Redesigning the DNA-Targeted chromophore in platinum-acridine anticancer agents: A structure-activity relationship study

DOI: 10.1002/chem.201404845

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Anticancer Drugs |Hot Paper|

Redesigning the DNA-Targeted Chromophore in Platinum–

Acridine Anticancer Agents: A Structure–Activity Relationship

Study

Amanda J. Pickard,^[a]Fang Liu,^[a]Thomas F. Bartenstein,^[a]Laura G. Haines,^[b]Keith E. Levine,^[b]

Gregory L. Kucera,^[c]and Ulrich Bierbach*^[a]

Abstract: Platinum–acridine hybrid agents show low-nano-

molar potency in chemoresistant non-small cell lung cancer

(NSCLC), but high systemic toxicity in vivo. To reduce the

promiscuous genotoxicity of these agents and improve their

pharmacological properties, a modular build–click–screen

approach was used to evaluate a small library of twenty

hybrid agents containing truncated and extended chromo-

phores of varying basicities. Selected derivatives were resyn-

thesized and tested in five NSCLC cell lines representing

large cell, squamous cell, and adenocarcinomas. 7-Amino-

benz[c]acridine was identified as a promising scaffold in

a hybrid agent (P1–B1) that maintained submicromolar ac-

tivity in several of the DNA-repair proficient and p53-mutant

cancer models, while showing improved tolerability in mice

by 32-fold compared to the parent platinum–acridine (P1–

A1). The distribution and DNA/RNA adduct levels produced

by the acridine- and benz[c]acridine-based analogues in NCI-

H460 cells (confocal microscopy, ICP-MS), and their ability to

bind G-quadruplex forming DNA sequences (CD spectrosco-

py, HR-ESMS) were studied. P1–B1 emerges as a less geno-

toxic, more tolerable, and potentially more target-selective

hybrid agent than P1–A1.

Introduction

Platinum-based drugs continue to be a mainstay in chemo-

therapy regimens for the treatment of a multitude of solid ma-

lignancies.^[1]Despite improvements in their pharmacological

properties and safety profile, many tumors are inherently in-

sensitive to this form of therapy. In several intractable forms of

cancer, such as non-small-cell lung cancer (NSCLC), current

platinum chemotherapies provide only a moderate survival

benefit both in the adjuvant and palliative setting.^[2]To extend

the spectrum of activity of the classical anticancer metal, plati-

num complexes have been introduced as reactive, electrophilic

components into multifunctional DNA-targeted therapies, such

as platinum–intercalator agents.^[3,4]

Figure 1. General structure of platinum–acridine hybrid agents.

by cisplatin^[3]and structurally related monofunctional-noninter-

calative DNA adducts.^[5]The hybrid adducts form at a signifi-

cantly higher rate and are a more severe form of DNA damage

than the classical cross-links.^[3]They effectively inhibit DNA syn-

thesis in treated cells and lead to stalled replication forks and

DNA double-strand breaks, which elicits responses from speci-

alized DNA damage recognition and repair modules.^[6]In addi-

tion, the hybrid adducts are significantly more potent inhibi-

tors of RNA polymerase II (Pol II)-mediated transcription than

the cross-links.^[7]Fluorescent post-labeling in whole lung

cancer cells recently revealed high levels of DNA adducts in all

phases of the cell cycle, both in the loosely packaged and the

condensed chromatin.^[8]The cumulative effects of this damage

result in cell cycle arrest in late G1/early S phase and efficiently

trigger apoptosis in NSCLC models representing a diverse

range of genetic backgrounds.^[9]Hybrids derived from the gen-

eral structure in Figure 1 effectively kill cancer cells at low-

nanomolar concentrations, which places them among the

The platinum–acridine agents represented by the general

structure in Figure 1 form mixed monofunctional-intercalative

DNA adducts distinct from the bifunctional cross-links induced

[a] Dr. A. J. Pickard, Dr. F. Liu, T. F. Bartenstein, Prof. U. Bierbach

Department of Chemistry, Wake Forest University

Winston-Salem, NC 27109 (USA)

E-mail: bierbau@wfu.edu

[b] L. G. Haines, Dr. K. E. Levine

RTI International, Research Triangle Park, NC 27709 (USA)

[c] Prof. G. L. Kucera

Department of Internal Medicine, Section on Hematology and Oncology

Wake Forest University Health Sciences

Winston-Salem, NC 27157 (USA)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/chem.201404845.

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most cytotoxic platinum-containing anticancer agents de-

scribed in the literature to date.^[6]

duced cationic charge at neutral physiological pH. In addition

to the desired effects on the target binding, reducing the ba-

sicity and hydrophilicity of the chromophore also has the po-

tential to improve important pharmacokinetic parameters of

the hybrids, such as uptake from circulation, membrane per-

meation, and tissue distribution.^[14]

Despite their promising performance in models of intracta-

ble cancers in vitro and in vivo, platinum–acridines show un-

favorable ADME^[10](absorption, distribution, metabolism, and

excretion) properties. They are highly hydrophilic, dicationic

molecules, which insufficiently accumulate in tumor tissue and

produce high levels of platinum in the kidneys and other

normal tissues when administered to mice.^[11]Like other cyto-

toxic agents, platinum–acridines exert their antitumor proper-

ties by indiscriminately damaging an essentially non-cancer-

specific target, chromosomal DNA,^[12]which likely also contrib-

utes to their low tolerability observed in test animals.^[11]Thus,

the remaining challenges with this type of anticancer agent re-

volve around improving its drug-like properties and reducing

its dose-limiting toxicities, while retaining clinically useful anti-

cancer activity.

Computational study of DNA-targeted chromophores

We used self-consistent reaction field DFT-B3LYP level calcula-

tions^[15]as a pre-screening platform to evaluate proton affini-

ties for a set of extended or truncated aromatic moieties de-

rived from the acridine scaffold. The virtual library included

substituted and unsubstituted 7-aminobenz[c]acridine (3–3d),

7-aminodibenz[b,h][1,6]naphthyridine (4–4b), 7-aminobenz[b]-

[1,10]phenanthroline (5–5b), and 7-aminodibenz[c,h]acridine

(6) (Figure 2). Computations were also performed on the

In the current study we used a combination of computation-

al and modular-synthetic library screening to devise a simple

strategy by which the pharmacological and target binding

properties of the platinum–acridines can be tuned. The ap-

proach involved attenuating the proton affinity and altering

the size of the hybrid agent’s intercalator moiety. Benz[c]acri-

dine was identified as a promising planar scaffold in a more

tolerable hybrid that maintains submicromolar activity in

a panel of NSCLC cell lines. Major differences between the acri-

dine- and benz[c]acridine-based hybrids emerged with respect

to their subcellular distribution and target binding levels.

Results

Design

In the new analogues the basic design of the hybrid agent

(Figure 1), in particular the monofunctional platinum and the

N-(2-aminoethyl)-N-alkylamidine linker connecting the plati-

num and intercalator moieties, was not changed. These previ-

ously optimized critical motifs allow strainless intercalation/p-

stacking of the chromophore with DNA bases adjacent to the

sites of platination.^[3]Systematic changes to the chromophore

were desired that reduce the electrostatically driven associa-

tion and promiscuous reactivity of the platinum–acridines with

nuclear DNA. The approach involved reducing the cationic

charge of the DNA-interacting chromophore and modifying its

shape and size. It consolidates two previously articulated con-

cepts: i) the design of “less-than-ideal” intercalators (deproton-

ated/neutral, asymmetric chromophores featuring fewer or

more than three fused aromatic rings), which reduces DNA af-

finity and enhances transcellular diffusion and tumor penetra-

tion,^[13]and ii) the design of compounds that interact with the

genome more selectively by targeting specific DNA structures

and associated processes critical for cancer cell viability (e. g.,

G-quadruplex-forming sequences).^[12]

Figure 2. Chromophores studied in computational screen.

parent 9-aminoacridine (2) and the truncated chromophore, 4-

aminoquinoline (1) (Figure 2). The final choice of modifications

and substitution patterns to achieve the desired electronic and

steric effects was based on chemical entities that are syntheti-

cally readily accessible. The proposed chromophores (except

for 4-aminoquinoline, 1) can be assembled by using simple

condensation and cyclization chemistries (see Scheme 1).

DFT calculations were performed for chromophores 1–6 in

their protonated (at the endocyclic nitrogen atom highlighted

in Figure 2) and unprotonated forms in a simulated dielectric.

