Synthesis and Biological Evaluation of Nitrated 7-, 8-, 9-, and 10-Hydroxyindenoisoquinolines as Potential Dual Topoisomerase i (Top1)-Tyrosyl-DNA Phosphodiesterase i (TDP1) Inhibitors

Article

pubs.acs.org/jmc

Synthesis and Biological Evaluation of Nitrated 7‑, 8‑, 9‑, and 10-

Hydroxyindenoisoquinolines as Potential Dual Topoisomerase I

(Top1)−Tyrosyl-DNA Phosphodiesterase I (TDP1) Inhibitors

Trung Xuan Nguyen,^†Monica Abdelmalak,^‡Christophe Marchand,^‡Keli Agama,^‡Yves Pommier,^‡

and Mark Cushman*^,†

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

Research, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States

^‡Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer

Institute, Bethesda, Maryland 20892-4255, United States

S

* Supporting Information

ABSTRACT: The structure−activity relationships and hit-to-lead

optimization of dual Top1−TDP1 inhibitors in the indenoisoqui-

noline drug class were investigated. A series of nitrated 7-, 8-, 9-,

and 10-hydroxyindenoisoquinolines were synthesized and eval-

uated. Several compounds displayed potent dual Top1−TDP1

inhibition. The 9-hydroxy series exhibited potencies and

cytotoxicities vs Top1 that surpassed those of camptothecin

(CPT), the natural alkaloid that is being used as a standard in the Top1-mediated DNA cleavage assay. One member of this

series was a more potent Top1 inhibitor at a concentration of 5 nM and produced a more stable ternary drug−DNA−Top1

cleavage complex than CPT.

INTRODUCTION

■

Human topoisomerase type IB (Top1) is an essential enzyme

for various cellular processes as it relaxes DNA supercoils and

resolves DNA topology so that genetic information can be

accessed, and critical events, including DNA replication and

transcription, can take place.^1,2The enzyme cuts a single strand

of DNA to form a transient complex that allows supercoils to

be dissipated, followed by religation of the broken strand.³The

cleavage complex can be trapped by Top1 poisons that

intercalate between the DNA base pairs at the cleavage site and

thereby inhibit the religation reaction. Collision of the

replication fork with the single-strand break then generates a

graph midpoint value obtained from cytotoxicity testing in the

Figure 1. Structure and activity of camptothecin (1). (a) See Table 1

for the Top1 inhibition grading rubric. (b) The MGM is the mean-

double-strand break, causing cell death.⁴Defective repair

explains the therapeutic eﬀect of Top1 inhibitors. DNA damage

produced by Top1 inhibitors can be reversed by a group of

enzymes that includes tyrosyl-DNA phosphodiesterase I

(TDP1), which hydrolyzes the phosphotyrosyl linkage between

degraded or denatured Top1 and the DNA at the cleavage site

to produce DNA 3′-phosphates and free peptides containing

terminal tyrosyl residue.⁵⁻⁸Polynucleotide kinase phosphatase

(PNKP) then hydrolyzes that 3′-phosphate and installs a

phosphate on the 5′-end on the other side of the break, and

DNA ligase III reseals the DNA.⁹Because TDP1 counteracts

the action of Top1 inhibitors, it is possible that TDP1

inhibitors could potentiate the cytotoxic eﬀects of Top1

inhibitors in combination anticancer drug therapy.⁹The

discovery of camptothecin (CPT, 1, Figure 1) as a selective

and potent Top1 poison led to the validation of Top1 as an

anticancer target,¹⁰and in turn attracted a considerable amount

National Cancer Institute’s panel of 60 human cancer cell lines.²⁹

of research eﬀort in the design and synthesis of Top1

inhibitors.^11,12As TDP1 plays an important role in the

maintenance of genomic stability¹³and is a key enzyme in

DNA repair machinery,¹⁴⁻¹⁷TDP1 is also being pursued as a

possible anticancer drug target.^9,15,18−26

TDP1-dependent repair pathways are normally redundant

with other DNA damage response pathways that are often

compromised in cancer cells. This provides a reason to expect

selective TDP1 inhibitor potentiation of Top1 cytotoxicity in

cancer cells vs normal cells. Moreover, checkpoint deﬁciencies

are common in cancer cells,²⁷and in these cases TDP1

Received: January 24, 2015

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Figure 2. Indenoisoquinoline Top1 inhibitors and their hydroxylated analogues.

Figure 3. (top) Proposed hydrogen bonding network of indenoisoquinoline 5 (red) in the DNA−Top1−indenoisoquinoline ternary complex.

(bottom) Hypothetical model of the ternary complex derived from the PDB ID 1SC7 crystal structure.³⁵Hydrogen bonds are shown as yellow lines.

The diagram is programmed for wall-eyed (relaxed) viewing.

becomes the main repair mechanism for removal of Top1-

mediated DNA damage.^5,9,28The goal of the present studies

was to design and synthesize potential anticancer agents that

would possess dual Top1−TDP1 inhibitory activities and

thereby potentially enhance anticancer activity and selectivity.

camptothecins, and have a long half-life in humans.³²Recently,

Cinelli et al. reported the synthesis of some hydroxylated

indenoisoquinolines that were utilized as synthetic standards in

the metabolism studies of 2 and 3 in human liver microsomes

in the presence of NADPH.³³Four of the synthetic compounds

were conﬁrmed as the actual metabolites of the two clinical

drugs based on matches in molecular masses and retention

times with the most abundant peaks of metabolites detected by

LC-MS/MS.³³Four other hydroxyindenoisoquinolines 8−11

(Figure 2) were not identiﬁed as actual metabolites, but they

exhibited attractive Top1 inhibition and antiproliferative

activities, especially the two 9-hydroxy-8-methoxy compounds

DESIGN RATIONALE

■

Two indenoisoquinoline Top1 inhibitors, indotecan (LMP400,

2) and indimitecan (LMP776, 3), are currently in phase I

clinical trials at the National Cancer Institute (Figure 2).^30,31

These compounds induce persistent Top1-linked DNA breaks,

overcome some of the drug resistance issues associated with the

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Figure 4. Hypothetical molecular model depicting a water-mediated hydrogen bond from indenoisoquinoline 5 (red) to the backbone of the

nonscissile DNA strand in ternary complex derived from the PDB ID 1K4T crystal structure.³⁶The diagram is programmed for wall-eyed (relaxed)

viewing.

10 and 11. Through molecular modeling, Cinelli rationalized

the lower potencies of their regioisomers (8-hydroxy-9-

methoxy, compounds 8 and 9) as a consequence of possible

steric and electronic clashes of the 9-methoxy group with the

nonscissile DNA strand in the ternary DNA−Top1-inhibitor

complex.³³However, independent studies reported by Morrell

et al. showed that placing a variety of bulky substituents (e.g.,

methoxycarbonyl, ethoxy, halogen) at the 9-position did not

result in a signiﬁcant attenuation or loss of Top1 inhibition or

antiproliferative activity of indenoisoquinolines when a nitro

group was present at the 3-position.³⁴⁻³⁶This observation led

to the hypothesis that the 3-nitro group exhibits a “steric-

rescuing” eﬀect, which is probably facilitated by its hydrogen

bonding to the Asn722 residue in the open environment of the

cleaved DNA strand, as observed in the hypothetical docking

model of compound 5 in the ternary complex (Figure 3),³⁵thus

moving the inhibitors away from the uncleaved DNA strand so

that bulky substituents could be tolerated in this crowded area.

Because both the 9-hydroxyl group and the 3-nitro group

enhance the anti-Top1 potency, the combination of these two

features in a single indenoisoquinoline molecule might create

exceptionally potent Top1 inhibitors. Although the nitro group

is a toxicophore, one of the objectives in the present case was to

synthesize very cytotoxic indenoisoquinolines that could be

linked to cancer cell-targeting moieties through the phenolic

hydroxyl groups, which would make the nitro group more

tolerable.

Morrell et al. reported a series of indenoisoquinolines bearing

a 3-nitro group on the A-ring and a 7- or 9-methoxy group on

the D-ring (compounds 4−7) as cytotoxic and potent Top1

inhibitors.^35,36The 9-methoxy group was shown to typically

improve cytotoxicity at the expense of Top1 inhibition, whereas

the 3-nitro group enhanced Top1 inhibition to a greater extent

than cytotoxicity.³⁵Although the importance of the 3-nitro

group to the biological activity of this drug class was

rationalized in detail by molecular modeling and electrostatic

potential calculations,³⁵explanations of the role of the 9-

methoxy group were not completely conﬁrmed. In particular,

electrostatic potential modeling revealed charge complemen-

tarity between the electron-rich, 9-position oxygen atom and

the outer edges of the nonscissile DNA base pairs in the Top1

cleavage complex, while the hypothetical binding model of 5

derived from the high resolution crystal structure of topotecan

(PDB ID: 1K4T, in which water molecules were present in the

intercalation site) showed a single bridging hydrogen bond

(2.78 Å) between a water molecule and the 9-methoxy group

(Figure 4).³⁶From these observations, it was concluded that

the optimal place to incorporate a methoxy group on the D-

ring was the 9-position (as in compounds 5−7).³⁶The

generally lower potencies of compounds in the 7-methoxy

series was rationalized as being the result of the 7-methoxy

group having deleterious steric interactions with the lactam side

chain, limiting the number of planar conformations (relative to

the 9-methoxy group) required to confer Top1 inhibition, and

increasing steric repulsion with the DNA base pairs of the

nonscissile DNA strand.³⁶However, Morrell did observe an

exception in that the 7-methoxy-3-nitroindenoisoquinoline 4

(Figure 2) was a potent Top1 inhibitor.

A hydroxyl group could possibly serve as a biologically active

replacement for the methoxy group. It might also provide a

point of attachment for the synthesis of prodrugs so that the

pharmacokinetics could be modulated and optimized. Hydroxy-

lated indenoisoquinolines might also oﬀer an advantage in

terms of solubility for intravenous formulation, which has been

a concern with this drug class. In the present studies, the

syntheses of 18 nitrated 7-, 8-, 9-, and 10-hydroxyindenoiso-

quinolines bearing a 3-nitro substituent were investigated

(Figure 5), all of which are analogues of the highly potent dual

Top1−TDP1 inhibitors 4−7,^34,37so as to optimize the dual

Top1−TDP1 inhibitory potency and gain more insight into the

importance of the location of the hydroxyl group on the overall

biological activities of this drug class.

CHEMISTRY

■

The indenoisoquinoline system can be constructed by several

methods: (1) condensation of indenoisochromenone with a

primary amine,³⁸(2) Suzuki−Miyaura cross-coupling reaction

followed by ring-closing metathesis,³⁹and (3) oxidative

cyclization of cis acid produced by the condensation of a

homophthalic anhydride and a Schiﬀ base.³⁴⁻³⁶The latter

methodology was chosen in this case to prepare all of the

proposed compounds because of the ease in synthesis of

intermediates and manipulation of starting materials to obtain

indenoisoquinolines bearing the desired substituents.

A. Synthesis of 8-Hydroxy-9-methoxy-3-nitroindenoi-

soquinolines and 9-Hydroxy-8-methoxy-3-nitroindenoi-

soquinolines. The synthesis of nitrated 9-hydroxy-8-methox-

yindenoisoquinolines 21, 23, and 25 is depicted in Scheme 1,

and the preparation of 8-hydroxy-9-methoxyindenoisoquino-

lines 32, 34, and 36 is shown in Scheme 2. Commercially

available homophthalic acid 12 was nitrated with fuming HNO₃

to provide the diacid 13, which underwent dehydration in AcCl

to provide anhydride 14. The reactive hydroxyl groups in

vanillin (15) and isovanillin (26) were protected with a benzyl

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slightly lower than those of morpholine compounds (44−52%).

Column chromatographic puriﬁcation of the crude mixtures

provided analytically pure products with much ease as

compared to the separation of the bromides 19 or 30 from

their regioisomers. These results supported the direct use of the

crude bromides 19 and 30 from the previous step.

Many attempts were made to achieve the debenzylation of

the protected compounds 20, 22, 24, 31, 33, and 35. 1,4-

Cyclohexadiene has been shown to be an eﬀective hydrogen

donor in catalytic transfer hydrogenation, a method that was

utilized to remove N-benzyloxycarbonyl, benzyl ester, and

benzyl ether protecting groups in peptide chemistry.⁴²

Disappointingly, attempted implementation of this method

on the benzyl-protected morpholine compound 31 in various

solvents (CHCl₃, EtOH, or glacial AcOH) at room temperature

was not successful in cleaving the benzyl group. In all cases,

starting material was recovered. Attempted debenzylation with

HBr in acetic acid (33 wt %) not only removed the benzyl

group in 31, but a subsequent Fisher esteriﬁcation of the

resulting phenol 32 also occurred to provide the acetate 37 as

the major product while the desired phenol 32 was only

obtained in minor amount (Scheme 4).

Treatment of the benzyl-protected starting materials 20, 22,

24, 31, 33, and 35 with aqueous HBr at 70 °C for 4−5 h,

followed by dilution with acetone and then concentration

(iterated three times), aﬀorded a mixture that was suitable for

vacuum ﬁltration to provide the desired phenols 21, 23, 25, 32,

34, and 36 in high yields (80−100%) and excellent purity. The

moderate yield of imidazole 23 (50%) was due to its higher

aqueous solubility. All of the starting materials produced

reaction mixtures as emulsions from which all of the products

precipitated. The products derived from vanillin (21, 23, and

25) were so ﬁnely powdered that the use of an HPLC ﬁlter

paper was crucial for their recovery, while a regular double-

layered ﬁlter paper worked well for those derived from

isovanillin (32, 34, and 36). The three-time iteration of

dilution with acetone and concentration was imperative for the

success of vacuum ﬁltration. For the syntheses of amines 25

and 36, corresponding azides 24 and 35 were subjected to a

Staudinger reduction with P(OEt)₃, followed by treatment with

aqueous HBr to achieve acidic hydrolysis and debenzylation

simultaneously in one pot.

B. Synthesis of 7-Hydroxy-3-nitroindenoisoquinolines

(Scheme 5). Commercially available salicylaldehyde 38 was O-

benzyl protected to give 39, which reacted with 3-bromopropyl-

amine to aﬀord Schiﬀ base 40. Condensation of 40 with

anhydride 14 in CHCl₃yielded cis acid 41, which upon

treatment with SOCl₂, followed by AlCl₃in 1,2-dichloroethane,

provided indenoisoquinoline bromide 42 in good yield (63%).

The ¹H NMR spectrum of 42 indicated excellent purity without

chromatographic puriﬁcation, and it also indicated that AlCl₃

induced debenzylation to aﬀord the free phenol. Interestingly,

the use of AlCl₃to simultaneously remove the benzyl group was

tried in connection with the chemistry described in Schemes 1

and 2, but in those cases it led to complicated mixtures and no

debenzylation products could be isolated. Displacement of the

bromide in 42 with morpholine, imidazole, or NaN₃, followed

by a Staudinger reduction of the azide intermediate and acidic

hydrolysis with methanolic HCl, provided the desired amines

43, 44, and 45, respectively. The pure products were isolated

without chromatographic puriﬁcation.

