Substituted dibenzo[c,h]cinnolines: Topoisomerase I-targeting anticancer agents

Bioorganic & Medicinal Chemistry 11 (2003) 1475–1491

Substituted Dibenzo[c,h]cinnolines: Topoisomerase I-Targeting

Anticancer Agents

Younong Yu,^aSudhir K. Singh,^aAngela Liu,^bTsai-Kun Li,^bLeroy F. Liu^b,c

and Edmond J. LaVoie^a,c,*

^aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA

^bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,

Piscataway, NJ 08854, USA

^cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA

Received 18 July 2002; accepted 24 October 2002

Abstract—Several substituted dibenzo[c,h]cinnolines were synthesized and evaluated for their potential to target topoisomerase I

and for their relative cytotoxic activity. Select benzo[i]phenanthridines are capable of stabilizing the cleavable complex formed with

topoisomerase I and DNA. This study was initiated to examine whether dibenzo[c,h]cinnolines, which are in essence aza analogues

of benzo[i]phenanthridines, possess similar pharmacological properties. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine

is one of the more potent benzo[i]phenanthridine derivatives in regard to topoisomerase I-targeting activity and cytotoxicity. The

structure–activity relationship observed with these substituted dibenzo[c,h]cinnolines parallels that observed for benzo[i]phenan-

thridine derivatives. Compared to similarly substituted benzo[i]phenanthridines, the dibenzo[c,h]cinnoline analogues exhibit more

potent topoisomerase I-targeting activity and cytotoxicity. The relative IC₅₀values obtained in assessing the cytotoxicity of 2,3-

dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline and 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine in the human

lymphoblastma cell line, RPMI8402, are 70 and 400 nM, respectively. In tumor cell lines selected for resistance to camptothecin and

known to express mutant topoisomerase I, benzo[i]phenanthridine derivatives were not cross-resistant. In contrast, similarly sub-

stituted dibenzo[c,h]cinnolines with signiﬁcant topoisomerase I-targeting activity did exhibit cross-resistance in these camptothecin-

resistant cell lines. The cytotoxicity of these dibenzo[c,h]cinnolines was not diminished in cells overexpressing the eﬄux transporter,

MDR1. These data indicate that substituted dibenzo[c,h]cinnolines can exhibit potent topoisomerase I-targeting activity and are

capable of overcoming the multi-drug resistance associated with this eﬄux transporter.

Introduction

antitumor activity of topoisomerase-targeting agents is

associated with their ability to stabilize the enzyme-

DNA cleavable complex. This drug-induced stabili-

zation of the enzyme–DNA cleavable complex eﬀectively

converts these enzymes into cellular poisons.

The topological state of DNA is regulated by DNA

topoisomerases, which perform this function through

the breaking and rejoining of DNA strands.^1À4Studies

have also demonstrated that these enzymes are involved

in controlling template supercoiling during RNA tran-

scription.^5,6There are two major subtypes of topo-

isomerases based upon diﬀerences in their initial

mechanisms. While the mechanism associated with

topoisomerase I (TOP1) involves the formation of a

single-strand DNA break, topoisomerase II (TOP2)

functions by creating a double-strand DNA break. The

Camptothecin and its structurally-related analogues are

among the more extensively studied agents that target

TOP1. Bi- and terbenzimidazoles,^7À10certain benzo[c]-

phenanthridine and protoberberine alkaloids and their

synthetic analogues,^11À15indolocarbazoles,¹⁶the fungal

metabolites bulgarein¹⁷and saintopin,¹⁸and indenoiso-

quinolines,^19,20phenazines,²¹and benzophenazines,²²

have been identiﬁed as TOP1-directed agents. Recently,

several benzo[i]phenanthridines have been identiﬁed

that exhibit potent activity as TOP1-targeting agents

and signiﬁcant cytotoxicity.^23,242,3-Dimethoxy-8,9-

methylenedioxybenzo[i]phenanthridine, 1 (Fig. 1), is

*Corresponding author at: Ernest Mario School of Pharmacy, 160

Frelinghuysen Road, Piscataway, NJ 08854, USA. Tel.: +1-732-445-

2674; fax: +1-732-445-6312; e-mail: elavoie@rci.rutgers.edu

http://www.rci.rutgers.edu /ꢀlayla/Faculty/LaVoie.htm

PII: S0968-0896(02)00604-1

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Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

among the more active benzo[i]phenanthridine derivatives

evaluated.

The present study explores the structure activity rela-

tionships of substituted dibenzo[c,h]cinnolines. Diben-

zo[c,h]cinnolines, which have incorporated into their

structure two adjacent heteroatoms, are structurally

similar to benzo[c]phenanthridines or benzo[i]phenan-

thridines The inﬂuence of substituents in both the

A- and D-ring of dibenzo[c,h]cinnolines on both TOP1-

targeting activity and cytotoxicity was investigated.

Among the compounds selected for synthesis and

pharmacological evaluation was 2,3-dimethoxy-8,9-

methylenedioxydibenzo[c,h]cinnoline, 4. To further

evaluate the structure–activity relationships within this

class of compounds, compounds 5–15 were synthesized

and their pharmacological activities evaluated (Chart 1).

The benzo[i]phenanthridine derivatives 2a and 2b were

previously selected for synthesis and pharmacological

evaluation based upon their structural similarity to

nitidine, 3. Compound 1, however, exhibited sig-

niﬁcantly greater cytotoxicity and TOP1-targeting

activity than either 2a or 2b. Substituents on the A- and

D-ring of benzo[i]phenanthridines can have a profound

eﬀect on pharmacological activity. It was assumed,

therefore, that each of these benzophenanthridines, 1–3,

act by adopting an orientation wherein a similar mole-

cular topology is achieved, but the relative position of

the heteroatom diﬀers. The presence of 2,3-dimethoxy

substituents in ring-A, a 8,9-methylenedioxy moiety in

ring-D, and a nitrogen heteroatom adjacent to the

benzo-ring that possesses the methylenedioxy sub-

stituent appear to deﬁne key structural elements that are

associated with the enhanced TOP1-targeting

activity and cytotoxicity observed with 1 relative to

other benzo[i]phenanthridine derivatives.^23,24

Chemistry

The synthetic method employed for the preparation of 5

is outlined in Scheme 1. Compound 16 was prepared

from 6,7-dimethoxy-a-tetralone. Using conditions as

employed in the Heck reaction, 6,7-dimethoxy-1,2-

dihydronaphthalene 17 was coupled with o-iodoni-

Figure 1. Structure of benzo[i]phenanthridines (1, 2a,b), nitidine (3),

2,3-dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline, 4, and the

numbering and ring designation of benzo[i]phenanthridines and

dibenzo[c,h]cinnolines.

Scheme 1. Preparation of 5: (i) H₃PO₄, DMF, 60 ^ꢁC; (ii)

Pd(PPh₃)₂Cl₂, NaOCOCH₃, DMA; (iii) DDQ, toluene, reﬂux; (iv) H₂,

10% Pd/C; (v) 48% HBr, NaNO₂, Cu powder.

Chart 1.

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1477

trobenzene to provide 18 in low yield (30%).²⁵Oxida-

tion of 18 with DDQ provided the naphthyl derivative

19, which was reduced to 2-(o-aminophenyl)-6,7-dime-

thoxynaphthalene, 20. Diazotization of 6-(2-amino-4,5-

dimethoxyphenyl)-2,3-dimethoxynaphthalene is known

to give 2,3,8,9-tetramethoxydibenzo[c,h]cinnoline in

approximately 20% yield.²⁶Similarly, diazotization of

20 provided 5 in 18% yield. The instability of several of

the intermediates, the low yields associated with several

of the initial reactions, and the general ineﬃciency of

this synthetic route prompted the development of an

alternative synthetic strategy.

of a mixture of 33 and 34. Diazotization of 30–33 pro-

vided the desired 2,3-dimethoxybenzo[c,h]cinnolines 6–9.

Treatment of 34 under similar conditions failed to pro-

vide 10. Hydrogenolysis of 9, however, did provide 10 in

good yield.

Select 8,9-methylenedioxydibenzo[c,h]cinnolines were

similarly prepared as illustrated in Scheme 3. Coupling

of trimethyl-4,5-methylenedioxy-2-nitrophenylstannane,

35, with 6,7-dimethoxy-2-triﬂuoromethanesulfonyloxy-

naphthalene (25), 2-bromo-6-methoxynaphthalene (36),

or 7-methoxy-2-triﬂuoromethanesulfonyloxynaphthalene

(37) provided 2-(2-nitrophenyl)naphthalenes 38–40.

Treatment of these 2-(2-nitrophenyl) naphthalenes with

hydrogen in the presence of 10% Pd/C provided aniline

derivatives 41–43. Diazotization of 41–43 gave the 8,9-

methylenedioxydibenzo[c,h]cinnolines, 4, 11 and 12 as

depicted in Scheme 3.

A substantially modiﬁed approach used for the pre-

paration of the 2,3-dimethoxydibenzo[c,h]cinnolines 6–10

is outlined in Scheme 2. Stille coupling proved to be an

eﬀective method for preparing 2-(2-nitrophe-

nyl)naphthalenes.²⁷Using variously substituted ortho-

nitroarylstannanes, Stille coupling of 21–24 with 6,7-

dimethoxy - 2 - triﬂuoromethanesulfonyloxynaphthalene

(25) provided the requisite 2-(2-nitrophenyl)naphthyl

derivatives, 26–29. These 2-(2-nitrophenyl)naphthyl

derivatives were readily converted to their aniline deri-

vatives, 30–34, using Pd/C and H₂. Under these condi-

tions, partial hydrogenolysis was observed with the

benzyloxy analogue 29, which resulted in the formation

An alternative route for the preparation of 12, 13 and

14 is outlined in Scheme 4. Nitration of 44–46 with 4

-methyl-4-nitro-2,3,5,6-tetrabromo-2,5-cyclohexadien-1-

one provided 47–49 in 35-58% yield.²⁸Using

N-phenyltriﬂuoromethanesulfonimide²⁹or triﬂuoro-

methanesulfonic anhydride, the triﬂates 50–52 were

prepared and coupled with trimethyl-4,5-methylene-

Scheme 2. Preparation of dibenzo[c,h]cinnolines, 6–10: (i) (CH₃)₃SnSn(CH₃)₃, Pd(PPh₃)₄, THF, reﬂux; (ii) Pd(PPh₃)₄, CuBr, THF; (iii) H₂, 10% Pd/

C, EtOAc; (iv) NaNO₂, AcOH, concd HCl; (v) H₂, 10% Pd/C, EtOAc.

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Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

dioxy-2-nitrophenyl stannane (35) to give the dinitro

intermediates, 53–55. Reduction of 53 and 54 with LAH

provided 12 and 13 in 75% and 42% yield, respectively.

Preparation of 14 was accomplished by treatment of 55

using Raney Ni in the presence of hydrazine.³⁰

sence of TOP1 and TOP2, respectively. The diben-

zo[c,h]cinnolines were evaluated for cytotoxicity toward

the human lymphoblast tumor cell line, RPMI8402 and

its camptothecin-resistant variant, CPT-K5, U937 and

its camptothecin-resistant variant U937/CR, as well as

KB3-1 and its variant, KBV-1, which overexpresses the

eﬄux transporter, P-glycoprotein (MDR1). These data

are provided in Table 2. Table 3 summarizes the cyto-

toxicity observed with 1 and 3–7 against the human

colon tumor cell line, HCT116, the human breast tumor

cell line, ZR-75-1, the human cervical tumor cell line,

HeLa, the human leukemia cell line, CEM and its TOP2

deﬁcient variant, CEM/V-1.

The phenolic dibenzo[c,h]cinnolines, 10 and 14, repre-

sent derivatives that could be utilized to form new ana-

logues or prodrugs that possess physico-chemical

properties for improving solubility, formulation, and

bioavailability. The synthesis of the 2-methoxy-3-

hydroxymethylcinnoline 15 was also targeted in an

eﬀort to identify a dibenzo[c,h]cinnoline derivative sui-

table for prodrug development. The synthetic approach

for the preparation of 15 is provided in Scheme 5. Cou-

pling of the ortho-nitrostannane 35 with 7-bromo-3-

methoxynaphthalene-2-carboxylic acid methyl ester

gave 57 in 55% yield. Reduction of the NO₂group of

compound 57 with Ra–Ni and hydrazine hydrate pro-

vided 58, which was converted to its hydroxymethyl

derivative 59 with LAH. Diazotization of 59 gave the

desired dibenzo[c,h]cinnoline, 15.

