 56.64; H, 8.33; N, 20.09.
(140 mg, 1.15 mmol), Pd2(dba)3 (45 mg, 5 mol %), P(t-Bu)3 (10% in
hexane, 242 mg, 12 mol %) and KF (191 mg, 3.3 mmol) in THF
(7 mL)was stirred at room temperature under argon atmosphere
for 72 h. The reaction mixture was filtered and the solid was
washed by THF (10 mL). The filtrate was concentrated and purified
by chromatography on silica gel eluting with hexane/benzene (3:2)
to give yellow crystal (382 mg, 92%); mp: 178–180 °C. 1H NMR
(CDCl3): d 7.62 (4H, m), 7.52 (3H, m), 7.32 (6H, m), 3.68 (2H, q,
J = 7.2 Hz), 2.88 (3H, s), 0.33 (3H, t, J = 7.2 Hz). 13C NMR (CDCl3):
d 176.2, 161.5, 160.8, 139.3, 138.6, 131.2, 131.1, 130.5, 129.7,
129.0, 128.5, 128.4, 128.3, 127.6, 127.1, 125.9, 125.5, 125.1,
122.8, 111.5, 60.1, 13.5, 12.8. MS (EI): m/z (%) 407 (M+, 100), 319
(17.91), 295 (12.85), 252 (13.86). Anal. Calcd for C27H21NO3: C,
79.59; H, 5.19; N, 3.44. Found: C, 79.94; H, 5.09; N, 3.57.
5.2. 2-[[[2-[[N,N-Bis[3-(N,N-dimethylamino)propyl]amino]-
carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]carbonyl]-1-methyl-
3-nitropyrrole (8)
To a solution of 6 (2.40 g, 7.38 mmol) in MeOH (175 mL), was
added Pd/C (5%) (2.50 g). The mixture was hydrogenated at 37–
40 psi at room temperature for 3 h. The catalyst was removed by
filtration and the solvent was removed in vacuo. To a solution of
the above residue in THF (40 mL), was added a solution of 3
(3.05 g, 11.73 mmol) within 10 min. The reaction mixture was stir-
red at room temperature for 3 h. Solvent was removed in vacuo
and the residue was then purified by chromatography on silica
gel eluting with MeOH/triethylamine (9/1) (Rf = 0.34) to give pale
yellow solid, 2.21 g (65%); mp: 125–127 °C. 1H NMR (CDCl3): d
9.12 (1H, s), 7.61 (1H, d, J = 1.8 Hz), 7.47 (1H, d, J = 1.8 Hz), 7.25
(1H, d, J = 1.8 Hz), 6.36 (1H, d, J = 1.8 Hz), 4.07 (3H, s), 3.71 (3H,
s), 3.54 (4H, t, J = 7.8 Hz), 2.34 (4H, t, J = 7.2 Hz), 2.28 (12H, s),
1.85 (4H, m). 13C NMR (CDCl3): d 164.1, 157.8, 135.2, 127.1,
126.8, 123.9, 121.3, 117.0, 107.8, 103.6, 57.1, 45.8, 45.6, 38.4,
36.1, 27.0; MS (CI): m/z (%) 462 (M+1, 100), 377 (19.91), 352
(14.52), 170 (67.70), 84 (28.51), 58 (17.97).
To a solution of 8 (100 mg, 0.2 mmol) in absolute ethanol (5 mL),
was added a solution of oxalic acid dihydrate (C2O4H2ꢃ2H2O) (57 mg,
0.45 mmol). The mixture was stirred at room temperature until no
more precipitate formed. The solid was filtered and washed with
absolute ethanol (5 mL ꢄ 5). The oxalic acid salt of 8 was obtained
after being dried in vacuo to yield a solid (109 mg, 90%), mp:
203–204 °C. Anal. Calcd for C22H35N7O4ꢃ1.5C2O4H2ꢃ0.5H2O: C,
49.58; H, 6.49; 16.19. Found: C, 49.81; H, 6.64; N, 16.05.