Figure 5. Proposed Top1−TDP1 inhibitors.

group. Benzylvanillin (16) and benzylisovanillin (27) reacted

with 3-bromopropylamine hydrobromide to give Schiﬀ bases

17 and 28, which upon condensation with anhydride 14 in

CHCl₃furnished cis acids 18 and 29, respectively, in good

yields with excellent diastereoselectivities. Treatment of 18 or

29 with SOCl₂(neat) provided a mixture of indenoisoquinoline

19 (from 18) or 30 (from 29) and their regioisomers, which

was evident by ¹H NMR spectroscopy and consideration of the

reaction mechanism (Scheme 3). Isomers 19 and 30 appeared

as the major products. The structure assignments were based

on the distinct splitting patterns of the D-ring hydrogens in the

¹H NMR spectra of the two regioisomers. The very similar R_f

values of the regioisomers led to lengthy and diﬃcult

puriﬁcations by ﬂash column chromatography. The yields of

pure 19 and 30 after two chromatographic puriﬁcations were

low (15−25%) and inconsistent. The low yields may be due to

the nitro group activating epimerization to the trans

diastereomers, which exist in pseudodiaxial conformations and

do not oxidize and cyclize in SOCl₂.^40,41

Treatment of cis acids 18 and 29 with SOCl₂alone resulted

in conversion to the acid chlorides, dehydrogenation, and

intramolecular Friedel−Crafts cyclization to provide the

aromatic indenoisoquinoline systems. Puriﬁcation of crude 19

or 30 by column chromatography (SiO₂) caused decom-

position of the products, and therefore the products were used

directly in the next step without further puriﬁcation.

Displacement of the terminal bromide in 19 or 30 with

morpholine or imidazole in 1,4-dioxane, or azide in DMSO,

yielded the benzyl-protected compounds 20, 22, and 24 (from

19), or 31, 33, and 35 (from 30), respectively. Many attempts

were made to optimize the yields of these S_N2 reactions. For

morpholine derivatives 20 and 31, the best yields (44−52%)

were obtained when the starting materials 19 or 30 were stirred

with morpholine for 16 h at room temperature. In contrast, the

syntheses of imidazole derivatives 22 and 33 required constant

heating at 70 °C for 16 h, and the yields (36−40%) were

C. Synthesis of 8- and 10-Hydroxy-3-nitroindenoiso-

quinolines. A similar approach was implemented to prepare 8-

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a

Scheme 1. Synthesis of Nitrated 9-Hydroxy-8-methoxyindenoisoquinolines

a

Reagents and conditions: (a) fuming HNO₃, 0−23 °C; (b) AcCl; reﬂux; (c) BnCl, DMF, K₂CO₃, 23 °C; (d) 3-bromopropylamine hydrobromide,

Et₃N, Na₂SO₄, CHCl₃, 23 °C; (e) CHCl₃, 0−23 °C; (f) SOCl₂, 0−23 °C; (g) morpholine, 1,4-dioxane, 23 °C; (h) aqueous HBr, 70 °C; (i)

imidazole, 1,4-dioxane, 70 °C; (j) NaN₃, DMSO, 23 °C; (k) (i) P(OEt)₃, benzene, reﬂux, (ii) aqueous HBr, 70 °C.

hydroxy-3-nitroindenoisoquinolines (Scheme 6). The commer-

cially available 3-hydroxybenzaldehyde 46 was protected with a

benzyl group to give 47, which was then converted to Schiﬀ

base 48 upon treatment with 3-bromopropylamine hydro-

bromide under basic conditions. Condensation of 48 with

anhydride 14 provided a mixture of the desired cis acid 49 and

its trans diastereomer. Boiling the mixture in CHCl₃, followed

by ﬁltration, helped remove the unwanted trans acid and

provide the pure cis acid 49 as a sole product. However,

treatment of 49 with SOCl₂0 to 23 °C, followed by AlCl₃in

reﬂuxing 1,2-dichloroethane, did not yield the desired bromide

50. Gratifyingly, a proﬁtable transformation was observed when

cis acid 49 was heated with SOCl₂(neat) at reﬂux for 4 h,

during which the solution turned to clear orange. Removal of

SOCl₂, followed by ﬁltering the residue and washing with ether,

Treatment of the mixture of bromides 51 and 52 with

morpholine and imidazole, followed by chromatographic

puriﬁcation, allowed the isolation of each pure morpholinyl

and imidazolyl 8- and 10-benzyloxy compounds 53, 54, 57, and

58 (Scheme 7). In all cases, the 8-substituted products eluted

ﬁrst (higher R_fvalue), and the two regioisomers were

1

distinguished based on the diﬀerences in H NMR multiplicity

of the three aromatic protons in the D-ring: the 8-substitiuted

products have 2 doublets and 1 singlet (or 1 doublet with a

small meta coupling constant), while the 10-substituted

products have 2 doublets and 1 multiplet. All of the benzyl-

protected materials were subjected to a 3 h debenzylation with

aqueous HBr (48 wt %) to provide the desired 8- and 10-

hydroxyindenoisoquinolines 55, 56, 59, and 60 in good yields

and purities. The azides 61 and 62, prepared by reaction of the

bromides 51 and 52 with sodium azide in DMSO at room

temperature, were subjected to a Staudinger reduction,

followed by simultaneous acidic hydrolysis and deprotection

1

provided an orange solid whose H NMR spectrum revealed

that it was an approximately 2:1, inseparable mixture of 8- and

10-(benzyloxy)indenoisoquinolines 51 and 52.

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a

Scheme 2. Synthesis of Nitrated 8-Hydroxy-9-methoxyindenoisoquinolines

a

Reagents and conditions: (a) BnCl, DMF, K₂CO₃, 23 °C; (b) 3-bromopropylamine hydrobromide, Et₃N, Na₂SO₄, CHCl₃, 23 °C; (c) anhydride 14,

CHCl₃, 0−23 °C; (d) SOCl₂, 0 to 23 °C; (e) morpholine, 1,4-dioxane, 23 °C; (f) aqueous HBr, 70 °C; (g) imidazole,1,4-dioxane, 70 °C; (h) NaN₃,

DMSO, 23 °C; (i) (i) P(OEt)₃, benzene, reﬂux, (ii) aqueous HBr, 70 °C.

Scheme 3. Formation of Two Regioisomers in the

Cyclization of Acid Chloride Intermediates

Scheme 4. Debenzylation of 31 with HBr in AcOH (33 wt

%)

with aqueous HBr, to provide pure HBr salts of amines 63 and

64 in excellent purity and moderate yields (Scheme 7).

D. Synthesis of 9-Hydroxy-3-nitroindenoisoquino-

lines (Scheme 8). 4-Benzyloxybenzaldehyde (65) is commer-

cially available or can be prepared easily by the methodology

shown in Scheme 5 for 2-benzyloxybenzaldehyde. Treatment of

65 with 3-bromopropylamine hydrobromide and Et₃N

provided Schiﬀ base 66, which upon condensation with

anhydride 14 produced cis acid 67 in good yield and excellent

F

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Scheme 5. Synthesis of 7-Hydroxy-3-

nitroindenoisoquinolines

Scheme 7. Synthesis of 8- and 10-Hydroxy-3-

nitroindenoisoquinolines

a

Reagents and conditions: (a) BnBr, DMF, K₂CO₃, 23 °C; (b) 3-

bromopropylamine hydrobromide, Et₃N, Na₂SO₄, CHCl₃, 23 °C; (c)

anhydride 14, CHCl₃, 0−23 °C; (d) (i) SOCl₂, 23 °C, (ii) AlCl₃, 1,2-

dichloroethane, 0−23 °C; (e) morpholine, 1,4-dioxane, 70 °C; (f)

imidazole, 1,4-dioxane, 70 °C; (g) (i) NaN₃, DMSO, 23 °C, (ii)

P(OEt)₃, benzene, reﬂux, (iii) HCl, MeOH, 70 °C.

a

Scheme 6

a

Reagents and conditions: (a) morpholine, 1,4-dioxane, 70 °C; (b)

HBr, H₂O, 70 °C; (c) imidazole, 1,4-dioxane, 70 °C; (d) NaN₃,

DMSO, 23 °C; (e) (i) P(OEt)₃, benzene, reﬂux, (ii) HBr, H₂O, 70 °C.

dichloroethane to provide the benzyl-free indenoisoquinoline

phenol 68 after aqueous workup. Treatment of 68 with

morpholine or imidazole in THF provided the corresponding

displacement products, which were then stirred in freshly made

methanolic HBr or HCl to aﬀord the HBr and HCl salts 69 and

70, respectively. This extra step was meant to facilitate later

biological testing because the salts were more soluble in DMSO

than the neutral compounds. The synthesis of amine 71 by the

previous methodology involving reduction of the azide

intermediate with P(OEt)₃in benzene (as shown Scheme 5)

was not successful due to complications in puriﬁcation and

isolation of the compound in solid form. The Staudinger

reaction was therefore reattempted by treating the azide

intermediate, obtained from 68, with PPh₃in THF (instead

of P(OEt)₃in benzene), followed by 4 h acidic hydrolysis with

methanolic HBr. This modiﬁcation provided the desired amine

71 in 32% yield with excellent purity.

a

Reagents and conditions: (a) BnBr, DMF, K₂CO₃, 23 °C; (b) 3-

bromopropylamine hydrobromide, Et₃N, Na₂SO₄, CHCl₃, 23 °C; (c)

anhydride 14, CHCl₃, 0 to 23 °C; (d) (i) SOCl₂, 0−23 °C, (ii) AlCl₃,

1,2-dichloroethane, reﬂux; (e) SOCl₂, reﬂux.

purity. Treatment of acid 67 with SOCl₂for 16 h at room

temperature yielded a yellow syrup upon removal of SOCl₂.

The yellow syrup was then treated with AlCl₃in 1,2-

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Scheme 8. Synthesis of 9-Hydroxy-3-

a

nitroindenoisoquinolines

Figure 6. Top1-mediated DNA cleavage induced by indenoisoquino-

lines 55, 56, 59, and 60. From left to right: lane 1, DNA alone; lane 2,

DNA + Top1; lane 3, CPT (1), 1 μM; lane 4, MJ-III-65, 1 μM; lanes

5−20, 55, 56, 59, and 60 at 0.1, 1.0, 10, and 100 μM, respectively.

Numbers and arrows on the left indicate arbitrary cleavage site

positions. MJ-III-65 is the positive indenoisoquinoline control.⁴⁵

a

Reagents and conditions: (a) 3-bromopropylamine hydrobromide,

Et₃N, Na₂SO₄, CHCl₃, 23 °C; (b) anhydride 14, CHCl₃, 0−23 °C; (c)

(i) SOCl₂, 23 °C, (ii) AlCl₃, 1,2-dichloroethane, 0−23 °C; (d) (i)

morpholine, THF, 70 °C, (ii) HBr, MeOH, 23 °C; (e) (i) imidazole,

THF, 70 °C, (ii) HCl, MeOH, 23 °C; (f) (i) NaN₃, DMSO, 23 °C,

(ii) PPh₃, THF, 70 °C, (iii) HBr, MeOH, 70 °C.

linkage between tyrosine and the 3′-end of a DNA substrate

(N14Y), thus preventing the generation of an oligonucleotide

with a free 3′-phosphate (N14P).²¹The anti-TDP1 potency, as

indicated by the disappearance of the gel band for N14P, was

recorded as IC₅₀based on the following semiquantitative scale:

0, IC₅₀> 111 μM; +, IC₅₀between 37 and 111 μM; ++, IC₅₀

between 12 and 37 μM; +++, IC₅₀between 1 and 12 μM; +++

+, IC₅₀< 1 μM. Representative gels demonstrating dose-

dependent Top1 and TDP1 inhibitory activities of some target

indenoisoquinolines are depicted in Figures 6 and 7,

respectively.

The antiproliferative activities of all of the target compounds

were evaluated in the National Cancer Institute’s Devel-

opmental Therapeutics Program screen (NCI-DTP) against 60

common human cancer cell lines (the “NCI-60”). All

compounds were initially tested in a one-dose prescreening

assay at moderately high concentration (10 μM), and those that

passed a cutoﬀ threshold were tested at ﬁve concentrations

ranging from 100 μM to 10 nM. The 50% growth-inhibitory

concentration (GI₅₀) in each cell line was determined by

interpolation of data points received from the ﬁve-dose screen.

The cytotoxicity of a compound was reported as a mean-graph

midpoint (MGM) of the average GI₅₀value across the entire

panel of 60 cell lines, where GI₅₀values that fell outside the

tested range (100 μM to 10 nM) were assigned as the maximal

(100 μM) and minimal (10 nM) drug concentrations,

respectively, used in the screening. Top1 and TDP1 inhibitory

potencies, and the antiproliferative activities of all target

compounds are collectively shown in Table 1. For comparison,

activities of compounds 1 (Figure 1), 4−5 and 8−11 (Figure 2)

are included.

BIOLOGICAL RESULTS AND DISCUSSION

■

All of the proposed compounds (collectively shown in Figure

5) were subjected to determinations of Top1 and TDP1

inhibitory potencies and antiproliferative activities. Top1

inhibition was recorded as the ability of a drug to poison

Top1 and induce enzyme-linked DNA breaks in the Top1-

mediated DNA cleavage assay.⁴³The anti-Top1 potency was

graded on the basis of visual inspection of the number and

intensities of the bands corresponding to DNA fragments and

was recorded on a semiquantitative scale relative to 1 μM

camptothecin (CPT, 1, Figure 1): 0, no inhibitory activity; +,

between 20% and 50% activity; ++, between 50% and 75%

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

equipotent; +++++, more potent. Ambiguous scores between

two values are designated with parentheses (e.g., ++(+) would

be between ++ and +++). The loss of potency at 100 μM of

drug concentration, as evidenced by the pale intensity or

disappearance of the DNA cleavage bands (Figure 6), results

from the drug intercalating into free DNA, rendering it a poorer

substrate for the Top1-catalyzed cleavage reaction. Some of the

indenoisoquinolines act as Top1 poisons (i.e., inhibiting the

DNA religation reaction) at low drug concentrations and as

Top1 suppressors (i.e., inhibiting the DNA cleavage reaction)

at high drug concentrations. The steric bulk of the substituent

on the lactam side chain seems to play a role in determining

whether a given indenoisoquinoline poisons or suppresses

Top1, with larger substituents showing a preference for DNA

intercalation into the binary Top1−DNA complex instead of

free DNA.⁴⁴In the present case, the 3-nitro group seems to

facilitate intercalation into free DNA so that 55, 56, 59, and 60,

with large substituents on the side chain, all act as Top1

suppressors at high drug concentrations (Figure 6).

As observed in Table 1, all of the compounds exhibited at

least some Top1 inhibitory activity. The nitrated 9-hydroxy-8-

methoxy series 21, 23, and 25 displayed only weak to moderate

anti-Top1 potencies (+ or ++) even though their 2,3-

dimethoxy analogues 10 and 11 were excellent Top1 inhibitors

(+++++). Furthermore, the nitrated 8-hydroxy-9-methoxy

TDP1 inhibition was measured as the ability of a drug to

inhibit the enzyme-catalyzed hydrolysis of the phosphodiester

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Figure 7. Inhibition of recombinant TDP1 hydrolysis of N14Y to N14P by indenoisoquinolines 21, 23, 25, 36, and 72. From left to right: lane 1,

DNA alone; lane 2, DNA + TDP1; lanes 3−22, compounds 21, 23, 25, 36, and 72 (each at 1.4, 4.1, 12.3, 37, and 111 μM). Bis(indenoisoquinoline)

72 (TDP1: +++) was used as the standard.

series 32, 34, and 36 showed a similar extent of Top1 inhibition

as observed in their 2,3-dimethoxy analogues 8 and 9 (++ or ++

+). In all cases, the nitrated compounds displayed a signiﬁcant

improvement in terms of cytotoxicity when compared to their

corresponding dimethoxy analogues, with the 9-hydroxy-8-

methoxy series 21, 23, and 25 possessing low nanomolar

antiproliferative potencies (MGM values of 16−21 nM).

