Novel angular benzophenazines: Dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents

J . Med. Chem. 2002, 45, 721-739

721

Novel An gu la r Ben zop h en a zin es: Du a l Top oisom er a se I a n d Top oisom er a se II

In h ibitor s a s P oten tia l An tica n cer Agen ts

Nigel Vicker,^†Luke Burgess,^†Irina S. Chuckowree,^†Rory Dodd,^†Adrian J . Folkes,^†David J . Hardick,^†

Timothy C. Hancox,^†Warren Miller,^†J ohn Milton,^†Sukhjit Sohal,^†Shouming Wang,^†Stephen P. Wren,^†

Peter A. Charlton,*^,‡Wendy Dangerfield,^‡Chris Liddle,^‡Prakash Mistry,^‡Alistair J . Stewart,^‡and

William A. Denny^§

Medicinal Chemistry and Pharmacology Departments, Xenova Ltd., 957 Buckingham Avenue, Slough, Berkshire,

SL1 4NL, U.K., and Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science,

The University of Auckland, Private Bag 92019, Auckland 1000, New Zealand

Received J uly 17, 2001

A series of substituted angular benzophenazines were prepared using a new synthetic route

via a novel regiocontrolled condensation of 1,2-naphthoquinones and 2,3-diaminobenzoic acids.

The synthesis and biological activity of this new series of substituted 8,9-benzo[a]phenazine

carboxamide systems are described. The analogues were evaluated against the H69 parental

human small cell lung carcinoma cell line and H69/LX4 resistant cell line which overexpresses

P-glycoprotein. Selected analogues were evaluated against the COR-L23 parental human non

small cell lung carcinoma cell line and the COR-L23/R resistant cell line which overexpresses

multidrug resistance protein. This series of novel angular benzophenazines were potent cytotoxic

agents in these cell lines and may be able to circumvent multidrug resistance mechanisms

which result in the lack of efficacy of many drugs in cancer chemotherapy. These compounds

show dual inhibition of topoisomerase I and topoisomerase II and thus target two key enzymes

responsible for the topology of DNA that are active at different points in the cell cycle. The

introduction of chirality into the carboxamide side chain of these novel benzophenazine

carboxamides has resulted in the discovery of a potent enantiospecific series of cytotoxic agents,

exemplified by 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-

methyl-ethyl)-amide, XR11576 ((R)-4j′′). In vivo activity has been demonstrated for 4-methoxy-

benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576,

after intravenous administration to female mice, and this compound has been selected as a

development candidate for further evaluation.

In tr od u ction

XR5000 (1) is an acridine derivative which acts as a

DNA intercalating agent, and the steric and electronic

effects that determine its cytotoxicity have recently been

discussed in detail.¹⁰To find a novel structural class of

second generation dual topoisomerase inhibitors, a

program of work was initiated to discover an orally

active dual inhibitor of topoisomerase I and II that

avoided MDR.

The topoisomerases are essential enzymes in the

regulation of DNA topology, which is required if cells

are to divide and proliferate,¹and are important cellular

targets for a number of successful chemotherapeutic

agents.¹Drugs that target topoisomerase II, for example

doxorubicin and etoposide, have been widely used in

cancer chemotherapy,²while those that specifically

target topoisomerase I, principally the camptothecin

analogues, have made an important impact more re-

cently for the treatment of colon cancer.³A shortfall of

many of these specific inhibitors of either topoisomerase

I or topoisomerase II is their inability to overcome

multidrug resistance (MDR).^4,5Several dual inhibitors

of topoisomerase I and II have been identified; these

include intoplicine,⁶XR5000 (DACA),⁷and TAS-103,⁸

all of which are in clinical evaluation. Recently it has

been suggested that TAS-103 predominantly acts as a

topoisomerase IIR inhibitor.⁹An advantage of both

XR5000 and TAS-103 is their ability to circumvent MDR

and to target two key enzymes that affect the topology

of DNA which are active at different points in the cell

cycle.

The phenazines are different chemically and structur-

ally from the acridines but have similar shape and have

been shown to fulfill the fundamental physicochemical

requirements for DNA intercalation.¹¹A strategy of

modifying the phenazine template to give structurally

novel dual topoisomerase inhibitors was adopted. The

* To whom correspondence should be addressed. Ph: +44 1753

706600. Fax: +44 1753 706607. E-mail: Peter_Charlton@Xenova.co.uk.

^†Medicinal Chemistry Department, Xenova Ltd.

^‡Pharmacology Department, Xenova Ltd.

^§Auckland Cancer Society Research Centre.

Published on Web 01/08/2002

722 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

Sch em e 1^a

a

Reagents: (a) SeO₂, AcOH; (b) EtOH, concentrated HCl; (c)

CDI, DMF, Et₃N, RNH₂or SOCl₂, DCM, Et₃N, RNH₂.

Sch em e 2^a

F igu r e 1.

synthesis and antitumor activity of substituted phen-

azine-1-carboxamides have previously been reported.¹¹

The substituent effects on phenazine-1-carboxamides (2)

have previously indicated that substitution at the

9-position gave improved cytotoxic potency in vitro when

evaluated against the L1210 and the P388 leukemia cell

lines.¹¹In addition, benzofusion at the 3,4-, 6,7-, and

8,9-positions, to give angular tetracyclic benzophen-

azines, indicated that the 8,9-benzo[a]phenazine-1-

carboxamide (3) was the most active compound in this

series when tested on these cell lines.¹¹

a

Reagents: (a) CuCl, Cu powder, N-ethylmorpholine, ethylene

glycol; (b) 2 M NaOEt, NaBH₄, EtOH; (c) CDI, DMF, Et₃N, RNH₂

or SOCl₂, DCM, Et₃N, RNH₂.

amines. The novel acids (5) were obtained from a

regiocontrolled condensation under acidic conditions

between 1,2-naphthoquinones (7) and 2,3-diaminoben-

zoic acids (6). The naphthoquinones (7) were in turn

prepared by oxidation of the corresponding tetralones

(8) using selenium dioxide as indicated in Scheme 1

below.

The dual topoisomerase I and II inhibitor Intoplicine

has a benzo-fused ring in a similar position to the

benzofusion in (3), and it has been shown in the

Intoplicine series that substitution in the 9-position with

a hydroxyl moiety gave improved biological activity in

vitro and in vivo when compared to the unsubstituted

analogue and was also shown to be essential for dual

inhibition of topoisomerase I and topoisomerase II.⁶It

was our postulate that novel substituted benzo[a]-

phenazine carboxamides would show similar trends in

biological activity and lead to the discovery of novel dual

inhibitors of the topoisomerases I and II.

In this paper we extend these studies to novel

substituted angular tetracyclic phenazines with solid

tumor activity by reporting the synthesis, biological

activity, and structure-activity relationships (SAR) for

substituted derivatives of (3) and the effects of modifying

the carboxamide side chain of (3) on these parameters.

Substituted benzo[a]-11-carboxamides (4) could also

be prepared according to the sequence shown in Scheme

2. An Ullman coupling of 2-bromo-3-nitrobenzoic acid

with naphthylamines (9) and subsequent reductive ring

closure of 10 to give 5, according to the method of Denny

et al.,¹¹followed by amide bond formation then gave the

desired compounds (4).

