Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase i (Tdp1)-topoisomerase i (Top1) inhibitors

Article

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Synthesis and Biological Evaluation of the First Dual Tyrosyl-DNA

Phosphodiesterase I (Tdp1)−Topoisomerase I (Top1) Inhibitors

Trung Xuan Nguyen,^†Andrew Morrell,^†Martin Conda-Sheridan,^†Christophe Marchand,^‡Keli Agama,^‡

Alun Bermingam,^§Andrew G. Stephen,^∥Adel Chergui,^‡Alena Naumova,^‡Robert Fisher,^∥

Barry R. O’Keefe,^§Yves Pommier,^‡and Mark Cushman*^,†

^†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer

Research, Purdue University, West Lafayette, Indiana 47907, United States

^‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255,

United States

^§Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Frederick, Maryland 21702,

United States

^∥Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702,

United States

ABSTRACT: Substances with dual tyrosyl-DNA phospho-

diesterase I−topoisomerase I inhibitory activity in one low

molecular weight compound would constitute a unique class of

anticancer agents that could potentially have signiﬁcant

advantages over drugs that work against the individual

enzymes. The present study demonstrates the successful

synthesis and evaluation of the ﬁrst dual Top1−Tdp1

inhibitors, which are based on the indenoisoquinoline

chemotype. One bis(indenoisoquinoline) had signiﬁcant

activity against human Tdp1 (IC₅₀= 1.52 0.05 μM), and

it was also equipotent to camptothecin as a Top1 inhibitor.

Signiﬁcant insights into enzyme−drug interactions were gained

via structure−activity relationship studies of the series. The

present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in

this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The

current study will facilitate future eﬀorts to optimize dual Top1−Tdp1 inhibitors.

(topotecan (2), irinotecan (3), and belotecan), as well as other

non-CPT Top1 inhibitors like indenoisoquinolines (indotecan

(4), and indimitecan (5)) (Figure 1), inhibit the religation rate

by selectively and reversibly binding to the Top1−DNA

interface.⁶This ultimately leads to cell death after collision of

the cleavage complex, with the replication fork resulting in

double-strand breakage.⁷⁻⁹Other naturally occurring DNA

lesions, such as strand breaks, abasic sites, base mismatches, and

certain oxidized or modiﬁed bases, can also induce stalled

Top1−DNA complexes via the misalignment of the 5′-hydroxyl

with the tyrosyl-DNA phosphodiester linkage, thus physically

blocking the Top1 religation reaction.^10,11Under these

conditions, cellular DNA metabolism results in repair of the

stalled Top1−DNA cleavage complex by DNA ligase, which

cannot work until the protein adduct is removed, and the

broken DNA strand is provided with termini consisting of a 5′-

phosphate on one end and a 3′-hydroxyl on the other end for

INTRODUCTION

■

Eukaryotic topoisomerase I (Top1) is an essential enzyme for

many critical cellular processes as it relaxes the double-helix

structure of DNA so that the stored genetic information can be

accessed during DNA replication, transcription, and repair.^1,2

The mechanism of action of Top1 starts with the nucleophilic

attack of the enzyme Tyr723 hydroxyl group on a

phosphodiester linkage in DNA, displacing the 5′-end to

become covalently attached to the 3′-end of DNA, thus forming

a “cleavage complex.”^2,3The religation reaction occurs faster

than cleavage so the equilibrium favors the uncleaved DNA

(Scheme 1).³

Under normal circumstances, the Top1−DNA cleavage

complex is a transitory intermediate in the Top1-catalyzed

reaction, as the broken DNA strand is quickly religated after a

local supercoil has been removed.¹⁻³However, Top1 can

become stalled in the DNA cleavage complex under a variety of

natural or unnatural conditions in which the rate of religation is

inhibited or reduced.^4,5For example, Top1 inhibitors, such as

camptothecin (CPT, 1) and its clinically used derivatives

Received: March 9, 2012

Published: April 26, 2012

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(3) DNA polymerase β replaces the missing DNA segment,

and ﬁnally, (4) DNA ligase III reseals the broken DNA.¹²

Tyrosyl-DNA phosphodiesterase I (Tdp1) has been shown

to be the only enzyme that speciﬁcally catalyzes the hydrolysis

of the phosphodiester bond between the catalytic Tyr723 of

Top1 and DNA-3′-phosphate.¹³Hence, Tdp1 is thought to be

associated with the repair of DNA lesions. The cellular

importance of Tdp1 also stems from the fact that it is

ubiquitous in eukaryotes and plays an important physiological

role, as the homozygous mutation H493R in its active site is

responsible for the rare autosomal recessive neurodegenerative

disease called spinocerebellar ataxia with axonal neuropathy

(SCAN1).¹⁴Tdp1 also has the ability to remove the 3′-

phosphoglycolate caused by oxidative DNA damage by

bleomycin¹⁵and repair trapped Top2−DNA cleavage com-

plexes.^16,17All this evidence suggests that Tdp1 assumes a

broader role in the maintenance of genomic stability. Hence,

this makes Tdp1 a rational anticancer drug development

target.^12,18

Scheme 1. Top1 in Action

Tdp1 is a member of the phospholipase D superfamily of

enzymes that catalyze the hydrolysis of a variety of

phosphodiester bonds in many diﬀerent substrates.¹⁹Crystallo-

graphic studies have revealed that human Tdp1 is composed of

two domains related by a pseudo-2-fold axis of symmetry.²⁰

Each domain contributes a histidine and a lysine residue to

form an active site that is centrally located at the symmetry

axis.²⁰Four additional residues, N283, Q294, N516, and E538,

are also positioned near the active site.²⁰The crystal structure

of Tdp1 in the quaternary complex with a vanadate ion, a

Top1-derived peptide, and a single-stranded DNA oligonucleo-

tide revealed an active site in which the DNA moiety occupies a

relatively narrow cleft rich in positive charges, while the peptide

moiety binds in another region of the active site characterized

by a relatively large, more open cleft that contains a mixed

charge distribution.²⁰⁻²²The trigonal bipyramidal geometry

exhibited by the vanadate implies an S_N2 mechanism for

nucleophilic attack on phosphate.^21,22Therefore, the mecha-

nism of action of Tdp1 is proposed to start with a nucleophilic

attack on the phosphotyrosyl bond by the catalytic H263

residue in the N-terminal domain, while the H493 residue in

the C-terminal domain acts as a general acid and donates a

proton to the tyrosine-containing peptide leaving group

(Scheme 2).²¹The resulting phosphoramide is stabilized by

hydrogen-bonding with catalytic K265 and K495. Hydrolysis of

this covalent intermediate occurs via a second S_N2 reaction by a

water molecule with the H493 residue acting as a general

base.²¹This proposed reaction step is supported by in vitro

studies showing that the SCAN1 H493R mutation leads to an

accumulation of Tdp1−DNA covalent intermediate.²³The ﬁnal

product in this process is a DNA molecule with a 3′-phosphate

end.¹²

Because the Top1−DNA phosphotyrosyl bond is buried

deep within the Top1−DNA complex and is inaccessible to

Tdp1,²⁴prior denaturation of the Top1−DNA complex or

proteolytic degradation of Top1 is required for the Tdp1

enzymatic activity.^25,26However, Tdp1 seems to be equally

eﬀective against many structural variations of DNA, including

single-strand, tailed duplex, and gapped duplex,^12,27,28although

the activity decreases as the oligonucleotide length is

shortened.^12,25These observations have implied that the

enzymatic activity of Tdp1 is inﬂuenced by the length of

Top1-derived polypeptide chain and the structure of the DNA

segment bound to Top1.^25,26Moreover, studies from SCAN1

Figure 1. Representative Top1 inhibitors.

DNA repair.¹²In detail, the overall process involves the

following steps: (1) tyrosyl-DNA phosphodiesterase I (Tdp1)

hydrolyzes the phosphotyrosyl linkage between degraded Top1

and DNA, (2) polynucleotide kinase phosphatase (PNKP)

hydrolyzes the resulting 3′-phosphate end and catalyzes the

phosphorylation of the 5′-hydroxyl end of the broken DNA

strand. This results in a broken DNA strand with termini

consisting of a 5′-phosphate and 3′-hydroxyl for DNA repair.

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Scheme 2. Tdp1 in Action¹²

cells provided evidence for Tdp1 participation in the repair of

Top1-mediated DNA damage^23,29,30and for the hyper-

sensitivity to camptothecin in human cells with a single defect

in Tdp1 activity.^15,31These observations suggest the possibility

of developing Tdp1 inhibitors that can potentiate the cytotoxic

eﬀects of Top1 inhibitors in anticancer drug therapy.¹²

To date, there are very few known Tdp1 inhibitors, and their

potencies and speciﬁcities leave much room for improvement

(Figure 2). For example, both vanadate and tungstate can

mimic the phosphate in the transition state, thus expressing

inhibition at millimolar concentrations.^20,21However, due to

poor speciﬁcity and hypersensitivity to all phosphoryl transfer

reactions, they cannot serve as pharmacological inhibitors.¹²

Other Tdp1 inhibitors are the aminoglycoside neomycin, which

has very low potency at IC₅₀= 8 mM,³²or furamidine (6),

which produces reversible and competitive inhibition of Tdp1

with an IC₅₀≈ 30 μM.³³However, 6 has additional targets

because of its DNA binding activities, which also makes

experimental data diﬃcult to interpret.³³The steroid NSC

88915 (7) was recently identiﬁed via high-throughput screening

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addition, previous studies reported the importance of the

sulfonate functional group in conferring the Tdp1 inhibitory

activity of steroid 7 (Figure 2) because this group mimics the

phosphotyrosyl bond in the Top1−DNA complex.³⁴This led

to the hypothesis that sulfonate analogues of the indenoiso-

quinolines in Table 1 might function as Tdp1 inhibitors. This

hypothesis was also supported by GOLD docking and energy

minimization of one hypothetical sulfonate (25, n = 3) in the

crystal structure of Tdp1 (1RFF) after deleting the

polydeoxyribonucleotide 5′-D(*AP*GP*TP*T)-3′, vanadate

(VO₄³⁻), and the Top1-derived peptide residues 720−727

(mutation L724Y). The docking results revealed a structure

that resembles the original structure: the tosylate moiety

matched the phosphotyrosine, the sulfonate was in place of the

vanadate, the alkyl chain played the “spacer” role of the

deoxyribose, and the indenoisoquinoline system overlapped

with a thymine of DNA (Figure 3). This result is in agreement

with those reported for steroid 7.³⁴Because a sulfonamide

bond is well-known to be more metabolically stable than a

sulfonate and the current compounds possess a structural

handle (amino group) that can be used to install a sulfonyl

linkage, a series of indenoisoquinoline sulfonates and

sulfonamides with diﬀerent linker lengths (n = 2−12) were

synthesized in order to investigate how the structures of these

indenoisoquinolines resemble the substrate of the Tdp1-

catalyzed reaction. Moreover, a previous study on steroid 7

indicated that diﬀerent substituents on the phenyl ring may

have positive eﬀects on Tdp1 inhibitory activity.³⁴Therefore,

four diﬀerent groups were placed at the sulfonamide position

(Figure 4).

Figure 2. Representative Tdp1 inhibitors.

as a potent and speciﬁc Tdp1 inhibitor with an IC₅₀= 7.7 μM.³⁴

However, this compound expressed some common pharmaco-

kinetic problems in cellular systems such as limited drug

uptake, poor cytotoxicity, and oﬀ-target eﬀects.³⁴Therefore,

there is an urgent need to design and develop potent Tdp1

inhibitors that can overcome these drawbacks. Furthermore,

potential anticancer agents that would possess both Tdp1 and

Top1 inhibitory activities are attractive because the two types of

activities could act synergistically.

TDP1 INHIBITOR DISCOVERY AND OPTIMIZATION

Screening of a focused library of indenoisoquinolines led to the

discovery of three potent Tdp1 inhibitors (Table 1). These

■

The ultimate goal of this project was to design and synthesize

compounds with dual Top1−Tdp1 inhibitory activities. Recent

studies have provided some evidence for a potential DNA

interaction of Tdp1 inhibitors.^19,34Prior studies demonstrated

that bis(indenoisoquinolines) monointercalate with DNA and

inhibit the DNA religation reaction by intercalating between

base pairs at the cleavage site in the ternary Top1−DNA−drug

cleavage complex.³⁷This led to the consideration of bis-

(indenoisoquinolines) as possible Tdp1 inhibitors. Figure 4

shows a summary of compounds proposed for synthesis.

Table 1. Tdp1 Inhibitory Activities of n-Alkylamino

Indenoisoquinolines

CHEMISTRY

■

Synthesis of Indenoisoquinoline Sulfonates. The key

starting material in the synthesis of all of the desired

compounds is lactone 11, which was synthesized based on

the procedure reported by Morrell et al.³⁸The reaction of 2-

carboxybenzaldehyde (8) and phthalide (9) in the presence of

sodium methoxide in methanol yielded the intermediate 10,

which formed lactone 11 upon cyclization under acidic

conditions in a one-pot synthesis carried out with the aid of

a Dean−Stark trap. The synthetic route to indenoisoquinoline

sulfonates 24−29 involved the preparation of n-hydroxyalkyl

indenoisoquinolines 18−23 from the reaction of lactone 11 and

n-aminoalcohols 12−17 for 3−6 h. The sulfonylation of

compounds 19−23 to aﬀord indenoisoquinoline sulfonates

25−29 was achieved in dichloromethane (Scheme 3).

However, the sulfonylation of 2-hydroxyethyl indenoisoqui-

noline 18 under the same conditions failed to yield the desired

sulfonate product 24 but instead gave the chloride analogue 30

or, in the presence of silver acetate, the acetate analogue 31

(Scheme 4). Silver acetate was initially meant to quench the

nucleophilic attack of the chloride ions in the hope of obtaining

the sulfonate 24, and the side product 31 was unexpected.

a

Compound-induced DNA cleavage due to Top1 inhibition is graded

by the following semiquantitative scale relative to 1 μM camptothecin

(1): 0, no inhibitory activity; +, between 20 and 50%, activity; ++,

between 50 and 75% activity; +++, between 75% and 95% activity; ++

++, equipotent. The 0/+ ranking is between 0 and +.

Tdp1-active n-alkylamino-containing indenoisoquinolines 77−

79 (n = 10−12), which had been synthesized previously by

Morrell et al.,³⁵did not display Top1 inhibition or were very

weak inhibitors, although other homologous compounds with

shorter linkers (69−71, n = 2−4) were good Top1 inhibitors.³⁶

This led to the idea that Tdp1 inhibition may potentially be

present in Top1 inhibitors with a shorter side chain. In

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Figure 3. Overlapped hypothetical structures of the binary complex Tdp1−indenoisoquinoline sulfonate 25 and the crystal structure of the

quaternary complex consisting of Tdp1−5′-D(*AP*GP*TP*T)-vanadate-3′-Top1-derived peptide residues 720−727 (mutation L724Y).³⁶Red,

indenoisoquinoline sulfonate; green, Tyr723; yellow, DT806; vanadate, fuchsia. The ﬁgure is programmed for wall-eyed (relaxed) viewing.

Figure 4. Compounds proposed for synthesis.

Two mechanisms are proposed for this transformation with

the sulfonate 24 assumed to be the ﬁrst intermediate (Scheme

5). In the ﬁrst proposed mechanism, 24 undergoes intra-

molecular cyclization and elimination of the tosylate group to

yield a 4,5-dihydro-3-oxazolium intermediate 32. Nucleophilic

attack of the chloride anion yields the substituted 2-chloroethyl

indenoisoquinoline 30. The presence of silver ion facilitates the

displacement of chloride by acetate to form 31. In the second

mechanism, chloride displaces the tosylate directly to form 31.

This mechanism does not explain why the alcohol 18

undergoes a unique reaction pathway.