The differences between the free energies of solvated proton-

ated and solvated unprotonated species (DG_proton.) were used

as a predictor of relative chromophore basicities.^[16]On the

basis of the calculated energies (Table 1), the nitrobenz[c]acri-

dine derivatives 3d and 3c can be expected to be the least

The new generation of compounds modeled from the plati-

num–acridine hybrid agents therefore display a truncated or

extended planar N-heterocyclic aromatic moiety and reduced

basicity of the endocyclic nitrogen, resulting in an overall re-

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Synthesis of selected chromo-

phores, library assembly, and

pre-screening

Introducing the selected chro-

mophores as components in

platinum–intercalator hybrids re-

quired installation of 2-methyla-

minoethyl side chains at the 4-,

9-, and 7-amino positions of the

aromatic systems to produce

precursors Q1, A1, and B1–B3

(Figure 3A), respectively. Mod-

ules Q1 and A1 were synthe-

sized from a selectively protect-

ed diamine linker and the appro-

priate phenoxy-substituted quin-

oline and acridine precursors

using

a

previously reported

Scheme 1. Synthesis of benz[c]acridine chromophores. a) POCl₃, 808C, b) (1) N-methylethylenediamine, dioxane,

reflux, (2) 2m NH₄OH; c) NaOH, phenol, 1208C; d) (1) N-methylethylenediamine, dioxane, reflux, (2) 2m NH₄OH; e)

(1) tert-butyl(2-aminoethyl)methylcarbamate, triethylamine, dioxane, reflux, (2) HCl/AcOH, (3) 2m NaOH.

scheme.^[18]The synthesis of the

benz[c]acridine derivatives, B1–

B3 (Scheme 1), commenced with

classical Ullman-type condensa-

basic chromophores in the series and 5–5b the most basic,

while the data suggests an intermediate basicity for the parent

acridine (2). Only chromophores predictably less basic than 9-

aminoacridine (pK_a!9.5^[17]), which would effectively reduce

the charge on the new hybrids at neutral physiological pH,

were considered as candidates for the assembly of a synthetic

library. To produce the desired structural and electronic diversi-

ty, the 7-aminobenz[c]acridine motifs 3, 3c, and 3d, along with

9-aminoacridine (2) and 4-aminoquinoline (1), were selected as

components in the synthesized intercalator modules.

tion of the appropriate substituted o-chlorobenzoic acid and

1-napthylamine, and subsequent cyclization of the condensa-

Table 1. Relative free energies of protonation calculated for model chro-

mophores.

[a]

Chromophore

DG_proton.(kcalmol^ꢀ1

)

3d

3c

4a

4b

4

3a

3b

1

6

3

2

5a

5b

5

ꢀ238.01*

ꢀ238.10*

ꢀ240.09

ꢀ240.89

ꢀ243.35

ꢀ243.58

ꢀ244.51

ꢀ244.89*

ꢀ245.65

ꢀ246.99*

ꢀ249.22*

ꢀ254.33

ꢀ255.27

ꢀ257.60

[a] Calculated for energy-optimized geometries in a self-consistent reac-

tion field mimicking the dielectric constant of water using density func-

tional theory (DFT). Proton affinities of structures increase from top to

bottom. Chromophores synthesized in this study are highlighted by as-

terisks. Free energies were defined as DG_proton.=DG_{(solv. BH+)}ꢀDG_{(solv. B)}. Be-

cause only relative energies between B and BH⁺are relevant, DG_(solv.

was not included in the calculations.

H+)

Figure 3. Chromophore modules (A), platinum modules (B), and micro-scale

“click” reactions (C) used in library assembly.

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tion products with POCl₃.^[19]Because the 7-position in the ni-

trobenz[c]acridines was activated for nucleophilic aromatic

substitution, both the 7-chloro and 7-phenoxy intermediates

readily and selectively reacted with the primary amino group

of N-methylethylenediamine to produce the desired linker-

modified chromophores B2 and B3. By contrast, the unsubsti-

tuted benz[c]acridine intermediates B1.2 and B1.3 were signifi-

cantly less reactive, and introduction of the (2-methylami-

no)ethyl linker to produce B1 required longer reaction times

and protection of the secondary amino group.

Four platinum–nitrile precursor complexes, P1–P4, were syn-

thesized as building blocks for assembling the library of hybrid

molecules (Figure 3B). The diam(m)ine nonleaving groups were

varied across the set of complexes as an additional means of

tuning the DNA binding of the

ing without isolation and purification of target compounds

(see Experimental Section for details).

The entire library was tested in NCI-H460 (large cell carcino-

ma, wild-type p53) and NCI-H520 (squamous cell carcinoma,

mutated p53) lung cancer cells. The chemosensitivity of the

two cell lines was assessed for three concentrations of the

newly generated platinum chromophores (P#-Q/A/B#) and

platinum-free chromophores (Q1, A1, B1, B2, and B3; 0.1, 1.0,

and 10 mm, 72 h incubations) using a colorimetric cell prolifera-

tion assay. (The precursor complexes P1, P2, P3, and P4, which

are unstable in aqueous media and biologically inactive,^[20]

were not screened.)

The resulting cell viabilities for treated cells are plotted rela-

tive to controls in Figure 4. Analysis of the two data sets re-

hybrids. Ammine (NH₃), ethane-

1,2-diamine (en), propane-1,3-di-

amine (pn), and N¹,N¹,N²,N²-tetra-

methylethane-1,2-diamine

(tmeda) were introduced as sub-

stitution-inert ligands. Previous

mechanistic studies have dem-

onstrated that the monofunc-

tional platinum moieties modi-

fied with pn (module P3) react

with DNA most rapidly, whereas

introduction of bulky tmeda

(module P4) as

a spectator

ligand almost completely elimi-

nates the formation of perma-

nent platinum–DNA adducts.^[3]

We rationalized these observa-

tions

for

platinum–amidine

model complexes by performing

DFT calculations on transition

states associated with both

aquation and nucleobase bind-

ing. On the basis of the free en-

Figure 4. Biological activity profiles of 20 compounds based on viabilities of drug-treated cells compared to un-

treated controls as monitored by the MTS assay. Compounds were tested in a) NCI-H460 cells and b) NCI-H520

cells dosed for 72 h with 0.1 mm drug (white bars), 1 mm drug (shaded bars) and 10 mm drug (solid bars) concen-

trations. Compounds chosen for further evaluation, along with their precursors, are highlighted as red bars. Plot-

ted data are the mean of three individual experiments ꢂthe standard deviations.

¼

ergies of activation (DG ) ex-

tracted from the modeled struc-

tures (see the Supporting Infor-

mation), the predicted trend in

reactivity is pn > NH₃ꢁ en >

tmeda, which is in agreement

with previous experimental findings.^[3]

veals the following trends: the hybrids and free chromophores

Next, we used a combinatorial assay designed for platinum–

acridines,^[20]which takes advantage of metal-facilitated amine–

nitrile addition chemistry, to assemble and pre-screen a small

library of target molecules. In the process, an amidine donor

group is formed, which establishes connectivity between the

chromophore and the platinum moiety (Figure 3C). Using the

five linker-modified chromophores and four platinum precur-

sors, twenty micro-scale reactions were assembled to generate

a library of twenty hybrid agents (Figure 3C). Reactions were

monitored by in-line high performance liquid chromatogra-

phy/electrospray mass spectrometry (LC-ESMS; see the Sup-

porting Information) and subjected to cell viability pre-screen-

generally perform better in NCI-H460 than in NCI-H520. In

both cell lines, the acridine-based hybrids containing P1, P2,

and P3 show the most pronounced cytotoxic response. They

are the only derivatives that show a response at the lowest

dose tested (0.1 mm). When grouped according to common

chromophore, the order of cell kill potential across the entire

five sets of compounds is A1 > B1 > B2 ꢁ B3 > Q1 in NCI-

H460, and A1 > B1 > B2 > B3 > Q1 in NCI-H520. Within each

set, hybrids containing P4 produce the lowest cytotoxicity,

consistent with their inability to produce permanent DNA ad-

ducts.^[3]Platinum–acridine hybrids generated from P1, P2, and

P3 are cytotoxic at concentrations two orders of magnitude

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lower than those observed for unmodified A1. For the plati-

num–benz[c]acridine hybrids containing B1, a less pronounced

enhancement is observed, and the opposite effect appears to

exist for hybrids derived from the nitrobenz[c]acridines B2 and

B3. On the basis of the preliminary structure–activity relation-

ships established in this assay and the aforementioned design

rationale, P1–A1, P1–B1, and P1–B2 were selected for scaled-

up synthesis and testing in an extended panel of NSCLC cell

lines.

dine system, shows a decrease of two orders of magnitude in

chromophore basicity. Modification of the benz[c]acridine

moiety with an electron-withdrawing 2-nitro group in P1–B2

results in another decrease in pK_aby 1.5 units. Thus, at close-

to-neutral pH, P1–A1 will exist as a fully protonated dication,

whereas P1–B1 and P1–B2 will form partially protonated and

fully deprotonated monocationic species, respectively.

The benz[c]acridine hybrids are noticeably less soluble than

P1–A1 in relevant aqueous buffers, most likely due to their re-

duced cationic charge and the increased lipophilicity of the

chromophores, which show a tendency to self-aggregate in so-

lution (for absorbance and fluorescence spectra, see the Sup-

porting Information). Because of the high degree of aggrega-

tion, attempts to determine reliable partition coefficients for

P1–B1 and P1–B2 in octanol/water were unsuccessful. Despite

these features, the chloride salts of both hybrid agents remain

sufficiently soluble (min. 200 mm in cell culture media when

solubilized with DMF or dmso).