In some cases the methyl ester of 5, formed from 5

under standard conditions, could be treated directly

with the relevant amine to give 4. A general synthesis

from protected amino acid derivatives of some of the

amines used to form 4 is outlined in Scheme 3 and is

applicable to racemic and enantiomerically pure com-

pounds.

The starting protected amino acid derivatives were

available after standard functional group manipulation.

Protection of the amino group as its butyloxycarbonyl

derivative was achieved using di-tert-butyl dicarbonate

and triethylamine in acetonitrile, and esterification of

the carboxylic acid group to give the methyl ester was

achieved using methanol HCl. These steps were high

yielding and typically >95%. Methyl ester reduction

mediated with diisobutylaluminum hydride (DIBAL) at

Ch em istr y

Substituted benzo[a]-11-carboxamides (4) were ob-

tained from amide bond formation between an activated

form of the corresponding acid (5) and the appropriate

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 723

Sch em e 3^a

protein (MRP). The activities of novel compounds de-

signed to examine the effects of substitutions in the

benzo[a]fused ring in these cell lines are indicated in

Table 1.

In the design of novel substituted benzo[a]phenazines,

the effects of substitution at the 2-4-positions were

examined in order to investigate any overlap with the

9-hydroxyl of Intoplicine. It has been previously indi-

cated by the 1-aza compound that substitution in the

1-position was least tolerant to substitution probably

due to the steric and electronic effects of this position

on the side chain and the phenazine nitrogen at the 12-

position.¹¹The activity of a series of fused heteroaro-

matic[a]phenazines which confirms this observation is

published elsewhere.¹²Substitution in the 2-4-positions

is tolerated with the 4-position in general being most

favored, providing the most potent compounds in this

seriessthe 4-OH 4f and 4-OMe 4c analogues. There

does not appear to be any major electronic effect of the

substituents on activity, with less than 3-fold difference

in activity with strongly electron withdrawing substit-

uents (e.g., NO₂, 4i) and strongly electron donating

substituents (e.g., OMe, 4c). This new finding that

activity is retained or improved by substitution at the

4-position gives scope for further structural modification

and SAR development.

As substitution at the 4-position had given the most

potent compounds, the tolerances to substitution at this

position were examined in detail and the cytotoxicity

of compounds in this series is listed in Tables 2 and 3.

SAR throughout are discussed in terms of activity on

the parental cell line compared with compound 4c.

Substitution with a wide range of functionality groups

such as thiomethyl 4j, halogen 4m -o, methyl 4p , nitrile

4q, ester 4r , hydroxymethyl 4t, and amino 4u was

tolerated with only marginal loss in activity (2-3-fold).

These results in general indicate that a variety of small

groups are tolerated in this region. In contrast the

corresponding methyl sulfone 4k , sulfonylamino 4l,

carboxylic acid 4s, dimethylamino 4w , acetylamino 4x,

and sulfonamide moieties 4y,z gave a dramatic loss in

a

Reagents: (a) DIBAL, toluene, - 78 °C; (b) Me₂NH‚HCl,

NaCNBH₃, NaOAc, MeOH; (c) 4 M HCl dioxan.

-78 °C gave the corresponding aldehyde without any

racemization of enantiopure compounds. Reductive ami-

nation of the aldehyde using dimethylamine in the

presence of sodium cyanoborohydride gave the required

N,N-dimethylamines. Removal of the tert-butyloxycar-

bonyl group was achieved in >95% yield using 4 M HCl

in dioxan. Where the R group in the side chain con-

tained a hydroxyl group, this was protected as the tert-

butyldimethylsilyl ether by reaction with tert-butyldi-

methylsilyl chloride in dichloromethane in the presence

of imidazole in quantitative yield. Removal of the tert-

butyldimethylsilyl group was simultaneous with the

removal of the butyloxycarbonyl group in step c using

4 M HCl in dioxan. The amines either synthesized or

commercial were coupled to an activated form of 5 to

form 4. Some functionality (R1-R6) in compounds 4 or

5 could be further modified to give compounds of the

same general structure.

Resu lts a n d Discu ssion

The cytotoxicity of compounds of general structure 4

was measured using the H69 parental (H69/P) human

small cell lung carcinoma cell line and the drug resistant

human small cell lung carcinoma cell line H69/LX4

which overexpresses P-glycoprotein (Pgp). The cytotox-

icity of compounds was also measured using the COR-

L23 parental (COR-L23/P) human non small cell lung

carcinoma cell line and also the drug resistant human

non small cell lung carcinoma cell line COR-L23/R

which overexpresses multidrug resistance associated

Ta ble 1. Substitution in the Benzo[a]fused Ring of 3 and 4a -i

H69/P

H69/LX4

L23/P

L23/R

compound

substituent

IC₅₀(nM)^a

Rf^b

IC₅₀(nM)^a

Rf^b

2.5

TAS-103

doxorubicin

topotecan

3

21.0 ( 7.7^c(n ) 45)

22.6 ( 8.3^c(n ) 45)

1.1 17.3 ( 4.2^c(n ) 18) 42.9 ( 22^c(n ) 17)

27.3 ( 30.6^c(n ) 22) 3874 ( 2791^c(n ) 22) 142

20.1 ( 7.0^c(n ) 19) 319.3 ( 145^c(n ) 16) 16

15.9 ( 5.3^c(n ) 20)

62.9 ( 30.0^c(n ) 20)

3.9 13.2 ( 3.6^c(n ) 20) 20.0 ( 4.1^c(n ) 20)

1.5

1.4

-

67

110

130

112

1.9 102

1.0 NT

140

NT

4a

4b

4c

4d

2-OMe

3-OMe

4-OMe

2-OH

141

35

86

185

49

282

1.3 68

1.4 14

3.3 NT

79

20

NT

1.2

1.4

-

4e

4f

4g

4h

3-OH

4-OH

2-NO₂

3-NO₂

4-NO₂

109

35

94

49

193

48

102

69

1.8 67

1.4 15

1.1 NT

1.4 NT

96

17

NT

1.4

1.1

-

4i

96

120

1.3 20

24

1.2

XR11576

23.0 ( 5.9^c(n ) 8)

29.0 ( 14.9^c(n ) 8)

1.2 10.1 ( 4.2^c(n ) 7)

12.1 ( 5.6^c(n ) 7)

a

Each assay was performed in quadruplicate with the number of determinations N g 2, and the results correspond to a mean value

b

where the IC₅₀) concentration of drug (nM) to reduce the cell number to 50%. The Rf is the resistance factor and is the ratio of the IC₅₀

on the resistant cell line over the IC₅₀on the parental cell line in the cell lines shown. NT (not tested). ^cMean ( SD.

724 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

Ta ble 2. Substitution at the 4-Position of the

Benzo[a]phenazine Carboxamides, 4j-4z

compound 4c, attachment to the phenazine nucleus is

by an oxygen linkage, and this enables rapid synthetic

access from a common intermediate 4f to a diverse set

of O-linked substituents at the 4-position to further

broaden SAR and examine substituent effects. The SAR

of analogues that are attached at the 4-position by

oxygen are listed in Table 3.