5.5. 3-(9-Anthracenyl)-N,N-bis[3-(N,N-dimethylamino)propyl]-
5-methyl-4-isoxazolecarboxamide (23)
To a suspension of anhydrous SmCl3 (0.21 g, 0.79 mmol) in THF
(6 mL) was added ethyl 3-(9-anthracenyl)-5-methyl-4-isoxazole-
carboxylate 17 (0.25 g, 0.72 mmol) in dry THF (5 mL). The slurry
was stirred under nitrogen at room temperature for 5.5 h and
was used in the following step.
To
a
solution of 3,30-iminobis(N,N-dimethylpropylamine)
(0.222 g, 1.19 mmol) in THF (10 mL) was added (CH3)3Al (2 M in
hexane, 0.75 mL) at 0 °C during 30 min. The resulting yellow solu-
tion was warmed to room temperature, and stirred for an addi-
tional 1 h.
The yellow-brown solution of the activated amino-lexitropsin
solution was transferred via cannula to the activated ester slurry,
at room temperature during 10 min. The mixture was then heated
to reflux for 8 h, after which time Na2SO4ꢃ10H2O (1.0 g) and cold
methanol (20 mL) were added to the reaction mixture in order to
quench the excessive (CH3)3Al. Solid was removed via centrifuga-
tion and a red solution was separated. The red solution was then
concentrated and purified by chromatography on silica gel (200–
400 mesh) eluting with methanol–30% NH4OH (95:5), to afford
23 as a yellow oil (0.32 g, 87%). 1H NMR (CDCl3): d 8.58 (1H, s),
8.06–8.00 (4H, m), 7.54–7.47 (4H, m), 3.06 (2H, t, J = 7.1 Hz), 2.74
(3H, s), 2.53 (2H, t, J = 7.1 Hz), 2.00 (6H, s), 1.87 (6H, s), 1.63 (2H,
t, J = 6.9 Hz), 1.45 (2H, t, J = 6.9 Hz), 1.11–0.95 (2H, m), 0.90–0.83
(2H, m). 13C NMR (CDCl3): d 170.8, 163.1, 157.8, 131.4, 131.1,
130.8, 130.3, 129.7, 129.3, 128.8, 128.7, 128.3, 127.1, 126.2,
125.8, 125.3, 121.9, 116.3, 56.2, 47.4, 46.4, 45.6, 45.4, 42.5, 26.7,
24.9, 11.5; MS (CI): m/z (%) 473 (M+1, 100), 188 (22.58), 58 (18.63).
To a solution of 23 (94 mg, 0.2 mmol) in absolute ethanol
(5 mL), was added a solution of oxalic acid dihydrate (C2O4H2ꢃ
2H2O) (57 mg, 0.45 mmol). The mixture was stirred at room tem-
perature until no more precipitate formed. The solid was filtered
and washed with absolute ethanol (5 mL ꢄ 5). The oxalic acid salt
of 23 was obtained after being dried in vacuo to yield a solid
(114 mg, 84%), mp: 185–186 °C. Anal. Calcd for C29H36N4O2ꢃ2-
C2H2O4ꢃ1.5H2O: C, 58.31; H, 6.38; N, 8.24. Found: C, 58.50; H,
6.12; N, 8.12.
5.3. 2-[[[2-[[[2-[[N,N-Bis[3-(N,N-dimethylamino)propyl]amino]-
carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]carbonyl]-1-methyl-
1H-pyrrol-4-yl]amino]carbonyl]-1-methyl-4-nitropyrrole (10)
To a solution of 8 (1.13 g, 2.45 mmol) in MeOH (90 mL), was
added Pd/C (5%) (2.00 g). The mixture was hydrogenated at 37–
40 psi at room temperature for 3 h. The catalyst was removed by
filtration and the solvent was removed in vacuo. To a solution of
the above residue in THF (30 mL), was added a solution of 3
(1.00 g, 3.90 mmol) within 10 min. The reaction mixture was stir-
red at room temperature of 3 h. Solvent was removed in vacuo
and the residue was then purified by chromatography on silica
gel eluting with MeOH/triethylamine (9/1) (Rf = 0.21) to give pale
yellow solid, (0.74 g, 52%); mp: 178–179 °C; 1H NMR (DMSO-d6):
d 10.29 (1H, s), 9.89 (1H, s), 8.18 (1H, d, J = 1.7 Hz), 7.58 (1H, d,
J = 1.7 Hz), 7.24 (2H, d, J = 1.7 Hz), 7.03 (1H, d, J = 1.7 Hz), 6.37 (1H,
d, J = 1.7 Hz); 3.96 (3H, s), 3.85 (3H, s), 3.58 (3H, s), 2.49 (4H, t,
J = 7.7 Hz), 2.15 (4H, t, J = 7.1 Hz), 2.10 (12H, s), 1.70–1.61 (4H, m).