Discussion

Previous studies demonstrated that the benzo[i]phenan-

thridine derivative 1 is an order of magnitude more

potent in its TOP1-targeting activity than 2a or 2b, as

determined by measuring cleavable complex formation

in the presence of enzyme and DNA. A further

enhancement in intrinsic TOP1-targeting activity was

observed with the dibenzo[c,h]cinnoline analogue 4,

which is approximately twice as potent as 1. The

importance of the methylenedioxy substituent to the

activity of these dibenzo[c,h]cinnolines as TOP1-target-

ing agents parallels previous observations noted in the

structure–activity relationships observed with benzo[i]-

Pharmacological evaluation

The relative topoisomerase-targeting activities (both

TOP1 and TOP2) of 1–15 are listed in Table 1. Figures 2

and 3 are representative of the DNA cleavage induced

by various substituted dibenzo[c,h]cinnolines in the pre-

Scheme 4. Alternative synthetic route for the preparation of 12–14: (i)

C₇H₃Br₄N₁O₃, Et₂O; (ii) (CF₃SO₂)₂O in pyridine or NaH,

(CF₃SO₂)₂NC₆H₅, THF; (iii) Pd(PPh₃)₄, LiCl, CuBr, THF; (iv) LAH,

Scheme 3. Preparation of 4, 11–12: (i) Pd(PPh₃)₄, CuBr, THF; (ii) H₂,

10% Pd/C, EtOAc; (iii) NaNO₂, AcOH, concd HCl.

.

THF; (v) NH₂NH₂H₂O, NaOH, EtOH.

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1479

phenanthridines. Removal of the methylenedioxy moi-

ety, as in 5, or its replacement by a methoxyl group, as

in the case of 7 and 8, resulted in a substantial loss of

activity. Consistent with structure–activity data

observed for benzo[c]phenanthridines and benzo[i]phe-

nanthridines, replacement of the methylenedioxy moiety

in Ring-D of 4 by two methoxyl groups, that is 2,3,8,9-

teramethoxydibenzo[c,h]cinnoline 6, a methoxyl and a

benzyloxy group 9, or a methoxyl and hydroxyl group

10, results in the complete loss of TOP1-targeting activ-

ity. These data clearly substantiate the importance of

the methylenedioxy moiety within the D-ring of these

dibenzo[c,h]cinnolines for retention of potent TOP1-

targeting activity.

camptothecin-resistant variant, U937/CR. Both of the

cell lines are resistant to camptothecin and have a func-

tional, but mutant TOP1 in which the cleavable com-

plex formed between enzyme and DNA is not stabilized

by camptothecin. The dibenzo[c,h]cinnoline derivative

with the greatest potency as a TOP1-targeting agent 4

was the most cytotoxic analogue to RPMI8402 and

U937 cells with IC₅₀values of 70 and 60 nM, respec-

Table 1. Topoisomerase-mediated DNA cleavage assays of diben-

zo[c,h]cinnoline derivatives and related compounds

Compd

Topo I-mediated

DNA cleavage^a

Topo II-mediated

DNA cleavage^b

1

2

2b

10

100

10

5

100

>1000

100

500

>1000

5000

300

>1000

1000

100

>1000

10

100

1

500

The eﬀect of altering substituents on the A-ring of these

benzo[c,h]cinnolines was also examined. Relative to 4,

removal of the 3-methoxyl substituent, as in 11, resulted

in a loss of activity. In contrast, removal of the 2-meth-

oxy substituent as in the case of 12, resulted in a sub-

stantial reduction in TOP1-targeting activity.

Nonetheless, at high concentrations of 12, stabilization

of cleavable complex formation was observed. Replace-

ment of the methoxyl group of 12 with either a benzy-

loxy group or a hydroxyl moiety as in the case of 13 or

14 resulted in the loss of signiﬁcant TOP1-targeting

activity. Compound 15 was synthesized in an eﬀort to

develop a dibenzo[c,h]cinnoline derivative that retained

TOP1-targeting activity, but possessed a hydroxyl

group that could serve as a handle for forming either a

derivative or prodrug with improved solubility. While

15 did retain activity as a TOP1-targeting agent, it was

substantially less active than 4.

3 (Nitidine)

4

5

6

7

8

9

10

11

12

13

14

>1000

500

>1000

1

15

CPT

VM-26

1

>1000

^aTopoisomerase I cleavage values are reported as REC, relative eﬀec-

tive concentration, that is concentrations relative to camptothecin

(CPT), whose value is arbitrarily assumed as 1, that are able to pro-

duce the same cleavage on the plasmid DNA in the presence of human

topoisomerase I.

^bTopoisomerase II cleavage values are reported as REC, relative

eﬀective concentration, that is concentrations relative to VM-26,

whose value is arbitrarily assumed as 1, that are able to produce the

same cleavage on the plasmid DNA in the presence of human topoi-

somerase II.

The cytotoxicity of 1–15 was evaluated in the human lym-

phoblast tumor cell line, RPMI8402 and its camptothecin-

resistant variant, CPT-K5. In addition, cytotoxicity was

also assessed in the leukemia cell line U937 and its

Scheme 5. Preparation of 3-hydroxymethyl-2-methoxy-8,9-methylenedioxydibenzo[c,h]cinnoline, 15: (i) Pd(PPh₃)₄, CuBr, THF; (ii) Ra–Ni,

NH₂NH₂, EtOH reﬂux; (iii) LAH, THF, rt; (iv) NaNO₂, AcOH, concd HCl.

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Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

tively. In both instances, signiﬁcant cross-resistance (a

>10-fold diﬀerence in cytotoxicity) was observed

between these parent cell lines and their respective

camptothecin-resistant variants, CPT-K5 and U937/

CR. Compounds 5, 7, and 8, in which the methylene-

dioxy group of 4 was removed or substituted by a single

methoxyl group at either the 8- or 9-position, were sub-

stantially less cytotoxic in both the RPMI8402 and

U937 tumor cell lines, with IC₅₀values ranging from 2.0

to 5.5 mM. In addition, the diﬀerence between the par-

ent cell lines and the camptothecin-resistant variants

was less pronounced than in the case of 4. This corre-

lates with their decreased potency as TOP1-targeting

agents. Nitidine, 3, is a TOP1 and TOP2-targeting

agent. As it can exert its cytotoxic eﬀect by targeting

TOP2, it is not surprising that its cross-resistance to

CPT-K5 is minimal. Those dibenzo[c,h]cinnoline deri-

vatives that were devoid of TOP1-targeting activity (6,

9, 10, 11, 13 and 14) also had comparatively weak

cytotoxic activities.

These studies were extended to an evaluation of the

relative cytotoxicity of 1–15 in KB3-1 tumor cell line

and a variant, KBV-1, which overexpresses the eﬄux

transporter, MDR-1 (P-glycoprotein). In these cell lines,

the more potent TOP1-targeting dibenzo[c,h]cinnolines

(4, 12 and 15) have IC₅₀values that range from 50 to

640 nM in these cell lines. For each of the dibenzo[c,h]-

cinnolines evaluated in this study, no signiﬁcant diﬀer-

ence in cytotoxicity (>10-fold) was observed between

these two cell lines. Therefore, these substituted diben-

zo[c,h]cinnolines are not substrates for this transporter

and are capable of overcoming this form of multi-drug

resistance.

The leukemia cell line CEM and its variants CEM/V1

and CEM/V2 are used to indicate whether certain cyto-

toxic agents are exerting their eﬀect by targeting TOP2.

A mutant, but functional TOP2 enzyme, is present in

CEM/V1 and CEM/V2 and is responsible for their

resistance to certain TOP2-targeting agents. Both VP-16

and VM-26 are known to target TOP2 and exhibit

resistance to CEM/V1 and CEM/V2 relative to their

parent cell line, CEM. As nitidine, 3, targets both TOP1

Table 3. Relative cytotoxicities of substituted dibenzo[c,h]cinnolines

Compound

Cytotoxicity IC₅₀(mM)^a

CEM CEM/V1 CEM/V5 HeLa HCT-116 ZR-75-1

1

0.1

3 (Nitidine) 0.16

0.4

1.0

0.04

4.1

0.2

0.8

0.06

6.2

0.2

0.07

0.04

4.7

0.3

1.9

0.04

2.8

0.2

0.6

0.03

3.2

4

5

0.03

1.5

6

15

>30

0.03

0.003

0.3

0.04

0.03

0.001

>30

0.07

0.004

9.4

2.1

0.3

>30

0.03

0.005

34

24

1.2

40

30

0.3

0.004

0.5

0.15

0.06

0.002

31

0.2

0.004

0.3

0.05

0.04

0.001

0.03

0.004

0.3

0.05

0.04

0.001

CPT^b

VP-16^b

VM-26^b

DOX^b

VBS^b

Figure 2. Stimulation of enzyme-mediated DNA cleavage by 1, 4, 5,

12, 15 and camptothecin (CPT) using human TOP1. The ﬁrst lane is

DNA control without enzyme. The second lane is the control with

enzyme alone. The rest of the lanes contain human TOP1 and serially

(10-fold each) diluted compound from 0.1 to 10 mM for compound 1,

and from 0.01 to 1.0 mM for CPT and compounds 4, 5, 12, and 15.

0.003

0.001

^aIC₅₀has been calculated after 4 days of continuous drug exposure.

^bCPT is camptothecin; VP-16 is etoposide; VM-26 is teniposide; DOX

is doxorubicin; VBS is vinblastine.

Table 2. Relative cytotoxicities of dibenzo[c,h]cinnolines

Compd

RPMI

CPT-K5

Cytotoxicity IC₅₀(mM)^a

U937

U937/CR

KB3-1

KBV-1

1

2a

0.38

4.5

7.6

0.4

0.07

5.5

0.36

10.8

9.7

4.0

5.5

0.13

2.9

3.0

0.8

0.06

3.8

0.11

5

5.3

3.5

7.5

20

0.31

5.5

12

0.06

0.05

6.4

0.24

1.8

19

2.5

0.12

3.5

2b

3 (Nitidine)

4

5

10

6

7

>30

2.0

>30

5

>30

2.0

>30

4.0

27

3.2

29

2.5

8

9

10

11

12

13

14

15

CPT

VM-26

4.0

39

>100

41

0.45

>10

10

0.32

0.005

0.22

28

61

>100

41

100

>10

0.38

61

3

67

>100

26

0.33

>10

7

0.065

0.006

0.03

7

58

>100

30

>10

0.1

0.65

0.01

3.3

28

>100

57

0.39

>10

10

0.21

0.01

0.02

2.5

75

>100

200

0.64

>10

0.17

0.03

2.3

0.28

^aIC₅₀has been calculated after 4 days of continuous drug exposure.

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1481

form as solvent, unless otherwise indicated. The chemi-

cal shifts were reported in d units downﬁeld from

tetramethylsilane (TMS). Coupling constants are repor-

ted in hertz (Hz). Melting points were determined with a

Thomas-Hoover Unimelt capillary melting point appa-

ratus. Infrared spectral data (IR) were obtained on a

Perkin–Elmer 1600 Fourier transform spectro-

photometer and are reported in cm^À1. Mass spectra

were obtained from Washington University Resource

for Biomedical and Bio-organic Mass Spectrometry in

the Department of Chemistry at Washington Uni-

versity, St. Louis, Mo. Tetrahydrofuran (THF) was

freshly distilled from sodium and benzophenone prior

to use. Tetrakis(triphenylphosphine)palladium(0) was

purchased from Aldrich Chemical Company (Milwau-

kee, WI) as bright yellow powder. 4-Methoxy-2-nitro-

bromobenzene, 36, was purchased from Aldrich

Chemical Company. The nitroarylstannanes, 21, 23,

and 35 were prepared as previously described.²⁷

Figure 3. Stimulation of enzyme-mediated DNA cleavage by 4, 10, 15,

and VM-26 (VM) using human TOP2. The ﬁrst lane is DNA control

without enzyme. The second lane is the control with enzyme alone.

The rest of the lanes contain human TOP2 and serially (10-fold each)

diluted compound from 0.01 to 0.1 mM for VM and 0.1 to 100 for

compounds 4, 10 and 15.

2,3-Dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline (4).

6-(2-Amino-4,5-methylenedioxyphenyl)-2,3-dimethoxy-

naphthalene (41) (40 mg, 0.13 mmol) was dissolved in

acetic acid (2 mL) and concentrated hydrochloric acid

(0.3 mL). The solution was cooled in an ice bath and

diazotized by the dropwise addition of a solution of

sodium nitrite (0.09 g in 1.5 mL of water). The resulting

diazonium solution was allowed to rise to room tem-

perature slowly and left overnight. To the resulting red

solution, which contained some precipitate, was added

50 mL of water. The mixture was extracted with ethyl

acetate (30 mLÂ3). The organic layers were combined

and washed with dilute sodium hydroxide solution, then

with water and brine. The ethyl acetate extracts were

dried (Na₂SO₄) and evaporated in vacuo. The crude

product was puriﬁed by column chromatography on

silica gel using 40:60 hexanes/ethyl acetate to give 4 (20

mg) in 48% yield; mp 292–294 ^ꢁC; ¹H NMR d 4.09 (3H,

s), 4.22 (3H, s), 6.24 (2H, s), 7.31 (1H, s), 7.80 (1H, s),

7.95 (1H, s), 8.00 (1H, d, J=9.2), 8.13 (1H, d, J=8.9),

9.14 (1H, s); ¹³C NMR d 56.5, 56.9, 98.1, 102.9, 104.6,

107.5, 107.9, 117.1, 119.6, 120.6, 126.9, 128.5, 131.7,

141.9, 145.6, 150.2, 151.2, 152.1; HRMS (EI) calcd for

C₁₉H₁₄N₂O₄m/z: 334.0953; found: 334.0946.

and TOP2, cross-resistance to these variant cell lines is

minimal. While 4 did exhibit weak activity as a TOP2-

targeting agent (ꢂ0.5% of that of nitidine), both 4 and

15 did not exhibit any cross-resistance in these variant

cell lines.

