Identification, synthesis, and biological evaluation of metabolites of the experimental cancer treatment drugs indotecan (LMP400) and indimitecan (LMP776) and investigation of isomerically hydroxylated indenoisoquinoline analogues as topoisomerase i poisons

Article

pubs.acs.org/jmc

Identiﬁcation, Synthesis, and Biological Evaluation of Metabolites of

the Experimental Cancer Treatment Drugs Indotecan (LMP400) and

Indimitecan (LMP776) and Investigation of Isomerically

Hydroxylated Indenoisoquinoline Analogues as Topoisomerase I

Poisons

Maris A. Cinelli,^†P. V. Narasimha Reddy,^†Peng-Cheng Lv,^†Jian-Hua Liang,^†Lian Chen,^‡Keli Agama,^§

Yves Pommier,^§Richard B. van Breemen,^‡and Mark Cushman*^,†

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

Purdue University, West Lafayette, Indiana 47907, United States

^‡Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The University of Illinois at Chicago, Chicago,

Illinois 60612, United States

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

United States

ABSTRACT: Hydroxylated analogues of the anticancer topoisomerase I (Top1)

inhibitors indotecan (LMP400) and indimitecan (LMP776) have been prepared because

(1) a variety of potent Top1 poisons are known that contain strategically placed

hydroxyl groups, which provides a clear rationale for incorporating them in the present

case, and (2) the hydroxylated compounds could conceivably serve as synthetic

standards for the identiﬁcation of metabolites. Indeed, incubating LMP400 and LMP776

with human liver microsomes resulted in two major metabolites of each drug, which had

HPLC retention times and mass fragmentation patterns identical to those of the

synthetic standards. The hydroxylated indotecan and indimitecan metabolites and

analogues were tested as Top1 poisons and for antiproliferative activity in a variety of

human cancer cell cultures and in general were found to be very potent. Diﬀerences in

activity resulting from the placement of the hydroxyl group are explained by molecular

modeling analyses.

topotecan (2) and irinotecan (3) are FDA-approved.^1,10,12

These compounds act by intercalating between the base pairs in

INTRODUCTION

■

Topoisomerases are nature’s ubiquitous solution for managing

the topology and torsional states of DNA. Topoisomerase I

(Top1) is an essential enzyme that relaxes supercoiled DNA so

the cleavage complex and binding at the Top1−DNA

interface,¹³where they “poison” the complex (prevent DNA

religation), resulting in persistent, covalent Top1-DNA lesions

that it may be replicated, transcribed, and repaired.¹⁻⁴The

that are then converted into irreversible double-strand breaks

enzyme acts through a nucleophilic tyrosine residue (Tyr723),

when they collide with the advancing replication machinery,

which nicks the phosphodiester backbone of double-stranded,

supercoiled DNA and forms a transient “cleavage complex” in

resulting in apoptosis.⁶⁻⁹Although potent, camptothecin

derivatives suﬀer from many shortcomings, including poor

which the 3′ end of the broken DNA strand is covalently linked

solubility, dose-limiting toxicity, pharmacokinetic limitations

to the enzyme. Within this “cleavage complex”, the scissile

(broken) strand undergoes “controlled rotation” around the

unbroken strand, a process that relaxes the DNA. The catalytic

cycle ends when the 5′ end of the scissile strand religates the

DNA and the enzyme is released. If this cycle is inhibited, DNA

damage ensues, which in turn activates DNA damage responses,

resulting from the instability of the E-ring lactone under

physiological pH, and binding of the lactone hydrolysis product

to plasma proteins.^10,14−16

The indenoisoquinolines were therefore developed as

therapeutic alternatives. In 1998 a COMPARE analysis^17,18

was performed on NSC 314622 (4), which indicated that it

may act in a manner similar to that of camptothecin and

leading to cell cycle arrest or the eventual triggering of

proapoptotic cascades.^1,5−9

derivatives. Indeed, this compound was found to be a Top1

As Topl is overexpressed and DNA damage responses are

inhibitor.¹⁹Since then, many optimization and SAR studies

defective in some human tumors, several Top1 inhibitors have

been developed as chemotherapeutic agents.^4,10Representative

examples are shown in Figure 1. The alkaloid camptothecin

Received: April 11, 2012

(1)¹¹is not used clinically, but its semisynthetic derivatives

A

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Representative Top1 inhibitors.

have produced potent indenoisoquinolines such as MJ-III-65

(5),²⁰⁻²⁴which inhibit Top1 through an intercalation and

interfacial mechanism similar to that of compound 1. Two of

these compounds, indimitecan (LMP776, 6) and indotecan

(LMP400, 7) were promoted into phase I clinical trials at the

National Cancer Institute.²⁵These compounds appear to be

stable and are powerful, cytotoxic Top1 poisons that induce

long-lasting DNA breaks and overcome the drug resistance

issues associated with the camptothecins.^20,26,27

Figure 2. Proposed metabolic pathways and potential metabolites of 6

and 7.

The metabolism of 6 and 7 is currently under investigation,

which has led to the synthesis of potential metabolites to be

used as synthetic standards for metabolism studies. As part of

this study, structural analogues of the proposed metabolites are

also being prepared and investigated for Top1 inhibitory

activity. It was proposed that the indenoisoquinolines 6 and 7

could be metabolically labile at several positions (Figure 2).

The methoxy groups of 6 and 7 are likely to be cleaved in vivo.

O-Dealkylation, catalyzed chieﬂy by hepatic cytochrome P450

enzymes, is a common and well-precedented metabolic process

that, for example, plays a signiﬁcant role in human metabolism

of the chemotherapeutic agents etoposide and teniposide,²⁸

opiates and opiate antagonists,^29,30and the topoisomerase

inhibitors berberine and Genz-644282.^31,32

tives,^33,34MDMA (“ecstasy”),³⁵and the designer drug

MDPPA.³⁶

Compounds 6 and 7 could also be substrates for metabolic

ketone reductases. This phase I process is observed in mice for

the related indenoisoquinoline oracin³⁷as well as for the

indenoisoquinoline rexinoid AM6-36 in human hepatocytes.³⁸

The synthesis of hydroxy and reduced analogues is also justiﬁed

by literature that is rich with potent Top1 inhibitors bearing

phenolic hydroxyls, including the active metabolite of 3 (SN-

38), the alkaloid fagaronine,¹⁰various other 10- and 11-

hydroxycamptothecins,^39,40indolocarbazoles,⁴¹and the dual

Top1/Top2 inhibitor TAS-103.⁴²Additionally, some 11-

hydroxy (reduced) indenoisoquinolines have been synthesized

and tested against Top1 with promising results,^43,44and 9-

hydroxyindenoisoquinolines have been assayed against Top2,⁴⁵

but to our knowledge, no A- or D-ring hydroxyindenoisoquino-

lines have been assayed against Top1. To this end, the

demethylenated/alkylated analogues 14a,b (Scheme 1) and

Methylenedioxy rings are also possible sites of metabolism

(likely via oxidation). Both demethylenation (to yield

catechols) and demethylenation/alkylation processes (to yield

o-methoxyphenols) are observed in rodent and human

metabolism of berberine,³¹safrole and piperonal deriva-

B

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

regioisomers 22a,b (Scheme 2), the demethylated analogues

35a,b (Scheme 4) and regioisomers 44a,b (Scheme 5), the 11-

hydroxyindenoisoquinolines 45a,b (Scheme 6), and the

catechols 52a,b (Scheme 7) were synthesized and evaluated

for Top1 inhibitory and antiproliferative activity and used as

standards for metabolism studies.

literature procedures.^50,51When conducted at low temperature,

the reaction provided the thermodynamically less stable cis

diastereomer 12 as a solid precipitate in good yield. Treatment

of 12 with thionyl chloride at room temperature, followed by

AlCl₃in dichloroethane, resulted in four reactions (acid

chloride formation, Friedel−Crafts cyclization, dehydrogen-

ation, and debenzylation) to yield the crude phenol 13.

Reaction of 13 with imidazole or morpholine in the presence of

sodium iodide in DMF yielded, respectively, the imidazolyl

compound 14a or the morpholinyl compound 14b.

CHEMISTRY

■

Indenoisoquinolines have been prepared by many routes,

including the condensation of primary amines with the

appropriately substituted isochromenones,⁴⁶Suzuki−Miyaura

cross-coupling followed by ring-closing metathesis,⁴⁷and the

oxidative cyclization of the cis acid condensation products

formed by the reaction of homophthalic anhydrides and

benzylidene Schiﬀ bases.⁴⁸The last route was envisioned for

the synthesis of hydroxyindenoisoquinolines, as it has a high

functional group tolerance and was previously used to

synthesize 6 and 7.²²Because of the presence of harsh and

oxidative intermediary steps, the phenol would have to be

unmasked at a late stage in the synthesis, and preparation of the

CD ring (indenone) system of 8-hydroxy-9-methoxyindeno-

isoquinolines (Scheme 1) thus began with benzylation of

isovanillin (8) to yield the ether 9. Condensation of this

aldehyde with 3-bromopropylamine aﬀorded the Schiﬀ base 10

in excellent yield. The isoquinolone (AB) ring system 12 was

synthesized from the condensation of 10 with 4,5-dimethoxy-

homophthalic anhydride (11),⁴⁹prepared by established

Analogously, 9-hydroxy-8-methoxyindenoisoquinolines were

prepared from vanillin (15, Scheme 2). Benzylation (to yield

a

Scheme 2

a

Scheme 1

a

Reagents and conditions: (a) BnBr, DMF, K₂CO₃, rt; (b) 3-

bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃, rt; (c) 11, CHCl₃, 10

°C to rt; (d) SOCl₂, rt; (e) imidazole or morpholine, NaI, DMF, 70

°C; (f), HBr, AcOH, H₂O, 55−70 °C.

16) and treatment with 3-bromopropylamine aﬀorded the

Schiﬀ base 17. Condensation with 11 likewise yielded the cis

acid 18. Treatment with thionyl chloride led to the benzyl-

protected phenol 19. Interestingly, adding 2 equiv of AlCl₃did

not produce a clean debenzylation as observed for the

regioisomer and gave only very low yields of the phenol 20

(Scheme 3). Compound 19 was therefore elaborated with

imidazole or morpholine to obtain the benzyl-protected

phenols 21a and 21b, respectively. Unfortunately, many

standard conditions for debenzylation failed, yielding either

unchanged starting material or decomposition products.

Aqueous hydrobromic acid in AcOH aﬀorded the free phenols

22a and 22b, but the poor solubility of the starting materials

a

Reagents and conditions: (a) BnBr, DMF, K₂CO₃, rt; (b) 3-

bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃; (c) CHCl₃, 10 °C to

rt; (d) (i) SOCl₂, rt, (ii) AlCl₃(2 equiv), 1,2-dichloroethane, rt; (e)

imidazole or morpholine, NaI, DMF, 70 °C.

C

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 3

Scheme 4

a

Reagents and conditions: (a) PMB-Cl, DMF, K₂CO₃, 70 °C; (b) 3-

bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃, rt; (c) 11, CHCl₃, 0

°C to rt; (d) SOCl₂, rt; (e) imidazole or morpholine, NaI, DMF, 70

°C or dioxane, reﬂux.

often resulted in competing demethylation and oxidation

processes, and this method was low-yielding and hard to

reproduce.

An alternative route (Scheme 3) was therefore devised. From

compound 15, O-PMB-vanillin (23) was prepared and

converted into the Schiﬀ base 24. Condensation with

compound 11 aﬀorded the cis acid 25. Treatment of compound

25 with SOCl₂again resulted in four reactions (acid chloride

formation, Friedel−Crafts acylation, dehydrogenation, and

PMB cleavage) to yield 20, which could be obtained on a

multigram scale. Reaction of 20 with imidazole or morpholine

and sodium iodide in DMF (the use of dioxane under argon

resulted in improved yields) led to 22a and 22b in acceptable

yields.

Several procedures were used to prepare A-ring demethy-

lated analogues. A similar benzyl protecting group strategy was

employed to prepare homophthalic anhydrides containing the

desired protected phenols, which was necessary in order to

avoid colored byproducts and other unidentiﬁed impurities

formed from the unprotected phenols in the presence of the

harsh acidic, basic, and oxidative conditions (Schemes 4 and 5).

Therefore, to prepare 2-hydroxy-3-methoxyindenoisoquino-

lines, homoisovanillic acid (26, Scheme 4) was ﬁrst benzylated

to yield compound 27.⁵²Ortho-hydroxymethylation aﬀorded

the homophthalide 28,⁵³which was saponiﬁed and oxidized

with KMnO₄to yield the diacid 29. The anhydride 30 was

prepared by heating the diacid in reﬂuxing acetyl chloride.

a

Reagents and conditions: (a) (i) KOH, BnCl, EtOH, reﬂux, (ii)

KOH, H₂O, reﬂux; (b) H₂CO, H₂O, HCl, AcOH, 45 °C; (c) (i)

KOH, H₂O, rt, (ii) KMnO₄, H₂O, 0 °C to rt, (iii) EtOH, reﬂux; (d)

AcCl, reﬂux; (e) 3-bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃, rt;

(f) CHCl₃, 0 °C to rt; (g) (i) SOCl₂, rt, (ii) AlCl₃(2 equiv) DCE, 0

°C; (h) imidazole or morpholine, NaI, DMF, 60 °C.

Compound 30 was condensed with the Schiﬀ base 32 derived

from piperonal (31) to yield the cis acid 33 in excellent yield.²¹

The use of aluminum chloride resulted in debenzylation

following the cyclization, but yields of the indenoisoquinoline

34 were very low (10−15%). A large amount of grayish

insoluble decomposition product formed, presumably via

competing demethylenation, oxidation, and complexation

processes. Nonetheless, 34 could be treated with sodium

iodide and imidazole or morpholine, and the analogues 35a and

35b were eventually obtained in pure form.

A similar route was used to prepare 3-hydroxy-2-methoxy-

indenoisoquinolines. Homovanillic acid (36, Scheme 5) was

hydroxylmethylated to yield compound 37.⁵⁴Benzylation

yielded 38, and a saponiﬁcation/oxidation sequence was

employed to prepare the homophthalic acid 39, which was

then converted into the anhydride 40. Condensation with 32

D

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 5

Scheme 6

a

Reagents and conditions: (a) NaBH₄, MeOH, 0 °C.

a

Scheme 7

a

Reagents and conditions: (a) H₂CO, H₂O, HCl, AcOH, 120 °C to rt;

(b) BnBr, K₂CO₃, acetone, 56 °C; (c) (i) KOH, H₂O, rt, (ii) KMnO₄,

H₂O, 0 °C to rt, (iii) EtOH, reﬂux; (d) AcCl, reﬂux; (e) 3-

bromopropylamine HBr, Et₃N, Na₂SO₄, CHCl₃, rt; (f) CHCl₃, 0 °C to

rt; (g) SOCl₂, rt; (h) AlCl₃(2 equiv), nitrobenzene, 90 °C; (i)

imidazole or morpholine, NaI, DMF, 70 °C.

aﬀorded the cis acid 41. Treatment of 41 with SOCl₂produced

the benzyl intermediate 42, but deprotection (here performed

with AlCl₃in nitrobenzene) was also low yielding (∼10%), as

observed for the regioisomer (vide supra). The resulting phenol

43 was elaborated as earlier described to yield the analogues

44a and 44b.

11-Hydroxy (keto-reduced) indenoisoquinoline analogues

45a and 45b were prepared via sodium borohydride reduction

of 6 and 7, respectively (Scheme 6). Finally, the catechols 52a

and 52b (Scheme 7) were also prepared using the benzyl

protecting group strategy. 3,4-Dihydroxybenzaldehyde (46)

was dibenzylated to yield compound 47 and converted into the

Schiﬀ base 48. Condensation with anhydride 32 aﬀorded the cis

acid 49, which was cyclized in cold SOCl₂to furnish the

intermediate indenoisoquinoline 50. Treatment with imidazole

a

Reagents and conditions: (a) BnBr, DMF, rt to 70 °C; (b) 3-

bromopropylamine HBr, Et₃N, Na₂SO₄, rt; (c) 11, CHCl₃, 0 °C to rt;

(d) SOCl₂, −4 °C to rt; (e) imidazole or morpholine, NaI, dioxane, 65

°C; (f) 48% HBr−H₂O, 70 °C (for 51a); (g) H₂, Pd/C, MeOH−THF

(for 51b).

or morpholine as described earlier yielded the protected

compounds 51a and 51b, respectively. Heating with aqueous

hydrobromic acid aﬀected the debenzylation of 51a (yielding

the catechol 52a), and atmospheric pressure hydrogenation of

51b aﬀorded the analogous compound 52b.

E

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

the loss of a methyl group, and comparison with the standards

identiﬁed this metabolite as the 3-desmethyl compound 44a.

An identical analysis performed for compound 7 (Figure 4)

yielded similar results; the most abundant metabolite M1 [12.3

METABOLISM STUDIES

■

Compounds 6 and 7 were incubated at 37 °C with pooled

human liver microsomes in the presence of NADPH (full

details in Experimental Section). After chilling and termination

of the reactions, the samples were centrifuged, and the

supernatants were analyzed by LC−MS and LC−tandem

electrospray mass spectrometry (LC−MS/MS). Indenoisoqui-

nolines 14a,b, 22a,b, 35a,b, 44a,b, 45a,b, and 52a,b were used

as synthetic standards.

In the case of both 6 and 7, positive-ion electrospray LC−

MS detected two major metabolites in their protonated forms.

The formation of these species required both liver microsomes

and NADPH, since omission of either produced no detectable

metabolites. In the case of 6, the most abundant metabolite M1

(Figure 3) was detected at a retention time of 12.8 min.

Figure 4. LC−MS retention times (a) and positive ion electrospray

ion tandem mass spectra fragmentation patterns (b) for metabolites

obtained upon incubation of 7 with human liver microsomes.

min, m/z 467.1823, C₂₅H₂₇N₂O₇(1.1 ppm)] matched a

synthetic standard of catechol 52b. Likewise, the structure of

metabolite M2 [13.6 min, m/z 465.1654, C₂₅H₂₅N₂O₇(−1.7

ppm)] was conﬁrmed as 3-desmethyl-LMP400 (44b) by

comparison of the retention time and fragmentation pattern

of the metabolite with those of the synthetic standard.