13C NMR (CDCl3): d 164.6, 159.2, 158.1, 135.4, 127.3, 126.7, 124.0,
123.6, 123.0, 121.8, 120.0, 117.6, 108.5, 102.9, 102.6, 57.1, 46.4,
45.6, 38.7, 37.2, 35.9, 27.1. MS (CI): m/z (%) 584 (M+1, 55.96), 397
(10.47), 275 (24.39), 153 (18.31), 149 (52.19), 122 (32.51), 107
(20.18), 101 (16.14), 81 (100). Anal. Calcd for C28H41N9O5: C,
57.62; H, 7.08; N, 21.60. Found: C, 57.23; H, 7.26; N, 21.24.
5.6. 3-(9-Anthracenyl)-N-[2-[[N,N-bis[3-(N,N-dimethylamino)-
propyl]amino]carbonyl]-1-methyl-1H-pyrrol-4-yl]-5-methyl-4-
isoxazolecarboxamide (24)
To a suspension of anhydrous SmCl3 (0.21 g, 0.79 mmol) in THF
(10 mL) was added 17 (0.25 g, 0.72 mmol) in dry THF (10 mL). The
slurry was stirred under nitrogen at room temperature for 5.5 h
and was used in the following step.
5.4. Ethyl 3-(100-phenyl-90-anthracenyl)-5-methyl-4-isoxazole-
carboxylate (20)
A suspension of 10% Pd/C (0.2 g) in a solution of 6 (0.385 g,
1.19 mmol) in methanol (30 mL), was stirred for 4.5 h under H2
pressure (37 psi) at room temperature. The catalyst was removed
A mixture of ethyl 3-(100-bromo-90-anthracenyl)-5-methyl-4-
isoxazolecarboxalate (420 mg, 1.02 mmol), phenyl boronic acid

X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
1679
by filtration, and the solvents removed in vacuo. The hydrogena-
tion product was dissolved into dry THF (15 mL) and (CH3)3Al
(2 M in hexane, 0.75 mL) was added at 0 °C during 30 min. The
mixture turned brown. The reaction was warmed to room temper-
ature, and stirred for an additional 1 h.
5.9. 3-(100-Chloro-90-anthracenyl)-N-[2-[[N-[3-(N,N-dimethyl-
amino)-propyl]amino]carbonyl]-1-methylpyrrol-4-yl]-5-methyl-
4-isoxazolecarboxamide (30)
By the same procedure as that described for 24, except for reflux-
ing for 18 h, from SmCl3 (393.5 g, 1.53 mmol), 18 (381.3 mg,
1.04 mmol), 10% Pd-C (310.6 mg), 12 (473.0 g, 1.86 mmol) and
(CH3)3Al (2 M in hexane, 2 mL), 30 was obtained as a pale yellow so-
lid (203.7 mg, 36%), mp: 208–210 °C. 1H NMR (CDCl3): d 8.65 (2H, dt,
J = 5.4, 0.6 Hz), 7.78 (1H, br), 7.73 (2H, dt, J = 5.4, 0.6 Hz), 7.69 (2H,
m), 7.57 (2H, m), 6.76 (1H, d, J = 1.2 Hz), 6.40 (1H, s), 5.19(1H, d,
J = 1.2 Hz), 3.72 (3H, s), 3.31 (2H, q, J = 3.6 Hz), 3.02 (3H, s), 2.36
(2H, t, J = 3.6 Hz), 2.08 (3H, s), 1.58 (2H, m). 13C NMR (CDCl3): d
176.5, 161.1, 157.7, 157.0, 132.8, 131.3, 128.5, 128.1, 127.6, 125.5,
125.4, 123.8, 120.2, 119.8, 118.3, 112.9, 101.5, 59.4, 45.4, 39.9,
36.5, 25.1, 13.8. MS (CI): m/z (%) 544.00 (M+, 65.88), 306.90
(58.70), 288.92 (24.86), 153.96 (100), 136.99 (90.87). Anal. Calcd
for C30H30ClN5O3: C, 66.23; H, 5.56; N, 12. 87. Found: C, 66.07; H,
5.50; N, 12.71.