Synthesis, physicochemical characterization, and biological

activity of selected target compounds

The three hybrids, P1–A1, P1–B1, and P1–B2, were generated

on a larger synthetic scale utilizing the same addition chemis-

try as shown in Figure 3. They contain an NH₃-substituted plati-

num moiety of intermediate reactivity and the same amidine-

modified linker group, but vary by the nature of the DNA-tar-

geted chromophores (Figure 5). All hybrids were isolated in

their monocationic form. The experimental pK_avalues for the

fully protonated, dicationic forms of the hybrids (Table 2 and

the Supporting Information) are in agreement with the trends

predicted from computational data and support the overall

design. Compound P1–A1 shows acid–base properties charac-

teristic of the 9-aminoacridine chromophore in platinum–acri-

dines (typical pK_arange: 9–10^[19]). By contrast, P1–B1, contain-

ing a significantly less basic, unsubstituted 7-aminobenz[c]acri-

IC₅₀values were determined for the three hybrids in large

cell carcinoma (NCI-H460), squamous cell carcinoma (NCI-

H520), and adenocarcinoma (NCI-H522, NCI-H1435, A549)

models of NSCLC (Table 2). For comparison, inhibitory concen-

trations were also determined for A1, B1, and B2 in selected

cell lines (Supporting Information). The data acquired for the

hybrids across the whole panel of cell lines confirms the gener-

al trend observed in the pre-screen, in which P1–A1 proved to

be the most cytotoxic and P1–B2 the least cytotoxic of the

three derivatives. P1–A1 shows the highest cytotoxicity in NCI-

H460 (wild-type p53) and NCI-H522 (mutated p53), in agree-

ment with previously reported data on chemically related hy-

brids.^[3,9a]IC₅₀values in the low nanomolar range are also ob-

served in A549. In NCI-H1435, a notoriously cisplatin-resistant

adenocarcinoma cell line,^[21]P1–A1 maintains submicromolar

activity. By contrast, P1–B1 and P1–B2 are less effective and re-

quire higher submicromolar and micromolar doses, respective-

ly, to achieve the same cell kill effect.

In NCI-H460 and NCI-H520 cells, P1–A1 shows greatly en-

hanced cytotoxicity compared to that of A1 by 520-fold and

100-fold, respectively. A less pronounced effect of approxi-

mately 4-fold is observed for P1–B1 when compared to B1,

whereas no significant difference in cytotoxicities exists be-

tween P1–B2 and B2. The largest fold variation in sensitivity of

a given cell line to the three hybrids is observed for A549

(2000-fold), while the smallest variation is observed for chemo-

Figure 5. Hybrid agents synthesized for further evaluation.

Table 2. IC₅₀values (mm) for platinum–chromophore hybrids in lung cancer cell lines^[a]and acid/base properties of their fully protonated, dicationic

forms.^[b]

Compd

NCI-H460^[c]

NCI-H520^[d]

NCI-H522^[d]

NCI-H1435^[d]

A549^[c]

pK_a

P1–A1

P1–B1

P1–B2

0.0052ꢂ0.0001 (520)^[e]

0.24ꢂ0.01 (3.8)

2.4ꢂ0.5 (0.9)

0.043ꢂ0.004 (100)

0.52ꢂ0.01 (3.5)

2.2ꢂ0.1 (1.5)

0.010ꢂ0.001

0.12ꢂ0.02

3.62ꢂ0.08

0.84ꢂ0.07

1.48ꢂ0.01

11.0ꢂ0.2

0.0065ꢂ0.0002

0.32ꢂ0.06

9.4ꢂ0.2

7.6ꢂ0.3

4.9ꢂ0.6

12.4ꢂ0.9

[a] Concentrations of compound that reduce cell viability by 50%, extracted from dose-response curves for 72 h drug incubations using a colorimetric

(MTS) cell proliferation assay. Values are means of two individual experiments performed in triplicate ꢂstandard deviations. [b] Determined from Hender-

son–Hasselbalch plots of UV-visible absorbance data (averages for three titrations ꢂstandard deviations; see the Supporting Information). [c] Wild-type

p53. [d] Mutated p53. [e] Values in parentheses are enhancement factors relative to Pt-free ligands A1, B1, and B2 (see Supporting Information).

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resistant NCI-H1435 (14-fold). For a specific compound, varia-

tions in IC₅₀values of up to 10-fold are observed across the

entire panel of cell lines, with the exception of compound P1–

A1, which proves to be 160-fold more active in NCI-H460 than

in NCI-H1435.

elles, including lysosomes, mitochondria, and endoplasmic re-

ticulum (ER). No nuclear staining was necessary to unequivo-

cally map the nuclei in cells in interphase, which could be

readily identified in the merged and bright field images.

In all analyzed samples, cells incubated with P1–B1 for 1, 3,

or 6 h showed higher fluorescence levels in the blue channel

than cells treated with P1–A1, consistent with a higher uptake

of the less basic and more lipophilic benz[c]acridine derivative.

P1–A1 and P1–B1 show a high degree of colocalization with

LysoTracker, indicating accumulation of the hybrids in this or-

ganelle (Figure 6A), but do not localize to the mitochondria

Distribution and nucleic acid binding in cancer cells

To gain insight into the intracellular distribution of the hybrids,

we used confocal fluorescence microscopy by taking advant-

age of the intrinsic blue fluorescence of the 9-aminoacridine

(in P1–A1) and 7-aminobenz[c]a-

cridine (in P1–B1) chromo-

phores. (The nitrobenzacridine

derivative, B2, was not sufficient-

ly fluorescent for this applica-

tion.) A known drawback of this

approach is that the fluores-

cence of the 9-aminoacridine

chromophore is significantly

quenched when intercalated be-

tween DNA base pairs,^[17]which

complicates the microscopic de-

tection of platinum-acridine as-

sociated with chromatin in the

cell’s nucleus. (A post-labeling

strategy has recently been de-

vised that facilitates the detec-

tion of adducts in the nuclear

DNA and subnuclear structures

in fixed cells.^[8]) By contrast, the

blue fluorescence of benz[c]acri-

dine in P1–B1, which is relatively

weak as a result of self-aggrega-

tion, is slightly enhanced in the

presence of calf thymus DNA

(see the Supporting Informa-

tion).

To study the two hybrid mole-

cules in intact cells, NCI-H460

cells were dosed with P1–A1

and P1–B1 at relatively high

concentrations (5 mm), but only

for a short period of time (max.

6 h). These conditions were nec-

essary to produce a detectable

fluorescence signal in target or-

ganelles without causing unde-

sired cell death and the cyto-

morphological consequences as-

sociated with it (degradation of

the nuclear envelope and frag-

mentation of the nuclear

DNA^[22]). Treated cells were then

co-stained with suitable fluores-

cent dyes to study accumulation

Figure 6. Subcellular distribution of compounds P1–A1 and P1–B1 in NCI-H460 cells. Confocal fluorescence mi-

croscopy images of cells co-stained with LysoTracker Red (A) and ER Tracker Red (B). Panel (C) shows a comparison

of colocalization images captured for cells treated with P1–A1 and P1–B1 for 6 h and co-stained with LysoTracker

Red. The arrows indicate blue-fluorescent intranuclear vesicular structures and regions of intense blue fluores-

of the hybrids in several organ- cence. Scale bars represent a distance of 10 mm.

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(see Supporting Information). The most intense blue fluores-

cence at each time point is detected for both hybrids in the ly-

sosomes. Lysosomal accumulation of P1–B1 is accompanied by

a pronounced decrease of LysoTracker-related red fluorescence

relative to levels in cells treated with P1–A1 (Figure 6A, lower

panel, and Supporting Information). This observation may indi-

cate that P1–B1, a protonable species at neutral cytosolic pH

(pK_a=7.6), but not P1–A1 (pK_a=9.4), increases the lysosomal

pH, resulting in reduced accumulation of the acidotropic Lyso-

Tracker dye (pK_a=7.5^[23]).

Cells incubated with P1–A1 for 1 h and subsequently treat-

ed with red-fluorescent ER Tracker to stain the endoplasmic re-

ticulum of the cytoplasm show acridine-related blue fluores-

cence confined to distinct areas identified as lysosomes (Fig-

ure 6B, upper panel). By contrast, P1–B1 produces significant

blue fluorescence in the area of the rough and smooth endo-

plasmic reticulum across the entire cytoplasm, in the perinu-

clear region, and in the nucleus (Figure 6B, lower panel). Con-

focal images captured of cells treated with P1–A1 and P1–B1

for 6 h and stained with LysoTracker show a high degree of

perinuclear clustering of lysosomes (Figure 6C). In these areas

a large number of blue-fluorescent vesicles are observed,

which are not stained by LysoTracker. From these vesicles,

which appear to localize to, and penetrate, the nuclear mem-

brane, emanate areas of intense blue fluorescence in the nu-

clear region, possibly indicating release of fluorescent hybrid

agent (see highlighted features in Figure 6C). While this causes

high levels of blue fluorescence across the entire nuclear

region in the case of P1–B1, relatively weaker P1–A1-related

fluorescence is observed only in contrast-enhanced images

captured after 6 h of continuous dosing in subnuclear struc-

tures previously identified as nucleoli^[8](Supporting Informa-

tion).