In both cases, these were the only metabolites detected, and

no matches were found with the other standards (14a,b, 22a,b,

and the regiomeric 2-desmethyl compounds 35a,b or 45a,b),

indicating that the predominant routes of human hepatic

metabolism for 6 and 7 are likely demethylenation and 3-O-

demethylation, as predicted by both the apparent metabolic

lability of these sites (Figure 2) and extensive literature

precedent. Catechol methylation, 2-O-demethyation, and

ketone reduction do not occur.

Figure 3. LC−MS retention times (a) and positive ion electrospray

ion tandem mass spectra fragmentation patterns (b) for metabolites

obtained upon incubation of 6 with human liver microsomes.

Accurate mass measurement provided an m/z of 448.1508,

which was within −0.2 ppm of the theoretical formula of

C₂₄H₂₂N₃O₆. This formula was consistent with the loss of a

methylene group, and LC−MS/MS comparison with the

synthetic standards conﬁrmed this metabolite as catechol 52a.

A second metabolite, M2, eluted at 13.2 min and produced an

m/z of 446.1361, which corresponded to the elemental

composition C₂₄H₂₀N₃O₆(2.0 ppm). This formula suggested

BIOLOGICAL EVALUATION OF

HYDROXYINDENOISOQUINOLINES

The indenoisoquinolines 14a,b, 22a,b, 35a,b, 44a,b, 45a,b, and

52a,b were tested for antiproliferative activity in the National

Cancer Institute’s developmental therapeutics assay (the “NCI-

60”) against cell lines derived from a variety of human tumors

(approximately 60 lines were used).^55,56After an initial one-

■

F

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Antiproliferative Potencies and Topoisomerase I Inhibitory Activities of Hydroxyindenoisoquinolines

a

cytotoxicity (GI₅₀in μM)

lung

HOP-62

colon

HCT-116

CNS

SF-539

melanoma

UACC-62

Ovarian

OVCAR-3

renal

SN12C

prostate

DU-145

breast

MCF-7

Top 1

c

b

compd

MGM

cleavage

d

1⁵⁷

0.01

0.03

35

0.01

41

0.01

0.22

73

0.02

0.01

1.58

<0.01

0.01

0.37

0.05

0.03

<0.01

0.02

0.040 0.0187

20.0 14

0.21 0.19

0.079 0.023

4.64 1.25

41.8 7.15

3.07 0.32

0.055 0.003

0.087 0.063

0.049

++++

++

4⁵⁷

1.3

4.2

68

37

5⁴⁶

0.02

0.10

<0.01

1.15

>100

2.34

<0.01

0.03

0.02

0.279

<0.01

0.02

1.35

2.40

0.229

0.407

0.04

0.03

0.5

<0.01

0.813

<0.01

0.155

>100

0.204

<0.01

0.02

++++

+++++

++(+)

+++

6⁴⁶

<0.01

1.78

<0.01

0.03

0.08

74.1

>100

8.32

0.02

0.03

0.07

0.871

0.03

1.95

4.79

0.407

0.501

7⁴⁶

14a

14b

22a

22b

35a

35b

44a

44b

45a

45b

52a

52b

>100

0.180

<0.01

0.02

0.331

0.07

0.282

<0.01

0.02

0.191

<0.01

0.02

<0.01

0.02

++++(+)

+++++

++(+)

++++

+++(+)

++

<0.01

0.257

<0.01

0.01

<0.01

0.335

<0.01

0.479

2.40

0.03

<0.01

0.195

<0.01

0.501

3.39

<0.01

0.257

<0.01

0.407

2.63

0.282

<0.01

0.372

0.776

<0.01

0.078

0.144

<0.01

0.427

0.646

0.02

0.412 0.005

0.043

0.056

0.549

2.09

1.66

3.16

++

0.04

0.01

0.03

0.100

0.224

++

0.371

0.148

0.065

0.490

0.050

0.602

++++

a

b

The cytotoxicity GI₅₀values are the concentrations corresponding to 50% growth inhibition. Mean-graph midpoint for growth inhibition of all

c

human cancer cell lines successfully tested, ranging from 10⁻⁸to 10⁻⁴M. Compound-induced DNA cleavage due to Top1 inhibition is graded by

the following rubric relative to 1 μM camptothecin: 0, no inhibitory activity; +, between 20% and 50% activity; ++, between 50% and 75% activity; +

d

++, between 75% and 95% activity; ++++, equipotent; +++++, more potent. For MGM GI₅₀values in which a standard error appears, the GI₅₀

values for individual cell lines are the average of two determinations; values without standard error are from one determination. The values for 1, 4, 5,

6, and 7 are from many determinations.

Figure 5. Top1-mediated DNA cleavage induced by indenoisoquinolines 22b, 14b, 35a, 44a, and 45a: lane 1, DNA alone; lane 2, Top1 + DNA; lane

3, 1, 1 μM; lane 4:, 5, 1 μM; lanes 5−24, 22b, 14b, 35a, 44a, and 45a at 0.1, 1, 10, and 100 μM, respectively, from left to right. Numbers and arrows

on the left indicate arbitrary cleavage site positions.

dose prescreening assay at moderately high concentration (10⁻⁵

M), selected compounds were tested at ﬁve concentrations

ranging from 10⁻⁸to 10⁻⁴M. Overall antiproliferative potential

is quantiﬁed as a mean-graph midpoint (MGM). This value can

be interpreted as a rough average GI₅₀across the whole assay,

where values that fall outside the tested concentration range

(<10⁻⁸or >10⁻⁴M) are respectively assigned either 10⁻⁸or

10⁻⁴M. These MGM values, along with GI₅₀values from

selected cell lines, are reported in Table 1. For completeness,

the MGM values for 1, 4, 5, 6, and 7 are also given.

Top1 inhibition was graded by the ability of a compound to

induce enzyme-linked DNA breakage and is reported on a

semiquantitative scale relative to 1 μM camptothecin: 0, no

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

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

activity; ++++, equipotent; +++++, more potent. Ambiguous

scores (e.g., between two values) are designated with

G

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 6. Minimized, top-ranked GOLD pose of compound 22b in ternary complex with DNA and Top1, constructed in SYBYL. The ligand is

colored in purple. Surrounding structures are colored by atom. Water molecules are depicted as red spheres, and hydrogens have been omitted. All

distances are measured from heavy atom to heavy atom. The diagram is programmed for wall-eyed (relaxed) viewing.

Figure 7. Minimized, top-ranked GOLD pose of compound 14b in ternary complex with DNA and Top1 (constructed in SYBYL). The ligand is

colored in cyan, surrounding structures are colored by element, and hydrogens have been omitted. All distances are measured from heavy atom to

heavy atom. The same water molecules from Figure 4 are shown as red spheres.

parentheses (e.g., ++(+) would be between ++ and +++).

These values are also reported in Table 1.

approximately half the anti-Top1 activity and their MGM

values are orders of magnitude greater than their isomeric

counterparts. Similar regiochemical eﬀects are reported for

other classes of Top1 inhibitors (indenoisoquinolines,

camptothecins, and aromathecins, among others).

What could be responsible for such a drastic eﬀect? A

molecular modeling and docking study was performed in an

attempt to rationalize this disparity. By use of GOLD, the

minimized structures of morpholine 22b and its regioisomer

14b were docked into the cleavage site of a mutant, solvated

Top1/topotecan crystal structure (modiﬁed from PDB code

1K4T) using the procedure previously described by Peterson et

al.⁴⁶The minimized, highest-ranked GOLD pose for

compound 22b is shown in Figure 6.

A major commonality among Top1 poisons is that a large

amount of steric bulk (e.g., multiple methoxy groups, halogens)

projecting toward the nonscissile strand often has deleterious

eﬀects on potency in general.^39,40,57Indeed, crystal structures of

several Top1 inhibitors⁵⁸reveal this region to be very “tight”

sterically, in agreement with the observation of a low functional

group tolerance. However, the 9-hydroxyl group of 22b appears

to be quite well accommodated here. The indenoisoquinoline is

calculated to bind in an intercalative mode identical to that

observed in the crystal structure of a related Top1−DNA−

indenoisoquinoline ternary complex,⁵⁸and the hydroxyl group

is involved in water-mediated hydrogen bonding with Lys374.

It is possible that with induced ﬁt, this hydroxyl could also

make contact with the nearby deoxyfuranose oxygen (here, 4.0

A representative example of Top1-linked DNA cleavage by

hydroxyindenoisoquinolines is shown in Figure 5. As observed

in the ﬁgure and listed in Table 1, all compounds tested

exhibited at least some Top1 inhibitory activity, with

compounds 22a, 22b, and 35a possessing excellent inhibitory

activity comparable to or greater than the clinical candidates 6

and 7. These compounds also have submicromolar antiprolifer-

ative potency (MGM values of 55 nM for 22a, 87 nM for 22b,

and 49 nM for 35a), comparable to that of 6 (79 nM), and

although they were weaker Top1 poisons, the conﬁrmed

metabolites 44a and 44b had MGM values of 43 and 56 nM,

respectively, which could serve to prolong the antitumor eﬀects

of 6 and 7 in vivo. A standard COMPARE analysis^17,18seeded

with the GI₅₀data for 22a, 22b, and 35a displayed moderate to

high correlations (Pearson coeﬃcients of 0.65−0.8) with the

cytotoxicity proﬁle of topotecan (2), indicating that it is likely

these compounds exert their cytotoxic or antiproliferative

eﬀects predominantly via Top1 inhibition. Generally, anti-Top1

activity and antiproliferative activity correlate well, with those

compounds that are better Top1 inhibitors displaying higher

potency in the NCI-60 screen.

Interestingly, the D-ring hydroxyindenoisoquinolines display

a striking correlation between regiochemistry, Top1 inhibition,

and cytotoxicity. While the 9-hydroxy-8-methoxyindenoisoqui-

nolines 22a and 22b are extremely potent, the regiomeric 8-

hydroxy-9-methoxyindenoisoquinolines 14a and 14b possess

H

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Å away), while the 8-methoxy group projects toward the major

groove of the complex.

supports this hypothesis as well: there is no diﬀerence (∼0.03

points) between the GoldScores for the top-ranked poses of

35b and 44b (and the average scores for the top four poses are

only ∼3 points apart), and the van der Waals and hydrogen-

bond contributions are approximately equal in both cases,

indicating that the diﬀerence in Top1 activity between these

compounds (lower than that observed between series 22 and

series 14) may not be due to placement of the hydroxyl group.

The literature supports this assumption as well: the

benzonaphthyridine Top1 poison Genz-644282 is very similar

in structure to 6 and 7, and its A-ring desemethyl metabolites

do not display any correlation between regiochemistry and

Top1 inhibition.³²

The lowest activity observed was from the 11-hydroxy

(reduced keto) analogues 45a and 45b. Although some potent

11-hydroxy and 11-alkoxyindenoisoquinolines have been

reported, the molecular modeling studies indicated that they

bind diﬀerently in the ternary cleavage complex, with an N-(p-

methoxybenzyl) group instead of the indenoisoquinoline ring

system intercalating between the base pairs.^43,44The fact that

the indenoisoquinoline oracin is deactivated by ketone

reduction is also consistent with the low potency of 45a and

45b.³⁷Overall, the evidence indicates that the ketone is

important for optimal binding of the indenoisoquinoline in the

DNA intercalative mode. The crystal structure of an

indenoisoquinoline−DNA−Top1 ternary complex (PDB code

1SC7)⁵⁸has demonstrated that the ketone hydrogen bonds to

the minor groove residue Arg364 (proposed to aid in the

stabilization of other bound inhibitors). The hydroxyl groups of

45a and 45b still can behave as hydrogen bond acceptors in our

models (not shown), so lack of hydrogen-bond-accepting

capability per se cannot be responsible for the decrease in

activity. Perhaps the reduction of the ketone changes the

electronics of the system, interfering with π−π stacking, or the

introduction of a tetrahedral carbon introduces nonplanarity

that could disrupt intercalation.

The highest ranked, energy minimized GOLD pose for

compound 14b is shown in Figure 7. The nonscissile side

appears to be signiﬁcantly more crowded, as the 9-methoxy

group now projects back toward the phosphodiester backbone,

and the network of water-mediated contacts is absent. The 8-

hydroxy group projects toward the solvent, where it hydrogen-

bonds with a nearby glutamate residue (Glu356). It is

conceivable that this contact could stabilize the binding of

14b, as its activity is not abolished completely relative to 22b,

and Glu356 is proposed to aid the binding of indolocarbazoles

and compound 2.⁵⁸Nonetheless, there is a sizable diﬀerence in

the GoldScores (67.9 for 14b vs 81.6 for 22b), and the average

diﬀerence in GoldScores between the top four poses for 22b

and 14b is approximately 16 points. As predicted by the model,

the diﬀerence in the hydrogen bonding term of the GoldScore

between the regioisomers is negligible (average of 10.3 for the

top four poses of 14b; 12.1 for the top four of 22b), but the

diﬀerence in the external van der Waals terms (a component of

the ﬁtness score that reﬂects a simple attractive/repulsive

potential)^59,60is signiﬁcant, 56 (average 53.3) for 14b versus 65

(average 62.6) for 22b, likely symbolizing a “less favorable” or

“more repulsive” ﬁt for the 9-methoxy isomer. Interestingly,

some 9-methoxyindenoisoquinolines are quite active against

Top1, although most of these compounds also contain a 3-nitro

group (which is well-documented to improve potency),^23,57

indicating that favorable A-ring electronics may somehow be

enough to oﬀset a negative steric eﬀect.

Additionally, perhaps the placement of 9-hydroxyl group is

important as well. In a similar study of camptothecins

performed by Wall et al.,⁴⁰most substitutions at position 11

of the camptothecin system attenuate the Top1 inhibitory

activity (Morrell and others hypothesize that this position is

roughly analogous to the indenoisoquinoline position 9^57,61).

One notable exception to this SAR is the active 11-

hydroxycamptothecin (53, Figure 8), which would point its

The catechols 52a and 52b are both also active against Top1,

although the imidazole 52a has only half the potency of the

morpholine 52b. Signiﬁcant diﬀerences between imidazole and

morpholine substituents were observed for other pairs in this

study (cf. 35a and 35b), although in the case of 35b vs 35a the

substituent eﬀects on potency are opposite those observed with

52a vs 52b.

CONCLUSIONS

■

Figure 8. 11-Hydroxycamptothecin.

A series of 8-, 9-, 2-, and 3-hydroxyindenoisoquinolines, 8,9-

dihydroxyindenoisoquinolines, and reduced keto analogues of

the clinical candidates 6 and 7 were designed based on the

rationale that (a) they are potential metabolites of indotecan

and indimitecan and could be useful as synthetic standards to

compare with the actual metabolites and (b) there are many

examples of potent hydroxylated/phenolic Top1 poisons in the

literature, so the hydroxyindenoisoquinolines might be of value

as anticancer drugs. To this end, the indenoisoquinolines were

prepared by the homopththalic anhydride/Schiﬀ base con-

densation method⁴⁹and were assayed against human Top1 and

a panel of human cancer cells. Interestingly, many of these

hydroxylated indenoisoquinolines are extremely potent Top1

poisons, especially those bearing the 9-hydroxy-8-methoxy

substitution pattern on the D ring. Molecular modeling

supports the observed activity, especially when compared to

the much less active 8-hydroxy-9-methoxyindenoisoquinolines.

A less signiﬁcant dependence of Top1 inhibitory potency on

phenolic hydroxyl toward the nonscissile strand as well. Further

support comes from a study of the regioisomers of

hydroxylated indolocarbazoles performed by Zembower et

al.⁶²Those compounds possessing a hydroxyl at position 9

(also roughly analogous to the numerically identical position of

indenoisoquinolines) are the most potent!⁶²

Interestingly, the regiochemical dependence is not observed

on the A-ring side, and activity here is variable. The 2-

hydroxyindenoisoquinoline 35a and 3-hydroxyindenoisoquino-

line 44b both possess signiﬁcant anti-Top1 activity, although

some diﬀerences between the morpholines and imidazoles are

observed. Assuming these compounds bind in ternary complex

as 22b and 14b are proposed to, the indenoisoquinoline A-ring

ﬁts into the scissile-strand cavity. The steric tolerance of this

spacious region is much higher, and it may render

regiochemical diﬀerences minimal to irrelevant. Docking

I

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

¹H NMR (CDCl₃) δ 9.82 (s, 1 H), 7.49−7.32 (m, 6 H), 7.02 (d, J =

regiochemistry was observed for A-ring hydroxyindenoisoqui-

nolines, a fact that is in agreement with our docking study.

Additionally, a metabolism study indicates that several of the

prepared hydroxyindenoisoquinolines are produced upon

incubation of the parent drugs with human liver microsomes

and NADPH. These data indicate that the primary routes of

metabolism are 3-demethylation and cleavage of the 8,9-

methylenedioxy group. Three additional points can be made

about the potential future signiﬁcance of this study: (1) the fact

that the metabolites are biologically active can be expected to

prolong the duration of antitumor activity expressed after drug

administration; (2) the biologically active hydroxylated

indenoisoquinolines provide convenient starting points for

further drug development, especially with regard to prodrugs,

since the phenolic hydroxyl groups provide convenient points

for attachment of prodrug modules; (3) the altered solubilities

of the hydroxylated derivatives may oﬀer advantages for iv

formulation.

