Gold(III) complexes of pyridyl- and isoquinolylamido ligands: Structural, spectroscopic, and biological studies of a new class of dual topoisomerase i and II inhibitors

Article

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Gold(III) Complexes of Pyridyl- and Isoquinolylamido Ligands:

Structural, Spectroscopic, and Biological Studies of a New Class of

Dual Topoisomerase I and II Inhibitors

Colin R. Wilson,^†Alexander M. Fagenson,^‡Wanvipa Ruangpradit,^‡Mark T. Muller,^‡

and Orde Q. Munro*^,†

^†School of Chemistry and Physics, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa

^‡College of Medicine, University of Central Florida, Biomolecular Research Annex, 12722 Research Parkway, Orlando, Florida

32826-3227, United States

S

* Supporting Information

ABSTRACT: The structures, spectroscopy, and cytotoxicity of four

novel nominally square-planar gold(III) chelates 1−4 with the general

formula cis-AuCl₂(X), where the ligand X is an anionic bidentate

pyridyl- or isoquinolylamido chelating agent, are described. The Au−

N

_amido, Au−N_pyridyl, and Au−N_isoquinolyldistances are 2.002(9)−

2.016(3), 2.01(1)−2.037(3), and 2.037(3) Å, respectively. Density

functional theory simulations aﬀorded accurate gold(III) coordination

geometries for 1−4 (bond distances and angles to within 5% of the X-

ray values), while accurate transition energies were limited to those

calculated in the UV spectral region. The complexes had variable

stability in dimethyl sulfoxide: compound 3 (relatively rigid) was

indeﬁnitely stable, compounds 1 and 2 (conformationally ﬂexible)

slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous

buﬀer). The isoquinolylamido derivative 4 was suﬃciently cytotoxic in the NCI-60 screen to undergo full ﬁve-dose testing.

Notably low GI₅₀(1.8, 2.3, and 3.2 μM) and IC₅₀(4.0, 9.8, and 15 μM) values were recorded for the OVCAR-3, IGROV1, and

SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known

anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison

zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The

Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts

as an interfacial poison of Top2 at low concentrations (<1 μM) and a catalytic inhibitor of the enzyme above 5 μM. Gel mobility

shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at

concentrations >5 μM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding

by the complex.

Chemical strategies for imparting redox stability to gold(III)

complexes typically involve the use of chelating and macrocyclic

ligands containing strong neutral or anionic σ-donor atoms (C,

N, and O to match the hard Au^IIIion). Tetradentate

macrocyclic ligands block ligand-exchange reactions at the

metal center, e.g., the binding of S-donor reducing ligands,

while powerful σ-donor chelating ligands transfer electron

density to the metal center and thus lower the reduction

potential of the Au^IIIion.²¹Examples of Au^III-stabilizing

chelating ligands are porphyrins [e.g., TPP (meso-tetraphenyl-

porphyrin dianion),^22,23terpy (2,2′;6′,2″-terpyridine),^21,24,25

glycylhistidinate,²⁶dithiocarbamates,²⁷cyclam (1,4,8,11-tetraa-

zacyclotetradecane),^21,28and damp (2-[(dimethylamino)-

methyl]phenyl)].²⁹⁻³¹Not all gold(III) complexes involving

INTRODUCTION

■

The ﬁrst report (1965)¹that DNA-targeting^2,3cisplatin

inhibited mitosis in bacteria and the subsequent clinical

deployment of cisplatin and its analogues^4,5as anticancer

metallodrugs^6,7have fueled the growth of multidisciplinary

studies on square-planar platinum(II) complexes⁸for several

decades.⁹⁻¹¹More recently, isoelectronic (d⁸) square-planar

gold(III) complexes have been featured in metallodrug

development eﬀorts as the quest for drug diversity and novel

compounds to overcome cisplatin-resistant tumor cell lines has

escalated.¹²⁻¹⁷Despite the expectation that gold(III) com-

plexes might act in a fashion similar to that of platinum(II)

complexes in biological systems, gold(III) complexes diﬀer

signiﬁcantly and are especially susceptible to reduction to Au^I

and colloidal gold, Au⁰.¹⁸Ligand design is thus crucial to

stabilizing the Au^IIIoxidation state for use in normal solutions

and physiological media.^19,20

Received: February 8, 2013

Published: July 1, 2013

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these chelating ligands are uniformly active against cancer. At

present, only a handful show signiﬁcant cytotoxicity:

[Au^III(bipyc-H)(OH)][PF₆] [bipyc-H, deprotonated 6-(1,1-

dimethylbenzyl)-2,2′-bipyridine],³²Au^III(dmamp)Cl₂[dmamp,

2-(dimethylaminomethyl)phenyl],^29,33[Au^III(DMDT)X₂] and

[Au^III(ESDT)X₂] (DMDT, N,N-dimethyldithiocarbamate;

ESDT, ethylsarcosinedithiocarbamate; X = Cl or Br),²⁷

[Au^III(Porph)]⁺(Porph, porphyrin dianion)³⁴⁻³⁹and

[Au^III_m(C^N^C)_mL]ⁿ⁺(m = 1−3; C^N^C, diphenylpyridine).⁴⁰

The biological targets, furthermore, are quite varied and include

DNA/topoisomerase I (Top1) for the cyclometalated C^N^C

gold(III) adduct^40,41and gold(III) porphyrins,²³protein targets

such as Bcl-2 and histone deacetylase (HDAC) for gold(III)

porphyrins,⁴²and proteasome inhibition for gold(III) dithio-

carbamates.^19,43

deprotonated ligands HL¹, HL², and HL⁴(Scheme 2). Single-

crystal X-ray structures were determined for H₂L⁴and the

Scheme 2. Structures of the Gold(III) Pyridyl- and

Isoquinolylamido Chelates Studied in This Work

Relatively few amidogold(III) complexes have been synthe-

sized and characterized or evaluated for their potential

cytotoxicity. In one of the earliest structure-focused studies of

these compounds, Cheung et al. reported the complex

Au^III(HL)Cl₂[H₂L, 2,2′-bis(2-pyridylcarboxyamide)-1,1′-bi-

naphthyl].⁴⁴Rather unexpectedly, the Au^IIIion was chelated

to half of the tetradentate ligand via amido and pyridyl N-donor

atoms, leaving a pair of cis-Cl⁻ions to complete the square-

planar coordination geometry and an unbound amide moiety.⁴⁴

This coordination mode, while not anticipated from the ligand’s

structure, presumably reﬂects bridge-impeded chelation of the

metal by all donor atoms or other factors related to substitution

of the Cl⁻ions. The modest cytotoxicity proﬁles and limited

solution stability under physiological conditions of a series of

tetradentate bis(amido)gold(III) chelates have recently been

reviewed alongside data for gold(III) porphyrins and Au^III-salen

derivatives.⁴⁵Of relevance to the present work, Yang and co-

workers reported that, out of a small series of three tridentate

amidogold(III) complexes, at least one compound, [Au-

(Quinpy)Cl]Cl [Quinpy, N-(8-quinolyl)pyridine-2-carboxami-

do], had reasonable solution stability, was capable of binding

calf-thymus DNA, and had quite promising cytotoxicity against

melanoma and lung cancer cell lines.⁴⁶These authors also

showed that a mono(amido)gold(III) complex, [Au(L-

N,N′)Cl₂] [L-N,N′, N-(4-methylphenyl)-2-pyridinecarboxa-

mide], reacted with 2 equiv of 5′-guanosine monophosphate

(5′-GMP) via substitution of the Cl⁻ions to give the bis(5′-

GMP) adduct.⁴⁷

gold(III) complexes 1−4. The ligands H₂L¹, H₂L², and HL³

have been described previously in the literature.⁴⁸⁻⁵¹Density

functional theory (DFT) simulations have been used to

delineate the electronic structures of 1−4. The cytotoxicity

proﬁles of compounds 2−4 were evaluated by the National

Cancer Institute (NCI) using their panel of 60 human cancer

cell lines. The cytotoxicity of compound 4 warranted further

investigation of its DNA aﬃnity and ability to target human

topoisomerase I and II (Top1 and Top2).

RESULTS AND DISCUSSION

■

From inception, a key objective of this work was to synthesize

structurally unique, predominantly planar cationic tetradentate

bis(amido)gold(III) complexes that could potentially act as

cytotoxic DNA intercalatorsa goal inspired in part by the

biochemistry and molecular biology of cationic gold(III)

porphyrins.²²The reaction of H₂Lⁿ(n = 1, 2, 4) with Au^III

was expected to give potentially cytotoxic cationic tetradentate

AuN₂N′₂trischelates of the metal (eq 1).

In this paper, we describe the synthesis and characterization

of novel H₂L⁴(Scheme 1) and the mono(amido)gold(III)

chelates formed by the deprotonated ligand L³and hemi-

n

H₂L + [Bu₄N][AuCl₄]

Scheme 1. Free Ligand Structures

n

⇌ [Au(L )]Cl + 2HCl + [Bu₄N]Cl

(1)

However, despite several attempts to synthesize the target salts

[Au(Lⁿ)]Cl (Scheme S1, Supporting Information), and in

consonance with the earlier literature on similar pyridylamido

complexes of gold(III),⁴⁴only the neutral cis-dichlorogold(III)

complexes 1−4 could be isolated. Evidently, the Au−Cl bonds

in this system are substitution-inert [in contrast to the

analogous palladium(II) complexes⁵²]. Notwithstanding the

intractable synthesis of the target AuN₂N′₂trischelates, the new

complexes 1−4 were deemed worthy of comprehensive

structural, spectroscopic, and cytotoxicity studies.

Molecular Structure of H₂L⁴. The X-ray crystal structure

of the novel ligand H₂L⁴is shown and brieﬂy discussed in the

Supporting Information (Figures S1 and S2) because the

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primary focus of this work is on the chemistry and molecular

biology of 1−4. However, one notable feature of the structure

of H₂L⁴is its markedly nonplanar conformation brought about

by an intramolecular hydrogen bond involving the N−H donor

of one amide group and the carbonyl O of the other. The ligand

is therefore not preorganized for tetradentate chelation of a

square-planar metal ion.

The Au−N_pyridineand Au−N_amidodistances for the pyridine-

based chelates 1−3 average 2.035(3) and 2.014(5) Å,

respectively; these values indicate crystallographically and

chemically distinct coordination interactions. Both N-donor

atom types (pyridine and amido) are sp²-hybridized; however,

as might be anticipated for an anionic N-donor atom (and,

consequently, a more powerful σ donor), the Au−N distance to

the amido N is more than 3 standard deviations shorter than

that to the pyridine N. This phenomenon has been observed in

the crystal structures of other amidogold(III) chelates.^53,54

Regarding the dissimilar Au−N bonds in pyridylamido chelates,

it is noteworthy that symmetric bipyridine (bipy) complexes of

gold(III) such as [AuCl₂(DMbipy)][PF₆] and

[AuCl₂(DMObipy)][PF₆], where DMbipy = 4,4′-dimethyl-

2,2′-bipyridine and DMObipy = 4,4′-dimethoxy-2,2′-bipyridine,

exhibit equivalent Au−N_pyridinedistances of 2.03(1) and 2.02(2)

Å, respectively.⁵⁵This conﬁrms the notion that the amido and

pyridine N atoms are chemically distinct (despite equivalent

hybridization), with the former acting as a strong σ donor and

the latter as a more balanced π donor/π acceptor,^56,57and that

this electronic distinction underpins dissymmetric coordination

of the Au^IIIion in the present compounds.

Molecular Structures of 1−3. The X-ray structures of 1−

3 are shown in Figure 1. The Au^IIIion is nominally square

The Au−Cl1 (trans to the amido group) and Au−Cl2 (trans

to the pyridine group) bond distances for 1−3 average

2.291(4) and 2.270(13) Å, respectively. Closer inspection of

the Au−Cl distances indicates that a signiﬁcant “trans

eﬀect”^58,59is structurally manifest in these complexes, as brieﬂy

noted in an earlier paper by Fan et al.⁵⁴More speciﬁcally, the

Au−Cl bond trans to the stronger σ-donor atom (N2, or

N_amido) is considerably longer than that trans to the pyridine N;

in the case of 3, this diﬀerence is as large as 0.034 Å and thus

statistically signiﬁcant relative to the standard uncertainties of

the experimental Au−Cl bond distances. The diﬀerence

between the coordination of chloride trans to the amido

group relative to the pyridine group in 1−3 becomes

particularly apparent if we compare the mean Au−Cl1 distance

above to the mean Au−Cl distance of 2.255(7) Å reported for

the symmetric bipy derivatives [AuCl₂(DMbipy)][PF₆] and

[AuCl₂(DMObipy)Cl₂][PF₆].⁵⁵These complexes typify the

expected metrics for the coordination of chloride trans to an

Au−N_pyridinebond in a symmetric ﬁve-membered chelate ring

system and highlight the marked elongation of the Au−Cl

bonds trans to the amido N donor in the present complexes

(especially 3). Interestingly, if the chelating σ-donor anion

coordinated to Au^IIIis switched to C, as in the complex

AuCl₂(2-phenylpyridine),⁶⁰then a very large structural trans

eﬀect occurs, with the Au−Cl distance trans to the Au−C bond

elongating beyond that observed here to 2.361(8) Å. As might

be anticipated, the structural trans eﬀect discussed above for

organometallic gold complexes is paralleled by a kinetic trans

eﬀect in the substitution reactions of the Au^IIIion. Anions such

as chloride coordinated trans to a powerful σ donor (e.g.,

Figure 1. Labeled views of single-crystal X-ray structures of gold(III)

chelates 1 (a), 2 (b), and 3 (c). H atoms are rendered as spheres of

uniform arbitrary radii, and bonds are represented as cylinders.