Compounds 1–6 and 15 were also evaluated together

with camptothecin, VP-16 (etoposide), VM-26 (tenipo-

side), doxorubicin, and vinblastine in several solid

tumor cell lines. In the human tumor cell lines, HeLa

(cervical), HCT-116 (colon), and breast (ZR-75-1), 4

exhibited signiﬁcant cytotoxicity with IC₅₀values ran-

ging from 30 to 40 nM. In the case of 15, IC₅₀values for

these cell lines ranged from 30 to 300 nM.

These data indicate that dibenzo[c,h]cinnolines can be

developed that (1) target TOP1, (2) exhibit signiﬁcant

cytotoxic activity, and (3) are capable of overcoming

MDR1-resistance. Compound 15 is a particularly inter-

esting dibenzo[c,h]cinnoline as its hydroxymethyl moi-

ety can serve as a synthetic handle for forming varied

derivatives and prodrugs so as to alter its physico-

chemical properties. This could be of advantage in

developing dibenzo[c,h]cinnolines to have properties

that may improve solubility, facilitate formulation, and

increase bioavailability.

2,3-Dimethoxydibenzo[c,h]cinnoline (5). 6-(2-Aminophe-

nyl)-2,3-dimethoxynaphthalene (20) (70 mg, 0.25 mmol)

was dissolved in 48% hydrobromic acid (4.25 mL),

cooled in ice-salt bath, and treated dropwise with stir-

ring with sodium nitrite (0.13 g) in water (2.2 mL).

Stirring was continued for 0.5 h., and copper powder

(0.5 g) was added to the cold solution with stirring. The

mixture was allowed to rise slowly to room temperature

and left overnight. The solid was ﬁltered oﬀ and washed

with hot chloroform. The chloroform solution was

washed with dilute sodium hydroxide solution, water,

dried (Na₂SO₄) and evaporated to give the crude pro-

duct. Chromatography on silica gel using 50:50 hex-

anes/ethyl acetate aﬀorded 5 (13 mg) in 18% yield; mp

Experimental

General

Column chromatography refers to ﬂash chromato-

graphy conducted on Silitech 32–63 um, 60A (ICN

Biomedicals, Eschwege, Germany) using the eluting

solvent systems as indicated. Proton (¹H NMR) and

carbon (¹³C NMR) nuclear magnetic resonance spectra

were obtained on a Varian Gemini-200 Fourier Trans-

form spectrometer. NMR spectra (200 MHz ¹H and

50 MHz ¹³C) were recorded using deuterated chloro-

201–203 ^ꢁC; H NMR d 4.11 (3H, s), 4.24 (3H, s), 7.37

1

(1H, s), 7.89–7.94 (2H, m), 8.14 (1H, d, J=8.9), 8.41

(1H, d, J=8.8), 8.61–8.66 (1H, m), 8.75–8.80 (1H, m),

9.24 (1H, s); ¹³C NMR d 56.1, 56.4, 104.0, 107.3, 112.3,

1482

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

116.5, 118.5, 121.7, 126.7, 128.6, 128.8, 131.1, 131.2,

131.9, 141.5, 146.3, 150.9, 151.0; HRMS (EI) calcd for

C₁₈H₁₄N₂O₂m/z: 290.1055; found: 290.1058.

cipitate, was added 50 mL of water. This mixture was

then extracted with ethyl acetate (40 mLÂ3). The organic

layers were combined and washed with dilute sodium

hydroxide solution, water and brine. The organic layer

was dried (Na₂SO₄) and evaporated in vacuo. The crude

product was puriﬁed by column chromatography on

silica gel using 35:65 hexanes/ethyl acetate to give 8 (16

mg) in 26% yield; mp 215–217 ^ꢁC; ¹H NMR d 4.07 (3H,

s), 4.09 (3H, s), 4.22 (1H, s), 7.29 (1H, s), 7.46 (1H, dd,

J₌9.1, 2.6), 7.72 (1H, d, J=2.5), 7.99 (1H, d, J=8.9),

8.20 (1H, d, J=9.0), 8.60 (1H, d, J=9.1), 9.17 (1H, s);

¹³C NMR d 56.3, 56.5, 56.9, 100.3, 104.6, 107.7, 117.0,

118.7, 121.3, 124.3, 127.0, 129.1, 131.4, 133.3, 141.8,

143.7, 151.2, 151.3, 162.0; HRMS (EI) calcd for

C₁₉H₁₆N₂O₃m/z: 320.1161; found: 320.1144.

2,3,8,9-Tetramethoxydibenzo[c,h]cinnoline (6). 6-(2-Amino-

4,5-dimethoxyphenyl)-2,3-dimethoxynaphthalene (30)

(11 mg, 0.033 mmol) was dissolved in acetic acid (0.6

mL) and concentrated hydrochloric acid (0.06 mL). The

solution was cooled in an ice bath and diazotized by the

dropwise addition of a solution of sodium nitrite (0.026

g in 0.5 mL of water). The resulting diazonium solution

was allowed to rise to room temperature slowly and left

overnight. To the resulting red solution, which con-

tained some precipitate, was added 30 mL of water. The

mixture was extracted with ethyl acetate (30 mLÂ3).

The organic layers were combined and washed with

1.0 N sodium hydroxide ﬁrst, water and brine. The ethyl

acetate extracts were dried (Na₂SO₄) and evaporated in

vacuo. The crude product was puriﬁed by column

chromatography on silica gel using 40:60 chloroform/

ethyl acetate to give the 6 (5 mg) in 44% yield; mp

9-Benzyloxy-2,3,8-trimethoxydibenzo[c,h]cinnoline (9).

6-(2 - Amino - 5 - benzyloxy - 4 - methoxyphenyl) - 2,3 -

dimethoxynaphthalene 33 (35 mg, 0.084 mmol) was dis-

solved in acetic acid (0.65 mL) and concentrated

hydrochloric acid (0.13 mL). The solution was cooled in

an ice bath and diazotized by the dropwise addition of a

solution of sodium nitrite (0.052 g in 0.52 mL of water).

The reaction mixture was allowed to warm slowly to

room temperature and left for 24 h. To the resulting red

solution containing some precipitate was added 50 mL

of water and the mixture was extracted with ethyl ace-

tate (30 mLÂ3). The organic layers were combined and

washed with dilute sodium hydroxide solution, water

and brine. The organic layer was dried (Na₂SO₄) and

evaporated in vacuo. The crude product was puriﬁed by

column chromatography on silica gel using 20:80 hex-

anes/ethyl acetate to give 9 (24 mg) in 67% yield; mp

>360 ^ꢁC (lit²⁶>360 ^ꢁC); H NMR d 4.09 (3H, s), 4.18

1

(6H, s), 4.23 (3H, s), 7.31 (1H, s), 7.74 (1H, s), 8.00 (1H,

s), 8.01 (1H, d, J=8.5), 8.20 (1H, d, J=8.9), 9.15 (1H,

s); ¹³C NMR d 56.5, 56.9, 99.9, 104.5, 107.9, 109.5,

116.9, 118.5, 118.9, 127.1, 128.4, 131.6, 141.8, 144.6,

151.1, 151.2, 152.0, 153.9; HRMS (EI) calcd for

C₂₀H₁₈N₂O₄m/z: 350.1267; found: 350.1266.

2,3,8-Trimethoxydibenzo[c,h]cinnoline (7). 6-(2-Amino-

4-methoxyphenyl)-2,3-dimethoxynaphthalene (31) (12

mg, 0.039 mmol) was dissolved in acetic acid (0.6 mL)

and concentrated hydrochloric acid (0.06 mL). The

solution was cooled in an ice bath and diazotized by the

dropwise addition of a solution of sodium nitrite (0.026

g in 0.5 mL of water). The resulting diazonium solution

was allowed to rise to room temperature slowly and left

overnight. To the resulting red solution which contained

some precipitate was added 30 mL of water and the

mixture was extracted with ethyl acetate (30 mLÂ3).

The organic layers were combined and washed with

dilute sodium hydroxide solution, water and brine. The

ethyl acetate extracts were dried (Na₂SO₄) and evapo-

rated in vacuo. The crude product was puriﬁed by column

chromatography on silica gel using 40:60 hexanes/ethyl

acetate to give 7 (5 mg) in 40% yield; mp 244–246 ^ꢁC;

¹H NMR d 4.10 (6H, s), 4.22 (3H, s), 7.32 (1H, s), 7.54

(1H, dd, J=9.1, 2.6), 8.04–8.08 (2H, m), 8.28 (1H, d,

J=8.9), 8.49 (1H, d, J=9.1), 9.16 (1H, s); ¹³C NMR d

56.3, 56.5, 56.9, 104.4, 107.9, 108.9, 116.9, 117.1, 119.5,

123.5, 124.5, 127.1, 128.4, 132.5, 141.8, 148.5, 151.1,

151.4, 160.5; HRMS (EI) calcd for C₁₉H₁₆N₂O₃m/z:

320.1161; found: 320.0384.

244–246 ^ꢁC; H NMR d 4.07 (3H, s), 4.14 (3H, s), 4.21

1

(3H, s), 5.40 (2H, s), 7.25 (1H, s), 7.37–7.60 (5H, m),

7.91 (1H, d, J=9.0), 7.97 (1H, s), 8.01 (1H, d, J=9.0),

9.11 (1H, s); ¹³C NMR d 56.5, 56.9, 56.9, 71.6, 101.7,

104.4, 107.8, 109.6, 116.8, 118.2, 118.8, 127.0, 128.0,

128.3, 128.9, 129.3, 131.5, 136.3, 141.6, 144.5, 151.0,

151.1, 152.3, 152.9; HRMS (EI) calcd for C₂₆H₂₂N₂O₄

m/z: 426.1580; found: 426.1577.

9-Hydroxy-2,3,8-trimethoxydibenzo[c,h]cinnoline

(10).

9-Benzyloxy-2,3,8-trimethoxydibenzo[c,h]cinnoline, 9,

(5 mg, 0.012 mmol) was hydrogenated overnight in ethyl

acetate (25 mL) at 26 lb/inch²using 10% palladium on

carbon (1.5 mg). The solution was passed through a

Celite 545 bed and the sicciate was washed with ethyl

acetate (10 mLÂ3). Concentration of the ethyl acetate

solution in vacuo gave the crude product. Chromato-

graphy using a 50:45:5 mixture of hexanes/ethyl acetate/

methanol as eluting solvent gave compound 10 (3 mg) in

1

76% yield; H NMR (DMSO-d₆) d 4.00 (3H, s), 4.10

(3H, s), 4.12 (3H, s), 7.66 (1H, s), 8.02 (2H, s), 8.21 (1H,

d, J=8.4), 8.38 (1H, d, J=8.9), 8.96 (1H, s); ¹³C NMR

(DMSO-d₆) d 55.9, 56.4, 103.1, 103.8, 108.5, 109.1,

117.4, 117.8, 118.0, 125.7, 128.1, 131.3, 140.4, 143.8,

150.5, 150.7, 151.3, 152.4; HRMS (EI) calcd for

C₁₉H₁₆N₂O₄m/z: 336.111; found: 336.1109.

2,3,9-Trimethoxydibenzo[c,h]cinnoline (8). 6-(2-Amino-

5-methoxyphenyl)-2,3-dimethoxynaphthalene (32) (60

mg, 0.20 mmol) was dissolved in acetic acid (1.5 mL) and

concentrated hydrochloric acid (0.3 mL). The solution

was cooled in an ice bath and diazotized by dropwise

addition of a solution of sodium nitrite (0.12 g in 1.2 mL

of water). The resulting diazonium solution was allowed

to rise to room temperature slowly and left overnight. To

the resulting red solution, which contained some pre-

2-Methoxy-8,9-methylenedioxydibenzo[c,h]cinnoline (11).

6-(2-Amino-4,5-methylenedioxyphenyl)-2-methoxynaph-

thalene 42 (170 mg, 0.58 mmol) was dissolved in acetic

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1483

acid (4.5 mL) and concentrated hydrochloric acid (0.9

mL). The solution was cooled in an ice bath and diazo-

tized by the dropwise addition of a solution of sodium

nitrite (0.36 g in 3.6 mL of water). The resulting diazo-

nium solution was allowed to warm slowly to room

temperature and left for 1 day. To the resulting red

solution containing some precipitate was added 50 mL

water and the mixture was extracted with ethyl acetate

(30 mLÂ3). The organic layers were combined and

washed with dilute sodium hydroxide, water and brine.