8.2 Hz, 1 H), 5.20 (s, 2 H), 3.97 (s, 3 H).

N-[3′-(Benzyloxy)-4′-methoxybenzylidene]-3-bromopro-

pan-1-amine (10). 3-Bromopropylamine hydrobromide (2.08 g, 9.49

mmol) was diluted with CHCl₃(10 mL). Compound 9 (2.00 g, 8.26

mmol) was added slowly as a solution in CHCl₃(10 mL) and

quantitatively transferred with 2 mL of the same solvent. Et₃N (0.917

g, 9.08 mmol) was added slowly, upon which the solution became

clear and colorless. Na₂SO₄(1.00 g, 5.21 mmol) was added, and the

mixture was stirred at room temperature for 24 h. Additional 3-

bromopropylamine salt (0.905 g, 4.13 mmol), Et₃N (0.500 g, 4.96

mmol), and approximately 0.500 g of Na₂SO₄were added. After a total

of 48 h, the mixture was washed with H₂O (3 × 70 mL) and saturated

aqueous NaCl (70 mL). The organic layer was dried over anhydrous

sodium sulfate and concentrated to yield a dark yellow syrup (2.86 g,

96%). IR (ﬁlm) 3291, 2932, 2837, 1645, 1601, 1511, 1431, 1265, 1137,

1

1024, 741, 697 cm⁻¹; H NMR (CDCl₃) δ 8.21 (s, 1 H), 7.49−7.31

(m, 6 H), 7.21 (dd, J = 2.1, 8.3 Hz, 1 H), 6.92 (d, J = 8.3 Hz, 1 H),

5.19 (s, 2 H), 3.93 (s, 3 H), 3.92 (s, 3 H), 3.73 (td, J = 1.0, 6.2 Hz, 2

H), 3.51 (t, J = 6.5 Hz, 2 H), 2.30−2.20 (m, 2 H); ESIMS m/z (rel

intensity) 362/364 (MH⁺, 97/100).

cis-[3-(Bromopropyl)amino)]-4-carboxy-3,4-dihydro-6,7-di-

methoxy-3-(3-benzyloxy-4-methoxyphenyl)-1(2H)-isoquino-

lone (12). Anhydride 11 (1.40 g, 6.30 mmol) was dissolved in CHCl₃

(35 mL), and the resulting solution was cooled to 0 °C. A solution of

compound 10 (2.40 g, 6.63 mmol) in cold CHCl₃(15 mL) was added

slowly over 5 min and quantitatively transferred with CHCl₃(5 mL).

Precipitate began forming after 30 min. The mixture was stirred for 2 h

at 0 °C and then warmed to room temperature and stirred for an

additional 2 h. The precipitate was ﬁltered out and washed with

CHCl₃(50 mL) to aﬀord the title compound as a white solid (2.81 g

77%) after drying: mp 193−195 °C (dec). IR (ﬁlm) 2933, 1733, 1619,

EXPERIMENTAL SECTION

■

General Procedures. Reagents and solvents were purchased from

commercial vendors and were used without further puriﬁcation.

Melting points were determined in capillary tubes using a Mel-Temp

apparatus and are not corrected. Infrared spectra were obtained as

ﬁlms on salt plates unless otherwise speciﬁed, using a Perkin-Elmer

Spectrum One FT-IR spectrometer, and are baseline-corrected. ¹H

NMR spectra were obtained at 300 or 500 MHz, using a Bruker

ARX300 and Bruker Avance 500 (QNP probe or TXI 5 mm/BBO

probe). ¹³C NMR spectra were obtained at 125 or 75 MHz. Mass

spectral analyses were performed at the Purdue University Campus-

Wide Mass Spectrometry Center. ESIMS was performed using a

FinniganMAT LCQ Classic mass spectrometer system. The electro-

spray ionization high resolution mass measurements were obtained in

the peak matching mode using a FinniganMAT XL95 (FinniganMAT

Corp., Bremen, Germany) mass spectrometer. The instrument was

calibrated to a resolution of 10 000 with a 10% valley between peaks

using the appropriate polypropylene glycol standards. EI/CIMS was

performed using a Hewlett-Packard Engine or GCQ FinniganMAT

mass spectrometer system. APCI-MS was performed using an Agilent

6320 trap mass spectrometer. Combustion microanalyses were

performed by Midwest Microlab LLC (Indianapolis, IN). Reported

values are within 0.4% of calculated values. HPLC was performed

using a Waters 1525 binary HPLC pump with a Waters 2487 dual

absorbance detector and an injection volume of 10 μL. A Sunrise C18

5 μm 100 Å reverse-phase column, with dimensions of 15 cm × 4.6

mm (ES Industries), was used for all HPLC experiments. The intensity

of the major peak in the analytical HPLC trace of each target

compound was ≥95% that of the combined intensities of all of the

peaks detected at 254 nm, and two diﬀerent solvent systems were used

to ascertain purity. Analytical thin-layer chromatography was

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

Compounds were visualized with both short and long wavelength UV

light. Silica gel ﬂash chromatography was performed using 40−63 μm

ﬂash silica gel.

1

1597, 1573, 1515, 1169, 1027, 696 cm⁻¹; H NMR (CDCl₃-DMSO-

d₆) δ 7.53 (s, 1 H), 7.19−7.13 (m, 5 H), 7.05 (s, 1 H), 6.53 (s, 3 H),

4.83 (d, J = 6.4 Hz, 1 H), 4.79 (s, 2 H), 4.44 (d, J = 6.2 Hz, 1 H), 3.81

(s, 3 H), 3.81−3.70 (m, 1 H), 3.73 (s, 3 H), 3.64 (s, 3 H), 3.30−3.20

(m, 2 H), 2.95−2.90 (m, 1 H), 2.01−1.80 (m, 2 H); APCI-MS m/z

(rel intensity) 540 (MH⁺− CO₂, 100). Anal. Calcd for

C₂₉H₃₀BrNO₇·H₂O: C, 57.81; H, 5.35; N, 2.32. Found: C, 57.57; H,

4.99; N, 2.32.

6-(3-Bromopropyl)-5,6-dihydro-8-hydroxy-2,3,9-trime-

thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13). The cis

acid 12 (1.00 g, 1.71 mmol) was diluted with SOCl₂(25 mL). The

dark reddish-brown mixture was stirred at room temperature for 2 h

and 20 min and was concentrated. The residue was dissolved in 1,2-

dichloroethane (40 mL), and aluminum chloride (0.479 g, 3.6 mmol)

was added. The mixture was stirred at room temperature for 20 h and

was quenched slowly by the addition of saturated aqueous NaHCO₃

(40 mL) and H₂O (50 mL). The aqueous and organic layers were

separated, and the aqueous phase was extracted with CHCl₃(2 × 50

mL). The organic phase was washed with H₂O (2 × 100 mL) and

saturated aqueous NaCl (100 mL), dried over anhydrous sodium

sulfate, and concentrated. The obtained residue was adsorbed onto

SiO₂(∼7 g) and puriﬁed by ﬂash column chromatography (SiO₂, ∼50

g), eluting with a gradient of 0.25% MeOH in CHCl₃to 1% MeOH in

CHCl₃to aﬀord the crude product as a pinkish solid (0.414 g, 51%)

after washing with 10% CHCl₃in ether (2 × 50 mL) and ether (50

mL) and drying: mp 248−252 °C. IR (ﬁlm) 3296, 1635, 1554, 1471,

1396, 1396, 1030, 871, 762, 761 cm⁻¹; ¹H NMR (CDCl₃) δ 8.04 (s, 1

H), 7.64 (s, 1 H), 7.31 (s, 1 H), 7.16 (s, 1 H), 7.95 (s, 1 H), 4.62 (t, J

= 7.5 H, 2 H), 4.06 (s, 3 H), 3.99 (s, 6 H), 3.65 (t, J = 6.5 Hz, 1 H),

2.50−2.40 (m, 2 H); EIMS m/z (rel intensity) 475 (M⁺, 100).

5,6-Dihydro-8-hydroxy-6-[3-(1H-imidazol-1-yl)propyl]-2,3,9-

trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (14a).

Compound 13 (0.300 g, 0.632 mmol), imidazole (0.517 g, 7.59

mmol), and NaI (1.14 g, 0.7.59 mmol) were diluted with anhydrous

DMF (90 mL), and the mixture was heated to 60 °C for 18 h. The

mixture was cooled and poured into H₂O (200 mL), and the solution

was extracted with CHCl₃(2 × 100, 1 × 50 mL). The burgundy-

colored organic layer was washed with H₂O (5 × 200 mL) and

Benzylisovanillin (9).⁶³Isovanillin (8, 3.00 g, 19.7 mmol) was

diluted with anhydrous DMF (50 mL). Benzyl bromide (3.47 g, 20.3

mmol) was added slowly, followed by K₂CO₃(6.54 g, 47.3 mmol).

The yellow mixture was rapidly stirred at room temperature for 2 h

and 15 min. The mixture was partitioned into an ether−water mixture

(1:1, 100 mL of each) and stirred. The organic and aqueous layers

were separated, and the aqueous layer was extracted with ether (1 × 50

mL, 2 × 25 mL). The combined organic layers were washed with H₂O

(2 × 50 mL) and saturated aqueous NaCl (50 mL), dried over

anhydrous sodium sulfate, and concentrated to yield a white

microcrystalline solid (4.43 g, 93%) after washing with hexanes (50

mL) and reﬁltering the ﬁltrate: mp 55−58 °C (lit.⁶³mp 61−62 °C).

J

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

saturated aqueous NH₄Cl (200 mL). The organic phase was dried over

anhydrous sodium sulfate, concentrated, and the residue was washed

with ether (100 mL) and ﬁltered. The precipitate was dissolved in

CHCl₃(50 mL), and the solution was concentrated and adsorbed

onto SiO₂(∼7 g) and puriﬁed by ﬂash column chromatography (SiO₂,

50 g), eluting with a gradient of 1% MeOH in CHCl₃to 4% MeOH in

CHCl₃, to yield a fuchsia-colored solid (0.112 g, 38%) after suspending

in DMF (10 mL) and precipitating with ether (150 mL) and washing

with ether (100 mL) and drying: mp 265−266 °C (dec). IR (ﬁlm)

3306, 2917, 1659, 1622, 1554, 1472, 1394, 1335, 1265, 1085, 1032,

1455, 1419, 1267, 1231, 1138, 1033, 743, 697 cm⁻¹; ¹H NMR

(CDCl₃) δ 8.22 (s, 1 H), 7.45−7.31 (m, 6 H), 7.10 (dd, J = 8.2, 1.8

Hz, 1 H), 6.90 (d, J = 8.2 Hz, 1 H), 5.21 (s, 2 H), 3.95 (s, 3 H), 3.74

(t, J = 6.2 Hz, 2 H), 3.51 (t, J = 6.3 Hz, 2 H), 2.30−2.20 (m, 2 H);

ESIMS m/z (rel intensity) 362/364 (MH⁺, 100/95).

cis-3-(4-Benzyloxy-3-methoxyphenyl)-N-(3-bromopropyl)-4-

carboxy-3,4-dihydro-6,7-dimethoxy-1(2H)-isoquinolone (18).

Compound 11 (1.93 g, 8.71 mmol) was diluted with CHCl₃(40

mL), and the mixture was cooled to 10 °C. The Schiﬀ base 17 (3.00 g,

8.28 mmol) was diluted in CHCl₃(20 mL, cooled to 10 °C), and this

solution was added slowly to the anhydride solution and transferred

with 5 mL of the same solvent. The mixture was stirred at 10 °C for 30

min and was then allowed to slowly warm to room temperature. After

3 h and 20 min, a precipitate had formed. This precipitate was

collected and washed with CHCl₃(30 mL) to yield a white solid (2.86

g, 59%) after drying: mp 172−175 °C. IR (ﬁlm) 2933, 1743, 1621,

1595, 1575, 1464, 1289, 1259, 1174, 1103, 1028, 908, 730, 697 cm⁻¹;

¹H NMR (CDCl₃) δ 7.72 (s, 1 H), 7.38−7.29 (m, 5 H), 7.09 (s, 1 H),

1

762, 688 cm⁻¹; H NMR (DMSO-d₆) δ 10.10 (br s, 1 H), 7.84 (s, 1

H), 7.73 (s, 1 H), 7.44 (s, 1 H), 7.27 (s, 1 H), 7.04 (s, 1 H), 6.99 (s, 1

H), 6.92 (s, 1 H), 4.37 (t, J = 7.2 Hz, 2 H), 4.20 (t, J = 7.0 Hz, 2 H),

3.88 (s, 3 H), 3.85 (s, 3 H), 3.84 (s, 3 H), 2.25−2.20 (m, 2 H); ESIMS

m/z (rel intensity) 462 (MH⁺, 100). Purity was estimated to be

∼100% by HPLC in both 90% MeOH−H₂O and 85% MeOH−H₂O.

Anal. Calcd for C₂₅H₂₃N₃O₆·H₂O: C, 62.62; H, 5.26; N, 8.76. Found:

C, 62.40; H, 4.85; N, 8.57.

5,6-Dihydro-8-hydroxy-2,3,9-trimethoxy-6-[3-(N-

morpholino)propyl]-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(14b). Compound 13 (0.090 g, 0.190 mmol) and NaI (0.200 g, 1.32

mmol) were diluted with anhydrous DMF (20 mL). Morpholine

(0.115 g, 1.32 mmol) was added, and the mixture was heated to 70 °C

for 18 h. The mixture was cooled and poured into H₂O (100 mL), and

the solution was extracted with CHCl₃(3 × 50 mL). The burgundy-

colored organic layer was washed with H₂O (4 × 100 mL). The

organic layer was dried over anhydrous sodium sulfate, concentrated,

and the resulting residue was adsorbed onto SiO₂(∼3 g) and puriﬁed

by ﬂash column chromatography (SiO₂, 26.0 g), eluting with a

gradient of 1% MeOH in CHCl₃to 2.5% MeOH in CHCl₃to yield a

mauve-colored solid (0.033 g, 36%) after dissolving in CHCl₃(5 mL)

and precipitating with ether (40 mL) and drying: mp 282−285 °C. IR

(ﬁlm) 2917, 1687, 1635, 1553, 1495, 1472, 1395, 1326, 1255, 1211,

6.71 (d, J = 8.9 Hz, 1 H), 6.60−6.57 (m, 2 H), 5.04 (s, 2 H), 5.00 (d, J

= 6.2 Hz, 1 H), 4.70 (d, J = 6.2 Hz, 1 H), 4.03−3.98 (m, 1 H), 3.96 (s,

3 H), 3.88 (s, 3 H), 3.63 (s, 3 H), 3.22−3.41 (m, 2 H), 3.22−3.15 (m,

2 H), 2.28−2.21 (m, 1 H), 2.18−2.08 (m, 1 H); ESIMS m/z (rel

intensity) 584/585 (MH⁺, 100/94). Anal. Calcd for C₂₉H₃₀BrNO₇: C,

59.60; H, 5.17; N, 2.40. Found: C, 59.49; H, 5.13; N, 2.34.

9-Benzyloxy-6-3-(bromopropyl)-5,6-dihydro-2,3,8-trime-

thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (19). Com-

pound 18 (1.00 g, 1.71 mmol) was diluted with SOCl₂(40 mL),

and the reaction mixture was stirred at room temperature, upon which

it became a bright reddish-purple color. TLC indicated completion

after 4.5 h. The SOCl₂was evaporated, and the residue was dissolved

in CHCl₃(40 mL) and quenched by the slow addition of saturated

aqueous NaHCO₃(50 mL). The layers were separated, and the

aqueous layer was extracted with CHCl₃(30 mL). The organic layers

were washed with H₂O (50 mL), and the aqueous layers were

extracted with CHCl₃(20 mL). The organic layers were ﬁnally washed

with saturated aqueous NaCl (50 mL), and the aqueous layers were

again extracted with CHCl₃(20 mL). The combined organic layers

were dried over anhydrous sodium sulfate, concentrated, and adsorbed

onto SiO₂(∼3.0 g). The residue was puriﬁed by ﬂash column

chromatography (SiO₂, 37.2 g), eluting with CHCl₃to yield a purple

solid (0.448 g, 45%) after dissolving in CHCl₃(30 mL) and

precipitating with ether (200 mL). The precipitate was ﬁltered and

1

1020, 867, 788, 757 cm⁻¹; H NMR (DMSO-d₆) δ 10.06 (br s, 1 H),

7.97 (s, 1 H), 7.46 (s, 1 H), 7.18 (s, 1 H), 7.06 (s, 1 H), 4.40−4.30 (m,

2 H), 3.89 (s, 3 H), 3.86 (s, 3 H), 3.84 (s, 3 H), 3.54 (s, 4 H), 2.50−

2.44 (m, 2 H, partially obscured by solvent peak), 2.36 (s, 4 H), 2.00−

2.90 (m, 2 H); ESIMS m/z (rel intensity) 481 (MH⁺, 100). Purity was

estimated to be 98.3% by HPLC in 95% MeOH−5% H₂O and 99% in

90% MeOH−10% H₂O. Anal. Calcd for C₂₆H₂₈N₂O₇·0.5 H₂O: C,

63.79; H, 5.97; N, 5.72. Found: C, 63.88; H, 5.78; N, 5.69.

Benzylvanillin (16).⁶⁴Vanillin (15, 3.00 g, 19.7 mmol) was

diluted with anhydrous DMF (50 mL). Benzyl bromide (3.47 g, 20.3

mmol) was added slowly, followed by K₂CO₃(6.54 g, 47.3 mmol).

The yellow mixture was rapidly stirred at room temperature for 2 h 20

min. The mixture was partitioned into an ether/water mixture (1:1,

100 mL of each) and stirred. The organic and aqueous layers were

separated, and the aqueous layer was extracted with ether (2 × 25 mL,

1 × 50 mL). The combined organic layers were washed with H₂O (1 ×

50 mL, 1 × 30 mL) and saturated aqueous NaCl (50 mL), dried over

anhydrous sodium sulfate, and concentrated to yield a white

microcrystalline solid (4.51 g, 95%) after washing with hexanes (50

mL) and reﬁltering the ﬁltrate: mp 51−54 °C (lit.⁶³mp 60−61 °C).

¹H NMR (CDCl₃) δ 9.84 (s, 1 H), 7.46−7.35 (m, 7 H), 7.00 (d, J =

1

washed with ether (40 mL): mp 223−226 °C (dec). The H NMR

indicated that the product contained some minor impurities, and no

further puriﬁcation or characterization was performed. This crude

material was used to prepare analogues. ¹H NMR (CDCl₃) δ 8.04 (s, 1

H), 7.62 (s, 1 H), 7.47−7.19 (m, 7 H), 5.23 (s, 2 H), 4.64 (t, J = 7.6

Hz, 2 H), 4.05 (s, 3 H), 4.03 (s, 3 H), 3.98 (s, 3 H), 3.71 (t, J = 6.0 Hz,

2 H), 2.40−2.40 (m, 2 H).

9-Benzyloxy-5,6-dihydro-6-[3-(1H-imidazol-1-yl)propyl]-

2,3,8-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(21a). Compound 19 (0.200 g, 0.354 mmol), NaI (0.360 g, 2.40

mmol), and imidazole (0.163 g, 2.40 mmol) were diluted with

anhydrous DMF (40 mL). The mixture was heated to 70 °C for 19 h

and cooled. The mixture was poured into H₂O (100 mL) and

extracted with CHCl₃(3 × 50 mL). The solution was washed with

H₂O (1 × 200 mL, 3 × 300 mL), and the aqueous layer was extracted

with CHCl₃(30 mL). The combined organic layers were dried over

anhydrous sodium sulfate, concentrated, and adsorbed onto SiO₂

(∼4.00 g). The residue was puriﬁed by ﬂash column chromatography

(SiO₂, ∼26.0 g), eluting with a gradient of 0.7% MeOH in CHCl₃to

2% MeOH in CHCl₃to yield a bright purple solid (0.103 g, 53%) after

washing with 1% CHCl₃in ether (60 mL) and ether (20 mL) and

drying: mp 236−240 °C. IR (ﬁlm) 2918, 1690, 1546, 1554, 1495,

1303, 1260, 1212, 1025, 863, 786 cm⁻¹; ¹H NMR (CDCl₃) δ 8.03 (s, 1

H), 7.64 (s, 1 H), 7.62 (s, 1 H), 7.46−7.33 (m, 5 H), 7.19 (s, 1 H),

7.11 (s, 1 H), 7.06 (s, 1 H), 6.79 (s, 1 H), 5.21 (s, 2 H), 4.58 (t, J = 6.4

Hz, 2 H), 4.24 (t, J = 6.7 Hz, 2 H), 4.05 (s, 3 H), 4.00 (s, 3 H), 3.86 (s,

3 H), 2.40−2.35 (m, 2 H); ESIMS m/z (rel intensity) 552 (MH⁺,

100).