By the same double activation procedure described for 23, after
the reaction work-up and separation, 24 was obtained as a pale
yellow solid (0.396 g 84%), mp: 109–111 °C. 1H NMR (CDCl3): d
8.63 (1H, s), 8.04 (2H, dd, J = 0.9, 8.3 Hz), 7.65 (2H, dd, J = 0.9,
8.3 Hz), 7.52–7.23 (4H, m), 6.56 (1H, d, J = 1.7 Hz), 6.40 (1H, s),
4.90 (1H, d, J = 1.7 Hz), 3.37 (3H, s), 3.21 (4H, t, J = 6.6 Hz), 2.94
(3H, s), 2.24–1.90 (16H, m), 1.60–1.45 (4H, m), 1.52 (2H, s). 13C
NMR (CDCl3): d 176.3, 163.9, 158.0, 157.9, 157.8, 157.6, 131.5,
131.2, 130.7, 129.2, 128.9, 128.4, 128.2, 127.9, 126.5, 125.8,
125.7, 125.3, 124.3, 120.7, 120.3, 116.1, 113.2, 57.2, 46.4, 45.8,
35.6, 26.6, 14.0. MS (CI): m/z (%) 595 (M+1, 100), 510 (6.09), 336
(19.88). Anal. Calcd for C35H42N6O3ꢃH2O: C, 68.60; H, 7.24; N,
13.71. Found: C, 68.33; H, 6.94; N, 13.61.
5.7. 3-(9-Anthracenyl)-N-[2-[[[2-[[N,N-bis[3-(N,N-dimethylamino)-
propyl]amino]carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]carbonyl]-
1-methyl-1H-pyrrol-4-yl]-5-methyl-4-isoxazolecarboxamide (25)
5.10. 3-(100-Br-90-anthracenyl)-N-[2-[[N-[3-(N,N-dimethylamino)-
propyl]amino]carbonyl]-1-methylpyrrol-4-yl]-5-methyl-4-
isoxazolecarboxamide (31)