The organic layer was dried (Na₂SO₄) and evaporated

in vacuo. The crude product was puriﬁed by column

chromatography on silica gel using 50:50 hexanes/ethyl

acetate to give 11 (20 mg) in 11% yield; mp 258–260 ^ꢁC;

¹H NMR d 4.01 (3H, s), 6.23 (2H, s), 7.30 (1H, J=2.6),

7.48 (1H, dd, J=9.1, 2.6), 7.75 (1H, s), 7.95 (1H, s), 8.00

(1H, d, J=9.2), 8.19 (1H, d, J=9.1), 9.62 (1H, d,

J=9.2); ¹³C NMR d 56.0, 98.0, 102.9, 107.5, 108.3,

119.1, 119.6, 119.7, 120.6, 125.9, 126.6, 132.3, 134.6,

142.5, 145.9, 150.2, 152.1, 160.2; HRMS (EI) calcd for

C₁₈H₁₂N₂O₃m/z: 304.0848; found: 304.0843.

added to a stirred solution of compound 54 (111 mg,

0.25 mmol) in dry THF (20 mL). The mixture was stir-

red at room temperature for 1 h. Excess LAH was

decomposed by sequential addition of 0.5 mL of water

and 0.5 mL of 10% NaOH and the reaction mixture

was ﬁltered through Celite 545. The ﬁltrate was con-

centrated in vacuo. The residue was extracted with

CHCl₃, washed with water, dried (Na₂SO₄) and con-

centrated in vacuo to provide 40 mg (42%) of 13 as a

solid: mp 246–247 ^ꢁC; ¹H NMR (DMSO-d₆) d 5.46 (2H,

s), 6.40 (2H, s), 7.36–7.62 (6H, m), 8.02 (1H s,), 8.12

(1H, d, J=8), 8.26 (1H, d, J=8), 8.38 (1H, s), 8.54 (1H,

d, J=8.2), 9.07 (1H, d, J=2.2); ¹³C NMR (DMSO-d₆) d

70.4, 99.3, 103.7, 105.3, 106.5, 117.9, 120.3, 121.1, 128.1,

128.5, 128.6, 129.2, 130.9, 132.7, 137.6, 141.4, 146.2,

151.1, 152.6, 159.1.

3-Hydroxy-8,9-methylenedioxydibenzo[c,h]cinnoline (14).

Sodium hydroxide (50 mg) and 10% Pd/C (10 mg) were

added to a solution of 55 (50 mg, 0.12 mmol) in EtOH

(30 mL). The reaction mixture was heated to reﬂux and

then hydrazine hydrate (0.2 mL) was added. The reac-

tion mixture was reﬂuxed for 1 h, allowed to cool to

room temperature, and the catalyst removed by ﬁl-

tration through Celite 545. The ﬁltrate was concentrated

in vacuo. Water was added and pH adjusted to 7 using

concd HCl. A yellow solid was ﬁltered, washed with

H₂O and allowed to dry. The solid was washed with

ether, and then chloroform to remove impurities, to

provide 18 mg of 14 (50% yield); mp >300 ^ꢁC; ¹H

NMR (DMSO-d₆) d 6.37 (2H, s), 7.26 (1H, dd, J=2.5,

9), 7.90 (1H, s), 8.19 (1H, d, J=2.5), 8.26 (1H, d, J=9),

8.32 (1H, s), 10.22 (1H, s); ¹³C NMR (DMSO-d₆) d

99.4, 101.0, 104.4, 106.6, 107.5, 116.5, 117.2, 120.5,

127.8, 129.8, 130.7, 134.2, 138.3, 151.7, 153.5, 158.1,

172.2.

3-Methoxy-8,9-methylenedioxydibenzo[c,h]cinnoline (12).

Method 1. 7-(2-Amino-4,5-methylenedioxyphenyl)-2-

methoxynaphthalene 43 (70 mg, 0.24 mmol) was dis-

solved in acetic acid (2.0 mL) and concentrated hydro-

chloric acid (0.4 mL). The solution was cooled in an ice

bath and diazotized by the dropwise addition of a solu-

tion of sodium nitrite (0.16 g in 1.6 mL of water). The

resulting diazonium solution was allowed to warm slowly

to room temperature and left overnight. To the resulting

red solution containing some precipitate was added 50

mL of water and the reaction mixture was extracted with

ethyl acetate (30 mLÂ3). The organic layers were com-

bined and washed with dilute sodium hydroxide, water

and brine. The organic layer was dried (Na₂SO₄) and

evaporated in vacuo. The crude product was puriﬁed by

column chromatography on silica gel using 55:45 hex-

anes/ethyl acetate to give 60 mg of 12 in 83% yield;

3-Hydroxymethyl-2-methoxy-8,9-methylenedioxydibenzo-

[c,h]cinnoline (15). Compound 59 (65 mg, 0.2 mmol)

was dissolved in a mixture of acetic acid (3 mL) and

concd HCl (1 mL). This mixture was cooled to 10 ^ꢁC. A

solution of NaNO₂(100 mg) in 2 mL of water was then

added dropwise. After the addition was complete, the

reaction was allowed to stir at room temperature for 24

h. Water was added to reaction mixture. The reaction

mixture was neutralized with NaOH to get a brown

solid. The precipitate was ﬁltered and washed with

water and allowed to dry. The solid was then washed

with ether and then chloroform to remove any impu-

rities, to provide 40 mg (59.7%) of 59; mp 219–220 ^ꢁC;

¹H NMR (DMSO-d₆) d 4.01 (3H, s), 4.79 (2H, s), 6.39

(2H, s), 7.63 (1H, s), 8.01 (1H, s), 8.26 (1H, d, J=8),

8.35 (1H, s), 8.64 (1H, d, J=8.2), 9.6 (1H, s); ¹³C NMR

(DMSO-d₆) d 56.4, 59.3, 99.0, 103.7, 106.2, 107.3, 119.3,

120.2, 120.7, 122.2, 124.8, 132.6, 133.7, 134.6, 142.0,

145.5, 150.8,152.7, 157.5.

Method 2. Lithium aluminum hydride (46 mg, 1.2

mmol) was added to a stirred solution of compound 53

(74 mg, 0.2 mmol) in diethyl ether (10 mL) and benzene

(10 mL). The mixture was stirred under reﬂux for 1 h.

After cooling to room temperature, the excess lithium

aluminum hydride was decomposed by sequential addi-

tion of 0.05 mL water, 0.05 mL 15% NaOH and 0.15 mL

water, and the reaction mixture ﬁltered through a Celite

545 bed. Evaporation of solvent in vacuo gave the crude

product, which was puriﬁed by column chromatography

using 50:50 hexanes/ethyl acetate mixture as eluting sol-

vent. The yield was 46 mg (75%); mp 259–261 ^ꢁC; H

1

NMR d 4.12 (3H, s), 6.24 (2H, s), 7.37 (1H, dd, J=8.8,

J=2.7), 7.80 (1H, s), 7.88 (1H, d, J=8.8), 7.96 (1H, s),

8.03 (1H, d, J=9.1), 8.09 (1H, d, J=9.0), 9.15 (1H, d,

J=2.7); ¹³C NMR d 56.3, 98.3, 102.9, 104.0, 107.5,

116.4, 120.4, 120.5, 121.1, 128.0, 130.0, 132.5, 133.3,

142.0, 146.1, 150.5, 152.1, 160.3; HRMS (EI) calcd for

C₁₈H₁₂N₂O₃m/z: 304.0848; found: 304.0852.

6,7-Dimethoxy-1-hydroxytetralin (16). A solution of

sodium borohydride (1 g, 26 mmol) in 10 mL of

methanol (caution: in forming this solution, heat was

generated and a signiﬁcant amount of H₂gas was gen-

erated) was added dropwise to a stirred solution of 6,7-

dimethoxy-1-tetralone (1 g, 4.9 mmol) in 20 mL

3-Benzyloxy-8,9-methylenedioxydibenzo[c,h]cinnoline (13).

Lithium aluminium hydride (37 mg, 1.0 mmol) was

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Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1

methanol. This mixture was stirred for 10 min, at which

time the reaction was complete as indicated by thin

layer chromatography. After the solvent was evapo-

rated, the residue was ﬁltered through a very short col-

umn using hexanes/ethyl acetate (70:30) as eluting

solvent. The eluent was concentrated in vacuo to give

1.0 g of 1-hydroxy-6,7-dimethoxytetralin 16 in 100%

yield; mp 169–171 ^ꢁC; ¹H NMR (DMSO-d₆) d 1.60–1.69

(2H, m), 1.83–1.89 (2H, m), 2.59–2.63 (2H, m), 3.71

(3H, s), 3.72 (3H, s), 4.46–4.51 (1H, m), 6.62 (1H, s),

6.95 (1H, s); ¹³C NMR (DMSO-d₆) d 19.5, 28.8, 32.7,

55.7, 66.3, 66.4, 111.7, 112.1, 128.7, 132.5, 147.1, 147.9.

141 ^ꢁC; H NMR d 4.00 (3H, s), 4.01 (3H, s), 7.12 (1H,

s), 7.14 (1H, s), 7.27 (1H, dd, J=8.4, 1.7), 7.46–7.62

(3H, m), 7.66 (1H, s), 7.72 (1H, d, J=8.4), 7.86 (1H, d,

J=8.1); ¹³C NMR d 56.4, 106.6, 107.1, 124.5, 124.6,

125.9, 127.3, 128.4, 129.3, 129.6, 132.7, 133.6, 137.0,

149.9, 150.5, 150.6; HRMS (EI) calcd for C₁₈H₁₅NO₄

m/z: 309.1001; found: 309.0999.

6-(2-Aminophenyl)-2,3-dimethoxynaphthalene (20). 6-(2-

Nitrophenyl)-2,3-dimethoxynaphthalene (19) (70 mg,

0.23 mmol) in ethyl acetate (45 mL) was hydrogenated

overnight at 40–45 lb/inch²using 10% palladium on

carbon (20 mg). The solution was passed through a

Celite 545 bed and the catalyst was washed with ethyl

acetate (3Â10 mL). Concentration in vacuo gave 20 (60

mg) in 99% yield; mp 145–147 ^ꢁC; ¹H NMR d 4.02 (3H,

s), 4.04 (3H, s), 6.82 (1H, d, J=8.0), 6.85–6.93 (1H, m),

7.16 (1H, s), 7.18 (1H, s), 7.20–7.26 (2H, m), 7.47 (1H,

dd, J=8.3, J=1.6), 7.78 (1H, d, J=8.8), 7.80 (1H, s);

¹³C NMR d 56.4, 106.7, 106.9, 116.1, 119.2, 126.1,

126.9, 127.3, 128.3, 128.7, 128.9, 129.9, 131.2, 135.8,

144.3, 150.2, 150.3; HRMS (EI) calcd for C₁₈H₁₇NO₂

m/z: 279.1259; found: 279.1267.

1,2-Dihydro-6,7-dimethoxynaphthalene (17). 6,7-Dime-

thoxy-1-hydroxytetralin, 16, (0.60 g, 2.9 mmol) was

dissolved in 15 mL of N,N-dimethylformamide. To this

solution was added orthophosphoric acid 0.25 mL (3.7

mmol). The reaction mixture was stirred at 60 ^ꢁC for 20

min until the reaction was complete as indicated by thin

layer chromatography. Ethyl acetate (50 mL) was added

and the reaction mixture was washed with water (60

mLÂ3). The organic layer was dried (Na₂SO₄) and eva-

porated. The crude product was puriﬁed by column

chromatography using a 80:20 mixture of hexanes/ethyl

acetate. The ﬁrst eluting compound was the desired

product 17. This reaction provided 0.55 g (100% yield)

Trimethyl(5-methoxy-2-nitrophenyl)stannane (22).

A

mixture of hexamethylditin (2.0 g, 6.13 mmol), 3-meth-

oxy-6-nitrobromobenzene (0.70 g, 3.0 mmol) and

Pd(PPh₃)₄(100 mg) in anhydrous THF (20 mL) was

heated to reﬂux under nitrogen until thin layer chroma-

tography no longer showed the presence of starting

material. After cooling to room temperature, THF was

evaporated and methylene chloride was added to the

residue. To this mixture, aqueous potassium ﬂuoride

(7.0 M, 1.5 mL) was added dropwise with vigorous stir-

ring. The mixture was passed through a Celite 545 bed

and the ﬁltrate washed with brine. The methylene chlo-

ride layer was dried (Na₂SO₄), ﬁltered and the solution

concentrated in vacuo. The residue was chromato-

graphed using a 125:2 mixture of hexanes/ethyl acetate

of 17; mp 35.5–36 ^ꢁC; H NMR d 2.24–2.32 (2H, m),

1

2.72 (2H, t, J=8.3), 3.86 (3H, s), 3.87 (3H, s), 5.92 (1H,

dt, J=9.6, 4.8), 6.38 (1H, d, J=9.6), 6.60 (1H, s), 6.66

(1H, s); ¹³C NMR d 23.7, 27.7, 56.5, 110.4, 112.0, 126.9,

127.4, 127.7, 128.4, 147.8, 148.1.