8.2 Hz, 1 H), 5.25 (s, 2 H), 3.95 (s, 3 H).

N-[4′-(Benzyloxy)-3′-methoxybenzylidene]-3-bromopro-

pan-1-amine (17). 3-Bromopropylamine hydrobromide (2.08 g, 9.49

mmol) was diluted with CHCl₃(10 mL). A solution of aldehyde 16

(2.00 g, 8.26 mmol) was added slowly as a solution in CHCl₃(12 mL)

and transferred quantitatively with the same solvent (3 mL). Et₃N

(0.917 g, 9.08 mmol) was added, upon which the suspension became

clear. Na₂SO₄(anhydrous, approximately 2.00 g) was added, and the

mixture was stirred at room temperature for 23 h. Additional 3-

bromopropylamine hydrobromide (0.904 g, 4.13 mmol) and Et₃N

(0.500 g, 4.96 mmol) were added, and the mixture was stirred for a

total of 44 h. The reaction mixture was diluted to a volume of 50 mL

with CHCl₃and was washed with H₂O (3 × 70 mL) and saturated

aqueous NaCl (50 mL). The organic phase was dried over anhydrous

sodium sulfate and concentrated to yield a yellow syrup (3.06 g, 100%

with residual solvent). IR (ﬁlm) 2935, 2840, 1645, 1600, 1585, 1511,

K

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

9-Benzyloxy-5,6-dihydro-2,3,8-trimethoxy-6-[3-(N-

morpholino)propyl]-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(21b). Compound 19 (0.100 g, 0.177 mmol) and NaI (0.178 g, 1.19

mmol) were diluted with anhydrous DMF (25 mL). Morpholine

(0.104 g, 1.19 mmol) was added, and the mixture was heated to 70 °C

for 2 h and then stirred for 17 h at room temperature. The mixture was

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

The organic phase was washed with H₂O (4 × 200 mL), dried over

anhydrous sodium sulfate, and adsorbed onto SiO₂(∼3.0 g). The

residue was puriﬁed by ﬂash column chromatography (SiO₂, ∼30 g),

eluting with a gradient of 0.2% MeOH in CHCl₃to 1% MeOH in

CHCl₃to yield a purple-brown solid (0.070 g, 69%) after washing with

2% CHCl₃in ether (50 mL) and drying: mp 252−254 °C. IR (ﬁlm)

2944, 1690, 1652, 1553, 1496, 1303, 1211, 1118, 1014, 918, 870, 786,

730 cm⁻¹; ¹H NMR (CDCl₃) δ 8.04 (s, 1 H), 7.65 (s, 1 H), 7.47−7.34

(m, 5 H), 7.21 (s, 2 H), 7.11 (s, 1 H), 5.23 (s, 2 H), 4.58 (d, J = 7.1

Hz, 2 H), 4.05 (s, 1 H), 3.99 (s, 3 H), 3.98 (s, 3 H), 3.70 (t, J = 4.6 Hz,

4 H), 2.59 (t, J = 6.8 Hz, 2 H), 2.55 (br s, 4 H), 2.13−2.07 (m, 2 H);

ESIMS m/z (rel intensity) 571 (MH⁺, 100). Anal. Calcd for

C₃₃H₃₄N₂O₆·H₂O: C, 67.33; H, 6.16; N, 4.76. Found: C, 67.28; H,

5.81; N, 4.80.

1496, 1394, 1261, 1210, 1116, 870, 786, 757 cm⁻¹; ¹H NMR (CDCl₃)

δ 8.05 (s 1 H), 7.64 (s, 1 H), 7.16 (s, 1 H), 7.02 (s, 1 H), 6.10 (br s, 1

H), 4.57 (t, J = 7.2 Hz, 2 H), 4.05 (s, 3 H), 4.01 (s, 3 H), 4.99 (s, 3 H),

3.70 (t, J = 4.5 Hz, 2 H), 2.60 (t, J = 7.1 Hz, 2 H), 2.40−2.50 (m, 4 H),

2.14−2.09 (m, 2 H); ESIMS m/z (rel intensity) 481 (MH⁺, 100).

Purity was estimated to be 97.5% by HPLC in 95% MeOH−5% H₂O

and 97.7% in 90% MeOH−10% H₂O. Anal. Calcd for C₂₆H₂₈N₂O₇: C,

64.99; H, 5.87; N, 5.83. Found: C, 64.92; H, 5.86; N, 5.75.

O-(4-Methoxybenzyl)vanillin (23).⁶⁵Vanillin (15, 3.00 g, 19.7

mmol) was diluted in dry DMF (55 mL). PMB-Cl (3.18 g, 20.9

mmol) was added, followed by anhydrous potassium carbonate (6.53

g, 47.3 mmol). The yellow mixture was heated to 70 °C for 3 h and

was then cooled and partitioned between ether and water (200 mL of

each). The layers were separated, and the aqueous layer was extracted

with ether (100 mL) and CHCl₃(1 × 100 mL, 2 × 50 mL). The

combined organic extracts were washed with H₂O (3 × 200 mL) and

saturated aqueous NaCl (100 mL). The organic phase was dried over

anhydrous sodium sulfate, ﬁltered to remove particulate matter, and

concentrated to yield a yellow residue. This residue was dissolved in

CHCl₃(20 mL), and addition of hexanes (175 mL) resulted in the

precipitation of a pale-yellow solid (4.82 g, 90%) which was washed

with hexanes (25 mL) and dried: mp 101−104 °C (lit.⁶⁵mp 100−102

°C). ¹H NMR (CDCl₃) δ 9.39 (s, 1 H), 7.42−7.36 (m, 4 H), 7.02 (d, J

= 8.0 Hz, 1 H), 6.93 (dd, J = 2.6, 6.7 Hz, 2 H), 5.18 (s, 2 H), 3.94 (s, 3

H), 3.81 (s, 3 H).

5,6-Dihydro-9-hydroxy-6-[3-(1H-imidazol-1-yl)propyl]-2,3,8-

trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22a,

Method 1). Compound 21a (0.090 g, 0.163 mmol) was diluted in

H₂O (30 mL), and the mixture was sonicated to ensure a

homogeneous suspension. A solution of HBr in AcOH (33%, 32

mL) was added slowly, and the mixture was heated at 65 °C for 2.5 h.

The mixture was a clear, reddish-purple color. The mixture was

concentrated, and H₂O (30 mL) was added to the residue. Saturated

aqueous NaHCO₃was added until the pH was neutral (an amount of

approximately 6 mL was needed). The suspension was extracted with

CHCl₃(3 × 50 mL), and the organic layers were washed with H₂O (4

× 200 mL) and dried over anhydrous sodium sulfate. The solution was

concentrated, adsorbed onto SiO₂(∼4.0 g), and the residue was

puriﬁed by ﬂash column chromatography (SiO₂, ∼27 g), eluting with a

gradient of 1.5% MeOH in CHCl₃to 4.5% MeOH in CHCl₃to yield

the title compound as a chalky, mauve-colored solid (0.023 g, 30%)

after washing with ether (30 mL) and drying: mp 254−257 °C (dec).

IR (ﬁlm) 2537, 1689, 1634, 1557, 1512, 1462, 1432, 1307, 1223, 1082,

N-[4′-(4″-Methoxybenzyloxy)-3′-methoxybenzylidene]-3-

bromopropan-1-amine (24). 3-Bromopropylamine HBr (1.85 g,

8.44 mmol) was diluted with CHCl₃(12 mL). Compound 23 (2.00 g,

7.34 mmol) was added as a solution in CHCl₃(15 mL) and was

quantitatively transferred with CHCl₃(3 mL). Et₃N (0.811 g, 8.7

mmol) was added slowly, which discharged the cloudiness of the

reaction mixture, followed by anhydrous Na₂SO₄(2.00 g, 14.1 mmol).

The mixture was stirred at room temperature for 24 h and diluted to

70 mL with CHCl₃. The mixture was washed with H₂O (70 mL), and

this aqueous phase was extracted with CHCl₃(50 mL). The combined

organic layers were washed with H₂O (2 × 50 mL) and saturated

aqueous NaCl (70 mL), dried over anhydrous sodium sulfate, and

concentrated to yield an orange oil (2.84 g, 99%) that solidiﬁed upon

standing: mp 78−80 °C. IR (ﬁlm) 2934, 2837, 1644, 1613, 1585,

1

1020, 872, 786 cm⁻¹; H NMR (DMSO-d₆) δ 10.00 (br s, 1 H), 7.89

1

1515, 1267, 1251, 1138, 1033 cm⁻¹; H NMR (CDCl₃) δ 8.22 (s, 1

(s, 1 H), 7.72 (s, 1 H), 7.49 (s, 1 H), 7.29 (s, 1 H), 7.00 (s, 1 H), 6.94

(s, 1 H), 6.90 (s, 1 H), 4.48 (t, J = 6.7 Hz, 2 H), 4.18 (t, J = 6.8 Hz, 2

H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 2.27−2.23 (m, 2 H);

ESIMS m/z (rel intensity) 462 (MH⁺, 100). Purity was estimated to

be 100% by HPLC in MeOH and 99.3% in 90% MeOH−10% H₂O.

Anal. Calcd for C₂₅H₂₃N₃O₆·0.5 H₂O: C, 63.82; H, 5.14; N, 8.93.

Found: C, 63.70; H, 4.96; N, 8.56.

H), 7.41−7.35 (m, 3 H), 7.11 (d, J = 8.0 Hz, 1 H), 6.92 (d, J = 8.1 Hz,

3 H), 5.13 (s, 2 H), 3.94 (s, 3 H), 3.81 (s, 3 H), 3.74 (t, J = 6.2 Hz, 2

H), 3.51 (d, J = 6.5 Hz, 2 H), 2.30−2.20 (m, 2 H); ESIMS m/z (rel

intensity) 392/394 (MH⁺, 100/97).

cis-3-(4-Methoxybenzyloxy-3-methoxyphenyl)-N-(3-bromo-

propyl)-4-carboxy-3,4-dihydro-6,7-dimethoxy-1(2H)-isoquino-

lone (25). Anhydride 11 (1.03 g, 4.64 mmol) was diluted with CHCl₃

(30 mL), and the solution was cooled to 0 °C. The Schiﬀ base 24

(1.19 g, 4.88 mmol) was added slowly as a precooled solution in

CHCl₃(20 mL) and quantitatively transferred with CHCl₃(3 mL).

The mixture was stirred at 0 °C for 2 h, followed by 2 h at room

temperature, upon which a precipitate had formed. Hexanes (12 mL)

were added, and the precipitate was collected, washed with 20%

hexanes in CHCl₃(50 mL), and dried to yield the title compound as a

white amorphous solid (2.22 g, 78%): mp 139−141 °C (dec). IR

(ﬁlm) 1996, 1722, 1619, 1592, 1573, 1251, 1172, 1138 cm⁻¹; ¹H NMR

(CDCl₃/DMSO-d₆) δ 7.63 (s, 1 H), 7.24 (d, J = 8.6 Hz, 2 H), 7.15 (s,

1 H), 6.81 (d, J = 8.6 Hz, 2 H), 6.63−6.51 (m, 3 H), 4.95−4.89 (m, 3

H), 4.54 (d, J = 6.4 Hz, 1 H), 4.00−3.88 (m, 1 H), 3.88 (s, 3 H), 3.81

(s, 3 H), 3.72 (s, 3 H), 3.60 (s, 3 H), 3.41−3.34 (m, 2 H), 3.10−3.00

(m, 1 H), 2.20−2.00 (m, 2 H); ESIMS m/z (rel intensity) 614/616

(MH⁺, 4.04/3.68), 534 (MH⁺− HBr, 100).

3-(Bromopropyl)-5,6-dihydro-9-hydroxy-2,3,8-trimethoxy-

5,11-dioxo-11H-indeno[1,2-c]isoquinoline (20). The cis acid 25

(4.71 g, 7.67 mmol) was diluted with SOCl₂(80 mL). The mixture

was stirred at room temperature for 5 h. The dark burgundy mixture

was then concentrated, and the residue was diluted with CHCl₃(40

mL). Ether (250 mL) was added, and the precipitate was collected,

washed with ether (100 mL), subjected to a second, identical

precipitation (20 mL of CHCl₃, 200 mL of ether, followed by washing

5,6-Dihydro-9-hydroxy-2,3,8-trimethoxy-6-[3-(N-

morpholino)propyl]-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(22b, Method 1). Compound 21b (0.074 g, 0.130 mmol) was diluted

in H₂O (60 mL), and the mixture was sonicated to ensure a

homogeneous suspension. A solution of HBr in AcOH (33%, 60 mL)

was added slowly, and the mixture was heated at 70 °C for 4 h. The

reddish-brown solution was concentrated, and the residue was diluted

with H₂O (100 mL). Saturated aqueous NaHCO₃was added until the

gas evolution ceased (approximately 30 mL was required). The

suspension was extracted with CHCl₃(2 × 50 mL, 1 × 25 mL), and

the organic layers were washed with H₂O (2 × 150 mL) and saturated

aqueous NaCl (150 mL) and dried over anhydrous sodium sulfate.

The solution was concentrated, adsorbed onto SiO₂(∼4 g) and the

residue was puriﬁed by ﬂash column chromatography (SiO₂, ∼25 g),

eluting with a gradient of 1% MeOH in CHCl₃to 2.5% MeOH in

CHCl₃to yield the title compound as a brown solid (0.035 g, 56%)

after washing with 2% CHCl₃in ether (50 mL) and ether (10 mL) and

drying: mp 258−260 °C (dec). Combining this with several reactions

run in parallel and repurifying the resulting material twice by ﬂash

column chromatography (SiO₂, ∼25 g), eluting with a gradient of 0.5%

MeOH in CHCl₃to 2.5% MeOH in CHCl₃, aﬀorded an analytically

pure sample (0.040 g) after dissolving in tetrahydrofuran (10 mL) and

precipitating with ether (50 mL). IR (ﬁlm) 2917, 1692, 1650, 1552,

L

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

with 100 mL of ether), and dried to aﬀord the crude product as a

reddish-brown solid (2.48 g, 68%). This material was used without any

further puriﬁcation. ¹H NMR (DMSO-d₆) δ 10.00 (br s, 1 H), 7.86 (s,

1 H), 7.46 (s, 1 H), 7.10 (s, 1 H), 6.93 (s, 1 H), 4.52 (t, J = 8.5 Hz, 2

H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.84 (s, 3 H), 3.74 (t, J = 3.6 Hz, 2 H),

2.35−2.25 (m, 2 H).

ﬁltration. Reﬁltration of the ﬁltrate aﬀorded additional product. A total

of 5.00 g (69%) was obtained, containing a small amount of impurities:

mp 105−110 °C (lit.⁵³mp 122 °C). H NMR (CDCl₃) δ 7.42−7.34

1

(m, 5 H), 6.76 (s, 1 H), 6.72 (s, 1 H), 5.25 (s, 2 H), 5.15 (s, 2 H), 3.89

(s, 3 H), 3.58 (s, 2 H).

4-(Benzyloxy)-2-(carboxymethyl)-5-methoxybenzoic Acid

(29). Compound 28 (5.00 g, 17.6 mmol) was diluted with a solution

of KOH (2.47 g, 44.0 mmol) in H₂O (250 mL). The mixture was

stirred at room temperature for 17 h. The clear orange solution was

cooled to 0 °C, and KMnO₄(6.21 g, 40.0 mmol) was added in

portions over 10 min. The mixture was stirred at 0 °C for 3 h and then

at room temperature for 48 h. Absolute EtOH (60 mL) was added,

and the mixture was heated at 75 °C for 30 min. The mixture was

ﬁltered hot to remove inorganic materials, and the ﬁlter cake was

washed with H₂O (100 mL). The ﬁltrate was concentrated to half its

volume and was washed with EtOAc (2 × 150 mL). The aqueous layer

was diluted with H₂O to a volume of 300 mL and, while stirring, was

made acidic (pH 1) by the addition of HCl (concentrated, 12 mL).

The mixture was chilled to 0 °C for 3 h, and the resultant white

precipitate was collected by vacuum ﬁltration, dried, triturated with

EtOAc−hexanes (70:30, 75 mL), and washed with EtOAc (30 mL) to

aﬀord an oﬀ-white solid (3.11 g, 56%): mp 199−200 °C. IR (neat)

3583, 3408, 2917, 1682, 1598, 1575, 1276, 1218, 1173, 1074, 666

5,6-Dihydro-9-hydroxy-6-[3-(1H-imidazol-1-yl)propyl]-2,3,8-

trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22a,

Method 2). Compound 20 (1.12 g, 2.35 mmol), imidazole (1.60 g,

23.5 mmol), and NaI (3.53 g, 23.5 mmol) were diluted with dioxane

(300 mL). The mixture was heated under argon to reﬂux for 29 h. The

mixture was cooled, concentrated, and the residue was dissolved in 5%

MeOH in CHCl₃(200 mL). This solution was washed with H₂O (300

mL), and the aqueous phase was exhaustively extracted with CHCl₃

(∼500 mL). The organic phase was washed with H₂O (approximately

2 × 600 mL) and dried over anhydrous sodium sulfate. The solution

was concentrated, adsorbed onto SiO₂(∼10 g), and puriﬁed by ﬂash

column chromatography (SiO₂, ∼50 g), eluting with a gradient of 1.5%

MeOH in CHCl₃to 5% MeOH in CHCl₃to yield a mauve-colored

solid (0.474 g, 44%) after dissolving in CHCl₃(10 mL), precipitating

with ether (300 mL), and washing with ether (50 mL). The analytical

data for this compound are identical to that prepared by method 1.