By the same procedure as that described for 24, from SmCl3
(0.21 g, 0.79 mmol), 17 (0.25 g, 0.72 mmol), 10% Pd/C (0.2 g), 8
(0.55 g, 1.19 mmol) and (CH3)3Al (2 M in hexane, 0.75 mL), 25
was obtained as a pale yellow solid (0.44 g 78%); mp: 118–120 °C
(dec). 1H NMR (CDCl3): d 8.72 (1H, s), 8.12 (2H, dd, J = 0.9,
8.2 Hz), 7.70 (2H, dd, J = 0.9, 8.2 Hz), 7.55–7.50 (4H, m), 7.19 (1H,
s), 7.09 (1H, d, J = 1.7 Hz), 6.41 (1H, s), 6.28 (1H, d, J = 1.7 Hz),
6.10 (1H, d, J = 1.7 Hz), 5.69 (1H, d, J = 1.7 Hz), 3.65 (3H, s), 3.61
(3H, s), 3.48 (4H, t, J = 7.8 Hz), 2.99 (3H, s), 2.28–2.10 (16H, m),
1.83–1.69 (4H, m), 1.52 (5H, s). 13C NMR (CDCl3): d 176.7, 164.3,
158.7, 158.5, 157.8, 131.5, 131.3, 130.9, 129.3, 129.0, 128.6,
128.4, 128.0, 126.6, 125.9, 125.7, 125.3, 124.3, 123.6, 121.2,
120.6, 120.5, 118.8, 116.7, 113.0, 103.3, 103.2, 103.0, 57.3, 46.4,
45.7, 36.9, 35.9, 26.8, 14.1. MS (FAB): m/z (%) 717 (M+1, 71), 530,
(13.19), 286 (15.85), 230 (12.43), 271 (12.37), 244 (59.35), 214
(20.21), 188 (12.72), 149 (61.28), 122 (49.88), 106 (16.22), 81
(100). Anal. Calcd for C41H48N8O4ꢃ2.5H2O: C, 64.63; H, 7.01; N,
14.71. Found: C, 64.77; H, 6.64; N, 15.08.
By the same procedure as that described for 30, from SmCl3
(481.5 mg, 1.87 mmol), 19 (341.6 mg, 0.83 mmol), 10% Pd/C
(490 mg), 12 (458.5 mg, 1.80 mmol) and (CH3)3Al (2 M in hexane,
2 mL), 31 was obtained as a yellow solid (280 mg, 61%), mp:
201.5–203.5 °C. 1H NMR (CDCl3): d 8.70 (2 H, d, J = 9 Hz), 7.71
(5H, m), 7.60 (2H, m), 7.57 (2H, m), 6.74 (1H, d, J = 1.5 Hz), 6.40
(1H, s, br), 5.24 (1H, d, J = 1.5 Hz), 3.74 (3H, s), 3.32 (2H, q,
J = 3.6 Hz), 3.04 (3H, s), 2.38 (2H, t, J = 3.6 Hz), 2.11 (6H, s), 1.61
(2H, m). 13C NMR (CDCl3): d 176.4, 161.0, 157.7, 157.0, 131.4,
130.3, 128.4, 128.0, 127.9, 127.0, 125.4, 123.8, 121.3, 119.7,
118.2, 112.9, 101.6, 59.3, 45.4, 39.7, 36.4, 25.2, 13.6. MS (CI): m/z
(%) 590.01 (M+1, 100.00), 588.02 (99.87), 542.97 (19.06), 485.88
(22.84), 321.85 (31.82), 242.96 (20.71), 153.99 (50.75), 136.00
(51.68), 129.09 (65.34), 84.38 (43.93). Anal. Calcd for
C30H30BrN5O3: C, 61.23; H, 5.14; N, 11.90. Found: C, 61.16; H,
5.29; N, 11.62.
5.11. 3-(100-Phenyl-90-anthracenyl)-N-[2-[[N-[3-(N,N-dimethyl-
amino)-propyl]amino]carbonyl]-1-methylpyrrol-4-yl]-5-
methyl-4-isoxazolecarboxamide (32)
5.8. 3-(9-Anthracenyl)-N-[2-[[[2-[[[2-[[N,N-bis[3-(N,N-dimethyl-
amino)propyl]amino]carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]-
carbonyl]-1-methyl-1H-pyrrol-4-yl]amino] carbonyl]-1-methyl-
1H-pyrrol-4-yl] ]-5-methyl-4-isoxazolecarboxamide (26)
By the same procedure as that described for 30, from SmCl3
(491.5 mg, 1.90 mmol), 19 (300.6 mg, 0.73 mmol), 10% Pd/C
(553 mg), 12 (500.5 mg, 1.97 mmol) and (CH3)3Al (2 M in hexane,
2 mL), 32 was obtained as a pale yellow solid, 130 mg (30%), mp:
126–128 °C. 1H NMR (CDCl3): d 7.68 (3H, m), 7.45 (10H, m), 6.49
(1H, s), 6.47 (1H, s), 5.37 (1H, s), 3.65 (3H, s), 3.25 (2H, q, 3.6Hz),
2.96 (3H, s), 2.27 (2H, t, J = 3.6 Hz), 2.01 (6H, s), 1.53 (2H, m). 13C
NMR (CDCl3): d 176.2, 161.5, 160.1, 157.3, 139.4, 138.8, 131.4,
131.1, 130.5, 129.7, 129.0, 128.5, 128.4, 128.3, 127.6, 127.1,
125.9, 125.5, 125.1, 123.8, 122.8, 119.8, 118.1, 111.5, 101.7, 60.1,
45.4, 39.6, 36.1, 25.0, 13.4. MS (EI): m/z (%) 587 (M+1, 39.04),
586 (M+, 100), 585 (M-1, 19.26), 484 (13.13), 320 (19.84). Anal.
Calcd for C36H35N5O3: C, 73.82; H, 6.02; N, 11.96. Found: C,
68.39; H, 5.85; N, 11.60.