Thermal ellipsoids are represented as 50% probability surfaces for 1

and 3 and 40% probability surfaces for 2.

planar in each complex with a pair of cis-Cl⁻ions coordinated

trans to the single pyridylamido chelating moiety of the ligand

in the case of 3 and one of the pair of such chelating groups in

1 and 2. The substituent groups attached to the deprotonated

metal-bound amide N atom (N2) in 1−3 adopt structure-

dependent conformations that have an essentially minimal

impact on the coordination geometry of the Au^IIIion in each

case (Table 1). The aryl-substituted derivative 3 has

coordination group parameters that are structurally and

statistically equivalent to the alkyl-substituted analogues 1 and

2, despite having a substituent that may conjugate with the

amide-based chelate ring.

−

C₆H₅) are kinetically more labile than those coordinated cis to

the “labilizing” ligand.⁶¹This reactivity pattern clearly correlates

with a longer, weaker Au−Cl bond trans to the strong σ-donor

ligand in the structures of such compounds. One might logically

expect that, for 1−3, substitution of at least one of the chloride

ions (with that trans to the amido group presumably being

more reactive) might occur with some nucleophiles.

The bond angles subtended at the metal ion in 1−3 (Table

1) are normal for ﬁve-membered ring chelates of Au^IIIwith two

N-donor atoms. The mean cis Cl−Au−Cl bond angle is

89.1(2)°; the mean cis N−Au−N angle is more acute,

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Table 1. Selected Crystallographic and DFT-Calculated Bond Distances and Bond Angles for 1−4

1

2

3

4

X-ray

DFT

X-ray

DFT

Bond Distances

X-ray

DFT

X-ray

DFT

Au1−N1

Au1−N2

Au1−Cl1

Au1−Cl2

2.036(5)

2.019(4)

2.290(2)

2.285(2)

1.211(7)

1.228(7)

1.345(8)

1.340(7)

2.07

2.04

2.34

2.3

2.037(3)

2.014(3)

2.287(1)

2.263(1)

1.230(5)

1.217(5)

1.341(5)

1.346(6)

2.07

2.04

2.34

2.3

2.031(4)

2.009(5)

2.295(2)

2.261(2)

1.21(1)

2.08

2.06

2.35

2.31

1.22

2.048(3)

2.035(3)

2.303(1)

2.270(1)

1.232(4)

1.240(6)

1.362(5)

1.391(5)

2.07

2.36

2.31

1.21

1.22

1.37

1.36

a

CO

1.22

1.35

1.22

1.35

b

CO

a

N−C

1.343(9)

1.36

b

N−C

Bond Angles

N1−Au1−Cl1

Cl1−Au1−Cl2

Cl2−Au1−N2

N2−Au1−N1

94.0(1)

94.5

89.6

95.3

80.6

113

116

117

94.85(9)

89.00(4)

94.90(9)

81.3(1)

94.5

89.3

95.6

80.6

113

116

117

114

95.3(1)

94.5

89.7

95.4

80.5

113

115

118

114

95.68(8)

89.42(2)

93.83(9)

81.1(1)

94.3

90.2

94.8

80.7

113

115

118

114

89.11(7)

95.8(1)

88.87(6)

94.4(1)

81.0(2)

81.5(2)

c

Au1−N1−C5

112.9(4)

115.3(4)

115.9(5)

113.6(5)

112.9(3)

115.0(3)

115.9(4)

113.7(3)

113.2(4)

115.2(4)

116.2(5)

113.9(6)

112.7(2)

116.0(2)

116.7(3)

113.0(3)

d

Au1−N2−C6

e

N1−C5−C6

f

N2−C6−C5

114

c

a

b

d

e

Metal-bound amide group. Free amide group. For 4, this angle is Au1−N1−C9. For 4, this angle is Au1−N2−C9. For 4, this angle is N1−C9−

f

C10. For 4, this angle is N2−C10−C9.

measuring 81.2(2)°. The latter bond angle, which deviates

signiﬁcantly from 90°, clearly reﬂects the geometric constraints

on metal-ion coordination imposed by the ﬁve-membered

chelate ring and compares favorably with equivalent values in

the literature, which range from 80.6(2)° for [AuCl₂(bipy)]-

[NO₃]⁶²to 81.42(7)° for AuCl₂(N-NP2C), where N-NP2C =

N-nonylpyridine-2-carboxamido anion.⁵³(Amidopyridine- and

bipy-type ligands have similar “bite” angles.) In each complex,

the cis N−Au−Cl bond angles are equivalent, averaging

95.0(7)° and 94.7(8)° for the angles to Cl1 and Cl2 for 1−3,

respectively.

Gold chelates 1−3 are polyfunctional in the sense that they

possess hydrogen-bond donor and acceptor groups as well as

aromatic ring systems. The supramolecular structures of the

compounds are accordingly both diverse and quite complex.

Largely because of the lack of cytotoxicity of 1−3 relative to 4

(as discussed later), brief comments on hydrogen-bonding and

π-stacking interactions in their crystal structures are given in the

Supporting Information (Figures S3−S12).

Crystal and Molecular Structure of 4. The X-ray

structure of 4 (Figure 2) is similar in its overall nominally

square-planar coordination geometry to the preceding com-

pounds and alike in conformation to 3 insofar as the dihedral

angle between the aryl ring (C11−C16) appended to the

metal-bound amido group (N2) and the 13-atom mean plane

of the isoquinolylamidogold(III) chelate (Au1, N1, C1−C10,

and N2) is near-orthogonal, measuring 80.7°. From Table 1,

the Au−N_amidoand Au−N_IQ, where IQ = isoquinoline, bond

distances of 4 follow the same pattern as the Au−N distances of

1−3, namely, that the bond to the amido group [Au1−N2,

2.035(3) Å] is 4 standard deviations (4σ) shorter than the bond

to the isoquinoline N [Au1−N1, 2.048(3) Å]. This, as with 1−

3, reﬂects the fact that the amido group N atom is a more

powerful σ donor (Lewis base) than the “pyridine” N atom of

the isoquinoline group and distinctly parallels the pK_a’s of the

two diﬀerent donor types: pK_a(N_amide) > 10;⁶³pK_a(N_IQ) ∼

5.5.⁶⁴The cis Au−Cl bonds of 4 exhibit the same asymmetry

observed in 1−3, namely, that the Au−Cl bond trans to the

amido N donor is signiﬁcantly longer (by 0.033 Å) than that

trans to the isoquinoline N atom. The magnitude of this

structural diﬀerence is equivalent, within 1σ, to that observed

for 3 and reﬂects a sizable trans eﬀect induced by the amido

group (vide supra). The absolute value of the Au1−Cl1 bond in

4 [2.303(1) Å] is ca. 5% longer than the mean distance for this

bond in 1−3 [2.291(3) Å], a relatively insigniﬁcant diﬀerence,

while the Au1−Cl2 bond distance trans to the isoquinoline N

atom exactly matches the average distance for this bond type in

chelates 1−3 [2.270(11) Å].

The coordination geometry of the Au^IIIion in compound 4 is

structurally distinct from that seen in 1−3 in a substantial

aspect. Speciﬁcally, the isoquinoline-based chelate system of 4

favors signiﬁcantly longer Au−N bonds. Thus, the Au−N_amido

bond of 4 measures 2.035(3) Å and is more than 4 standard

deviations (4σ) longer than the mean Au−N_amidobond of 1−3

[2.014(5) Å]; similarly, the Au−N_IQbond of 4 [2.048(3) Å] is

more than 4σ longer than the mean Au−N_pyridinebond distance

for 1−3 [2.035(3) Å]. Note that this structural diﬀerence exists

despite the pK_aof pyridine (5.2) being roughly the same as that

for isoquinoline (5.5).⁶⁴Elongation of the Au−N bonds in 4

reﬂects coordination of the Au^IIIion by a poorer σ-donor and/

or π-acceptor ligand, with all other structural parameters being

equivalent. Inspection of the chelate ring angles for 1−4 (Table

1) conﬁrms that pyridyl- and isoquinolylamido chelate ring

systems are indeed metrically equivalent (the N2−Au1−N1,

Au1−N1−C5, Au1−N2−C6, N1−C5−C6, and N2−C6−C5

bond angles diﬀer by <1° across the series for each chemically

unique bond angle). Note that isoquinoline itself is a slightly

stronger-ﬁeld ligand than pyridine because of its marginally

higher pK_a, or Lewis basicity, and better π-acceptor ability in

square-planar complexes of nickel(II).⁶⁵The underlying

reasons for elongation of the Au−N bond distances in 4 are

therefore likely to be electronic, as opposed to structural, in

origin but are clearly more complicated than a simplistic

interpretation based on extrapolation of the electronic

properties of isoquinoline complexes of nickel(II) to the

present system. Notably, the DFT-calculated Au−N_amidobond

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2b). The metal-free isoquinoline rings of two neighboring

molecules of 4 stack with exactly parallel ring planes about a

center of inversion to form a relatively tight interaction with a

mean-plane separation of 3.523 Å that is well within the 3.35−

3.8 Å range expected for π-stacked aryl rings.⁶⁶If the

isoquinoline atom N4 is used as a frame of reference, then

the geometry of the interacting isoquinoline rings is the typical

oﬀset antiparallel arrangement (N4 atoms pointing in opposite

directions) for aromatic N-heterocycles.⁶⁷This particular

ring···ring arrangement evidently represents energetically

favorable dipole alignments in the two-ring systems. The

distance between the centers of gravity of the two isoquinoline

rings, Cg1···Cg1ⁱ, measures 3.694 Å such that the lateral shift of

the ring systems is 1.111 Å. As is lucidly presented and

discussed by Janiak,⁶⁷the laterally displaced ring systems seen

in 4 would permit optimal π−σ attraction between stacked

isoquinoline groups in addition to normal London forces of

attraction. Isoquinoline and quinoline ring systems are, of

course, well-known for their π-stacking ability and are often key

pharmacophores prevalent in many anticancer,⁶⁸⁻⁷⁰antiviral,⁷¹

and antitrypanosome⁷²compounds with a mechanism of action

(MOA) involving DNA binding by intercalation.⁷³Of special

relevance to the present work, we note that the clinically

deployed anticancer drug camptothecin (CPT; a quinoline

alkaloid) poisons human Top1 by DNA intercalation at a 5′-

TA-3′ dinucleotide site targeted for covalent binding by the

enzyme during its catalytic cycle.⁷⁴A ﬁnal point on the

structure of the π-stacked dimer of 4 is that, in addition to π−π

interactions between the isoquinoline rings, a symmetry-related

pair of unconventional C−H···Cl hydrogen bonds between an

isoquinoline ring hydrogen donor (H22) and the coordinated

chloride ion acceptor of the neighboring molecule (Cl2ⁱ)

stabilizes the interaction. In short, the auxiliary (metal-free)

isoquinoline ring of 4 is clearly a potentially useful functional

group that may engage in interactions with biological targets

such as DNA by π stacking as well as hydrogen bonding.

IR and NMR Spectroscopy. For the three bis(amide)

ligands H₂L¹, H₂L², and H₂L⁴and their corresponding

mono(chelate) gold(III) complexes 1, 2, and 4, the single

amide carbonyl band, ν(CO), observed in the IR spectrum of

the free ligand splits into two separate signals after chelation of

gold(III) by one of the pair of amide groups. From Table 2, the

CO stretching mode of the metal-free amide group, ν₂(CO),

occurs at a higher frequency than that for the metal-bound

amide group, ν₁(CO). The frequency drop for the metal-bound

amide group is largest for 2 (Δν_2,1= 22 cm⁻¹) and smallest for

Figure 2. (a) Labeled thermal ellipsoid view (50% probability

surfaces) of the low-temperature X-ray structure of 4. Bonds are

rendered as cylinders and H atoms as spheres of uniform arbitrary

radii. The solvent (diethyl ether) has been omitted for clarity. (b)

Partially labeled view of the centrosymmetric π-stacked dimer formed

by 4. The isoquinoline ring planes are parallel; the distance between

the isoquinoline ring centers of gravity (Cg1···Cg1ⁱ) is indicated. The

mean-plane separation (MPS) and lateral shift (LS) of the stacked

isoquinoline rings (calculated from the coordinates of Cg1 and Cg1ⁱ)

are depicted graphically on the triangle to the right of the top-down

view of the dimer (lower left). The dimer is additionally stabilized by

Table 2. Experimental and DFT-Calculated IR Data (Amide

a,b

i

Carbonyl Group Region) for 1−4

C−H···Cl hydrogen bonds: H₂₂···Cl₂, 2.960(1) Å; C₂₂···Cl₂, 3.864(4)

i

Å; C₂₂−H₂₂···Cl₂, 159.5(3)°. Crystallographic symmetry code: (i) −x,

ν₁(CO)/cm⁻¹

ν₂(CO)/cm⁻¹

Δν_2,1/cm⁻¹

1 − y, 1 − z .

expt

DFT

diﬀ

expt

DFT

diﬀ

expt DFT

1

2

3

4

1647

1645

1643

1652

1757

1767

1772

110

112

124

120

1667

na

1795

1789

na

128

122

na

20

22

na

16

38

32

na

19

distances parallel the experimental trend, consistent with a

fundamental diﬀerence between the pyridyl- and isoquinolyla-

mido chelate ring systems.