6-(2-Nitrophenyl)-7,8-dihydro-2,3-dimethoxynaphthalene

(18). Pd(PPh₃)₂Cl₂(840 mg, 1.2 mmol) and sodium

acetate (200 mg, 2.4 mmol) were added to a solution of

1,2-dihydro-6,7-dimethoxynaphthalene, 17, (700 mg, 3.7

mmol) and 1-iodo-2-nitrobenzene (925 mg, 3.7 mmol) in

N,N-dimethylacetamide (50 mL). The mixture was stir-

red under nitrogen at 140 ^ꢁC overnight, and then con-

centrated in vacuo. Ethyl acetate (60 mL) was added to

the residue and washed with distilled water (50 mL).

The organic layer was separated and ﬁltered through a

Celite 545. The organic layer was then washed with

brine, dried (Na₂SO₄) and concentrated in vacuo. The

residue was chromatographed to give 18 (330 mg) in

29% yield; mp 139–141 ^ꢁC; ¹H NMR d 2.51 (2H, t,

J=8.1), 2.92 (2H, t, J=8.1), 3.87 (3H, s), 3.90 (3H, s),

6.45 (1H, s), 6.67 (1H, s), 6.73 (1H, s), 7.38–7.45 (2H,

m), 7.54–7.62 (1H, m), 7.88–7.93 (1H, m); ¹³C NMR d

28.5, 28.5, 56.6, 111.0, 111.7, 124.9, 127.0, 127.1, 128.1,

128.3, 131.3, 133.3, 135.7, 138.7, 148.0, 148.9; HRMS

(EI) calcd for C₁₈H₁₇NO₄m/z: 311.1157; found:

311.1145.

1

to give 22 (200 mg) in 21% yield; H NMR d 0.34 (9H,

s), 3.91 (3H, s), 6.92 (1H, dd, J=9.1, 2.7), 7.13 (1H, d,

J=2.8), 8.33 (1H, d, J=9.1); ¹³C NMR d-7.1, 56.2,

114.2, 122.4, 127.0, 143.6, 146.8, 164.1.

3-Methoxy-6-nitrobromobenzene. Nitric acid (70%, 5

mL) was placed in a 25 mL round-bottomed ﬂask.

Concentrated sulphuric acid (4 mL) was then added

dropwise with stirring. The mixture was kept cool dur-

ing the addition by immersing the ﬂask in an ice bath. 3-

Methoxybromobenzene (4 g, 21.5 mmol) was then

introduced dropwise. The reaction mixture was then

heated to 50 ^ꢁC and stirred for 5 h. After cooling, the

mixture was poured into 100 mL of cold water and

extracted with ethyl acetate (30 mLÂ3). The organic

layers were combined and washed with water (50

mLÂ4) and brine. The ethyl acetate layer was dried

(Na₂SO₄) and concentrated in vacuo. The crude product

was puriﬁed by column chromatography on silica gel

using 95:5 hexanes/ethyl acetate. The ﬁrst compound

that eluted from the column was 3-methoxy-4-nitro-

6-(2-Nitrophenyl)-2,3-dimethoxynaphthalene (19). 6-(2-

Nitrophenyl)-7,8-dihydro-2,3-dimethoxynaphthalene (18)

(100 mg, 0.32 mmol) was reﬂuxed overnight in toluene

(20 mL) with DDQ (109 mg, 0.48 mmol). The reaction

mixture was allowed to cool to room temperature and

was then ﬁltered through a Celite 545 bed. The ﬁltrate

was evaporated to dryness to give the crude product and

chromatographed on silica gel using 80:20 hexanes/ethyl

acetate to aﬀord 19 (90 mg) in 91% yield; mp 139–

1

bromobenzene (1.2 g) in 24% yield; H NMR d 3.96

(3H, s), 7.17 (1H, dd, J=8.6, 1.9), 7.24 (1H, d, J=1.9),

7.75 (1H, d, J=8.6); ¹³C NMR d 57.3, 117.6, 124.0,

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1485

127.4, 129.1, 142.3, 154.0. The second compound elut-

ing from the column was the desired 3-methoxy-6-

nitrobromobenzene (1.5 g, 30% yield); mp 43–45 ^ꢁC; ¹H

NMR d 3.89 (3H, s), 6.91 (1H, dd, J=9.1, 2.7), 7.21

(1H, d, J=2.7), 7.98 (1H, d, J=9.1); ¹³C NMR d 56.7,

114.0, 117.3, 120.6, 128.5, 163.2.

149.5, 149.5; HRMS (EI) calcd for C₁₄H₁₃O₂Br m/z:

292.0099; found: 292.0085.

5-Bromo-2-methoxyphenol. To a solution of 5-bromo-2-

methoxybenzaldehyde (2.4 g, 11.2 mmol) in 50 mL

CH₂Cl₂, m-chloroperoxybenzoic acid (70–75%, 7.0 g,

containing 28.4 mmol m-CPBA) was added and the

mixture was stirred at ambient temperature for 2 days.

The reaction was quenched with aqueous saturated

NaHCO₃solution and extracted with ethyl acetate (50

mLÂ3). The organic extract was dried (Na₂SO₄) and

ﬁltered through silica gel. Evaporation of the solvent

Trimethyl(3-benzyloxy-4-methoxy-6-nitrophenyl)stannane

(24). A mixture of hexamethylditin (2 g, 6.13 mmol), 3-

benzyloxy-4-methoxy-6-nitro-bromobenzene (1.4 g, 4.14

mmol) and Pd(PPh₃)₄(200 mg) in anhydrous THF (40

mL) was heated to reﬂux under nitrogen for 2 days.

After cooling to room temperature, THF was evapo-

rated and methylene chloride was added to the residue.

To this mixture, aqueous potassium ﬂuoride (7.0 M, 1.5

mL) was added dropwise with vigorous stirring. The

mixture was passed through a Celite 545 bed and the

ﬁltrate washed with brine. The methylene chloride layer

was dried (Na₂SO₄), ﬁltered and evaporated in vacuo.

The residue was chromatographed using a 90:10

mixture of hexanes/ethyl acetate to give 24 (1.16 g) in

ꢁ

1

gave the product (2.1 g) in 92% yield; mp 62–64 C; H

NMR d 3.87 (3H, s), 6.71 (1H, d, J=8.6), 6.97 (1H, dd,

J₁=8.6, J₂=2.4), 7.07 (1H, d, J=2.4); ¹³C NMR d 56.6,

112.4, 113.8, 118.3, 123.3, 146.4, 147.0.

6,7-Dimethoxy-2-triﬂuoromethanesulfonyloxynaphthalene

(25).

3,4-Dihydro-6,7-dimethoxy-2-triﬂuoromethane-

sulfonyloxynaphthalene (200 mg, 0.59 mmol) was

reﬂuxed overnight in toluene (30 mL) with DDQ (166

mg, 0.73 mmol). This reaction mixture was allowed to

cool to room temperature and then ﬁltered through

Celite 545. The ﬁltrate was concentrated in vacuo to

give the crude product. Chromatography on silica gel

using a 80:20 hexanes/ethyl acetate aﬀorded 25 (190 mg)

in 95% yield; ¹H NMR d 4.00 (6H, s), 7.10 (1H, s), 7.12

(1H, s), 7.21 (1H, dd, J=8.9, J=2.5), 7.58 (1H, d,

J=2.5), 7.71 (1H, d, J=8.9); ¹³C NMR d 56.4, 106.6,

106.6, 109.7, 116.1, 117.9, 118.2, 122.5, 128.9, 129.0,

129.8, 146.6, 150.9, 151.3; HRMS (EI) calcd for

C₁₃H₁₁O₅F₃S m/z: 336.0279; found: 336.0288.

66% yield; mp 81–83 ^ꢁC; H NMR d 0.27 (9H, s), 3.97

1

(3H, s), 5.29 (2H, s), 7.04 (1H, s), 7.36–7.44 (5H, m),

7.91 (1H, s); ¹³C NMR d À7.2, 56.8, 71.6, 108.0, 119.6,

127.8, 128.9, 129.3, 132.6, 136.4, 146.9, 150.2, 153.3;

HRMS (EI) calcd for C₁₇H₂₁NO₄Sn m/z: 408.0258;

found: 408.0243.

3-Benzyloxy-4-methoxy-6-nitrobromobenzene. 3-Benzyl-

oxy-1-bromo-4-methoxybenzene (1 g, 3.4 mmol) was

dissolved in 50 mL acetic acid in a 100 mL round-bot-

ꢁ

tomed ﬂask and cooled to 0 C using an ice bath. 2.5

mL of nitric acid (70%) in 6 mL of acetic acid was

added dropwise. The reaction mixture was allowed to

slowly rise to room temperature. After 3 h no starting

material was detected by thin layer chromatography.

Evaporation of acetic acid gave the crude product,

which was ﬁltered through a short silica gel column

using a 80:20 mixture of hexanes/ethyl acetate to give 3-

benzyloxy-4-methoxy-6-nitrobromobenzene (1.15 g) in

quantitative yield; mp 134–135 ^ꢁC; ¹H NMR d 3.93 (3H,

s), 5.19 (2H. s), 7.17 (1H, s), 7.38–7.45 (5H, m), 7.57

(1H, s); ¹³C NMR d 57.0, 72.0, 107.7, 109.7, 118.7, 128.,

129.1, 129.4, 135.5, 142.4, 149.2, 152.4; HRMS (EI)

calcd for C₁₄H₁₂NO₄Br m/z: 336.9950; found: 336.9941.

3,4-Dihydro-6,7-dimethoxy-2-triﬂuoromethanesulfonyl-

oxynaphthalene. A solution of 6,7-dimethoxy-2-tetra-

lone (250 mg, 1.2 mmol) in THF (5 mL) was added to a

suspension of sodium hydride (60 wt% in mineral oil,

75 mg, 1.9 mmol) in THF (10 mL) cooled by ice bath.

This mixture was stirred for 0.5 h. A solution of

N-phenyltriﬂuoromethanesulfonimide (500 mg, 1.4

mmol) in THF (5 mL) was then added, and the reaction

stirred at 0 ^ꢁC for 9 h. After concentration in vacuo, the

residue was chromatographed using 80:20 hexanes/ethyl

acetate

to

give

3,4-dihydro-6,7-dimethoxy-2-

triﬂuoromethanesulfonyloxynaphthalene (300 mg) in

73% yield as a pale yellow liquid at room temperature;

¹H NMR d 2.66 (2H, t, J=8.5), 3.00 (2H, t, J=8.4),

3.86 (3H, s), 3.88 (3H, s), 6.40 (1H, s), 6.62 (1H, s), 6.68

(1H, s); ¹³C NMR d 27.1, 28.9, 56.5, 111.3, 111.7, 115.9,

118.7, 122.3, 124.0, 126.0, 148.2, 148.9, 149.3; HRMS

(EI) calcd for C₁₃H₁₃O₅F₃S m/z: 338.0436; found:

338.0453.

3-Benzyloxy-1-bromo-4-methoxybenzene. A solution of

5-bromo-2-methoxyphenol (2.0 g, 10 mmol) and a-bro-

motoluene (2.6 g, 15.3 mmol) in CH₃CN (30 mL) and

acetone (25 mL) was treated with potassium carbonate

(2.1 g, 15.2 mmol). The resulting mixture was heated to

reﬂux under nitrogen for 18 h. After cooling to room

temperature, the reaction mixture was ﬁltered through a

Celite 545 bed. The acetone was removed in vacuo and

50 mL ethyl acetate was added to the residue. The ethyl

acetate solution was washed with water, brine, dried

(Na₂SO₄), and then evaporated in vacuo. The residue

was chromatographed using a 90:10 mixture of hexanes/

ethyl acetate to give 3-benzyloxy-1-bromo-4-methoxy-

benzene (2.77 g) in 96% yield; mp 70–71 ^ꢁC; ¹H NMR d

3.86 (3H, s), 5.12 (2H. s), 7.77 (1H, J=9.2), 7.04–7.08

(2H, m), 7.33–7.48 (5H, m); ¹³C NMR d 56.7, 71.7,

113.0, 113.5, 117.7, 124.5, 127.9, 128.6, 129.1, 136.9,

6-(4,5-Dimethoxy-2-nitrophenyl)-2,3-dimethoxynaphtha-

lene (26). Tetrakis(triphenylphosphine)palladium (0)

(80 mg) and cuprous bromide (16 mg) were added to a

solution of 6,7-dimethoxy-2-triﬂuoromethanesulfonyl-

oxynaphthalene 25 (220 mg, 0.66 mmol) and tri-

methyl(nitroaryl)stannane 21 (220 mg, 0.64 mmol) in

THF (25 mL) at room temperature and stirred for 0.5 h.

The mixture was then reﬂuxed under N₂overnight.