Although the apparent enhancement of cytotoxicity brought

about by the presence of the 3-nitro group was in agreement

with our initial hypothesis, the stark contrasts in Top1

inhibition among these very similar analogues was rather

surprising for several reasons.

First, the electron-withdrawing nature of the 3-nitro group is

proposed to confer biological activity to the indenoisoquino-

lines because it facilitates the π−π stacking by enhancing

charge-transfer interactions between the aromatic indenoiso-

quinoline ring system and the ﬂanking DNA base pairs, and it

also provides a favorable hydrogen bond to the Asn722 residue

of Top1 in the spacious area provided by the cleaved DNA

strand, thus moving the inhibitor away from the crowded

environment of the uncleaved DNA strand. As a result,

favorable A-ring electronics may oﬀset the deleterious steric

eﬀect of bulky substituents at the 9-position that clash with the

unbroken DNA strand.³⁴⁻³⁶As noted above, Morrell et al.

reported that 3-nitroindenoisoquinolines bearing bulky groups

like methoxycarbonyl, ethoxy, or halogen (bromine, iodine) at

the 9-position exhibited excellent Top1 inhibitory potencies (+

++ to ++++).³⁴

the hypothesis that for the 2,3-dimethoxy series, the 9-methoxy

group protrudes near the phosphodiester backbone of the

uncleaved DNA strand, thus representing a less favorable ﬁt as

compared to the 8-methoxy group.³³However, for the 3-nitro

series, the results were completely opposite because the 9-

hydroxy-8-methoxy compounds 21, 23, and 25 were not better

but were even somewhat less active than their 8-hydroxy-9-

methoxy regioisomers 32, 34, and 36.

Third, the anti-Top1 potency and antiproliferative activity

generally correlate well, with better Top1 inhibitors also

displaying higher cytotoxicity. However, this was not the case

for the nitrated 9-hydroxy-8-methoxy series 21, 23, and 25

because these relatively weak Top1 inhibitors (+ or ++)

exhibited cytotoxicities (16−21 nM) 4-fold higher than their

strong Top1 inhibitor (+++++) dimethoxy analogues 10 and

11 (55−87 nM). The reason for the lack of a strong correlation

between Top1 inhibition and antiproliferative activity observed

for the nitrated series remains unknown, but it is probably a

complicated consequence of several factors, including pharma-

cokinetics (cellular permeability, solubility, and metabolism)

and possible oﬀ-target eﬀects.

All of the nitrated hydroxyindenoisoquinolines that do not

possess a D-ring methoxy group displayed similar or better

Top-1 inhibitory potency than their analogues with a methoxy

group. In contrast to the 9-hydroxy-8-methoxy series, a strong

correlation between Top1 inhibition and antiproliferative

activity was observed for these compounds. Indeed, the order

of Top1 inhibition and cytotoxicity went from the 9-hydroxyl

series 68−71 as the most active and cytotoxic (Top1 inhibition

++++ or more, MGM 14−117 nM) to the 8-hydroxyl series 55,

59, and 63 (+++ to ++++, 56−407 nM), and ﬁnally to the

equipotent 7-hydroxyl series 43−45 and the 10-hydroxyl series

56, 60, and 64 (++ to +++ for both series, 234 to 3550 nM for

the 10-hydroxyl) as the least active and least cytotoxic. The 7-

hydroxyl series 43−45 had very low cytotoxicities in the initial

one-dose testing at 10 μM that did not pass the predetermined

cutoﬀ value to warrant the subsequent ﬁve-dose testing

required for the GI₅₀determination in the NCI-60.

Second, even though this eﬀect did not enhance Top1

inhibition, it was expected to at least retain, instead of

drastically attenuate the activity, as shown by the drastic drop

in the nitrated 9-hydroxy-8-methoxy series 21, 23, and 25 (+ or

++) compared to their dimethoxy analogues 10 and 11 (++++

+). Such a decrease in Top1 inhibition is even more striking if

one takes into account that for the 2,3-dimethoxy series, the 9-

hydroxy-8-methoxy compounds 10 and 11 are much more

potent than their 8-hydroxy-9-methoxy regioisomers 8 and 9.

Rationalizing this observation through docking studies led to

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Interestingly, the 9-hydroxyl series 68−71 displayed potencies

and cytotoxicities comparable to or even greater than

camptothecin (1, ++++, 40 nM) and the 3-nitro-9-methoxy

standard 5 (+++++, 27 nM). Therefore, the 9-hydroxyl group

fulﬁlled our initial mission of replacing the 9-methoxy group in

compounds 5−7 with a reactive functional group that could be

derivatized to produce prodrugs or linked to targeting moieties

or antibodies for improvement of selectivity for cancer cells vs

normal cells. The better overall activities of the 8- and 9-

hydroxyl series as compared to the 7- and 10-hydroxyl series

suggest that the 8- and 9-positions are optimal for forming

favorable interactions between the D-ring hydroxyl group and

their surroundings in the ternary cleavage complex. In

agreement with the docking results reported by Morrell (Figure

4),³⁶the 8- and 9-hydroxyl groups are within a reasonable

distance to form a water-mediated hydrogen bond from the

oxygen atom to the phosphodiester DNA backbone, and the

electron-rich oxygen atom also provides an electrostatic

potential that is complementary to the outer edges of the

nonscissile DNA strand. Furthermore, because the 9-hydroxyl

group can be considered to be directly across from and in

resonance with the 3-nitro group, the slightly higher activities

observed for the 9-hydroxyl series might result from resonance

contributors involving donation of electron density from the

phenol to the nitro group. Within each series, the position of

the hydroxyl group in the D-ring is a more important

determinant of Top1 inhibitory activity than the identity of

the amine group in the side chain.

Interestingly, the indenoisoquinoline bromide 68, a member

of the 9-hydroxyl series, was a more potent Top1 poison (++++

+) than CPT (1, ++++). Indeed, the following two points are

apparent from the inspection of the gels in Figure 8: (1) 68

trapped Top1 cleavage complexes at a concentration as low as 5

nM, whereas CPT had lesser activity at that concentration, and

(2) in contrast to the other indenoisoquinolines reported in

this study, the bromide 68 did not intercalate into free DNA to

suppress the cleavage reaction at a concentration as high as 100

μM. It might be reasonable to pose the question of whether the

superior potency of 68 came about as a result of its DNA

alkylation via displacement of the bromide group in the

inhibitor. To answer this question, the kinetics of ternary

cleavage complex reversal for 68 were studied, and the results

showed that it formed drug−Top1−DNA cleavage complexes

that did not reverse as quickly as CPT at their common

cleavage sites (70, 92, 97, and 119) (Figure 9). The ternary

cleavage complex complexes induced by 68 were quite

persistent but did reverse over time, while those induced by

CPT reversed much faster. Obviously 68 forms more stable

cleavage complexes than CPT. The decrease in intensity of the

cleavage bands induced by 68 over time also suggest that the

drug did not alkylate or form an irreversible ternary cleavage

complex.

Figure 8. Comparison of Top1 inhibitory potencies between

camptothecin (CPT, 1) and indenoisoquinoline bromide 68 in a

dose-dependent Top1-mediated DNA cleavage. From left to right:

lane 1, DNA alone; lane 2, DNA + Top1; lanes 3−16, CPT (1) and 68

at 0.005, 0.01, 0.05, 0.1, 1.0, 10, and 100 μM, respectively.

analogues, with one single exception of the imidazole 34

possessing an anti-TDP1 potency (++) comparable to its

amino analogue 36. This trend also consistently applied across

the whole series of hydroxyindenoisoquinolines, with or

without a methoxy. This observation is in agreement with the

hypothesis that the positively charged ammonium cation is of

importance in orienting the ammonium group toward the

hydrophilic region in the TDP1 active site so that the aromatic

indenoisoquinoline ring system could reside in close proximity

and form favorable contacts with some key residues in the

catalytic region (H263, K265, N283, H493, K495, and N516),

thus conferring TDP1 inhibitory activities to the indenoisoqui-

nolines.³Moreover, it is also apparent from Table 1 that most

indenoisoquinolines with a morpholine or imidazole side chain

are inactive against TDP1 (0 activity), with the imidazole 34 as

the single exception (++ activity). Similar observations

regarding indenoisoquinoline TDP1 inhibitors were reported

by Conda-Sherian³⁷and Lv,⁴⁶in which compounds bearing a

bulky group on the lactam side chain (e.g., morpholine,

imidazole, Boc-protected amino, N-methylpiperidine) showed

no TDP1 inhibition.

Table 1 reveals that most nitrated hydroxyindenoisoquino-

lines were either inactive or weakly active against TDP1 with 0

or + activity. However, there were also compounds that

exhibited good anti-TDP1 potencies (++ to +++), with the 8-

hydroxyindenoisoquinoline amine 63 displaying the highest

activity (+++). With an excellent anti-Top1 potency of ++++,

amine 63 is the most potent dual Top1−TDP1 inhibitor

reported in the present study. A thorough inspection of TDP1

inhibition within each series in Table 1 revealed an apparent

trend: compounds with a primary amine side chain consistently

exhibited higher potencies than their morpholine and imidazole

When compared to the lead compounds 4−5, the target

compounds were, in general, somewhat weaker TDP1

inhibitors. The primary amines 25, 36, 45, 63, 64, and 71

were all TDP1 inhibitors. The most advantageous position for a

single hydroxyl group in the D-ring for the highest TDP1

inhibitory activity is at C-8, followed by C-7 and C-10, which

confer equal activity. Placing the hydroxyl group at C-9 is the

least eﬀective.

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Figure 9. Kinetics of reversal assay of Top1-mediated DNA cleavage complexes induced by CPT vs bromide 68. The forward reaction was

terminated with 0.35 M NaCl.

hydroxyl group in the D-ring is at C-8. The 8-hydroxyindenoi-

soquinoline amine 63 is the most potent dual Top1−TDP1

inhibitor reported in the present studies (++++ for Top1 and +

++ for TDP1). Its activities are also comparable to the

bis(indenoisoquinoline) 72 (Figure 7), which is the most

potent dual Top1−TDP1 inhibitor. These results detail

successful potency optimization of phenolic indenoisoquino-

lines as Top1−TDP1 inhibitors.

CONCLUSION

■

A series of 18 nitrated 7-, 8-, 9-, and hydroxyindenoisoquino-

lines were designed as dual Top1−TDP1 inhibitors based on

the ideas that (1) they are the demethylated analogues of the

lead compounds 4 and 5, both of which are potent dual Top1−

TDP1 inhibitors, (2) the hydroxyl group might serve as a

replacement of the methoxy group in the lead compounds 4

and 5 and provide a point of attachment for prodrug modules,

targeting moieties, or antibodies, and (3) both the 3-nitro

group in the A-ring and the hydroxyl group in the D-ring of

indenoisoquinolines were previously shown separately to

enhance both Top1 inhibitory and antiproliferative activities

of Top1 poisons. All of the target compounds were synthesized

using the condensation of homophthalic anhydride and Schiﬀ

bases bearing appropriate substituents, and were evaluated for

dual Top1−TDP1 inhibition and antiproliferative activities.

The 9-hydroxyl series comprised of compounds 68−71 was the

most active Top1 inhibitors in the series. Among them,

bromide 68 exhibited many unique features: (1) its anti-Top1

potency and cytotoxicity surpassed those of CPT, (2) it acted

as a Top1 poison and was not a Top1 suppressor at a

concentration as high as 100 μM, whereas many other

indenoisoquinolines start to suppress DNA cleavage at this

concentration, (3) it induced a drug−DNA−Top1 ternary

cleavage complex that reversed slower than that of

camptothecin, and (4) there was no evidence of DNA

alkylation. All the indenoisoquinolines bearing a terminal

amine on their lactam side chains had TDP1 inhibitory

activities, while their morpholine and imidazole analogues were

generally inactive. The relative TDP1 inhibitory potencies of

amines 45 (++), 63 (+++), 64 (++), and 71 (+) indicate that

the most advantageous location for installation of a single

EXPERIMENTAL SECTION

■

General. Solvents and reagents were purchased from commercial

vendors and were used without further puriﬁcation. Melting points

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

were uncorrected. Infrared spectra were obtained as ﬁlms on KBr

pellets with CHCl₃as the solvent, using a PerkinElmer 1600 series or

1

Spectrum One FTIR spectrometer, and were baseline-corrected. H

NMR spectra were recorded at 300 or 500 MHz, using Bruker

ARX300 or Bruker Avance 500 spectrometers with a QNP probe or

TXI 5 mm/BBO probe, respectively. Mass spectral analyses were

performed at the Purdue University Campus-Wide Mass Spectrometry

Center. ESI-MS studies were performed using a FinniganMAT XL95

(FinniganMAT Corp., Bremen, Germany) mass spectrometer. The

instrument was calibrated to a resolution of 10000 with a 10% valley

between peaks using the appropriate polypropylene glycol standards.

EI/CI-MS studies were performed using a Hewlett-Packard Engine or

GCQ FinniganMAT mass spectrometer. APCI-MS studies were

performed using an Agilent 6320 ion trap mass spectrometer.

Combustion microanalyses were performed at Midwest Microlab,

LLC (Indianapolis, IN). All reported values were within 0.4% of

calculated values. Analytical thin layer chromatography was carried out

on Baker-ﬂex silica gel IB2-F plastic-backed TLC plates. Compounds

were visualized with both short and long wavelength UV light and

ninhydrin staining unless otherwise speciﬁed. Silica gel ﬂash column

chromatography was performed using 40−63 μM ﬂash silica gel.

Analytical HPLC studies were performed using a Waters 1525 binary

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HPLC pump with a Waters 2487 dual wavelength absorbance detector

and an injection volume of 10 μL. A Sunrise C₁₈5 μM 100 Å reverse-

phase column with dimensions of 15 cm × 4.6 mm (ES Industries)

was used for all analytical HPLC experiments. 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 unless

otherwise speciﬁed. All yields refer to isolated compounds.

room temperature for 16 h. The red solution was evaporated to

dryness. The resulting residue was diluted with CHCl₃(50 mL) and

treated slowly with saturated aqueous NaHCO₃(100 mL). The

mixture was stirred at room temperature for 10 min, and the two layers

were separated. The aqueous layer was extracted with CHCl₃(100 mL

× 2). The combined extract was washed with H₂O (100 mL × 3) and

brine (100 mL). The organic layer was dried over anhydrous Na₂SO₄,

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

column chromatography (SiO₂), eluting with CHCl₃to provide the

product 19 as a reddish-brown solid (722 mg, 50%); mp 218−220 °C

4-Nitrohomophthalic Acid.⁴⁷Homophthalic acid (12, 10.0 g, 55.5

mmol) was slowly added to fuming HNO₃(35 mL) at 0 °C. After the

addition was complete, the clear-yellow solution was warmed to room

temperature, and stirring was continued for 5 h, during which a white

precipitate was formed. Ice was slowly added to the cloudy mixture

until the volume was doubled. The mixture was sonicated until the ice

melted, and then it was ﬁltered. The residue was washed with cold

H₂O to aﬀord the product 13 as an amorphous white solid (8.83 g,

71%): mp 219−220 °C (lit.⁴⁷217 °C). ¹H NMR (300 MHz, DMSO-

d₆) δ 8.61 (d, J = 2.6 Hz, 1 H), 8.37 (dd, J = 2.6 and 5.6 Hz, 1 H), 7.67

(d, J = 8.4 Hz, 1 H), 4.10 (s, 2 H).