An independent study of the feasibility of displacing the

chloride by acetate was done by mixing and stirring chloride 30

and silver acetate in dichloromethane without the presence of

any base at room temperature for 24 h. The result showed that

30 was incompletely converted to the acetate 31 and,

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a

formation of the very explosive di- and triazidomethane

(from CHCl₃) during workup,³⁹an alternative route was

utilized to make 17 under safer and milder conditions using the

Scheme 3. Synthesis of Indenoisoquinoline Sulfonates

́

classical Delepine reaction (Scheme 6B). In this method,

urotropine 34 was alkylated by the 7-bromoalcohol 33 to

produce a quaternary hexamethylenetetramine salt 35, which

produced the 7-aminoalcohol 17 in good yield upon acidic

hydrolysis in ethanol. Another advantage of this method is that

intermediate 17 requires no puriﬁcation and can react with

lactone 11 to aﬀord 23 in very high purity.

Synthesis of Indenoisoquinoline Sulfonamides. All the

desired n-alkylamino indenoisoquinoline hydrochloride salts

69−79 were synthesized based on the procedures reported by

Morrell et al.³⁵as follows: the diamines 36−46 were Boc

protected at one end by using a 5:1 diamine:Boc₂O ratio. The

mono-Boc-protected products 47−57 were treated with lactone

11 in a 1:1 ratio to give the Boc-protected indenoisoquinolines

58−68 in high yields, which, upon deprotection of the amines

in methanolic HCl, provided the indenoisoquinoline hydro-

chloride salts 69−79. All of the indenoisoquinoline sulfona-

mides with methyl (80−90), phenyl (91−101), p-methyl-

phenyl (102−112), and p-bromophenyl (113−123) substitu-

tents were synthesized in good to excellent yields by gently

heating each hydrochloride salt at 70 °C with a corresponding

sulfonating reagent (mesyl, benzenesulfonyl, tosyl, or p-

bromobenzenesulfonyl chloride) in 1:2 ratio in the presence

of triethylamine (2 equiv) for 16 h (Scheme 7).

A diﬀerent synthetic route to the indenoisoquinoline

sulfonamides was investigated as follows: (1) the sulfonamide

group was incorporated into the linker chain by having the

sulfonating reagents (mesyl, benzenesulfonyl, tosyl, or p-

bromobenzenesulfonyl chloride) react with diamines in a 1:5

molar ratio, with the procedure for these reactions being similar

to that of making mono-Boc-protected diamines, (2) heating

the monosulfonated diamines with lactone 11 in a 1:1 molar

ratio under reﬂux to obtain the desired sulfonamides (Scheme

8). The advantage of this approach is that the introduction and

removal of the protecting Boc group were skipped, thus

providing a shorter route to make sulfonamides with hypo-

thetically higher overall yields. However, the apparent weakness

is that derivatization of side chains at the sulfonamide end

would be impossible. Nevertheless, this idea was ﬁrst tested by

the reaction of N-(3-aminopropyl)-4-methylbenzenesulfona-

mide (124) with lactone 11 to give sulfonamide 103 in 70%

yield. Following this success, N-(11-aminoundecyl)-4-methyl-

benzenesulfonamide (125) and N-(11-aminoundecyl)-

benzenesulfonamide (126) were synthesized to use in the

next reaction with lactone 11. However, the condensation

occurred very slowly in CHCl₃and a signiﬁcant amount of

starting materials was observed even after 48 h of heating at

reﬂux. Then benzene was employed to help remove H₂O from

the reaction mixture in order to force the equilibrium to the

product side, but the reaction provided only about 30% yield of

crude product after 2 days of heating in the presence of a

Dean−Stark trap and starting materials were still present in the

mixture. Hence, the original method was utilized to make all of

the desired sulfonamides 80−123.

a

Reagents and conditions: (a) NaOMe, MeOH, EtOAc, 65 °C; (b)

HCl, PTSA, benzene, reﬂux; (c) CHCl₃, reﬂux; (d) TsCl, DMAP,

CH₂Cl₂, Et₃N, rt.

Scheme 4. Formation of the Undesired Chloride (30) and

Acetate (31) Analogues

surprisingly, to alcohol 18. This result implied the sensitivity of

acetate 31 to hydrolysis and its instability in solution.

7-Hydroxyheptyl indenoisoquinoline 23 could be prepared

from lactone 11 and the commercially available 7-amino-1-

heptanol (17) as depicted in Scheme 3. However, due to the

very high cost of 17 (and any n-aminoalcohol with more than

six methylene units), several alternative routes were considered

for making 23. Aminoalcohol 17 can be readily prepared from

the reaction of its low-cost 7-bromo analogue 33 with sodium

azide in CHCl₃to aﬀord an azido intermediate, which yields 17

upon reduction with Fe in aqueous ammonia (Scheme 6A).

However, because this route poses an explosion hazard due to

the instability of the azido compound and the possible

Synthesis of Bisindenoisoquinolines and Other Com-

pounds. The goal of this project was to achieve dual Top1−

Tdp1 inhibition, and this was attempted by making

sulfonamides from substrates that have been shown to be

active against Top1 by previous biological assays (Figure 5).⁴⁰

The presence of a bulky substituent at position 9 on the D-ring

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Scheme 5. Proposed Mechanisms for the Formation of Chloride 30 and Acetate 31

of the indenoisoquinoline system attenuates Top1 inhibition,⁴⁰

as seen in compounds 127 and 129 (see structure 127 in Figure

5 for indenoisoquinoline numbering). Nevertheless, to gain a

quick look into the eﬀects of various substituents on the A- and

D-rings of the indenoisoquinoline system to Tdp1 inhibitory

activity, all the starting materials 127−131 were subjected to

similar reaction conditions to obtain the corresponding

sulfonamides 132−136 (Figure 5). In addition, three bis-

(indenoisoquinlines) 140−142 were prepared by heating 2

equiv of lactone 11 with diamines 137−139 at reﬂux for 16 h.

Polyamino bis(indenoisoquinline) 145 was synthesized based

on the procedure reported by Morrell et al.³⁷(Scheme 9).

assay is represented in Figure 6, and representative gels

demonstrating dose-dependent Tdp1 inhibition by some

indenoisoquinoline amine hydrochlodrides are depicted in

Figure 7.

Additionally, all indenoisoquinoline sulfonates and sulfona-

mides were tested for Top1 cleavage complex poisoning in a

Top1-mediated DNA cleavage assay.⁴¹The potency of a

compound against Top1 is correlated to the intensities of the

bands corresponding to DNA fragments in this assay and is

graded by the following semiquantitative scale relative to 1 μM

camptothecin: 0, no inhibitory activity; +, between 20% and

50% activity; ++, between 50% and 75% activity; +++, between

75% and 95% activity; ++++, equipotent. The Tdp1 and Top1

activities of all target compounds are represented in Figure 8.

The indenoisoquinoline amine hydrochlorides 69−79 were

shown to be Tdp1 inhibitors (IC₅₀= 13 to 55 μM). Despite

being less active than steroid 7 (Figure 2, IC₅₀= 7.7 μM), their

potencies were retained in whole cell extract while the steroid

expressed oﬀ-target eﬀects and lost its potency in cellular

environments.³⁴Notably, compounds with longer linkers (n =

10−12) showed higher Tdp1 inhibition than those with shorter

BIOLOGICAL RESULTS AND DISCUSSION

■

Tdp1 inhibitory activity was measured by the drug’s ability to

inhibit the hydrolysis of the phosphodiester linkage between

tyrosine and the 3′-end of the DNA substrate and to prevent

the generation of an oligonucleotide with a free 3′-phosphate

(N14P).³⁴Therefore, the disappearance of the gel band for

N14P indicated Tdp1 inhibition. The Tdp1 catalytic gel-based

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One member of this amino series (70, n = 3) was subjected

to surface plasmon resonance (SPR) assays based on reported

procedures³⁴in order to gain a more detailed understanding of

the Tdp1−inhibitor interaction and to measure the aﬃnity of

70 to Tdp1 (Figure 9). The binding of the inhibitor to the

surface-bound Tdp1 induces an increase in resonance units

from the initial baseline until a steady-state phase of this

interaction is achieved as depicted by a plateau.³⁴The decrease

in resonance units corresponds to the dissociation or

reversibility of the interaction.³⁴This result demonstrates

direct binding of 70 to Tdp1. This compound was also

evaluated using a ﬂuorescence resonance energy transfer

(FRET) assay and was found to be a competitive inhibitor

with K_i= 3.19 μM (Figure 10). Steroid 7, which binds Tdp1

directly, is also competitive with the DNA substrate for the

Tdp1 active site.³⁴

Scheme 6. Alternative Routes to Synthesize 17

All indenoisoquinoline sulfonates and sulfonamides (com-

pounds 80−123) were inactive against Tdp1 (IC₅₀> 111 μM).

This result was unexpected because extensive studies on steroid

7 clearly emphasized the importance of the sulfonyl ester

moiety for Tdp1 inhibition,³⁴and our initial modeling (Figure

3) also suggested a good mimic of the phosphotyrosine linkage

by the sulfonate group, which is similar to how steroid 7

resembles this group. As the sulfonate or sulfonamide moiety

was attached to the terminal amino group, the activity was

completely abolished independently of the linker length (n =

2−12). Similarly, sulfonamides 132−136 were also inactive

against Tdp1. Although steric clashes between the sulfonate or

sulfonamide group with components of the binding pocket

might be responsible for this result, this explanation is not

satisfactory because even when a short side chain is present

such as in compound 80, which bears a relatively small mesylate

group on an ethyl linker, the activity drops drastically. In the

case of the amino series (compounds 69−79), the ligand-

binding site seems to accommodate a long side chain quite well.

Therefore, steric clashes could only explain the inactivity of

compounds with very long and bulky side chains. A diﬀerent

factor must have been the cause of the signiﬁcant attenuation of

Tdp1 inhibition upon sulfonylation of the amino series even

though this was completely diﬀerent in case of steroid 7.

Because of the lack of a crystal structure of Tdp1 in complex

with an inhibitor, no ﬁrm conclusion can be drawn as to where

a ligand binds and how it inhibits Tdp1 activity, and molecular

modeling failed to provide an answer to the question of

sulfonate and sulfonamide inactivities in the present series.

Among the four bis(indenoisoquinolines) synthesized and

tested, only the polyamino bis(indenoisoquinoline) 145

side chains, while the Top1 inhibition trend was the opposite. It

is known that the optimal length of the side chain at the lactam

position in Top1 indenoisoquinoline inhibitors is three

methylene units (propyl).^35,40Molecular modeling indicated

that an increase in hydrophobicity as a direct result of increased

linker length in these Top1 inhibitors caused negative

interactions with the hydrated binding pocket of the Top1−

DNA cleavage complex.³⁵That explained the decrease in Top1

inhibition of this series as the number of methylene units

increases. This is, however, not the case for Tdp1 inhibition.

Although the present results for Tdp1 inhibition do not show a

linear correlation of chain length and potency, they indicate

that the longer chains may gain favorable hydrophobic

interactions in the binding site of Tdp1. Additionally,

hypothetical models show that the lactam side chain protrudes

toward the major groove of DNA and causes minimal steric

clashes within the binding pocket in the Top1−DNA cleavage

complex.³⁵Similar conclusions can be drawn for Tdp1: because

no signiﬁcant reduction in activity was observed in the series

with increasing chain length, the lactam side chain must be well

accommodated. Indenoisoquinolines with n = 3 and 4

(compounds 70 and 71) with IC₅₀= 22−29 μM, MGM GI₅₀

= 0.16−0.32 μM,³⁵Top1 inhibition “+++” are the most potent

representatives of the 69−79 series tested for dual Top1−Tdp1

inhibitory activity. These also represent a new chemotype of

fully synthetic small-molecule Tdp1 inhibitors.

displayed low micromolar Tdp1 inhibition, with an IC₅₀

=

1.5 μM and 1.9 μM against rec and WCE Tdp1, respectively.

Three other bis(indenoisoquinolines) 140−142, which were

made because of the initial positive results from the amino

series with 10−12 methylene linkers, were inactive. This result

provides additional support to the hypothesis that the

protonation of the amino groups may give favorable charge-

complementary interactions within the Tdp1 binding pocket,

thereby conferring Tdp1 inhibition. The polyamino bis-

(indenoisoquinoline) 145 is currently the most potent dual

Top1−Tdp1 inhibitor, displaying Top1 inhibition (“++++”)

equipotent to campothecin,³⁷excellent antiproliferative po-

tency (MGM GI₅₀= 0.394 μM),³⁷and also excellent inhibitory

activity against Tdp1 in both human rec. and WCE Tdp1 with

IC₅₀= 1.5 and 1.9 μM, respectively.

Surprisingly, the hydroxyl analogues (compound 18−23)

were inactive against Tdp1. Despite being similar in ability to

form hydrogen bonds, the discrepancy in Tdp1 potency when

going from the amino group to the hydroxyl group in this series

lends support to the hypothesis that hydrogen bonding may not

be a predominant factor that determines Tdp1 inhibitory

activity. Moreover, it seems reasonable to consider these amino

inhibitors to be protonated at physiological pH, and this

positively charged state, which is not possible in the hydroxyl

series 18−23, seems to be an important requirement for Tdp1

inhibition. This rationale is in agreement with a previous report

that the terminal amino group in this series is important for

cellular cytotoxicity although not absolutely necessary for Top1

inhibition.³⁵

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a

Scheme 7. Synthesis of Indenoisoquinoline Sulfonamides

a

Reagents and conditions: (a) Boc₂O, CHCl₃, rt; (b) 11, CHCl₃, reﬂux; (c) 3 M HCl in MeOH, rt; (d) RSO₂Cl, Et₃N, CHCl₃, 70 °C.

the polymethylene moiety linking the amino group to the

heterocycle may also contribute to activity. The polyamino

bis(indenoisoquinoline) 145 is currently the most potent dual

Top1−Tdp1 inhibitor. This encouraging result has much

signiﬁcance because: (1) this class of indenoisoquinoline

compounds serves as the ﬁrst evidence that having Top1 and

Tdp1 inhibitory activity in one single small molecule is in fact

possible, (2) the unique structural features of indenoisoquino-

lines allow much room for manipulation so the pharmacoki-

netics (absorption, distribution, and excretion) can be

modulated and optimized in ways that are not possible for

other types of Tdp1 inhibitors, (3) they represent lead

molecules for development of new dual Top1−Tdp1 inhibitory

agents, and (4) they provide a set of inhibitory ligands that

CONCLUSION

■

A series of bis(indenoisoquinolines) and indenoisoquinolines

with amino, sulfonate, and sulfonamide side chains have been

synthesized to evaluate the hypothesis that dual Top1−Tdp1

inhibition can be achieved in a single compound. In contrast

with the reported importance of the sulfonyl ester moiety to the

Tdp1 inhibition, all sulfonates and sulfonamides were inactive

while the free amines at various linker lengths displayed good

to excellent inhibition. Among them, two compounds, 70 and

71, were potent against both Tdp1 and Top1, and the overall

series represents the ﬁrst dual Top1−Tdp1 inhibitors ever

reported. Signiﬁcant insights for future lead optimization were

deduced: (1) hypothetical charge-complementary interactions

between protonated amino groups within the Tdp1 active site

may contribute to high potency, and (2) the hydrophobicity of

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Scheme 8. Alternative Synthetic Route to Indenoisoquinoline Sulfonamides

with EtOAc (50 mL × 3). The organic layers were combined and

extracted with 1 N NaOH (50 mL × 3). The aqueous layers were

combined and acidiﬁed with concentrated HCl until pH 1 to give a red

solution. The acidic mixture was extracted with ethyl acetate (50 mL ×

3) and washed with brine (50 mL). The organic layers were dried over

anhydrous MgSO₄, ﬁltered, and concentrated to yield the intermediate

10. The crude intermediate 10 was dissolved in benzene (125 mL),

followed by an addition of TsOH·H₂O (100 mg). The resulting

mixture was heated for 7 h at reﬂux in a ﬂask aﬃxed with a Dean−

Stark trap. The solution was cooled to room temperature,

concentrated, diluted with CHCl₃(150 mL), and washed with satd

NaHCO₃(50 mL × 3) and brine (50 mL). The organic layer was

dried over anhydrous Na₂SO₄and concentrated to yield the desired

product as an orange solid (1.69 g, 93%); mp 254−256 °C (lit.³⁸257

°C). ¹H NMR (300 MHz, CDCl₃) δ 8.39 (d, J = 7.8 Hz, 1 H), 8.31 (d,

J = 8.2 Hz, 1 H), 7.84 (t, J = 7.5 Hz, 1 H), 7.61 (d, J = 6.9 Hz, 1 H),

7.55−7.39 (m, 4 H).

could possibly be crystallized in complex with Tdp1, which

would facilitate the structure-based drug design approach.