Figure 7. Amounts of DNA (a) and RNA (b) extracted from untreated NCI-

H460 cells and cells treated with 1 mm P1–A1 or P1–B1 for 3 h (black) and

6 h (white). These data were used to determine the Pt/nucleic acid ratio (c)

in DNA after 3 h (black) and 6 h (shaded black), and in RNA after 3 h (gray)

and 6 h (shaded gray) of continuous dosing. Data with one asterisk (*) dis-

plays no statistical difference from control at 95% confidence (a=0.05).

Data with two asterisks (**) displays no statistical difference at 95% confi-

dence (a=0.05), but displays a statistical difference at 90% confidence

(a=0.10) when using a two-tailed t-test assuming unequal variances.

of approximately 70% relative to control cells at the 6 h time

point is observed for both P1–A1 and P1–B1 (Figure 7b), cor-

roborating the notion that both hybrids interfere with the tran-

scription machinery or cause post-transcriptional degradation

of RNA in treated cells.

Analysis of the microscopic images confirms that both P1–

A1 and P1–B1 rapidly enter lung cancer cells and accumulate

at high levels in the lysosomes. The fluorescence intensity ob-

served for P1–B1 is significantly higher than for P1–A1 in the

nucleus and in the cytoplasm, suggesting major differences in

subcellular levels of the two hybrids. The results also suggest

that lysosome-like vesicles may be involved in the cellular traf-

ficking of the hybrid agents and their delivery to the nucleus.

Because fluorescence intensities cannot be directly correlat-

ed with the levels of irreversible damage in chromatin (Pt ad-

ducts) produced by P1–A1 and P1–B1, we extracted and puri-

fied the nuclear DNA from treated NCI-H460 cells and quanti-

fied the platinum content by inductively-coupled plasma mass

spectrometry (ICP-MS). For comparison, the fraction of cellular

RNA was also recovered and analyzed. Cells were dosed with

P1–A1 and P1–B1 at the same time points applied in the

imaging study, but at a lower concentration (1 mm for 3 and

6 h). The amounts of DNA extracted from cells treated with the

hybrids, in particular for the 6 h time point, do not vary signifi-

cantly from the controls (Figure 7a). This observation confirms

the absence of apoptotic cell death in the population of treat-

ed cells, which would have resulted in major fragmentation

and loss of DNA during the extraction procedure. By contrast,

a more dramatic time-dependent reduction in extractable RNA

In DNA samples analyzed by ICP-MS, a rapid and time-de-

pendent accumulation of platinum is observed for P1–A1 (Fig-

ure 7c) at levels consistent with data reported previously for

platinum–acridines.^[24]Quite unexpectedly, accumulation of

platinum at similar high levels was also observed in the ex-

tracted RNA for P1–A1. A different scenario is observed for

P1–B1, for which concentrations of DNA-associated platinum

in cells treated for 3 and 6 h were not statistically different

from control levels. Although P1–B1 does not seem to accu-

mulate in chromosomal DNA, it is possible that this agent se-

lectively targets noncanonical DNA motifs in the genome at

levels undetectable by ICP-MS. Likewise, only slightly higher

levels of platinum than in controls were detected for P1–B1 in

RNA.

Interactions with G-quadruplex DNA

The cumulative data from the experiments performed in

cancer cells suggest that chromosomal dsDNA may not be the

primary cellular target of the benz[c]acridine containing phar-

macophore and that it may trigger cell death by alternate

mechanisms (see also the Discussion). One potential target of

the benz[c]acridine derivatives is G-quadruplex (G4)-forming

DNA, which is ubiquitous in cancer-related genomic sequences

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Figure 8. Interaction of chromophores and the corresponding hybrid agents with G-quadruplex forming sequences (a) (10 mm), and new chromophore and

platinum-chromophore hybrid agents. Circular dichroism (CD) spectra (b–d) of in the interaction between unfolded TEL24 (black dashed line) and increasing

equivalents of a) A1, b) B1, and c) B2 to a final ratio of 5 equiv drug to one quadruplex (thick black line) (10 mm tetrabutylammonium phosphate/1 mm EDTA

buffer, pH 6.75). CD spectra were also acquired of P1–A1 (red), P1–B1 (blue), and P1–B2 (green) incubated with pre-folded G-quadruplex-forming sequences

(same buffer with 100 mm KCl added) for e) TEL24, f) NUC23A, g) NUC23P, and h) NUC25M. i) Induced circular dichroism (ICD) spectra for the chromophore

region of P1–A1 (red), P1–B1 (blue), and P1–B2 (green) in complex with pre-folded TEL24. For representative structures of G4 topologies, see the Supporting

Information.

and considered a preclinically validated anticancer target.^[25]It

is possible to shift the p–p stacking potential of DNA intercala-

tive molecules from classical Watson–Crick to these non-canon-

ical DNA structures. Favorable stacking with the G4 motifs is

typically observed for molecules containing extended, asym-

metric aromatic moieties, such as quindoline^[26]and ellipti-

cine,^[27]which are structurally similar to the benz[c]acridine

scaffold, or with chromophores bearing side chains that disfa-

vor insertion into the DNA duplex (e. g., in 3,6-disubstituted

acridines^[28]).

show characteristic features of parallel (major component) and

antiparallel (minor component, except for B2 in NUC23A) G4

DNA (see the Supporting Information), suggesting that multi-

ple topologies, or mixed topologies with parallel and antiparal-

lel strand polarities, exist in solution. Among the 12 titrations,

addition of B1 into a solution of TEL24 showed the most dis-

tinct change in ellipticities and gave the cleanest conversion

from unfolded single-strand into a single G4 structure, based

on distinct isoelliptic points. The CD features of TEL24 in com-

plex with B1 are unique among all combinations tested, partic-

ularly the strong negative bands at ~260 and ~310 nm. While

the former band is a characteristic feature of the antiparallel

basket conformation of the human telomeric sequence ob-

served in Na⁺solution,^[32]the band at longer wavelength most

likely arises from induced circular dichroism (ICD) or exciton-

coupled benz[c]acridine chromophores,^[33]which absorb in that

region (see the Supporting Information).

We have used a combination of circular dichroism (CD) spec-

troscopy and high-resolution electrospray mass spectrometry

(HR-ESMS) to study the interactions of P1–A1, P1–B1, and P1–

B2, as well as the platinum-free precursors A1, B1, and B2 with

selected G4-forming model sequences. They include a 24-mer

representing the human telomeric repeat ([T₂AG₃]_n),^[29]as well

as three of the guanine-rich sequences found in the genes en-

coding 45S pre-ribosomal RNA (rDNA),^[30]which are transcribed

by RNA polymerase I (Pol I) in the cell’s nucleolus (Figure 8a).^[31]

The ability of A1, B1, and B2 to induce the G4 structure in

each of the sequences was first tested in an unbiased fashion

in a Na⁺- and K⁺-free buffer. In titrations with the three chro-

mophores, the random-coil form of TEL24 is converted into

a quadruplex structure. While A1 produces a mixture of paral-

lel and antiparallel topologies, as indicated by the positive CD

bands at ~260 nm and ~290 nm, respectively,^[32]B1 and B2

stabilize antiparallel forms of the sequence (Figure 8b–d). CD

spectra recorded for the titrations of the three rDNA sequences

Hybrids P1–A1, P1–B1, and P1–B2 were also incubated with

the four sequences in physiologically relevant K⁺buffer, in

which TEL24 and NUC23A form a parallel/antiparallel hybrid

structure^[32]and a mixture of the two forms, respectively (Fig-

ure 8e, f). Under these conditions, NUC23P and NUC25M exist

predominantly in a parallel conformation. P1–A1 causes a no-

ticeable perturbation in the CD bands of TEL24 and NUC23A in

favor of the parallel structural component, whereas P1–B1 and

P1–B2 effect relatively minor structural changes (Figure 8e–h).

To demonstrate differences in the binding modes in TEL24, ICD

spectra were recorded in the ligand region where the p–p

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transitions of the aromatic chromophores are observed (325–

500 nm; Figure 8i). The acridine chromophore in P1–A1 does

not show significant ICD features in complex with the telomer-

ic G-quadruplex, consistent with multiple non-specific binding

modes giving rise to inefficient coupling between the DNA’s

and ligand’s transition dipoles. An entirely different situation is

observed for P1–B1 and P1–B2, which produce strong positive

ICD bands for the transition centered at ~400 nm and

~450 nm (short-axis p–p transition), respectively, and a weaker

negative band around 350 nm (long-axis p–p transition). This

CD signature has been used as a diagnostic of coplanar orien-

tation of the intercalator and adjacent nucleobases in cova-

lent-intercalative DNA adducts,^[34]which strongly supports effi-

cient p–p stacking interactions between the G4 tetrad and

benz[c]acridine chromophores.