5,6-Dihydro-9-hydroxy-2,3,8-trimethoxy-6-[3-(N-

morpholino)propyl]-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(22b, Method 2). Compound 20 (0.100 g, 0.211 mmol) and NaI

(0.189 g, 1.27 mmol) were diluted with dry DMF (50 mL). The

mixture was heated to 65 °C, and morpholine (0.111 g, 1.27 mmol)

was added. After 18 h at 65 °C, the mixture was cooled and poured

into H₂O (120 mL). The mixture was extracted with CHCl₃(3 × 75

mL) and the organic phase was washed with H₂O (5 × 250 mL) and

saturated aqueous NH₄Cl (250 mL). The extract was dried over

anhydrous sodium sulfate. The solution was concentrated, adsorbed

onto SiO₂(∼3 g), and the product was puriﬁed by ﬂash column

chromatography (SiO₂, ∼20 g), eluting with a gradient of 0.5% MeOH

in CHCl₃to 2% MeOH in CHCl₃. The product was suspended in

CHCl₃(3 mL), precipitated with ether (100 mL), and washed with

ether (20 mL) to yield a brown solid (0.045 g, 44%). The analytical

data for this compound are identical to that prepared by method 1.

O-Benzylhomoisovanillic Acid (27).⁵²KOH (3.93 g, 70.0

mmol) was diluted with absolute EtOH (100 mL), and the mixture

was warmed to 55 °C, upon which the mixture became a clear

solution. Homoisovanillic acid (26, 5.00 g, 27.4 mmol) was added in

small portions, and the mixture was heated to gentle reﬂux under a

continuous argon ﬂow. Benzyl chloride (7.49 g, 60.0 mmol) was added

as a solution in absolute EtOH (10 mL) dropwise over 15 min and was

quantitatively transferred with EtOH (3 mL). The mixture was heated

at reﬂux for 2 h following addition, upon which a cloudy precipitate

formed. A solution of KOH (1 g) in H₂O (20 mL) was added to the

mixture, and reﬂux was continued for 20 min. The solution was cooled

to room temperature and stirred for 1 h before it was concentrated to

¹/₃of its original volume. The resulting residue was diluted with H₂O

1

cm⁻¹; H NMR (DMSO-d₆) δ 12.3 (br s, 1 H), 7.47−7.36 (m, 6 H),

7.07 (s, 1 H), 5.11 (s, 2 H), 3.86 (s, 2 H), 3.77 (s, 3 H). One of the

carboxyl protons is not visible because of exchange with residual water.

¹³C NMR (DMSO d₆, 125 MHz) δ 172.9, 168.0, 150.7, 147.4, 136.8,

131.3, 128.8, 128.4, 128.4, 122.5, 117.2, 114.1, 70.2, 55.9; ESIMS m/z

(rel intensity) 339.2 (MNa⁺, 100); negative ion 315.1 [(M − H⁺)⁻,

100)]. Anal. Calcd for C₁₇H₁₆O₆·0.5H₂O: C, 62.77; H, 5.27. Found: C,

62.60; H, 4.99.

6-(Benzyloxy)-7-methoxy-4H-isochromene-1,3-dione (30).

Compound 29 (2.25 g, 7.11 mmol) was diluted with acetyl chloride

(40 mL), and the mixture was heated at reﬂux for 2.5 h. The mixture

was then concentrated to yield a yellow solid (2.12 g, 100%): mp

160−165 °C. IR (neat) 2939, 1741, 1515, 1277, 1224, 1028 cm⁻¹; ¹H

NMR (CDCl₃) δ 7.60 (s, 1 H), 7.42−7.26 (s, 5 H), 6.71 (s, 1 H), 5.24

(s, 2 H), 4.00 (s, 2 H), 3.96 (s, 3 H); ¹³C NMR (CDCl₃, 125 MHz) δ

164.9, 160.6, 154.4, 149.7, 135.2, 128.8, 128.7, 128.4, 127.1, 113.6,

111.5, 110.3, 71.0, 56.2, 33.9; CIMS m/z (rel intensity) 299 (MH⁺,

40), 209 (100), 181 (100).

N-[3′,4′-(Methylenedioxy)benzylidene]-3-bromopropan-1-

amine (32).²¹3-Bromopropylamine hydrobromide (3.35 g, 15.3

mmol) was diluted with CHCl₃(15 mL). Piperonal (31, 2.0 g, 13.3

mmol) was added slowly as a solution in CHCl₃(15 mL) and

quantitatively transferred with 5 mL of the same solvent. Et₃N (1.6 g,

16.0 mmol) was added slowly, upon which the solution became clear

and colorless. Na₂SO₄(3.00 g, 21.1 mmol) was added, and the mixture

was stirred at room temperature for 27 h. The mixture was diluted

with CHCl₃to a volume of 70 mL and washed with H₂O (70 mL).

The aqueous layer was extracted with CHCl₃(50 mL), and the

combined organic layers were washed with H₂O (2 × 70 mL) and

saturated aqueous NaCl (70 mL). The organic layer was dried over

anhydrous sodium sulfate and concentrated to yield a yellow oil (3.44

g, 96%). ¹H NMR (CDCl₃) δ 8.21 (s, 1 H), 7.34 (d, J = 0.9 Hz, 1 H),

7.13 (dd, J = 0.9, 7.9 Hz, 1 H), 6.85 (d, J = 7.9 Hz, 1 H), 6.01 (s, 2 H),

3.73 (t, J = 6.3 Hz, 2 H), 3.52 (t, J = 6.4 Hz, 2 H), 2.30−2.20 (m, 2 H).

cis-6-(Benzyloxy)-N-(3-bromopropyl)-4-carboxy-3,4-dihy-

dro-7-methoxy-3-(3,4-methylenedioxyphenyl)-1(2H)-isoqui-

nolone (33). Compound 30 (2.09 g, 7.03 mmol) was dissolved in

CHCl₃(65 mL), and the solution was cooled to 0 °C. A solution of

Schiﬀ base 32 (2.00 g, 7.40 mmol) in CHCl₃(15 mL) was cooled to 0

°C and added slowly via addition funnel to the anhydride. Within

minutes, a yellow precipitate began to form. The mixture was stirred

for 2 h at 0 °C and then warmed to room temperature, where it was

stirred for 2 h. The precipitate was collected via vacuum ﬁltration and

washed with CHCl₃(100 mL) to yield the desired product as a cream-

colored solid (2.63 g, 66%) after drying: mp 198−199 °C (dec). IR

(ﬁlm) 2972, 2937, 1742, 1592, 1570, 1259, 1226, 1168 cm⁻¹; ¹H NMR

(DMSO-6₆) δ 12.96 (br s, 1 H), 8.31 (s, 1 H), 7.53−7.35 (s, 5 H),

(400 mL) and made acidic (pH 1) by the addition of HCl

(concentrated, 5 mL). The resulting precipitate was collected, dried,

and recrystallized from 40% acetone in ether to yield the product as an

oﬀ-white solid (5.68 g, 76%) after drying: mp 111−114 °C (lit.⁶³mp

124−125 °C). ¹H NMR (DMSO-d₆) δ 12.4 (br s, 1 H), 7.45−7.32 (m,

5 H), 6.96 (s, 1 H), 6.91 (d, J = 9.6 Hz, 1 H), 6.80−6.79 (m, 1 H),

5.01 (s, 2 H), 3.72 (s, 3 H), 3.45 (s, 2 H).

6-(Benzyloxy)-7-methoxy-1H-isochromen-3(4H)-one (28).⁵⁴

Compound 27 (7.00 g, 25.7 mmol) was diluted with glacial AcOH

(90 mL). Formalin (37% formaldehyde in H₂O, 24 mL) was added,

followed by HCl (concd, 6 mL). The mixture was heated at 45 °C for

22 h, cooled, and poured into H₂O (300 mL). The cloudy suspension

was extracted with CHCl₃(3 × 100 mL). The organic layers were

washed with saturated aqueous NaHCO₃(3 × 150 mL) and H₂O (3 ×

200 mL). The combined aqueous layers were extracted with CHCl₃

(100 mL). The organic layers were dried over anhydrous sodium

sulfate and concentrated. The resulting pale yellow oil was dissolved in

CHCl₃(10 mL), and hexanes (100 mL) were added slowly to

precipitate a white amorphous solid, which was collected by vacuum

M

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.26 (s, 1 H), 6.80 (d, J = 8 Hz, 1 H), 6.57 (dd, J = 1.4, 8.1 Hz, 2 H),

6.46 (s, 1 H), 5.95 (s, 2 H), 5.03−5.00 (m, 3 H), 4.69 (d, J = 6 Hz, 1

H), 3.88−3.82 (s, 1 H), 3.82 (s, 3 H), 3.56−3.50 (s, 2 H), 2.95−2.88

(m, 1 H), 2.14−1.96 (m, 2 H); ¹³C NMR (DMSO, 75 MHz) δ 171.5,

163.6, 151.4, 148.8, 148.0, 147.8, 137.4, 132.0, 129.4, 129.1, 127.9,

122.6, 122.4, 113.2, 110.9, 108.9, 108.6, 102.1, 71.0, 62.4, 56.4, 48.3,

45.5, 33.4, 31.9; ESIMS m/z (rel intensity) 488.5 (MH⁺− HBr, 100).

Anal. Calcd for C₂₈H₂₆BrNO₇·H₂O: C, 57.35; H, 4.81; N, 2.39. Found:

C, 57.34; H, 4.46; N, 2.42.

dissolved in anhydrous DMF (10 mL), and NaI (0.314 g, 2.09 mmol)

and morpholine (0.18 mL, 2.06 mmol) were added. The mixture was

heated to 80 °C for 26 h under argon. After the evaporation of the

solvent, the residue was partitioned between chloroform (60 mL) and

water (30 mL). The organic layer was washed with water (30 mL) and

puriﬁed by silica gel column chromatography (chloroform−MeOH,

10:0.5), which aﬀorded the product (0.020 g, 25%): mp 228−230 °C.

IR (neat) 3583, 2918, 2852, 1701, 1634, 1610, 1470, 1389, 1270, 1115,

1033, 666 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.90 (s, 1 H), 7.46 (s,

1 H), 7.16 (s, 1 H), 6.97 (s, 1 H), 6.05 (s, 2 H), 4.61 (t, J = 7.2 Hz, 2

H), 4.05 (s, 3 H), 3.70−3.63 (m, 5 H), 2.51 (t, J = 6.8 Hz, 2 H), 2.43

(br, 4 H), 1.96−1.91 (m, 2 H); ¹³C NMR (CDCl₃, 125 MHz) δ 187.3,

160.7, 152.5, 150.5, 148.5, 146.2, 138.5, 131.7, 126.9, 124.9, 124.6,

122.2, 109.3, 107.3, 105.8, 103.9, 102.1, 66.8, 56.2, 53.4, 41.0, 25.5;

ESIMS m/z (rel intensity) 465.1 (MH⁺, 100); negative ion 463.2 [(M

− H⁺) ⁻, 100]; HRESIMS m/z 465.1659 (MH⁺), calcd for

C₂₅H₂₅N₂O₇465.1662. Anal. Calcd for C₂₅H₂₄N₂O₇: C, 64.65; H,

5.21; N, 6.03. Found: C, 64.69; H, 5.22; N, 6.18.

6-(3-Bromopropyl)-5,6-dihydro-2-hydroxy-3-methoxy-8,9-

methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(34). Compound 33 (1.35 g, 2.38 mmol) was diluted with SOCl₂(30

mL). The mixture was stirred at room temperature for 3 h and 40 min.

The dark red mixture was concentrated, and the residue was

suspended in 1,2-dichloroethane (200 mL). Aluminum chloride

(0.475 g, 0.357 mmol) was added, and the mixture was stirred at 0

°C for 10 min. Additional AlCl₃(0.237 g, 1.78 mmol) was then added,

and the mixture was sonicated for 1.5 h at 0 °C. The mixture was

poured into a mixture of ice and saturated aqueous NaHCO₃(200

mL). The mixture was extracted with CHCl₃(4 × 150 mL), and the

organic layer was ﬁltered to remove aluminum salts. The organic layer

was washed with H₂O (3 × 200 mL), dried over anhydrous sodium

sulfate, concentrated, and adsorbed onto SiO₂(∼5 g). The residue was

puriﬁed by ﬂash column chromatography (SiO₂, ∼40 g), eluting with

CHCl₃to yield a purple-gray solid (0.108 g, 10%) after suspending in

CHCl₃(10 mL), precipitating with ether (100 mL), and washing with

ether (20 mL): mp 276−278 °C. IR (ﬁlm) 3368, 2918, 1681, 1652,

7-Hydroxy-6-methoxyisochroman-3-one (37).⁵⁴Glacial

AcOH (25 mL) was added to 36 (0.96 g, 5.3 mmol). The solution

was heated to 90 °C, and concentrated HCl (0.9 mL) and formalin

(37% formaldehyde in H₂O, 0.9 mL) were added rapidly, and then

heating was stopped immediately. After 1.5 h at room temperature, ice

(20 g) and H₂O (20 mL) were added to the solution. The organic

material was extracted with CHCl₃(4 × 50 mL), washed with

saturated aqueous NaHCO₃(2 × 40 mL) and saturated aqueous NaCl

(50 mL), and the solvent was evaporated to yield the crude product

(0.81 g). Recrystallization from CH₂Cl₂−ether gave pure 37 as a white

powder (0.36 g, 35%): mp 178−179 °C (lit.⁵⁴174−177 °C). ¹H NMR

(DMSO-d₆, 300 MHz) δ 9.05 (s, 1 H), 6.88 (s, 1 H), 6.75 (s, 1 H),

5.18 (s, 2 H), 3.74 (s, 3 H), 3.62 (s, 2 H).

7-(Benzyloxy)-6-methoxyisochroman-3-one (38). A mixture

of 37 (0.160 g, 0.82 mmol), anhydrous K₂CO₃(500 mg, 3.62 mmol),

and benzyl bromide (0.25 mL, 2.08 mmol) in dry acetone was heated

at 56 °C for 4 h. The inorganic compounds were ﬁltered oﬀ and

washed with acetone. Evaporation of the ﬁltrate aﬀorded a residue that

was partitioned between CHCl₃(30 mL) and H₂O (30 mL). The

organic layer was dried over anhydrous sodium sulfate, concentrated,

and the residue was puriﬁed by ﬂash column chromatography (SiO₂,

∼25 g), eluting with a gradient of 10% EtOAc−hexanes to 25%

EtOAc−hexanes to yield the product (0.132 g, 57%) as a white

powder: mp 129−130 °C (lit.⁵⁴137−138 °C). ¹H NMR (CDCl₃, 500

MHz) δ 7.31−7.43 (m, 5 H), 6.74 (s, 1 H), 6.73 (s, 1 H), 5.19 (s, 2

H), 5.14 (s, 2 H), 3.89 (s, 3 H), 3.62 (s, 2 H).

5-(Benzyloxy)-2-(carboxymethyl)-4-methoxybenzoic Acid

(39). A solution of compound 38 (2.00 g, 7.00 mmol) and KOH (2

g, 36.0 mmol) in H₂O (60 mL) was stirred at 0 °C, and KMnO₄(3.30

g, 20.1 mmol) was added slowly. After the mixture was allowed to

stand for 24 h at room temperature, excess KMnO₄was destroyed by

addition of EtOH (20 mL) and by heating at 60−70 °C for 0.5 h. The

solution was ﬁltered and concentrated to a small volume under

reduced pressure. Acidiﬁcation with concentrated HCl (to pH 2) gave

a precipitate that was collected. Recrystallization from acetone gave the

pure product (0.675 g, 31%) as a yellow powder: mp 236−237 °C

(lit.⁶⁶238 °C). ¹H NMR (DMSO-d₆, 300 MHz) δ 12.35 (s, 2 H), 7.50

(s, 1 H), 7.33−7.42 (m, 5 H), 6.93 (s, 1 H), 5.08 (s, 2 H), 3.83 (s, 2

H), 3.80 (s, 3 H).

1

1615, 1491, 1432, 1389, 1304, 1267, 1033 cm⁻¹; H NMR (DMSO-

d₆) δ 10.44 (s, 1 H), 7.81 (s, 1 H), 7.47 (s, 1 H), 7.35 (s, 1 H), 7.05 (s,

1 H), 6.17 (s, 2 H), 4.50 (t, J = 6.9 Hz, 2 H), 3.84 (s, 3 H), 3.73 (t, J =

6.5 Hz, 2 H), 2.30−2.20 (m, 2 H); ESIMS m/z (rel intensity) 458/460

(MH⁺, 9/8.5), 378 (MH⁺− HBr, 100).

5,6-Dihydro-2-hydroxy-6-3-(1H-imidazol-1-yl)propyl)-3-me-

thoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]-

isoquinoline (35a). Compound 34 (0.212 g, 0.463 mmol), imidazole

(0.315 g, 4.63 mmol), and NaI (0.693 g, 4.63 mmol) were diluted with

dry DMF (60 mL), and the mixture was heated at 60 °C for 20 h.

Additional imidazole (0.200 g, 2.94 mmol) was added, and the mixture

was heated for an additional 40 min. The mixture was cooled and

poured into H₂O (200 mL). CHCl₃(200 mL) was added, and an

insoluble suspension formed. This suspension was saved, and the

remaining aqueous layer was extracted with CHCl₃(2 × 200 mL).