EXPERIMENTAL SECTION

■

General. Solvents and reagents were purchased from commercial

vendors and were used without any further puriﬁcation. Melting points

were determined using capillary tubes with a Mel-Temp apparatus and

were uncorrected. Infrared spectra were obtained using KBr pellets

using CHCl₃as the solvent. IR spectra were recorded using a Perkin-

1

Elmer 1600 series or Spectrum One FTIR spectrometer. H NMR

spectra were recorded at 300 MHz using a Bruker ARX300

spectrometer with a QNP probe. Mass spectral analyses were

performed at the Purdue University Campus-Wide Mass Spectrometry

Center. ESI-MS studies were performed using a FinniganMAT LCQ

Classic mass spectrometer. EI/CI-MS studies were performed using a

Hewlett-Packard Engine or GCQ FinniganMAT mass spectrometer.

APCI-MS studies were carried out using an Agilent 6320 ion trap mass

spectrometer. Combustion microanalyses were performed at Midwest

Microlab, LLC. All reported values are within 0.4% of the calculated

values. Analytical thin layer chromatography was carried out on Baker-

ﬂex silica gel IB2-F plates, and compounds were visualized with short

wavelength UV light and ninhydrin staining. Silica gel ﬂash

chromatography was performed using 230−400 mesh silica gel.

HPLC analyses were performed on a Waters 1525 binary HPLC

pump/Waters 2487 dual λ absorbance detector system using a 5 μM

C₁₈reverse phase column. Purities of biologically important

compounds were ≥95%. For purities estimated by HPLC, the major

peak accounted for ≥95% of the combined total peak area when

monitored by a UV detector at 254 nm. All yields refer to isolated

compounds.

General Procedure for the Preparation of n-Hydroxyalkyl

Indenoisoquinolines 18−22. n-Aminoalcohols 12−16 (0.50 g) in

CHCl₃(10 mL) were added to a solution of lactone 11 (1.0 equiv) in

CHCl₃(50 mL). The reaction mixtures were heated at reﬂux for 3−6

h with stirring and then washed with H₂O (50 mL × 2) and brine (50

mL). The organic layers were dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with 5% MeOH in CHCl₃, to provide

the products 18−22 in high purity.

6-(2-Hydroxyethyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (18).⁴²The general procedure provided the desired product as

a red solid (1.10 g, 62%); mp 201−204 °C (lit.⁴²200−201 °C). H

1

NMR (300 MHz, CDCl₃) δ 8.68 (d, J = 8.1 Hz, 1 H), 8.32 (d, J = 8.3

Hz, 1 H), 7.74 (dt, J = 1.2 and 7.1 Hz, 1 H), 7.62 (dd, J = 1.2 and 7.0

Hz, 2 H), 7.48−7.37 (m, 3 H), 4.76 (t, J = 5.8 Hz, 2 H), 4.21 (q, J =

5.6 Hz, 2 H), 2.60 (m, J = 5.3 Hz, 1 H). ESI-MS m/z (rel intensity)

292 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 292.0974; found,

292.0978. HPLC purity: 96.6% (MeOH, 100%), 96.2% (MeOH−

H₂O, 90:10).

Benz[d]indeno[1,2-b]pyran-5,11-dione (11).³⁸Sodium metal

(3.678 g, 0.160 mol) was cut into small pieces and added to MeOH

(40 mL) to make a 4 M methanolic solution of sodium methoxide,

which was then added to a solution of 2-carboxybenzaldehyde (8)

(1.000 g, 6.661 mmol) and phthalide (9) (0.893 g, 6.661 mmol) in

ethyl acetate (20 mL). The mixture was stirred and heated at 70 °C for

16 h to yield an orange solution, which was then concentrated, diluted

with H₂O (100 mL), acidiﬁed with 10% HCl until pH 1, and extracted

6-(3-Hydroxypropyl)-5H-indeno[1,2-c]isoquinoline-

5,11(6H)-dione (19).⁴²The general procedure provided the desired

product as a red solid (1.20 g, 98%); mp 173−175 °C (lit.⁴²170−171

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a

Figure 5. Ring-substituted indenoisoquinoline sulfonamides. The cytotoxicity GI₅₀values are the concentrations corresponding to 50% growth

inhibition. The MGM is the mean graph midpoint for growth inhibition of all 60 human cancer cell lines successfully tested, ranging from 10⁻⁸to

10⁻⁴molar, where values that fall outside the range were taken as 10⁻⁸and 10⁻⁴molar. ^bCompound-induced DNA cleavage due to Top1 inhibition is

graded by the following semiquantitative relative to 1 μM camptothecin (1): 0, no inhibitory activity; +, between 20 and 50%, activity; ++, between

50 and 75% activity; +++, between 75% and 95% activity; ++++, equipotent. The 0/+ ranking is between 0 and +.

°C). ¹H NMR (300 MHz, CDCl₃) δ 8.75 (d, J = 8.2 Hz, 1 H), 8.38 (d,

J = 8.1 Hz, 1 H), 7.79−7.65 (m, 3 H), 7.53−7.41 (m, 3 H), 4.76 (t, J =

6.4 Hz, 2 H), 3.75 (q, J = 6.0 Hz, 2 H), 3.26 (t, J = 6.3 Hz, 1 H), 2.20

(m, 2 H). ESI-MS m/z (rel intensity) 328 (MNa⁺, 61). HRMS (+ESI)

calcd for MH⁺, 306.1130; found, 306.1127. HPLC purity: 99.5%

(MeOH, 100%), 98.6% (MeOH−H₂O, 90:10).

6-(5-Hydroxypentyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (21).⁴³The general procedure provided the desired product as

an orange solid (1.45 g, 90%); mp 144−146 °C (lit.⁴³146−148 °C).

¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.35 (d, J =

8.4 Hz, 1 H), 7.75 (dt, J = 1.2 and 7.0 Hz, 1 H), 7.65 (dd, J = 1.4 and

4.9 Hz, 1 H), 7.49−7.38 (m, 4 H), 4.56 (t, J = 7.7 Hz, 2 H), 3.74 (q, J

= 5.3 Hz, 2 H), 1.98 (m, 2 H), 1.74−1.60 (m, 4 H), 1.42 (t, J = 4.8 Hz,

1 H). ESI-MS m/z (rel intensity) 334 (MH⁺, 100). HRMS (+ESI)

calcd for MH⁺, 334.1443; found, 334.1445. HPLC purity: 97.9%

(MeOH, 100%), 95.4% (MeOH−H₂O, 90:10).

6-(4-Hydroxybutyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (20).⁴³The general procedure provided the desired product as

a red solid (1.23 g, 95%); mp 165−166 °C (lit.⁴³160−162 °C). H

1

NMR (300 MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.3

Hz, 1 H), 7.76 (dt, J = 1.2 and 8.2 Hz, 1 H), 7.65 (dd, J = 1.4 and 6.7

Hz, 1 H), 7.59 (d, J = 7.6 Hz, 1H), 7.50−7.38 (m, 3 H), 4.62 (t, J = 7.7

Hz, 2 H), 3.84 (q, J = 6.0 Hz, 2 H), 2.07 (m, 2 H), 1.86 (m, 2 H), 1.70

(t, J = 5.3 Hz, 1 H). ESI-MS m/z (rel intensity) 320 (MH⁺, 42).

HRMS (+ESI) calcd for MH⁺, 320.1287; found, 320.1289. HPLC

purity: 99.1% (MeOH, 100%), 96.2% (MeOH−H₂O, 90:10).

6-(6-Hydroxyhexyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (22). The general procedure provided the desired product as a

red solid (1.24 g, 84%); mp 139−141 °C. IR (ﬁlm) 3425, 1765, 1698,

1

1663, 1611, 1550, 1504, 1427, 1317, 758 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.2 Hz, 1 H), 7.75 (dt,

J = 1.3 and 7.4 Hz, 1 H), 7.65 (dd, J = 1.0 and 7.2 Hz, 1 H), 7.50−7.37

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a

Scheme 9. Preparation of Bisindenoisoquinolines

a

Reagents and conditions: (a) CHCl₃, reﬂux, 72 h; (b) Et₃N, Boc₂O, rt, 16 h; (c) TFA, rt.

(m, 4 H), 4.56 (t, J = 7.7 Hz, 2 H), 3.70 (q, J = 5.7 Hz, 2 H), 3.50 (d, J

= 4.9 Hz, 1 H), 1.96 (m, 2 H), 1.66−1.45 (m, 6 H). ESI-MS m/z (rel

intensity) 370 (MNa⁺, 100). HRMS (+ESI) calcd for MNa⁺, 370.1419;

found, 370.1424. HPLC purity: 97.6% (MeOH, 100%), 99.0%

(MeOH−H₂O, 90:10).

6-(7-Hydroxyheptyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (23). Bromoalcohol 33 (0.975 g, 5.0 mmol) and urotropine

(34, 0.771 g, 5.5 mmol) were dissolved in CHCl₃(20 mL). The

mixture was stirred at reﬂux for 5 h and allowed to stand overnight to

induce the formation of quaternary salt 35. The salt was ﬁltered and

added to a 2 M ethanolic HCl solution (15 mL). The reaction mixture

was warmed up gently with a heat gun and swirled to produce white

NH₄Cl precipitate, which was removed by ﬁltration. The mother

liquor was concentrated in vacuo and diluted in H₂O (20 mL),

followed by cooling in an ice bath. The solution was made strongly

alkaline (pH 13) with 6 M NaOH, extracted with diethyl ether (25 mL

× 3), and washed with brine (25 mL). The ethereal layers were

combined, dried over anhydrous Na₂SO₄, and concentrated to aﬀord a

yellowish oil of crude 17. The crude 17 was dissolved in CHCl₃(20

Figure 6. Schematic representation of the Tdp1 gel-based assays using

recombinant Tdp1.

Figure 7. Representative gels showing concentration-dependent inhibition of endogenous Tdp1 in whole cell extract by indenoisoquinoline amine

hydrochloride inhibitors. Concentrations were 0.5, 1.4, 4.1, 12.3, 37, and 111 μM.

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Figure 8. Inhibitory activities of target compounds against Tdp1 and Top1.

mL) and added to a solution of lactone 11 (1 equiv of 33) in CHCl₃

(50 mL). The reaction mixture was heated at reﬂux for 18 h,

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with EtOAc−hexane in a gradient of

concentration ratios from 5:3 to 7:3 to provide the desired product as

a ﬁne red powdery solid (0.95 g, 52%); mp 132−135 °C. IR (ﬁlm)

1

3468, 1776, 1698, 1663, 1611, 1550, 1504, 1427, 1318, 756 cm⁻¹. H

NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 8.1

Hz, 1 H), 7.72 (t, J = 7.0 Hz, 1 H), 7.65 (d, J = 6.4 Hz, 1 H), 7.49−

7.40 (m, 4 H), 4.54 (t, J = 7.8 Hz, 2 H), 3.67 (d, J = 3.4 H, 2 H), 1.94−

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4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)butyl-

4-methylbenzenesulfonate (26). The general procedure provided

the desired product as an orange solid (129 mg, 87%); mp 160−163

°C. IR (ﬁlm) 1757, 1695, 1667, 1612, 1550, 1504, 1428, 1348, 1174,

1

753 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.0 Hz, 1 H),

8.32 (d, J = 7.4 Hz, 1 H), 7.78−7.70 (m, 3 H), 7.66 (d, J = 7.0 Hz, 1

H), 7.50−7.42 (m, 4 H), 7.33 (d, J = 8.2 Hz, 2 H), 4.55 (t, J = 6.8 Hz,

2 H), 4.16 (t, J = 5.9 Hz, 2 H), 2.42 (s, 3 H), 1.98−1.88 (m, 4 H).

ESIMS m/z (rel intensity) 496 (MNa⁺, 100). HRMS (+ESI) calcd for

MNa⁺, 496.1195; found, 496.1201. HPLC purity: 98.4% (MeOH,

100%), 99.2% (MeOH−H₂O, 90:10).

5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)pentyl-

4-methylbenzenesulfonate (27). The general procedure provided

the desired product as an orange solid (119 mg, 81%); mp 163−165

°C. IR (ﬁlm) 1697, 1661, 1609, 1549, 1503, 1427, 1355, 1174, 755

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.34

(d, J = 7.8 Hz, 1 H), 7.79−7.70 (m, 3 H), 7.66 (d, J = 6.8 Hz, 1 H),

7.50−7.39 (m, 4 H), 7.34 (d, J = 8.1 Hz, 2 H), 4.51 (t, J = 7.8 Hz, 2

H), 4.09 (t, J = 6.1 Hz, 2 H), 2.43 (s, 3 H), 1.91−1.75 (m, 4 H), 1.65−

1.57 (m, 2 H). ESI-MS m/z (rel intensity) 488 (MH⁺, 100). HRMS

(+ESI) calcd for MH⁺, 488.1532; found, 488.1539. HPLC purity:

97.1% (MeOH, 100%), 98.0% (MeOH−H₂O, 90:10).

Figure 9. Direct binding of 70 to Tdp1 by surface plasmon resonance

spectroscopy. Variable concentrations of 70 (33, 11, 3.7, 1.2, and 0.4

μM) were injected over amine coupled Tdp1 protein. The compound

rapidly reaches equilibrium and then completely dissociates.

6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)hexyl-

4-methylbenzenesulfonate (28). The general procedure provided

the desired product as an orange solid (123 mg, 85%); mp 146−149

°C. IR (ﬁlm) 1767, 1693, 1662, 1610, 1550, 1503, 1428, 1357, 1176,

1.86 (m, 2 H), 1.62−1.43 (m, 8 H), 1.30 (m, 1 H). ESI-MS m/z (rel

intensity) 362 (MH⁺, 17). HRMS (+ESI) calcd for MH⁺, 362.1756;

found, 362.1760. Anal. Calcd for C₂₃H₂₃NO₃·0.2H₂O: C, 75.68; H,

6.46; N, 3.84. Found: C, 75.51; H, 6.32; N, 3.62.

1

757 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.2 Hz, 1 H),

8.34 (d, J = 7.9 Hz, 1 H), 7.80−7.70 (m, 3 H), 7.65 (d, J = 6.8 Hz, 1

H), 7.49−7.38 (m, 4 H), 7.36 (d, J = 8.0 Hz, 2 H), 4.51 (t, J = 7.6 Hz,

2 H), 4.07 (t, J = 6.3 Hz, 2 H), 2.44 (s, 3 H), 1.88 (m, 2 H), 1.73 (m, 2

H), 1.57 (m, 4 H). ESI-MS m/z (rel intensity) 502 (MH⁺, 100).

HRMS (+ESI) calcd for MH⁺, 502.1688; found, 502.1694; HPLC

purity: 98.0% (MeOH, 100%), 96.8% (MeOH−H₂O, 90:10).

General Procedure for the Preparation of Indenoisoquino-

line Sulfonates 25−29 and Chloride 30. A solution of Et₃N (2

equiv) in CH₂Cl₂(1 mL) and DMAP (0.2 equiv) was added to

solutions of the n-alkylhydroxy indenoisoquinolines 19−23 (100 mg)

in CH₂Cl₂(10 mL). The solutions were stirred at room temperature

for 5 min, and tosyl chloride (2 equiv) was added. The reaction

mixtures were stirred at room temperature for 16 h, quenched with aq

3 M HCl (50 mL), and washed with H₂O (50 mL), satd NaHCO₃(50

mL), and brine (50 mL). The organic layers were dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated, puriﬁed by ﬂash

column chromatography (SiO₂), eluting with EtOAc (3−5%) in

CHCl₃, to provide the indenoisoquinoline sulfonates 25−29 in high

purity after trituration with diethyl ether (20 mL).