After cooling, THF was evaporated and ethyl acetate

(30 mL) was added to the residue. The solution was

1486

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

washed with water. The organic layer was separated and

passed through a Celite 545 bed to remove suspended

particles. The organic layer was then washed with brine,

dried (Na₂SO₄), and evaporated in vacuo. The residue

was chromatographed using a 60:40 mixture of hexanes/

ethyl acetate to give 26 (120 mg) in 54% yield; mp 190–

stirred for 0.5 h. The mixture was then reﬂuxed under N₂

for 2 days. After cooling, THF was evaporated and ethyl

acetate (30 mL) was added to the residue. The solution

was washed with water. The organic layer was separated

and passed through a Celite 545 bed to remove suspended

particles. The organic layer was then washed with brine,

dried (Na₂SO₄), and evaporated in vacuo. The residue

was chromatographed using a 70:30 mixture of hexanes/

ethyl acetate to give 29 (230 mg) in 35% yield; mp 151–

153 ^ꢁC; ¹H NMR d 3.98 (3H, s), 3.99 (3H, s), 4.01 (3H, s),

5.20 (2H. s), 6.94 (1H, s), 7.10 (1H, s), 7.14 (1H, s), 7.18

(1H, dd, J=8.4, 1.8), 7.36–7.43 (5H, m), 7.54 (1H, d,

J=1.5), 7.56 (1H, s), 7.68 (1H, d, J=8.3); ¹³C NMR d

56.4, 57.0, 71.7, 106.7, 107.0, 108.7, 116.4, 124.9, 125.8,

127.0, 128.0, 128.9, 129.0, 129.2, 129.5, 131.8, 134.5,

136.2, 141.9, 149.0, 150.4, 151.9; HRMS (EI) calcd for

C₂₆H₂₃NO₆m/z: 445.1525; found: 445.1355.

192 ^ꢁC (lit²⁶189–190); H NMR d 3.95 (3H, s), 3.99

1

(6H, s), 4.01 (3H, s), 6.86 (1H, s), 7.12 (1H, s), 7.14 (1H,

s), 7.23 (1H, dd, J=8.4, 1.8), 7.56 (1H, s), 7.61 (1H, d,

J=1.7), 7.70 (1H, d, J=8.4); ¹³C NMR d 56.4, 56.9,

106.7, 107.0, 108.3, 114.4, 124.9, 125.7, 127.0, 129.0,

129.5, 132.2, 134.6, 141.6, 148.4, 150.4, 152.7.

6-(5-Methoxy-2-nitrophenyl)-2,3-dimethoxynaphthalene

(27). Tetrakis(triphenylphosphine)palladium (0) (80

mg) and cuprous bromide (6 mg) were added to a solu-

tion

of

6,7-dimethoxy-2-triﬂuoromethanesulfonyl-

oxynaphthalene 25 (200 mg, 0.60 mmol) and

trimethyl(nitroaryl)stannane 22 (200 mg, 0.64 mmol) in

THF (25 mL) at room temperature and the mixture was

stirred for 0.5 h. The mixture was then reﬂuxed under

N₂overnight. After cooling, THF was evaporated and

ethyl acetate (30 mL) was added to the residue. The

solution was washed with water. The organic layer was

separated and passed through a Celite 545 bed to

remove suspended particles. The organic layer was

then washed with brine, dried (Na₂SO₄), and evapo-

rated in vacuo. The residue was chromatographed

using a 75:25 mixture of hexanes/ethyl acetate to give

a mixture of two compounds with similar R_fvalues.

This mixture was used for the next step without fur-

ther puriﬁcation.

6-(2-Amino-4,5-dimethoxyphenyl)-2,3-dimethoxynaphtha-

lene (30). 6-(4,5-Dimethoxy-2-nitrophenyl)-2,3-dime-

thoxynaphthalene 26 (12 mg, 0.033 mmol) was

hydrogenated overnight in ethyl acetate (20 mL) at

40ꢀ45 lb/inch²using 10% palladium on carbon (10 mg)

as catalyst. The reaction solution was passed through a

Celite 545 bed and the catalyst was washed with ethyl

acetate (10 mLÂ3). Concentration of the solution in

vacuo gave 30 (11 mg) in 100% yield; mp 200–202 ^ꢁC

(lit²⁶201–202 ^ꢁC); H NMR d 3.84 (3H, s), 3.89 (3H, s),

1

4.01 (3H, s), 4.02 (3H, s), 6.41 (1H, s), 6.80 (1H, s), 7.14

(1H, s), 7.15 (1H, s), 7.43 (1H, dd, J=8.4, J=1.6), 7.75

(1H, d, J=1.5), 7.75 (1H, d, J=8.4); ¹³C NMR d 56.4,

57.2, 101.3, 106.6, 106.8, 115.2, 119.9, 126.2, 126.8, 127.3,

128.5, 129.9, 135.7, 138.0, 142.7, 149.8, 150.1, 150.3.

6-(4-Methoxy-2-nitrophenyl)-2,3-dimethoxynaphthalene

(28). Tetrakis(triphenylphosphine)palladium (0) (60

mg) and cuprous bromide (10 mg) were added to a

solution of 6,7-dimethoxy-2-triﬂuoromethanesulfonyloxy-

naphthalene 25 (150 mg, 0.45 mmol) and trimethyl(ni-

troaryl)stannane 23 (140 mg, 0.45 mmol) in THF (20

mL) at room temperature and the mixture was stirred

for 0.5 h. The mixture was then reﬂuxed under N₂for 36

h. After cooling, THF was evaporated and ethyl acetate

(30 mL) was added to the residue. The solution was

washed with water. The organic layer was separated and

passed through a Celite 545 bed to remove suspended

particles. The organic layer was then washed with brine,

dried (Na₂SO₄), and evaporated in vacuo. The residue

was chromatographed using a 70:30 mixture of hexanes/

6-(2-Amino-5-methoxyphenyl)-2,3-dimethoxynaphthalene

(31). Crude 6-(5-methoxy-2-nitrophenyl)-2,3-dimethoxy-

naphthalene 27 (100 mg, approximately 90%) was

hydrogenated overnight in ethyl acetate (40 mL) at

40ꢀ45 lb/inch²using 10% palladium on carbon (30 mg)

as catalyst. The reaction solution was passed through a

Celite 545 bed and the catalyst was washed with ethyl

acetate (10 mLÂ3). Concentration in vacuo gave the

crude product. The residue was chromatographed using

a 50:50 mixture of hexanes/ethyl acetate to give 31 (66

mg); mp 158–160 ^ꢁC; ¹H NMR d 3.57 (2H, s), 3.79 (3H,

s), 4.01 (3H, s), 4.03 (3H, s), 6.77–6.84 (3H, m), 7.15

(1H, s), 7.16 (1H, s), 7.45 (1H, dd, J=8.3, 1.8), 7.75–

7.79 (2H, m); ¹³C NMR d 56.3, 56.4, 106.6, 106.9, 114.8,

116.4, 117.4, 126.0, 126.8, 127.3, 128.7, 129.4, 129.8,

135.8, 137.9, 150.2, 150.3, 153.6; HRMS (EI) calcd for

C₁₉H₁₉NO₃m/z: 309.1365; found: 309.1375.

1

ethyl acetate to give 28 (60 mg) in 43% yield; H NMR

d 3.92 (3H, s), 4.01 (3H, s), 4.02 (3H, s), 7.12–7.26 (4H,

m), 7.39–7.46 (2H, m), 7.61 (1H, d, J=1.7), 7.71 (1H, d,

J=8.4); ¹³C NMR d 56.4, 106.6, 107.0, 109.5, 119.2,

124.8, 125.9, 127.2, 129.0, 129.4, 129.6, 133.5, 150.3,

150.4, 159.5; HRMS (EI) calcd for C₁₉H₁₇NO₅m/z:

339.1107; found: 339.1108.

6-(2-Amino-4-methoxyphenyl)-2,3-dimethoxynaphthalene

(32). 6-(4-Methoxy-2-nitrophenyl)-2,3-dimethoxynaph-

thalene 28 (18 mg, 0.053 mmol) was hydrogenated

overnight in ethyl acetate (20 mL) at 40ꢀ45 lb/inch²

using 10% palladium on carbon (10 mg) as catalyst. The

reaction solution was ﬁltered through Celite 545 and the

sicciate was washed with ethyl acetate (10 mLÂ3).

Concentration in vacuo gave the crude product. The

residue was chromatographed using a 60:40 mixture of

hexanes/ethyl acetate to give 32 (14 mg) in 85% yield;

¹H NMR d 3.83 (3H, s), 4.01 (3H, s), 4.03 (3H, s), 6.37

6-(5-Benzyloxy-4-methoxy-2-nitrophenyl)-2,3-dimethoxy-

naphthalene (29). Tetrakis(triphenylphosphine)palladium

(0) (200 mg) and cuprous bromide (20 mg) were added to

a

solution of 6,7-dimethoxy-2-triﬂuoromethane-

sulfonyloxynaphthalene 25 (500 mg, 1.49 mmol) and tri-

methyl(nitroaryl)stannane 24 (950 mg, 2.25 mmol) in

THF (40 mL) at room temperature and the mixture was

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1487

(1H, d, J=2.5), 6.45 (1H, dd, J=8.3, 2.4), 7.12–7.16

(3H, m), 7.42 (1H, dd, J=8.4, 1.7), 7.72–7.77 (2H, m);

¹³C NMR d 55.7, 56.4, 101.6, 104.8, 106.6, 106.8, 121.5,

126.4, 126.8, 127.2, 128.5, 129.9, 132.0, 135.6, 145.3,

150.0, 150.3, 160.5; HRMS (EI) calcd for C₁₉H₁₉NO₃

m/z: 309.1365; found: 309.1355.

solution of 25 (500 mg, 1.5 mmol) and the trimethyl(ni-

troaryl)stannane 35, (0.6 g, 1.83 mmol) in THF (30 mL)

at room temperature and stirred for 0.5 h. The mixture

was then reﬂuxed under N₂overnight. After cooling,

THF was evaporated and ethyl acetate was added to the

residue. The solution was washed with water. The

organic layer was ﬁltered through Celite 545 and then

washed with brine, dried (Na₂SO₄), and evaporated in

vacuo. The residue was chromatographed using a 70:30

mixture of hexanes/ethyl acetate to give 38 in 42% yield;

¹H NMR d 3.92 (3H, s), 6.19 (2H, s), 6.76 (1H, s), 7.12

(1H, d, J=2.5), 7.18 (1H, d, J=8.3), 7.28 (1H, dd,

J=9.0, 2.3), 7.70 (1H, s), 7.85 (1H, d, J=9.2), 7.93 (1H,

d, J=8.4); ¹³C NMR d 56.08, 100.59, 103.93, 106.31,

110.70, 121.54, 124.07, 126.47, 128.71, 129.55, 130.33,

130.99, 131.23, 142.83, 148.92, 152.07, 160.68.

6-(2-Amino-5-benzyloxy-4-methoxyphenyl)-2,3-dimethoxy-

naphthalene (33) and 6-(2-amino-5-hydroxy-4-methoxy-

phenyl)-2,3-dimethoxynaphthalene (34). Compound 29

(50 mg, 0.112 mmol) was hydrogenated in ethyl acetate

(40 mL) at 30 lb/inch²using 10% palladium on carbon

(15 mg) as catalyst for 16 h. The solution was passed

through a Celite 545 bed and the catalyst was washed

with ethyl acetate (10 mLÂ3). Concentration of the

ethyl acetate solution in vacuo gave the crude product.

Column chromatography was performed using a 35:65

mixture of hexanes/ethyl acetate as eluting solvent to

give compounds 33 and 34.

6-(4,5-Methylenedioxy-2-nitrophenyl)-2-methoxynaph-

thalene (39). Tetrakis(triphenylphosphine)palladium(0)

(120 mg) and cuprous bromide (20 mg) were added to a

solution of 2-bromo-6-methoxynaphthalene (0.3 g, 1.27

mmol) and trimethyl(3,4-methylenedioxy-6-nitrophe-

nyl)stannane 35 (0.45 g, 1.37 mmol) in THF (30 mL) at

room temperature and the mixture stirred for 0.5 h. The

mixture was then reﬂuxed under N₂for 16 h. After

cooling, THF was evaporated and 50 mL ethyl acetate

was added to the residue. The solution was washed with

water, ﬁltered through Celite 545, then washed with

brine, dried (Na₂SO₄), and evaporated in vacuo. The

residue was chromatographed using a 80:20 mixture of

hexanes/ethyl acetate to give the desired product 39

6-(2-Amino-5-benzyloxy-4-methoxyphenyl)-2,3-dimethoxy-

naphthalene (33). The higher R_fmaterial on thin layer

chromatography proved to be compound 33. The yield

was 34 mg (73%); ¹H NMR d 3.90 (3H, s), 4.01 (3H, s),

4.02 (3H, s), 5.08 (2H, s), 6.42 (1H, s), 6.87 (1H, s), 7.12

(1H, s), 7.15 (1H, s), 7.33–7.48 (6H, m), 7.71–7.75 (2H,

m); ¹³C NMR d 56.4, 56.4, 56.5, 73.1, 101.5, 106.6,

106.8, 119.1, 120.0, 126.2, 126.8, 127.3, 128.2, 128.2,

128.5, 128.9, 129.9, 135.6, 138.2, 138.9, 141.7, 150.1,

150.3, 150.9; HRMS (EI) calcd for C₂₆H₂₅NO₄m/z:

415.1784; found: 415.1775.