1

(dec). IR (ﬁlm) 1677, 1611, 1504, 1427, 1336, 1300, 746 cm⁻¹. H

NMR (300 MHz, CDCl₃) δ 9.15 (d, J = 2.4 Hz, 1 H), 8.78 (d, J = 9.0

Hz, 1 H), 8.47 (dd, J = 2.5 and 6.7 Hz, 1 H), 7.44−7.36 (m, 6 H), 7.31

(d, J = 1.8 Hz, 1 H), 5.26 (s, 2 H), 4.71 (t, J = 7.0 Hz, 2 H), 4.06 (s, 3

H), 3.74 (t, J = 5.7 Hz, 2 H), 2.51 (m, 2 H). ESI-MS m/z (rel

intensity) 549/551 ([MH]⁺, 42/53). HRMS (+ESI) calcd for

C₂₇H₂₁BrN₂O₆MH⁺, 549.0661; found, 549.0672.

9-(Benzyloxy)-8-methoxy-6-(3-morpholinopropyl)-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11[6H]-dione (20). Bromide 19 (320 mg,

0.58 mmol) and morpholine (304 mg, 3.49 mmol) were diluted in 1,4-

dioxane (30 mL). The mixture was stirred at room temperature for 16

h and then evaporated to dryness. The resulting residue was diluted

with H₂O (100 mL) and extracted with CHCl₃(50 mL × 3). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with a gradient of MeOH in CHCl₃

(2% to 4%) to provide the product 20 as a brown solid (140 mg,

44%); mp 233−234 °C (dec). IR (ﬁlm) 1673, 1612, 1557, 1507, 1428,

1333, 1300, 667 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 9.15 (d, J = 2.5

Hz, 1 H), 8.75 (d, J = 9.0 Hz, 1 H), 8.45 (dd, J = 2.4 and 6.5 Hz, 1 H),

7.47−7.35 (m, 5 H), 7.31 (s, 1 H), 7.21 (s, 1 H), 5.25 (s, 2 H), 4.63 (t,

J = 7.3 Hz, 2 H), 4.01 (s, 3 H), 3.66 (m, 4 H), 2.60 (t, J = 6.7 Hz, 2

H), 2.46 (m, 4 H), 2.14 (m, 2 H). ESI-MS m/z (rel intensity) 556

([MH]⁺, 100). HRMS (+ESI) calcd for C₃₁H₂₉N₃O₇MH⁺, 556.2084;

found, 556.2076.

9-Hydroxy-8-methoxy-6-(3-morpholinopropyl)-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione Hydrobromide (21).

Compound 20 (50 mg, 0.090 mmol) was diluted in aqueous HBr

(48 wt %, 35 mL), and the mixture was heated at 70 °C for 5 h, during

which it turned to a black emulsion. The cooled mixture was

concentrated to remove HBr. The concentrate was then diluted with

acetone (10 mL) and concentrated again. This procedure was done

three times. The ﬁnal mixture was ﬁltered through an HPLC ﬁlter

paper, and the residue was washed with acetone and CHCl₃to provide

the desired product 21 as a black solid (47.5 mg, 97%); mp >400 °C.

IR (ﬁlm) 3206, 1697, 1641, 1614, 1558, 1506, 1427, 1335, 792 cm⁻¹.

¹H NMR (300 MHz, CDCl₃) δ 10.45 (s, 1 H), 9.49 (s, 1 H), 8.87 (s, 1

4-Nitrohomophthalic Anhydride (14).⁴⁸Diacid 13 (8.83 g, 39.2

mmol) was diluted in acetyl chloride (30 mL), and the mixture was

stirred and heated at reﬂux for 4 h. The yellow solution was then

evaporated to dryness. The resulting residue was ﬁltered and washed

with 50% CHCl₃in hexane to provide the product 14 as pale-yellow

solid (5.75 g, 71%); mp 147−148 °C (lit.⁴⁸154−155 °C). H NMR

1

(300 MHz, DMSO-d₆) δ 8.66 (s, 1 H), 8.55 (d, J = 8.1 Hz, 1 H), 7.73

(d, J = 7.9 Hz, 1 H), 4.41 (s, 2 H).

Benzylvanillin (16).⁴⁹Vanillin (15, 5.00 g, 32.9 mmol), benzyl

bromide (5.90 g, 34.5 mmol), and K₂CO₃(9.08 g, 65.7 mmol) were

diluted in DMF (50 mL). The yellow mixture was stirred at room

temperature for 2 h, poured into a solution of Et₂O−H₂O (200 mL,

1:1), and stirred for 10 min. The ethereal layer was separated. The

aqueous layer was extracted with Et₂O (50 mL × 2). The combined

extract was washed with H₂O (100 mL × 3) and brine (100 mL). The

organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated to dryness. The crude residue was washed with hexane

to provide the pure product 16 as a white solid (7.91 g, 99%); mp 49−

51 °C (lit.⁴⁹61 °C). H NMR (300 MHz, CDCl₃) δ 9.84 (s, 1 H),

1

7.44−7.36 (m, 7 H), 7.00 (d, J = 8.2 Hz, 1 H), 5.25 (s, 2 H), 3.95 (s, 3

H).

N-[4′-(Benzyloxy)-3′-methoxybenzylidene]-3-bromopropyl-1-

amine (17).³³Benzylvanillin 16 (3.00 g, 12.4 mmol), 3-bromopropyl-

amine hydrobromide (3.12 g, 14.2 mmol), Et₃N (1.39 g, 13.6 mmol),

and Na₂SO₄(3.52 g, 24.8 mmol) were diluted in CHCl₃(100 mL).

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

washed with H₂O (100 mL × 3) and brine (100 mL). The organic

layer was dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to

provide the product 17 as a yellow syrup (4.49 g, 100% + residual

solvent). IR (ﬁlm) 2937, 2841, 1646, 1602, 1587, 1512, 1456, 1415,

1

1270, 1233, 743 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.22 (s, 1 H),

H), 8.65 (d, J = 8.8 Hz, 1 H), 8.55 (d, J = 8.2 Hz, 1 H), 7.23 (s, 1 H),

7.08 (s, 1 H), 4.63 (m, 2 H), 4.00−3.95 (m, 7 H), 3.61 (m, 4 H), 3.10

(m, 2 H), 2.28 (m, 2 H). ESI-MS m/z (rel intensity) 447 (MH⁺, 89).

HRMS (+ESI) calcd C₂₄H₂₃N₃O₇for MH⁺, 466.1614; found,

466.1618; HPLC purity: 100% (MeOH, 100%), 97.8% (MeOH−

H₂O, 90:10). Anal. Calcd for C₂₄H₂₄BrN₃O₇·0.1H₂O: C, 52.59; H,

4.45; N, 7.67. Found: C, 52.28; H, 4.24; N, 7.30.

7.45−7.30 (m, 6 H), 7.10 (dd, J = 1.7 and 6.4 Hz, 1 H), 6.90 (d, J =

8.2 Hz, 1 H), 5.20 (s, 2 H), 3.95 (s, 3 H), 3.74 (t, J = 6.1 Hz, 2 H),

3.51 (t, J = 6.5 Hz, 2 H), 2.27 (m, 2 H).

cis-3-[4-(Benzyloxy)-3-methoxyphenyl]-N-(3-bromopropyl)-4-car-

boxy-3,4-dihydro-7-nitro-1(2H)-isoquinolone (18). Schiﬀ base 17

(7.48 g, 20.7 mmol) was diluted in CHCl₃(50 mL) and cooled to 0

°C, and anhydride 14 (4.28 g, 20.7 mmol) was added. The red mixture

was stirred at 0 °C for 2 h and then at room temperature for 3 h. The

mixture was ﬁltered, and the residue was washed with 50% CHCl₃in

hexane to aﬀord the product 18 as a white solid (7.55 g, 64%); mp

145−146 °C. IR (ﬁlm) 3079, 1748, 1621, 1520, 1493, 1418, 1349,

1177, 755 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 9.06 (d, J = 2.4 Hz, 1

H), 8.36 (dd, J = 2.5 and 6.0 Hz, 1 H), 7.87 (d, J = 8.8 Hz), 7.35−7.28

(m, 5 H), 6.71 (d, J = 8.9 Hz, 1 H), 6.50 (m, 2 H), 5.13 (d, J = 6.4 Hz,

1 H), 5.04 (s, 2 H), 4.80 (d, J = 6.4 Hz, 1 H), 4.04 (m, 1 H), 3.63 (s, 3

H), 3.48 (m, 2 H), 3.28 (m, 1 H), 2.31 (m, 1 H), 2.18 (m, 1 H). ESI-

MS m/z (rel intensity) 569/571 ([MH]⁺, 27/28). HRMS (+ESI)

calcd for C₂₇H₂₅BrN₂O₇MH⁺, 569.0923; found. 569.0932.

6-(3-(1H-Imidazol-1-yl)propyl)-9-(benzyloxy)-8-methoxy-3-nitro-

5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (22). Bromide 19 (100

mg, 0.182 mmol) and imidazole (124 mg, 1.82 mmol) were diluted in

1,4-dioxane (30 mL). The mixture was heated at 70 °C for 16 h and

then evaporated to dryness. The resulting residue was diluted with

H₂O (100 mL) and extracted with CHCl₃(50 mL × 3). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with 4% MeOH in CHCl₃to provide

the product 22 as a brown solid (36.2 mg, 37%); mp 235−236 °C

1

(dec). IR (ﬁlm) 1662, 1612, 1555, 1424, 1291, 746 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 9.17 (d, J = 2.3 Hz, 1 H), 8.77 (d, J = 9.0 Hz, 1

H), 8.48 (dd, J = 2.4 and 6.6 Hz, 1 H), 7.61 (s, 1 H), 7.46−7.30 (m, 5

H), 7.12 (s, 1 H), 7.05 (s, 1 H), 6.85 (s, 1 H), 5.24 (s, 2 H), 4.61 (t, J

9-(Benzyloxy)-6-(3-bromopropyl)-8-methoxy-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11(6H)-dione (19). Cis acid 18 (1.50 g, 2.63

mmol) was diluted in SOCl₂(50 mL) and the mixture was stirred at

M

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Article

= 6.8 Hz, 2 H), 4.27 (t, J = 6.5 Hz, 2 H), 3.86 (s, 3 H), 2.42 (m, 2 H).

ESI-MS m/z (rel intensity) 537 (MH⁺, 100). HRMS (+ESI) calcd for

C₃₀H₂₄N₄O₆MH⁺, 537.1774; found, 537.1784.

concentrated to dryness. The crude residue was washed with hexane to

provide the pure product 27 as a white solid (7.34 g, 92%); mp 48−49

°C (lit.⁵⁰61−62 °C). H NMR (300 MHz, CDCl₃) δ 9.82 (s, 1 H),

1

6-(3-(1H-Imidazol-1-yl)propyl)-9-hydroxy-8-methoxy-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione Hydrobromide (23).

Compound 22 (50 mg, 0.093 mmol) was diluted in aqueous HBr

(48 wt %, 35 mL) and the mixture was heated at 70 °C for 5 h, during

which it turned to a brown emulsion. The cooled mixture was

concentrated to remove HBr. The concentrate was then diluted with

acetone (10 mL) and concentrated again. This procedure was done

three times. The ﬁnal mixture was ﬁltered through an HPLC ﬁlter

paper, and the residue was washed with acetone and CHCl₃to provide

the desired product 23 as a pale-brown solid (24.6 mg, 50%); mp >400

°C. IR (ﬁlm) 3398, 1680, 1609, 1557, 1492, 1429, 1385, 1338, 859

7.48−7.46 (m, 4 H), 7.39−7.36 (m, 3 H), 7.01 (d, J = 8.5 Hz, 1 H),

5.20 (s, 2 H), 3.97 (s, 3 H).

N-[3′-(Benzyloxy)-4′-methoxybenzylidene]-3-bromo-1-propyl-

amine (28).³³Benzylisovanillin 27 (3.00 g, 12.4 mmol), 3-

bromopropylamine hydrobromide (3.12 g, 14.2 mmol), Et₃N (1.39

g, 13.6 mmol), and Na₂SO₄(3.52 g, 24.8 mmol) were diluted in

CHCl₃(100 mL). The mixture was stirred at room temperature for 16

h and then washed with H₂O (100 mL × 3) and brine (100 mL). The

organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated to provide the product 28 as a yellow syrup (4.49 g,

100% + residual solvent). IR (ﬁlm) 2935, 2837, 1642, 1602, 1583,

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 10.43 (s, 1 H), 9.11 (s, 1 H),

1

1512, 1437, 1265, 1137, 741 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

8.86 (s, 1 H), 8.65 (d, J = 9.2 Hz, 1 H), 8.54 (d, J = 6.5 Hz, 1 H), 7.83

(s, 1 H), 7.68 (s, 1 H), 7.23 (s, 1 H), 7.08 (s, 1 H), 4.60 (m, 2 H), 4.37

(m, 2 H), 3.97 (s, 3 H), 2.50 (m, 2 H, under the water peak). ESI-MS

m/z rel intensity) 466 (MH⁺, 100). HRMS (+ESI) calcd for

C₂₃H₁₈N₄O₆MH⁺, 447.1305; found, 447.1303. HPLC purity: 100%

(MeOH, 100%), 96.7% (MeOH−H₂O, 90:10). Anal. Calcd for

C₂₃H₁₉BrN₄O₆·0.5H₂O: C, 51.51; H, 3.76; N, 10.45. Found: C, 51.33;

H, 3.46; N, 10.30.

8.21 (s 1 H), 7.49−7.44 (m, 3 H), 7.40−7.33 (m, 3 H), 7.21 (dd, J =

6.4 and 1.8 Hz, 1 H), 6.92 (d, J = 8.2 Hz, 1 H), 5.20 (s, 2 H), 3.93 (s, 3

H), 3.73 (t, J = 5.4 Hz, 2 H), 3.51 (t, J = 6.5 Hz, 2 H), 2.26 (m, 2 H).

cis-3-[3-(Benzyloxy)-4-methoxyphenyl]-N-(3-bromopropyl)-4-car-

boxy-3,4-dihydro-7-nitro-1(2H)-isoquinolone (29). Schiﬀ base 28

(4.486 g, 12.38 mmol) was diluted in CHCl₃(50 mL), and the mixture

was cooled to 0 °C for 10 min. Anhydride 14 (2.56 g, 12.38 mmol)

was then added. The red mixture was stirred at 0 °C for 2 h and then

at room temperature for 3 h. The mixture was ﬁltered, and the residue

was washed with 50% CHCl₃in hexane to provide the product 29 as a

white solid (4.867 g, 69%); mp 165−167 °C. IR (ﬁlm) 3078, 1754,

6-(3-Azidopropyl)-9-(benzyloxy)-8-methoxy-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11(6H)-dione (24). Bromide 19 (150 mg, 0.273

mmol) and NaN₃(178 mg, 2.73 mmol) were diluted in DMSO (50

mL), and the mixture was stirred at room temperature for 16 h. The

deep-red solution was diluted with H₂O (100 mL) and extracted with

CHCl₃(50 mL × 3). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated, adsorbed onto SiO₂,

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

CHCl₃, to provide the product 24 as a deep-red solid (36.2 mg, 26%);

mp 205−207 °C (dec). IR (ﬁlm) 2090, 1673, 1610, 1579, 1502, 1427,

1

1630, 1519, 1352, 1259, 1272, 1142, 701 cm⁻¹. H NMR (300 MHz,

CDCl₃) δ 8.99 (d, J = 2.3 Hz, 1 H), 8.28 (dd, J = 8.4 and 2.5 Hz, 1 H),

7.74 (d, J = 8.4 Hz, 1 H), 7.41−7.35 (m, 6 H), 6.70 (d, J = 8.4 Hz, 1

H), 6.48 (d, J = 8.5 Hz, 1 H), 6.38 (d, J = 2.0 Hz, 1 H), 5.23 (d, J = 14

Hz, 1 H), 5.04 (s, 2 H), 4.64 (d, J = 6.5 Hz, 1 H), 3.94 (m, 1 H), 3.83

(s, 3 H), 3.43 (m, 2 H), 3.22 (m, 1 H), 2.20 (m, 1 H), 2.10 (s, 1 H).