By the same procedure as that described for 24, from SmCl3
(0.21 g, 0.79 mmol), 17 (0.25 g, 0.72 mmol), 10% Pd–C (0.2 g), 10
(0.693 g, 1.19 mmol) and (CH3)3Al (2 M in hexane, 0.75 mL), 26
was obtained as a pale yellow solid (0.37 g, 56%), mp: 131–
133 °C (dec). 1H NMR (CDCl3): d 8.56 (1H, s), 7.96 (2H, dd, J = 0.8,
8.1 Hz), 7.62 (2H, dd, J = 0.8, 8.1 Hz), 7.46–7.30 (4H, m), 7.08 (1H,
d, J = 1.8 Hz), 6.98 (1H, s), 6.89 (1H, d, J = 1.8 Hz), 6.59 (1H, s),
6.38 (1H, d, J = 1.8 Hz), 6.28 (1H, d, J = 1.8 Hz), 6.23 (1H, d,
J = 1.8 Hz), 6.20 (1H, d, J = 1.8 Hz), 5.78 (1H, s), 3.83 (3H, s), 3.58
(3H, s), 3.54 (3H, s), 3.44 (4H, t, J = 7.2 Hz), 2.91 (3H, s), 2.16–2.10
(16H, m), 1.72–1.65 (4H, m), 1.53 (3H, s) 13C NMR (CDCl3): d
179.6, 176.6, 176.0, 175.6, 170.8, 169.8, 159.6, 159.3, 158.5,
158.0, 131.4, 130.7, 129.1, 128.2, 128.1, 127.7, 126.3, 125.5,
124.9, 123.7, 123.5, 122.2, 121.9, 120.5, 120.1, 119.4, 118.9,
117.0113.3, 104.3, 103.9, 103.4, 57.0, 46.1, 45.4, 36.8, 36.7, 35.6,
13.8. MS (FAB): m/z (%) 839 (M+1, 43), 717 (11.64), 286 (29.71),
277 (15.81), 271 (25.40), 244 (100), 214 (31.05), 149 (87.91), 123
(64.75), 85 (86.34). Anal. Calcd for C47H54N10O5ꢃ1.5H2O) C, 65.18;
H, 6.63; N, 16.17. Found: C, 65.37; H, 6.56; N, 15.83.
5.12. In vitro anti-tumor assays
The NCI’s in vitro anti-tumor screen (Alley, Scudiero, et al.,
1988; Boyd, 1989; Boyd and Paull, 1995)13,50,51 consists of 60 hu-
man tumor cell lines against which compounds 22–33 are tested
at a minimum of five concentrations at 10-fold dilutions. A 48 h

1680
X. Han et al. / Bioorg. Med. Chem. 17 (2009) 1671–1680
28. Han, X.; Twamley, B.; Natale, N. R. J. Heterocycl. Chem. 2003, 40, 539.
29. Han, X.; Natale, N. R. J. Heterocycl. Chem. 2001, 38, 415.
30. Eliadis, A.; Phillips, D. R.; Reiss, J. A.; Skorobogaty, A. J. Chem. Soc., Chem.
Commun. 1988, 1049.
continuous drug exposure protocol is used, and a sulforhodamine B
(SRB) protein assay is used to estimate cell viability or growth.
31. Bai, R. L.; Paull, K. D.; Herald, C. L.; Malspeis, L.; Pettit, G. R.; Hamel, E. J. Biol.
Chem. 1991, 266, 15882.
Acknowledgments
32. Paull, K. D.; Lin, C. M.; Malspeis, L.; Hamel, E. Cancer Res. 1992, 52, 3892.
33. Mergny, J. L.; Helene, C. Nature Med. 1998, 4, 1366.
34. Mergny, J. L.; Mailliet, P.; Lavelle, F.; Riou, J. F.; Laoui, A. Anti-Cancer Drug Des.
1999, 14, 327.
N.R.N. thanks the National Institute of Neurological Disease and
Stroke for Grant NS38444 and P20RR015583. We would like to
thank the University of Idaho Research Council, and the National
Cancer Institute for financial support. Dr. Dan Zaharevitz assisted
us with the COMPARE calculation and interpretation. X.H., C.L.
and K.C.R., acknowledge the Malcolm and Carol Renfrew Scholar-
ship Endowment, University of Idaho. C.L. and K.C.R. also thank
P20RR16454.