The supramolecular structure of 4 is dominated by

unconventional C−H···O and C−H···Cl hydrogen bonds

(Figure S12 in the Supporting Information), as with 1−3.

These interactions allow 4 to form one-dimensional hydrogen-

bonded chains parallel to the c axis of the unit cell. More

interesting, however, is the structure of the centrosymmetric

hydrogen-bond-stabilized π-stacked dimer formed by 4 (Figure

1668

1791

123

a

Abbreviations: ν₁(CO) and ν₂(CO) are the stretching frequencies for

the metal-bound and free amide carbonyl groups, respectively; Δν_2,1is

the diﬀerence between ν₂and ν₁, i.e., ν₂−ν₁; expt, experimental

frequency; diﬀ, diﬀerence between the DFT-calculated and exper-

b

imental frequencies; na, not applicable. Experimental wavenumbers

are accurate to 1 cm⁻¹.

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4 (Δν_2,1= 16 cm⁻¹). The DFT-calculated frequencies for the

CO modes of the metal-bound and free amide groups are

between 110 and 128 cm⁻¹higher than the experimental

frequencies. A mean scaling factor of 0.933 may therefore be

used to correct the DFT-simulated spectra for assigning

experimental IR bands. This scaling factor, which accounts

for phase diﬀerences between the solid-state and gas-phase

spectra as well as intrinsic shortfalls of the theoretical method,

is similar to that widely used for simulations at the B3LYP/6-

31G(d) level of theory (0.961).⁷⁵The DFT-calculated values of

Δν_2,1are in reasonably good agreement with the experimental

values, with the degree of similitude best for compound 4.

Moreover, the gas-phase DFT-calculated values of ν₁(CO)

decrease linearly with increasing C−O bond distance for 1−4

(Figure S13 in the Supporting Information). This is the

expected trend as the C−O bond order decreases. A similar

correlation does not exist for the experimental frequencies and

bond distances, possibly because of inherent limitations in the

accuracy of the ambient-temperature X-ray data for 1−3.

An explanation for the shift to lower wavenumber for the

metal-bound amide band, ν₁(CO), relative to the free amide

band is that the resonance hybrid structure for the chelate lies

somewhere between the two resonance forms A (⁻N−CO)

and B (NC−O⁻) illustrated in Scheme 3. Because a decrease

distinct amide groups) are consistent with previous spectral

data reported for a comparable mono(amido) chelate of

gold(III).⁴⁴

1

The H NMR spectrum for the free ligand H₂L⁴exhibits an

“all-aromatic” proton spectrum with signals spanning the 7−11

ppm range (Figure 3). The chemically and magnetically

Scheme 3. Illustration of Bond Resonance Involving the

a

Metalbound Amide Group of 4 (by Way of Example)

a

Lone pairs of electrons are shown for the amide group to aid

visualization of the two resonance forms. The group R represents the

chemically distinct remainder of the structure of complex 4 (Scheme

1

Figure 3. H NMR spectra (500.01 MHz) of symmetric H₂L⁴and its

asymmetric mono(amido)gold(III) complex, 4 (inset structure).

Signal integrals and peak assignments are based on the schemes for

H₂L⁴and 4 given in the Experimental Section. Arrows track the

splittings and shifts for protons a and f that accompany chelation of

Au^IIIand the loss of symmetry in 4 relative to the free ligand.

Intramolecular hydrogen bonds and a midrange C−H··· π interaction

are shown with dashed lines and interaction distances (structure inset).

The spectra were recorded in DMSO-d₆at 303 K.

2).

in the C−O bond order is expected to reduce the frequency of

the normal mode of vibration for the carbonyl group (Figure

S13 in the Supporting Information), the experimental data

suggest that the resonance hybrid structures of chelates 1−4 lie

slightly closer to resonance form B than form A, consistent with

the behavior previously deduced by Rosenberg for several

peptide chelates of copper(II).⁷⁶Resonance form B has a

higher N−C bond order, or more NC double-bond

character, than form A. The X-ray data for 4 (Table 1)

conﬁrm that the N−C bond for the metal-bound amido group

[1.362(5) Å] is shorter than that for the metal-free amide group

[1.391(5) Å], consistent with a more delocalized electronic

structure for the amido group of the chelate ring and,

consequently, a slightly higher N−C bond order. One caveat

here is that the DFT-calculated C−N bond distances for the

metal-bound and free amide groups of 4 are essentially

equivalent and do not follow the experimental data.

Furthermore, when averaged over functional group type, the

X-ray data (Table S2 in the Supporting Information) do not

collectively delineate statistically meaningful diﬀerences in the

mean C−O and N−C bond distances for the metal-bound and

free amide groups of 1−4 because of intrinsic resolution limits

in the diﬀraction data. The IR data in the carbonyl frequency

region for 1, 2, and 4 (i.e., independent CO modes for the

equivalent amide NH protons of the free ligand resonate

farthest downﬁeld (10.90 ppm). The relatively narrow line

width (2.33 Hz) of the amide NH signal indicates negligible

dynamic proton exchange in solution at ambient temperature

(as expected for nonacidic protons). Analysis of the intra-

molecular hydrogen bonds of H₂L⁴(Figure S2 in the

Supporting Information) shows that the amide NH atom

H100 is involved in a three-center (bifurcated) hydrogen bond

in which the adjacent isoquinoline N (N1) and carbonyl O

(O1) atoms act as hydrogen-bond acceptors for the N−H

donor. The amide NH atom H200 is hydrogen-bonded to the

adjacent isoquinoline atom N4. Even if some rotational

exchange of the two isoquinolylamido moieties occurs in

solution, the NH protons of H₂L⁴are expected to be rigorously

hydrogen-bonded throughout such a process, consistent with

an intrinsically narrow line width. Interestingly, the amide NH

protons of H₂L¹and H₂L²have broader line widths than that

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observed for H₂L⁴and, furthermore, exhibit 1:2:1 triplet

patterns indicative of spin−spin coupling to the ¹⁴N

nucleus.⁴⁸⁻⁵¹The aromatic nature of the bridge between the

amide groups of H₂L⁴presumably negates ¹H−¹⁴N spin

coupling in this compound.

Supporting Information). The spin−spin coupling constants,

¹J_HN, are 6.08(2) and 6.19(6) Hz for 1 and 2, respectively. The

line widths of the NH proton resonances of 1 and 2 are

comparable, averaging 4.6(4) and 4.9(7) Hz, respectively; both

are broader than the Lorentzian line width (2.24 Hz) for the

NH proton of 4. As noted above for H₂L⁴, the lack of ¹⁴N−¹H

spin coupling in the case of 4 presumably reﬂects the eﬀect of

the aromatic bridge (benzene ring) between the amide groups,

which negates spin coupling (this is certainly the case at 303

K). A ﬁnal point regarding the NMR spectra for 1, 2, and 4 is

that time-dependent spectral changes were evident in dimethyl

sulfoxide (DMSO). Speciﬁcally, complexes 1 and 2 showed

clear evidence of demetalation over a period of ca. 1 month

(Figure S21 in the Supporting Information), while complex 4

formed an insoluble brown precipitate after 10 days, possibly a

new compound or an aggregated species. Compound 3, on the

other hand, was stable.

As far as the diamagnetic gold(III) complexes are concerned,

1

the H and ¹³C NMR spectra of 1−3 are fully assigned in the

Experimental Section and displayed in Figures S14−S19 in the

1

Supporting Information. A more detailed analysis of the H

NMR spectrum of the gold(III) complex 4 is given below and

in Figure 3 for two reasons: ﬁrst, the ligand is novel and,

second, the solid-state conformation of 4 is evidently

maintained in ﬂuid solution, which is of signiﬁcance when

interpreting biological data for this complex in solution. Before

1

closer inspection of the H NMR spectrum of 4, an important

general observation for the metal chelates 1, 2, and 4 is that the

2-fold structural and magnetic symmetry of the free ligand is

lost upon chelation of the Au^IIIion by only one of two

otherwise equivalent amide groups. This leads to doubling-up

of the number independent ¹H and ¹³C signals for each

gold(III) complex relative to the free ligand, with with the only

exception being the signal from one, as opposed to two, amide

NH protons (deprotonation of the metal-bound amide group

accounting for the loss of the second amide proton). From

Figure 3, it is evident that the amide proton (k) in the gold(III)

complex exhibits a minor (0.07 ppm) upﬁeld shift relative to

the free ligand. More marked, however, is the splitting and

dramatic shifts of the isoquinoline α-CH proton resonances

(i.e., those adjacent to the isoquinoline N atom, a and q) upon

chelation of the metal ion. From the X-ray structure of 4, CH

proton a (atom H1) forms an intramolecular hydrogen bond to

the closest chloride ligand (Cl1). The interaction is

characterized by the following distances and angle: H1···Cl1,

2.71 Å; C1−H1, 0.95 Å; C1−H1···Cl1, 121.1°. The eﬀect of

this particular interaction is marked deshielding of the proton

(labeled a in both spectral traces), which culminates in a 0.76

ppm downﬁeld shift in the signal for 4 relative to the chemical

shift of the proton in the free ligand. The analogous proton on

the metal-free isoquinoline group of 4, proton q, is chemically

and magnetically distinct from proton a. Because of the spatial

location of proton q directly over the metal-bound isoquinoline

ring containing N1 (at a fairly loose ring centroid-to-proton

distance of 4.33 Å), proton q is shielded by the ring current of

the ring system. The magnitude of the upﬁeld shift is 0.52 ppm.

Equivalent protons f in the free ligand split into two single-

integral signals (f and l) in complex 4. These protons are only

marginally aﬀected by metalation of the ligand, consistent with

an essentially unchanged environment relative to the free

ligand. The remaining isoquinoline ring protons of 4 split into

independent signals consistent with the fact that only one

isoquinoline ring serves as a ligand to Au^IIIand all symmetry is

lost in the metal complex. For example, metalation splits the 4-

fold-degenerate doublet at 8.25 ppm of the free ligand (protons

b and e) into four independent doublets (b, e, m, and p);

protons b and e belonging to the metal-bound isoquinoline ring

exhibit a ≥0.23 ppm downﬁeld shift relative to the free ligand

and reﬂect, in broad terms, the eﬀect that polarization of the

ring by the metal ion has on ¹H nuclear shielding in the present

system.

DFT Simulations and Electronic Spectroscopy. In this

section, we describe selected structural and electrostatic data

obtained from DFT simulations on 1−4 with a speciﬁc

emphasis on compound 4 (because of its moderate cytotoxicity

and thus potential as a prototypical metallodrug candidate).

Table 1 compares DFT-calculated bond distances and bond

angles for 1−4, notably those of the chelate ring and ligands to

Au^III, to the analogous structural parameters of the X-ray

structures. The mean diﬀerences between the calculated and

observed bond distances of the ligand donor atoms to the Au^III

ion of each structure are 0.029(16), 0.037(13), 0.051(6), and

0.037(11) Å for 1−4, respectively. The degree of similitude

between the calculated and observed structures is even higher

for the CO and amide N−C bonds of 1−4 with the mean

diﬀerences measuring 0.003(9), 0.001(7), 0.010(7), and

−0.018(14) Å, respectively.

A visual comparison of the match between the DFT-

calculated and X-ray structures of 4 is shown in Figure 4a.

Superposition of the structures by least-squares minimization of

the diﬀerences between the non-H atoms of the isoquinoline

rings, the Au^IIIand Cl⁻ions, as well as the C and N atoms of

the amido chelates reﬂects good agreement between the DFT-

calculated and experimental structures (RMSD, 0.0593 Å).

(Similar structural agreement is observed for 1−3; Figure S22

in the Supporting Information.) The structural superposition of

Figure 4a conﬁrms the accuracy of the DFT method in the ﬁrst

instance and, second, highlights the fact that the conformations

of the auxiliary isoquinolylamido moieties in the two structures

diﬀer rather signiﬁcantly. The nonequivalent positioning of the

isoquinoline rings may be traced to the fact that the in vacuo

DFT-calculated structures do not take into account the

hydrogen bonding and π stacking between the metal-free

isoquinoline rings of the X-ray structure, i.e., conformational

perturbations induced by lattice interactions, as discussed above

(Figure 2b).