Discussion

The 7-aminobenz[c]acridine chromophore was identified as

a promising new building block in platinum–intercalator

hybrid agents, which may help overcome several of the draw-

backs of the platinum–acridines. The goal was to generate

a less genotoxic derivative of the parent platinum–acridine

pharmacophore. As an additional benefit, the desired molecule

would also act by an altered mechanism at the cellular and

molecular level, thereby potentially overcoming resistance in

repair-proficient cancers. Unsubstituted benz[c]acridine (B1) in

combination with a monofunctional DNA platinating moiety

was able to produce submicromolar cell kill in chemoresistant

forms of lung cancer. Hybrids containing substituted nitro-

benz[c]acridine B3 and quinoline Q1 were not pursued further

because they showed poor activity, low solubility, and/or no

significant advantages over the platinum-free chromophores.

NSCLC cell lines characterized by wild-type p53 protein were

exquisitely sensitive to the acridine-based hybrid, P1–A1. P1–

A1 was approximately 50-fold more potent than P1–B1 in NCI-

H460 and A549 cells (Table 2). This observation suggests that

a p53-mediated mechanism^[36]is an important contributor to

the cell kill effected by this highly DNA damaging agent. Char-

acteristically, the cytotoxic advantage of P1–A1 is less pro-

nounced in DNA repair-proficient cells harboring a p53 muta-

tion where relatively higher IC₅₀values are observed (Table 2).

The cell kill potential of P1–B1 appears to be affected by these

parameters to a lesser extent, and the new derivative proved

to be even more cytotoxic in NCI-H522 than in NCI-H460 de-

spite the p53 status and the higher DNA repair activity ob-

served in the former cell line.^[21]P1–B1, while significantly less

cytotoxic than P1–A1, clearly outperforms cisplatin in cell lines

representing this form of cancer.^[9,37]

To confirm adduct formation in the quadruplex-forming se-

quences, samples of the pre-folded G4 structures incubated

with hybrid agent (Figure 8e–i) were also analyzed by HR-

ESMS. The spectra recorded in negative-ion mode provide evi-

dence of substitution of the chloro ligand on platinum by nu-

cleobase nitrogen in all of the sequences for which formation

of 1:1 adducts was predicted from CD/ICD data. (For mass

spectra and assignments of molecular ions, see the Supporting

Information.) On the basis of peak intensities and assuming in-

source dissociation of the resulting platinum–DNA coordinative

bonds is negligible or occurs to a similar extent for the three

hybrids, P1–A1 and P1–B1 generally produce the highest level

of binding, whereas P1–B2 forms adducts at a much lower

yield or proves to be completely unreactive (see the Support-

ing Information). Thus, the combined CD and HR-MS data con-

firm that the hybrids form coordinative adducts with the G-

quadruplex structures in which the planar chromophores are

p-stacked with the guanine tetrads.

Equilibrium dialysis experiments were also conducted to es-

tablish the selectivity of the chromophores (and their platinum

derivatives) for G4 versus double-stranded DNA. Unfortunately,

these experiments were unsuccessful due to irreversible aggre-

gation and deposition of the benz[c]acridine derivatives on all

components of the microdialysis apparatus and membranes.

Lung adenocarcinomas are often highly resistant to

common cytotoxics,^[2b,38]such as cisplatin (e. g., NCI-H1435),

and show high expression levels of antiapoptotic proteins.^[36,39]

On the other hand, the treatment of squamous cell lung can-

cers (e. g., NCI-H520), which show genomic alterations similar

to basal (triple-negative) breast cancer and squamous head

and neck cancer, continues to rely on traditional systemic

therapies.^[40]Unlike the more common adenocarcinomas, they

are notoriously insensitive to common molecularly targeted

therapies, such as inhibitors targeted at epidermal growth

factor receptor tyrosine kinase.^[41]P1–B1 may be a suitable

candidate for tackling these aggressive phenotypes.

In vivo toxicity

The acridine-based hybrid agents have previously shown rela-

tively low toxicity in non-transformed cells,^[35]but were quite

toxic when administered to mice.^[11]The target tissue and cell-

type specificity of the systemic toxicity caused by platinum–ac-

ridines are unknown. To determine if the design of P1–B1

indeed translates into improved in vivo tolerability, a dose es-

calation study was performed in mice. No signs of toxicity and

no weight loss were observed at a dose of 3.2 mgkg^ꢀ1when

administered intraperitoneally (i.p.) on five consecutive days

(see the Supporting Information). By contrast, the maximum

tolerated dose (MTD) for P1–A1 was found to be 0.1 mgkg^ꢀ1

using the same dosing scheme.^[11]These results confirm that

the simple structural modification of extending the aromatic

chromophore is able to reduce the acute toxicity of this type

of hybrid molecule by 32-fold.

The acridine- and benz[c]acridine-based agents share some

common features but also show some critical differences at

the cellular level. Our imaging data demonstrate that the lyso-

somes are a major target organelle of both agents. Excessive

sequestration of drugs into this acidic organelle has been

linked to multidrug resistance, but may also lead to lysosomal

injury, resulting in cell death.^[42]P1–A1 and P1–B1 contain

weakly basic chromophores, making them prime candidates

for lysosomal accumulation. Contributions of lysosome disrup-

tion to the cell death, in particular for the presumably less gen-

otoxic benz[c]acridine derivative, cannot be ruled out.^[43]On

the basis of confocal images, lysosome-type vesicles may also

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be involved in the subcellular trafficking of the hybrid mole-

cules and their delivery across the nuclear membrane.^[44]

cant damage in therapeutically useful G-quadruplex-forming

sequences.

Previous studies on the subcellular distribution of the plati-

num-acridines have shown that while these agents produce

approximately 60-fold higher intracellular levels than cisplatin

they result in 5-fold higher DNA adduct levels than the clinical

drug.^[24]This observation suggests that a major fraction of the

hybrid agents must be present in the cytosol or be associated

with cytoplasmic organelles, which is consistent with their ly-

sosomal accumulation observed in the current study. P1–A1 in

the nucleus of NCI-H460 cells is only very weakly blue fluores-

cent as a consequence of the quenched fluorescence of DNA-

associated 9-aminoacridine, and ICP-MS detection was required

to demonstrate its accumulation in this target organelle. P1–

B1 produced significantly higher fluorescence levels in cyto-

plasmic structures and membranes and also significantly more

intense blue staining of the cells’ nuclei than compound P1–

A1, but ICP-MS was unable to detect platinum in extracted nu-

clear DNA. A plausible explanation for this observation would

be that P1–B1 enters the nucleus but does not accumulate in

chromatin because DNA adduct formation is disfavored. Alter-

natively, it may be reasoned that the blue fluorescence and

platinum do not colocalize due to decomposition of the

hybrid agent, resulting in cleavage of the platinum–benz[c]acri-

dine linkage prior to entering the nucleus. No such fragmenta-

tion, however, has been observed for relevant conditions in an-

alytical assays mimicking the cell incubations performed with

P1–B1, and this possibility can be firmly ruled out.

In conclusion, we have investigated structure–activity rela-

tionships in novel derivatives of platinum–acridines containing

truncated and extended forms of the DNA-targeted 9-amino-

acridine intercalating moiety. Replacement of acridine with

a benz[c]acridine moiety profoundly alters the target binding

properties, cellular pharmacology, and in vivo tolerability of

this type of agent. If the improved safety offsets the relative

loss in cytotoxic potential observed for P1–B1 and results in

a more favorable therapeutic window, this derivative should

have an advantage over P1–A1 in vivo. In addition to redesign-

ing the chromophore, introduction of cleavable, lipophilic “pro-

tecting” groups may turn the benz[c]acridine-based agent into

an even more tolerable prodrug (as demonstrated for the anal-

ogous acridine-based agents).^[50]Finally, potential non-DNA-

mediated mechanisms and mechanisms involving non-canoni-

cal nucleic acid structures remain to be investigated.

Experimental Section

Reagents and general procedures

All reagents were used as obtained from commercial sources with-

out further purification unless indicated otherwise. Compounds

A1,^[18]P1,^[11]and tert-butyl(2-aminoethyl)methylcarbamate^[18]were

synthesized according to reported procedures. Synthetic (desalted)

oligodeoxyribonucleotides were purchased from IDT, Inc. (Coral-

1

ville, IA). H NMR and proton-decoupled ¹³C spectra of the target

The reduced reactivity of P1--B1 relative to P1–A1 with nu-

cleic acids is also reflected in the RNA binding levels. Despite

the difference in nucleic acid affinity, both analogues show the

same effect on the total RNA content of treated cells, which

was unexpected. RNA itself is generally not considered a pri-

mary cancer target of platinum-based therapies, although in-

tracellular platinum levels in this biomolecule are usually

high.^[45]A recent mechanistic study of platinum–acridines

using a yeast-based chemical genomics platform indicated that

severe DNA damage is the major cause of cell kill produced by

these agents.^[6]It also demonstrated that cytotoxic action on

non-DNA targets involved in RNA metabolism as well as on

components of the translational machinery (ribosomal subu-

nits) compromise cell viability.^[6]The distinct nucleic acid

damage profile of P1–B1 in cancer cells warrants further exper-

imentation to determine if RNA-mediated mechanisms may be

involved in its cell kill.

compounds and intermediates were recorded on Bruker Advance

300 or Bruker DRX-500 instruments. Chemical shifts (d) are report-

ed in parts per million (ppm) relative to the internal standard tetra-

methylsilane (TMS). In-line LC-ESMS analyses were performed on

an Agilent 1100 LC/MSD ion trap mass spectrometer equipped

with an electrospray source. HR-ESMS was performed on a Thermo

Scientific LTQ Orbitrap XL equipped with an electrospray source.