Replacement of methyl 4c by ethyl 4a ′ does not result

in any loss of potency. However, the isopropyl 4b′

derivative loses ∼12-fold activity, indicating a steric

constraint in this region, and this is supported by the

loss of cytotoxicity for benzyl analogue 4c′. In addition,

the propargyl 4d ′ and the cyanomethoxy 4e′ compounds

are equipotent with the 4c analogue, confirming that

small groups with directional functionality are well

tolerated. The 3-cyanopropoxy moiety is also tolerated

(4f′), and this coupled with the result above in Table 2

for the cyanomethylamino compound 4v supports this

hypothesis.

The ethylamide 4h ′ is ∼2-fold less potent, and the

acetyloxy compound 4g′ retains activity but may be

metabolized to the potent phenolic analogue 4f within

the cell.

These data also show that the methoxyacetyl ana-

logue 4l′ is equipotent and the methoxycarboxamide 4m ′

is 2.5-fold less active. A dramatic loss of activity is

observed for the methoxyethylcarboxylate 4k ′, and the

methoxycarboxylic acid 4n ′ loses 100-fold activity again

showing acidic functionality is disfavored.

The ethoxymethoxy 4i′ and ethoxymorpholino 4p ′

analogues lose 2-3-fold activity, and the ethoxyhydroxy

analogue 4j′ loses 8-9-fold activity. The propyloxydi-

methylamino compound 4o′ loses ∼3-fold activity, and

this together with the ethoxymorpholino result 4p ′

indicates that basic groups can be tolerated in this

region.

The results in Tables 2 and 3 show that the most

potent compounds tend to have small substituents with

a directional effect in a limited region, and although

larger groups are tolerated, the positioning of function-

ality on them and the directional effects are more

limited.

To investigate the effect of disubstitution in the benzo-

[a]fused ring, the 3,4-dimethoxy analogue 4q′ was

prepared. This was found to be ∼12-fold less potent than

the parent 4c (Table 4). Further disubstitution in the

benzo[a]fused ring was not examined. Previously Denny

had shown in the phenazine series that substitution in

the 6- and 7-positions (5,6-positions in this series) was

disfavored, so work concentrated on the substituent

effects at the 8-, 9-, and 10-positions.

A methyl substituent at the 8-position 4r ′ occupies a

position similar to that of the 8-methyl group in In-

toplicine, and it was found that this compound was only

∼2-fold less active than the parent. The introduction of

Cl 4s′ and OMe 4u ′ at the 9-position gave analogues

approximately equipotent with the parent 4c, showing

substitution at this position was tolerated. The 9-bromo

analogue 4t′ lost ∼4.5-fold activity, indicating there may

be some size constraint in this region.

H69/P

IC₅₀

H69/LX4

IC₅₀

substituent

R

compound

(nM)^a

Rf^b

1.1

142

3.9

1.4

0.8

2.8

>7.8

0.7

1.4

2.2

0.8

1.5

0.9

-

3.6

1.7

TAS-103

doxorubicin

21

27

22

3874

62

49

94

topotecan

4c

16

35

4-OMe

4j

4k

4l

4m

4n

4-SMe

121

1364

638

111

117

77

4-SO₂Me

4-SO₂NHMe

4-F

4-Cl

4-Br

3813

>5000

78

159

171

98

147

4o

4p

4q

4r

4s

4t

4u

4v

4w

4x

4-Me

4-CN

117

101

103

>5000

117

111

103

778

468

2133

2137

4-CO₂Me

4-CO₂H

4-CH₂OH

4-NH₂

4-NHCH₂CN

4-NMe₂

4-NHCOMe

4-NHSO₂Me

4-N(SO₂Me)₂

91

>5000

423

193

155

925

1.5

1.2

>5000

4987

>5000

>10.7

4y

4z

2.3

>2.3

a,b

See footnotes of Table 1.

Ta ble 3. Oxygen Linked 4-Substituted Benzo[a]phenazine

Carboxamides, 4a ′-4p ′

H69/P H69/LX4

substituent

R

IC₅₀

compound

(nM)^a

Rf^b

1.1

142

3.9

TAS-103

doxorubicin

topotecan

4f

21

27

16

22

3874

62

4-OH

35

48

49

1.4

3.7

2.1

1.6

1.5

0.9

1.5

2.8

1.6

1.4

0.8

0.9

3.1

3.7

>1.3

0.8

2.4

4c

4-OMe

35

4a ′

4b′

4c′

4d ′

4-OEt

49

182

874

882

61

4-OⁱPr

420

556

40

4-OBn

4-OCH₂CCH

4-OCH₂CN

4e′

28

24

58

67

4f′

4g′

4-OCH₂CH₂CH₂CN

4-OCOMe

39

24

4h ′

4i′

4j′

4k ′

4l′

4m ′

4n ′

4o′

4p ′

4-OCONHEt

4-OCH₂CH₂OMe

4-OCH₂CH₂OH

4-OCH₂CO₂Et

4-OCH₂COMe

4-OCH₂CONH₂

4-OCH₂CO₂H

4-OCH₂CH₂CH₂NMe₂

4-OCH₂CH₂morpholino

64

90

303

587

45

103

124

235

506

138

328

>5000

91

89

3844

116

99

236

a,b

See footnotes of Table 1.