3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

propyl-4-methylbenzenesulfonate (25).⁴⁴The general procedure

provided the desired product as a red solid (132.5 mg, 88%); mp 177−

179 °C (lit.⁴⁴180−182 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J

= 8.1 Hz, 1 H), 8.31 (d, J = 7.5 Hz, 1 H), 7.83 (d, J = 8.3 Hz, 2 H),

7.73−7.63 (m, 3 H), 7.49−7.34 (m, 5 H), 4.62 (t, J = 7.9 Hz, 2 H),

4.31 (t, J = 5.7 Hz, 2 H), 2.46 (s, 3 H), 2.34 (m, 2 H). ESI-MS m/z

(rel intensity) 482 (MNa⁺, 100). HRMS (+ESI) calcd for MNa⁺,

482.1038; found, 482.1041. HPLC purity: 98.6% (MeOH, 100%),

99.4% (MeOH−H₂O, 90:10).

7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)heptyl-

4-methylbenzenesulfonate (29). The general procedure provided

the desired product as a red solid (109 mg, 76%); mp 123−126 °C. IR

(ﬁlm) 1775, 1698, 1664, 1611, 1550, 1504, 1428, 1358, 1176, 758

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.0 Hz, 1 H), 8.35

(d, J = 7.6 Hz, 1 H), 7.80−7.70 (m, 3 H), 7.65 (d, J = 6.8 Hz, 1 H),

7.49−7.39 (m, 4 H), 7.35 (d, J = 8.1 Hz, 2 H), 4.51 (t, J = 7.7 Hz, 2

H), 4.05 (t, J = 6.3 Hz, 2 H), 2.44 (s, 3 H), 1.90 (m, 2 H), 1.67 (m, 2

H), 1.49 (m, 2 H), 1.40−1.37 (m, 4 H). ESI-MS m/z (rel intensity)

516 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 516.1845; found,

516.1848. HPLC purity: 95.4% (MeOH, 100%), 98.3% (MeOH−

H₂O, 90:10).

6-(2-Chloroethyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione (30).⁴²The general procedure provided the chloride product as

a purple solid (105 mg, 99%); mp 210−212 °C (lit.⁴²197−199 °C).

¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J =

8.2 Hz, 1 H), 7.78 (dt, J = 1.2 and 8.1 Hz, 1 H), 7.67 (d, J = 6.8 Hz, 1

H), 7.59 (d, J = 7.4 Hz, 1 H), 7.52−7.42 (m, 3 H), 4.86 (t, J = 7.4 Hz,

2 H), 3.97 (t, J = 7.7 Hz, 2 H). Probe EIMS m/z (rel intensity) 309

Figure 10. Competitive inhibition of 70 against recombinant Tdp1 measured by FRET assay.

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(M⁺, 33). HRMS (EI) calcd for M⁺, 309.0557; found, 309.0560.

HPLC purity: 95.3% (MeOH, 100%), 97.3% (MeOH−H₂O, 90:10).

2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin6(11H)-yl)ethyl

Acetate (31). Et₃N (34.7 mg, 0.343 mmol), CH₃COOAg (57.2 mg,

0.343 mmol), and TsCl (65.4 mg, 0.343 mmol) were added to the

solution of 18 (50 mg, 0.172 mmol) in CH₂Cl₂(50 mL). The reaction

mixture was stirred at room temperature for 16 h and then washed

with satd NaHCO₃(50 mL) and brine (50 mL). The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated, adsorbed onto SiO₂,

and puriﬁed by ﬂash column chromatography (SiO₂), eluting with

EtOAc−CHCl₃(2:8) to aﬀord the desired product as a red solid (38.5

mg, 67%); mp 221−224 °C. IR (ﬁlm) 1738, 1693, 1655, and 1506

J = 6.8 Hz, 1 H), 4.70 (t, J = 6.0 Hz, 2 H), 3.25 (q, J = 6.5 Hz, 2 H),

3.00 (s, 3 H), 2.23−2.23 (m, 2 H). APCI-MS m/z (rel intensity) 383

(MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 383.1066; found,

383.1069. HPLC purity: 99.1% (MeOH, 100%), 96.7% (MeOH−

H₂O, 90:10).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)butyl)-

methanesulfonamide (82). The crude product was eluted with

EtOAc−CHCl₃(7:3) to aﬀord the desired product as an orange solid

(42.2 mg, 81%); mp 193−194 °C. IR (ﬁlm) 3272, 2346, 1694, 1655,

1611, 1549, 1504, 1318, 1152, 756 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)

δ 8.72 (d, J = 7.8 Hz, 1 H), 8.34 (d, J = 8.3 Hz, 1 H), 7.74 (t, J = 6.8

Hz, 1 H), 7.66 (d, J = 6.9 Hz, 1 H), 7.50−7.40 (m, 4 H), 4.60 (m, 3

H), 3.34 (q, J = 6.6 Hz, 2 H), 3.00 (s, 3 H), 2.05 (m, 2 H), 1.85 (m, 2

H). ESI-MS m/z (rel intensity) 397 (MH⁺, 100). HRMS (+ESI) calcd

for MH⁺, 397.1222; found, 397.1231. HPLC purity: 98.4% (MeOH,

100%), 97.2% (MeOH−H₂O, 90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

pentyl)methanesulfonamide (83). The crude product was eluted

with EtOAc−CHCl₃(6:4) to aﬀord the desired product as an orange

solid (44.0 mg, 79%); mp 150−152 °C. IR (ﬁlm) 3275, 1698, 1660,

1611, 1550, 1504, 1318, 1150, 756 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)

δ 8.72 (d, J = 8.0 Hz, 1 H), 8.34 (d, J = 8.0 Hz, 1 H), 7.76 (td, J = 7.1

and 1.1 Hz, 1 H), 7.65 (d, J = 6.9 Hz, 1 H), 7.50−7.41 (m, 4 H), 4.57

(t, J = 7.4 Hz, 2 H), 4.48 (t, J = 5.9 Hz, 1 H), 3.24 (q, J = 6.4 Hz, 2 H),

2.97 (s, 3 H), 2.00 (m, 2 H), 1.76 (m, 2 H), 1.65 (m, 2 H). ESI-MS m/

z (rel intensity) 411 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺,

411.1379; found, 411.1381. HPLC purity: 99.7% (MeOH, 100%),

98.5% (MeOH−H₂O, 90:10).

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.75 (d, J = 8.0 Hz, 1 H), 8.37

(d, J = 7.9 Hz, 1 H), 7.75 (t, J = 5.8 Hz, 2 H), 7.67 (dd, J = 1.1 and 5.8

Hz, 1 H), 7.51−7.41 (m, 3 H), 4.84 (t, J = 6.2 Hz, 2 H), 4.57 (t, J = 6.1

Hz, 2 H), 1.94 (s, 3 H). Probe CIMS m/z (rel intensity) 334 (MH⁺,

100). HRMS (EI) calcd for M⁺, 333.1001; found, 333.1005. HPLC

purity: 97.3% (MeOH, 100%), 98.0% (MeOH−H₂O, 90:10).

Compound 47−79 were synthesized based on the procedures

reported by Morrell at al.³⁵Purities of biologically tested

indenoisoquinoline amine hydrochlorides 69−79 were ≥95% by

HPLC (Table 2).

Table 2. Purities of Indenoisoquinoline Amine

Hydrochlorides 69−79 by HPLC

purity by HPLC

compd

MeOH, 100%

MeOH−H₂O, 90:10

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)hexyl)-

methanesulfonamide (84). The crude product was eluted with

EtOAc−CHCl₃(5:5) to aﬀord the desired product as an orange solid

(42.7 mg, 77%); mp 160−161 °C. IR (ﬁlm) 3355, 1697, 1646, 1608,

1548, 1504, 1452, 1364, 1258, 764 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)

δ 8.72 (d, J = 8.2 Hz, 1 H), 8.36 (d, J = 7.6 Hz, 1 H), 7.76 (dd, J = 7.0

and 1.2 Hz, 1 H), 7.65 (d, J = 6.8 Hz, 1 H), 7.50−7.41 (m, 4 H), 4.56

(t, J = 7.6 Hz, 1 H), 4.44 (m, 1 H), 3.20 (q, J = 6.5 Hz, 2 H), 2.97 (s, 3

H), 1.93 (m, 2 H), 1.67 (m, 2 H), 1.54 (m, 4 H). ESI-MS m/z (rel

intensity) 425 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 425.1538;

found, 425.1532. HPLC purity: 99.8% (MeOH, 100%), 98.3%

(MeOH−H₂O, 90:10).

69

70

71

72

73

74

75

76

77

78

79

97.8

96.7

98.5

98.3

98.1

95.7

96.9

95.5

99.4

98.7

99.2

97.6

98.0

100

97.8

100

98.5

95.8

98.8

96.6

98.5

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

heptyl)methanesulfonamide (85). The crude product was eluted

with EtOAc−CHCl₃(6:4) to aﬀord the desired product as an orange

solid (33 mg, 60%); mp 159−162 °C. IR (ﬁlm) 3227, 1687, 1646,

General Procedure for the Preparation of Indenoisoquino-

line Sulfonamides 80−123. Indenoisoquinoline salts 69−79 (50

mg, 0.107−0.153 mmol) were dissolved in CHCl₃(15 mL), followed

by the addition of Et₃N (2 equiv) in CHCl₃(1 mL). The solutions

were stirred at room temperature for 5 min. Mesyl, benzenesulfonyl,

tosyl, or p-bromobenzenesulfonyl chloride (2 equiv) were then added.

The mixtures were heated at reﬂux for 16 h and then washed with satd

NaHCO₃(50 mL) and brine (50 mL). The organic layers were dried

over anhydrous Na₂SO₄, ﬁltered, and concentrated, and puriﬁed by

ﬂash column chromatography (SiO₂), eluting with EtOAc−CHCl₃to

provide the indenoisoquinoline sulfonamide products in high purity.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-

methanesulfonamide (80). The crude product was eluted with

EtOAc−CHCl₃(4:6) to aﬀord the desired product as an orange solid

(38.7 mg, 69%); mp 250−255 °C. IR (ﬁlm) 3313, 1705, 1653, 1609,

1549, 1502, 1418, 1321, 1129, 758 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.59 (d, J = 8.0 Hz, 1 H), 8.23 (d, J = 8.0 Hz, 1 H),

7.89−7.82 (m, 2 H), 7.58−7.47 (m, 5 H), 4.61 (t, J = 6.7 Hz, 2 H),

3.42 (m, 2 H), 2.91 (s, 3 H). Probe CIMS m/z (rel intensity) 369

(MH⁺, 100). HRMS (EI) calcd for M⁺, 368.0831; found, 368.0835.

HPLC purity: 96.0% (MeOH, 100%), 96.3% (MeOH−H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

propyl)methanesulfonamide (81). The crude product was eluted

with EtOAc−CHCl₃(6:4) to aﬀord the desired product as an orange

solid (47.1 mg, 84%); mp 201−203 °C. IR (ﬁlm) 3203, 1696, 1646,

1

1609, 1547, 1505, 1429, 1308, 1144, 754 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.2 Hz, 1 H), 7.73 (dd,

J = 1.3 and 6.9 Hz, 1 H), 7.65 (d, J = 6.7 Hz, 1 H), 7.50−7.39 (m, 4

H), 4.54 (t, J = 7.8 Hz, 2 H), 4.24 (m, 1 H), 3.19 (q, J = 6.8 Hz, 2 H),

2.97 (s, 3 H), 1.91 (m, 2 H), 1.63−1.43 (m, 8 H). ESI-MS m/z (rel

intensity) 461 (MNa⁺, 100). HRMS (+ESI) calcd for MNa⁺, 461.1511;

found, 461.1518. Anal. Calcd for C₂₄H₂₆N₂O₄S·0.75H₂O: C, 63.77; H,

6.13; N, 6.20. Found: C, 63.73; H, 6.06; N, 5.86.

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)octyl)-

methanesulfonamide (86). The crude product was eluted with

EtOAc−CHCl₃(6:4) to aﬀord the desired product as an orange solid

(41.3 mg, 75%); mp 151−155 °C. IR (ﬁlm) 3214, 1767, 1699, 1645,

1

1612, 1551, 1504, 1426, 1315, 1142, 760 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 8.0 Hz, 1 H), 7.75 (t, J

= 7.1 Hz, 1 H), 7.66 (d, J = 6.8 Hz, 1 H), 7.50−7.39 (m, 4 H), 4.54 (t,

J = 8.2 Hz, 2 H), 4.24 (m, 1 H), 3.17 (q, J = 6.9 Hz, 2 H), 2.96 (s, 3

H), 1.90 (m, 2 H), 1.57−1.26 (m, 10 H). ESI-MS m/z (rel intensity)

453 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 453.1848; found,

453.1853. Anal. Calcd for C₂₅H₂₈N₂O₄S: C, 66.35; H, 6.24; N, 6.19.

Found: C, 66.34; H, 6.31; N, 5.81.

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

nonyl)methanesulfonamide (87). The crude product was eluted

with EtOAc−CHCl₃(5:5) to aﬀord the desired product as an orange

solid (54.2 mg, 82%); mp 154−156 °C. IR (ﬁlm) 3211, 1700, 1645,

1550, 1506, 1426, 1314, 1142, 759 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)

δ 8.73 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 7.7 Hz, 1 H), 7.73 (t, J = 8.2

1610, 1549, 1504, 1428, 1317, 1137, 757 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.32 (d, J = 8.1 Hz, 1 H), 7.77 (t, J

= 7.0 Hz, 1 H), 7.65 (d, J = 6.5 Hz, 1 H), 7.55−7.38 (m, 4 H), 5.73 (t,

1

4471

dx.doi.org/10.1021/jm300335n | J. Med. Chem. 2012, 55, 4457−4478

Journal of Medicinal Chemistry

Article

Hz, 1 H), 7.66 (d, J = 6.2 Hz, 1 H), 7.50−7.40 (m, 4 H), 4.54 (t, J =

8.0 Hz, 2 H), 4.21 (m, 1 H), 3.17 (q, J = 6.9 Hz, 2 H), 2.96 (s, 3 H),

1.90 (m, 2 H), 1.63−1.23 (m, 12 H). ESI-MS m/z (rel intensity) 467

(MH⁺, 50). HRMS (+ESI) calcd for MH⁺, 467.2005; found, 467.2007.

Anal. Calcd for C₂₆H₃₀N₂O₄S·0.2H₂O: C, 66.41; H, 6.52; N, 5.96.

Found: C, 66.18; H, 6.39; N, 5.70.