(0.29 g) in 71% yield; mp 165–167 ^ꢁC; H NMR d 3.94

1

6-(2-Amino-5-hydroxy-4-methoxyphenyl)-2,3-dimethoxy-

naphthalene (34). The lower R_fmaterial on thin layer

chromatography proved to be compound 34. The yield

was 6 mg (16%); IR (KBr) 3432, 2937, 2364, 1625, 1508,

(3H, s), 6.14 (2H, s), 6.88 (1H, s), 7.16–7.21 (2H, m),

7.31 (1H, dd, J=8.5, 1.9), 7.47 (1H, s), 7.67–7.77 (3H,

m); ¹³C NMR d 55.9, 103.5, 105.9, 106.2, 111.7, 119.9,

126.9, 127.0, 127.6, 129.2, 130.1, 133.8, 133.9, 134.5,

143.4, 147.5, 151.5, 158.6; HRMS (EI) calcd for

C₁₈H₁₃NO₅m/z: 323.0794; found: 323.0788.

1

1459, 1252, 1232 cm^À1; H NMR d 3.88 (3H, s), 4.01

(3H, s), 4.02 (3H, s), 6.40 (1H, s), 6.85 (1H, s), 7.12 (1H,

s), 7.15 (1H, s), 7.41 (1H, dd, J=8.4, 1.7); 7.73 (1H, d,

J=1.5), 7.74 (1H, d, J=8.3); ¹³C NMR d 56.4, 100.5,

106.6, 106.8, 116.9, 121.1, 126.3, 126.8, 127.3, 128.5,

129.9, 135.5, 137.2, 139.1, 147.1, 150.1, 150.3.

7-(4,5-Methylenedioxy-2-nitrophenyl)-3-methoxynaphth-

alene (40). Tetrakis(triphenylphosphine)palladium(0)

(120 mg) and cuprous bromide (20 mg) were added to a

solution of 7-methoxy-2-triﬂuoromethanesulfonyl-

oxynaphthalene 37 (336 mg, 1.1 mmol) and tri-

methyl(nitroaryl)stannane 35 (300 mg, 0.92 mmol) in

THF (30 mL) at room temperature and the mixture was

stirred for 0.5 h. The mixture was then reﬂuxed under

N₂overnight. After cooling, THF was evaporated in

vacuo and ethyl acetate (30 mL) was added to the resi-

due. The solution was washed with water. The organic

layer was ﬁltered through Celite 545 bed, then washed

with brine, dried (Na₂SO₄), and evaporated in vacuo.

The residue was chromatographed using a 80:20 mix-

ture of hexanes/ethyl acetate to give 40 (100 mg) in 34%

yield; ¹H NMR d 3.92 (3H, s), 6.15 (2H, s), 6.88 (1H, s),

7.13–7.23 (3H, m), 7.48 (1H, s), 7.64 (1H, s), 7.74–7.81

(2H, m); ¹³C NMR d 55.8, 103.5, 105.9, 106.5, 111.6,

119.8, 124.1, 126.0, 128.5, 128.7, 129.8, 133.9, 135.0,

136.6, 143.4, 147.6, 151.5, 158.6; HRMS (EI) calcd for

C₁₈H₁₃NO₅m/z: 323.0794; found: 323.0787.

7-Methoxy-2-triﬂuoromethanesulfonyloxynaphthalene (37).

A solution of 7-methoxy-2-naphthol (0.75 g, 4.3 mmol)

in THF (10 mL) was added to a suspension of sodium

hydride (60 wt%, 205 mg, 5.1 mmol) in THF (10 mL)

cooled by ice bath and stirred for 1.5 h. A solution of N-

phenyltriﬂuoromethanesulfonimide (1.55 g, 4.34 mmol)

in THF (10 mL) was then added, and the reaction mix-

ture was stirred for 9 h. After evaporation of the solvent

in vacuo, the residue was mixed with silica gel (4 g) and

then chromatographed using 500:18 hexanes/ethyl ace-

tate to give 37 (1.19 g) in 90% yield; mp 34 ^ꢁC (lit³¹

34 ^ꢁC); H NMR 3.93 (3H, s), 7.13–7.25 (3H, m), 7.65

1

(1H, d, J=2.5), 7.77 (1H, d, J=9.1), 7.83 (1H, d, J=8.8);

¹³C NMR d 55.9, 106.3, 116.1, 117.5, 118.5, 120.8, 122.5,

128.3, 129.9, 130.7, 135.4, 148.3, 159.4; HRMS (EI) calcd

for C₁₂H₉SO₄F₃m/z: 306.0174; found: 306.0176.

6-(4,5-Methylenedioxy-2-nitrophenyl)-2,3-dimethoxynaph-

thalene (38). Tetrakis(triphenylphosphine)palladium(0)

(126 mg) and cuprous bromide (21 mg) were added to a

6-(2-Amino-4,5-methylenedioxyphenyl)-2,3-dimethoxy-

naphthalene (41). 6-(4,5-Methylenedioxy-2-nitrophe-

1488

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

nyl)-2,3-dimethoxynaphthalene (38) (25 mg) was hydro-

genated overnight in ethyl acetate (40 mL) at 40–45 lb/

inch²under palladium catalysis (10 wt % on activated

carbon, 20 mg). The solution was passed through a Celite

545 bed and the catalyst was washed with ethyl acetate

(3Â10 mL). Concentration in vacuo gave the crude pro-

duct. Chromatography using 60:40 hexanes/ethyl acetate

the eluting solvent to yield 380 mg of 47 (35%); mp 130–

131 ^ꢁC (lit²⁶130 ^ꢁC); H NMR d 3.96 (3H, s), 7.04 (1H,

1

d, J=9.0), 7.10 (1H, dd, J=9.0, 2.6), 7.67 (1H, d,

J=8.9), 7.87 (1H, d, J=8 .9), 8.37 (1H, d, J=2.5); ¹³C

NMR d 56.0, 104.3, 116.8, 117.3, 124.2, 129.4, 131.5,

139.6, 160.4, 162.8.

1

gave 41 (15 mg) in 66% yield; H NMR d 4.01 (3H, s),

2-Hydroxy-7-benzyloxy-1-nitronaphthalene (48). 7-Ben-

zyloxy-2-naphthol³²(2.5 g, 10.0 mmol) was dissolved in

100 mL of dry ether. To this solution was added 4-

methyl-4-nitro-2,3,5,6-tetrabromo-2,5-cyclohexadien-1-

one²⁶(4.69 g, 10 mmol) as a solid. The color of the

solution became red and eventually dark brown. The

reaction was continued for 3 h at room temperature.

The reaction mixture was concentrated in vacuo. The

residue was chromatographed using a 85:15 mixture of

hexanes and ethyl acetate to provide 1.6 g (55%) of 48;

4.02 (3H, s), 5.91 (2H, s), 6.40 (1H, s), 6.73 (1H, s), 7.13

(1H, s), 7.15 (1H, s), 7.39 (1H, dd, J=8.2, 1.8), 7.72 (1H,

s), 7.74 (1H, d, J=8.5); ¹³C NMR d 56.4, 98.3, 101.2,

106.6, 106.8, 110.7, 120.5, 126.3, 127.0, 127.3, 128.5,

129.9, 135.7, 138.9, 141.1, 148.0, 150.1, 150.3; HRMS

calcd for C₁₉H₁₇NO₄: 323.1157; found: 323.1170.

6-(2-Amino-4,5-methylenedioxyphenyl)-2-methoxynaphth-

alene (42). 6-(4,5-Methylenedioxy-2-nitrophenyl)-2-

methoxynaphthalene 39 (260 mg, 0.81 mmol) was hydro-

genated overnight in ethyl acetate (35 mL) at 40–45 lb/

inch²using 10% palladium on carbon (70 mg) as catalyst.

The reaction solution was ﬁltered through Celite 545 and

the sicciate was washed with ethyl acetate (10 mLÂ3).

The ﬁltrate was concentrated and the residue chromato-

graphed using a 3:1 mixture of hexanes/ethyl acetate to

give 42 (180 mg) in 76% yield; mp 130–132 ^ꢁC; ¹H NMR

d 3.56 (2H, s), 3.95 (3H, s), 5.92 (2H, s), 6.40 (1H, s), 6.75

(1H, s), 7.17–7.22 (2H, m), 7.51 (1H, dd, J=8.5, 1.6),

7.73–7.82 (3H, m); ¹³C NMR d 55.8, 98.3, 101.3, 106.1,

110.7, 119.7, 120.3, 127.8, 128.3, 128.6, 129.6, 129.9,

134.0, 135.1, 139.0, 141.2, 148.1, 158.3; HRMS (EI) calcd

for C₁₈H₁₅NO₃m/z: 293.1052; found: 293.1051.

mp 177–178 ^ꢁC; H NMR d 5.22 (2H, s), 7.10 (d, 1H,

1

J=8), 7.21 (1H, dd, J=4, 10.2), 7.38–7.56 (5H, m), 7.74

(1H, d, J=8.0), 7.93 (1H, d, J=8.2), 8.56 (1H, s,), 12.42

(1H, s).

2-Hydroxy-7-acetoxy-1-nitronaphthalene (49). 7-Acet-

oxy-2-naphthol³³(1.01 gm, 5.0 mmol) was dissolved in

100 mL of dry ether. To this solution was added 4-

methyl-4-nitro-2,3,5,6-tetrabromo-2,5-cyclohexadien-1-

one²⁶(2.35 g, 5.0 mmol) as a solid. The color of the

solution became red and eventually dark brown. The

reaction was continued for 3 h at room temperature.

The reaction mixture was concentrated in vacuo and the

crude material was puriﬁed by ﬂash chromatography

eluting with hexanes/ethyl acetate (85:15), to provide

1

7-(2-Amino-4,5-methylenedioxyphenyl)-2-methoxynaph-

thalene (43). 7-(4,5-Methylenedioxy-2-nitrophenyl)-2-

methoxynaphthalene 40 (100 mg, 0.31 mmol) was hydro-

genated overnight in ethyl acetate (35 mL) at 40–45 lb/

inch²using 10% palladium on carbon (30 mg) as catalyst.

The reaction solution was ﬁltered through Celite 545 and

the sicciate washed with ethyl acetate (10 mLÂ3). The

ﬁltrate was concentrated in vacuo and the residue chro-

matographed using a 75:25 mixture of hexanes/ethyl ace-

tate to give 43 (75 mg) in 83% yield; ¹H NMR d 3.64 (2H,

s), 3.94 (3H, s), 5.92 (2H, s), 6.40 (1H, s), 6.76 (1H, s),

7.15–7.20 (2H, m), 7.40 (1H, dd, J=8.3, 1.7), 7.75–7.85

(3H, m); ¹³C NMR d 55.8, 98.4, 101.3, 106.3, 110.7, 119.4,

120.3, 125.8, 127.4, 128.3, 128.7, 129.7, 135.3, 138.0,

139.0, 141.2, 148.2, 158.5; HRMS (EI) calcd for

C₁₈H₁₅NO₃m/z: 293.1052; found: 293.1052.

715 mg (58%) of 49; H NMR d 2.41 (3H, s), 7.21 (1H,

d, J=4), 7.35 (1H, d, J=4), 7.83 (1H, d, J=8.0), 8.02

(1H, d, J=8.2 ), 8.76 (1H, s), 12.38 (1H, s).

7-Methoxy-1-nitro-2-triﬂuoromethanesulfonyloxynaphtha-

lene (50). A solution of compound 49 (380 mg, 2.24

mmol) in THF (15 mL) was added to a suspension of

sodium hydride (60 wt% in mineral oil, 90 mg, 2.25

mmol) in THF (10 mL) at 0 ^ꢁC and the mixture stirred

for 0.5 h.

A

solution of N-phenyltriﬂuoro-

methanesulfonimide (800 mg, 2.24 mmol) in THF

(10 mL) was then added, and the reaction stirred at 0 ^ꢁC

for 8 h. After concentration in vacuo, the residue

was chromatographed using 85:15 hexanes/ethyl acetate

to give 50 (526 mg) containing approximately 10%

N-phenyltriﬂuoromethanesulfonamide.

2-Hydroxy-7-methoxy-1-nitronaphthalene (47). 7-Meth-

oxy-2-naphthol (871 mg, 5 mmol) was dissolved in 40 mL

of dry ether. To this solution was added 4-methyl-4-nitro-

2,3,5,6-tetrabromo-2,5-cyclohexadien-1-one²⁶(2.33 g,

5 mmol) as a solid. The color of the solution slowly

became red, and eventually dark red with some dark

precipitate adhering to the inside surface of the ﬂask.

The reaction continued for 2.5 h at room temperature.

Evaporation of the solvent gave the crude product. To

the residue was added 20 mL of methanol/water (80/20).

The reaction mixture was ﬁltered and the sicciate was

washed with methanol/water (80/20). The ﬁltrate was

then evaporated under vacuum and chromatographed

using a 90:10 mixture of hexanes and ethyl acetate as

7- Benzyloxy-1-nitro-2-triﬂuoromethanesulfonyloxynaph-

thalene (51). To a cold solution of 48 (147 mg, 0.5

mmol) in dry pyridine (4 mL), triﬂuoromethanesulfonic

anhydride (0.08 mL, 0.5 mmol) was added dropwise at

0 ^ꢁC and stirring was continued for 30 min at 0 ^ꢁC. Then

the ice bath was removed and the reaction was stirred at

room temperature overnight. The reaction mixture was

poured on ice-water. A light yellow solid separated

which was ﬁltered, washed with water and dried to

provide 155 mg of 51 (70% yield).