cytotoxicity (>10-fold). The extent to which functionality

is tolerated is dependent on the size and direction of

the electronic effects of the functionality, with strongly

acidic groups being disfavored. The cyanomethylamino

compound 4v only lost ∼3-fold activity, indicating that

the electronic effects of small linear functionality is

allowed in this region. All of the compounds in Table 2

are less cytotoxic than the 4-methoxy analogue 4c. In

Methoxy substitution at the 10-position 4v′ resulted

in >75-fold loss of cytotoxicity, indicating the impact of

substitution at this position on the geometry of the side

chain at the 11-position. This effect is emphasized

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 725

Ta ble 4. Substituent Effects at the 3-, 8-, 9-, and 10-Positions

of the 4-Methoxy-benzo[a]phenazine Carboxamides 4q′-4z′

Ta ble 5. Varation of the Amide Side Chain at the 11-Position

of the 4-Methoxy-benzo[a]phenazine Carboxamides 4a ′′-4s′′

H69/P H69/LX4

substituent

R

IC₅₀

H69/P

IC₅₀

H69/LX4

IC₅₀

compound

(nM)^a

Rf^b

1.1

compound

substituent

(nM)^a

Rf^b

1.1

TAS-103

doxorubicin

topotecan

4c

4a ′′

4b′′

4c′′

4d ′′

4e′′

4f′′

4g′′

4h ′′

4i′′

4j′′

4k ′′

4l′′

4m ′′

4n ′′

4o′′

21

27

16

22

3874 142

62

49

TAS-103

doxorubicin

topotecan

4c

4q′

4r ′

4s′

4t′

4u ′

4v′

21

27

16

35

436

87

20

159

19

2640

19

103

34

22

3874

62

3.9

142

-(CH₂)₂NMe₂

-(CH₂)₃NMe₂

-CH₂CH(NMe₂)CH₂NMe₂

-(CH₂)₂NEt₂

-(CH₂)₂piperidino

35

1.4

1.3

3.8

2.4

2.1

>9.7

1.7

5.5

1.9

2.4

1.4

1.2

1.5

1.1

0.7

1.1

1.2

1.1

3.9

1.4

1.8

1.9

1.0

0.6

1.3

1.4

1.3

1.0

0.85

1.1

470

126

412

156

515

597

614

40

597

480

1001

330

>5000

1004

3378

77

4-OMe

3,4-di-OMe

49

804

168

21

100

25

3619

25

107

29

4-OMe, 8-Me

4-OMe, 9-Cl

4-OMe, 9-Br

4,9-di-OMe

4,10-di-OMe

4,10-di-OH

4-OMe, 10-OH

4-OMe, 10-NH₂

4-OMe, 10-NHMe

-CH₂CH(OH)CH₂NH₂

-(CH₂)₂morpholino

-(CH₂)₂N(CH₂CH₂OH)₂

-(CH₂)₂pyrrolidino

-(CH₂)₂NHMe

-CH(Me)CH₂NMe₂

-CH₂CH(Me)NMe₂

-C(Me)₂CH₂NMe₂

-CH(Et)CH₂NMe₂

-CH(ⁱPr)CH₂NMe₂

-CH(Bn)CH₂NMe₂

-CH(CH₂OH)CH₂NMe₂

-CH(CH₂CH₂OH)CH₂NMe₂

-CH(CO₂Me)CH₂NMe₂

-CH(CO₂H)CH₂NMe₂

4w ′

4x′

4y′

99

30

234

44

126

642

118

25

513

28

101

435

107

35

478

23

26

468

211

4z′

127

143

a,b

See footnotes of Table 1.

further in the 4,10-dihydroxy analogue 4w ′, which is

equipotent with the parent. In XR5000 the postulated

active conformation has an internal hydrogen bond

between the amide carbonyl and the protonated acridine

nitrogen. In the phenazine series, however, with a much

4p ′′

4q′′

4r ′′

4s′′

>5000 >10

>5000 >23

a,b

See footnotes of Table 1.

piperidino (4d ′′), morpholino (4f′′), and N(CH₂CH₂OH)₂

(4g′′) all resulted in reduced activity. An extended side

chain analogue (4b′′) with a dimethylamino group â to

the amide lost significant activity, and a primary amino

group at the terminus with an hydroxyl group â to the

amide (4e′′) also gave a dramatic loss of potency. The

pyrrolidino (4h ′′) and methylamino (4i′′) compounds

were equiactive and ∼3-fold less active than the parent

4c. These results show that there is a steric constraint

at the side chain terminus with small hydrophobic

groups being favored and hydrophilic substituents not

being well tolerated. The dimethylamino moiety at the

side chain terminus linked by a two-carbon unit to the

amide was optimal in this series. This important

observation led to the investigation of substituent effects

in the ethylamide side chain.

lower pK_a, a hydrogen bond exists between the amide

N-H and the phenazine nitrogen, the amide proton in

the NMR spectrum having a chemical shift of ∼10 ppm.

In the 10-methoxy substituted analogue, the chemical

shift of the amide proton in the NMR spectrum is ∼6

ppm, indicating that it is not hydrogen bonded in the

active conformation as evidenced from the dramatic loss

of potency.

This theory is reinforced by the introduction of

hydroxyl 4x′, amino 4y′, and methylamino 4z′ groups

at the 10-position. These changes would enable an

internal hydrogen bond to form with the side chain

carbonyl, and these analogues are within a 1-3-fold

activity range of the parent 4c.

Having studied the effects of substitution at the 10-

position on activity with the dimethylaminoethylamide

side chain, the SAR of side chain variation was explored

in more detail. This work was conducted using the

template from analogue 4c shown below, and the effects

of side chain variation are discussed in comparison to

the dimethylaminoethylamide side chain. The results

are summarized in Table 5.

Extending the chain length by one carbon unit (4a ′′)

resulted in ∼13-fold loss of activity, and this clearly

indicates the importance of chain length and the posi-

tioning of the basic side chain for activity. Replacement

of the dimethylamino moiety with diethylamino (4c′′),

Introduction of a methyl group R 4j′′ or R 4k ′′ to the

amide resulted in compounds that were equipotent and

∼3-fold less active, respectively. However, the gem-

dimethyl analogue 4l′′ lost significant activity (∼12-

fold), showing this amount of steric bulk was not

tolerated R to the amide group. The above observations

led to substitution R to the amide being extended to a

range of substituents with diverse stereoelectronic ef-

fects, with compounds being made in racemic form in

the first instance.

The steric tolerance R to the amide was examined by

replacement of the methyl by ethyl (4m ′′), isopropyl

(4n ′′), and benzyl (4o′′). The ethyl compound 4m ′′ was

∼3-fold less active, the isopropyl 4n ′′ was equiactive,

and the benzyl 4o′′ was ∼14-fold less active. It appears

there is reasonable tolerance in this region as the

isopropyl result 4n ′′ indicates, but larger groups such

as benzyl 4o′′ and the result from the gem dimethyl

726 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

Ta ble 6. Enantiospecific Side Chain Variation at the

11-Position of 4-Methoxy-benzo[a]phenazine Carboxamides

Ta ble 7. Pharmacokinetics in XR11576 in Female Balb/c Mice

Balb/c mice

admistration

20 mg/kg iv

0.8 ( 0.3

64 ( 6

6.3 ( 0.6

0.0044 (

0.0002

50 mg/kg iv

2.9 ( 0.8

156 ( 9

6.4 ( 0.4

0.0030 (

0.00034

50 mg/kg po

0.35 ( 0.29

114 ( 30

N/A

0.002 (

0.001

C

_max(µg/mL)

AUC (µg‚min/mL)

Cl (mL/min)

K

_el(min^-1

)

V

F (%)

_ss(L)

1.3 ( 0.4

1.9 ( 0.6

N/A

72 ( 25

hydroxymethyl 4p ′′ and the 2-hydroxyethyl analogues

4u ′′ were synthesized as the (S)-enantiomers, and as

predicted, the potent activity was retained.

Selected compounds were studied for their ability to

stabilize cleavable complexes in the presence of either

topoisomerase I or II as described previously.⁷The

presence of cleavable complexes was indicated by an

increase in the number and intensity of bands observed

on the gel after electrophoresis and autoradiography.

From these data it can be concluded that a representive

set of potent cytotoxic novel angular phenazines are dual

inhibitors of topoisomerase I and II.⁷As an example,

compound (R)-4j′′ inhibited both topoisomerase I and

II in a dose-dependent manner between 0.03 and 1 µM.⁷

The mechanism of inhibition of topoisomerase I and

II is unknown for these compounds. However, it has

recently been shown that an acridine-4-carboxamide

intercalates into duplex DNA with a specific hydrogen

bonded interaction to the N7 of guanine in the major

groove.¹³This study showed that a single hydrogen bond

is formed by the protonated N,N-dimethylamino group

of the N-(2-(dimethylamino)ethyl)-4-carboxamide side

chain to N7 of one of the guanine residues at the

intercalation site. The change in electrostatic potential

in the major groove resulting from the neutralization

of the partial negative charge on guanine by the

protonated N,N-dimethylamino group, together with the

steric blocking of the groove by the side chain, is likely

to be important in the inhibition of the topoisomerases.