These layers were washed with H₂O (4 × 400 mL), saturated aqueous

NH₄Cl (200 mL), dried over anhydrous sodium sulfate, and

concentrated. The residue was washed with ether (50 mL), dissolved

in CHCl₃(50 mL), concentrated, and adsorbed onto SiO₂(3 g). This

residue was puriﬁed by ﬂash column chromatography (SiO₂, 60 g),

eluting with a gradient of 0.5% MeOH in CHCl₃to 7% MeOH in

CHCl₃to aﬀord a purple solid. Additional product was obtained by

drying the CHCl₃-insoluble suspension over anhydrous sodium sulfate

and concentrating the mixture. This solid was washed with H₂O (80

mL) and puriﬁed by dissolving in DMF (5 mL) and precipitating with

ether (50 mL). The solid (0.054 g, 26%) was washed with 10% CHCl₃

in ether (50 mL) and ether (20 mL) and dried. To produce an

analytically pure sample (0.034 g), the solid was adsorbed onto SiO₂

(3 g) and puriﬁed by ﬂash column chromatography (SiO₂, 20 g),

eluting with a gradient of 2% MeOH in CHCl₃to 5% MeOH in

CHCl₃and then washing the product with 10% CHCl₃in ether (20

mL) and ether (20 mL): mp 269−272 °C (dec). IR (ﬁlm) 3391, 3117,

2916, 1691, 1637, 1605, 1488, 1304, 1225, 1091, 1028 cm⁻¹; ¹H NMR

(DMSO-d₆) δ 10.46 (br s, 1 H), 7.81 (s, 1 H), 7.76 (s, 1 H), 7.47 (s, 1

H), 7.27 (s, 1 H), 7.04 (s, 1 H), 6.98 (s, 1 H), 6.92 (s, 1 H), 6.16 (s, 2

H), 4.40−4.30 (m, 2 H), 4.29 (t, J = 6.1 Hz, 2 H), 2.20−2.10 (m, 2

H); ESIMS m/z (rel intensity) 446.3 (MH⁺, 100); HRESIMS m/z

446.1347 (MH⁺), calcd for C₂₄H₂₀N₃O₆446.1352. Anal. Calcd for

C₂₄H₁₉N₃O₄·1.25H₂O: C, 61.60; H, 4.63; N, 8.98. Found: C, 61.67; H,

4.27; N, 8.79.

7-(Benzyloxy)-6-methoxyisochroman-1,3-dione (40). Acetyl

chloride (4.0 mL) was added to 39 (0.175 g, 0.550 mmol). The

solution was heated at reﬂux for 2 h and was concentrated to yield an

oily residue. Recrystallization from CHCl₃−ether gave pure 40 as a

yellow powder (0.157 g, 91%): mp 107−109 °C (dec). IR (ﬁlm) 2940,

1

1710, 1601, 1517, 1272, 1221, 1015 cm⁻¹; H NMR (CDCl₃, 300

MHz) δ 7.63 (s, 1 H), 7.26−7.47 (m, 5 H), 6.70 (s, 1 H), 5.18 (s, 2

H), 4.05 (s, 2 H), 3.96 (s, 3 H); CIMS m/z (rel intensity) 299 (MH⁺,

100).

cis-7-(Benzyloxy)-N-(3-bromopropyl)-4-carboxy-3,4-dihy-

dro-6-methoxy-3-(3,4-methylenedioxyphenyl)-1(2H)-isoqui-

nolone (41). A solution of the anhydride 40 (0.276 g, 0.92 mmol)

dissolved in CHCl₃(3 mL) was added dropwise to a solution of the

5,6-Dihydro-2-hydroxy-3-methoxy-8,9-methylenedioxy-6-3-

[(morpholino)propyl)]-5,11-dioxo-11H-indeno[1,2-c]-

isoquinoline (35b). Compound 34 (0.080 g, 0.17 mmol) was

N

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Schiﬀ base 32 (0.191 g, 0.92 mmol) in CHCl₃(3 mL). After 24 h,

hexanes (5 mL) were added to the mixture, and the precipitate that

formed was ﬁltered oﬀ and washed with a mixture of ethyl acetate (5

mL) and CHCl₃(5 mL) to aﬀord an impure mixture containing the

desired product. This mixture was puriﬁed by ﬂash column

chromatography (SiO₂, ∼25 g), eluting with a gradient of 10%

MeOH in CHCl₃to yield the product as a yellow powder (0.188 g,

36%): mp 195−197 °C (dec). IR (ﬁlm) 2942, 1712, 1677, 1601, 1578,

1526, 1220, 1178 cm⁻¹; ¹H NMR (DMSO, 300 MHz) δ 7.78 (s, 1 H),

7.32−7.50 (m, 5 H), 7.08 (s, 1 H), 6.60−6.65 (m, 2 H), 6.48 (s, 1 H),

5.90 (s, 2 H), 5.22 (s, 2 H), 4.97−4.99 (d, J = 6.00 Hz, 1 H), 4.68−

4.71 (d, J = 6.48 Hz, 1 H), 3.95−3.97 (m, 1 H), 3.88 (s, 3 H), 3.45 (m,

2 H), 3.11−3.13 (m, 1 H), 2.13−2.24 (m, 2 H); ESIMS m/z (rel

intensity) 488.3 (MH⁺− HBr, 100).

5,6-Dihydro-3-hydroxy-2-methoxy-8,9-methylenedioxy-6-3-

[(morpholino)propyl)]-5,11-dioxo-11H-indeno[1,2-c]-

isoquinoline (44b). Compound 43 (1.23 g, 2.74 mmol) and NaI

(4.93 g, 32.88 mmol) were diluted with anhydrous DMF (40 mL).

Morpholine (2.87 mL, 32.88 mmol) was added, and the mixture was

heated to 65 °C for 22 h under an argon atmosphere. The mixture was

cooled, poured into H₂O (200 mL), and extracted with CHCl₃(4 ×

100 mL). The organic phase was washed with H₂O (200 mL) and

saturated aqueous NaHCO₃(200 mL) and was dried over anhydrous

sodium sulfate. The solution was concentrated, adsorbed onto SiO₂

(∼5 g), and puriﬁed by ﬂash column chromatography (SiO₂, ∼60 g),

eluting with a gradient of 1% MeOH in CHCl₃to 5% MeOH in

CHCl₃to yield the title compound as a purple solid (0.481 g, 38%):

mp 321−323 °C (dec). IR (ﬁlm) 3244, 2962, 2811, 1687, 1655, 1503,

1286, 1111, 1035, 861 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.05 (s, 1

H), 7.75 (s, 1 H), 7.44 (s, 1 H), 6.91 (s, 1 H), 6.09 (s, 2 H), 4.47−4.52

(m, 2 H), 4.09 (s, 3 H), 3.74−3.77 (m, 4 H), 2.52−2.56 (m, 6 H),

2.00−2.04 (m, 2 H); ESIMS m/z (rel intensity) 465.2 (MH⁺, 100);

HRESIMS m/z 465.1665 (MH⁺), calcd for C₂₅H₂₄N₂O₇465.1662.

Anal. Calcd for C₂₅H₂₄N₂O₇·H₂O: C, 62.23; H, 5.43; N, 5.81. Found:

C, 62.14; H, 5.00; N, 5.70.

6-(3-Bromopropyl)-5,6-dihydro-3-benzyloxy-2-methoxy-

8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]-

isoquinoline (42). The cis acid 41 (1.00 g, 1.76 mmol) was diluted in

SOCl₂(45 mL), and the mixture was stirred at room temperature for

20 h. The solvent was removed under reduced pressure, and the

organic material was extracted with chloroform (3 × 20 mL), washed

with saturated aqueous NaHCO₃(2 × 20 mL) and saturated aqueous

NaCl (20 mL), and then concentrated. The obtained residue was

adsorbed onto SiO₂(∼10 g) and puriﬁed by ﬂash column

chromatography (SiO₂, ∼30 g), eluting with 1.25% MeOH in

CHCl₃to yield the solid product (0.404 g, 42%): mp 234−236 °C.

5,6-Dihydro-11-hydroxy-6-(3-imidazolylpropyl)-2,3-methyl-

enedioxy-5-oxo-11H-indeno[1,2-c]isoquinoline (45a). Indenoi-

soquinoline 6 (0.170 g, 0.370 mmol) was dissolved in MeOH (50

mL). NaBH₄(0.025 g, 0.740 mmol) was added at 0 °C, and the

reaction mixture was stirred at room temperature for 3 h. The mixture

was concentrated. CHCl₃(150 mL) was added, and the mixture was

washed with water (2 × 100 mL). The CHCl₃was removed on a

rotary evaporator and the crude mixture was puriﬁed by ﬂash column

chromatography (SiO₂), eluting with 10% MeOH in CHCl₃to yield

the title compound (0.110 g, 64%) as white solid: mp >280 °C. IR

1

IR (ﬁlm) 3421, 1698, 1644, 1483, 1309, 1282, 1036, 698 cm⁻¹; H

NMR (CDCl₃, 300 MHz) δ 8.04 (s, 1 H), 7.70 (s, 1 H), 7.29−7.50

(m, 5 H), 7.27 (s, 1 H), 7.07 (s, 1 H), 6.09 (s, 2 H), 5.24 (s, 2 H),

4.55−4.60 (m, 2 H), 4.04 (s, 3 H), 3.78−3.82 (t, J = 6.0 Hz, 2 H),

2.33−2.38 (m, 2 H); ESIMS m/z (rel intensity) 468.5 (MH⁺− HBr,

100).

1

(KBr) 3583, 1635, 1610, 1481, 1292, 1258, 1034, 750, 666 cm⁻¹; H

6-(3-Bromopropyl)-5,6-dihydro-3-hydroxy-2-methoxy-8,9-

methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(43). The benzylated indenoisoquinoline 42 (1.73 g, 3.16 mmol) was

dissolved in nitrobenzene (30 mL), and AlCl₃(0.839 g, 6.32 mmol)

was added. The mixture was stirred at room temperature for 15 min

and then heated at 90 °C for 45 min. The mixture was cooled and

poured into a mixture of ice and saturated aqueous NaHCO₃(80 mL).

The mixture was extracted with CHCl₃(4 × 100 mL) and the organic

layer was ﬁltered to remove aluminum salts. The organic layer was

washed with H₂O (3 × 100 mL), dried over anhydrous sodium sulfate,

concentrated, and adsorbed onto SiO₂(∼5 g). The residue was

puriﬁed by ﬂash column chromatography (SiO₂, ∼50 g), eluting with a

gradient of 0.25% MeOH in CHCl₃to 1% MeOH in CHCl₃to aﬀord

the crude product as a pinkish solid (0.230 g, 16%): mp 226−228 °C

(dec). IR (ﬁlm) 3446, 2346, 1698, 1638, 1483, 1394, 1310, 1215, 1034

NMR (CDCl₃+ CD₃OD, 300 MHz) δ 7.49 (s, 1 H), 7.43 (s, 1 H),

7.24 (s, 1 H), 7.03 (s, 1 H), 6.91 (s, 1 H), 6.87 (s, 1 H), 6.55 (s, 1 H),

5.90 (d, J = 1.6 Hz, 2 H), 5.25 (s, 1 H), 4.40 (m, 1 H), 4.21 (m, 1 H),

4.04 (m, 2 H), 3.89 (s, 3 H), 3.80 (s, 3 H), 2.13 (m, 2 H); ¹³C NMR

(CDCl₃+ CD₃OD, 75 MHz) δ 162.7, 153.7, 148.6, 148.2, 147.5,

142.4, 138.0, 136.6, 129.8, 128.6, 128.1, 120.2, 118.8, 116.8, 107.8,

106.2, 103.0, 101.78, 101.72, 71.1, 55.8, 55.6, 44.5, 40.8, 30.9; ESIMS

(m/z, relative intensity) 462 (MH⁺, 100); HRESIMS m/z 462.1670

(MH⁺), calcd for C₂₅H₂₃N₃O₆462.1665. Purity by HPLC was

estimated to be 99.4% in 85% MeOH−H₂O and 99.5% in 90%

MeOH−H₂O.

5,6-Dihydro-11-hydroxy-2,3-methylenedioxy-6-(3-morpho-

linylpropyl)-5-oxo-11H-indeno[1,2-c]isoquinoline (45b). Inden-

oisoquinoline 7 (0.1 g, 0.209 mmol) was dissolved in MeOH (30 mL).

NaBH₄(0.014 g, 0.418 mmol) was added at 0 °C, and the reaction

mixture was stirred at room temperature for 3 h. The mixture was

concentrated. CHCl₃was added (100 mL), and the solution was

washed with water (2 × 50 mL). The CHCl₃was removed on a rotary

evaporator and the crude mixture was puriﬁed by ﬂash column

chromatography (SiO₂), eluting with 5% MeOH in CHCl₃to give the

title compound (0.55 g, 55%) as white solid: mp 228−229 °C. IR

(KBr) 3325, 2956, 1610, 1629, 1553, 1483, 1294, 1257, 1037, 665

cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.33 (s, 1 H), 7.23 (s, 1 H), 7.12

(s, 1 H), 7.09 (s, 1 H), 6.06 (s, 1 H), 6.02 (s, 1 H), 5.29 (s, 1 H), 4.31

(m, 1 H), 4.05 (s, 3 H), 3.72 (m, 4 H), 3.63 (s, 3 H), 3.42 (m, 1 H),

2.40 (m, 6 H), 1.76 (m, 2 H); ¹³C NMR (CDCl₃, 75 MHz) δ 162.3,

153.5, 148.2, 147.4, 142.9, 138.2, 129.3, 128.7, 119.6, 116.9, 107.9,

106.5, 102.9, 102.8, 101.7, 77.2, 71.6, 66.7, 56.2, 56.1, 55.4, 54.0, 42.5,

25.9; ESIMS (m/z, relative intensity) 481 (MH⁺, 100); HRESIMS m/

z 481.1972 (MH⁺), calcd for C₂₆H₂₈N₂O₇481.1975. Purity by HPLC

was estimated to be 98.3% in 95% MeOH−H₂O and 97.2% in 40%

MeOH−H₂O

1

cm⁻¹; H NMR (DMSO, 300 MHz) δ 9.86 (s, 1 H), 7.85 (s, 1 H),

7.46 (s, 1 H), 7.35 (s, 1 H), 7.05 (s, 1 H), 6.17 (s, 2 H), 4.48 (m, 2 H),

3.89 (s, 3 H), 3.82−3.87 (t, J = 6.18 Hz, 2 H), 2.19 (m, 2 H); ESIMS

m/z (rel intensity) 458/460 (MH⁺, 28/27).

5,6-Dihydro-3-hydroxy-6-3-(1H-imidazol-1-yl)propyl)-2-me-

thoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]-

isoquinoline (44a). Compound 43 (0.210 g, 0.46 mmol) and NaI

(0.82 g, 5.52 mmol) were diluted with anhydrous DMF (15 mL).

Imidazole (0.375 g, 5.52 mmol) was added, and the mixture was

heated to 70 °C for 24 h under an argon atmosphere. Ice (30 g) was

added, and the solution was extracted with CHCl₃(5 × 50 mL). The

organic layer was washed with saturated NaHCO₃(3 × 30 mL), water

(3 × 30 mL) and dried over anhydrous sodium sulfate. The solution

was concentrated, adsorbed onto SiO₂(∼5 g), and puriﬁed by ﬂash

column chromatography (SiO₂, ∼60 g), eluting with a gradient of 1%

MeOH in CHCl₃to 5% MeOH in CHCl₃to yield the product as a

dark brown solid (0.045 g, 28%): mp >350 °C. IR (ﬁlm) 3446, 1693,

1642, 1484, 1431, 1394, 1309, 1225, 1033, 659 cm⁻¹; ¹H NMR

(DMSO, 300 MHz) 9.88 (s, 1 H), 7.88 (m, 2 H), 7.48 (s, 1 H), 7.32

(s, 1 H), 7.09 (s, 1 H), 7.05 (s, 1 H), 6.99 (s, 1 H), 6.17 (s, 2 H), 4. 38

(m, 2 H), 4.17 (m, 2 H), 3.89 (s, 1 H), 2.18 (m, 2 H); ESIMS m/z (rel

intensity) 446.5 (MH⁺, 100); HRESIMS m/z 446.1355 (MH⁺), calcd

for C₂₄H₁₉N₃O₆446.1352. Purity was estimated to be 95.2% by HPLC

in both 95% MeOH−H₂O and 85% MeOH−H₂O.

3,4-Dibenzyloxybenzaldehyde (47).⁶⁷Compound 46 (3.00 g,

21.7 mmol) was diluted with dry DMF (50 mL). Benzyl bromide

(7.65 g, 44.7 mmol) was added slowly, followed by anhydrous K₂CO₃

(9.60 g, 69.4 mmol). The mixture was stirred at room temperature for

2 h. Additional K₂CO₃(2.4 g, 17.3 mmol) was added, and the mixture

was heated to 70 °C for 30 min and then cooled to room temperature.

O

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

The mixture was partitioned between H₂O and ether (120 mL each).

The organic layer was separated, and the water layer was extracted

with ether (3 × 50 mL). The pooled organic layers were washed with

H₂O (2 × 50 mL) and saturated aqueous NaCl (50 mL). The pale,

straw-colored extracts were dried over anhydrous sodium sulfate and

concentrated to yield a cream-colored solid (6.57 g, 95%) after

washing with hexanes (75 mL) and drying: mp 87−88.5 °C (lit⁶⁷mp

85−87 °C). ¹H NMR (CDCl₃) δ 9.81 (s, 1 H), 7.49−7.31 (m, 12 H),

7.04 (d, J = 8.3 Hz, 1 H), 5.27 (s, 2 H), 5.22 (s, 2 H).

heated at 70 °C for 20 h. The reaction mixture was cooled to room

temperature and concentrated. H₂O (75 mL) was added, and the

mixture was extracted with CHCl₃(2 × 100 mL). The combined

organic layer was washed with saturated aqueous NaCl (50 mL). The

organic layer was concentrated and the crude mixture was puriﬁed by

silica gel ﬂash column chromatography, eluting with 2% MeOH in

CHCl₃to yield the product (0.320 g, 67%) as a purple solid: mp 235−

236 °C. IR (KBr) 2961, 1689, 1648, 1589, 1553, 1493, 1433, 1398,

1300, 1206, 1021, 747 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.97 (s, 1

H), 7.57 (s, 1 H), 7.51 (s, 1 H), 7.47−7.33 (m, 10 H), 7.20 (s, 1 H),

7.06 (s, 1 H), 6.95 (s, 1 H), 6.72 (s, 1 H), 5.23 (s, 2 H), 5.19 (s, 2 H),

4.35 (t, J = 6.3 Hz, 2 H), 4.02 (s, 3 H), 3.96 (m, 2 H), 3.94 (s, 3 H),

1.98 (m, 2 H); ESIMS m/z (rel intensity) 628 (MH⁺, 100), 1255

(2MH⁺, 26). Anal. Calcd for C₃₈H₃₃N₃O₆: C, 72.71; H, 5.30; N, 6.69.