All NMR and LC-ESMS spectra were processed by using the Mes-

tReNova Suite (version 8.1.2) equipped with MS plugins. HR-ESMS

data was processed with Xcalibur 2.1 (Thermo Scientific). The ana-

lytical purity of target compounds was determined by reverse-

phase HPLC in conjunction with product analysis by ESMS and was

found to be greater than 95% in all cases (see the Supporting In-

formation). For the preparation of biological buffers, biochemical

grade chemicals (Fisher/Acros) were used. HPLC grade solvents

were used for all HPLC and mass spectrometry experiments. All re-

agents were used as obtained from commercial sources without

further purification unless indicated otherwise. Stock solutions of

platinum complexes in DMF (DMSO for P1–B2) were stored at

ꢀ208C and brought to room temperature immediately before use.

Finally, there has been a longstanding interest in targeting

anticancer agents to G-quadruplex DNA with the goal of trig-

gering apoptotic signals in a more cancer-cell specific way.^[25]

The discovery that platinum-acridines produce adducts in the

human telomeric G-quadruplex at a faster rate than in

dsDNA^[46]prompted us here to also explore the newly de-

signed chromophores as G4-directed ligands. We demonstrat-

ed that benz[c]acridine alone is able to induce the G-quadru-

plex structure in various target sequences and, when intro-

duced into hybrid agents, promotes platination of the DNA

secondary structure without disrupting the G4 tetrad. It is un-

clear if this reactivity is sufficiently selective to produce signifi-

Computational studies

Chromophore models were built using the Gaussview5 program.^[47]

Optimizations and single-point energy calculations were performed

with the Gaussian 09 (G09) software package.^[48]All geometries

were fully optimized in the gas phase at the gradient-corrected

DFT level using the spin-unrestricted B3LYP functional and the 6–

311G** basis set.^[15]Vibrational frequencies were used to confirm

that the optimized structures had converged to their local minima

and the equilibrium structures contained no imaginary frequencies.

Single-point energy calculations were performed using the self-

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consistent reaction field (SCRF) approach,^[49]which simulates a con-

stant dielectric constant of e=78.3553 for water. All models of

platinum complexes and transition states were built, optimized,

and calculated according to a previously published procedure.^[7]

lytical purity: ꢃ 95% by LC-ESMS. ¹H NMR (300 MHz, [D₆]DMSO):

d=9.47 (d, J=2.5 Hz, 1H), 9.30 (d, J=7.7 Hz, 1H), 8.36 (dd, J=9.4,

2.4 Hz, 1H), 8.19 (d, J=9.4 Hz, 1H), 8.10 (d, J=9.4 Hz, 1H), 8.01–

7.92 (m, 1H), 7.85–7.66 (m, 3H), 3.96 (t, J=6.3 Hz, 2H), 2.93 (t, J=

6.2 Hz, 2H), 2.33 ppm (s, 3H); ¹³C NMR (75 MHz, [D₆]DMSO): d=

153.42, 149.57, 149.19, 141.41, 133.98, 130.76, 130.65, 129.36,

127.49, 126.74, 125.39, 123.87, 122.56, 122.35, 121.10, 114.94,

111.93, 50.96, 48.58, 35.62 ppm; UV/Vis (BPES buffer, pH 7.2, 1%

Synthesis of N¹-methyl-N²-(quinolin-4-yl)ethane-1,2-diamine

(Q1)

A mixture of 4-phenoxyquinoline (Q1.1) (504 mg, 2.28 mmol), tert-

butyl(2-aminoethyl)methylcarbamate (773 mg, 4.43 mmol), and

freshly distilled triethylamine (1 mL) in anhydrous dioxane (1 mL)

was heated at reflux for 72 h. The solvent was removed, and the

residue was stirred overnight in 4m HCl. The reaction mixture was

partitioned between a 10% NaOH and chloroform. The chloroform

layer was washed with saturated aqueous NaCl and 2m NaOH,

treated with activated carbon, dried over sodium sulfate, and fil-

tered through a Celite pad. Evaporation of the solvent afforded Q1

as a light yellow oil, which was dried in a vacuum. Yield: 273 mg,

60%. Analytical purity: ꢃ 95% by LC-ESMS. ¹H NMR (300 MHz,

[D₆]DMSO): d=8.38 (d, J=5.3 Hz, 1H), 8.29–8.11 (m, 1H), 7.90–7.71

(m, 1H), 7.59 (ddd, J=8.3, 6.7, 1.4 Hz, 1H), 7.50–7.32 (m, 1H), 7.04

(s, 1H), 6.47 (d, J=5.4 Hz, 1H), 3.35 (t, J=6.5 Hz, 2H), 2.79 (t, J=

6.4 Hz, 2H), 2.34 ppm (s, 3H); ¹³C NMR (75 MHz, [D₆]DMSO): d=

150.58, 149.85, 148.20, 128.92, 128.55, 123.64, 121.51, 118.74, 98.10,

49.36, 41.95, 35.75 ppm; UV/Vis (BPES buffer, pH 7.2, 1% SDS): l_max

SDS): l_max=441 nm, e=4851m^ꢀ1cm^ꢀ1

.

Synthesis of N¹-methyl-N²-(10-nitrobenz[c]acridin-7-yl)ethane-

1,2-diamine (B3)

A

mixture of 10-nitro-7-chlorobenz[c]acridine (B3.2) (251 mg,

0.81 mmol) and N-methylethylenediamine (141 mL, 1.62 mmol) was

stirred at reflux in anhydrous dioxane (4 mL) for 24 h. After the

mixture had cooled to room temperature, the dioxane was re-

moved by rotary evaporation and the residue was suspended in

2m NaOH. After 2 h of stirring, the dark red solid was collected by

vacuum filtration and washed with 2m NH₄OH. The crude product

was partitioned between chloroform and saturated NaCl. The

chloroform layer was washed with 2m NH₄OH, treated with activat-

ed carbon, dried over sodium sulfate, and filtered through a Celite

pad. Rotary evaporation of the solvent afforded B3 as a red solid,

which was dried in a vacuum. Yield: 229 mg, 81%. Analytical

purity: ꢃ 95% by LC-ESMS. ¹H NMR (300 MHz, [D₆]DMSO): d=

9.39–9.23 (m, 1H), 8.82 (d, J=2.5 Hz, 1H), 8.65 (d, J=9.4 Hz, 1H),

8.31–8.02 (m, 2H), 8.04–7.87 (m, 1H), 7.88–7.68 (m, 3H), 3.87 (t, J=

6.2 Hz, 2H), 2.86 (t, J=6.2 Hz, 2H), 2.30 ppm (s, 3H);¹³C NMR

(75 MHz, [D₆]DMSO): d=151.31, 148.87, 147.48, 146.22, 133.57,

130.75, 129.25, 127.54, 126.91, 126.21, 125.14, 125.06, 124.70,

121.23, 120.30, 115.16, 113.34, 51.22, 48.80, 35.64 ppm; UV/Vis

=339 nm, e=13008m^ꢀ1cm^ꢀ1

.

Synthesis of N¹-(benz[c]acridin-7-yl)-N²-methylethane-1,2-di-

amine (B1)

A mixture of 7-phenoxybenz[c]acridine (B1.3) (918 mg, 2.86 mmol),

tert-butyl(2-aminoethyl)methylcarbamate (778 mg, 4.46 mmol), an-

hydrous triethylamine (400 mL, 2.87 mmol) in anhydrous dioxane

(2 mL) was stirred at reflux for five days. The solvent was removed

by rotary evaporation and the remaining residue stirred overnight

in 4 mL of a 3:1 mixture of concentrated glacial acetic acid and

concentrated hydrochloric acid. The acid was removed by rotary

evaporation and the residue was neutralized with 2m NaOH. The

reaction mixture was partitioned between the 2m NaOH solution

and chloroform. The chloroform layer was washed with saturated

NaCl and 2m NaOH, treated with activated carbon, dried over

sodium sulfate, and filtered through a Celite pad. Rotary evapora-

tion of the solvent afforded B1 as a brown oil, which was dried in

a vacuum. Yield: 778 mg, 90%. Analytical purity: ꢃ 95% by LC-

ESMS. ¹H NMR (300 MHz, CDCl₃): d=9.54–9.41 (m, 1H), 8.32–8.18

(m, 2H), 8.01 (d, J=9.4 Hz, 1H), 7.87–7.53 (m, 5H), 7.47 (ddd, J=

8.3, 6.7, 1.3 Hz, 1H), 6.05 (s, 1H), 3.84–3.73 (m, 2H), 2.94–2.84 (m,

2H), 2.53 ppm (s, 3H); ¹³C NMR (75 MHz, [D₆]DMSO): d=151.23,

147.50, 147.23, 133.38, 131.27, 129.47, 129.40, 128.55, 127.36,

126.41, 124.94, 123.31, 123.27, 123.08, 121.67, 118.09, 112.67, 51.42,

BPES Buffer, pH 7.2 with 1% SDS): l_max=403 nm, e=6922m^ꢀ1cm^ꢀ1

.