As many of the phenazines described here have the

same or a similar 11-carboxamide side chain to the

analogue described as well as a flat tetracyclic moiety,

intercalation in the minor groove and additional binding

to the major groove in guanine rich regions is a

suggested mechanism for the inhibition of the topo-

isomerases by these compounds.

a,b

See footnotes of Table 1.

compound 4l′′ above indicate that if the side chain

adopts an unfavorable conformation, then potency is

reduced. The effect of introducing hydrophilic groups in

this region was examined, and both the hydroxymethyl

4p ′′ and hydroxyethyl 4q′′ moieties gave compounds at

least equipotent with the parent 4c. Introduction of

ester 4r ′′ and acid 4s′′ groups lead to significant loss of

activity and were inactive on the resistant cell line. The

results from these substitutions have led to the impor-

tant observation that substitution with both hydropho-

bic and hydrophilic moieties is tolerated, enabling the

physicochemical properties of the molecule to be varied.

From the SAR in this novel series of angular phen-

azines, the enantiomerically pure compound (R)-4j′′

(XR11576) was a dual inhibitor of topoisomerase I and

II and a potent cytotoxic agent in all the cell lines in

which it was tested. The results in the H69 and L23

parental and resistant cell lines compared to known

topoisomerase inhibitors are summarized in Table 1.

Due to XR11576 having a favorable profile in vitro to

other compounds in this series and known topo-

isomerase inhibitors it was chosen for pharmacokinetic

and xenograft studies in vivo. The plasma pharmaco-

kinetics following intravenous and oral administration

of XR11576 in female mice is summarized in Table 7.

These results indicate that XR11576 has an oral

bioavailability of ∼72% in female mice and demon-

strates linear pharmacokinetics after iv dosing. In

addition, XR11576 exhibits a relatively large volume of

distribution and low clearance.

Having examined the tolerances in the racemic series,

the enantiomerically pure compounds were synthesized

for some of the more potent analogues in the racemic

series. These analogues are listed in Table 6.

The (R)-enantiomer of the methyl analogue 4j′′ was

more potent than its racemate, and the (S)-enantiomer

which was only ∼80% optically pure was ∼4-fold less

active than the pure (R)-enantiomer. This indicated the

importance of the chirality at this center and the

directional positioning of the substituent on activity and

is to our knowledge the first report of side chain chirality

in DNA intercalating agents having an important

influence on biological activity. The (R)-enantiomer of

the isopropyl compound 4n ′′ was also more active than

its racemate and the cyclic (R)-N-methylpyrrolidine

compound 4t′′ 3-4-fold less active compared to 4j′′.

Knowing these chirality and directionality effects, the

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 727

available, they were prepared using standard literature meth-

ods. The following tetralones were prepared: 5-methyl-1-

tetralone;¹⁵5-fluoro-1-tetralone;¹⁵5,6-dimethoxy-1-tetralone;¹⁴

5-bromo-1-tetralone;¹⁶5-cyano-1-tetralone was prepared from

5-bromo-1-tetralone by treatment with copper(I) cyanide in

DMF under standard conditions;¹⁷5-chloro-1-tetralone was

prepared from 5-amino-1-tetralone¹⁸by diazotization under

standard conditions, followed by treatment with copper(I)

chloride;¹⁹5-ethoxy-1-tetralone was prepared from 5-hydroxy-

1-tetralone by alkylation with ethyl iodide under standard

conditions; 5-methylsulfanyl-1-tetralone was also prepared

from 5-hydroxy-1-tetralone as described in the literature;²⁰

5-methylsulfonyl-1-tetralone was prepared from 5-methylsul-

fanyl-1-tetralone using MCPBA under standard conditions;²¹

and 7-nitro-1-tetralone.²²These tetralones were oxidized to the

corresponding 1,2-naphthoquinones using the procedure de-

scribed above.

Gen er a l Meth od for th e Syn th esis of Su bstitu ted

Ben zo[a ]p h en a zin e-11-ca r boxylic Acid s. 4-Meth oxy-ben -

zo[a ]ph en azin e-11-car boxylic acid. A mixture of 5-methoxy-

[1,2]naphthoquinone¹⁴(1.98 g, 9.3 mmol), 2,3-diamino-benzoic

acid, diacetate salt,²³(4.03 g, 14.8 mmol), and concentrated

hydrochloric acid (2.2 mL) was heated to reflux in ethanol (20

mL) for 4 h. The reaction mixture was cooled and the

precipitate collected by filtration and washed with ethanol and

ether to yield the title compound as a beige solid (2.74 g, 97%).

¹H NMR (DMSO-d₆) δ 4.05 (s, 3H), 7.50 (d, 1H), 7.84-7.87

(m, 1H), 7.99 (d, 1H), 8.08-8.10 (m, 1H), 8.41-8.49 (m, 3H),

8.63 (d, 1H). MS (DCI/NH₃) m/e 305 (M + H)⁺.

Using a H69/P Xenograft, the effect of XR11576 on

tumor growth and body weight of female nude tumor

bearing mice was examined. The results of these studies

are shown in Figure 1.

Data in Figure 1 is expressed as mean ( SEM. The

effect of treatment on tumor volume is plotted as a

percentage of that seen on day 0 (To) which is the start

of treatment. The results indicate that XR11576 given

q7d×3 at 30 and 45 mg/kg iv evoked a dose related

tumor growth delay in this model.

Su m m a r y

The discovery and SAR of a novel series of substituted

benzo[a]phenazine carboxamides that are potent cyto-

toxic agents on a number of human cancer cell lines has

been described. These compounds have been designed

and shown to act as dual inhibitors of topoisomerase I

and II and thus target two key enzymes that affect the

topology of DNA at different points of the cell cycle. The

ability of these compounds to overcome potential mul-

tidrug resistance mechanisms has been demonstrated

by their cyctotoxicity on Pgp and MRP overexpressing

cell lines. One of the most potent compounds on all cell

lines examined is XR11576, and this compound has been

shown to be orally bioavailable and has demonstrated

tumor growth delay in female mice by both the oral and

iv routes. The novel substituted benzo[a]phenazine

carboxamide XR11576 has been selected as a candidate

for further evaluation.

The following substituted benzo[a]phenazine-11-carboxylic

acids were prepared by the general method above.

4-Meth yl-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (32%).

¹H NMR (DMSO-d₆) δ 2.79 (s, 3H), 7.85-7.78 (m, 2H), 8.11-

8.05(m, 2H), 8.47-8.44 (m, 2H), 8.51 (dd, 1H), 9.02 (d, 1H).

4-F lu or o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (31%).

¹H NMR (DMSO-d₆) δ 7.80 (m, 1H), 7.95 (m, 1H), 8.11-8.06

(m, 2H), 8.38 (d, 1H), 8.43 (dd, 1H), 8.51 (dd, 1H), 8.97 (d, 1H).

3,4-Dim eth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid

(74%). ¹H NMR (CDCl₃) δ 3.98 (s, 3H), 4.04 (s, 3H), 7.76 (d,

1H), 7.95 (d, 1H), 8.05 (t, 1H), 8.37 (d, 1H), 8.48 (d, 2H), 8.79

(d, 1H).