Found: C, 72.30; H, 5.29; N, 6.62.

8,9-Dibenzyloxy-5,6-dihydro-2,3-dimethoxy-6-[3-(N-

morpholino)propyl-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(51b). Compound 50 (0.250 g, 0.392 mmol) was dissolved in dioxane

(50 mL). NaI (0.466 g, 3.92 mmol) and morpholine (0.271 g, 3.12

mmol) were added at room temperature, and the reaction mixture was

heated at 65 °C for 20 h. The reaction mixture was cooled to room

temperature and concentrated. H₂O (75 mL) was added, and the

mixture was extracted with CHCl₃(2 × 100 mL). The combined

organic layer was washed with saturated aqueous NaCl (50 mL). The

organic layer was concentrated and the crude mixture was puriﬁed by

ﬂash column chromatography (SiO₂), eluting with 1% MeOH in

CHCl₃to yield the product (0.150 g, 60%) as a brown solid: mp 208−

210 °C. IR (KBr) 2827, 1676, 1649, 1612, 1591, 1552, 1496, 1380,

1300, 1254, 1206, 1117, 870, 697 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)

δ 8.01 (s, 1 H), 7.60 (s, 1 H), 7.46 (m, 10 H), 7.22 (s, 1 H), 7.03 (s, 1

H), 5.24 (s, 1 H), 5.23 (s, 1 H), 4.39 (m, 2 H), 4.03 (s, 3 H), 3.95 (s, 3

H), 3.62 (m, 4 H), 2.41 (m, 6 H), 1.87 (m, 2 H); ESIMS m/z (rel

intensity) 647 (MH⁺, 100). Anal. Calcd for C₃₉H₃₈N₂O₇·0.75H₂O: C,

70.95; H, 6.03; N, 4.24. Found: C, 71.04; H, 5.86; N, 4.23

N-[3′,4′-(Dibenzyloxy)benzylidene]-3-bromopropan-1-

amine (48). 3-Bromopropylamine hydrobromide (0.791 g, 3.61

mmol) was diluted with CHCl₃(5 mL). Compound 47 (1.00 g, 3.14

mmol) was added slowly as a solution in CHCl₃(6 mL) and

quantitatively transferred with 3 mL of the same solvent. Et₃N (0.348

g, 3.45 mmol) was added slowly, upon which the solution became

clear and colorless. Na₂SO₄(1.20 g, 6.24 mmol) was added, and the

mixture was stirred at room temperature for 22 h. The mixture was

diluted to a volume of 30 mL with CHCl₃and was washed with H₂O

(3 × 40 mL) and saturated aqueous NaCl (40 mL). The organic layer

was dried over anhydrous sodium sulfate and concentrated to yield a

dark yellow syrup (1.36 g, 99%). IR (ﬁlm) 3031, 2840, 1344, 1600,

1

1582, 1509, 1454, 1432, 1383, 1268, 1135, 1022, 735, 691 cm⁻¹; H

NMR (CDCl₃) δ 8.20 (s, 1 H), 7.50−7.33 (m, 11 H), 7.20 (dd, J =

1.6, 8.3 Hz, 1 H), 6.96 (d, J = 8.3 Hz, 1 H), 5.21 (s, 4 H), 3.73 (td, J =

1.2, 6.2 Hz, 2 H), 3.51 (t, J = 6.6 Hz, 2 H), 2.30−2.22 (m, 2 H);

ESIMS m/z (rel intensity) 438/440 (MH⁺, 100/92).

cis-[3′,4′-(Dibenzyloxy)phenyl]-N-3-(bromopropyl)]-4-car-

boxy-3,4-dihydro-6,7-dimethoxy-3-1(2H)-isoquinolone (49).

Anhydride 11 (1.0 g, 4.50 mmol) was diluted with CHCl₃(30 mL),

and the solution was cooled to 0 °C. The Schiﬀ base 48 (1.97 g, 4.50

mmol) was added slowly as a precooled solution in CHCl₃(20 mL)

and quantitatively transferred with CHCl₃(3 mL). The mixture was

stirred at 0 °C for 2 h, followed by 10 h at room temperature, upon

which a precipitate had formed. Hexanes (50 mL) were added, and the

precipitate was collected, washed with 20% hexanes in CHCl₃(50

mL), and dried to yield the title compound as a light yellow

amorphous solid (2.34 g, 79%): mp 168−169 °C (dec). IR (KBr)

3583, 2937, 1736, 1597, 1513, 1426, 1286, 1265, 1138, 1023, 665

6-(3-(1H-Imidazol-1-yl)propyl)-8,9-dihydroxy-2,3-dime-

thoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (52a).

Compound 51a (0.100 g, 0.159 mmol) was diluted in 48% aqueous

HBr (25 mL), and the solution was heated at 70 °C for 15 h. After the

mixture was cooled to room temperature, chloroform and acetone (10

mL each) were added to the reaction mixture and removed on the

rotary evaporator three times. The leftover solid in water was cooled to

0 °C and then ﬁltered oﬀ. The solid was washed with 10% MeOH in

chloroform (25 mL) and with acetone (50 mL) to aﬀord catechol 52a

(0.068 g, 81%) as a light brown solid: mp 273−274 °C. IR (KBr)

3230, 1688, 1628, 1577, 1557, 1425, 1303, 1247, 865, 782, 665 cm⁻¹;

¹H NMR (DMSO-d₆, 300 MHz) δ 9.14 (s, 1 H), 7.91 (s, 1 H), 7.84 (s,

1

cm⁻¹; H NMR (CDCl₃+ DMSO-d₆, 300 MHz) δ 7.53 (s, 1 H),

7.30−7.20 (m, 10 H), 7.10 (s, 1 H), 6.70 (s, 1 H), 6.63 (s, 1 H), 4.94

(s, 2 H), 4.90 (d, J = 7.2 Hz, 1 H), 4.83 (s, 2 H), 4.50 (d, J = 7.4 Hz, 1

H), 3.83 (s, 3 H), 3.76 (s, 3 H), 3.33 (m, 2 H), 2.93 (m, 2 H), 2.00

(m, 2 H); ESIMS m/z (rel intensity) 660/662 (MH⁺, 100/97). Anal.

Calcd for C₃₅H₃₄BrNO: C, 63.64; H, 5.19; N, 2.12. Found: C, 63.46;

H, 5.21; N, 2.12.

8,9-Dibenzyloxy-6-(3-bromopropyl)-5,6-dihydro-2,3-dime-

thoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (50). Com-

pound 49 (1.00 g, 1.51 mmol) was cooled to −4 °C and then diluted

with SOCl₂(25 mL) and stirred for 3 h. After that the reaction mixture

was stirred at room temperature for an additional 4 h, upon which it

had become a bright reddish-purple color. The SOCl₂was evaporated.

The residue was dissolved in CHCl₃(40 mL), and the solution was

neutralized by the slow addition of saturated aqueous NaHCO₃(40

mL). The layers were separated, and the aqueous layer was extracted

with CHCl₃(30 mL). The organic layers were washed with H₂O (50

mL) and saturated aqueous NaCl (50 mL). The combined organic

layers were dried over anhydrous sodium sulfate and concentrated.

The residue was puriﬁed by ﬂash column chromatography (SiO₂),

eluting with CHCl₃to yield a reddish-purple solid (0.610 g, 63%): mp

214−215 °C. IR (KBr) 2939, 1688, 1643, 1613, 1591, 1552, 1494,

1 H), 7.67 (s, 1 H), 7.47 (s, 1 H), 7.07 (s, 1 H), 6.94 (s, 1 H), 4.42 (m,

4 H), 3.88 (s, 3 H), 3.84 (s, 3 H), 2.33 (m, 2 H); ¹³C NMR (DMSO-

d₆, 125 MHz) δ 190.1, 161.6, 154.4, 154.3, 149.0, 148.3, 146.5, 135.5,

128.3, 127.8, 126.6, 121.9, 119.8, 115.7, 112.8, 111.6, 107.7, 106.2,

102.1, 55.6, 55.4, 46.5, 40.8, 29.9; ESIMS m/z (rel intensity) 448

(MH⁺, 100); HRESIMS m/z 448.1514 (MH⁺), calcd for C₂₄H₂₂N₃O₆

448.1509. Purity was estimated to be 98.1% by HPLC in 85% MeOH/

1% TFA−15% H₂O and 98.3% in 70% MeOH/1% TFA−30% H₂O.

8,9-Dihydroxy-2,3-dimethoxy-6-(3-morpholinopropyl)-5H-

indeno[1,2-c]isoquinoline-5,11(6H)-dione (52b). Compound 51b

(0.125 g, 0.195 mmol) was dissolved in a mixture of MeOH and THF

(20 and 10 mL) and hydrogenated (with a balloon) in the presence of

10% Pd−C (10 mg) for 24 h. The Pd−C was ﬁltered oﬀ and the

ﬁltrate was concentrated and puriﬁed by ﬂash column chromatography

(SiO₂), eluting with 7% MeOH in CHCl₃to provide the product 52b

(0.041 g, 47%) as brown solid: mp 285−287 °C. IR (KBr) 3217, 2927,

1645, 1614, 1591, 1550, 1520, 1471, 1354, 1322, 1253, 1216, 870, 785,

1

1432, 1301, 1265, 1207, 1015, 737, 665 cm⁻¹; H NMR (CDCl₃, 300

MHz) δ 7.99 (s, 1 H), 7.58 (s, 1 H), 7.46−7.29 (m, 10 H), 7.20 (s, 1

H), 7.18 (s, 1 H), 5.26 (s, 2 H), 5.22 (s, 2 H), 4.51 (t, J = 7.3 Hz, 2 H),

4.02 (s, 3 H), 3.95 (s, 3 H), 3.58 (t, J = 6.2 Hz, 2 H), 2.33 (m, 2 H);

EIMS m/z (rel intensity) 639/641 (M⁺, 2/2).

8,9-Dibenzyloxy-5,6-dihydro-6-[3-(1H-imidazol-1-yl)propyl]-

2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

(51a). Compound 50 (0.500 g, 0.782 mmol) was dissolved in dioxane

(75 mL). NaI (0.971 g, 6.52 mmol) and imidazole (0.443 g, 6.52

mmol) were added at room temperature, and the reaction mixture was

1

657 cm⁻¹; H NMR (CDCl₃+ CD₃OD, 300 MHz) δ 7.88 (s, 1 H),

7.49 (s, 1 H), 7.01 (s, 1 H), 6.90 (s, 1 H), 4.36 (m, 2 H), 3.94 (s, 1 H),

3.88 (s, 1 H), 3.68 (t, J = 4.4 Hz, 2 H), 2.51 (m, 6 H), 2.02 (m, 2 H);

¹³C NMR (DMSO-d₆, 125 MHz) δ 190.2, 162.0, 154.8, 154.5, 149.4,

148.7, 146.8, 128.6, 128.2, 126.9, 116.1, 113.1, 111.9, 108.1, 106.5,

102.4, 63.7, 55.9, 55.8, 53.7, 51.4, 41.1, 23.9; ESIMS m/z (rel

intensity) 467 (MH⁺, 100); HRESIMS m/z 467.1824 (MH⁺), calcd

P

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

for C₂₅H₂₇N₄O₇467.1818. Purity was estimated to be 97.7% by HPLC

in 85% MeOH/1% TFA−15% H₂O and 98.8% in 70% MeOH/1%

TFA−30% H₂O.

Metabolism of LMP400 and LMP776 by Human Liver

Microsomes.³⁸Pooled human liver microsomes were purchased

from Life Technologies (Grand Island, NY). Other chemicals were

purchased from Sigma-Aldrich (St. Louis, MO). All organic solvents

were HPLC grade or higher and were purchased from Fisher Scientiﬁc

(Hanover Park, IL).

Compound 7 (LMP400) or 6 (LMP776) (10 μM) was incubated at

37 °C with pooled human liver microsomes (15 donors, mixed

gender) containing 1 mg/mL of microsomal protein and 50 mM

phosphate buﬀer at pH 7.4 in a total volume of 200 μL. After a 5 min

preincubation, 1 mM NADPH was added to initiate reaction, and the

mixture was incubated for an additional 60 min. The reaction was

stopped by chilling the mixture on ice and by addition of 20 μL of ice-

cold acetonitrile/water/formic acid (86:10:4, v/v/v) to precipitate

proteins. Samples were centrifuged. Supernatants were removed,

evaporated to dryness under nitrogen, and the residues were dissolved

in the mobile phase prior to analysis using liquid chromatography−

mass spectrometry (LC−MS) and LC−tandem mass spectrometry

(LC−MS/MS). Control incubations were identical except for the

elimination of microsomal protein or NADPH.

LC−MS and LC−MS/MS. Analyses of LMP400 or LMP776 and

their metabolites were carried out using a Waters (Milford, MA) 2690

HPLC system equipped with a Waters Xterra 2.1 mm × 100 mm C₁₈

column. The solvent system consisted of a linear gradient from 0.1%

formic acid in water to methanol as follows: 20−45% methanol over

10 min, 45−90% methanol over 1 min, and isocratic 90% methanol for

another 2 min. The column was equilibrated with 20% methanol for at

least 10 min between analyses. The ﬂow rate was 0.2 mL/min, and the

column temperature was 33 °C. The HPLC was interfaced with a high

resolution Waters Q-TOF Synapt hybrid quadrupole/time-of-ﬂight

mass spectrometer, and positive ion electrospray was used for sample

ionization. For accurate mass measurements, leucine enkephalin ([M +

H]⁺of m/z 556.2771) was introduced postcolumn as a lock mass. The

mass accuracy obtained was <5 ppm. Data were acquired from m/z

100−700. Tandem mass spectra were acquired at a collision energy of

20 eV using argon as the collision gas at a pressure of 2.0 × 10⁻⁵mbar.

Topoisomerase I Mediated DNA Cleavage Reactions. Human

recombinant Top1 was puriﬁed from baculovirus as previously

described.⁶⁸DNA cleavage reactions were prepared as previously

reported²⁶with the exception of the DNA substrate.⁶⁹Brieﬂy, a 117 bp

DNA oligonucleotide (Integrated DNA Technologies) encompassing

the previously identiﬁed Top1 cleavage sites in the 161 bp fragment

from pBluescript SK(−) phagemid DNA was employed. This 117 bp

oligonucleotide contains a single 5′-cytosine overhang, which was 3′-

end-labeled by ﬁll-in reaction with [α-³²P]dGTP in React 2 buﬀer (50

mM Tris-HCl, pH 8.0, 100 mM MgCl₂, 50 mM NaCl) with 0.5 units

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

10 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 the addition of two volumes of loading dye

(80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA,

0.1% xylene cyanol, and 0.1% bromophenol blue). Aliquots of each

reaction were subjected to 20% denaturing PAGE. Gels were dried and

visualized by using a phosphoimager and ImageQuant software

(Molecular Dynamics). For simplicity, cleavage sites were numbered

as previously described in the 161 bp fragment.⁶⁸

21.3419, y = −3.9888, z = 28.2163). The ligand was then deleted.

Indenoisoquinolines to be modeled were constructed in SYBYL. Atom

types were assigned using SYBYL atom typing. Hydrogens were added,

and the ligands were minimized by conjugate gradient method using

the MMFF94s force ﬁeld with MMFF94 charges, a distance-

dependent dielectric function, and a 0.01 kcal mol⁻¹Å⁻¹energy

gradient convergence criterion. Each ligand was docked into the

mutant crystal structure using GOLD 3.2 using default parameters, and

the coordinates were deﬁned by the crystal structure as described

above. The top four poses for each ligand were examined (because

lower-ranked poses with much lower GoldScores were sometimes

“ﬂipped” or rotated binding modes). The highest-ranked poses for

these ligands were merged into the crystal structure, and the entire

complex was subsequently subjected to minimization using a standard

Powell method, the MMFF94s force ﬁeld and MMFF94 charges, a

distance-dependent dielectric function, and a 0.05 kcal mol⁻¹Å⁻¹

energy gradient convergence criterion. During the minimization, the

ligand and a 7 Å sphere surrounding the ligands were allowed to move

while the structures outside this sphere were frozen in an aggregate.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@

purdue.edu.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was facilitated by the National Institutes of Health

(NIH) through support with Research Grant UO1 CA89566.

M.A.C. thanks Dr. Karl Wood for consultation and valuable

discussions. Joe Fornefeld of Midwest Microlab and Dr. Martin

Conda-Sheridan (Purdue University, IN) provided additional

assistance. This project has also been funded in whole or in part

with federal funds from the National Cancer Institute, National

I n s t i t u t e s o f H e a l t h , u n d e r C o n t r a c t N o .

HHSN261200800001E. The content of this publication does

not necessarily reﬂect the views or policies of the Department

of Health and Human Services, nor does mention of trade

names, commercial products, or organizations imply endorse-

ment by the U.S. Government.

ABBREVIATIONS USED

■

APCI, atmospheric pressure chemical ionization; GOLD,

Genetic Optimisation for Ligand Docking (program);

MDMA, 3,4-methylenedioxy-N-methylamphetamine; MDPPA,

3′,4′-methylenedioxy-α-pyrrolidinopropiophenone; MGM,

mean-graph midpoint; MMFF94, Merck Molecular Force

Field 94; NCI-60, National Cancer Institute 60 cell line

screening assay; Top1, topoisomerase 1

REFERENCES

■

(1) Pommier, E.; Topoisomerase, I. Inhibitors: Camptothecins and

Beyond. Nat. Rev. Cancer 2006, 6, 789−802.

(2) Stewart, L.; Redinbo, M. R.; Qiu, X.; Hol, W. G. J.; Champoux, J.

J. A Model for the Mechanism of Human Topoisomerase I. Science

1998, 279, 1534−1541.

(3) Wang, J. C. Cellular Roles of DNA Topoisomerases: A Molecular

Perspective. Nat. Rev. Mol. Cell Biol. 2002, 3, 430−440.

(4) Pommier, Y. DNA Topoisomerase I Inhibitors: Chemistry,

Biology, and Interfacial Inhibition. Chem. Rev. 2009, 109, 2894−2902.