35. Kerwin, S. M. Curr. Pharm. Des. 2000, 6, 441.
36. Cuesta, J.; Read, M. A.; Neidle, S. Mini-Revi. Med. Chem. 2003, 3, 11.
37. Youngquist, R. S.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 2565.
38. White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1997, 4, 569.
39. Arola, A.; Vilar, R. Curr. Top. Med. Chem. 2008, 8, 1405.
40. Wakefield, B. J.; Wright, D. J. Adv. Heterocycl. Chem. 1979, 25, 147.
41. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J. Cancer
Res. 1988, 48, 589.
42. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91.
43. (a) Mitani, S.; Kamata, H.; Fujiwara, M.; Aoki, N.; Tango, T.; Fukuchi, K.; Oka, T.
Clin. Exp. Med. 2001, 1, 105; (b) Broers, Jos L. V.; Viallet, Jean; Jensen, Sandra M.;
Harvey, Pass; Travis, William D.; Minna, John D.; Linnoila, R. Ilona. Am. J. Res.
Cell Mol. Biol. 1993, 9, 33; (c) Arango, D.; Mariadason, J. M.; Wilson, A. J.; Yang,
W.; Corner, G. A.; Nicholas, C.; Aranes, M. J.; Augenlicht, L. H. Br. J. Cancer 2003,
89, 1757; (d) Simonsson, T.; Pecinka, P.; Kubista, M. Nucleic Acids Res. 1998, 26,
1167.
44. (a) Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott, B. J.; Mayo, J. G.;
Malspeis, L.; Grever, M. R. Life Sci. 1995, 57, 131; (b) Bollini, M.; Casal, J. J.;
Bruno, A. M. Bioorg. Med. Chem. 2008, 16, 8003.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.056.
References and notes
1. Hurley, L. H. J. Med. Chem. 1989, 32, 2027.
45. Han, F. X.; Wheelhouse, R. T.; Hurley, L. H. J. Am. Chem. Soc. 1999, 121, 3561.
46. Wozczk, M.; Grzesiak, E.; Kowalczyk, D.; Ostaszewski, R. Proc. SPIE 2002, 4749,
259.
2. Damia, G.; Broggini, M. Eur. J. Cancer 2004, 40, 2550.
3. Di Marco, A. Antibiot. Chemother. 1978, 23, 216.
4. Dervan, P. B. Science 1986, 232, 464.
47. Kumar, C. V.; Asuncion, E. H. J. Am. Chem. Soc. 1993, 115, 8541.
48. Samudrala, R.; Zhang, X.; Wadkins, R. M.; Mattern, D. L. Bioorg. Med. Chem.
2007, 15, 186.
5. Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215.
6. Bailly, C.; Chaires, J. B. Bioconjugate Chem. 1998, 9, 513.
7. Cech, T. R. Nature 1988, 332, 777.
49. Zhuang, X.; Tang, J.; Hao, Y.; Tan, Z. J. Mol. Recognit. 2007, 20, 386.
50. http://dtp.nci.nih.gov/docs/compare/compare_intro.html. Last accessed on
Nov. 25, 2008.
8. Zahler, A. M.; Williamson, J. R.; Cech, T. R.; Prescott, D. M. Nature 1991, 350, 718.
9. Cech, T. R. Angew. Chem. 2000, 39, 34.
10. Phan, A. T.; Mergny, J.-L. Nucleic Acids Res. 2002, 30, 4618.
11. Verner, E. J.; Oliver, B. J.; Schlicksupp, L.; Natale, N. R. Heterocycles 1990, 31, 327.
12. Zhou, P.; Mosher, M. D.; Taylor, W. D.; Crawford, G. A.; Natale, N. R. Bioorg. Med.
Chem. Lett. 1997, 7, 2455.
51. Boyd, M. R.; Paull, K. D.; Rubinstein, L. R. In Cytotoxic Anticancer Drugs: Models
and Concepts for Drug Discovery and Development; Vleriote, F. A., Corbett, T. H.,
Baker, L. H., Eds.; Kluwer Academic: Hingham, MA, 1992; p 11.