ESI-MS m/z (rel intensity) 445 ([MH − COOH − Br]⁺, 100). HRMS

(+ESI) calcd for C₂₇H₂₅BrN₂O₇[MH − COOH − Br]⁺, 445.1763;

found, 445.1771.

1

847 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 9.14 (d, J = 2.3 Hz, 1 H),

8.75 (d, J = 9.0 Hz, 1 H), 8.45 (dd, J = 2.3 and 6.7 Hz, 1 H), 7.48−

7.35 (m, 5 H), 7.28 (s, 1 H), 5.26 (s, 2 H), 4.61 (t, J = 6.9 Hz, 2 H),

4.07 (s, 3 H), 3.79 (t, J = 5.7 Hz, 2 H), 2.16 (m, 2 H). ESI-MS m/z

(rel intensity) 512 (MH⁺, 100). HRMS (+ESI) calcd for C₂₇H₂₁N₅O₆

MH⁺, 512.1570; found, 512.1576.

8-(Benzyloxy)-6-(3-bromopropyl)-9-methoxy-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11[6H]-dione (30). Cis acid 29 (1.50 g, 2.63

mmol) was diluted in SOCl₂(50 mL) at 0 °C and stirred for 16 h,

during which the mixture warmed to room temperature. The red

solution was evaporated to dryness, and the residue was diluted with

CHCl₃(50 mL) and treated slowly with saturated NaHCO₃(100

mL). The mixture was stirred at room temperature for 10 min, and the

two layers were separated. The aqueous layer was extracted with

CHCl₃(100 mL × 2). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated, adsorbed onto SiO₂,

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

CHCl₃to provide the product 30 as a greenish-brown solid (578 mg,

40%); mp 239−240 °C. IR (ﬁlm) 1671, 1613, 1557, 1507, 1488, 1335,

1297, 746 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 9.15 (d, J = 3.3 Hz, 1

H), 8.78 (d, J = 9.0 Hz, 1 H), 8.47 (dd, J = 6.7 and 2.4 Hz, 1 H), 7.44−

7.31 (m, 5 H), 6.91−6.88 (m, 2 H), 5.30 (s, 2 H), 4.59 (t, J = 7.9 Hz, 2

H), 4.02 (s, 3 H), 3.62 (t, J = 6.0 Hz, 2 H), 2.35 (m, 2 H). APCI-MS

m/z (rel intensity) 549/550 ([MH]⁺, 100). HRMS (+EI/CI) calcd for

C₂₇H₂₁BrN₂O₆MH⁺, 549.0661; found, 549.0655.

8-(Benzyloxy)-9-methoxy-6-(3-morpholinopropyl)-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11[6H]-dione (31). Bromide 30 (87 mg,

0.16 mmol) and morpholine (83 mg, 5.2 mmol) were diluted in 1,4-

dioxane (30 mL). The mixture was stirred at room temperature for 16

h and then evaporated to dryness. The resulting residue was diluted

with H₂O (100 mL) and extracted with CHCl₃(50 mL × 3). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with a gradient of MeOH in CHCl₃

(0% to 2%), to provide the product 31 as a deep-purple solid (46 mg,

52%) after being triturated with ether; mp 229−230 °C. IR (ﬁlm)

3399, 1698, 1672, 1614, 1556, 1330, 1304 cm⁻¹. ¹H NMR (300 MHz,

CDCl₃) δ 9.14 (d, J = 2.3 Hz, 1 H), 8.76 (d, J = 9.0 Hz, 1 H), 8.45 (dd,

J = 6.7 and 2.4 Hz, 1 H), 7.42−7.35 (m, 5 H), 7.31 (s, 1 H), 7.12 (s, 1

6-(3-Aminopropyl)-9-hydroxy-8-methoxy-3-nitro-5H-indeno[1,2-

c]isoquinoline-5,11(6H)-dione Hydrobromide (25). Azide 24 (30 mg,

0.059 mmol) was diluted in benzene (50 mL), and triethyl phosphite

(29.2 mg, 0.176 mmol) was added. The mixture was heated at reﬂux

for 16 h and then allowed to cool to room temperature. Aqueous HBr

(48 wt %, 30 mL) was added, and the mixture was heated at 70 °C for

5 h, during which it turned to a brown/red emulsion. The cooled

mixture was concentrated to remove benzene and HBr. The

concentrate was then diluted with acetone (10 mL) and concentrated

again. This procedure was done three times. The ﬁnal mixture was

ﬁltered through an HPLC ﬁlter paper, and the residue was washed

with acetone and CHCl₃to provide the desired product 25 as a brown

solid (26.0 mg, 93%); mp 285−287 °C (dec). IR (ﬁlm) 3243, 2848,

1

1705, 1641, 1614, 1562, 1488, 1336, 1207, 1133, 868 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 10.41 (s, 1 H), 8.83 (d, J = 2.3 Hz, 1 H), 8.60 (d,

J = 9.0 Hz, 1 H), 8.51 (dd, J = 2.5 and 6.5 Hz, 1 H), 7.74 (br s, 3 H),

7.19 (s, 1 H), 7.03 (s, 1 H), 4.58 (m, 2 H), 3.98 (s, 3 H), 3.01 (m, 2

H), 2.14 (m, 2 H). ESI-MS m/z (rel intensity) 396 (MH⁺, 100).

HRMS (+ESI) calcd for C₂₀H₁₉N₃O₆MH⁺, 396.1196; found,

396.1199. HPLC purity: 100% (MeOH, 100%), 98.6% (MeOH−

H₂O, 90:10). Anal. Calcd for C₂₀H₁₈BrN₃O₆: C, 50.44; H, 3.81; N,

8.82. Found: C, 50.13; H, 3.75; N, 8.59.

Benzylisovanillin (27).⁵⁰Isovanillin 26 (5.00 g, 32.9 mmol), benzyl

bromide (5.90 g, 34.5 mmol), and K₂CO₃(9.08 g, 65.7 mmol) were

diluted in DMF (50 mL). The yellow mixture was stirred at room

temperature for 2 h and then poured into a solution of Et₂O−H₂O

(200 mL, 1:1) and stirred for 5 min. The ethereal layer was separated.

The aqueous layer was extracted with Et₂O (50 mL × 2). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

N

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H), 5.30 (s, 2 H), 4.47 (t, J = 7.3 Hz, 2 H), 4.03 (s, 3 H), 3.55 (m, 4

H), 2.41 (m, 6 H), 1.88 (m, 2 H). ESI-MS m/z (rel intensity) 556

([MH]⁺, 100). HRMS (+ESI) calcd for C₃₁H₂₉N₃O₇MH⁺, 556.2084;

found, 556.2082. HPLC purity: 99.2% (MeOH, 100%), 99.0%

(MeOH−H₂O, 90:10).

anhydrous Na₂SO₄, ﬁltered, and concentrated, adsorbed onto SiO₂,

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

CHCl₃to provide the product 35 as a brown solid (40.5, 29%); mp

222−224 °C (dec). IR (ﬁlm) 2099, 1713, 1666, 1614, 1508, 1340, 751

1

cm⁻¹. H NMR (300 MHz, CDCl₃) δ 9.14 (d, J = 2.2 Hz, 1 H), 8.77

8-Hydroxy-9-methoxy-6-(3-morpholinopropyl)-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione Hydrobromide (32).

Compound 31 (25 mg, 0.045 mmol) was diluted in aqueous HBr

(48 wt %, 30 mL), and the mixture was heated at 70 °C for 5 h, during

which it turned to a brown emulsion. The cooled mixture was

concentrated to remove HBr. The concentrate was then diluted with

acetone (10 mL) and concentrated again. This procedure was done

three times. The ﬁnal mixture was ﬁltered under vacuum, and the

residue was washed with acetone and CHCl₃to provide the desired

product 32 as a pale plum-colored solid (24.6 mg, 100%); mp 288−

289 °C. IR (ﬁlm) 3259, 1682, 1608, 1556, 1506, 1390, 1334, 1275,

(d, J = 9.0 Hz, 1 H), 8.46 (dd, J = 2.4 and 6.5 Hz, 1 H), 7.44−7.37 (m,

5 H), 7.31 (s, 2 H), 5.33 (s, 2 H), 4.52 (t, J = 7.1 Hz, 2 H), 4.02 (s, 3

H), 3.67 (t, J = 5.9 Hz, 2 H), 2.01 (m, 2 H). ESI-MS m/z (rel

intensity) 512 (MH⁺, 28).

6-(3-Aminopropyl)-8-hydroxy-9-methoxy-3-nitro-5H-indeno[1,2-

c]isoquinoline-5,11(6H)-dione Hydrobromide (36). Azide 35 (30 mg,

0.059 mmol) was diluted in benzene (50 mL), and triethyl phosphite

(29.2 mg, 0.176 mmol) was added. The mixture was heated at reﬂux

for 16 h and then allowed to cool to room temperature. Aqueous HBr

(48 wt %, 30 mL) was added, and the reaction mixture was heated at

70 °C for 5 h, during which it turned to a brown/red emulsion. The

cooled mixture was concentrated to remove benzene and HBr. The

concentrate was then diluted with acetone (10 mL) and concentrated

again. This procedure was done three times. The ﬁnal mixture was

ﬁltered under vacuum, and the residue was washed with acetone and

CHCl₃to provide the desired product 36 as a brown solid (24.6 mg,

88%); mp 338−340 °C (dec). IR (ﬁlm) 3218, 2853, 1678, 1609, 1555,

1472, 1336, 1178, 855 cm⁻¹. ¹H NMR (300 MHz, CDCl₃) δ 10.34 (s,

1 H), 8.85 (d, J = 2.5 Hz, 1 H), 8.66 (d, J = 9.0 Hz, 1 H), 8.55 (dd, J =

2.5 and 6.4 Hz, 1 H), 7.73 (br s, 3 H), 7.28 (s, 2 H), 4.49 (m, 2 H),

3.92 (s, 3 H), 2.99 (m, 2 H), 2.08 (m, 2 H). ESI-MS m/z (rel

intensity) 396 (MH⁺, 44). HRMS (+ESI) calcd for C₂₀H₁₇N₃O₆MH⁺,

396.1196; found, 396.1191. HPLC purity: 100% (MeOH, 100%),

98.6% (MeOH−H₂O, 90:10). Anal. Calcd for C₂₀H₁₈BrN₃O₆·0.5H₂O:

C, 49.50; H, 3.95; N, 8.66. Found: C, 49.39; H, 3.77; N, 8.48.

2-(Benzyloxy)benzaldehyde (39).⁵¹Compound 38 (5.00 g, 0.041

mol) was diluted in DMF (30 mL), followed by addition of benzyl

bromide (7.70 g, 0.045 mol) and K₂CO₃(11.3 g, 0.082 mmol). The

yellow mixture was stirred at room temperature for 3 h until it turned

cloudy white. The mixture was then diluted in H₂O (100 mL) and

extracted with ether (50 mL × 3). The combined extract was washed

with H₂O (50 mL × 3) and brine (50 mL). The organic layer was

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to yield the

1

1209, 861 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 10.28 (s, 1 H), 9.58

(s, 1 H), 8.84 (d, J = 2.4 Hz, 1 H), 8.65 (d, J = 8.9 Hz, 1 H), 8.55 (dd,

J = 2.5 and 6.5 Hz, 1 H), 7.30 (s, 1 H), 7.26 (s, 1 H), 4.50 (m, 2 H),

3.92 (s, 3 H), 3.63 (m, 4 H), 3.45 (m, 4 H), 3.10 (m, 2 H), 2.24 (m, 2

H). ESI-MS m/z (rel intensity) 466 (MH⁺, 100). HRMS (+ESI) calcd

for C₂₄H₂₃N₃O₇MH⁺, 466.1614; found, 466.1606. HPLC purity:

98.0% (MeOH, 100%), 97.2% (MeOH−H₂O, 90:10). Anal. Calcd for

C₂₄H₂₄BrN₃O₇: C, 52.76; H, 4.43; N, 7.69. Found: C, 52.51; H, 4.39;

N, 7.35.

6-(3-(1H-Imidazol-1-yl)propyl)-8-(benzyloxy)-9-methoxy-3-nitro-

5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (33). Bromide 30 (100

mg, 0.182 mmol) and imidazole (124 mg, 1.82 mmol) were diluted in

1,4-dioxane (30 mL). The mixture was heated at 70 °C for 16 h and

then evaporated to dryness. The resulting residue was diluted with

H₂O (100 mL) and extracted with CHCl₃(50 mL × 3). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with 4% MeOH in CHCl₃, to provide

the product 33 as a brown solid (35.0 mg, 36%); mp 236−237 °C

1

(dec). IR (ﬁlm) 1671, 1612, 1557, 1507, 1334, 1299 cm⁻¹. H NMR

(300 MHz, CDCl₃) δ 9.14 (d, J = 2.3 Hz, 1 H), 8.76 (d, J = 9.0 Hz, 1

H), 8.47 (dd, J = 2.4 and 6.6 Hz, 1 H), 7.52 (s, 1 H), 7.38−7.36 (m, 4

H), 7.32 (m, 2 H), 7.08 (s, 1 H), 6.94 (s, 1 H), 6.83 (s, 1 H), 5.25 (s, 2

H), 4.40 (t, J = 6.5 Hz, 2 H), 4.03 (s, 3 H), 4.01 (t, J = 7.1 Hz, 2 H),

2.00 (m, 2 H). ESI-MS m/z (rel intensity) 537 (MH⁺, 100). HRMS

(+ESI) calcd for C₃₀H₂₄N₄O₆MH⁺, 537.1774; found, 537.1780.

6-(3-(1H-Imidazol-1-yl)propyl)-8-hydroxy-9-methoxy-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione Hydrobromide (34).

Compound 33 (30 mg, 0.056 mmol) was diluted in aqueous HBr

(48 wt %, 30 mL) and the mixture was stirred at 70 °C for 5 h, during

which it turned to a brown emulsion. The cooled mixture was

concentrated to remove HBr. The concentrate was then diluted with

acetone (10 mL) and concentrated again. This procedure was done

three times. The ﬁnal mixture was ﬁltered under vacuum, and the

residue was washed with acetone and CHCl₃to provide the desired

product 34 as a pale plum-colored solid (23.6 mg, 80%); mp 285−286

°C. IR (ﬁlm) 3301, 1669, 1616, 1562, 1334, 1265, 1165, 854 cm⁻¹. ¹H

NMR (300 MHz, CDCl₃) δ 10.27 (s, 1 H), 9.12 (s, 1 H), 8.80 (d, J =

2.2 Hz, 1 H), 8.62 (d, J = 8.9 Hz, 1 H), 8.52 (dd, J = 2.3 and 6.7 Hz, 1

H), 7.83 (s, 1 H), 7.68 (s, 1 H), 7.22 (s, 2 H), 4.48 (m, 2 H), 4.42 (t, J

= 6.7 Hz, 2 H), 3.91 (s, 3 H), 2.41 (m, 2 H). ESI-MS m/z (rel

intensity) 447 (MH⁺, 100). HRMS (+ESI) calcd for C₂₃H₁₈N₄O₆

MH⁺, 447.1305; found, 447.1308. HPLC purity: 97.8% (MeOH,

100%), 97.3% (MeOH−H₂O, 90:10). Anal. Calcd for C₂₃H₁₉BrN₄O₆·

0.1H₂O: C, 52.21; H, 3.66; N, 10.59. Found: C, 51.91; H, 3.55; N,

10.32.