Synthesis of [PtCl(NH₃)₂(N-(2-(acridin-9-ylamino)ethyl)-N-meth-

ylpropionimidamide)]Cl (P1–A1)

(An improved procedure for this previously synthesized^[11]deriva-

tive is described.) A mixture of [PtCl(EtCN)(NH₃)₂]Cl (P1) (107 mg,

0.30 mmol) and N¹-(acridin-9-yl)-N²-methylethane-1,2-diamine (A1)

(81 mg, 0.32 mmol) in anhydrous DMF (2 mL) was stored at ꢀ208C

for 72 h. Activated carbon was added to the DMF solution, which

was then filtered through a 0.2 mm membrane into vigorously

stirred anhydrous diethyl ether. The yellow precipitate was recov-

ered by vacuum filtration, washed excessively with diethyl ether,

and dried in a vacuum to afford P1–A1 as a yellow solid. Yield:

162 mg, 88%. Analytical purity: ꢃ 95% by LC-ESMS. ¹H NMR

(500 MHz, [D₄]MeOH): d=8.25 (d, J=8.6 Hz, 2H), 7.92–7.55 (m,

5H), 7.35 (dd, J=9.6, 7.5 Hz, 2H), 4.06 (t, J=6.4 Hz, 2H), 3.74 (t, J=

6.3 Hz, 2H), 3.19–2.86 (m, 5H), 1.27 ppm (t, J=7.5 Hz, 4H);

¹³C NMR (75 MHz, [D₄]MeOH): d=171.62, 155.25, 132.36, 129.24,

126.62, 125.96, 123.66, 29.08, 11.53 ppm; HR-ESMS (positive-ion

mode): m/z for C₁₉H₂₈ClN₆Pt [M]⁺, calcd: 570.1706; found: 570.1073

(tolerance: 0.549 ppm).

49.02, 35.66 ppm; UV/Vis (BPES buffer, pH 7.2, 1% SDS): l_max

427 nm, e=10071m^ꢀ1cm^ꢀ1

=

.

Synthesis of N¹-methyl-N²-(9-nitrobenz[c]acridin-7-yl)ethane-

1,2-diamine (B2)

Synthesis of [PtCl(NH₃)₂(N-(2-(benz[c]acridin-7-ylamino)ethyl)-

N-ethylpropionimidamide)]Cl (P1-B1)

A

mixture of 9-nitro-7-chlorobenz[c]acridine (B2.3) (298 mg,

0.81 mmol) and N-methylethylenediamine (150 mL, 1.72 mmol) was

stirred at reflux in anhydrous dioxane (10 mL) for 24 h. After the

mixture had cooled to room temperature, the solvent was re-

moved by rotary evaporation and the residue was suspended in

2m NaOH. After 2 h of stirring, a dark red solid was collected by

vacuum filtration and washed with 2m NH₄OH to afford B2 as

a red solid, which was dried in a vacuum. Yield: 280 mg, 99%. Ana-

A mixture of [PtCl(EtCN)(NH₃)₂]Cl (P1) (251 mg, 0.70 mmol) and N¹-

(benz[c]acridin-7-yl)-N²-methylethane-1,2-diamine (B1) (325 mg,

1.02 mmol) in anhydrous DMF (1.5 mL) was stirred at 48C for four

days and then slowly warmed to room temperature and allowed

to stir for an additional 24 h. The solvent was removed by vacuum

distillation at 358C, and the resulting brown residue was redis-

Chem. Eur. J. 2014, 20, 16174 – 16187

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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper

solved in a minimal amount of anhydrous MeOH. The solution was

treated with a small amount of activated carbon, filtered through

a 0.2 mm membrane into vigorously stirred anhydrous diethyl

ether, and allowed to stir for 24 h. A yellow precipitate formed,

which was recovered by vacuum filtration, washed with diethyl

ether, and dried in a vacuum to afford P1–B1 as a yellow solid.

Yield: 385 mg, 83%. Analytical purity: ꢃ 90% by LC-ESMS. Prior to

biological testing P1–B1 was subjected to additional purification

by dissolving the product in anhydrous CH₂Cl₂, passing the solu-

tion through a celite pad, and recovering batches of precipitated

product after reducing the solvent volume by rotary evaporation.

10 mm in DMF and then serially diluted with media to a final (non-

toxic) DMF content of less than 1%. The cytotoxicity of the 20

combinations was assessed at chromophore/platinum concentra-

tions of 0.1 mm, 1.0 mm, and 10 mm in NCI-H460 and NCI-H520 cells

using a previously reported colorimetric cell proliferation assay.^[20]

Cell culture

The human NSCLC cell lines, NCI-H460 (large cell), NCI-H520 (squa-

mous cell), NCI-H522, NCI-H1435, and A549 (adenocarcinomas)

were obtained from the American Type Culture Collection (Rock-

ville, MD, USA). NCI-H460, NCI-H520, and NCI-H522 cells were cul-

tured in RPMI-1640 media (HyClone) supplemented with 10% fetal

bovine serum (FBS), 10% penstrep (P&S), 10% l-glutamine, and

1.5 gL^ꢀ1NaHCO₃. A549 cells were cultured in HAM’s F12 K media

(Gibco) with the same additives as above. NCI-H1435 cells were

cultured in serum-free 1:1 DMEM/F12 media (Gibco) containing

2.436 gL^ꢀ1NaHCO₃, 0.02 mgmL^ꢀ1insulin, 0.01 mgmL^ꢀ1transferrin,

25 nm sodium selenite, 50 nm hydrocortisone, 1 ngmL^ꢀ1epidermal

growth factor, 0.01 mm ethanolamine, 0.01 mm phosphorylethanol-

amine, 100 pM triiodothyronine, 0.5% (w/v) bovine serum albumin

(BSA), 10 mm HEPES, 0.5 mm sodium pyruvate, and an extra

2 mmL-glutamine (final concentration 4.5 mm). Cells were incubat-

ed at a constant temperature at 378C in a humidified atmosphere

containing 5% CO₂and were subcultured every 2–3 days to main-

tain cells in logarithmic growth. The slowly dividing NCI-H1435

cells were subcultured every 7 days.

Analytical purity:

ꢃ

95% by LC-ESMS. ¹H NMR (500 MHz,

[D₄]MeOH): d=9.24 (d, J=5.3 Hz, 1H), 8.35–8.22 (m, 1H), 8.23–8.12

(m, 1H), 8.00 (d, J=9.3 Hz, 1H), 7.92–7.79 (m, 1H), 7.79–7.43 (m,

5H), 3.87 (t, J=6.6 Hz, 2H), 3.66 (t, J=6.6 Hz, 2H), 2.92 (d, J=

35.3 Hz, 7H), 1.22 ppm (t, J=7.6 Hz, 3H); ¹³C NMR (126 MHz,

[D₄]MeOH): d=169.94, 151.27, 147.66, 134.07, 129.71, 128.69,

128.36, 127.48, 126.56, 124.82, 124.52, 124.07, 122.59, 120.54,

118.78, 113.90, 10.19 ppm; HR-ESMS (positive-ion mode): m/z for

C₂₃H₃₀ClN₆Pt [M]⁺, calcd: 620.1863; found: 620.1862 (tolerance:

0.134 ppm).

Synthesis of [PtCl(NH₃)₂(N-methyl-N-(2-((9-nitrobenz[c]acridin-

7-yl)amino)ethyl)propionimidamide)]Cl 0.33 DMF (P1–

B2·0.33DMF)

This derivative was generated by the same procedure as P1–B1.

Starting from P1 (203 mg, 0.57 mmol) and N¹-methyl-N²-(9-nitro-

benz[c]acridin-7-yl)ethane-1,2-diamine (B2) (297 mg, 0.86 mmol) in

anhydrous DMF (5 mL), 166 mg (41%) of the DMF solvate of P1–

Cytotoxicity assay

The cytotoxicity studies were carried out according to a standard

protocol using the Celltiter 96 aqueous nonradioactive cell prolifer-

ation assay kit (Promega, Madison, WI). Stock solutions (10 mm) of

the test compounds were prepared in DMF, except for P1–B2

which was prepared in DMSO, and serially diluted with media prior

to incubation with cancer cells. In all cases, cells were incubated

with drug for 72 h before quantifying cell viability using the MTS

assay. Relative cell viability was determined from the viability of

treated and untreated (control) cells. IC₅₀values were calculated

from sigmoidal curve fits using the appropriate dose-response

equation in GraphPad Prism (GraphPad Software, La Jolla, CA).