Figure 4b illustrates the NBO-calculated partial charge

distribution for 4. Three features are noteworthy: (1) The

fractional charge on the Au^IIIion (+0.76 e) is high, despite the

presence of three anionic ligands and a net charge of zero for

the molecule. This localization of positive charge in the

molecule is clearly likely to favor electrostatic interactions with

electron-rich systems such as the π bonds of heterocyclic

nucleobases or the sugar−phosphate backbone of dsDNA as

well as increase the polarity of the compound. (2) The

presence of the Au^IIIion evidently polarizes the coordinated

amide group and isoquinoline rings relative to those of the

Interestingly, the signals for the amide NH protons of 1 and

2 (but not 4) exhibit triplet patterns (1:2:1 intensity ratio)

consistent with spin−spin coupling to the ¹⁴N nucleus (I = 1)

of the amide N atom in each case (Figure S20 in the

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Figure 4. (a) Superposition (least-squares ﬁt) of the DFT-calculated

and X-ray structures of 4. The root-mean-square deviation (RMSD)

for the ﬁt (coordination sphere and chelated isoquinolylamido moiety)

is indicated. (b) Selected NBO-calculated partial charges for

compound 4. Charges involving the chelate ring and coordinated

Cl⁻ions are emphasized.

metal-free half of the molecule (partial charge values typically

become more positive by up to 0.03 e in the metal-bound

isoquinoline ring). (3) The metal-free amide group clearly has

both the orientation and partial charge separation to favor

hydrogen bonding with suitable partners such as a neighboring

molecule in the solid state (e.g., as shown in Figure S12 in the

Supporting Information) or suitable biomolecules in living cells.

Collectively, complexes 1−4 display similar fractional charge

distributions; the partial charges on the metal ion, donor atoms,

and amide N, C, and O atoms are essentially equivalent to

within 3% of an electron (Table S4 in the Supporting

Information). Perhaps the only signiﬁcant variation in

otherwise marginally variant electron populations for the four

complexes is the electron density found in the nominally vacant

5d_{x −y}orbital, which measures 1.343, 1.448, 1.330, and 1.637 e

for complexes 1−4, respectively. The somewhat higher electron

density for 4 is also reﬂected in the total 5d-orbital electron

density, which follows the order 2 (9.203 e) < 3 (9.217 e) ≅ 1

(9.218 e) < 4 (9.250 e). These data suggest that the

isoquinolylamido chelating ligand of 4 (as opposed to the

pyridylamido chelating ligands of 1−3) is an inherently better σ

donor than the less extensively conjugated heterocyclic

congener.

Figure 5. (a) Superposition of the experimental electronic spectrum of

4 (CH₂Cl₂, 295 K, solid black line) and the TD-DFT-calculated

spectrum (30 states, CH₂Cl₂solvent continuum, broken red line).

Calculated states are depicted as vertical lines with intensities given by

their oscillator strengths. The DFT-calculated spectral envelope is

scaled by a factor of 0.7664 to match the intensity of the experimental

spectrum at 295 nm. The LMCT band (lowest-energy transition) has

a calculated wavelength of 635 nm. (b) Selected MO energy levels for

4 (left) alongside plots of the four frontier MOs of the complex (right)

and the transformed Cartesian axes for assignment of the d-orbital

components of each MO. Abbreviations: H, HOMO (MO 135); L,

LUMO (MO 136); LMCT, ligand-to-metal charge transfer; Δ_1/2, half-

width at half-height. (c) Experimental electronic spectrum of 4 from

part a deconvoluted into eight constituent Voigt functions after

background subtraction (R²= 0.998). Component band maxima and

their standard uncertainties are indicated; band widths and amplitudes

are given in Table S6 in the Supporting Information.

2

Parts a and c of Figure 5 highlight the calculated and

experimental electronic spectra of 4 (spectra for 1−3 are

available in Figures S23−S25 in the Supporting Information).

More speciﬁcally, Figure 5a compares the spectrum calculated

using time-dependent DFT (TD-DFT)⁷⁷methods to the

experimental spectral envelope; Figure 5b illustrates some of

the key molecular orbitals (MOs) involved in the transitions

that collectively account for the spectrum of the complex, and

Figure 6c shows the experimental spectrum of 4 deconvoluted

into eight constituent Voigt functions after background

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π*, π → dσ*, and π → π*/dπ* transitions clustered at 285 and

346 nm in the calculated spectrum.

The DFT-calculated and experimental electronic spectra of 4

(and compounds 1−3 in general) are, however, poorly matched

in the visible region (making deﬁnitive band assignments

somewhat more diﬃcult). The problem is that the experimental

spectrum of 4 is characterized by a rather broad and featureless

absorption envelope that maximizes at ca. 470 nm (Figure 5a);

this maximum is without a DFT-calculated counterpart and, as

shown by the deconvoluted band proﬁle in Figure 5c,

comprises at least three main transitions (437, 472, and 516

nm; bands 5−7, respectively). The two more intense

components labeled band 6 (472 nm) and band 7 (516 nm)

most probably correspond to the two most intense visible-

region LMCT bands of the calculated spectrum (429 and 635

nm), both of which involve excitations from π-symmetry MOs

localized on the ligand framework to the LUMO comprising

2

mainly the 5d_{x −y}orbital and σ* components on the ligand

donor atom sets. If the lowest-energy band in the experimental

spectrum (516 nm) corresponds to the HOMO → LUMO

transition, then it is clear that the DFT-calculated energy of this

band is too low by a rather substantial 119 nm. The DFT-

calculated LMCT transition at 429 nm is then 43 nm too high

in energy relative to the experimental band with suﬃcient

intensity to match this assignment at 472 nm (i.e., band 6).

Despite these obvious disagreements between the calculated

and experimental electronic spectra of 4 and the ensuing

diﬃculty in assigning the experimental bands, it is noteworthy

that the three bands that make up the visible-region spectra of 4

have band widths, Δ_1/2, that are ≥1.5 times broader than those

determined for the π → π* bands below 370 nm in the

spectrum. This observation coupled with the molar absorptiv-

ities of the visible bands (ε > 1000 M⁻¹cm⁻¹) conﬁrms their

LMCT character.

NCI Cytotoxicity Screens. Compounds 2−4 were

accepted for initial single-dose NCI-60 cytotoxicity screens

(10⁻⁵M test agent concentration) by the NCI (Bethesda,

MD); compound 1 was not selected owing to its structural

homology with 2. Of the three compounds screened, only 4

was suﬃciently cytotoxic in the single-dose assay to warrant a

full ﬁve-dose NCI screen spanning the concentration range

10⁻⁸−10⁻⁴M. (Cytotoxicity data for 2−4 over the NCI’s panel

of 60 human cancer cell lines are available in the Supporting

Information.) From the one-dose screens, the mean growth

percentages were 105, 97, and 60% for compounds 2−4,

respectively. From the ﬁve-dose screen of 4, the lowest IC₅₀

values were for the ovarian cancer cell lines OVCAR-3 (4.0

μM) and IGROV1 (9.8 μM) and the colon cancer cell line SW-

620 (15 μM). Figure 6 compares the best GI₅₀values for

compound 4 with analogous data for clinically deployed

anticancer drugs that have deﬁnitive MOAs. Mean cytotoxicity

parameters for compound 4 obtained for each of the nine

classes of human cancer in the NCI-60 screen are listed in

Table 3. Collectively, the data indicate that colon and ovarian

cancer cell lines are, on average, the most susceptible to the

gold(III) complex with GI₅₀and IC₅₀values below 10 and 20

μM, respectively. Several noteworthy points emerge from the

comparative data of Figure 6: (1) The cytotoxicity of 4

compares favorably with that of cisplatin (a DNA cross-linking

agent and guanine-N7 binder) and etoposide (a nonintercalat-

ing topoisomerase II, Top2, poison). (2) With the exception of

CPT, neither 4 nor any of the other drugs exhibit low

nanomolar GI₅₀values. The growth inhibition data for 4, both

Figure 6. Selected NCI cytotoxicity data for 4 and clinically employed

anticancer drugs with known MOAs. The bar chart plots the

concentrations at which 50% growth inhibition occurs (GI₅₀) against

the human cancer cell line for the indicated compounds. Tall bars

correspond to more active growth inhibition by the test agents. The

inset illustrates the normalized dose−response proﬁles for two of the

relevant cancer cell lines with compound 4. Abbreviations: LE,

leukemia; LC, nonsmall lung cancer; CO, colon cancer; CNS, central

nervous system cancer; ME, melanoma; OV, ovarian cancer; RE, renal

cancer; PR, prostate cancer; BR, breast cancer.

subtraction. Transition assignments for the more important

excited states of 4 are listed in Table S5 in the Supporting

Information. As a whole, the electronic spectra for gold(III)

compounds 1−4 are characterized by one or more intense π →

π* bands between 270 and 330 nm and multiple, weaker

ligand-to-metal charge-transfer (LMCT) bands in the region

330−600 nm. The agreement between the DFT-calculated and

experimental spectra is best for compounds 3 and 4, with the

notable exception of the lowest-energy LMCT band (HOMO

→ LUMO), which is uniformly predicted to be more intense

and of longer wavelength (by as much as 150 nm in the case of

3) than the experimental band proﬁle. Refocusing on

compound 4, parts a and c of Figure 5 indicate that the

calculated transition energies for 4 closely match the

experimental spectrum below 330 nm in the region dominated

by intraligand π → π* transitions (with the caveat that the

calculated intensities are ca. 23% higher than those measured

experimentally). Notably, the calculated transition energies

involving the excited states with the largest oscillator strengths

(302 and 323 nm) fall within 2 nm of the band maxima for the

peaks deduced by deconvolution of the experimental spectrum

of the complex (bands 2 and 3). Similarly, the lower-intensity

deconvoluted bands at 284 and 343 nm (bands 1 and 4) in the

experimental spectrum match the energies of the weaker π →

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Table 3. Summary of Cytotoxicity Parameters for

Compound 4 from a Five-Dose Screen against the NCI’s

a

Panel of 60 Human Cancer Cell Lines

cancer

N

GI₅₀/μM

IC₅₀/μM

LC₅₀/μM

leukemia

lung

6

13(10)

11(4)

24(18)

31(11)

13(4)

>100

9

92(77)

85(68)

86(70)

>100

colon

7

8.9(2.7)

11(4)

CNS

6

32(17)

27(15)

18(6)

melanoma

ovarian

renal

8

14(5)

7

7.2(2.5)

8.6(2.6)

8.3(4.1)

7.7(2.8)

10(4)

60(24)

66(44)

70(42)

87(71)

78(57)

8

21(10)

19(10)

26(10)

23(11)

prostate

breast

2

6

b

average

59

a

Abbreviations: N, number of cell lines within each cancer category;

GI₅₀, compound concentration eﬀecting 50% growth inhibition; IC₅₀,

compound concentration eﬀecting 100% growth inhibition; LC₅₀,

compound concentration that induces 50% cell death. Total number

of cell lines used. Estimated standard deviations are given in

parentheses; large values indicate variable susceptibility of a speciﬁc

group of cell lines to the test compound (see Table S7 in the

Supporting Information).

b

Figure 7. Multivariate hierarchical cluster analysis of the −log(GI₅₀)

data from the NCI-60 screen of compound 4 and 24 anticancer

compounds with known in vivo targets. The dendrogram was

produced using the group average method (average linkages) and

Minkowski distances.

on average and for the most sensitive cancer cell lines, therefore

do not compare favorably with the ultrahigh cytotoxicity

displayed by CPT (the archetypal Top1 poison). (3) A

commercially successful anticancer compound need not have

submicromolar GI₅₀values. For example, 5-ﬂuorouracil displays

only moderate cytotoxicity (the GI₅₀values for many cell lines

exceed 10 μM), yet the drug is a widely prescribed

antineoplastic antimetabolite.^78,79As noted above, the NCI’s

panel of seven ovarian cancer cell lines was the most sensitive

to 4. Upon further reﬂection, 4 behaves similarly to the

structurally related cis-dichlorogold(III) dithiocarbamates re-

ported by Ronconi et al.,²⁷with both compound classes

exhibiting IC₅₀values in the low micromolar range for ovarian

cancer.

An important question is whether the GI₅₀values acquired in

the NCI-60 screen for a compound with an unknown MOA

may be used to statistically delineate a probable MOA or

cellular target for the compound. We have used hierarchical

cluster analysis (Figure 7) with the set of GI₅₀values for 4 and

several anticancer drugs with well-known MOAs to determine a

likely target for 4 in vivo. As highlighted in Figure 7, compound

4 is equivalent to zorubicin, a human topoisomerase IIα

(Top2) poison, and, moreover, clusters with the topoisomerase

IB (Top1) poisons topotecan, 9-methoxycamptothecin, and

CPT. The statistical data therefore strongly suggest that

compound 4 exerts its cytotoxic eﬀect by inhibiting Top2

and/or Top1. Note that despite 4 having some structural

similarity to cisplatin (a pair of cis-chloride ligands bound to a

d⁸transition-metal ion), the compound exhibits a cytotoxicity

proﬁle dissimilar to that of cisplatin. One reason for this is that

the chloride ligands in 4 are substitution-inert, reducing the

likelihood of aquation of the metal ion under physiological

conditions and thus subsequent coordination of the gold(III)

complex to DNA.

inhibitors; drugs that inhibit the enzyme by noncovalent

binding of the protein or the enzyme’s target DNA sequence),

experimental methods capable of delineating between the two

types of MOAs ultimately need to be used.