Exp er im en ta l Section

Meth od s a n d Ma ter ia ls. Reagents, starting materials, and

solvents were purchased from common commercial suppliers

and used as received or distilled from the appropriate drying

agent. Reactions requiring anhydrous conditions were per-

formed under an atmosphere of nitrogen or argon. Precoated

4-Br om o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (54%).

¹H NMR (DMSO-d₆) δ 7.87 (t, 1H), 8.08 (t, 1H), 8.20 (d, 1H),

8.26 (m, 1H), 8.33 (m, 1H), 8.48 (m, 2H), 9.24 (d, 1H).

4-Cya n o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (63%).

¹H NMR (DMSO-d₆) δ 8.10-8.18 (m, 2H), 8.33 (d, 1H), 8.43

(m, 2H), 8.48-8.54 (m, 2H), 9.48 (d, 1H). MS (DCI/NH₃) m/e

300 (M + H)⁺.

4-Ch lor o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (36%).

¹H NMR (DMSO-d₆) δ 7.93 (t, 1H), 8.08 (dd, 1H), 8.11 (d, 1H),

8.19 (d, 1H), 8.43, (dd, 1H), 8.52, (t, 2H), 9.17, (d, 1H).

4-Meth a n esu lfon yl-ben zo[a ]p h en a zin e-11-ca r boxylic

Acid (30%). ¹H NMR (DMSO-d₆) δ 8.16 (m, 2H), 8.32 (m, 1H),

8.43 (d, 1H), 8.56 (m, 2H), 9.03 (d, 1H), 9.61 (d, 1H). MS (DCI/

NH₃) m/e 353 (M + H)⁺.

Ben zo[a ]p h en a zin e-4,11-d ica r boxylic Acid 4-m eth yl

Ester (28%). ¹H NMR (DMSO-d₆) δ 4.03 (s, 3H), 8.10 (m, 2H),

8.20 (d, 1H), 8.44 (m, 2H), 8.53 (d, 1H), 9.47 (d, 1H).

4-Eth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (76%).

¹H NMR (DMSO-d₆) δ 1.52 (q, 3H), 4.33 (t, 2H), 7.50 (d, 1H),

7.85-7.90 (m, 1H), 8.00 (d, 1H), 8.08-8.11 (m, 1H), 8.45-8.55

(m, 3H), 8.68 (d, 1H).

2-Nitr o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid was pre-

pared from 7-nitro[1,2]naphthoquinone and 2,3-diamino-ben-

zoic acid, diacetate salt using the previously described meth-

ods²³for the synthesis of benzo[a]phenazine-11-carboxylic

acids. The 7-nitro[1,2]naphthoquinone was prepared from

7-nitrotetralone.²²The acid was used without further purifica-

tion.

aluminum backed silica gel 60 F₂₅₄plates with

a layer

thickness of 0.25 mm were used for thin-layer chromatography,

and the stationary phase for preparative column chromatog-

raphy using medium pressure was silica gel 60, mesh size 40-

60 µm from E. Merck, Darmstadt, Germany.

NMR spectra were obtained using a Bruker ACF 400

operating at 400 MHz, and the (¹H) ppm were calibrated to

residual CHCl₃in CDCl₃at 7.26 ppm. Mass spectra were

obtained in the indicated mode using a Finnigan SSQ 710L

machine. Melting points were determined using an electro-

thermal 9100 series apparatus.

The compound purity on all compounds tested in biological

systems was assessed as being >95% using HPLC using a

water/acetonitrile gradient at 30 °C on a Waters symmetry

C₁₈150 mm × 4.6 mm column and by NMR spectroscopy.

Microanalyses were performed on a representation of com-

pounds by MEDAC Ltd. U.K. Where analyses are indicated

only by the symbols of the elements, results obtained were

within 0.4% of the theoretical values.

Ch em istr y. Gen er a l Meth od for th e P r ep a r a tion of 1,2-

Na p h th oqu in on es. 1,2-Naphthoquinones were prepared from

the corresponding substituted R-tetralones by oxidation with

selenium dioxide in acetic acid using the method of Bekaert.¹⁴

In a typical example, commercially available 5-methoxy-1-

tetralone (15.35 g, 87.1 mmol) and ground selenium dioxide

(24.2 g, 217.75 mmol) in glacial acetic acid (160 mL) were

warmed at 65 °C for 7 h. The mixture was then cooled to room

temperature and the acetic acid removed in vacuo. The residue

was treated with ethyl acetate (100 mL) and filtered through

Celite and a plug of silica eluting with ethyl acetate, the

organics were concentrated, and the product precipitated on

standing at 4 °C overnight to yield the product as a red solid

(11.1 g, 68%). If the required tetralones were not commercially

4-Hyd r oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid . To

a solution of 4-methoxy-benzo[a]phenazine-11-carboxylic acid

(441 mg, 1.45 mmol) in dichloromethane (30 mL) cooled to 0

°C was added a 1.0 M solution of boron tribromide in

dichloromethane (7.25 mL, 7.25 mmol). The reaction mixture

728 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

was allowed to warm to room temperature and then stirred

for 16 h. The mixture was then poured onto ice/water (20 mL)

yielding 4-hydroxy-benzo[a]phenazine-11-carboxylic acid as a

red/brown solid which was collected by filtration and air-dried

(230 mg, 55%). ¹H NMR (DMSO-d₆) δ 7.35 (d, 1H), 7.75 (m,

1H), 7.92 (d, 1H), 8.08 (m, 1H), 8.47-8.55 (m, 4H), 10.72 (br,

s, 1H).

1H). This mixture was used directly in the next step without

purification without further purifcation. The mixture of 4-chlo-

rosulfonyl-benzo[a]phenazine-11-carboxylic acid and 3-chloro-

sulfonyl-benzo[a]phenazine-11-carboxylic acid (182 mg, 0.48

mmol) was dissolved in dichloromethane (5 mL). To this was

added a 40% solution of methylamine in water (5 mL), and

the reaction mixture was stirred vigorously for 4 h. The

reaction mixture was then poured onto dichloromethane,

acidified (2 N HCl), extracted into dichloromethane, and dried

(MgSO₄) and the solvent removed in vacuo to give a ∼1:1

mixture of the title compounds (160 mg, 0.44 mmol, 91%). The

two isomers were separated after further chemical modifica-

4-Ben zyloxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid . A

mixture of 4-hydroxy-benzo[a]phenazine-11-carboxylic acid (80

mg, 0.28 mmol), sodium hydroxide (34 mg, 0.85 mmol), and

benzyl bromide (100 µL) in ethanol (2 mL) was heated to reflux

for 4 h. The reaction mixture was then cooled, diluted with

ethyl acetate (15 mL), washed with 1M HCl, and dried

(MgSO₄) ,and the solvent was removed in vacuo to yield the

1

tion. H NMR (CDCl₃/MeOH-d₄) includes δ 2.55 (s, 3H), 2.61

(s, 3H), 9.22 (d, 1H), 9.38 (s, 1H).