1

B2 were recovered. Analytical purity: ꢃ 95% by LC-ESMS. H NMR

(500 MHz, [D₆]DMSO): d=9.49–9.38 (m, 1H), 9.31 (d, J=8.0 Hz,

1H), 8.40 (dd, J=9.4, 2.4 Hz, 1H), 8.20 (dd, J=32.7, 9.5 Hz, 2H),

8.07–7.67 (m, 4H), 6.13 (s, 1H), 4.29 (s, 3H), 3.98 (d, J=24.8 Hz,

5H), 3.81 (t, J=7.1 Hz, 2H), 3.02 (s, 5H), 1.24 ppm (s, 3H); ¹³C NMR

(126 MHz, [D₆]DMSO): d=168.81, 153.25, 149.54, 149.42, 141.89,

134.07, 130.95, 130.85, 129.58, 127.69, 126.98, 125.46, 124.46,

122.55, 122.27, 121.18, 115.40, 112.64 ppm; HR-ESMS (positive-ion

mode): m/z for C₂₃H₂₉O₂ClN₇Pt [M]⁺, calcd: 665.1719; found:

665.1701 (tolerance: 2.766 ppm).

For synthetic details of new precursors and intermediates, see the

Supporting Information.

Confocal microscopy

NCI-H460 cells were seeded into poly-d-lysine coated glass-bottom

Petri dishes (MatTeck Corporation, Ashland, MD, USA) with

10⁵cellsmL^ꢀ1suspended in 2 mL of medium per dish. Cells were

incubated overnight and then treated with P1–A1, P1–B1 (5 mm),

or medium for controls for 1, 3, and 6 h. After treatment, medium

was removed and the cells were stained with 75 nm of LysoTracker

Red DND-99 (Invitrogen), 1 mm of ER-Trackerꢂ Red (BODIPYꢃ TR

Glibenclamide, Invitrogen), and 100 nm MitoTracker Deep Red TM

(Invitrogen) in 2 mL pre-warmed Hank’s Balanced Salt Solution

(HBSS) at 378C for 30 min. After staining with each organelle

marker, solutions were replaced with fresh medium. Images were

collected using a Zeiss LSM 710 confocal microscope (Carl Zeiss Mi-

croImaging, Thornwood, NY) using a 63x (PLAN APO, 1.2 NA) ob-

jective lens. All images were acquired in multi-track configuration

mode to minimize excitation cross talk and emission bleed-

through. The fluorescence of P1–A1 (blue), P1–B1 (blue), Lyso-

Tracker Red DND-99, ER-Tracker Red, and MitoTracker Deep Red,

was excited/collected at 405/477, 440/470, 561/591, 561/636, or

633/661 nm, respectively. ZEN software (blue edition, Carl Zeiss,

2011) was used for image processing. Confocal image planes for

Combinatorial library

Stock solutions of the chromophores (Q1, A1, B1, B2, B3) and the

platinum complexes (P1, P2, P3, P4) were prepared in dry DMF at

a concentration of 300 mm. To 0.6 mL micro-centrifuge tubes were

added 20 mL of each chromophore, and the solutions were cooled

to ꢀ208C. Aliquots of the platinum stock solutions/suspensions

were homogenized by pipetting and added directly to the cooled

chromophores. Mixtures were incubated at 48C for seven days in

a benchtop multi-tube vortexer. Small aliquots were removed from

each reaction and diluted with methanol containing 0.1% (v/v)

formic acid to generate 15 mm samples for in-line LC-ESMS analysis

of precursor conversion and product identity. Chromatographic

separations were performed with a 2.1ꢁ30 mm Rapid Resolution

ZORBAX StableBond C-18 (3.5 mm) column, which was maintained

at 408C. The LC-ESMS analysis was performed on an Agilent

1100 LC/MSD ion trap mass spectrometer with an autosampler

thermostatted at 48C using optimized separation and ionization

conditions for each sample (see the Supporting Information). Prior

to dosing cancer cells, micro-scale reactions were first diluted to

Chem. Eur. J. 2014, 20, 16174 – 16187

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16185

ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper

each channel were not contrast-adjusted (unless indicated) or oth-

erwise changed. Panels were assembled and annotated without

any additional manipulation of images in Adobe Photoshop CS2.

prepared in the same buffer supplemented with 100 mm KCl.

Stock solutions of A1, B1, B2, P1–A1, and P1–B1 were prepared in

anhydrous DMF to a final concentration of 1 mm. P1–B2 was dis-

solved in DMSO. Samples were mixed for 30 s and allowed to equi-

librate for an additional 30 s before spectra were acquired. To

allow proper folding of the sequences into G-quadruplexes, sam-

ples were heated to 958C for 10 min and slowly cooled overnight

in a dry bath heat block. Pre-folded DNA sequences were incubat-

ed with 1.05 equivalents of hybrid agents for 48 h at 378C. CD

spectra were recorded of each untreated DNA sample and each

treated sample using the acquisition parameters described above.

CD spectra in the ligand (ICD) region of each hybrid agent in com-

plex with TEL24 were recorded in the 325–500 nm range at a con-

centration of 65 mm (strand).

Extraction of cellular nucleic acids

The nucleic acid content was extracted from cells and purified

using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Alameda, CA,

USA). 1.5 million exponentially-growing NCI-H460 cells were

seeded into 60 mm cell culture dishes with 2 mL of media and

were allowed to attach for 24 h. Cells were dosed with 1 mm P1–

A1 or P1–B1 at 378C for 3 and 6 h. Experiments were performed

in triplicate for each derivative and time point. Cells were washed

three times in cold PBS, trypsinized, and removed from the dishes

using PBS washes. Samples were centrifuged at 1500 rpm for

5 min at 48C to generate cell pellets, which were resuspended in

5 mL of cold PBS and again centrifuged at 1500 rpm for 5 min at

48C. The supernatant was removed and the DNA/RNA was extract-

ed from the pellets according to the manufacturer’s protocol. The

concentration of recovered nucleic acids and the purity of each

sample were determined from absorbances at 260 and 280 nm

(triplicate readings).

HR-ESMS analysis of platinum-modified G-quadruplex se-

quences

All HR-ESMS spectra were recorded on a Thermo Scientific LTQ Or-

bitrap XL. Unmodified and platinum-modified G-quadruplex se-

quences in annealing buffer were desalted using Amicon Ultra-

0.5 mL centrifugal filters with a 3 kDa molecular weight cut-off (Mil-

lipore Corporation). Samples were diluted to a final concentration

of 1 mm in 50% 5 mm ammonium acetate in water (pH 7.2)/50%

5 mm ammonium acetate in methanol and introduced into the

electrospray ionization source by direct injection utilizing the same

solvent system at a flow rate of 100 mLmin^ꢀ1. Spectra were record-

ed in negative-ion mode. Blank injections were run between sam-

ples to avoid carryover.

ICP-MS analysis

The concentration of Pt was determined by quadrupole inductively

coupled mass spectrometry (ICP-MS). Prior to analysis, samples

were subjected to a rigorous digestion procedure in the presence

of high-purity, Ultrex-grade nitric (HNO₃) and hydrochloric (HCl)

acids (J.T. Baker, Phillipsburg, NJ). Nominal 0.200 mL aliquots of

each acid were added to the samples directly in 15 mL centrifuge

tubes (NUNC, Rochester, NY), along with 0.600 mL of approximate-

ly 18-MW quality deionized water (DI H₂O), obtained from a Pure

Water Solutions (Hillsborough, NC) system. Samples were then

capped and placed in a controlled temperature water bath (VWR,

Model 750HT) maintained at 908C. After four hours, samples were

removed from the bath and were allowed to cool to room temper-

ature before adding of DI H₂O to a total volume of 4 mL. An ali-

quot (3 mL) of each digested sample was transferred to an ICP-MS

acid-washed autosampler tube and was fortified to contain a nomi-

nal 10 ngmL^ꢀ1concentration of bismuth (Bi) internal standard.

Prior to the sample analysis, the Thermo X-Series II ICP-MS (Wal-

tham, MA, USA) was calibrated with Pt-containing acid matrix

matched standards ranging from 0.001 to 5.00 ng PtmL^ꢀ1. Single

element Pt and Bi stock solutions traceable to the National Insti-

tute of Standards and Technology (NIST) were obtained from

a commercial vendor (High Purity Standards, Charleston, SC) and

were used to prepare all solutions. After calculating pg Pt/mg DNA

values, few outliers were identified using Grubb’s outlier test at

90% confidence and removed from the data set. For additional de-

tails of the sample analysis and quality control, see the Supporting

Information.

Acknowledgements

This work was supported by the National Institutes of Health

(Grant CA101880) and Wake Forest Innovations. HR-ESMS spec-

tra were acquired on a Thermo Scientific LTQ Orbitrap instru-

ment (NSF grant 947028). Computations were performed on

the Wake Forest University DEAC Cluster, a centrally managed

resource with support provided in part by the University. We

thank Dr. Marcus W. Wright and Tiffany K. West for technical as-

sistance.

Keywords: anticancer drugs · cellular target · cytotoxicity ·

DNA adducts · intercalation

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Redesigning the DNA-Targeted chromophore in platinum-acridine anticancer agents: A structure-activity relationship study

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