Topoisomerase IIα Inhibition Assays. Because 4 targets

Top2 (Figure 7), we have used a Top2 enzyme inhibition assay

(equivalent to that developed and marketed by TopoGEN,

Inc.) to gauge the dose−response function for the test agent

with the target enzyme. The agarose gel shown in Figure 8a

clearly indicates that concentrations of 4 < 50 nM result in

essentially undetectable quantities of linear DNA cleavage

product and do not inhibit decatenation of kDNA by Top2.

However, for [4] in the range of 50 nM to 1 μM (lanes 8−11),

there is a clear nonlinear increase in the amount of linear DNA

with increasing gold(III) chelate concentration. The dose−

response function maximizes at [4] = 1 μM before

commencing a nonlinear decrease with increasing [4] over

the range of 5−50 μM (lanes 12−15). The overall dose−

response function is therefore biphasic with a skewed bell-like

shape. The bell-shaped dependence of the linear DNA product

concentration upon increasing [4] is well-ﬁtted by eq 2

(Experimental Section), a simple summation of two consec-

utive opposing (positive and negative) dose−response

functions. The ﬁrst binding event, marked by its midpoint A

on the graph, has K_D1∼ 240 nM²and a Hill coeﬃcient, n_H1, of

2.4(7) (i.e., ∼2 within 1σ). The Hill coeﬃcient of 2 is

consistent with two binding sites for the gold(III) complex in

the ﬁrst equilibrium.⁸⁰The second binding event, midpoint B

on the graph, has K_D2∼ 8.6 μM and a Hill coeﬃcient, n_H2, of

1.2(2), with the latter value for the Hill coeﬃcient reﬂecting a

single binding site for 4 in the second equilibrium.

The biphasic dose−response function, dual-equilibrium

dissociation constants, and Hill coeﬃcients above clearly signify

a switch in the reaction mechanism from compound 4 behaving

as an IFP at low concentrations to the compound operating as a

CIC at higher concentrations. Mechanistically, the ﬁrst phase is

consistent with two molecules of 4 noncovalently binding to an

intermediate Top2−DNA covalent cleavage complex at its two

nick sites (K_D1) in a manner akin to that recently elucidated for

etoposide (VP-16) from the X-ray structure of the quaternary

Conﬁrmation of the molecular MOA of compound 4 implied

by the hierarchical cluster analysis requires that in vitro enzyme

inhibition assays with both Top1 and Top2 be conducted in the

ﬁrst instance. Because topoisomerase inhibitors may be divided

into IFPs (interfacial poisons; drugs that irreversibly trap

DNA−enzyme covalent complexes) and CICs (catalytic

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The mechanistic switch with increasing [4] to a second phase

requires that the gold(III) chelate has an additional target with

a lower binding constant (K_D2) than insertion of the compound

at the Top2−DNA cleavage complex nick sites (K_D1). As

discussed later, 4 binds weakly to DNA, probably by

intercalation (but only at higher concentrations), and it is

therefore conceivable that weak intercalative binding at (or

near) the DNA base sequence targeted by Top2 as [4]

increases competes with nick-site binding and ultimately

dominates (by preventing the enzyme from associating with

its substrate) at elevated concentrations of the gold(III) chelate.

In eﬀect, catalytic inhibition of Top2 wins out over poisoning of

the enzyme at higher concentrations of 4 such that the yield of

linear reaction product declines for [4] > 1 μM (lanes 11−15).

Note that catalytic inhibition of Top2 becomes clearly evident

even at [4] = 500 nM (lane 10) because the yield of NOC

DNA is markedly reduced at this concentration. Furthermore,

untransformed catenated DNA (nonmigrating in 1% agarose

gel) has sharply accumulated in the well, a pattern that becomes

even more marked in the 1−50 μM concentration range for 4

(lanes 11−15). On the basis of the published catalytic

mechanism for Top2⁸²and the crystal structure of the VP-

16-poisoned enzyme,⁸¹a simpliﬁed mechanistic scheme (Figure

8b) may be used to adequately account for the present data.

Dual-mode IFP catalytic inhibitors of Top2 with hallmark

bell-shaped dose−response curves have been reported by

others, e.g., with compounds such as 9-hydroxyellipticine⁸³and

adriamycin (doxorubicin),^84,85but often the data are not

suitable for the quantiﬁcation of stepwise K_Dvalues as we have

been able to do with 4. Our interpretation of the dual-mode

reaction of 4 with Top2 is thus consistent with earlier

viewpoints on similar biphasic inhibition of the enzyme with

polycyclic organic compounds. Moreover, the enzyme inhib-

ition data fully support the MOA of 4 identiﬁed from the in

vivo cytotoxicity data (GI₅₀values) by hierarchical cluster

analysis.

Topoisomerase IB Inhibition Assays. Hierarchical cluster

analysis of the NCI data for 4 (Figure 7) showed that, in

addition to being equivalent to zorubicin (a Top2 IFP), 4

behaves similarly (not identically) to topotecan, a Top1 IFP.

The most widely used Top1 inhibition assay gauges the ability

of Top1 to relax SC plasmid DNA with increasing drug

concentration. However, with a possible mixed mode of

inhibition (IFP and CIC), the standard assay is complicated

by the fact that 4 is also a DNA intercalator, as described below.

As a result, the topoisomer distribution of reaction products is

altered by the test drug, which complicates data interpretation.

As detailed in the Experimental Section, we have modiﬁed the

standard Top1 inhibition assay so that it may be used to

distinguish between an IFP and a catalytic inhibitor of Top1.

The new assay is based on the fact that, at low salt

concentrations and high enzyme loadings, the probability of

forming a 2:1 (Top1)₂−DNA covalent cleavage complex during

enzyme turnover increases. In the presence of an IFP of the

enzyme, which traps the covalent cleavage complexes by

binding at the enzyme-bound nick sites on adjacent DNA

strands, the fraction of doubly nicked DNA present in the

system will increase. Upon quenching of the reaction followed

by PK digestion to remove covalently bound Top1 from the

DNA, this yields an increase in the fraction of linear DNA in

the reaction product mixture (provided that some of the nick

sites on adjacent strands are close enough that the circular

DNA opens up to its linear form with fairly short, nonannealing

Figure 8. (a) Electrophoretic analysis (1% agarose gel, 0.5 μg mL⁻¹

ethidium bromide, EB) of a topoisomerase II (Top2) decatenation

assay employing kinetoplast DNA (kDNA, 200 ng μL⁻¹, 30 ng well⁻¹)

as the substrate, an enzyme concentration of 2 units (U) per μL,

etoposide (VP-16) as the Top2 poison control, and compound 4 as

the test agent over a broad concentration range. All annotated lanes,

unless otherwise indicated, contain Top2. The inset plots the relative

peak areas (proportional to the concentration) for the highlighted

linear DNA bands. The solid line shows a ﬁt of the dose−response

data to eq 2. (Independent functions ﬁtting parts A and B of the

skewed bell-shaped curve are shown as broken gray lines.) (b)

Biphasic inhibition of Top2 by 4. The normal catalyzed reaction of

Top2 runs from left to right: Top2 forms a covalent cleavage complex

(Top2-DNA^CC) with catenated DNA (DNA^cat) as the substrate,

allowing the passage of a remote DNA strand through the double-

stranded break in the covalently anchored DNA within the cleavage

complex. Top2 reseals the cleaved DNA strand prior to release of

circular DNA. Repetition of the reaction converts catenated DNA into

decatenated DNA (DNA^decat), with the latter comprising supercoiled

(SC) and nicked-open-circular (NOC) DNA. Compound 4 behaves as

an IFP that traps the covalent cleavage complex at low concentrations

(section A of the dose−response curve, K_D1). At higher concentrations

of 4 (section B of the dose−response curve, K_D2), competitive binding

of the gold(III) chelate to the DNA substrate blocks Top2 from

recognizing its substrate, leading to catalytic inhibition of the enzyme.

Abbreviation: PK, proteinase K.

(drug)₂−DNA−enzyme covalent cleavage complex.⁸¹Figure 8a

shows that, as the concentration of the gold(III) chelate

increases from 50 nM to 1 μM, the trapped (i.e., poisoned)

fraction of the intermediate DNA−enzyme covalent cleavage

complex increases during the catalytic cycle of the enzyme, as

expected, accounting for the increase in the amount of linear

DNA product isolated from the reaction.

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sticky ends). In essence, under these assay conditions, the

activity of Top1 is substantially enhanced, signiﬁcantly

increasing the probability of trapping the cleavage intermediate,

even in the absence an IFP. To prove that the cleavages we

detect are due to Top1, a control with CPT (Figure 9, lane 5)

Top 2 (with K_Dvalues in the micromolar range). The ability of

4 to act as an IFP of Top2 at low concentrations (K_Din the

nanomolar range) is, however, unique to its mode of action

with Top2. This likely reﬂects the speciﬁc three-dimensional

constraints of the pair of nick sites in the Top2−DNA covalent

cleavage complex,⁸¹which evidently ﬁt the structure of 4 yet are

diﬀerent from the single nick site of the Top1−DNA cleavage

complex.⁷⁴Despite having obtained X-ray structural data for 4

(vide supra) and an understanding of the compound’s ability to

form π−π stacks, speculation as to how 4 might intercalate at

the nick sites of the Top2−DNA cleavage complex is

unwarranted with the data at hand. Interestingly, Wang et

al.⁸⁷recently reported a series of cationic and neutral

platinum(II) poly(pyridine) complexes with acetylide nucleo-

bases as variable coligands that were either Top2 or dual Top2/

Top1 inhibitors. Changes in the ligand structure modulated

both the activity level and mechanism of inhibition (IFP or

CIC) of Top2 by the compounds. In the present study, 4

evidently behaves similarly to the above platinum(II)

complexes.

DNA Binding by 4. From the reactions of 4 with Top1 and

Top2, catalytic inhibition of both enzymes at [4] > 1 μM

appears to reﬂect binding of the compound to the enzyme’s

DNA substrate (in a fashion similar to that of doxorubicin and

its analogues).⁸⁴We employed electrophoretic mobility shift

assays (EMSAs) with pHOT1 plasmid DNA and varying

concentrations of 4, EB (a cationic DNA intercalator control),

and m-AMSA (a neutral DNA intercalator control⁸⁸) to test

this hypothesis and to further investigate the interaction of 4

with DNA. In these experiments, the plasmid DNA substrate

was equilibrated with the test compounds for 15 min at 37 °C

in essentially the same buﬀer solution as that used for the Top1

and Top2 inhibition assays. The gel in Figure 10a shows that

both plasmid DNA bands (SC DNA, form I; NOC DNA, form

II) exhibit reduced mobility with increasing concentrations of

both EB and 4. The SC DNA band (form I) is aﬀected most by

intercalation of EB and by the binding of 4, with compound 4

inducing a 6% mobility shift of SC DNA at a concentration of

50 μM (Figure 10b). In comparison, the cationic DNA

intercalator control compound (EB) engenders a 21% mobility

shift of SC DNA at a concentration of 50 μM. The marked

diﬀerence in the magnitude of the mobility shift for the SC

DNA band probably reﬂects several factors including the charge

diﬀerences for the two compounds, their intrinsic DNA binding

aﬃnities, the extent and sites of intercalation, as well as their

inherently diﬀerent structures.

We tested the hypothesis that the charge of the DNA

intercalator is important in determining the magnitude of the

mobility shift by performing the analogous EMSA for m-AMSA,

a neutral DNA intercalator control compound (Figure 10d,e).

This compound had the smallest eﬀect (3.9% shift) on the

mobility of SC DNA, and the band broadening observed with

EB and 4 was virtually absent. One interpretation of this result

is that, although 4 is overall a neutral compound, it does

contain the Au^IIIcation, which, as shown by our DFT

simulations, retains a fractional positive charge of ca. 0.76 e

on the Au^IIIcenter, making it polar and thus allowing it to have

a similar (but understandably smaller) eﬀect on the topology

(and thus mobility) of SC DNA relative to the cationic

intercalator EB. In a nutshell, the results displayed in Figure 10

suggest that 4 weakly intercalates DNA at relatively high

concentrations (>5 μM) and that this likely underpins catalytic

inhibition of Top1 and Top2 by the compound. Interestingly,

Figure 9. Electrophoretic analysis of a “zero-salt, enzyme-rich”

topoisomerase IB (Top1) cleavage assay employing SC plasmid

DNA (pHOT1, 188 ng μL⁻¹) as the substrate, an enzyme

concentration of 50 U μL⁻¹(in all annotated lanes except the ﬁrst),

CPT as the Top1 poison control, and compound 4 over a wide

concentration range, maximizing at 25 μM. The inset plots the relative

concentrations of NOC and SC DNA in lanes 9−13. The gel (1%

agarose) contains EB (0.5 μg mL⁻¹). Abbreviations: L, NOC, SC, and

RX represent linear, nicked-open circular, supercoiled, and relaxed

DNA, respectively.

shows that this potent IFP results in double-stranded DNA

breaks because of the nested action of Top1 molecules. In

support of this, previous work showed that endogenous Top1

has the ability to “cluster” at catalytic sites on genomic DNA in

situ;⁸⁶thus, at high input levels of enzyme in the presence of an

IFP like CPT, nested single-stranded DNA cleavages give rise

to double-stranded breaks.