1

product 39 as a colorless liquid (8.69 g, 100%). H NMR (300 MHz,

CDCl₃) δ 10.57 (s, 1 H), 7.88 (d, J = 2.0 Hz, 1 H), 7.86 (d, J = 1.6 Hz,

1 H), 7.54−7.38 (m, 5 H), 7.07−7.05 (m, 2 H), 5.20 (s, 2 H).

N-[2-(Benzyloxy)benzylidene]-3-bromo-1-propylamine (40). 3-

Bromopropylamine hydrobromide (3.56 g, 16.2 mmol) was diluted

in CHCl₃(30 mL) and Et₃N (1.64 g, 16.2 mmol). The mixture was

stirred until the salt dissolved completely, and then compound 39

(3.00 g, 14.1 mmol) and Na₂SO₄(4.02 g, 28.3 mmol) were added.

The mixture was stirred at room temperature for 16 h, diluted with

CHCl₃(100 mL), and then washed with H₂O (100 mL × 3) and brine

(100 mL). The organic layer was dried over anhydrous Na₂SO₄,

ﬁltered, and concentrated to yield the product 40 as a yellow syrup

(4.69 g, 100%). IR (ﬁlm) 2894, 1637, 1599, 1452, 1245, 754 cm⁻¹. ¹H

NMR (300 MHz, CDCl₃) δ 8.82 (s, 1 H), 7.96 (d, J = 7.7 Hz, 1 H),

7.43−7.34 (m, 6 H), 7.00 (m, 2 H), 5.14 (s, 2 H), 3.78 (dt, J = 0.9 and

5.4 Hz, 2 H), 3.52 (t, J = 6.6 Hz, 2 H), 2.28 (m, 2 H). ESI-MS m/z

(rel intensity) 354/356 (MNa⁺, 99/100).

cis-4-Carboxy-3,4-dihydro-N-(3-bromopropyl)-3-[2-(benzyloxy)-

phenyl]-7-nitro-1(2H)-isoquinolone (41). Schiﬀ base 40 (4.69 g,

0.014 mmol) was diluted in CHCl₃(50 mL) at 0 °C, and anhydride 14

(2.92 g, 0.014 mmol) was added. The red mixture was stirred at 0 °C

for 1 h and then at room temperature for 3 h. The cloudy orange

mixture was ﬁltered, and the residue was washed with 50% CHCl₃in

hexane to provide the product 41 as a pale-yellow solid (3.48 g, 46%);

mp 145−147 °C. IR (ﬁlm) 3075, 1749, 1621, 1579, 1525, 1486, 1353,

6-(3-Azidopropyl)-8-(benzyloxy)-9-methoxy-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11(6H)-dione (35). Bromide 30 (150 mg, 0.273

mmol) and NaN₃(178 mg, 2.73 mmol) were diluted in DMSO (30

mL), and the mixture was stirred at room temperature for 16 h. The

deep-red solution was diluted with H₂O (100 mL) and extracted with

CHCl₃(50 mL × 3). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

1

1162, 760 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 8.65 (d, J = 2.6

Hz, 1 H), 8.38 (dd, J = 2.6 and 5.9 Hz, 1 H), 7.73 (dd, J = 0.7 and 8.0

Hz, 1 H), 7.46−7.31 (m, 5 H), 7.22 (m, 1 H), 7.05 (d, J = 8.3 Hz, 1

H), 6.74 (d, J = 3.9 Hz, 2 H), 5.74 (d, J = 6.9 Hz, 1 H), 5.05 (s, 2 H),

3.88 (m, 1 H), 3.51 (m, 3 H), 3.01 (m, 1 H), 2.11 (m, 1 H), 1.96 (m, 1

H); ESI-MS m/z (rel intensity) 415 ([MH − COOH − Br]⁺, 100).

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Journal of Medicinal Chemistry

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HRMS (+ESI) calcd for C₂₆H₂₃BrN₂O₆(MH − COOH − Br)⁺,

415.1658; found, 415.1654.

then concentrated to dryness. The residue was triturated with acetone,

ﬁltered, and washed with acetone to provide the product 45 as a

yellow solid (56.5 mg, 60%); mp 272−274 °C (dec). IR (ﬁlm) 3283,

6-(3-Bromopropyl)-7-hydroxy-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (42). Cis acid 41 (0.50 g, 0.93 mmol)

was diluted in SOCl₂(30 mL), and the mixture was stirred at room

temperature for 5 h. The resulting yellow solution was evaporated to

dryness. The yellow syrup was diluted in 1,2-dichloroethane (30 mL)

at 0 °C, followed an addition of AlCl₃(0.37 g, 2.78 mmol), and the

mixture was stirred for 15 min. Stirring was continued at room

temperature for 7 h. The black mixture was then diluted with CHCl₃

(50 mL) and washed with cold 6 N HCl (100 mL). The aqueous layer

was extracted with CHCl₃(50 mL × 2). The combined extract was

washed with H₂O (100 mL × 3) and brine (100 mL). The yellow

organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated to dryness. The residue was triturated with ether,

ﬁltered, and washed with excess ether to provide the product 42 in

high purity as a yellow amorphous solid (253 mg, 63%); mp 172−175

1

1712, 1671, 1603, 1509, 1484, 1341, 1284, 760 cm⁻¹. H NMR (300

MHz, DMSO-d₆) δ 9.41 (d, J = 9.3 Hz, 1 H), 9.00 (d, J = 2.6 Hz, 1 H),

8.70 (dd, J = 2.6 and 6.6 Hz, 1 H), 8.03 (d, J = 8.1 Hz, 1 H), 7.86 (br s,

3 H), 7.81 (t, J = 7.4 Hz, 1 H), 7.61 (d, J = 7.3 Hz, 1 H), 7.51 (t, J =

7.3 Hz, 1 H), 4.47 (t, J = 6.5 Hz, 2 H), 2.87 (m, 2 H), 2.32 (m, 2 H).

ESI-MS m/z (rel intensity) 366 (MH⁺, 100). HRMS (+ESI) calcd for

C₁₉H₁₅N₃O₅MH⁺, 366.1090; found, 366.1093. HPLC purity: 96.4%

(MeOH, 100%), 98.1% (MeOH−H₂O, 90:10).

3-(Benzyloxy)benzaldehyde (47).⁵²Compound 46 (5.00 g, 0.041

mol) was diluted in DMF (50 mL), followed by addition of benzyl

bromide (7.70 g, 0.045 mol) and K₂CO₃(11.3 g, 0.082 mmol). The

yellow mixture was stirred at room temperature for 3 h until it turned

cloudy white. The mixture was then diluted in H₂O (100 mL) and

extracted with ether (50 mL × 3). The combined extract was washed

with H₂O (50 mL × 3) and brine (50 mL). The organic layer was

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to yield a

pale-yellow syrup, which solidiﬁed upon standing at room temper-

ature. The solid was washed with hexane and ﬁltered to provide the

1

°C. IR (ﬁlm) 1719, 1674, 1606, 1516, 1342 cm⁻¹. H NMR (300

MHz, CDCl₃) δ 9.58 (d, J = 9.3 Hz, 1 H), 9.29 (d, J = 2.6 Hz, 1 H),

8.61 (dd, J = 2.6 and 6.7 Hz, 1 H), 8.01 (dd, J = 1.2 and 7.1 Hz, 1 H),

7.70 (dt, J = 1.3 and 7.2 Hz, 1 H), 7.53−7.42 (m, 2 H), 4.72 (t, J = 7.2

Hz, 2 H), 3.50 (t, J = 5.9 Hz, 1 H), 2.69 (m, 2 H). ESI-MS m/z (rel

intensity) 351 ([MH − HBr]⁺, 100).

1

product 47 as a white solid (8.69 g, 100%); mp 43−45 °C. H NMR

(300 MHz, CDCl₃) δ 9.98 (s, 1 H), 7.49−7.35 (m, 8 H), 7.28 (d, J =

2.5 Hz, 1 H), 5.13 (s, 2 H).

7-Hydroxy-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (43). Bromide 42 (70 mg, 0.16 mmol)

and morpholine (71 mg, 0.81 mmol) were diluted in 1,4-dioxane (30

mL). The yellow mixture was heated at 70 °C for 16 h. The solution

was then diluted with H₂O (100 mL) and extracted with CHCl₃(50

mL × 3). The combined extract was washed with H₂O (100 mL × 3)

and brine (100 mL). The organic layer was dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated to dryness. The residue was

triturated with acetone, ﬁltered, and washed with ether to provide the

product 43 as a brown solid (68.7 mg, 97%); mp 216−218 °C. IR

(ﬁlm) 3413, 1719, 1675, 1606, 1515, 1342, 1117 cm⁻¹. ¹H NMR (300

MHz, DMSO-d₆) δ 9.39 (d, J = 9.3 Hz, 1 H), 8.99 (d, J = 2.4 Hz, 1 H),

8.68 (dd, J = 2.6 and 6.5 Hz, 1 H), 8.12 (d, J = 8.3 Hz, 1 H), 7.79 (t, J

= 7.5 Hz, 1 H), 7.59 (d, J = 8.3 Hz, 1 H), 7.50 (t, J = 7.5 Hz, 1 H),

4.60 (m, 2 H), 3.17 (m, 4 H), 2.04−1.98 (m, 8 H). ESI-MS m/z (rel

intensity) 436 (MH⁺, 100). HRMS (+ESI) calcd for C₂₃H₂₁N₃O₆

MH⁺, 436.1509; found, 436.1505. HPLC purity: 95.0% (MeOH,

100%), 96.7% (MeOH−H₂O, 90:10).

N-[3-(Benzyloxy)benzylidene]-3-bromo-1-propylamine (48). 3-

Bromopropylamine hydrobromide (6.45 g, 29.4 mmol) was diluted

in CHCl₃(50 mL) and Et₃N (2.98 g, 29.4 mmol). The mixture was

stirred for 5 min, and then compound 47 (5.00 g, 23.6 mmol) and

Na₂SO₄(6.69 g, 4.71 mmol) were added. The mixture was stirred at

room temperature for 16 h and then washed with H₂O (100 mL × 3)

and brine (100 mL). The organic layer was dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated to yield the product 48 as a pale-

yellow syrup (7.57 g, 97%). IR (ﬁlm) 2847, 1646, 1580, 1445, 1263,

1

689 cm⁻¹. H NMR (300 MHz, CDCl₃) δ 8.31 (s, 1 H), 7.46−7.28

(m, 8 H), 7.08 (dd, J = 1.0 and 1.4 Hz, 1 H), 5.11 (s, 2 H), 3.78 (dt, J

= 0.9 and 5.3 Hz, 2 H), 3.52 (t, J = 6.5 Hz, 2 H), 2.29 (m, 2 H). ESI-

MS m/z (rel intensity) 332/334 (MH⁺, 100/99).

cis-3-[3-(Benzyloxy)phenyl]-N-(3-bromopropyl)-4-carboxy-3,4-di-

hydro-7-nitro-1(2H)-isoquinolone (49). Schiﬀ base 48 (4.57 g, 13.8

mmol) was diluted in CHCl₃(50 mL) at 0 °C, and anhydride 14 (2.85

g, 13.8 mmol) was added. The red mixture was stirred at 0 °C for 1 h

and then at room temperature for 2 h. The cloudy orange mixture was

ﬁltered, and the residue was diluted in CHCl₃(50 mL) and heated at

reﬂux for 20 min. The cloudy mixture was then ﬁltered and washed

with hot CHCl₃to provide the product 49 as an oﬀ-white solid (5.08

g, 68%); mp 166−167 °C. IR (ﬁlm) 3082, 1749, 1628, 1586, 1524,

1268, 1181, 698 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 8.59 (d, J =

2.4 Hz, 1 H), 8.25 (dd, J = 2.4 and 5.9 Hz, 1 H), 7.46 (d, J = 8.4 Hz, 1

H), 7.36−7.26 (m, 5 H), 7.20 (t, J = 7.9 Hz, 1 H), 6.87 (dd, J = 2.2

and 5.9 Hz, 1 H), 6.71 (s, 1 H), 6.63 (d, J = 7.8 Hz, 1 H), 5.10 (d, J =

5.4 Hz, 1 H), 5.00 (s, 2 H), 4.14 (m, 1 H), 3.81 (q, J = 6.7 H, 1 H),

3.59 (t, J = 7.1 Hz, 2 H), 2.96 (m, 1 H), 2.21 (m, 2 H). ESI-MS m/z

(rel intensity) 495/497 ([MH − COOH]⁺, 91/97). HRMS (+ESI)

calcd for C₂₆H₂₃BrN₂O₆(MH − COOH)⁺, 495.0919; found,

495.0911.

6-(3-(1H-Imidazol-1-yl)propyl)-7-hydroxy-3-nitro-5H-indeno[1,2-

c]isoquinoline-5,11(6H)-dione (44). Bromide 42 (100 mg, 0.23

mmol) and imidazole (79 mg, 1.16 mmol) were diluted in 1,4-

dioxane (30 mL). The yellow mixture was heated at 70 °C for 16 h.

The solution was then diluted with H₂O (100 mL) and extracted with

CHCl₃(50 mL × 3). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated to dryness. The residue

was triturated with acetone, ﬁltered, and washed with ether to provide

the product 44 as a yellow solid (77.2 mg, 79%); mp 173−175 °C. IR

1

(ﬁlm) 1717, 1675, 1606, 1515, 1342, 1119, 756 cm⁻¹. H NMR (300

MHz, DMSO-d₆) δ 9.38 (d, J = 9.3 Hz, 1 H), 8.97 (d, J = 2.5 Hz, 1 H),

8.67 (dd, J = 2.5 and 6.7 Hz, 1 H), 7.85 (d, J = 7.7 Hz, 1 H), 7.76 (t, J

= 7.4 Hz, 1 H), 7.56 (d, J = 8.0 Hz, 1 H), 7.38 (t, J = 8.5 Hz, 1 H),

4.37 (m, 2 H), 4.14 (m, 2 H). ESI-MS m/z (rel intensity) 417 (MH⁺,

100). HRMS (+ESI) calcd for C₂₂H₁₆N₄O₅MH⁺, 417.1199; found,

417.1202.

6-(3-Aminopropyl)-7-hydroxy-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione Hydrochloride (45). Bromide 42 (100

mg, 0.23 mmol) and NaN₃(45 mg, 0.70 mmol) were diluted in

DMSO (30 mL). The yellow mixture was stirred at room temperature

for 16 h. The solution was then diluted with H₂O (100 mL) and

extracted with CHCl₃(50 mL × 3). The combined extract was washed

with H₂O (100 mL × 3) and brine (100 mL). The organic layer was

dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to dryness.

The residue was diluted in benzene (30 mL), and triethyl phosphite

(116 mg, 0.70 mmol) was added. The yellow solution was heated at

reﬂux for 16 h and then cooled to room temperature. Methanolic HCl

(3 M, 40 mL) was added. The mixture was heated at 70 °C for 3 h and

Mixture of 8- and 10-(Benzyloxy)-6-(3-bromopropyl)-3-nitro-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione (51 and 52). Cis acid 49

(1.48 g, 2.86 mmol) was diluted in SOCl₂(50 mL), and the mixture

was heated at reﬂux for 4 h. The resulting red solution was evaporated

to dryness. The residue was triturated with ether and ﬁltered to

1

provide an approximately 2:1 (by H NMR) mixture of 8- and 10-

(benzyloxy)indenoisoquinolines (51 and 52, respectively) as an

orange solid (0.32 g, 22%). Identities of the products were determined

1

by H NMR. The product mixture was subjected to the next S_N2

reaction without further puriﬁcation.