7.39 (m, 7 H), 4.83 (t, J = 6.2 Hz, 1 H), 4.54 (t, J = 7.5 Hz, 2 H), 3.14

(q, J = 6.5 Hz, 2 H), 1.98 (m, 2 H), 1.78 (m, 2 H). ESI-MS m/z (rel

intensity) 459 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 459.1379;

found, 459.1375. HPLC purity: 98.5% (MeOH, 100%), 98.3%

(MeOH−H₂O, 90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

pentyl)benzenesulfonamide (94). The crude product was eluted

with EtOAc−CHCl₃(2:8) to aﬀord the desired product as an orange

solid (55.8 mg, 84%); mp 194−195 °C. IR (ﬁlm) 3284, 1728, 1699,

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

decyl)methanesulfonamide (88). The crude product was eluted

with EtOAc−CHCl₃(3:7) to aﬀord the desired product as an orange

solid (45.3 mg, 83%); mp 115−117 °C. IR (ﬁlm) 3297, 1697, 1663,

1

1662, 1609, 1548, 1504, 1446, 1326, 1261, 1161, 758 cm⁻¹. H NMR

1

1611, 1551, 1505, 1428, 1351, 1175, 759 cm⁻¹. H NMR (300 MHz,

(300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.4 Hz, 1

H), 7.87 (dd, J = 1.5 and 5.4 Hz, 2 H), 7.76 (td, J = 1.3 and 7.0 Hz, 1

H), 7.65 (d, J = 6.8 Hz, 1 H), 7.57−7.38 (m, 7 H), 4.66 (t, J = 6.1 Hz,

1 H), 4.52 (t, J = 7.5 Hz, 2 H), 3.05 (q, J = 6.3 Hz, 2 H), 1.91 (m, 2

H), 1.67 (m, 2 H), 1.58 (m, 2 H). ESI-MS m/z (rel intensity) 473

(MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 473.1535; found,

473.1532. HPLC purity: 99.5% (MeOH, 100%), 98.7% (MeOH−

H₂O, 90:10).

CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 7.5 Hz, 1 H), 7.72 (t, J

= 6.9 Hz, 1 H), 7.65 (d, J = 6.3 Hz, 1 H), 7.49−7.40 (m, 4 H), 4.53 (t,

J = 7.9 Hz, 2 H), 4.20 (br s, 1 H), 3.16 (q, J = 6.8 Hz, 2 H), 2.95 (s, 3

H), 1.93 (m, 2 H), 1.56−1.18 (m, 14 H). ESI-MS m/z (rel intensity)

481 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 481.2161; found,

481.2165. HPLC purity: 95.7% (MeOH, 100%), 96.2% (MeOH−

H₂O, 90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

undecyl)methanesulfonamide (89). The crude product was eluted

with EtOAc−CHCl₃(5:5) to aﬀord the desired product as an orange

solid (48.7 mg, 89%); mp 136−137 °C. IR (ﬁlm) 3299, 1699, 1658,

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

hexyl)benzenesulfonamide (95). The crude product was eluted

with EtOAc−CHCl₃(2:8) to aﬀord the desired product as an orange

solid (57.8 mg, 91%); mp 150−152 °C. IR (ﬁlm) 3272, 1698, 1663,

1

1612, 1577, 1504, 1424, 1312, 1134, 761 cm⁻¹. H NMR (300 MHz,

1503, 1427, 1320, 1159, 756 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

CDCl₃) δ 8.72 (d, J = 8.2 Hz, 1 H), 8.35 (d, J = 8.1 Hz, 1 H), 7.75 (t, J

= 7.3 Hz, 1 H), 7.65 (d, J = 6.3 Hz, 1 H), 7.49−7.40 (m, 4 H), 4.53 (t,

J = 7.8 Hz, 2 H), 4.25 (br s, 1 H), 3.16 (q, J = 6.6 Hz, 2 H), 2.96 (s, 3

H), 1.90 (m, 2 H), 1.61−1.25 (m, 16 H). ESI-MS m/z (rel intensity)

495 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 495.2318; found,

495.2322. HPLC purity: 97.4% (MeOH, 100%), 100% (MeOH−H₂O,

90:10).

8.73 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 7.6 Hz, 1 H), 7.89 (dd, J = 6.7

and 1.6 Hz, 2 H), 7.76 (t, J = 8.2 Hz, 1 H), 7.66 (d, J = 6.9 Hz, 1 H),

7.58−7.41 (m, 7 H), 4.56−4.48 (m, 3 H), 3.03 (q, J = 6.3 Hz, 2 H),

1.88 (m, 2 H), 1.56−1.48 (m, 6 H). ESI-MS m/z (rel intensity) 509

(MNa⁺, 100). HRMS (+ESI) calcd for MNa⁺, 509.1511; found,

509.1521. HPLC purity: 97.9% (MeOH, 100%), 97.4% (MeOH−

H₂O, 90:10).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

dodecyl)methanesulfonamide (90). The crude product was eluted

with EtOAc−CHCl₃(5:5) to aﬀord the desired product as an orange

solid (58.0 mg, 89%); mp 113−117 °C. IR (ﬁlm) 3268, 1698, 1662,

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

heptyl)benzenesulfonamide (96). The crude product was eluted

with EtOAc−CHCl₃(2:8) to aﬀord the desired compound as an

orange product (37.8 mg, 75%); mp 178−181 °C. IR (ﬁlm) 3281,

1

1610, 1550, 1504, 1427, 1318, 1151, 759 cm⁻¹. H NMR (300 MHz,

1698, 1669, 1608, 1548, 1504, 1446, 1324, 1160, 759 cm⁻¹. H NMR

CDCl₃) δ 8.71 (d, J = 8.0 Hz, 1 H), 8.35(d, J = 8.0 Hz, 1 H), 7.74 (t, J

= 7.7 Hz, 1 H), 7.64 (d, J = 6.5 Hz, 1 H), 7.49−7.40 (m, 4 H), 4.52 (t,

J = 7.7 Hz, 2 H), 4.26 (m, 1 H), 3.16 (dd, J = 13 and 6.7 Hz, 2 H),

2.95 (s, 3 H), 1.89 (m, 14 H). APCI-MS m/z (rel intensity) 509

(MH⁺, 100). HRMS (+ESI) calcd for MNa⁺, 531.2294; found,

531.2291. HPLC purity: 99.2% (CH₃CN, 100%), 98.3% (CH₃CN−

H₂O, 90:10).

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.37 (d, J = 8.0 Hz, 1

H), 7.88 (dd, J = 0.92 and 6.8 Hz, 2 H), 7.66 (t, J = 6.7 Hz, 1 H), 7.57

(d, J = 6.9 Hz, 1 H), 7.54−7.43 (m, 7 H), 4.52 (t, J = 6.8 Hz, 3 H),

3.01 (q, J = 6.6 Hz, 2 H), 1.87 (m, 2 H), 1.49−1.25 (m, 8 H). ESI-MS

m/z (rel intensity) 501 (MH⁺, 45). HRMS (+ESI) calcd for MNa⁺,

523.1668; found, 523.1672. Anal. Calcd for C₂₉H₂₈N₂O₄S·0.7H₂O: C,

67.87; H, 5.77; N, 5.46. Found: C, 67.50; H, 5.40; N, 5.37.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-

benzenesulfonamide (91). The crude product was eluted with

EtOAc−CHCl₃(2:8) to aﬀord the desired product as an orange solid

(47.8 mg, 72%); mp 246−249 °C. IR (ﬁlm) 3233, 1702, 1652, 1610,

1551, 1505, 1425, 1342, 1157, 756 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.54 (d, J = 8.1 Hz, 1 H), 8.18−8.13 (m, 2 H), 7.80−7.72

(m, 4 H), 7.58−7.48 (m, 7 H), 4.55 (t, J = 6.9 Hz, 2 H), 3.23 (t, J =

6.8 Hz, 2 H). ESI-MS m/z (rel intensity) 431 (MH⁺, 13). HRMS

(+ESI) calcd for MH⁺, 431.1066; found, 431.1073. HPLC purity:

95.9% (MeOH, 100%), 98.0% (MeOH−H₂O, 90:10).

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

octyl)benzenesulfonamide (97). The crude product was eluted

with EtOAc−CHCl₃(2:8) to aﬀord the desired compound as an

orange product (36.1 mg, 72%); mp 136−140 °C. IR (ﬁlm) 3271,

1

1698, 1663, 1504, 1427, 1320, 1159, 756 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.0 Hz, 1 H), 7.88 (d,

J = 6.9 Hz, 2 H), 7.70 (t, J = 7.0 Hz, 1 H), 7.66 (d, J = 6.7 Hz, 1 H),

7.61−7.38 (m, 7 H), 4.52 (t, J = 7.8 Hz, 2 H), 4.36 (m, 1 H), 3.00 (q, J

= 6.8 Hz, 2 H), 1.91 (m, 2 H), 1.47−1.25 (m, 10 H). ESI-MS m/z (rel

intensity) 1051 (M₂Na⁺, 100). HRMS (+ESI) calcd for MH⁺,

515.2005; found, 515.2014; HPLC purity: 99.4% (MeOH, 100%),

99.3% (MeOH−H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

propyl)benzenesulfonamide (92). The crude product was eluted

with EtOAc−CHCl₃(2:8) to aﬀord the desired product as a red solid

(57.2 mg, 88%); mp 216−218 °C. IR (ﬁlm) 3282, 1655, 1611, 1550,

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

nonyl)benzenesulfonamide (98). The crude product was eluted

with EtOAc−CHCl₃(6:4) to aﬀord the desired compound as an

1

1502, 1446, 1317, 1161, 748 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

1

8.72 (d, J = 8.0 Hz, 1 H), 8.31 (d, J = 8.1 Hz, 1 H), 7.90−7.88 (m, 2

H), 7.78 (d, J = 8.3 Hz, 1 H), 7.65 (d, J = 5.9 Hz, 1 H), 7.51−7.38 (m,

7 H), 5.96 (t, J = 5.0 Hz, 1 H), 4.67 (t, J = 6.2 Hz, 2 H), 3.01 (q, J =

4.8 Hz, 2 H), 2.15 (m, 2 H). APCI-MS m/z (rel intensity) 445 (MH⁺,

100). HRMS (+ESI) calcd for MNa⁺, 467.1042; found, 467.1047.

HPLC purity: 95.8% (MeOH, 100%), 95.1% (MeOH−H₂O, 95:5).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

butyl)benzenesulfonamide (93). The crude product was eluted

with EtOAc−CHCl₃(3:7) to aﬀord the desired product as a red solid

(57.2 mg, 89%); mp 184−186 °C. IR (ﬁlm) 3222, 1700, 1652, 1614,

1551, 1507, 1427, 1324, 1164, 753 cm⁻¹. ¹H NMR (300 MHz, CDCl₃)

δ 8.71 (d, J = 8.0 Hz, 1 H), 8.32 (d, J = 8.0 Hz, 1 H), 7.89 (d, J = 6.9

Hz, 2 H), 7.75 (t, J = 7.1 Hz, 1 H), 7.65 (d, J = 7.1 Hz, 1 H), 7.56−

orange solid (58.2 mg, 78%); mp 162−164 °C. H NMR (300 MHz,

CDCl₃) δ 8.73 (d, J = 7.9 Hz, 1 H), 8.36 (d, J = 8.4 Hz, 1 H), 7.87 (d,

J = 7.0 Hz, 2 H), 7.84−7.43 (m, 9 H), 4.53 (t, J = 8.1 Hz, 2 H), 4.38

(m, 1 H), 2.99 (q, J = 6.9 Hz, 2 H), 1.89 (m, 2 H), 1.57−1.25 (m, 12

H). ESI-MS m/z (rel intensity) 551 (MNa⁺, 100). HRMS (+ESI)

calcd for MH⁺, 529.2161; found, 529.2159. HPLC purity: 100%

(MeOH, 100%), 96.0% (MeOH−H₂O, 90:10).

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

decyl)benzenesulfonamide (99). The crude product was eluted

with EtOAc−CHCl₃(15:85) to aﬀord the desired product as an

orange solid (54.6 mg, 88%); mp 128−130 °C. IR (ﬁlm) 3274, 1698,

1

1667, 1611, 1550, 1505, 1428, 1320, 1160, 757 cm⁻¹. H NMR (300

MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.2 Hz, 1 H),

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7.88 (dd, J = 1.5 and 5.3 Hz, 2 H), 7.73 (dt, J = 1.3 and 7.0 Hz, 1 H),

7.66 (d, J = 6.2 Hz, 1 H), 7.57−7.40 (m, 7 H), 4.53 (t, J = 7.6 Hz, 2

H), 4.37 (t, J = 6.1 Hz, 1 H), 2.99 (q, J = 6.7 Hz, 2 H), 1.90 (m, 2 H),

1.52−1.24 (m, 14 H). APCI-MS m/z (rel intensity) 543 (MH⁺, 100).

HRMS (+APCI) calcd for MNa⁺, 565.2137; found, 565.2142. HPLC

purity: 100% (MeOH, 100%), 99.4% (MeOH−H₂O, 90:10).

H), 7.76−7.70 (m, 3 H), 7.65 (d, J = 6.8 Hz, 1 H), 7.50−7.38 (m, 4

H), 7.30−7.27 (m, 2 H), 4.55 (m, 3 H), 3.02 (q, J = 6.4 Hz, 2 H), 2.41

(s, 3 H), 1.90 (m, 2 H), 1.65 (m, 2 H), 1.54 (m, 2 H). ESI-MS m/z

(rel intensity) 487 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺,

487.1692; found, 487.1688. HPLC purity: 99.2% (MeOH, 100%),

98.1% (MeOH−H₂O, 90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

undecyl)benzenesulfonamide (100). The crude product was

eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired compound as

a red solid (53.4 mg, 87%); mp 170−172 °C. IR (ﬁlm) 3289, 1698,

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)hexyl)-

4-methylbenzenesulfonamide (106). The crude product was

eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as a

red solid (60.2 mg, 92%); mp 146−149 °C. IR (ﬁlm) 3583, 1688,

1

1669, 1609, 1548, 1445, 1326, 1160, 760 cm⁻¹. H NMR (300 MHz,

1652, 1608, 1500, 1419, 1314, 1151, 759 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 8.4 Hz, 1 H), 7.88 (dd,

J = 1.6 and 6.9 Hz, 2 H), 7.76 (dt, J = 1.2 and 7.1 Hz, 1 H), 7.66 (d, J =

6.2 Hz, 1 H), 7.61−7.39 (m, 7 H), 4.53 (t, J = 8.1 Hz, 2 H), 4.38 (m, 1

H), 2.99 (q, J = 6.9 Hz, 2 H), 1.89 (m, 2 H), 1.57−1.25 (m, 16 H).

ESI-MS m/z (rel intensity) 557 (MH⁺, 100). HRMS (+ESI) calcd for

MH⁺, 557.2474; found, 557.2477. HPLC purity: 98.8% (MeOH,

100%), 100% (MeOH−H₂O, 90:10).

CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.35 (d, J = 8.1 Hz, 1 H), 7.76−

7.71 (m, 3 H), 7.66 (d, J = 6.7 Hz, 1 H), 7.50−7.40 (m, 4 H), 7.30−

7.27 (m, 2 H), 4.53 (m, 3 H), 3.00 (q, J = 6.2 Hz, 2 H), 2.42 (s, 3 H),

1.88 (m, 2 H), 1.53−1.48 (m, 6 H). ESI-MS m/z (rel intensity) 501

(MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 501.1848; found,

501.1860. HPLC purity: 96.4% (MeOH, 100%), 97.6% (MeOH−

H₂O, 90:10).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

dodecyl)benzenesulfonamide (101). The crude product was

eluted with EtOAc−CHCl₃(7:3) to aﬀord the desired compound as

an orange solid (62.8 mg, 86%); mp 128−130 °C. IR (ﬁlm) 3275,

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

heptyl)-4-methylbenzenesulfonamide (107). The crude product

was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired compound

as an orange solid (41.5 mg, 80%); mp 173−175 °C. IR (ﬁlm) 3275,

1

1698, 1662, 1610, 1504, 1426, 1319, 1159, 756 cm⁻¹. H NMR (300

1696, 1663, 1609, 1575, 1549, 1503, 1426, 1321, 1158, 780 cm⁻¹. H

MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 8.1 Hz, 1 H),

7.87 (d, J = 7.0 Hz, 2 H), 7.84−7.42 (m, 9 H), 4.52 (t, J = 7.6 Hz, 2

H), 4.39 (m, 1 H), 2.98 (q, J = 6.7 Hz, 2 H), 1.89 (m, 2 H), 1.59−1.21

(m, 12 H). ESI-MS m/z (rel intensity) 571 (MH⁺, 100). HRMS

(+ESI) calcd for MH⁺, 571.2631; found, 571.2628. Anal. Calcd for

C₃₄H₃₈N₂O₄S: C, 71.55; H, 6.71; N, 4.91. Found: C, 71.70; H, 6.82;

N, 5.09.

NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 7.7 Hz, 1 H), 8.36 (d, J = 8.4

Hz, 1 H), 7.76−7.73 (m, 3 H), 7.65 (d, J = 5.9 Hz, 1 H), 7.50−7.45

(m, 4 H), 7.31−7.29 (m, 2 H), 4.49 (m, 3 H), 2.96 (q, J = 6.5 Hz, 2

H), 2.42 (s, 3 H), 1.87 (m, 2 H), 1.49−1.25 (m, 8 H). ESI-MS m/z

(rel intensity) 537 (MNa⁺, 100). HRMS (+ESI) calcd for MNa⁺,

537.1824; found, 537.1819. Anal. Calcd for C₃₀H₃₀N₂O₄S·0.7H₂O: C,

68.34; H, 6.00; N, 5.31. Found: C, 67.97; H, 5.75; N, 4.96.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-

4-methylbenzenesulfonamide (102). The crude product was

eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as an

orange solid (57.4 mg, 84%); mp 272−274 °C. IR (ﬁlm) 3209, 1702,

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

octyl)-4-methylbenzenesulfonamide (108). The crude product

was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired compound

as an orange solid (36.0 mg, 70%); mp 136−139 °C. IR (ﬁlm) 3272,

1

1646, 1611, 1552, 1506, 1427, 1322, 1147, 763 cm⁻¹. H NMR (300

1698, 1663, 1611, 1550, 1504, 1428, 1320, 1159, 757 cm⁻¹. H NMR

MHz, DMSO-d₆) δ 8.55 (d, J = 8.0 Hz, 1 H), 8.18 (d, J = 7.6 Hz, 1 H),

8.01 (m, 1 H), 7.83−7.76 (m, 2 H), 7.58−7.48 (m, 6 H), 7.26 (d, J =

8.1 Hz, 2 H), 4.53 (t, J = 7.0 Hz, 2 H), 3.24 (t, J = 6.7 Hz, 2 H), 2.29

(s, 3 H). ESI-MS m/z (rel intensity) 443 ([M − H]⁻, 100). HRMS

(+ESI) calcd for MH⁺, 445.1222; found, 445.1220. HPLC purity:

98.6% (MeOH, 100%), 96.4% (MeOH−H₂O, 90:10).

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 8.1 Hz, 1

H), 7.75−7.70 (m, 3 H), 7.66 (d, J = 6.9 Hz, 1 H), 7.50−7.40 (m, 4

H), 7.32 (d, J = 8.0 Hz, 2 H), 4.53 (t, J = 8.2 Hz, 2 H), 4.31 (m, 1 H),

2.97 (t, J = 6.7, 2 H), 2.42 (s, 3 H), 1.88 (m, 2 H), 1.56−1.19 (m, 10

H). ESI-MS m/z (rel intensity) 529 (MH⁺, 78). HRMS (+ESI) calcd

for MH⁺, 529.2161; found, 529.2155. HPLC purity: 97.7% (MeOH,

100%), 96.7% (MeOH−H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

propyl)-4-methylbenzenesulfonamide (103). The crude product

was eluted with EtOAc−CHCl₃(3:7) to aﬀord the desired product as

an orange solid (58.8 mg, 87%); mp 213-216 °C. IR (ﬁlm) 3294, 1705,

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

nonyl)-4-methylbenzenesulfonamide (109). The crude product

was eluted with EtOAc−CHCl₃(7:3) to aﬀord the desired compound

as an orange product (61.3 mg, 80%); mp 159−160 °C. IR (ﬁlm)

3276, 1769, 1698, 1663, 1610, 1549, 1504, 1427, 1320, 1158, 758

1

1638, 1609, 1548, 1501, 1422, 1327, 1163, 758 cm⁻¹. H NMR (300

MHz, DMSO-d₆) δ 8.58 (d, J = 8.1 Hz, 1 H), 8.21 (d, J = 8.4 Hz, 1 H),

7.82−7.74 (m, 3 H), 7.65−7.63 (m, 2 H), 7.59−7.50 (m, 4 H), 7.33−

7.31 (m, 2 H), 4.49 (t, J = 7.5 Hz, 2 H), 2.93 (m, 2 H), 2.33 (s, 3 H),

1.92 (m, 2 H). ESI-MS m/z (rel intensity) 459 (MH⁺, 100). HRMS

(+ESI) calcd for MH⁺, 459.1379; found, 459.1384. HPLC purity:

100% (MeOH, 100%), 99.4% (MeOH−H₂O, 90:10).

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36

(d, J = 7.4 Hz, 1 H), 7.75−7.70 (m, 3 H), 7.66 (d, J = 6.8 Hz, 1 H),

7.49−7.39 (m, 4 H), 7.32−7.29 (m, 2 H), 4.53 (t, J = 7.9 Hz, 2 H),

4.33 (t, J = 7.1 Hz, 1 H), 2.96 (m, 2 H), 2.42 (s, 3 H), 1.88 (m, 2 H),

1.51−1.26 (m, 12 H). ESI-MS m/z (rel intensity) 1107 (M₂Na⁺, 18).

HRMS (+ESI) calcd for MH⁺, 543.2318; found, 543.2307. Anal. Calcd

for C₃₂H₃₄N₂O₄S·0.2H₂O: C, 70.36; H, 6.35; N, 5.13. Found: C,

70.22; H, 6.35; N, 4.97.

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)butyl)-

4-methylbenzenesulfonamide (104). The crude product was

eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as an

orange solid (59.7 mg, 90%); mp 190−192 °C. IR (ﬁlm) 3272, 1699,

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

decyl)-4-methylbenzenesulfonamide (110). The crude product

was eluted with EtOAc−CHCl₃(10:90) to aﬀord the desired product

as a red solid (49.4 mg, 78%); mp 133−135 °C. IR (ﬁlm) 3263, 1699

1

1661, 1606, 1545, 1501, 1424, 1312, 1154, 759 cm⁻¹. H NMR (300

MHz, CDCl₃) δ 8.71 (d, J = 8.1 Hz, 1 H), 8.32 (d, J = 7.5 Hz, 1 H),

7.76−7.70 (m, 3 H), 7.64 (d, J = 6.9 Hz, 1 H), 7.50−7.41 (m, 4 H),

7.30−7.26 (m, 2 H), 4.76 (t, J = 6.3 Hz, 1 H), 4.54 (t, J = 7.5 Hz, 2 H),

3.11 (q, J = 6.5 Hz, 2 H), 2.40 (s, 3 H), 1.98 (m, 2 H), 1.76 (m, 2 H).

ESI-MS m/z (rel intensity) 473 (MH⁺, 100). HRMS (+ESI) calcd for

MNa⁺, 495.1355; found, 495.1362. HPLC purity: 96.4% (CH₃CN,

100%), 97.1% (CH₃CN−H₂O, 90:10).

1

1663, 1611, 1550, 1504, 1428, 1320, 1159, 758 cm⁻¹. H NMR (300

MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 7.4 Hz, 1 H),

7.76−7.71 (m, 3 H), 7.66 (d, J = 6.3 Hz, 1 H), 7.50−7.40 (m, 4 H),

7.32−7.30 (m, 2 H), 4.53 (t, J = 7.7 Hz, 2 H), 4.33 (t, J = 5.9 Hz, 1 H),

2.97 (q, J = 6.8 Hz, 2 H), 2.43 (s, 3 H), 1.92 (m, 2 H), 1.55−1.19 (m,

14 H). APCI-MS m/z (rel intensity) 557 (MH⁺, 100). HRMS

(+APCI) calcd for MH⁺, 557.2474; found, 557.2478. HPLC purity:

97.0% (MeOH, 100%), 98.8% (MeOH−H₂O, 90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

undecyl)-4-methylbenzenesulfonamide (111). The crude prod-

uct was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-

pentyl)-4-methylbenzenesulfonamide (105). The crude product

was eluted with EtOAc−CHCl₃(15:85) to aﬀord the desired product

as an orange solid (61.5 mg, 93%); mp 187−188 °C. IR (ﬁlm) 3273,

1

1694, 1671, 1609, 1548, 1504, 1426, 1325, 1159, 758 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 7.4 Hz, 1

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compound as an orange solid (45.0 mg, 71.4%); mp 170−172 °C. IR

(ﬁlm) 3289, 1698, 1669, 1609, 1575, 1548, 1505, 1445, 1326, 1160,

a red solid (68.5 mg, 93%); mp 188−190 °C. IR (ﬁlm) 3199, 1760,

1

1698, 1636, 1610, 1574, 1503, 1458, 1330, 1160, 757 cm⁻¹. H NMR

1

760 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.0 Hz, 1 H),

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 7.5 Hz, 1

H), 7.76−7.71 (m, 3 H), 7.66−7.64 (m, 3 H), 7.50−7.39 (m, 4 H),

4.74 (t, J = 6.2 Hz, 1 H), 4.54 (t, J = 7.2 Hz, 2 H), 3.03 (q, J = 6.4 Hz,

2 Hz), 1.89 (m, 2 H), 1.57−1.48 (m, 6 H). ESI-MS m/z (rel intensity)

565/567 (MH⁺, 91/100). HRMS (+ESI) calcd for MNa⁺, 587.0616;

found, 587.0610. HPLC purity: 96.6% (MeOH, 100%), 98.2%

(MeOH−H₂O, 90:10).

8.36 (d, J = 8.0 Hz, 1 H), 7.76−7.70 (m, 3 H), 7.65 (d, J = 6.4 Hz, 1

H), 7.49−7.40 (m, 4 H), 7.32−7.29 (m, 2 H), 4.53 (t, J = 7.5 Hz, 2

H), 4.33 (br s, 1 H), 2.97 (q, J = 6.6 Hz, 2 H), 2.43 (s, 3 H), 1.90 (m,

2 H), 1.58−1.23 (m, 16 H). ESI-MS m/z (rel intensity) 571 (MH⁺,

100). HRMS (+ESI) calcd for MH⁺, 571.2631; found, 571.2625.

HPLC purity: 96.7% (MeOH, 100%), 96.7% (MeOH−H₂O, 95:5).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

dodecyl)-4-methylbenzenesulfonamide (112). The crude prod-

uct was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired

compound as an orange solid (55 mg, 73%); mp 108−112 °C. IR

(ﬁlm) 3275, 1698, 1666, 1611, 1550, 1504, 1428, 1320, 1160, 757

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

heptyl)-4-bromobenzenesulfonamide (118). The crude product

was eluted with EtOAc−CHCl₃(5:95) to aﬀord the desired product as

a red solid (70.5 mg, 96%); mp 160−161 °C. IR (ﬁlm) 3290, 1699,

1

1661, 1610, 1550, 1503, 1427, 1320, 1161, 757 cm⁻¹. H NMR (300

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.2 Hz, 1 H), 8.36

MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 7.7 Hz, 1 H),

7.76−7.70 (m, 3 H), 7.66−7.63 (m, 3 H), 7.50−7.39 (m, 4 H), 4.69 (t,

J = 6.1 Hz, 1 H), 4.51 (t, J = 7.7 Hz, 2 H), 3.01 (q, J = 6.7 Hz, 2 Hz),

1.90 (m, 2 H), 1.53−1.36 (m, 8 H). ESI-MS m/z (rel intensity) 579/

581 (MH⁺, 92/100). HRMS (+ESI) calcd for MNa⁺, 579.0953; found,

579.0959. HPLC purity: 98.3% (MeOH, 100%), 98.9% (MeOH−

H₂O, 90:10).

(d, J = 7.0 Hz, 1 H), 7.75−7.63 (m, 4 H), 7.49−7.29 (m, 6 H), 4.53 (t,

J = 7.3 Hz, 2 H), 4.27 (m, 1 H), 2.96 (q, J = 6.7 Hz, 2 H), 2.42 (s, 3

H), 1.90 (m, 2 H), 1.57−1.22 (m, 18 H). ESI-MS m/z (rel intensity)

607 (MNa⁺, 30). HRMS (+ESI) calcd for MNa⁺, 607.2607; found,

607.2604. Anal. Calcd for C₃₅H₄₀N₂O₄S: C, 71.89; H, 6.89; N, 4.79.

Found: C, 71.49; H, 6.97; N, 4.83.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

ethyl)-4-bromobenzenesulfonamide (113). The crude product

was eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as

an orange solid (64.2 mg, 82%); mp 260−263 °C. IR (ﬁlm) 3208,

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

octyl)-4-bromobenzenesulfonamide (119). The crude product

was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired product as

an orange solid (67.6 mg, 78%); mp 119−121 °C. IR (ﬁlm) 3272,

1

1707, 1645, 1611, 1551, 1506, 1427, 1324, 1146, 827 cm⁻¹. H NMR

1

1698, 1662, 1611, 1550, 1504, 1428, 1331, 1163, 757 cm⁻¹. H NMR

(300 MHz, DMSO-d₆) δ 8.56 (d, J = 8.0 Hz, 1 H), 8.22 (m, 2 H),

7.80−7.69 (m, 4 H), 7.64−7.61 (m, 2 H), 7.56−7.48 (m, 4 H), 4.54 (t,

J = 6.4 Hz, 2 H), 3.26 (t, J = 6.7 Hz, 2 H). ESI-MS m/z (rel intensity)

507/509 ([M − H]⁻, 78/100). HRMS (−ESI) calcd for [M − H]⁻,

507.0014; found, 507.0017. HPLC purity: 97.7% (MeOH, 100%),

95.3% (MeOH−H₂O, 90:10).

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.1 Hz, 1 H), 8.36 (d, J = 8.4 Hz, 1

H), 7.74−7.71 (m, 3 H), 7.67−7.64 (m, 3 H), 7.47−7.43 (m, 4 H),

4.53 (t, J = 7.7 Hz, 2 H), 4.43 (t, J = 6.4 Hz, 1 H), 3.00 (q, J = 6.8 Hz,

2 H), 1.91 (m, 2 H), 1.55−1.20 (m, 10 H). ESI-MS m/z (rel intensity)

593/595 (MH⁺, 100/91). HRMS (+ESI) calcd for MH⁺, 593.1110;

found, 593.1105. Anal. Calcd for C₃₀H₂₉BrN₂O₄S: C, 60.71; H, 4.92;

N, 4.72. Found: C, 60.69; H, 5.01; N, 4.67.

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

propyl)-4-bromobenzenesulfonamide (114). The crude product

was eluted with EtOAc−CHCl₃(9:1) to aﬀord the desired product as

an orange solid (40.2 mg, 52%); mp 241−243 °C. IR (ﬁlm) 3303,

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

nonyl)-4-bromobenzenesulfonamide (120). The crude product

was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired product as

an orange solid (52.2 mg, 61%); mp 143−146 °C. IR (ﬁlm) 3288,

1

1762, 1696, 1653, 1609, 1549, 1505, 1427, 1325, 1166, 751 cm⁻¹. H

NMR (300 MHz, DMSO-d₆) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.28 (d, J =

7.6 Hz, 1 H), 7.79−7.74 (m, 3 H), 7.66−7.39 (m, 7 H), 6.11 (t, J = 6.7

Hz, 1 H), 4.67 (t, J = 6.0 Hz, 2 H), 3.01 (q, J = 6.6 Hz, 2 H), 2.18 (m,

2 H). ESI-MS m/z (rel intensity) 523/525 (MH⁺, 100/93). HRMS

(+ESI) calcd for MH⁺, 523.0327; found, 523.0335. HPLC purity:

95.4% (MeOH, 100%), 95.7% (MeOH−H₂O, 95:5).

1

1697, 1662, 1610, 1550, 1504, 1427, 1320, 1162, 757 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 8.2 Hz, 1

H), 7.74−7.71 (m, 3 H), 7.66−7.63 (m, 3 H), 7.49−7.40 (m, 4 H),

4.53 (t, J = 6.4 Hz, 3 H), 2.99 (q, J = 6.8 Hz, 2 H), 1.91 (m, 2 H),

1.58−1.25 (m, 12 H). ESI-MS m/z (rel intensity) 607/609 (MH⁺, 88/

100). HRMS (+ESI) calcd for MH⁺, 607.1266; found, 607.1263. Anal.