1

4-Dim eth ylsu lfa m oyl-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid a n d 3-d im eth ylsu lfa m oyl-ben zo[a ]p h en a zin e-11-

ca r boxylic a cid (168 mg, 91%) were prepared in an analogous

manner by reaction of dimethylamine with the mixture of

4-chlorosulfonyl-benzo[a]phenazine-11-carboxylic acid and

3-chlorosulfonyl-benzo[a]phenazine-11-carboxylic acid (180 mg,

0.47 mmol). The two isomers were separated after further

chemical modification. NMR (DMSO-d₆) of mixture includes

δ 9.06 (d, 1H), 9.42 (s, 1H), 2.93 (s, 6H).

4-Nitr o-ben zo[a ]ph en azin e-11-car boxylic acid an d 3-n i-

tr o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid . Concentrated

sulfuric acid (5 mL) and concentrated nitric acid (5 mL) were

mixed together at 0 °C. To this mixture was added benzo[a]-

phenazine-11-carboxylic acid (100 mg, 0.36 mmol), and the

reaction mixture was allowed to warm slowly to room tem-

perature. After 24 h the reaction mixture was poured onto

water yielding a yellow precipitate, this was collected by

filtration to yield a 4:1 mixture of 4-nitro-benzo[a]phenazine-

11-carboxylic acid and 3-nitro-benzo[a]phenazine-11-carboxylic

acid (84%). The two isomers were separated after further

chemical modification. ¹H NMR (DMSO-d₆) includes δ 9.82 (d,

1H), 9.46 (d, 1H), 1:4 ratio. MS (DCI/NH₃) m/e 320 (M + H)⁺.

9-Br om o-4-m et h oxy-b en zo[a ]p h en a zin e-11-ca r b oxyl-

ic Acid . To a solution of 4-methoxy-benzo[a]phenazine-11-

carboxylic acid (100 mg, 0.33 mmol) in chloroform (7 mL) was

added dropwise bromine (5 mL). Stirring was continued for

48 h. The chloroform and bromine were removed in vacuo, and

the residue was purified by flash chromatography to give a

yellow gum (25 mg, 20%). ¹H NMR (CDCl₃) δ 3.97 (s, 3H), 5.76

(d, 1H), 6.11 (d, 1H), 6.99 (d, 1H), 7.79 (d, 1H), 7.95 (t, 1H),

8.33 (dd, 1H), 8.85 (dd, 1H), 14.46 (br. s, 1H).

crude title compound as a brown solid (50 mg, 47%). H NMR

(DMSO-d₆) includes δ 5.35 (s, 2H). This compound was used

directly in the next step without further purification.

The following compounds were prepared in an analogous

manner from 4-hydroxy-benzo[a]phenazine-11-carboxylic acid

using the appropriate alkylating reagent.

4-P r op-2-yn yloxy-ben zo[a ]ph en azin e-11-car boxylic acid

was prepared using propargyl bromide (64%).

4-Isobu toxy-ben zo[a ]ph en azin e-11-car boxylic acid was

prepared using isobutyl bromide (13%). The above materials

were used without further purification.

4-(2-Meth oxy-eth oxy)-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid was prepared using 2-bromoethyl methyl ether (40%).

¹H NMR (DMSO-d₆) δ 3.40 (s, 3H), 3.85 (t, 2H), 4.40 (t, 2H),

7.51 (d, 1H), 7.87 (t, 1H), 8.01 (d, 1H), 8.10 (t, 1H), 8.50 (m,

3H), 8.67 (d, 1H).

4-Eth oxyca r bon ylm eth oxy-ben zo[a ]p h en a zin e-11-ca r -

boxylic a cid was prepared using ethyl bromoacetate (78%).

Sodium ethoxide in dry ethanol was used for this reaction. ¹H

NMR (CDCl₃) δ 1.28 (t, 3H), 4.25 (q, 2H), 4.81 (s, 2H), 7.07 (d,

1H), 7.64 (t, 1H), 7.84 (dd, 1H), 7.90 (d, 1H), 8.38 (m, 2H), 8.59

(d, 1H), 9.04 (d, 1H).

4-[2-(ter t-Bu tyl-d im eth yl-sila n yloxy)-eth oxy]-ben zo[a ]-

p h en a zin e-11-ca r boxylic a cid was prepared using tert-

butyl-(2-iodo-ethoxy)-dimethylsilane (6%). ¹H NMR (CDCl₃) δ

0.00 (s, 6H), 0.80 (s, 9H), 4.00 (t, 2H), 4.19 (t, 2H), 7.21 (d,

1H), 7.65 (t, 1H), 7.83 (d, 1H), 7.90 (t, 1H), 8.41 (dd, 1H), 8.52

(d, 2H), 8.82 (dd, 1H), 15.75 (br. s, 1H). The tert-Butyl-(2-iodo-

ethoxy)-dimethyl-silane was prepared using standard proce-

dures from 2-iodo-ethanol (99%). ¹H NMR (CDCl₃) δ 0.00 (s,

6H), 0.82 (s, 9H), 3.12 (t, 2H), 3.74 (t, 2H).

4-Am in o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid a n d

3-Am in o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid . To the

4:1 mixture of 4-nitro-benzo[a]phenazine-11-carboxylic acid

and 3-nitro-benzo[a]phenazine-11-carboxylic acid (52 mg, 0.16

mmol) in ethanol (5 mL) was added ammonium chloride

solution (3 mL) and indium (cat.). The reaction mixture was

heated to reflux, then cooled, and filtered through a bed of

Celite. The filtrate was diluted with water, extracted into

dichloromethane, and dried (MgSO₄) and the solvent removed

in vacuo to yield the title compounds as a mixture (48 mg,

Ben zo[a ]p h en a zin e-11-ca r boxylic Acid Meth yl Ester .

A mixture of 1,2-naphthoquinone (2.0 g, 12.6 mmol) and 2,3-

diamino-benzoic acid, diacetate salt (3.79 g, 13.9 mmol) was

heated to reflux in acetic acid (30 mL) for 2 h. The reaction

mixture was cooled and the solvent removed in vacuo to yield

a gum. This was purified using flash chromatography to yield

a 2:1 mixture of the desired title compound to the undesired

benzo[a]phenazine-8-carboxylic acid (885 mg). The mixture of

benzo[a]phenazine-11-carboxylic acid and benzo[a]phenazine-

8-carboxylic acid (885 mg) was heated to reflux in a mixture

of methanol (40 mL) and acetyl chloride (920 µL) for 90 min.

The reaction mixture was then cooled slowly to yield the title

compound as a single isomer which was collected by filtration

1

0.15 mmol, 94%). H NMR (DMSO-d₆) δ 6.95 (d, 1H), 7.55 (t,

1H), 7.75 (m, 2H), 8.05 (m, 2H), 8.20 (d, 1H), 8.65 (d, 1H). The

mixture was used directly in the next step and the constituents

separated after further modification.

1

(377 mg, 11%). H NMR (DMSO-d₆) δ 4.21 (s, 3H), 7.95-8.01

2-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid . To

a solution of 8-amino-naphthalen-2-ol (8.00 g, 50 mmol) in dry

DMF (80 mL) was carefully added sodium hydride (60%

dispersion in mineral oil, 3.2 g, 222 mmol). After being stirred

for 4 h, the reaction mixture was cooled in an ice bath and

methyl iodide (3.13 mL) was added dropwise. The reaction

mixture was then stirred at room temperature for 3 days.