8-(Benzyloxy)-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (53) and 10-(Benzyloxy)-6-(3-morpho-

linopropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione

(54). A mixture of bromides 51 and 52 (150 mg, 0.29 mmol) and

morpholine (151 mg, 1.73 mmol) was diluted in 1,4-dioxane (30 mL),

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Journal of Medicinal Chemistry

Article

and the mixture was heated at 70 °C for 16 h. The mixture was then

evaporated to dryness. The resulting residue was diluted with CHCl₃

(100 mL) and washed with H₂O (100 mL × 4) and brine (100 mL).

The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with a gradient of MeOH in CHCl₃

(0% to 1%), to provide the 8-benzyloxy product (53) as an orange

solid (60.3 mg, 40%) and the 10-benxyloxy product (54) as a red solid

(48.6 mg, 32%).

CHCl₃(100 mL) and washed with H₂O (100 mL × 3) and brine (100

mL). The organic layer was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated, adsorbed onto SiO₂, and puriﬁed by ﬂash column

chromatography (SiO₂), eluting with a gradient of MeOH in CHCl₃

(0−2%) to provide the 8-benzyloxy product (57) as an orange solid

(35.6 mg, 37%) and the 10-benxyloxy product (58) as an orange solid

(17.8 mg, 18%).

8-Benzyloxy (57): mp 265−267 °C. IR (ﬁlm) 1670, 1613, 1560,

1

1505, 1339, 1229, 842 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 8.89

8-Benzyloxy (53): mp 250−252 °C. IR (ﬁlm) 1669, 1615, 1560,

(d, J = 2.4 Hz, 1 H), 8.76 (d, J = 9.0 Hz, 1 H), 8.59 (d, J = 9.1 Hz, 1

H), 7.78 (s, 1 H), 7.64 (d, J = 8.2 Hz, 1 H), 7.52 (d, J = 7.0 Hz, 2 H),

7.44−7.35 (m, 3 H), 7.26 (m, 2 H), 7.17 (d, J = 8.0 Hz, 1 H), 6.90 (m,

1 H), 5.28 (s, 2 H), 4.51 (m, 2 H), 4.16 (m, 2 H), 2.26 (m, 2 H). ESI-

MS m/z (rel intensity) 507 (MH⁺, 100). HRMS (+ESI) calcd for

MH⁺, 507.1668; found, 507.1662.

10-Benzyloxy (58): mp 260−261 °C. IR (ﬁlm) 1671, 1612, 1562,

1502, 1454, 1331, 1264, 840 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ

8.88 (m, 1 H), 8.78 (d, J = 9.1 Hz, 1 H), 8.54 (dd, J = 2.4 and 6.6 Hz,

1 H), 7.80 (br s, 1 H), 7.55 (d, J = 7.6 Hz, 2 H), 7.47−7.31 (m, 6 H),

6.94 (m, 2 H), 5.33 (s, 2 H), 4.48 (t, J = 7.6 Hz, 2 H), 4.26 (t, J = 6.6

Hz, 2 H), 2.24 (m, 2 H). ESI-MS m/z (rel intensity) 507 (MH⁺, 100).

HRMS (+ESI) calcd for C₂₉H₂₂N₄O₅MH⁺, 507.1668; found,

507.1673.

1

1508, 1337, 1114, 841 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 9.17

(d, J = 2.4 Hz, 1 H), 8.87 (d, J = 8.9 Hz, 1 H), 8.48 (dd, J = 2.4 and 6.5

Hz, 1 H), 7.65 (d, J = 8.0 Hz, 1 H), 7.45−7.41 (m, 6 H), 6.94 (dd, J =

1.7 and 6.4 Hz, 1 H), 5.16 (s, 2 H), 4.62 (t, J = 7.2 Hz, 2 H), 3.58 (t, J

= 4.4 Hz, 4 H), 2.56 (t, J = 6.1 Hz, 2 H), 2.44 (m, 4 H), 2.06 (m, 2 H).

ESI-MS m/z (rel intensity) 526 (MH⁺, 100). HRMS (+ESI) calcd for

C₃₀H₂₇N₃O₆MH⁺, 526.1978; found, 526.1984.

10-Benzyloxy (54): mp 254−256 °C. IR (ﬁlm) 1678, 1610, 1558,

1332, 1117, 746 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 9.18 (d, J =

2.4 Hz, 1 H), 8.95 (d, J = 9.0 Hz, 1 H), 8.49 (dd, J = 2.5 and 6.5 Hz, 1

H), 7.54−7.48 (m, 3 H), 7.45−7.34 (m, 4 H), 7.10 (d, J = 8.5 Hz, 1

H), 5.35 (s, 2 H), 4.65 (t, J = 7.9 Hz, 2 H), 3.71 (t, J = 4.4 Hz, 4 H),

2.60 (t, J = 6.2 Hz, 2 H), 2.49 (m, 4 H), 2.08 (m, 2 H). ESI-MS m/z

(rel intensity) 526 (MH⁺, 100). HRMS (+ESI) calcd C₃₀H₂₇N₃O₆for

MH⁺, 526.1978; found, 526.1974.

6-(3-(1H-Imidazol-1-yl)propyl)-8-hydroxy-3-nitro-5H-indeno[1,2-

c]isoquinoline-5,11(6H)-dione Hydrobromide (59). Compound 57

(41 mg, 0.081 mmol) was diluted in aqueous HBr (30 mL, 48 wt %),

and the mixture was heated at 70 °C for 3 h. The red mixture was

diluted in CHCl₃(10 mL) and acetone (10 mL) and then

concentrated in vacuo. This procedure was done 3 times. The ﬁnal

concentrate was ﬁltered through an HPLC ﬁlter paper, and the residue

was washed with acetone and CHCl₃to provide the product 59 as a

bright-orange solid (36.3 mg, 90%); mp 329−331 °C (dec). IR (ﬁlm)

8-(Hydroxy)-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (55). Compound 53 (55 mg, 0.10

mmol) was diluted with aqueous HBr (48 wt %, 30 mL), and the

mixture was heated at 70 °C for 3 h. The red mixture was diluted with

CHCl₃(10 mL) and acetone (10 mL) and then concentrated in vacuo.

This procedure was done three times. The ﬁnal concentrate was

ﬁltered through an HPLC ﬁlter paper, and the residue was washed

with acetone and CHCl₃to provide the desired product 55 as an

orange solid (51.6 mg, 96%); mp >400 °C. IR (ﬁlm) 3353, 1668, 1615,

1562, 1506, 1435, 1338, 1258, 847 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 10.87 (s, 1 H), 9.48 (br s, 1 H), 8.90 (d, J = 2.4 Hz, 1 H),

8.77 (d, J = 9.0 Hz, 1 H), 8.60 (dd, J = 2.4 and 6.6 Hz, 1 H), 7.56 (d, J

= 8.1 Hz, 1 H), 7.25 (s, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 4.56 (m, 2 H),

3.98 (m, 2 H), 3.64 (m, 2 H), 3.44 (m, 4 H), 3.09 (m, 2 H), 2.25 (m, 2

H). ESI-MS m/z (rel intensity) 436 (MH⁺, 100). HRMS (+ESI) calcd

for C₂₃H₂₁N₃O₆MH⁺, 436.1509; found, 436.1514. HPLC purity:

98.0% (MeOH, 100%), 97.4% (MeOH−H₂O, 90:10). Anal. Calcd for

C₂₃H₂₂BrN₃O₆·0.75H₂O: C, 52.14; H, 4.47; N, 7.93. Found: C, 51.79;

H, 4.19; N, 7.64.

10-(Hydroxy)-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (56). Compound 54 (45 mg, 0.086

mmol) was diluted with aqueous HBr (48 wt %, 30 mL), and the

mixture was heated at 70 °C for 3 h. The red mixture was diluted with

CHCl₃(10 mL) and acetone (10 mL) and then concentrated in vacuo.

This procedure was done 3 times. The ﬁnal concentrate was ﬁltered

through an HPLC ﬁlter paper, and the residue was washed with

acetone and CHCl₃to provide the desired product 56 as a brown solid

(19 mg, 43%); mp 314−316 °C (dec). IR (ﬁlm) 2917, 1688, 1611,

1557, 1506, 1430, 1344, 1172, 802 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 10.72 (s, 1 H), 9.48 (br s, 1 H), 8.91 (d, J = 2.4 Hz, 1 H),

8.81 (d, J = 8.9 Hz, 1 H), 8.61 (dd, J = 2.5 and 6.5 Hz, 1 H), 7.50 (t, J

= 7.8 Hz, 1 H), 7.39 (d, J = 7.7 Hz, 1 H), 7.09 (d, J = 8.4 Hz, 1 H),

4.56 (m, 2 H), 3.98 (m, 2 H), 3.63 (m, 2 H), 3.43 (m, 4 H), 3.09 (m, 2

H), 2.26 (m, 2 H). ESI-MS m/z (rel intensity) 436 (MH⁺, 100).

HRMS (+ESI) calcd for C₂₃H₂₁N₃O₆MH⁺, 436.1509; found,

436.1500. HPLC purity: 98.8% (MeOH, 100%), 98.3% (MeOH−

H₂O, 90:10). Anal. Calcd for C₂₃H₂₂BrN₃O₆: C, 53.50; H, 4.29; N,

8.14. Found: C, 53.24; H, 4.29; N, 7.91.

1

3064, 1669, 1615, 1562, 1506, 1340, 1255, 835 cm⁻¹. H NMR (300

MHz, DMSO-d₆) δ 10.84 (s, 1 H), 9.04 (s, 1 H), 8.89 (d, J = 2.4 Hz, 1

H), 8.76 (d, J = 9.0 Hz, 1 H), 8.60 (dd, J = 2.5 and 6.5 Hz, 1 H), 7.80

(s, 1 H), 7.63 (s, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.18 (s, 1 H), 6.89 (d,

J = 9.5 Hz, 1 H), 4.54 (m, 2 H), 4.40 (t, J = 6.6 Hz, 2 H), 2.42 (m, 2

H). ESI-MS m/z (rel intensity) 417 (MH⁺, 100). HRMS (+ESI) calcd

for C₂₂H₁₆N₄O₅MH⁺, 417.1199; found, 417.1195. Anal. Calcd for

C₂₂H₁₇BrN₄O₅·0.5H₂O: C, 52.19; H, 3.58; N, 11.07. Found: C, 51.82;

H, 3.44; N, 10.67.

6-(3-(1H-Imidazol-1-yl)propyl)-10-hydroxy-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11(6H)-dione Hydrobromide (60). Compound

58 (27 mg, 0.053 mmol) was diluted in aqueous HBr (30 mL, 48 wt

%), and the mixture was heated at 70 °C for 3 h. The red mixture was

diluted in CHCl₃(10 mL) and acetone (10 mL) and then

concentrated in vacuo. This procedure was done 3 times. The ﬁnal

concentrate was ﬁltered through an HPLC ﬁlter paper, and the residue

was washed with acetone and CHCl₃to provide the product 60 as a

bright-orange solid (25.3 mg, 95%); mp 337−339 °C (dec). IR (ﬁlm)

3347, 2946, 1694, 1666, 1615, 1563, 1503, 1428, 1336, 1287, 852

cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 10.70 (s, 1 H), 9.11 (s, 1 H),

8.90 (d, J = 2.4 Hz, 1 H), 8.79 (d, J = 8.9 Hz, 1 H), 8.60 (dd, J = 2.5

and 6.5 Hz, 1 H), 7.83 (s, 1 H), 7.68 (s, 1 H), 7.46 (t, J = 7.9 Hz, 1 H),

7.28 (d, J = 7.4 Hz, 1 H), 7.08 (d, J = 8.5 Hz, 1 H), 4.54 (m, 2 H), 4.42

(t, J = 7.1 Hz, 2 H), 2.39 (m, 2 H). ESI-MS m/z (rel intensity) 417

(MH⁺, 100). HRMS (+ESI) calcd for C₂₂H₁₆N₄O₅MH⁺, 417.1199;

found, 417.1203. Anal. Calcd for C₂₂H₁₇BrN₄O₅·0.25H₂O: C, 52.66;

H, 3.52; N, 11.16. Found: C, 52.81; H, 3.49; N, 10.79.

6-(3-Aminopropyl)-8-hydroxy-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione Hydrobromide (63) and 6-(3-Amino-

propyl)-10-hydroxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-

dione Hydrobromide (64). A mixture of bromides 51 and 52 (100 mg,

0.19 mmol) and NaN₃(63 mg, 0.96 mmol) was diluted in DMSO (30

mL), and the mixture was stirred at room temperature for 16 h. The

mixture was then diluted with H₂O (100 mL) and extracted with

CHCl₃(50 mL × 3). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, concentrated, and puriﬁed by ﬂash

column chromatography (SiO₂), eluting with CHCl₃to provide azides

6-(3-(1H-Imidazol-1-yl)propyl)-8-(benzyloxy)-3-nitro-5H-indeno-

[1,2-c]isoquinoline-5,11(6H)-dione (57) and 6-(3-(1H-Imidazol-1-

yl)propyl)-10-(benzyloxy)-3-nitro-5H-indeno[1,2-c]isoquinoline-

5,11(6H)-dione (58). A mixture of bromides 51 and 52 (100 mg, 0.19

mmol) and imidazole (131 mg, 1.9 mmol) was diluted in 1,4-dioxane

(30 mL), and the mixture was heated at 70 °C for 16 h. The mixture

was then concentrated to dryness. The resulting residue was diluted in

Q

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Journal of Medicinal Chemistry

Article

1

61 and 62 as orange solids. Both compounds were diluted separately

in benzene (20 mL) and triethyl phosphite (36 mg, 0.22 mmol) and

heated at reﬂux for 16 h. Aqueous HBr (48 wt %, 20 mL) was added to

the cooled solutions, and stirring was continued at reﬂux for 4 h,

during which orange precipitates formed. The two mixtures were

concentrated, washed with acetone (10 mL) and CHCl₃(10 mL), and

concentrated again. The mixtures were then ﬁltered and the residues

were washed thoroughly with acetone and CHCl₃to provide the

amines 63 (32 mg, 37% in two steps) and 64 (28 mg, 33% in two

steps) as orange solids.

IR (ﬁlm) 3273, 1659, 1613, 1531, 1345, 1270, 755 cm⁻¹. H NMR

(300 MHz, DMSO-d₆) δ 10.82 (s, 1 H), 8.83 (d, J = 2.1 Hz, 1 H), 8.61

(d, J = 9.0 Hz, 1 H), 8.51 (d, J = 9.2 Hz, 1 H), 7.74 (d, J = 8.6 Hz, 1

H), 6.99 (s, 1 H), 6.89 (d, J = 8.6 Hz, 1 H), 4.54 (m, 2 H), 3.78 (t, J =

6.3 Hz, 2 H), 2.33 (m, 2 H). ESI-MS m/z (rel intensity) 428/430 (M⁺,

99/100). HRMS (+ESI) calcd for C₁₉H₁₃BrN₂O₅M⁺, 428.0008;

found, 428.0000.