Calcd for C₃₁H₃₁BrN₂O₄S: C, 61.28; H, 5.14; N, 4.61. Found: C,

61.22; H, 5.16; N, 4.57.

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

butyl)-4-bromobenzenesulfonamide (115). The crude product

was eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as

a red solid (63.7 mg, 84%); mp 176−177 °C. IR (ﬁlm) 3257, 1698,

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

decyl)-4-bromobenzenesulfonamide (121). The crude product

was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired product as

an orange solid (65.2 mg, 77%); mp 160−163 °C. IR (ﬁlm) 3262,

1

1663, 1611, 1576, 1503, 1427, 1332, 1163, 757 cm⁻¹. H NMR (300

MHz, CDCl₃) δ 8.71 (d, J = 8.1 Hz, 1 H), 8.32 (d, J = 8.0 Hz, 1 H),

7.76−7.70 (m, 3 H), 7.67−7.63 (m, 3 H), 7.49−7.40 (m, 4 H), 4.98 (t,

J = 6.3 Hz, 1 H), 4.54 (t, J = 7.3 Hz, 2 H), 3.14 (q, J = 6.4 Hz, 2 Hz),

1.99 (m, 2 H), 1.80 (m, 2 H). ESI-MS m/z (rel intensity) 537/539

(MH⁺, 89/100). HRMS (+ESI) calcd for MH⁺, 537.0484; found,

537.0489. HPLC purity: 99.4% (MeOH, 100%), 98.1% (MeOH−

H₂O, 90:10).

1

1696, 1658, 1609, 1548, 1503, 1426, 1321, 1158, 753 cm⁻¹. H NMR

(500 MHz, CDCl₃) δ 8.72 (d, J = 8.0 Hz, 1 H), 8.35 (d, J = 8.0 Hz, 1

H), 7.74−7.71 (m, 3 H), 7.66−7.64 (m, 3 H), 7.48−7.40 (m, 4 H),

4.52 (t, J = 7.0 Hz, 2 H), 4.40 (m, 1 H), 2.97 (q, J = 6.5 Hz, 2 H),

1.91−1.88 (m, 2 H), 1.54−1.22 (m, 14 H). ESI-MS m/z (rel intensity)

621/623 (MH⁺, 100/99.8). HRMS (+ESI) calcd for MH⁺, 621.1423;

found, 621.1430. Anal. Calcd for C₃₂H₃₃BrN₂O₄S: C, 61.83; H, 5.35;

N, 4.51. Found: C, 61.95; H, 5.40; N, 4.71.

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

pentyl)-4-bromobenzenesulfonamide (116). The crude product

was eluted with EtOAc−CHCl₃(15:85) to aﬀord the desired product

as an orange solid (68.0 mg, 91%); mp 169−171 °C. IR (ﬁlm) 3271,

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

undecyl)-4-bromobenzenesulfonamide (122). The crude prod-

uct was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired product

as an orange solid (60.6 mg, 86%); mp 150−153 °C. IR (ﬁlm) 3310,

1

1698, 1661, 1611, 1576, 1505, 1428, 1332, 1163, 757 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 7.3 Hz, 1

H), 7.74−7.70 (m, 3 H), 7.66−7.61 (m, 3 H), 7.49−7.41 (m, 4 H),

4.73 (t, J = 6.0 Hz, 1 H), 4.54 (t, J = 7.2 Hz, 2 H), 3.04 (q, J = 6.4 Hz,

2 Hz), 1.92 (m, 2 H), 1.68 (m, 2 H), 1.53 (m, 2 H). ESI-MS m/z (rel

intensity) 551/553 (MH⁺, 100/98). HRMS (+ESI) calcd for MH⁺,

551.0640; found, 551.0652; HPLC purity: 98.3% (MeOH, 100%),

98.2% (MeOH−H₂O, 90:10).

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

hexyl)-4-bromobenzenesulfonamide (117). The crude product

was eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as

1

1705, 1661, 1608, 1548, 1504, 1422, 1328, 1156, 757 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 8.73 (d, J = 8.0 Hz, 1 H), 8.36 (d, J = 8.2 Hz, 1

H), 7.75−7.71 (m, 3 H), 7.66−7.61 (m, 3 H), 7.49−7.38 (m, 4 H),

4.54 (t, J = 7.5 Hz, 2 H), 4.40 (t, J = 6.1 Hz, 1 H), 2.99 (q, J = 6.8 Hz,

2 H), 1.90 (m, 2 H), 1.56−1.23 (m, 16 H). ESI-MS m/z (rel intensity)

635/637 (MH⁺, 100/92). HRMS (+ESI) calcd for MH⁺, 635.1579;

found, 635.1584. Anal. Calcd for C₃₃H₃₅BrN₂O₄S: C, 62.36; H, 5.55;

N, 4.41. Found: C, 62.15; H, 5.62; N, 4.44.

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Article

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-

dodecyl)-4-bromobenzenesulfonamide (123). The crude prod-

uct was eluted with EtOAc−CHCl₃(6:4) to aﬀord the desired product

as an orange solid (65.3 mg, 78%); mp 118−120 °C. IR (ﬁlm) 3275,

N-(3-(2,3-Dimethoxy-5,11-dioxo-5H-indeno[1,2-c]-

isoquinolin-6(11H)-yl)propyl)benzenesulfonamide (136). The

crude product was eluted with EtOAc−CHCl₃(4:6) to aﬀord the

desired product as an orange solid (58.9 mg, 94%); mp 249−250 °C.

IR (ﬁlm) 3166, 1701, 1626, 1612, 1589, 1554, 1513, 1478, 1426, 1335,

1

1698, 1664, 1611, 1550, 1504, 1428, 1332, 1164, 757 cm⁻¹. H NMR

1

(300 MHz, CDCl₃) δ 8.72 (d, J = 8.3 Hz, 1 H), 8.35 (d, J = 8.1 Hz, 1

H), 7.73−7.63 (m, 6 H), 7.49−7.40 (m, 4 H), 4.53 (t, J = 7.8 Hz, 2

H), 4.41 (t, J = 5.9 Hz, 1 H), 2.99 (q, J = 6.7 Hz, 2 H), 1.90−1.87 (m,

2 H), 1.58−1.21 (m, 18 H). ESI-MS m/z (rel intensity) 649/651

(MH⁺, 100/80). HRMS (+ESI) calcd for MH⁺, 649.1736; found,

649.1728. Anal. Calcd for C₃₄H₃₇BrN₂O₄S·0.5H₂O: C, 62.00; H, 5.82;

N, 4.25. Found: C, 61.71; H, 5.62; N, 4.15.

1260, 1171, 1093, 1021, 789 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

8.12 (s, 1 H), 7.91 (dd, J = 6.8 and 1.6 Hz, 2 H), 7.64 (s, 1 H), 7.60 (d,

J = 6.6 Hz, 1 H), 7.52−7.37 (m, 6 H), 6.12 (t, J = 6.9 Hz, 1 H), 4.65 (t,

J = 5.9 Hz, 2 H), 4.07 (s, 3 H), 4.03 (s, 3 H), 3.05 (q, J = 6.3 Hz, 2 H),

2.13 (m, 2 H). ESI-MS m/z (rel intensity) 505 (MH⁺, 100). HRMS

(+ESI) calcd for MH⁺, 505.1433; found, 505.1423. HPLC purity:

100% (MeOH, 100%), 100% (MeOH−H₂O, 90:10).

General Procedure for the Preparation of Indenoisoquino-

line Sulfonamides 132−136. Indenoisoquinoline salts 127−131

(50 mg, 0.088−0.102 mmol), prepared previously via the reported

literature procedures,⁴⁰were dissolved in CHCl₃(15 mL), Et₃N (2

equiv) in CHCl₃(1 mL) was added, and the solutions were stirred at

room temperature for 5 min. Benzenesulfonyl chloride (2 equiv) was

added. The mixtures were heated at reﬂux for 3 h and then washed

with satd NaHCO₃(50 mL) and brine (50 mL). The organic layer was

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated, adsorbed

onto SiO₂, and puriﬁed by ﬂash column chromatography (SiO₂),

eluting with EtOAc−CHCl₃to provide the indenoisoquinoline

sulfonamide products 132−136 in high purity.

General Procedure for the Preparation of Bisindenoisoqui-

nolines 140−142. Diamines 137−139 (100 mg, 1 equiv) were

dissolved in CHCl₃(10 mL) and added to a solution of lactone 11 (2

equiv) in CHCl₃(20 mL). The mixtures were stirred at reﬂux for 16 h

and then concentrated, adsorbed onto SiO₂, and puriﬁed with ﬂash

column chromatography (SiO₂), eluting with CHCl₃to aﬀord the

products 140−142 as orange or red solids.

6,6′-(Decane-1,10-diyl)bis(5H-indeno[1,2-c]isoquinoline-

5,11[6H]-dione) (140). The general procedure provided the product

as an orange solid (223 mg, 64%); mp 201−202 °C. IR (ﬁlm) 1762,

1657, 1609 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J = 8.1 Hz,

2 H), 8.35 (d, J = 8.2 Hz, 2 H), 7.75 (d, J = 8.3 Hz, 2 H), 7.64 (d, J =

6.4 Hz, 2 H), 7.49−7.39 (m, 8 H), 4.53 (t, J = 7.9 Hz, 4 H), 1.90 (m, 4

H), 1.57 (m, 4 H), 1.54−1.38 (m, 8 H). ESI-MS m/z (rel intensity)

633 (MH⁺, 57). HRMS (+ESI) calcd for MH⁺, 633.2753; found,

633.2763. Anal. Calcd for C₄₂H₃₆N₂O₄: C, 79.72; H, 5.73; N, 4.43.

Found: C, 79.53; H, 5.76; N, 4.39.

N-(3-(3-Nitro-5,11-dioxo-9-phenyl-5H-indeno[1,2-c]-

isoquinolin-6(11H)-yl)propyl)benzenesulfonamide (132). The

crude product was eluted with EtOAc−CHCl₃(2:8) to aﬀord the

desired product as an orange solid (22.9 mg, 37%); mp 246−247 °C.

IR (ﬁlm) 3318, 1706, 1658, 1615, 1553, 1501, 1448, 1383, 1336, 1153,

1

749 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 8.79 (d, J = 2.2 Hz, 1

6,6′-(Undecane-1,11-diyl)bis(5H-indeno[1,2-c]isoquinoline-

5,11[6H]-dione) (141). The general procedure provided the product

as an orange solid (223 mg, 64%); mp 201−202 °C. IR (ﬁlm) 1764,

H), 8.66 (d, J = 9.0 Hz, 1 H), 8.54 (dd, J = 6.5 and 2.4 Hz, 1 H), 7.89−

8.75 (m, 8 H), 7.61−7.45 (m, 6 H) 4.50 (m, 2 H), 3.02 (m, 2 H), 1.97

(m, 2 H). ESI-MS m/z (rel intensity) 566 (MH⁺, weak). HRMS

(+ESI) calcd for MH⁺, 566.1386; found, 566.1378. HPLC purity:

100% (MeOH, 100%), 99.1% (MeOH−H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-[1,3]dioxolo[4,5-g]indeno[1,2-c]-

isoquinolin-6(11H)-yl)propyl)benzenesulfonamide (133). The

crude product was eluted with EtOAc−CHCl₃(2:8) to aﬀord the

desired product as an orange solid (51.3 mg, 81%); mp 225−226 °C.

IR (ﬁlm) 3223, 1700, 1636, 1608, 1562, 1499, 1467, 1329, 1174, 765

cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 7.88−7.76 (m, 4 H), 7.60 (d,

J = 5.3 Hz, 1 H), 7.58−7.47 (m, 7 H), 6.19 (s, 2 H), 4.43 (t, J = 7.3

Hz, 2 H), 2.95 (q, J = 6.0 Hz, 2 H), 1.90 (m, 2 H). APCI-MS m/z (rel

intensity) 489 (MH⁺, 100). HRMS (+APCI) calcd for MNa⁺,

511.0940; found, 511.0949. HPLC purity: 100% (MeOH, 100%),

99.9% (MeOH−H₂O, 90:10).

1

1736, 1702, 1655 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.71 (d, J =

8.1 Hz, 2 H), 8.35 (d, J = 7.7 Hz, 2 H), 7.82 (d, J = 8.2 Hz, 2 H), 7.64

(d, J = 6.5 Hz, 2 H), 7.48−7.38 (m, 8 H), 4.52 (t, J = 8.1 Hz, 4 H),

1.92 (m, 4 H), 1.58 (m, 4 H), 1.41−1.33 (m, 10 H). ESI-MS m/z (rel

intensity) 647 (MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 647.2910;

found, 647.2908. Anal. Calcd for C₄₃H₃₈N₂O₄: C, 79.85; H, 5.92; N,

4.33. Found: C, 79.80; H, 5.99; N, 4.29.

6,6′-(Dodecane-1,12-diyl)bis(5H-indeno[1,2-c]isoquinoline-

5,11[6H]-dione) (142). The general procedure provided the product

as a red solid (135 mg, 82%); mp 226−228 °C. IR (ﬁlm) 1694, 1660,

1609 cm⁻¹. ¹H NMR (300 MHz, CDCl₃): δ 8.71 (d, J = 8.0 Hz, 2 H),

8.35 (d, J = 8.1 Hz, 2 H), 7.74 (t, J = 7.5 Hz, 2 H), 7.64 (d, J = 6.9 Hz,

2 H), 7.48−7.37 (m, 8 H), 4.52 (t, J = 7.9 Hz, 4 Hz), 1.90 (m, 4 H),

1.53 (m, 4 H), 1.40−1.31 (m, 12 H). ESI-MS m/z (rel intensity) 661

(MH⁺, 100). HRMS (+ESI) calcd for MH⁺, 661.3066; found,

661.3054. HPLC purity: 99.7% (CH₃CN, 100%), 97.0% (CH₃CN−

H₂O, 95:5).

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]-

isoquinoline)-(6-propyl-tert-BOCamino)}propane (144).³⁷Tet-

ramine 143 (100 mg, 0.53 mmol) was diluted in CHCl₃(10 mL) and

added to a solution of lactone 11 (276 mg, 1.12 mmol) in CHCl₃(40

mL). The reaction mixture was stirred at reﬂux for 72 h. Et₃N (270

mg, 2.66 mmol) and Boc₂O (360 mg, 1.65 mmol) were then added to

the cooled mixture, and stirring continued at room temperature for 16

h. The mixture was concentrated, adsorbed on to SiO₂, and puriﬁed

with ﬂash column chromatography (SiO₂), eluting with EtOAc−

hexane (2:8) and then with MeOH−CHCl₃(3:97) to provide the

product as an orange solid (352 mg, 78%); mp 84−86 °C (lit.³⁷86−

88 °C). ¹H NMR (CDCl₃) δ 8.63 (d, J = 7.9 Hz, 2 H), 8.24 (d, J = 7.6

Hz, 2 H), 7.65 (t, J = 7.3 Hz, 2 H), 7.55 (d, J = 6.9 Hz, 2 H), 7.41−

7.30 (m, 8 H), 4.49 (br s, 4 H), 3.44 (br s, 4 H), 3.27 (t, J = 6.3 Hz, 4

H), 2.08 (br s, 4 H), 1.86 (br s, 2 H), 1.41 (br s, 18 H).