Figure 9 shows the reaction products generated in this

modiﬁed Top1 inhibition assay. Lane 2 of the EB gel indicates

that Top1 relaxes pHOT1 plasmid DNA in the absence of an

added inhibitor to give a heightened concentration of NOC

DNA and relaxed-circular DNA (RX DNA), as expected from

our working hypothesis above. In the presence of a high

concentration of a known and potent Top1 IFP (50 μM CPT,

lane 5), only NOC DNA and linear DNA are obtained from the

reaction. (RX DNA is absent because of complete poisoning of

Top1 at this dose of the control compound.)

Lanes 7−13 reﬂect the reaction products with increasing

concentrations of 4 up to 25 μM; at no point is linear DNA

evident. Notably, the yield of NOC DNA decreases in a

sigmoidal fashion as [4] increases, while the fraction of

untransformed SC DNA substrate increases with the sigmoidal

mirror image curve. The assay clearly identiﬁes 4 as a catalytic

inhibitor of Top1 with a K_Dor IC₅₀value of ca. 4 μM.

Overview of the MOA of 4. The Top2 and Top1 enzyme

inhibition assays (Figures 8 and 9) indicate that 4 targets both

DNA-regulating enzymes in similar, yet diﬀerent ways. Catalytic

inhibition through DNA binding (see below) is evidently the

common catalytic inhibition mechanism for both Top1 and

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Supporting Information, it is clear that DNA-bound 4 induces

partial negative supercoiling of relaxed pHOT1 plasmid DNA

in the presence of Top1. This conﬁrms that 4 is a weaker DNA

intercalator than EB but, nonetheless, an intercalator capable of

catalytically inhibiting both Top1 and Top2. Importantly,

because not all DNA intercalators inhibit these DNA-regulating

enzymes, we conclude that 4 must intercalate, even if only

partially, at a base pair at or near the target base sequences

recognized by Top1 and Top2.

Stability of 4 in Biological Buﬀers. The moderate

cytotoxicity of 4 in vivo (Table 3) suggests that the loss of 4 to

physical, chemical, or cellular (e.g., drug eﬄux) processes might

lower its eﬀective concentration over the time course of cell

growth experiments. From the time-dependent decrease in the

absorbance at 295 nm for 4 (Figure S30 in the Supporting

Information; pH 7.34, 37 °C), the monomeric gold(III)

complex clearly undergoes slow, biphasic precipitation. The

process commences after a ca. 5−6 h induction period and is

essentially complete after 48 h. Notably, the rate constants were

independent of the NaCl concentration (from 35.5 to 1000

mM); the averaged values were k₁= 0.184(6) h⁻¹and k₂=

3.4(1.6) × 10⁻³h⁻¹. Importantly, hydrolysis of the Au−Cl

bonds of 4 appears not to occur for two reasons: (1) such a

process would show a dependence on [NaCl], contrary to

observation, and (2) substitution of the chloride ligands bound

to the Au^IIIion would be expected to shift the LMCT bands in

the electronic spectrum. Our spectral data are consistent with

the Au−Cl bonds of 4 remaining intact for more than 72 h in

solution. Similar solution stability was recently reported for cis-

dichlorogold(III) derivatives of 2-(2′-pyridyl)benzimidazole.⁹⁰

That said, on the time scale of the NCI’s cytotoxicity screens

(48 h), precipitation of 4 will compete with cellular uptake of

the compound and may ultimately lower the eﬀective

cytotoxicity of the compound.

CONCLUSIONS

Figure 10. (a) EMSA of double-stranded pHOT1 plasmid DNA as a

function of the concentration of 4 and the cationic DNA intercalator

EB control. DNA band identities: I, SC DNA; II, NOC DNA. (b)

Two-dimensional peak deconvolution of lanes 5 and 10−14 of the gel

shown in part a. (c) Two-dimensional peak deconvolution of lanes 1−

4 of the gel shown in part a. (d) EMSA of double-stranded pHOT1

plasmid DNA as a function of the concentration of the neutral DNA

intercalator m-AMSA [m-amsacrine or 4′- (acridinylamino)-

methanesulfon-m-anisidide]. (e) Two-dimensional peak deconvolution

of lanes 1−4 of the gel shown in part d.

■

In summary, and before enumerating the key conclusions of

this work, four neutral AuCl₂(X) derivatives 1−4, where X is an

anionic bidentate pyridyl- or quinolylamido chelate, were

synthesized and characterized by X-ray diﬀraction, DFT

simulations, and a range of spectroscopic techniques.

Compounds 2−4 were screened for their cytotoxicity against

the NCI’s panel of 60 human cancer cell lines and the MOA of

the most active compound (quinolylamido chelate 4)

elucidated using statistical methods coupled with in vitro

enzyme inhibition assays. DNA and topoisomerases IB and IIα

are the principal cellular targets of the compound.

The main conclusions of this study are the following. (1)

Although the ligands for 1, 2, and 4 are potentially tetradentate,

they fail to substitute all four Cl⁻ions when reacted with

[AuCl₄]⁻salts, leading to bidentate cis-dichlorogold(III)

complexes. (2) Compounds 1−4 exhibit a signiﬁcant structural

trans eﬀect induced by the amido N donor, which favors

elongation of the diametric Au−Cl bond in each case. (3) DFT

calculations showed that signiﬁcant residual fractional cationic

charge resides on the Au^IIIions of 1−4 (ca. +0.8 e), which is

evidently important for the interaction of 4 with DNA. (4)

Compound 4 was moderately cytotoxic in a ﬁve-dose NCI-60

screen (mean IC₅₀over 60 cell lines ∼38 μM). (5) Hierarchical

cluster analysis methods indicated that 4 is mechanistically

equivalent to the doxorubicin analogue zorubicin, a Top2

poison, and mechanistically similar to topotecan, a Top1

poison. (6) Using a Top2 inhibition assay, we have shown that

DNA-intercalating cytotoxic gold(III) porphyrins also inhibit

Top1, but a deﬁnitive mechanism is still lacking.²³

With all DNA-binding compounds, the question arises as to

whether the compound is an intercalator or a groove binder.

There are diﬀerent ways to answer this question, with one of

the simplest (other than an EMSA) being a DNA winding

assay.⁸⁹In this experiment (Figure S29 in the Supporting

Information), fully relaxed plasmid DNA was equilibrated with

4 prior to reaction with Top1. As highlighted by Palchaudhuri

and Hergenrother,⁸⁹a DNA intercalator will bind to the relaxed

plasmid DNA generated by the action of Top1 and then induce

negative supercoiling of the substrate. In the case of a strong

DNA intercalator, this ideally results in the appearance of

topoisomer bands on the gel with increasing intercalator

concentration and ultimately only SC DNA at the highest

compound dose. Nonintercalative compounds yield a negative

result in the assay. From the data in Figure S29 in the

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1

RNH−CO−R). H NMR (500 MHz, DMSO-d₆, 303.15 K): δ 10.90

(s, 2H, g), 9.36 (s, 2H, a), 8.73 (s, 2H, f), 8.25 (d, 4H, J = 9.0 Hz, e, b),

7.90 (ddd, 2H, J₁= 9.4 Hz, J₂= 4.3 Hz, J₃= 1.2 Hz, d), 7.87 (dd, 2H, J₁

= 5.9 Hz, J₂= 3.6 Hz, h), 7.82 (ddd, 2H, J₁= 9.3 Hz, J₂= 4.3 Hz, J₃=

1.1 Hz, c), 7.33 (dd, 2H, J₁= 6.0 Hz, J₂= 3.6 Hz, i). ¹³C NMR (123

MHz, DMSO-d₆, 303.15 K): δ 163.62 (C-10), 152.13 (C-1), 143.76

(C-9), 135.91 (C-11), 132.01 (C-5), 131.67 (C-7), 129.92 (C-4),

129.89 (C-2), 128.59 (C-3), 128.37 (C-6), 126.07 (C-12), 125.68 (C-

13), 121.04 (C-8).

4 is an IFP of Top2 at low concentrations (K_D∼ 240 nM) and

a catalytic inhibitor of the enzyme at higher concentrations (K_D

∼ 9 μM). (7) A new enzyme inhibition assay capable of

distinguishing between IFPs and catalytic inhibitors of Top1

was developed and used to prove that 4 is a catalytic inhibitor,

but not an IFP, of Top1. (8) Last, catalytic inhibition of Top1

and Top2 by 4 evidently reﬂects DNA intercalation by the

compound. Going forward, structural derivatives or formula-

tions (e.g., micelles) of 4 less susceptible to precipitation from

aqueous solutions may have improved cytotoxicity and thus

prospects for further development.

General Synthetic Procedure for Metal Complexes. A

solution of the ligand (5.29 × 10⁻⁴mol) in DCM (15 mL) was

added to a solution of K[AuCl₄] (5.29 × 10⁻⁴mol) in methanol (10

mL). This solution was then stirred while sodium acetate (6.00 × 10⁻⁴

mol) in methanol (10 mL) was added dropwise prior to heating the

reaction under reﬂux for 2 h. The solution was then cooled to room

temperature and allowed to stir overnight to aﬀord an orange-red

precipitate, which was ﬁltered oﬀ and washed with methanol (2 × 10

mL).

EXPERIMENTAL SECTION

■

General Procedures. All reagents were used as received from

Aldrich unless otherwise stated. All reactions were carried out under

atmospheric conditions. Pyridine, methanol, dimethyl sulfoxide

(DMSO), and nitromethane were Chromosolve HPLC-grade solvents.

Dichloromethane (DCM) was distilled over calcium hydride. Gold

pellets were received as a loan from Mintek (Randburg, South Africa)

as part of project AuTEK Biomed. Electronic spectra were recorded

with a Shimadzu UV-1800 spectrometer (2 nm slit width) using

varying solvents in 1.0-cm-path-length quartz cuvettes. Fourier

transform infrared spectra were obtained on a Bruker Alpha

spectrometer (36 scans, spectral resolution = 1.0 cm⁻¹) with a

diamond ATR accessory for studying microcrystalline samples. ¹H and

¹³C NMR spectra were recorded using saturated solutions in DMSO-

d₆or chloroform-d₁using either a 500 MHz Bruker Avance III

spectrometer equipped with an 11.7 T magnet and a 5 mm BBO probe

or a 400 MHz Bruker Avance III spectrometer equipped with a 9.4 T

magnet and a 5 mm BBOF probe. The solvent signal was used as the

reference. All spectra were recorded at 303.15 K unless otherwise

stated. Proton and carbon signal assignments were made from

consideration of the DFT-calculated nuclear shielding tensors (vide

infra) in conjunction with the primary experimental NMR spectra

(one-dimensional ¹H and ¹³C, COSY, HSQC, HMBC, DEPT-90, and

DEPT-135). KyPlot 5.0.2 (KyensLab, Inc.)⁹¹was used for all graphing

and curve-ﬁtting. Fityk 0.9.8 was used for spectral peak deconvolution

and envelope ﬁtting.⁹²

AuCl₂(HL¹), Compound 1. cis-Dichloro{(pyridin-2-ylcarbonyl)[2-

[(pyridin-2-ylcarbonyl)amino]ethyl]azanido}gold(III), Yield: 72%

(orange powder). TOF MS ES: m/z 558.9976 (calcd, m/z

558.9979), [(M + Na)⁺]. Anal. Calcd for C₁₄H₁₃AuCl₂N₄O₂: C,

31.30; H, 2.44; N, 10.43. Found: C, 30.95; H, 2.42; N, 10.16. UV−vis

[DMSO; λ_max, nm (ε, M⁻¹cm⁻¹)]: 312 (5.1 × 10³), 453 (1.4 × 10²).

IR (powder, cm⁻¹): 3360 (s, ν(N−H), R−NH−COR), 1645 (s,

ν(CO), RNH−CO−R), 1602 (s, ν(CO), RN−CO−R). ¹H NMR

(400 MHz, DMSO-d₆, 303.15 K): δ 9.32 (d, 1H, J = 3.04 Hz, a), 8.88

(t, 1H, J = 6.08(2) Hz, g), 8.56 (d, 1H, J = 2.04 Hz, k), 8.48 (t, 1H, J =

7.64 Hz, c), 8.03 (t, 1H, J = 6.82 Hz, b), 7.92 (m, 3H, d, h, i), 7.54 (m,

1H, j), 3.72 (t, 2H, J = 5.42 Hz, e), 3.57 (m, 2H, f). ¹³C NMR (100

MHz, DMSO-d₆, 303.15 K): δ 171.66 (C-6), 164.73 (C-9), 150.60 (C-

11), 149.40 (C-10), 148.70 (C-5), 145.51 (C-1), 145.31 (C-3), 138.06

(C-2), 130.33 (C-4), 128.65 (C-14), 126.73 (C-13), 122.19 (C-12),

46.35 (C-7), 40.23 (C-8).