(5) Pommier, Y.; Barcelo, J. A.; Rao, V. A.; Sordet, O.; Jobson, A. G.;

Thibaut, L.; Miao, Z. H.; Seiler, J. A.; Zhang, H.; Marchand, C.;

Agama, K.; Nitiss, J. L.; Redon, C. Repair of Topoisomerase I-

Docking and Modeling Studies. The Top1 crystal structure for

docking was prepared, and the docking protocol was validated as

previously described.⁴⁶The ternary complex centroid coordinates for

docking were deﬁned using the ligand in the Top1/topotecan crystal

structure (PDB code 1K4T) as the center of the binding pocket (x =

Q

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Mediated DNA Damage. Prog. Nucleic Acid Res. Mol. Biol. 2006, 81,

179.

(24) Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K.

W.; Cushman, M.; Pommier, Y. Differential Induction of Topoisomer-

ase I-DNA Cleavage Complexes by the Indenoisoquinoline MJ-III-65

(NSC 706744) and Camptothecin: Base Sequence Analysis and

Activity against Camptothecin-Resistant Topoisomerase I. Cancer Res.

2003, 63, 7428−7435.

(25) A Phase I Study of Indenoisoquinolines LMP400 and LMP776

in Adults with Relapsed Solid Tumors and Lymphomas. http://

clinicaltrials.gov/ct2/show/study/NCT01051635 (accessed February

17, 2012).

(26) Antony, S.; Agama, K. K.; Miao, Z. H.; Takagi, K.; Wright, M.

H.; Robles, A. I.; Varticovski, L.; Nagarajan, M.; Morrell, A.; Cushman,

M.; Pommier, Y. Novel Indenoisoquinolines NSC 725776 and NSC

724998 Produce Persistent Topolsomerase I Cleavage Complexes and

Overcome Multidrug Resistance. Cancer Res. 2007, 67, 10397−10405.

(27) Chrencik, J. E.; Staker, B. L.; Burgin, A. B.; Pourquier, P.;

Pommier, Y.; Stewart, L.; Redinbo, M. R. Mechanisms of

Camptothecin Resistance by Human Topoisomerase I Mutations. J.

Mol. Biol. 2004, 339, 773−784.

(6) Bailly, C. Topoisomerase I Poisons and Suppressors as

Anticancer Drugs. Curr. Med. Chem. 2000, 7, 39−58.

(7) Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. Camptothecin

Induces Protein-Linked DNA Breaks via Mammalian DNA Topo-

isomerase I. J. Biol. Chem. 1985, 260, 14873−14878.

(8) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A.

B.; Stewart, L. The Mechanism of Topoisomerase I Poisoning by a

Camptothecin Analogue. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15387−

15392.

(9) Sordet, O.; Khan, Q. A.; Kohn, K. W.; Pommier, Y. Apoptosis

Induced by Topoisomerase Inhibitors. Curr. Med. Chem.: Anti-Cancer

Agents 2003, 3, 271−290.

(10) Teicher, B. Next Generation of Topoisomerase I Inhibitors:

Rationale and Biomarker Strategies. Biochem. Pharmacol. 2008, 75,

1262−1271.

(11) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail,

A. T.; Sim, G. A. Plant Antitumor Agents. I. The Isolation and

Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor

Inhibitor from Camptotheca acuminata. J. Am. Chem. Soc. 1966, 88,

3888−3890.

(12) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Camptothecin:

Current Perspectives. Bioorg. Med. Chem. 2004, 12, 1585−1604.

(13) Pommier, Y.; Marchand, C. Interfacial Inhibitors: Targeting

Macromolecular Complexes. Nat. Rev. Drug Discovery 2012, 11, 25−

36.

(28) Relling, M. V.; Nemec, J.; Schuetz, E. G.; Schuetz, J. D.;

Gonzalez, F. J.; Korzekwa, K. R. O-Demethylation of Epipodophyllo-

toxins Is Catalyzed by Human Cytochrome P450 3A4. Mol. Pharmacol.

1994, 45, 352−358.

(29) Kupfer, A.; Schmid, B.; Pfaff, G. Pharmacogenetics of

̈

Dextromethorphan O-Demethylation in Man. Xenobiotica 1986, 16,

421−433.

(30) Paar, W. D.; Frankus, P.; Dengler, H. J. The Metabolism of

Tramadol by Human Liver Microsomes. Clin. Invest. 1992, 70, 708−

710.

(14) Schaeppi, U.; Fleischman, R. W.; Cooney, D. A. Toxicity of

Camptothecin (NSC-100880). Cancer Chemother. Rep., Part 3 1974, 5,

25−36.

(31) Qiu, F.; Zhu, Z. Y.; Kang, N.; Piao, S. J.; Qin, G. Y.; Yao, X. S.

Isolation and Identification of Urinary Metabolites of Berberine in Rats

and Humans. Drug Metab. Dispos. 2008, 36, 2159−2165.

(32) Sooryakumar, D.; Dexheimer, T. S.; Teicher, B. A.; Pommier, Y.

Molecular and Cellular Pharmacology of the Novel Noncamptothecin

Topoisomerase I Inhibitor Genz-644282. Mol. Cancer Ther. 2011, 10,

1490−1499.

(33) Benedetti, M. S.; Malnoe, A.; Broillet, A. L. Absorption,

Metabolism, and Excretion of Safrole in the Rat and Man. Toxicology

1977, 7, 69−83.

(15) Luzzio, M. J.; Besterman, J. M.; Emerson, D. L.; Evans, M. G.;

Lackey, K.; Leitner, P. L.; McIntyre, G.; Morton, B.; Myers, P. L.; Peel,

M.; Sisco, J. M.; Sternbach, D. D.; Tong, W. Q.; Truesdale, A.;

Uehling, D. E.; Vuong, A.; Yates, J. Synthesis and Antitumor-Activity

of Novel Water-Soluble Derivatives of Camptothecin as Specific

Inhibitors of Topoisomerase-I. J. Med. Chem. 1995, 38, 395−401.

(16) Mi, Z.; Burke, T. G. Differential Interactions of Camptothecin

Lactone and Carboxylate Forms with Human Blood Components.

Biochemistry 1994, 33, 10325−10336.

(17) Paull, K. D.; Hamel, E.; Malspeis, L. Prediction of Biochemical

Mechanism of Action from the in Vitro Antitumor Screen of the

National Cancer Institute. In Cancer Chemotherapeutic Agents; Foye,

W. O., Ed.; American Chemical Society: Washington, DC, 1995; pp

9−45.

(18) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,

D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and Analysis of

Patterns of Differential Activity of Drugs Against Human Tumor Cell

Lines: Development of Mean Graph and COMPARE Algorithm. J.

Natl. Cancer Inst. 1989, 81, 1088−1092.

(19) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier,

Y. Protein-Linked DNA Strand Breaks Induced by NSC 314622, a

Novel Noncamptothecin Topoisomerase I Poison. Mol. Pharmacol.

1998, 54, 50−58.

(20) Pommier, Y.; Cushman, M. The Indenoisoquinoline Non-

camptothecin Topoisomerase I Inhibitors: Update and Perspectives.

Mol. Cancer Ther. 2009, 8, 1008−1014.

(21) Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony,

S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis and

Anticancer Activity of Simplified Indenoisoquinoline Topoisomerase

I Inhibitors Lacking Substituents on the Aromatic Rings. J. Med. Chem.

2004, 47, 5651−5661.

(22) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.;

Kohlhagen, G.; Agama, K.; Hollingshead, M.; Pommier, Y.;

Cushman, M. Synthesis and Evaluation of Indenoisoquinoline

Topoisomerase I Inhibitors Substituted with Nitrogen Heterocycles.

J. Med. Chem. 2006, 49, 6283−6289.

(34) Klungsoyr, J.; Scheline, R. R. Metabolism of Piperonal and

Piperonyl Alcohol in the Rat with Special Reference to the Scission of

the Methylenedioxy Group. Acta Pharm. Suec. 1984, 21, 67−72.

(35) Lim, H. K.; Foltz, R. L. In Vitro and in Vivo Metabolism of 3,4-

(Methylenedioxy)amphetamine in the Rat: Identification of Metabo-

lites Using an Ion Trap Detector. Chem. Res. Toxicol. 1988, 1, 370−

378.

(36) Springer, D.; Fritschi, G.; Maurer, H. H. Metabolism and

Toxicological Detection of the New Designer Drug 3′,4′-Methyl-

enedioxy-alpha-pyrrolidinopropiophenone Studied in Urine Using Gas

Chromatography−Mass Spectrometry. J. Chromatogr, B: Anal. Technol.

Biomed. Life Sci. 2003, 793, 377−388.

(37) Novotna, R.; Wsol, V.; Xiong, G.; Maser, E. Inactivation of the

Anticancer Drugs Doxorubicin and Oracin by Aldo-keto Rductase

(AKR) 1C3. Toxicol. Lett. 2008, 181, 1−6.

(38) Chen, L.; Conda-Sheridan, M.; Reddy, P. V. N.; Morrell, A.;

Park, E. J.; Kondratyuk, T. P.; Pezzuto, J. M.; van Breemen, R.;

Cushman, M. Identification, Synthesis, and Biological Evaluation of

the Metabolites of 3-Amino-6-(3′-aminopropyl)-5H-indeno[1,2-c]-

isoquinoline-5,11-(6H)dione (AM6-36), a Promising Rexinoid Lead

Compound for the Development of Cancer Chemotherapeutic and

Chemopreventive Agents. J. Med. Chem. 2012, 55, 5965−5981.

(39) Wall, M. E.; Wani, M. C.; Nicholas, A. W.; Manikumar, G.; Tele,

C.; Moore, L.; Truesdale, A.; Leitner, P.; Besterman, J. M. Plant

Antitumor Agents. 30. Synthesis and Structure−Activity of Novel

Camptothecin Analogs. J. Med. Chem. 1993, 36, 2689−2700.

(40) Wani, M. C.; Nicholas, A. W.; Manikumar, G.; Wall, M. E. Plant

Antitumor Agents. 25. Total Synthesis and Antileukemic Activity of

Ring A Substituted Camptothecin Analogues. Structure−Activity

Correlations. J. Med. Chem. 1987, 30, 1774−1779.

(23) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,

M. Synthesis of Nitrated Indenoisoquinolines as Topoisomerase I

Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3659−3663.

R

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(41) Bailly, C.; Riou, J. F.; Colson, P.; Houssier, C.; Rodrigues-

Pereira, E.; Prudhomme, M. DNA Cleavage by Topoisomerase I in the

Presence of Indolocarbazole Derivatives of Rebeccamycin. Biochemistry

1997, 36, 3917−3929.

(42) Fortune, J. M.; Velea, L.; Graves, D. E.; Utsugi, T.; Yamada, Y.;

Osheroff, N. DNA Topoisomerases as Targets for the Anticancer Drug

TAS-103: DNA Interactions and Topoisomerase Catalytic Inhibition.

Biochemistry 1999, 38, 15580−15586.

(43) Van, H. T. M.; Le, Q. M.; Lee, K. Y.; Lee, E. S.; Kwon, Y.; Kim,

T. S.; Le, T. N.; Lee, S. H.; Cho, W. J. Convenient Synthesis of

Indeno[1,2c]isoquinolines as Constrained Forms of 3-Arylisoquino-

lines and Docking Study of a Topoisomerase I Inhibitor into DNA-

Topoisomerase I Complex. Bioorg. Med. Chem. Lett. 2007, 17, 5763−

5767.

(44) Khadka, D. B.; Cho, W. J. 3-Arylisoquinolines as Novel

Topoisomerase I Inhibitors. Bioorg. Med. Chem. 2011, 19, 724−734.

(45) Ryckebusch, A.; Garcin, D.; Lansiaux, A.; Goossens, J. F.;

Baldeyrou, B.; Houssin, R.; Bailly, C.; Henichart, J. P. Synthesis,

Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition

Properties of Novel Indeno[2,1-c]quinolin-7-one and indeno[1,2-

c]isoquinolin-5,11-dione Derivatives. J. Med. Chem. 2008, 51, 3617−

3629.

(46) Peterson, K. E.; Cinelli, M. A.; Morrell, A. E.; Mehta, A.;

Dexheimer, T. S.; Agama, K.; Antony, S.; Pommier, Y.; Cushman, M.

Alcohol-, Diol-, and Carbohydrate-Substituted Indenoisoquinolines as

Topoisomerase I Inhibitors: Investigating the Relationships Involving

Stereochemistry, Hydrogen Bonding, and Biological Activity. J. Med.

Chem. 2011, 54, 4937−4953.

(47) Lebrun, S.; Couture, A.; Deniau, E.; Grandclaudon, P. Suzuki−

Miyaura Cross-Coupling and Ring-Closing Metathesis: A Strategic

Combination to the Synthesis of Indeno[1,2-c]isoquinolin-5,11-

diones. Tetrahedron Lett. 2011, 52, 1481−1484.

(48) Cushman, M.; Gentry, J.; Dekow, F. W. Condensation of Imines

with Homophthalic Anhydrides. A Convergent Synthesis of cis- and

trans-13-Methyltetrahydroprotoberberines. J. Org. Chem. 1977, 42,

1111−1116.

(59) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.

Development and Validation of a Genetic Algorithm for Flexible

Docking. J. Mol. Biol. 1997, 267, 727−748.

(60) Jones, G.; Willett, P.; Glen, R. C. Molecular Recognition of

Receptor Sites Using a Genetic Algorithm with a Description of

Desolvation. J. Mol. Biol. 1995, 245, 43−53.

(61) Cinelli, M. A.; Morrell, A. E.; Dexheimer, T. S.; Agama, K.;

Agrawal, S.; Pommier, Y.; Cushman, M. The Structure−Activity

Relationships of A-Ring-Substituted Aromathecin Topoisomerase I

Inhibitors Strongly Support a Camptothecin-like Binding Mode.

Bioorg. Med. Chem. 2010, 18, 5535−5552.

(62) Zembower, D. E.; Zhang, H. P.; Lineswala, J. P.; Kuffel, M. J.;

Aytes, S. A.; Ames, M. M. Indolocarbazole Poisons of Human

Topoisomerase I: Regioisomeric Analogues of ED-110. Bioorg. Med.

Chem. Lett. 1999, 9, 145−150.

(63) Duclos, R. I.; Tung, J. S.; Rapoport, H. A High-Yield

Modification of the Pschorr Phenanthene Synthesis. J. Org. Chem.

1984, 49, 5243−5246.

(64) Guthrie, D. A.; Frank, A. W.; Purves, C. B. Studies in the

Polyoxyphenol Series. XI. Synthesis of Papaverine and Papaveraldine

by the Pomeranz−Fritsch Method. Can. J. Chem. 1955, 33, 729−742.

(65) Ploypradith, P.; Cheryklin, P.; Niyomtham, N.; Bertoni, D. R.;

Ruchirawat, S. Solid-Supported Acids as Mild and Versatile Reagents

for the Deprotection of Aromatic Ethers. Org. Lett. 2007, 9, 2637−

2640.

(66) Prasad, D.; Chaudhury, D. N. Synthesis of 3,4-Dihydro-7-

hydroxy-6-methoxyisocoumarin. J. Indian Chem. Soc. 1962, 39, 672−

676.

(67) Bolek, D. J.; Guetschow, M. Preparation of 4,6,3′,4′-

Tetrasubstituted Aurones via Aluminum Oxide-Catalyzed Condensa-

tion. J. Heterocycl. Chem. 2005, 42, 1399−1403.

(68) Pourquier, P.; Ueng, L.-M.; Fertala, J.; Wang, D.; Park, H.-J.;

Essigmann, J. M.; Bjornsti, M.-A.; Pommier, Y. Induction of Reversible

Complexes Between Eukaryotic DNA Topoisomerase I and DNA-

Containing Oxidative Base Damages. 7,8-Dihydro-8-oxoguanine and

5-Hydroxycytosine. J. Biol. Chem. 1999, 274, 8516−8523.

(69) Dexheimer, T. S.; Pommier, Y. DNA Cleavage Assay for the

Identification of Topoisomerase I Inhibitors. Nat. Protoc. 2008, 3,

1736−1750.

(49) Cushman, M.; Cheng, L. Stereoselective Oxidation by Thionyl

Chloride Leading to the Indeno[1,2-c]isoquinoline System. J. Org.

Chem. 1978, 43, 3781−3783.

(50) Finkelstein, J.; Brossi, A. 6.7-Dimethoxy-3-isochromanone. Org.

Synth. 1988, 6, 471.

(51) Rastogi, S. N.; Bindra, J. S.; Anand, N. α-Methyldopa in a Rigid

Framework: 3-Amino-3-methyl-6,7-dihydroxy-3,4-dihydroxycoumarins

and 2-Amino-6,7-dihydroxytetralin-2-carboxylic Acids. Indian J. Chem.

1971, 9, 1175−1182.

(52) Nonni, A. J.; Dence, C. W. The Synthesis of 1.2-Diarylpropane-

1,3-diols via the Ivanoff Reaction. J. Wood Chem. Technol. 1982, 2,

161−168.

(53) Patra, A.; Ghosh, G.; Mukhopadhyay, P. K. Studies on Some

Azlactones and Isochromanones. Synthesis and Spectral Character-

istics. J. Indian Chem. Soc. 1987, 64, 414−417.

(54) Spangler, R. J.; Beckmann, B. G.; Kim, J. H. A New Synthesis of

Benzocyclobutenes. Thermal and Electron Impact Induced Decom-

position of 3-Isochromanones. J. Org. Chem. 1977, 42, 2989−2996.

(55) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;

Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New

Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. J.

Natl. Cancer Inst. 1990, 82, 1107−1112.

(56) Boyd, M. R.; Paull, K. D. Some Practical Considerations and

Applications of the National Cancer Institute in Vitro Anticancer Drug

Discovery Screen. Drug Dev. Res. 1995, 34, 91−109.

(57) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.;

Pommier, Y.; Cushman, M. Optimization of the Indenone Ring of

Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem. 2007,

50, 4388−4404.

(58) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.;

Zembower, D.; Stewart, L.; Burgin, A. B. Structures of Three Classes

of Anticancer Agents Bound to the Human Topoisomerase I-DNA

Covalent Complex. J. Med. Chem. 2005, 48, 2336−2345.

S

dx.doi.org/10.1021/jm300519w | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Identification, synthesis, and biological evaluation of metabolites of the experimental cancer treatment drugs indotecan (LMP400) and indimitecan (LMP776) and investigation of isomerically hydroxylated indenoisoquinoline analogues as topoisomerase i poisons

DOI: 10.1021/jm300519w

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm300519w

Full text of DOI:10.1021/jm300519w

Products guided by the article

R&D Labs maybe for 1415806-75-5

Relevant to this article

Hot Product