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]-

isoquinoline)-6-propylamino}propane Bis(triﬂuoroacetate)

(145).³⁷Boc-protected bis(indenoisoquinoline) 144 (352 mg, 0.41

mmol) was diluted in CF₃COOH (30 mL), and the mixture was

stirred at room temperature for 2 h. The reaction mixture was

concentrated, and the resultant solid was triturated with chloroform

and ﬁltered to provide the product as an orange solid (247 mg, 93%);

M e t h y l 2 , 3 - D i m e t h o x y - 5 , 1 1 - d i o x o - 6 - ( 3 -

(phenylsulfonamido)propyl)-6,11-dihydro-5H-indeno[1,2-c]-

isoquinoline-9-carboxylate (134). The crude product was eluted

with EtOAc−CHCl₃(4:6) to aﬀord the desired product as a deep-

purple solid (53.7 mg, 88%); mp 249−251 °C. IR (ﬁlm) 3244, 1727,

1

1645, 1611, 1554, 1511, 1482, 1321, 1259, 1161, 802, 764 cm⁻¹. H

NMR (300 MHz, CDCl₃) δ 8.15−8.12 (m, 2 H), 8.07 (s, 1 H), 7.92

(d, J = 7.1 Hz, 2 H), 7.63 (s, 1 H), 7.56−7.45 (m, 4 H), 6.04 (t, J = 6.4

Hz, 1 H), 4.65 (t, J = 6.1 Hz, 2 H), 4.07 (s, 3 H), 4.03 (s, 3 H), 3.97 (s,

3 H), 3.08 (q, J = 6.2 Hz, 2 H), 2.12 (m, 2 H). APCI-MS m/z (rel

intensity) 563 (MH⁺, 100). HRMS (+APCI) calcd for MNa⁺,

585.1308; found, 585.1302. HPLC purity: 100% (MeOH, 100%),

99.9% (MeOH−H₂O, 90:10).

N-(3-(3-Nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6-

(11H)-yl)propyl)benzenesulfonamide (135). The crude product

was eluted with EtOAc−CHCl₃(2:8) to aﬀord the desired product as

an orange solid (28.6 mg, 46%); mp 258−259 °C. IR (ﬁlm) 3214,

1699, 1656, 1613, 1553, 1503, 1453, 1423, 1381, 1324, 1259, 1158,

802, 746 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 8.88 (d, J = 2.4 Hz,

1 H), 8.74 (d, J = 8.9 Hz, 1 H), 8.60 (dd, J = 6.5 and 2.4 Hz, 1 H),

7.91 (t, J = 8.0 Hz, 2 H), 7.82 (dd, J = 6.7 and 1.5 Hz, 2 H), 7.66−7.57

(m, 6 H), 4.51 (t, J = 8.0 Hz, 2 H), 3.01 (q, J = 6.2 Hz, 2 H), 1.96 (m,

2 H). APCI-MS m/z (rel intensity) 490 (MH⁺, 100). HRMS (+APCI)

calcd for MH⁺, 490.1073; found, 490.1070. HPLC purity: 97.1%

(MeOH, 100%), 100% (MeOH−H₂O, 90:10).

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mp 224−226 °C (lit.³⁷225−227 °C). H NMR (DMSO-d₆) δ 8.59−

Determination of the Mechanism of Inhibition of Compound 70.

Mechanistic characterization of compound 70 was carried out using a

FRET assay employing a custom designed substrate (Bermingham et

al. manuscript in preparation). The assay followed the real-time

observation of reaction time-course data, permitting an accurate

measure of the reaction rate in the presence of an inhibitor. The

mechanism of inhibition of 70 was observed by measuring the rate of

the Tdp1 catalyzed reaction under a matrix of varying substrate and

inhibitor concentrations. In the assay, Tdp1 FRET substrate was

present as a dilution series ranging from 2.25 to 0.035 μM over eight

2-fold steps. Compound 70 was present at three concentrations

equaling 0.33 × IC₅₀, 1.0 × IC₅₀, and 3.0 × IC₅₀. A “no inhibitor”

sample was also included to allow measurement of the rate of reaction

in the absence of inhibition.

Immediately prior to executing the assay, stocks of Tdp1 enzyme,

Tdp1 FRET substrate and compound 70 were created at 3× their ﬁnal

desired assay concentrations. Five μL of compound 70 stock dilutions

were combined with 5 μL of a 1.5 nM Tdp1 stock in appropriate wells

in a low volume 384-well plate (Greiner 784900. Greiner, Monroe,

NC) and allowed to incubate on ice for 1 h to ensure complete

binding equilibrium. After incubation, 5 μL of the 3× Tdp1 FRET

substrate dilution series was added to the plate to initiate the reaction.

The assay plate was placed in a Tecan Saﬁre plate reader (Tecan US,

Durham, NC) and time-course data observed for 1 h at excitation and

emission wavelengths of 525 nm (bandwidth = 5) and 545 nm

(bandwidth = 5) respectively for all wells. The ﬁnal assay volume was

15 μL, with Tdp1 present at a ﬁnal ﬁxed concentration of 500 pM for

all wells. Experimental data was plotted as reaction rate versus

substrate concentration for all inhibitor concentrations and analyzed

using models for competitive, noncompetitive (pure and mixed) and

uncompetitive inhibition (eqs A−D, respectively) using GraphPad

Prism (Graphpad, La Jolla, CA). To identify the most appropriate

mechanistic model describing the inhibition data, Akaike’s Information

Criterion (Akaike, H., 1973) was used. Information theory and an

extension of the maximum likelihood principle in ref 47 was employed.

Equations. A. Competitive Inhibition.

1

8.57 (m, 6 H), 8.22 (d, J = 8.1 Hz, 2 H), 7.86−7.78 (m, 4 H), 7.62−

7.49 (m, 8 H), 4.60 (t, J = 6.4 Hz, 4 H), 3.08 (br s, 4 H), 2.96 (br s, 4

H), 2.15 (br s, 4 H), 1.90 (m, 2 H). HPLC purity: 97.7% (MeOH−

H₂O, 80:20), 97.6% (MeOH−H₂O, 70:30).

Biological Tests. Topoisomerase I-Mediated DNA Cleavage

Reactions. Human recombinant Top1 was puriﬁed from

baculovirus as previously described.⁴⁵DNA cleavage reactions

were prepared as previously reported with the exception of the

DNA substrate.⁴⁶Brieﬂy, a 117-bp DNA oligonucleotide

(Integrated DNA Technologies) encompassing the previously

identiﬁed Top1 cleavage sites in the 161-bp fragment from

pBluescript SK(−) phagemid DNA was employed. This 117-bp

oligonucleotide contains a single 5′-cytosine overhang, which

was 3′-end-labeled by ﬁll-in reaction with [α-³²P]dGTP in React

2 buﬀer (50 mM Tris-HCl, pH 8.0, 100 mM MgCl₂, 50 mM

NaCl) with 0.5 unit of DNA polymerase I (Klenow fragment,

New England BioLabs). Unincorporated [³²P]dGTP was

removed using mini Quick Spin DNA columns (Roche,

Indianapolis, IN), and the eluate containing the 3′-end-labeled

DNA substrate was collected. Approximately 2 nM radiolabeled

DNA substrate was incubated with recombinant Top1 in 20 μL

of reaction buﬀer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5

mM MgCl₂, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for

20 min in the presence of various concentrations of

compounds. The reactions were terminated by adding SDS

(0.5% ﬁnal concentration) followed by the addition of two

volumes of loading dye (80% formamide, 10 mM sodium

hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1%

bromphenol blue). Aliquots of each reaction mixture were

subjected to 20% denaturing PAGE. Gels were dried and

visualized by using a phosphoimager and ImageQuant software

(Molecular Dynamics). For simplicity, cleavage sites were

numbered as previously described in the 161-bp fragment.⁴⁵

Gel-Based Assay Measuring the Inhibition of Recombinant Tdp1.

A 5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a

3′-phosphotyrosine (N14Y) was generated as described by Dexheimer

et al.³⁴The DNA substrate was then incubated with 5 pM

recombinant Tdp1 in the absence or presence of inhibitor for 15

min at room temperature in a buﬀer containing 50 mM Tris HCl, pH

7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and

0.01% Tween-20. Reactions were terminated by the addition of 1

volume of gel loading buﬀer [99.5% (v/v) formamide, 5 mM EDTA,

0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue].

Samples were subjected to a 16% denaturing PAGE and gels were

exposed after drying to a PhosphorImager screen (GE Healthcare).

Gel images were scanned using a Typhoon 8600 (GE Healthcare), and

densitometric analyses were performed using the ImageQuant software

(GE Healthcare).

V

_max[S]

ν =

[I]

K_i

[S] + K_m1 +

(

)

Where V_maxis maximum reaction velocity, [S] is the substrate

concentration, K_mis the Michaelis constant, and K_iis the inhibition

constant.

B. Pure Noncompetitive Inhibition.

V

_max[S]

v =

[I]

K_i

([S] + K_m) 1 +

(

)

Where K_iis the inhibition constant in the presence or absence of

Gel-Based Assay Measuring the Inhibition of Endogenous

Human Tdp1 in Whole Cell Extract.¹⁷DT40 knockout cells (1

×10⁷) for chicken Tdp1 and complemented with human Tdp1 were

collected, washed, and centrifuged. Cell pellet was then resuspended

with 100 μL of CellLytic M cell lysis reagent (SIGMA-Aldrich C2978).

After 15 min, the lysate was centrifuged at a 12000g for 10 min, and

the supernatant was transferred to a new tube. Protein concentration

was determined using a nanodrop spectrophotometer (Invitrogen),

and the whole cell extract was stored at −80 °C. The 5′-[³²P]-labeled

single-stranded N14Y DNA oligonucleotide containing a 3′-phospho-

tyrosine (see above) was incubated with 1−5 μg/mL of whole cell

extract in the absence or presence of inhibitor for 15 min at room

temperature in the same assay buﬀer used for recombinant Tdp1 (see

section above). Reactions were then treated similarly to the

recombinant enzyme containing samples (see section above).

All compounds were ﬁrst tested in gel-based assays for Tdp1

inhibition using recombinant (rec) human Tdp1, and only the active

compounds were tested for human Tdp1 inhibition in whole cell

extract (WCE). Furamidine (Figure 2) was included in all experiments

as positive control.

substrate.

C. Mixed Noncompetitive Inhibition.

V

_max[S]

v =

[I]

K_ies

[I]

K_ie

[S] 1 +

+ K_m1 +

(

)

(

)

Where K_ieis the inhibition constant for binding to the enzyme in

the absence of substrate, and K_iesthe inhibition constant for binding to

the ES complex.

D. Uncompetitive Inhibition.

V

_max[S]

v =

[I]

[S] 1 +

+ K_m

(

)

K_ies

Where K_iesis the inhibition constant for binding to the ES complex.

Surface Plasmon Resonance Analysis. Binding experiments

were performed on a Biacore T100 instrument (GE, Piscataway, NJ).

Tdp1 was amine coupled to a CM5 sensor chip (GE Healthcare,

Piscataway NJ). Coupling reagents [N-ethyl-N′-(3-

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dx.doi.org/10.1021/jm300335n | J. Med. Chem. 2012, 55, 4457−4478

Journal of Medicinal Chemistry

Article

dimethylaminopropyl)carbodiimide] (EDC), N-hydroxysuccinimide

(NHS), and ethanolamine were purchased from GE Heathcare,

(Piscataway, NJ). Neutravidin was obtained from Pierce. To protect

the amine groups within the active site from modiﬁcation, Tdp1 was

bound with a 14-base oligonucleotide before coupling to the surface.

Speciﬁcally, 1 μM Tdp1 was incubated with 2 μM of a 14-base

oligonucleotide containing a phosphate group at the 3′ end

(GATCTAAAAGACTT) in 10 mM sodium acetate pH 4.5 for 20

min. The CM5 chip surface was activated for 7 min with 0.1 M NHS

and 0.4 M EDC at a ﬂow rate of 20 μL/min and Tdp1−

oligonucleotide mixture was injected until approximately 4000 RU’s

was attached. Activated amine groups were quenched with an injection

of 1 M solution of ethanolamine pH 8.0 for 7 min. Any bound

oligonucleotide was removed by washing the surface with 1 M NaCl. A

reference surface was prepared in the same manner without coupling

of Tdp1. Compound 70 was diluted into running buﬀer [10 mM

Hepes, 150 mM NaCl, 0.01% tween 20 (v/v), 5% DMSO (v/v) pH

7.5] and injected over all ﬂow cells at 30 μL/min at 25 °C. Following

compound injections, the surface was regenerated with a 30 s injection

1 M NaCl, a 30 s injection of 50% DMSO (v/v), and a 30 s running

buﬀer injection. Each cycle of compound injection was followed by

buﬀer cycle for referencing purposes. A DMSO calibration curve was

included to correct for refractive index mismatches between the

running buﬀer and compound dilution series.

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Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy.

Anticancer Agents Med. Chem 2008, 8, 381−389.

AUTHOR INFORMATION

Corresponding Author

*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@

■

purdue.edu.

(13) Yang, S. W.; Burgin, A. B., Jr.; Huizenga, B. N.; Robertson, C.

A.; Yao, K. C.; Nash, H. A. An Eukaryotic Enzyme that can Disjoin

Dead-End Covalent Complexes between DNA and Type I Top-

oisomerases. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11534−11539.

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A. M.; Armstrong, D.; Mao, Y.; Quiocho, F. A.; Roa, B. B.; Nakagawa,

M.; Stockton, D. W.; Lupski, J. R. Mutation of Tdp1, Encoding a

Topoisomerase I-Dependent DNA Damage Repair Enzyme, in

Spinocerebellar Ataxia with Axonal Neuropathy. Nature Genet. 2002,

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Povirk, L. F. Deﬁciency in 3′-Phosphoglycolate Processing in Human

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(Tdp1). Nucleic Acids Res. 2005, 33, 289−297.

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Mielke, C.; Interthal, H.; Pouliot, J. J.; Boege, F.; Marko, D. Tdp1

Overexpression in Human Cells Counteracts DNA Damage Mediated

by Topoisomerases I and II. J. Biol. Chem. 2004, 279, 55618−55625.

(17) Murai, J.; Huang, S. N.; Das, B. B.; Dexheimer, T. S.; Takeda, S.;

Pommier, Y. Tyrosyl-DNA phosphodiesterase 1 (TDP1) Repairs

DNA Damages Induced by Topoisomerases I and II, and Base

Alkylation in Vertebrate Cells. J. Biol. Chem. 2012, 287, 12848−12857.

(18) Marchand, C.; Lea, W. A.; Jadhav, A.; Dexheimer, T. S.; Austin,

C. P.; Inglese, I.; Pommier, Y.; Simeonov, A. Identiﬁcation of

Phosphotyrosine Mimetic Inhibitors of Human Tyrosyl-DNA

Phosphodiesterase I by a Novel AlphaScreen High-Throughput

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Crystal Structure of Human Tyrosyl-DNA Phosphodiesterase, Tdp1.

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was made possible by the National Institutes of

Health (NIH) through support with Research Grant UO1

CA89566, and by a Purdue Research Foundation Grant. This

research was also supported in part by the Intramural Research

Program of the NIH, National Cancer Institute, Center for

Cancer Research. This project has been funded in part with

federal funds from the National Cancer Institute, National

I n s t i t u t e s o f H e a l t h , u n d e r C o n t r a c t n o .

HHSN261200800001E. The content of this publication does

not necessarily reﬂect the views or policies of the Department

of Health and Human Services, nor does mention of trade

names, commercial products, or organizations imply endorse-

ment by the U.S. Government.

ABBREVIATIONS USED

■

APCI-MS, atmospheric-pressure chemical ionization mass

spectrometry; CI/EI-MS, chemical ionization/electron impact

mass spectrometry; CPT, camptothecin; DMAP, 4-dimethyla-

minopyridine; DMSO-d₆, dimethyl-d₆sulfoxide; ESI-MS,

electrospray ionization mass spectrometry; HRMS, high

resolution mass spectrometry; PTSA, p-toluenesulfonic acid;

SCAN1, spinocerebellar ataxia with axonal neuropathy; Tdp1,

tyrosyl-DNA phosphodiesterase I; TFA, triﬂuoroacetic acid;

Top1, topoisomerase type I; TsCl, p-toluenesulfonyl chloride

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Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase i (Tdp1)-topoisomerase i (Top1) inhibitors

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