Ligand Synthesis. The free ligands HL¹, H₂L², and H₂L³were

prepared according to literature procedures and gave consistent

spectroscopic data.⁴⁸⁻⁵¹

Ligand H₂L⁴. To a solution of isoquinolinecarboxylic acid (2.00 g,

1.624 × 10⁻²mol) in dry pyridine (15 mL) was added 1,2-

diaminobenzene (0.878 g, 8.122 × 10⁻³mol) in pyridine (5 mL). The

mixture was stirred for 5 min, during which time a white precipitate

formed. The mixture was then heated to 110 °C prior to the dropwise

addition of triphenylphosphite (5.041 g, 1.624 × 10⁻²mol), which was

followed by continuous stirring at 110 °C for 4 h. The solution was

then allowed to cool and stand overnight, during which time the

product (N,N′-benzene-1,2-diyldiisoquinoline-3-carboxamide, H₂L⁴)

crystallized out of solution. The product was isolated by ﬁltration and

washed with diethyl ether (2 × 25 mL) and distilled water (2 × 25

mL) to obtain a white powder. X-ray-quality crystals were obtained by

diﬀusion of diethyl ether into a saturated solution of H₂L⁴in DCM.

Yield: 65% (colorless crystals).

AuCl₂(HL²), Compound 2. cis-Dichloro{(pyridine-2-yl-carbonyl)[3-

[(pyridin-2-ylcarbonyl)amino]propyl]azanido}gold(III). Yield: 78%

(orange powder). TOF MS ES: m/z 573.0134 (calcd, m/z

573.0135), [(M + Na)⁺]. Anal. Calcd for C₁₅H₁₅AuCl₂N₄O₂: C,

32.69; H, 2.74; N, 10.16. Found: C, 32.57; H, 2.69; N, 10.02. UV−vis

[DMSO; λ_max, nm (ε, M⁻¹cm⁻¹)]: 310 (3.9 × 10³), 449 (1.2 × 10²).

IR (powder cm⁻¹): 3392 (s, ν(N−H), R-NH−COR), 1666 (s, ν(CO),

RNHCO−R), 1643 (s, ν(CO), RN-CO-R). ¹H NMR (400 MHz,

DMSO-d₆, 303.15 K): δ 9.29 (d, 1H, J = 2.85 Hz, a), 8.81 (t, 1H, J =

6.19(6) Hz, h), 8.61 (m, 1H, i), 8.50 (t, 1H, J = 7.72 Hz, b), 8.00 (m,

4H, c, d, k, l), 7.58 (m, 1H, j), 3.56 (m, 2H, e), 3.36 (m, 2H, g), 1.86

(m, 2H, f). ¹³C NMR (100 MHz, DMSO-d₆, 303.15 K): δ 170.96 (C-

6), 163.55 (C-10), 149.95 (C-15), 148.82 (C-11), 148.28 (C-5),

145.03 (C-1), 144.91 (C-3), 137.77 (C-13), 129.82 (C-2), 128.18 (C-

4), 126.34 (C-14), 121.72 (C-12), 44.49 (C-7), 36.31 (C-9), 29.94 (C-

8).

TOF MS ES: m/z 419.1507 (calcd, m/z 419.1508), M⁺. Anal. Calcd

for C₂₆H₁₈N₄O₂: C, 74.63; H, 4.34; N, 13.39. Found: C, 74.73; H,

4.46; N, 13.57. UV−vis [CH₂Cl₂; λ_max, nm (ε, M⁻¹cm⁻¹)]: 232.5 (1.6

× 10⁵), 289 (4.6 × 10⁴), 310 (3.4 × 10⁴), 324.5 (2.8 × 10⁴). IR

(powder, cm⁻¹): 3337 (s, ν(N−H), R−NH−COR), 1689 (s, ν(CO),

7902

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Inorganic Chemistry

Article

AuCl₂(L³), Compound 3. cis-Dichloro[(4-methoxyphenyl)(pyridin-

2-ylcarbonyl)azanido]gold(III). Yield: 69% (dark red powder). TOF

MS ES: m/z 494.9947 (calcd, m/z 494.9941), M⁺. Anal. Calcd for

C₁₃H₁₁AuCl₂N₄O₂: C, 31.54; H, 2.24; N, 5.66. Found: C, 31.73; H,

2.10; N, 5.32. IR (powder cm⁻¹): 2947, 3063, 3105 (s, ν(CH),

OCH₃), 1643 (s, ν(CO), RN-CO-R). UV−vis [CH₂Cl₂; λ_max, nm (ε,

M⁻¹cm⁻¹)]: 265 (9.5 × 10³), 333 (4.9 × 10³), 400 (3.3 × 10³), 470

T = 296(2) K, R1 (wR2) = 0.0485 (0.1263) for 3035 unique data with

I > 2σ(I), R1 (wR2) = 0.0629 (0.1377) for all 21333 data (R_int

=

0.0378), goodness of ﬁt (based on F²) = 1.064.

X-ray data for 1: C₁₄H₁₃AuCl₂N₄O₂, fw = 537.15 amu, a = 7.477

(5) Å, b = 10.210(5) Å, c = 11.023(5) Å, α = 85.443(5)°, β =

79.313(5)°, γ = 72.383(5)°, V = 787.9(7) Å³, triclinic, P1, Z = 2, D =

̅

c

2.264 g cm⁻³, μ = 9.689 mm⁻¹, T = 296(2) K, R1 (wR2) = 0.0335

(0.0816) for 3043 unique data with I > 2σ(I), R1 (wR2) = 0.0396

(0.0834) for all 6130 data (R_int= 0.0402), goodness of ﬁt (based on

F²) = 1.010.

1

(3.3 × 10³). H NMR (400 MHz, DMSO-d₆, 303.15 K): δ 9.36 (d,

1H, J = 2.86 Hz, a), 8.55 (t, 1H, J = 7.73 Hz, c); 8.08 (m, 2H, b, d),

7.20 (m, 2H, e), 6.91 (m, 2H, f), 3.78 (s, 3H, g). ¹³C NMR (100 MHz,

DMSO-d₆, 303.15 K): δ 169.83 (C-6), 158.26 (C-10), 148.26 (C-5),

144.98 (C-1), 144.98 (C-3), 137.14 (C-7), 130.04 (C-8), 130.01 (C-

2), 128.44 (C-4), 113.36 (C-9), 55.24 (C-11).

X-ray data for 2: C₁₅H₁₅AuCl₂N₄O₂, fw = 551.18 amu, a =

11.188(5) Å, b = 10.192(5) Å, c = 15.132(5) Å, β = 93.350(5)°, V =

1722.5(13) Å³, monoclinic, P2₁/n, Z = 4, D_c= 2.125 g cm⁻³, μ = 8.866

mm⁻¹, T = 296(2) K, R1 (wR2) = 0.0317 (0.0740) for 4240 unique

data with I > 2σ(I), R1 (wR2) = 0.0553 (0.0803) for all 6154 data (R_int

= 0.0454), goodness of ﬁt (based on F²) = 0.960.

X-ray data for 3: C₁₃H₁₁AuCl₂N₂O₂, fw = 495.10 amu, a =

12.971(5) Å, b = 8.701(5) Å, c = 14.068(5) Å, β = 114.723(5)°, V =

1442.2(11) Å³, monoclinic, P2₁/n, Z = 4, D_c= 2.280 g cm⁻³, μ =

10.572 mm⁻¹, T = 296(2) K, R1 (wR2) = 0.0361 (0.0990) for 2494

unique data with I > 2σ(I), R1 (wR2) = 0.0403 (0.1018) for all 14511

data (R_int= 0.0451), goodness of ﬁt (based on F²) = 1.051.

AuCl₂(HL⁴), Compound 4. The same general procedure used above

was employed for the synthesis of 4, cis-dichloro{(isoquinolin-3-

ylcarbonyl)[2-[(isoquinolin-3-ylcarbonyl)amino]phenyl]azanido}gold-

(III). However, the postmetalation reaction mixture was ﬁltered and

the ﬁltrate allowed to evaporate to dryness in a beaker to aﬀord solid 4

as well as an unidentiﬁed byproduct. The solid product was dissolved

in DCM and the insoluble byproduct removed by ﬁltration. The

solution of 4 in DCM was layered in test tubes with diethyl ether to

aﬀord clean 4 as a dark-red powder. X-ray-quality crystals were

obtained by the slow diﬀusion of diethyl ether into a saturated solution

of 4 in nitromethane. Yield: 30% (dark-red powder). TOF MS ES: m/

z 685.0463 (calcd, m/z 685.0472), M⁺. Anal. Calcd for

C₂₆H₁₇AuCl₂N₄O₂: C, 45.57; H, 2.50; N, 8.18. Found: C, 45.81; H,

2.61; N, 8.03. IR (powder cm⁻¹): 3324 (s, ν(N−H), R−NH−COR),

1668 (s, ν(CO), RNH−CO−R), 1652 (s, ν(CO), RN−CO−R). UV−

vis [CH₂Cl₂; λ_max, nm (ε, M⁻¹cm⁻¹)]: 240 (1.2 × 10⁵), 247 (1.0 ×

X-ray data for 4·OEt₂: C₂₆H₁₇AuCl₂N₄O₂·C₄H₁₀O, fw = 759.42

amu, a = 15.990(5) Å, b = 12.445(5) Å, c = 15.602(5) Å, β =

104.487(5)°, V = 3006.0(18) Å³, monoclinic, P2₁/c, Z = 4, D_c= 1.678

g cm⁻³, μ = 5.109 mm⁻¹, T = 100(2) K, R1 (wR2) = 0.0393 (0.0904)

for 7698 unique data with I > 2σ(I), R1 (wR2) = 0.0595 (0.0961) for

all 45814 data (R_int= 0.0577), goodness of ﬁt (based on F²) = 0.955.

Molecular Simulations. All DFT calculations were performed

using the multiprocessor version of Gaussian 09W⁹⁶with the

HSEH1PBE^97,98hybrid functional and the 6-311G(d,p) basis

set^99,100for all atoms except Au, for which the Los Alamos eﬀective

core potential basis set LanL2DZ^101,102was implemented. An f-type

polarization function on Au (α = 0.20) was additionally incorporated.

Spectral properties were calculated with standard procedures in

Gaussian 09W. Speciﬁcally, NMR shielding tensors were determined

by GIAO theory^103,104with DMSO as the solvent (self-consistent-ﬁeld

reaction method based on the conductor-like polarizable continuum

model^105,106). Electronic spectra were calculated using the TD-DFT

method (analytical gradients)^107,108with 30 excited states in a DCM

solvent continuum. Partial atomic charges were calculated using the

natural population analysis routine of NBO version 3.¹⁰⁹

NCI Cytotoxicity Screens. Compounds 2−4 were accepted by the

NCI (Bethesda, MD) for single-dose screens at a concentration of

10⁻⁵M against their panel of 60 diﬀerent human tumor cell lines,

enabling growth percentages of each cell line to be determined in the

presence of the test compounds. Compound 4 proceeded further to a

ﬁve-dose NCI-60 screen spanning the concentration range of 10⁻⁸−

10⁻⁵M, allowing the determination of GI₅₀, TGI, and LC₅₀values for

the compound. The methodology followed and deﬁnitions of the

foregoing cytotoxicity parameters are available on the NCI’s

Developmental Therapeutics Program (DTP) web site.¹¹⁰The set

of 60 GI₅₀values measured for 4 were compared (in negative log

format) to the analogous publicly available DTP data for 25 well-

known anticancer drugs that have established MOAs. More

speciﬁcally, the data were analyzed using KyPlot⁹¹with a statistical

multivariate cluster analysis algorithm (group average method,

Minkowski distances) to calculate a dendrogram on which the

analyzed set of anticancer drugs cluster according to similarities in

their GI₅₀proﬁles and thus MOAs.