52. Li, C.; Twamley, B.; Natale, N. R. Acta Crystallogr., Sect. E 2006, E62, o854.
53. Li, C.; Twamley, B.; Natale, N. R. J. Heterocycl. Chem. 2008, 45, 259.
54. Goodsell, D.; Dickerson, R. E. J. Med. Chem. 1986, 29, 727.
55. Goodsell, D. S.; Ng, H. L.; Kopka, M. L.; Lown, J. W.; Dickerson, R. E. Biochemistry
1995, 34, 16654.
13. Boyd, M. R. Principles Pract. Oncol. 1989, 3, 1.
14. Sausville, E. A.; Johnson, J. I.; Cragg, G. M.; Decker, S. Cancer Drug Discovery and
Development: New Paradigms for a New Millennium. In Anticancer Agents,
Frontiers in Cancer Chemotherapy; Ojima, I., Vite, G. D., Altmann, K., Eds.; ACS
Symposium Series 796; ACS: Washington, DC, 2001; p 1.
15. Han, H.; Hurley, L. H. Trends Pharmacol. Sci. 2000, 21, 136.
16. Hurley, L. H.; Wheelhouse, R. T.; Sun, D.; Kerwin, S. M.; Salazar, M.; Ferdoroff, O.
Y.; Han, F. X.; Han, H.; Zbicka, E.; Von Hoff, D. D. Pharmacol. Ther. 2000, 85, 141.
17. Neidle, S. Chem. Br. 2000, 36, 27.
18. Neidle, S.; Kelland, L. R. Anti-Cancer Drug Des. 1999, 14, 341.
19. Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 11593.
20. Mehta, A. K.; Shayo, Y.; Vankayalapati, H.; Hurley, L. H.; Schaefer, J. Biochemistry
2004, 43, 11953.
21. Gendler, P. L.; Rapoport, H. J. Med. Chem. 1981, 24, 33.
22. Nishiwaki, E.; Tanaka, S.; Lee, H.; Shibuya, M. Heterocycles 1988, 27, 1945.
23. Mosher, M. D.; Natale, N. R. J. Hetereocycl. Chem. 1995, 32, 779.
24. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 4171.
25. Mosher, M. D.; Natale, N. R.; Vij, A. Acta Crystallogr., Sect. C 1996, C52, 2513.
26. Shen, Z. Q.; Wang, F. S.; Hu, Z. Y.; Yu, F. S.; Qian, B. G. J. Polym. Sci., Polym. Chem.
Ed. 1980, 18, 3345.
56. Zamponi, G.; Stotz, S. C.; Staples, R. J.; Rogers, T. A.; Nelson, J. K.; Hulubei, V.;
Blumenfeld, A.; Natale, N. R. J. Med. Chem. 2003, 46, 87.
57. Parkinson, G. N.; Lee, M. P.; Neidle, S. Nature 2002, 417, 876.
58. Clark, G. R.; Pytel, P. D.; Squire, C. J.; Neidle, S. J. Am. Chem. Soc. 2003, 125, 4066.
59. (a) Kim, N. M.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho,
P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science
1994, 266, 2011; (b) Gomez, D.; Mergny, J.-L.; Riou, J.-F. Cancer Res. 2002, 62,
3365.
60. Li, C.; Rider, K.; Fusco, W.; Natale, N. R.; Crawford, R. Synthesis and Activity of
Anti-cancer Compounds that Target G-quadruplex DNA, 227th ACS National
Meeting, Anaheim, CA, March 28–April 1, 2004. MEDI 142. See also, Chemical &
Engineering News, March 8, 2004, p. TECH-76.
61. DeCian, A.; Cristofari, G.; Reichenbach, P.; De Lemos, E.; Monchaud, D.;
Teulade-Fichou, M.; Shin-ya, K.; Lacroix, L.; Lingner, J.; Megny, J. PNAS 2007,
104, 17347.
62. (a) Rosu, F.; Gabelica, V.; Houssier, C.; Colson, P.; Pauw, E. D. Rapid Commun.
Mass Spectrom. 2002, 16, 1729; (b) Rosu, F.; Gabelica, V.; Shin-ya, K.; De Pauw,
E. Chem. Commun. 2003, 2702.
27. Han, X.; Li, C.; Rider, K. C.; Blumenfeld, A.; Twamley, B.; Natale, N. R.
Tetrahedron Lett. 2002, 43, 7673.