7-Acetoxy-1-nitrotriﬂuoromethanesulfonyloxynaphthalene

(52). To a cold solution of 49 (705 mg, 2.85 mmol) in

dry pyridine (5 mL), triﬂuoromethanesulfonic anhy-

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

1489

dride (0.48 mL, 2.85 mmol) was added dropwise at 0 ^ꢁC

with stirring. After being allowed to stir for 30 min at

0 ^ꢁC, the mixture was allowed to warm to room tem-

perature. The reaction mixture was poured on ice-water.

A light yellow solid separated which was ﬁltered washed

with water and dried to provide 975 mg (90% yield) of

545. The organic layer was then washed with brine, dried

(Na₂SO₄), and concentrated in vacuo. The residue was

chromatographed using a mixture of hexanes/ethyl acetate

(80:15) to provide 130 mg (40% yield) of 55; mp 166–

167 ^ꢁC; ¹H NMR d 2.38 (3H, s), 6.19 (2H, dd, J=1.5, 2.5),

6.75 (1H, s), 7.33 (1H, s), 7.37 (1H, s), 7.45 (1H, dd, J=2.5,

11.5), 7.72 (1H, s), 8.02 (t, 1H, J=9); ¹³C NMR d 21.1,

103.4, 103.6, 105.7, 105.9, 109.6, 110.2, 113.8, 123.7, 125.3,

126.2, 127.8, 129.9, 130.9, 131.4, 148.7, 151.1, 151.8, 169.2.

52; mp 108–109 ^ꢁC; H NMR d 2.42 (3H, s), 7.48–7.68

1

(3H, m), 8.12 (1H, d, J=12), 8.24 (1H, d, J=12).

6-(4,5-Methylenedioxy-2-nitrophenyl)-2,3-dimethoxy-5-

nitronaphthalene (53). Tetrakis(triphenylphosphine)-

palladium(0) (100 mg) and cuprous bromide (20 mg)

were added to a solution of 7-methoxy-1-nitro-2-tri-

ﬂuoromethanesulfonyloxynaphthalene 50 (366 mg, 1.04

mmol) and trimethyl(nitroaryl)stannane 35 (500 mg, 1.52

mmol) in THF (30 mL) at room temperature and the

mixture was stirred for 0.5 h. The mixture was then

reﬂuxed under N₂overnight. After cooling, THF was

evaporated and ethyl acetate (30 mL) was added to the

residue. The solution was washed with water. The

organic layer was ﬁltered through Celite 545 bed and

then washed with brine, dried (Na₂SO₄), and evaporated

in vacuo. The residue was chromatographed using a

70:30 mixture of hexanes/ethyl acetate to give 53 (160

mg) in 42% yield; mp 187–189 ^ꢁC; IR (KBr) 2925, 1628,

1526, 1487, 1364, 1332, 1265, 1230 cm^À1; ¹H NMR d 3.92

(3H, s), 6.19 (2H, s), 6.76 (1H, s), 7.12 (1H, d, J=2.5),

7.18 (1H, d, J=8.3), 7.28 (1H, dd, J=9.0, 2.3), 7.70 (1H,

s), 7.85 (1H, d, J=9.2), 7.93 (1H, d, J=8.4); ¹³C NMR d

56.1, 100.6, 103.9, 106.3, 110.7, 121.5, 124.1, 126.5, 128.7,

129.6, 130.3, 131.0, 131.2, 142.8, 148.9, 152.1, 160.7.

7-Bromo-3-methoxynaphthalene

2-carboxylic

acid

methyl ester³²(56). A mixture of 6-bromo-3-hydroxy-2-

naphthoic acid (5.0 g, 18.0 mmol), anhydrous K₂CO₃

(12.9 gm) and dimethyl sulfate (4.5 g, 41 mmol) in dry

acetone (150 mL) were reﬂuxed for 4 h. The reaction

mixture was cooled and water (5 mL) was added. The

reaction mixture was then stirred for 2 h to allow for

hydrolysis of any remaining dimethyl sulfate. The reac-

tion mixture was then ﬁltered and the ﬁltrate con-

centrated in vacuo. The residue was dissolved in CHCl₃

and washed several times with water, was dried,

(Na₂SO₄) and the solvent removed in vacuo to aﬀord

4.6 g (83.6%) of (56) as a solid; mp 166–167 ^ꢁC.

2-Acetoxy-6-(4,5-methylenedioxy-2-nitrophenyl)-3-meth-

oxynaphthalene (57). Tetrakis(triphenylphosphine)-

palladium(0) (280 mg) and cuprous bromide (150 mg)

were added to a solution of 56 (2.95 g, 10 mmol) and the

trimethyl(nitroaryl)stannane 35 (3.55 g, 10.6 mmol) in

THF (200 mL). The reaction mixture was heated to

reﬂux for 96 h under N₂. After allowing the reaction

mixture to cool, THF was removed in vacuo. The residue

was dissolved in 150 mL of chloroform. The solution was

washed with water. The organic layer was separated and

ﬁltered through Celite 545. The organic layer was then

washed with brine, dried (Na₂SO₄) and concentrated in

vacuo. The residue was chromatographed using hexanes/

ethyl ace_ꢁtate (85:15) to give 2.1 g of 57 (55.3% yield); mp

2-(4,5-Methylenedioxy-2-nitrophenyl)-7-methoxy-1-nitro-

Tetrakis(triphenylphosphine)-

naphthalene

(54).

palladium(0) (90 mg), lithium chloride (240 mg) and

cuprous bromide (60 mg) were added to a solution of 51

(886 mg, 2.0 mmol) and trimethyl(nitroaryl)stannane 35

(804 mg, 2.4 mmol) in THF (200 mL) and the reaction

mixture was heated to reﬂux for 24 h under N₂. After

cooling, THF was evaporated and chloroform (100 mL)

was added to the residue. The solution was washed with

water. The organic layer was separated, ﬁltered through

Celite 545 and then washed with brine, dried (Na₂SO₄),

and concentrated in vacuo. The residue was chromato-

graphed using a mixture of hexanes/ethyl acetate _ꢁ(85:15)

1

186–187 C; H NMR d 3.97 (3H, s), 4.03 (3H, s), 6.17

(2H, s), 6.87 (1H, s), 7.25 (1H, d, J=4), 7.39 (1H, dd,

J=2, 8), 7.50 (1H, s), 7.73 (1H, d, J=4), 7.78 (1H, s),

8.30 (1H, s); ¹³C NMR d 52.3, 56.2, 103.1, 105.6, 106.9,

111.2, 122.6, 126.8, 127.5, 128.6, 132.9, 134.3, 135.5,

143.0, 147.4, 151.2, 156.4, 166.6.

1

to provide 520 mg (58.5%) of 54; mp 151–152 C; H

2-Acetoxy-6-(2-Amino-4,5-methylenedioxyphenyl)-3-meth-

oxynaphthalene (58). To a reﬂuxing solution of 57 (760

mg, 2.0 mmol) and Ra–Ni (200 mg) in ethanol (50 mL),

hydrazine hydrate (2.5 mL) was added dropwise. After

the addition was complete, the reaction mixture was

heated to reﬂux for 2 h. The mixture was ﬁltered

through Celite 545. The ﬁltrate was concentrated in

vacuo to provide 700 mg (99% yield) of solid; mp 192–

193 ^ꢁC; ¹H NMR d 3.98 (3H, s), 4.04 (3H, s), 5.94 (2H, s),

6.40 (2H, s), 6.78 (1H, s), 7.28 (1H, d, J=2), 7.61 (1H, dd,

J=2, 8), 7.80 (1H, s), 7.86 (1H, d, J=4), 8.32 (1H, s).

NMR d 5.2 (2H, s), 6.19 (2H, t, J=2.2), 6.77 (1H, s),

7.21 (1H, dd, J=1.4, 8.4), 7.34 (1H, s), 7.36 (6H, m),

7.65 (1H, s), 7.88 (1H, d, J=9.2), 7.95 (1H, d, J=8.4);

¹³C NMR d 70.5, 101.7, 103.5, 105.9, 110.3, 121.3,

123.9, 126.1, 127.9, 128.3, 128.4, 128.0, 129.3, 130.0,

130.7, 130.8, 136.1, 142.5, 145.2, 148.5, 151.7, 159.4.

2-(4,5-Methylenedioxy-2-nitrophenyl)-7-acetoxy-1-nitro-

naphthalene

(55).

Tetrakis(triphenylphosphine)-

palladium(0) (23 mg), lithium chloride (126 mg) and

cuprous bromide (15 mg ) were added to a solution of 52

(379 mg, 1.0 mmol) and trimethyl(nitroaryl)stannane 35

(402 mg, 2.4 mmol) in THF (200 mL) and reaction mixture

was heated to reﬂux for 72 h under N₂. After cooling, THF

was evaporated and chloroform (100 mL) was added to

the residue. The solution was washed with water. The

organic layer was separated and ﬁltered through Celite

6-(2-Amino-4,5-methylenedioxyphenyl)-2-hydroxymethyl-

3-methoxynaphthalene (59). To a solution of 58 (702

mg, 2.0 mmol) in dry THF (60 mL), LAH (115 mg, 3.0

mmol) was added in portions with stirring at room

temperature under N₂. The reaction was stirred at room

temperature for 2 h. Excess LAH was decomposed with

1490

Y. Yu et al. / Bioorg. Med. Chem. 11 (2003) 1475–1491

a few drops of 10% NaOH solution. The reaction mix-

ture was ﬁltered through Celite 545 and the sicciate was

washed with THF. The ﬁltrate was concentrated in

vacuo and dried (Na₂SO₄) to provide 470 mg (73%

concentrations of drug, and MTT assays were per-

formed at the end of the fourth day. Each assay was

performed with a control that did not contain any drug.

All assays were performed at least twice in six replicate

wells.

yield) of 58 as a light yellow solid; mp 143–144 ^ꢁC; H

1

NMR d 4.00 (3H, s), 4.83 (2H, s), 5.92 (2H, s), 6.40 (1H,

s), 6.73 (1H, s), 7.15 (1H, s), 7.50 (1H, dd, J=1.4, 8.2),

7.75–7.81 (3H, m); ¹³C NMR d 55.5, 62.1, 98.1, 100.9,

105.1, 110.3, 120.1, 127.1, 127.6, 127.9, 128.1, 129.0,

131.2, 133.1, 135.0, 138.2, 140.9, 147.7, 156.2.

Acknowledgements

We are grateful to Washington University Resource for

Biomedical and Bio-Organic Mass Spectrometry for

providing mass spectral data and for the partial support

of this facility by the National Institutes of Health

(Grant No. P41RR0954). This study was supported by

AVAX Technologies, Inc, (E.J.L.) and Grant CA39662

(L.F.L.) and Grant CA077433 (L.F.L.) from the

National Cancer Institute.

Topoisomerase-mediated DNA cleavage assays. Human

topoisomerase I was expressed in Escherichia coli and

isolated as a recombinant fusion protein using a T7

expression system as described previously.¹³Recombi-

nant human topoisomerase IIa was isolated and pur-

iﬁed as previously described.³⁴Plasmid YepG was also

puriﬁed by the alkali lysis method followed by phenol

deproteination and CsCl/ethidium isopycnic centrifuga-

tion method as described.³⁵The end-labeling of the

plasmid was accomplished by digestion with a restric-

tion enzyme followed by end-ﬁlling with Klenow poly-

merase as previously described.³⁶The cleavage assays

were performed as previously reported.⁸The drug and

the DNA in presence of topoisomerase I was incubated

for 30 min at 37 ^ꢁC. The reactions were terminated by

the addition of 5 mL of 5% SDS and 1 mg/mL protein

kinase K with an additional 1 h of incubation at 37 ^ꢁC.

Samples were then alkali denatured by the addition of

NaOH, EDTA, sucrose, and bromophenol blue to ﬁnal

concentrations of 75 mM, 2.5%, and 0.05 mg/mL,

respectively, prior to loading onto a neutral agarose gel.

After development of the gels, typically 24-h exposure

was used to obtain autoradiograms outlining the extent

of DNA fragmentation. Topoisomerase I-mediated

DNA cleavage values are reported as REC, Relative

Eﬀective Concentration, i.e., concentrations relative to

camptothecin, whose value is arbitrarily assumed as 1.0,

that are able to produce the same cleavage on the plas-

mid DNA in the presence of human topoisomerase I.

Topoisomerase II-mediated DNA cleavage values are

reported as REC, Relative Eﬀective Concentration,

wherein potency was based upon the relative amount of

drug needed to induce approximately 10% DNA frag-

mentation. REC values for topoisomerse II-mediated

cleavage are reported relative to VM-26, whose value is

arbitrarily set as 1.0.

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Article Doi

Substituted dibenzo[c,h]cinnolines: Topoisomerase I-targeting anticancer agents

DOI: 10.1016/S0968-0896(02)00604-1

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