1

10⁵), 295 (3.0 × 10⁴), 326 (2.2 × 10⁴), 470 (2.9 × 10³). H NMR

(500.013 MHz, DMSO-d₆, 303.15 K): δ 10.85 (s, 1H, k), 10.33 (s, 1H,

a), 9.10 (s, 1H, q), 8.86 (d, 1H, j), 8.84 (s, 1H, l), 8.69 (s, 1H, f), 8.53

(d, 1H, J = 8.14 Hz, b), 8.48 (d, 1H, J = 8.00 Hz, e), 8.29 (t, 1H, J =

7.75 Hz, c), 8.22 (d, 1H, J = 8.14 Hz, m), 8.15 (t, 1H, J = 7.61 Hz, i),

8.12 (d, 1H, J = 8.24 Hz, p), 7.88 (t, 1H, J = 7.49 Hz, n), 7.78 (t, 1H, J

= 7.54 Hz, o), 7.48 (d, 1H, J = 7.93 Hz, g), 7.47 (t, 1H, J = 7.50 Hz, d),

7.22 (t, 1H, J = 7.74 Hz, h). ¹³C NMR (100 MHz, DMSO-d₆, 303.15

K): δ 170.47 (C-17), 162.13 (C-10), 152.28 (C-26), 150.88 (C-1),

143.40 (C-18), 139.43 (C-9), 137.41 (C-16), 136.75 (C-4), 135.91

(C-11), 135.41 (C-7), 134.31 (C-20), 132.49 (C-14), 132.15 (C-22),

131.78 (C-15), 130.43 (C-5), 130.10 (C-2), 130.01 (C-23), 129.78

(C-25), 129.14 (C-3), 129.11 (C-21), 128.62 (C-24), 128.38 (C-19),

128.37 (C-12), 124.15 (C-13), 120.75 (C-8), 119.79 (C-6).

X-ray Crystallography. Single crystals of H₂L⁴and 1−4 were

epoxy-mounted on glass ﬁbers and used for data collection on an

Oxford Diﬀraction Xcalibur2 CCD four-circle diﬀractometer equipped

with an Oxford Instruments CryoJet. The data were collected at room

temperature unless otherwise stated, with Mo Kα (λ = 0.71073 Å)

radiation at a crystal-to-detector distance of 50 mm using ω scans at θ

= 29.389°, with varying exposure times taken at 2.01 kW X-ray power

(0.75° frame widths). The data were reduced with the program

CrysAlis RED⁹³using outlier rejection, scan speed scaling, and standard

Lorentz and polarization correction factors. Unless otherwise stated,

direct methods (SHELXS-97⁹⁴running in Olex2⁹⁵) were used to solve

the structures. All non-H atoms were located in the E map and reﬁned

anisotropically with SHELXL-97.⁹⁴H atoms in each of the structures

were idealized (riding model) with standard SHELXL-97 parameters,

unless otherwise stated.

Topoisomerase II Inhibition Assay. Human topoisomerase IIα

(Top2) catalyzed dsDNA decatentation reactions were performed

with puriﬁed kinetoplast DNA (kDNA) and puriﬁed enzyme

(TopoGEN Inc., Port Orange, FL) following the manufacturer’s

published method. The resulting agarose gels were destained in

deionized water for 15 min and imaged with UV transillumination

(302 nm) on a Syngene ChemGenius system followed by

quantiﬁcation and analysis with ImageJ 1.46r.¹¹¹

X-ray data for H₂L⁴: C₂₆H₁₈N₄O₂, fw = 418.44 amu, a = 5.7773 (2)

Å, b = 13.3867 (5) Å, c = 26.7245 (9) Å, β = 93.836 (3)°, V = 2062.22

(13) Å³, monoclinic, P2₁/n, Z = 4, D_c= 1.348 g cm⁻³, μ = 0.088 mm⁻¹,

7903

dx.doi.org/10.1021/ic400339z | Inorg. Chem. 2013, 52, 7889−7906

Inorganic Chemistry

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The variation in the linear DNA product concentration as a function

of the logarithm of [4] was analyzed using the dual dose−response

function below, eq 2,

vortex mixing. A 5.0 μL aliquot of each solution was pipetted into a

precast 1% EB−agarose gel (to give 20 ng of DNA per lane) prior to

electrophoresis at 50 V for 1 h in a 1X TBE-EB buﬀer. (Both the gel

and electrophoresis buﬀer contained EB at a ﬁnal concentration of 0.5

mg mL⁻¹; this permits optimal resolution of DNA cleavage products.)

The gel was brieﬂy destained and imaged as described above (Top2

inhibition assay).

⎡

⎢

⎣

⎤

⎥

⎦

1

y = 100

−

n_H2

n_H1

10^C

1 +

DNA Binding Assay. The signiﬁcant cytotoxicity proﬁle of 4 and

the presence of isoquinoline groups in its structure (i.e., known DNA

intercalator groups) warranted an assessment of its DNA binding

aﬃnity. Standard spectroscopic titrations of the metal complexes with

calf-thymus DNA in phosphate-buﬀered 5% DMSO solutions (pH 7.0,

25 °C) gave irreproducible binding isotherms. EMSAs were required

to reproducibly detect an interaction between 4 and DNA. For these

experiments, 6.3 ng μL⁻¹of negatively supercoiled pHOT1 plasmid

DNA (TopoGEN, Inc.) was used as the dsDNA substrate. Reaction

solutions were prepared by mixing 1.0 μL of a 188 ng μL⁻¹pHOT1

stock solution, 3.0 μL of pH 7.9 1X TGS buﬀer [100 mM Tris-HCl, 10

mM EDTA, 1.5 M NaCl, 1 mM spermidine, 1% BSA, and 50% (v/v)

glycerol], and 25.0 μL of deionized water in 1 mL polyethylene

microcentrifuge tubes. To these solutions were added 1.0 μL aliquots

of standard solutions of EB (a cationic DNA intercalator control) or

m-AMSA (a neutral DNA intercalator control) in DMSO (molecular

biology grade, Sigma) to give ﬁnal control compound concentrations

of 500 nM, 5.0 μM, and 50 μM. For 4, additions of 1.0 μL aliquots of

appropriate freshly made standard solutions of the test compound in

DMSO were made to an analogous series of pHOT1-containing

reaction solutions (29.0 μL total volumes) to give ﬁnal compound

concentrations ranging from 5.0 nM to 50 μM. The solutions were

incubated at 37 °C for 15 min before adding 6.0 μL of a 6X

electrophoresis loading dye (50% aqueous glycerol and 0.025%

bromophenol blue) and mixing by vortex centrifugation. Aliquots from

each dyed solution (5.0 μL) were loaded on a freshly cast 1% agarose

gel and electrophoresed in 1X TBE buﬀer (88 mM boric acid, 88 mM

Tris base, 10 mM EDTA, pH 7.8) at 50 V for 60 min. The gel was

rinsed in deionized water and stained for 10 min in an aqueous 1X

TBE-buﬀered EB solution (0.5 μg mL⁻¹) prior to imaging as described

above (Top2 inhibition assay).

(_K)

D2

D1

(2)

where y is proportional to the concentration of linear DNA reaction

product(s) (represented as % abundance relative to the DNA band

with the largest area on the gel), C is the molar concentration of 4, and

K_D1and K_D2are the drug or ligand equilibrium dissociation constants

for the ﬁrst and second consecutive inhibition steps, respectively. As

lucidly pointed out by Prinz,⁸⁰the above K_Dvalues are equivalent to

IC₅₀values (inhibitor concentrations giving 50% inhibition) for the

enzyme. The associated Hill coeﬃcients for the two steps are n_H1and

n_H2; these coeﬃcients give the number of ligands bound in each step

and thus the number of binding sites per enzyme or macromolecular

target, provided that (i) the reaction is in a dilute solution with

[enzyme] ≪ [inhibitor] so that the enzyme concentration does not

aﬀect the K_Dvalues,¹¹²(ii) the inhibitor is nonaggregating over the

concentration range of the study,¹¹³and (iii) the inhibitor does not

operate by nonspeciﬁc allosteric denaturation of the enzyme.⁸⁰The

nonlinear least-squares ﬁt of the experimental data to eq 1 (Figure 9a)

had the following optimized parameters: K_D1, 2.4(1.1) × 10⁻⁷M²; n_H1

,

2.5(7); K_D2, 8.6(1.5) × 10⁻⁶M; n_H2, 1.2(3); R²= 0.987.

Topoisomerase I Inhibition Assay. Inhibition of Top1-catalyzed

relaxation of negatively supercoiled pHOT1 plasmid DNA was studied

by two methods: (1) conventional DNA unwinding assays as designed,

marketed, and distributed by TopoGEN Inc. and (2) a new DNA

cleavage assay contrived to discriminate between a Top1 catalytic

inhibitor and a Top1 IFP. The basis of this new method is simple: at

low NaCl concentrations and elevated Top1 concentrations (typically

10 times higher than those employed in a standard Top1 inhibition

assay), Top1−DNA covalent cleavage complexes with a higher than

1:1 stoichiometry are favored. This results in elevated levels of NOC

DNA product when plasmid DNA is used as the substrate in the

absence of a Top1 poison. In the presence of a Top1 poison, elevated

levels of NOC DNA and linear DNA (normally absent in a standard

Top1 inhibition assay) are observed. The linear DNA product in this

case arises from cleavages that occur in close proximity on both strands

of the DNA substrate ostensibly caused by a 2:1 enzyme−DNA

stoichiometry at the hexadecameric Top1 cleavage site that is

engineered into the pHOT1 plasmid substrate. Because Top1 poisons

trap the covalent cleavage intermediates in the catalytic cycle of the

enzyme, Top1 IFPs are identiﬁable by high levels of both NOC and

linear DNA reaction products. In contrast, catalytic inhibitor

compounds (CICs) do not trap the intermediate covalent DNA−

enzyme cleavage complexes and merely lead to dose-dependent

reduction of the yield of NOC DNA. Importantly, CICs are

distinguished by the absence of linear DNA products in this assay.

Cleavage reactions were carried out in 1 mL polyethylene

microcentrifuge tubes with 50 U μL⁻¹of Top1 (TopoGEN, Inc.)

and 188 ng μL⁻¹of supercoiled pHOT1 plasmid DNA (TopoGEN,

Inc.) in a master mix of 1X TGS buﬀer [100 mM Tris-HCl, pH 8.0, 10

mM ethylenediaminetetraacetic acid (EDTA), 1 mM spermidine, 1%

bovine serum albumin, 5% glycerol, and 0 mM NaCl] at a total initial

volume of 29 μL. To each of these solutions was added a 1.0 μL

aliquot of an appropriate standard solution of 4 dissolved in molecular

biology grade DMSO (Sigma). Final concentrations of 4 ranged from

5 nM to 25 μM in a ﬁnal reaction volume of 30 μL. Two controls were

included: (1) a reaction in the presence of 50 μM CPT (Top1 IFP)

and (2) a zero-drug control (1.0 μL of DMSO added to 29 μL of the

master mix). Reactions were prepared on ice and initiated by

incubation for 30 min at 37 °C. Following incubation, reactions were

stopped by the addition of 3.0 μL of 10% sodium dodecyl sulfate and

digested with proteinase K (0.5 mg mL⁻¹) for 30 min at 37 °C. To

each solution was added 6 μL of 6X DNA electrophoresis loading dye

(50% aqueous glycerol and 0.025% bromophenol blue) prior to brief

ASSOCIATED CONTENT

* Supporting Information

■

S

Figures S1−S30 containing additional crystallographic and

spectroscopic data as well as one-dose NCI cytotoxicity screens

for 1−3 (ﬁve-dose data for 4 are included as an addendum),

Tables S1−S7 listing further structural, DFT, and spectroscopic

data for one or more of 1−4, and ﬁnal X-ray crystallographic

models in a multipart CIF ﬁle. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: munroo@ukzn.ac.za.

■

Author Contributions

C.R.W. synthesized and characterized the compounds and

wrote parts of the Experimental Section and draft manuscript;

A.M.F. and W.R. performed the topoisomerase assays; O.Q.M.

performed the EMSAs; M.T.M. directed the molecular biology

experiments; O.Q.M. produced the ﬁgures, wrote the ﬁnal

manuscript, and directed the chemistry. All authors have given

their approval of the ﬁnal version of the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

O.Q.M. and C.R.W. thank the University of KwaZulu-Natal for

facilities and funding. C.R.W. thanks the NRF (South Africa)

■

7904

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Inorganic Chemistry

Article

(25) Hollis, L. S.; Lippard, S. J. J. Am. Chem. Soc. 1983, 105, 4293−

4299.

for a Ph.D. bursary. Special thanks go to AuTEK Biomed (Dr.

Judy Coates) for providing signiﬁcant ﬁnancial resources for

running this project over several years and for seeding the

foundations of our current metallodrug discovery initiative at

UKZN. A.M.F., W.R., and M.T.M. thank the University of

Central Florida (UCF) for research facilities and funding.

Furthermore, all authors thank TopoGEN, Inc. (Port Orange,

FL), for kindly providing puriﬁed enzymes, kDNA, plasmid

DNA, and assay kits that made the pivotal molecular biology

component of this study possible. O.Q.M. extends his deepest

gratitude to the NCI (Bethesda, MD), speciﬁcally the DTP, for

their expert cytotoxicity screening service and to Craig

Grimmer (UKZN) for all of his help with the NMR

experiments. Finally, O.Q.M. thanks the Fulbright Association

for a 2011−2012 Visiting Fulbright Scholarship to the USA and

M.T.M. for his friendship and generosity while hosting him at

UCF.

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Gold(III) complexes of pyridyl- and isoquinolylamido ligands: Structural, spectroscopic, and biological studies of a new class of dual topoisomerase i and II inhibitors

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