9-Hydroxy-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione Hydrobromide (69). Phenol 68 (50 mg,

0.12 mmol) and morpholine (51 mg, 0.58 mmol) were diluted in THF

(20 mL). The red mixture was heated at 70 °C for 16 h. The cooled

solution was diluted with 3 M methanolic HBr (20 mL) and stirred at

room temperature for 4 h. The deep-red solution was concentrated

and ﬁltered through an HPLC ﬁlter paper. The residue was washed

with CHCl₃to provide the product 69 as a deep-brown solid (49 mg,

Amine 63: mp 353−355 °C (dec). IR (ﬁlm) 3248, 1666, 1613,

1

1595, 1507, 1480, 1388, 1341, 1295, 864 cm⁻¹. H NMR (300 MHz,

DMSO-d₆) δ 10.86 (s, 1 H), 8.87 (d, J = 2.4 Hz, 1 H), 8.74 (d, J = 9.0

Hz, 1 H), 8.58 (dd, J = 2.6 and 6.4 Hz, 1 H), 7.73 (br s, 3 H), 7.53 (d,

J = 8.0 Hz, 1 H), 7.24 (d, J = 1.5 Hz, 1 H), 6.88 (dd, J = 1.5 and 6.5

Hz, 1 H), 4.55 (t, J = 6.7 Hz, 2 H), 3.00 (m, 2 H), 2.11 (m, 2 H). ESI-

MS m/z (rel intensity) 366 (MH⁺, 14); HRMS (+ESI) calcd for

C₁₉H₁₅N₃O₅MH⁺, 366.1090; found, 366.1087. HPLC purity: 97.1%

(MeOH, 100%).

1

83%); mp 295−297 °C (dec). H NMR (300 MHz, DMSO-d₆) δ

10.87 (s, 1 H), 9.53 (br s, 1 H), 8.86 (d, J = 2.4 Hz, 1 H), 8.66 (d, J =

9.0 Hz, 1 H), 8.56 (dd, J = 2.4 and 6.5 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1

H), 7.04 (d, J = 2.4 Hz, 1 H), 6.93 (dd, J = 2.3 and 6.0 Hz, 1 H), 4.52

(m, 2 H), 3.98 (m, 2 H), 3.64 (m, 2 H), 3.38 (m, 4 H), 3.08 (m, 2 H),

2.21 (m, 2 H). ESI-MS m/z (rel intensity) 436 (MH⁺, 100). HRMS

(+ESI) calcd for C₂₃H₂₁N₃O₆MH⁺, 436.1509; found, 436.1508. Anal.

Calcd for C₂₃H₂₂BrN₃O₆: C, 53.50; H, 4.29; N, 8.14. Found: C, 53.51;

H, 4.23; N, 7.98.

Amine 64: mp 337−339 °C (dec). ¹H NMR (300 MHz, DMSO-d₆)

δ 10.69 (s, 1 H), 8.90 (d, J = 2.4 Hz, 1 H), 8.79 (d, J = 8.9 Hz, 1 H),

8.60 (dd, J = 2.6 and 6.4 Hz, 1 H), 7.71 (br s, 3 H), 7.49−7.44 (m, 1

H), 7.39−7.35 (m, 1 H), 7.08 (d, J = 8.3 Hz, 1 H), 4.55 (t, J = 6.8 Hz,

2 H), 3.01 (m, 2 H), 2.10 (m, 2 H). MALDI-MS m/z (rel intensity)

366 (MH⁺, 100). HRMS (+ESI) calcd for C₁₉H₁₅N₃O₅MH⁺,

366.1090; found, 366.1109. HPLC purity: 96.5% (MeOH, 100%).

N-[4-(Benzyloxy)benzylidene]-3-bromo-1-propylamine (66). 3-

Bromopropylamine hydrobromide (3.56 g, 16.2 mmol) was diluted

in CHCl₃(50 mL) and Et₃N (1.64 g, 16.2 mmol). The mixture was

stirred for 5 min, and then compound 65 (3.00 g, 14.1 mmol) and

Na₂SO₄(4.02 g, 28.3 mmol) were added. The mixture was stirred at

room temperature for 16 h and then washed with H₂O (100 mL × 3)

and brine (100 mL). The organic layer was dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated to yield the product 66 as a pale-

yellow syrup (4.69 g, 100% + residual solvent). IR (ﬁlm) 2839, 1645,

6-(3-(1H-Imidazol-1-yl)propyl)-7-hydroxy-3-nitro-5H-indeno[1,2-

c]isoquinoline-5,11(6H)-dione hydrochloride (70). Bromide 68 (70

mg, 0.16 mmol) and imidazole (67 mg, 0.98 mmol) were diluted in

THF (20 mL). The red mixture was heated at 70 °C for 16 h. The

cooled solution was diluted with 3 M methanolic HCl (20 mL) and

stirred at room temperature for 4 h. The deep-red mixture was

concentrated and ﬁltered through an HPLC ﬁlter paper. The residue

was washed with CHCl₃to aﬀord the product 70 as a brown solid

(42.6 mg, 58%); mp 323−325 °C (dec). IR (ﬁlm) 1668, 1611, 1503,

1428, 1334, 1263 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 9.38 (d, J =

9.3 Hz, 1 H), 8.97 (d, J = 2.5 Hz, 1 H), 8.67 (dd, J = 2.5 and 6.7 Hz, 1

H), 7.85 (d, J = 7.7 Hz, 1 H), 7.76 (t, J = 7.4 Hz, 1 H), 7.56 (d, J = 8.0

Hz, 1 H), 7.38 (t, J = 8.5 Hz, 1 H), 4.37 (m, 2 H), 4.14 (m, 2 H). ESI-

MS m/z (rel intensity) 417 (MH⁺, 100). HRMS (+ESI) calcd for

C₂₂H₁₆N₄O₅MH⁺, 417.1199; found, 417.1202. HPLC purity: 100%

(MeOH, 100%), 100% (MeOH−H₂O, 70:30).

1

1605, 1509, 1246, 1166, 830 cm⁻¹. H NMR (300 MHz, CDCl₃) δ

8.26 (s, 1 H), 7.68 (dd, J = 1.8 and 6.9 Hz, 2 H), 7.45−7.33 (m, 5 H),

7.02 (dd, J = 1.9 and 6.9 Hz, 2 H), 5.11 (s, 2 H), 3.74 (dt, J = 0.9 and

6.2 Hz, 2 H), 3.51 (t, J = 6.6 Hz, 2 H), 2.27 (m, 2 H). ESI-MS m/z

(rel intensity) 332/334 (MH⁺, 100/97).

6-(3-Aminopropyl)-9-hydroxy-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione Hydrobromide (71). Bromide 68 (100

mg, 0.23 mmol) and NaN₃(75 mg, 1.15 mmol) were diluted in

DMSO (30 mL) and stirred at room temperature for 16 h. The

solution was then diluted with H₂O (100 mL) and extracted with

CHCl₃(50 mL × 3). The combined extract was washed with H₂O

(100 mL × 3) and brine (100 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated to provide the crude

intermediate azide. The compound was then diluted in THF (20 mL),

followed by addition of PPh₃(181 mg, 0.69 mmol), and the mixture

was heated at reﬂux for 16 h. The cooled deep-brown solution was

diluted with 3 M methanolic HBr (20 mL), and stirring was continued

at 70 °C for 4 h. The bright-red solution was evaporated, rediluted in

CHCl₃(10 mL), and let sit for 16 h at 0 °C, during which a precipitate

formed. The solution was then ﬁltered through an HPLC ﬁlter paper,

and the residue was washed thoroughly with CHCl₃to provide the

product 71 (33.1 mg, 32%) as a deep-red solid; mp 360−362 °C

cis-4-Carboxy-3,4-dihydro-N-(3-bromopropyl)-3-[4-(benzyloxy)-

phenyl]-7-nitro-1(2H)-isoquinolone (67). Schiﬀ base 66 (4.69 g, 14.1

mmol) was diluted in CHCl₃(50 mL) at 0 °C, and anhydride 14 (2.80

g, 13.5 mmol) was added. The red mixture was stirred at 0 °C for 2 h,

and then at room temperature for 2 h. The cloudy orange mixture was

ﬁltered, and the residue was washed with 50% CHCl₃in hexane to

provide the product 67 as an oﬀ-white solid (5.18 g, 71%); mp 140−

1

141 °C. IR (ﬁlm) 3076, 1727, 1630, 1525, 1347, 1187, 738 cm⁻¹. H

NMR (300 MHz, DMSO-d₆) δ 8.71 (d, J = 2.6 Hz, 1 H), 8.39 (dd, J =

2.6 and 6.0 Hz, 1 H), 7.92 (d, J = 8.2 Hz, 1 H), 7.40−7.30 (m, 5 H),

6.92−6.83 (m, 4 H), 5.19 (d, J = 6.4 Hz, 1 H), 5.03 (d, J = 6.3 Hz, 1

H), 4.98 (s, 2 H), 3.90 (m, 1 H), 3.59 (m, 2 H), 3.03 (m, 1 H), 2.16

(m, 1 H), 2.04 (m, 1 H). ESI-MS m/z (rel intensity) 415 ([MH −

COOH − Br]⁺, 100). HRMS (+ESI) calcd for C₂₆H₂₃BrN₂O₆MH⁺,

539.0818; found, 539.0812.

6-(3-Bromopropyl)-9-hydroxy-3-nitro-5H-indeno[1,2-c]-

isoquinoline-5,11(6H)-dione (68). Cis acid 67 (1.00 g, 1.85 mmol)

was diluted in SOCl₂(50 mL), and the mixture was stirred at room

temperature for 5 h. The resulting yellow solution was evaporated to

dryness. The yellow syrup was diluted in 1,2-dichloroethane (40 mL)

at 0 °C, followed an addition of AlCl₃(0.75 g, 5.56 mmol), and the

mixture was stirred for 15 min. Stirring was continued at room

temperature for 8 h. The black mixture was then diluted with CHCl₃

(50 mL) and THF (30 mL) and washed with cold 6 N HCl (100 mL).

The aqueous layer was extracted with CHCl₃(50 mL × 2). The

combined extract was washed with H₂O (100 mL × 3) and brine (100

mL). The red organic layer was dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated to dryness. The residue was triturated with CHCl₃,

ﬁltered, and washed with excess CHCl₃to provide the product 68 in

high purity as a deep-red solid (262 mg, 33%); mp 281−283 °C (dec).

1

(dec). H NMR (300 MHz, DMSO-d₆) δ 10.83 (s, 1 H), 8.83 (d, J =

2.3 Hz, 1 H), 8.61 (d, J = 9.0 Hz, 1 H), 8.52 (dd, J = 2.5 and 6.4 Hz, 1

H), 7.74−7.67 (m, 4 H), 6.99 (d, J = 2.3 Hz, 1 H), 6.91 (dd, J = 2.2

and 6.1 Hz, 1 H), 4.52 (t, J = 7.0 Hz, 2 H), 2.99 (m, 2 H), 2.08 (m, 2

H). APCI-MS m/z (rel intensity) 366 ([MH − NH₃]⁺, 100). HRMS

(+ESI) calcd for C₁₉H₁₅N₃O₅MH⁺, 366.1090; found, 366.1082;

HPLC purity: 99.6% (MeOH, 100%).

Topoisomerase I-Mediated DNA Cleavage Reactions. Human

recombinant Top1 was puriﬁed from baculovirus as previously

described.⁵³DNA cleavage reactions were performed as previously

reported with the exception of the DNA substrate.³¹Brieﬂy, a 117-bp

DNA oligonucleotide (Integrated DNA Technologies) encompassing

the previously identiﬁed Top1 cleavage sites in the 161-bp fragment

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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, and 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 room

temperature for 20 min in the presence of various concentrations of

compounds. The reactions were terminated by adding SDS (0.5% ﬁnal

concentration), followed by addition of two volumes of loading dye

(80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA,

0.1% xylene cyanol, and 0.1% bromophenol blue). Aliquots of each

reaction mixture were subjected to 20% denaturing PAGE. Gels were

dried and visualized by using a phosphorimager and ImageQuant

software (Molecular Dynamics). For simplicity, cleavage sites were

numbered as previously described in the 161-bp fragment.⁵³

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.²¹The DNA substrate was then incubated with 5

pM recombinant TDP1 in the absence or presence of inhibitor for 15

min at room temperature in reaction buﬀer [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 an 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 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).

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 endorsement by the U.S. Government.

ABBREVIATIONS USED

■

APCI-MS, atmospheric-pressure chemical ionization mass

spectrometry; CPT, camptothecin; DMSO-d₆, dimethyl-d₆

sulfoxide; EI/CI-MS, electron impact/chemical ionization

mass spectrometry; ESI-MS, electrospray ionization mass

spectrometry; HRMS, high resolution mass spectrometry;

MALDI-MS, matrix-assisted laser desorption ionization mass

spectrometry; PMB-Cl, p-methoxybenzyl chloride; TDP1,

tyrosyl-DNA phosphodiesterase I; Top1, human topoisomerase

type IB

REFERENCES

■

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ASSOCIATED CONTENT

* Supporting Information

SMILES molecular formula strings. This material is available

free of charge via the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@

purdue.edu.

■

(8) Debethune, L.; Kohlhagen, G.; Grandas, A.; Pommier, Y.

Processing of Nucleopeptides Mimicking the Topoisomerase I−DNA

Covalent Complex by Tyrosyl-DNA phosphodiesterase. Nucleic Acids

Res. 2002, 30, 1198−1204.

Notes

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The authors declare the following competing ﬁnancial

interest(s): Mark Cushman is on the Board of Directors and

is an investor in Linus Oncology, Inc., which has licensed

indenoisoquinoline intellectual property owned by Purdue

University. Neither Linus Oncology, Inc., nor any other

commercial company sponsored or provided other direct

ﬁnancial support to the author or his laboratory for the research

reported in this article. The remaining authors have no

competing and/or relevant ﬁnancial interest(s) to disclose.

(12) Pfister, T. D.; Reinhold, W. C.; Agama, K.; Gupta, S.; Khin, S.

A.; Kinders, R. J.; Parchment, R. E.; Tomaszewski, J. E.; Doroshow, J.

H.; Pommier, Y. Topoisomerase I Levels in the NCI-60 Cancer Cell

Line Panel Determined by Validated ELISA and Microarray Analysis

and Correlation with Indenoisoquinoline Sensitivity. Mol. Cancer Ther.

2009, 8, 1878−1884.

(13) Takashima, H.; Boerkoel, C. F.; John, J.; Saifi, G. M.; Salih, M.

A. M.; Armstrong, D.; Mao, Y. X.; 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, 32, 267−272.

ACKNOWLEDGMENTS

■

This work was made possible by the National Institutes of

Health (NIH) through support with research grants

U01CA089566 and P30CA023168 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 (Z01-BC006161). This

project has been funded in part with federal funds from the

National Cancer Institute, National Institutes of Health, under

contract no. HHSN261200800001E. The content of this

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DOI: 10.1021/acs.jmedchem.5b00136

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(14) Murai, J.; Huang, S. Y. N.; Das, B. B.; Dexheimer, T. S.; Takeda,

S.; Pommier, Y. Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs

DNA Damage Induced by Topoisomerases I and II and Base

Alkylation in Vertebrate Cells. J. Biol. Chem. 2012, 287, 12848−12857.

(15) Marchand, C.; Lea, W. A.; Jadhav, A.; Dexheimer, T. S.; Austin,

C. P.; Inglese, J.; Pommier, Y.; Simeonov, A. Identification of

Phosphotyrosine Mimetic Inhibitors of Human Tyrosyl-DNA

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DOI: 10.1021/acs.jmedchem.5b00136

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Article Doi

Synthesis and Biological Evaluation of Nitrated 7-, 8-, 9-, and 10-Hydroxyindenoisoquinolines as Potential Dual Topoisomerase i (Top1)-Tyrosyl-DNA Phosphodiesterase i (TDP1) Inhibitors

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