Water (10 mL) was then added, and the volatiles were removed

in vacuo. The residue was dissolved in chloroform, washed with

water, and dried (MgSO₄) and the solvent removed in vacuo

to yield a dark oil which was purified using flash chromatog-

raphy eluting with chloroform to yield 7-methoxy-naphthalen-

1-ylamine as a dark brown liquid (2.92 g, 16.9 mmol).

Treatment of 7-methoxy-naphthalen-1-ylamine (1.19 g, 6.88

mmol) with 2-bromo-3-nitro-benzoic acid (1.8 g, 6.88 mmol)

(m, 2H), 8.02-8.09 (m, 1H), 8.10-8.11 (m, 1H), 8.35 (d, 1H),

8.47(d, 1H), 8.57 (d, 1H), 9.06 (d, 1H), 9.53-9.55 (m, 1H). This

compound was identical spectroscopically to the title compound

prepared by a different route.¹¹

4-Meth ylsu lfa m oyl-ben zo[a ]p h en a zin e-11-ca r boxylic

Acid a n d 3-Meth ylsu lfa m oyl-ben zo[a ]p h en a zin e-11-ca r -

boxylic Acid . Benzo[a]phenazine-11-carboxylic acid methyl

ester (220 mg, 0.72) was heated under nitrogen to 180 °C in

chlorosulfonic acid (2 mL) for 6 h. The reaction was then cooled

and poured onto ice/water and the pale yellow solid collected

by filtration to give approximately a 1:1 mixture of 4-chloro-

sulfonyl-benzo[a]phenazine-11-carboxylic acid and 3-chloro-

sulfonyl-benzo[a]phenazine-11-carboxylic acid (182 mg, 0.48

mmol). ¹H NMR (DMSO-d₆) includes δ 9.30 (s, 1H), 9.41 (d,

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 729

according published methods,¹¹to yield 2-(7-methoxy-naph-

thalen-1-ylamino)-3-nitro-benzoic acid (0.47 6, 21%), and

reductive cyclization of 7-methoxy-naphthalen-1-ylamine using

sodium borohydride¹¹yielded the desired title compound (43%).

¹H NMR (DMSO-d₆) δ 4.02 (s, 3H), 7.56 (dd, 1H), 7.86 (d, 1H),

8.10-8.04 (m, 2H), 8.23 (d, 1H), 8.42 (dd, 1H), 8.48 (dd, 1H),

8.55 (d, 1H), 14.44 (s, 1H). MS (DCI/NH₃) m/e 305 (M + H)⁺.

3-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid was

prepared in an analogous manner starting from 5-amino-2

naphthol to give the title compound. ¹H NMR (DMSO-d₆) δ

3.99 (s, 3H), 7.55 (dd, 1H), 7.67 (d, 1H), 7.96-8.10 (m, 2H),

8.23 (d, 1H), 8.51 (d, 2H), 8.94 (d, 1H), 14.60 (br. s, 1H). MS

(DCI/NH₃) m/e 305 (M + H)⁺.

The amines used to couple to a derivative of the substituted

benzo[a]phenazine-11-carboxylic acids above were either com-

mercially available or synthesized by known methods as

outlined below.

4-Aza-^DL-leu cin e Meth yl Ester Dih ydr och lor ide. Metha-

nol (150 mL) was saturated with anhydrous hydrogen chloride

gas, and to this solution was added 4-aza-^DL-leucine (4.86 g,

23.7 mmol). The reaction mixture was stirred overnight at

room temperature. The solvent was removed in vacuo to yield

1

the title compound (5.2 g, 100%). H NMR (d₃-MeOD) δ 3.16

(s, 6H), 3.71 (m, 1H), 3.96 (td, 1H), 4.04 (s, 3H), 4.78 (t, 1H).

N¹,N¹-Dim eth yl-bu ta n e-1,2-d ia m in e Dih yd r och lor id e.

Methyl N-(tert-butoxycarbonyl)-2-aminobutyrate was prepared

as described in the literature.²⁶Dropwise treatment over 45

min of methyl N-(tert-butoxycarbonyl)-2-aminobutyrate (4.34,

20 mmol) in toluene (50 mL) with 1 M diisobutyl aluminum

hydride in toluene (50 mL, 50 mmol) at -78 °C followed by

stirring for a further 45 min yielded N-(tert-butoxycarbonyl)-

4,10-Dim eth oxy-ben zo[a ]ph en azin e-11-car boxylic Acid.

2-Amino-6-methoxy-3-nitro-benzoic acid methyl ester was

prepared using a procedure analogous to that described in the

literature.²⁴Hydrolysis of 2-amino-6-methoxy-3-nitro-benzoic

acid methyl ester (1.00 g, 4.42 mmol) was achieved using

potassium hydroxide in refluxing ethanol for 2 h to yield

2-amino-6-methoxy-3-nitro-benzoic acid (773 mg, 82%). Hy-

drogenation of the 2-amino-6-methoxy-3-nitro-benzoic acid (1.0

g, 4.72 mmol) group was performed in acetic acid/water over

10% palladium on carbon catalyst on a Parr apparatus at 50

psi using hydrogen to yield 2,3-diamino-6-methoxy-benzoic acid

(730 mg, 52%). 2,3-Diamino-6-methoxy-benzoic acid (729 mg,

2.41 mmol) was reacted with 5-methoxy-[1,2]naphthoquinone

as described in the general method to yield the desired title

2-aminobutanal (2.58 g, 76%) as previously described.²⁷

A

mixture of N-(tert-butoxycarbonyl)-2-aminobutanal (1.85 g,

13.1 mmol), dimethylamine hydrochloride (1.61 g, 19.7 mmol),

sodium acetate (1.21 g, 14.8 mmol), and sodium cyanoboro-

hydride (0.83 g, 13.2 mmol) in methanol was stirred at room

temperature for 24 h. The pH was adjusted to 6-7 using acetic

acid and monitored during the reaction. The reaction mixture

was then concentrated in vacuo, and the residue was dissolved

in ethyl acetate and washed with water. The organic layer was

dried (MgSO₄) and the solvent removed in vacuo to yield a

colorless oil, which was purified on silica eluting with ethyl

acetate 70% and hexane 30% to yield the desired dimethyl-

amine derivative as a pale oil (0.56 g, 3.3 mmol). To this

compound (260 mg, 1.53 mmol) was added a 4.0 M solution of

HCl in dioxane (2 mL) carefully, and the reaction mixture was

stirred for 90 min. The reaction mixture was then concentrated

in vacuo to yield the desired title compound as an off-white

1

compound (628 mg,78%). H NMR (DMSO-d₆) δ 4.05 (s, 3H),

4.10 (s, 3H), 7.45 (d, 1H), 7.78-7.82 (m, 1H), 7.92 (d, 1H), 8.03

(d, 1H), 8.36-8.40 (m, 2H), 8.78 (d, 1H).

4-Meth oxy-8-m eth yl-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic Acid . 2,3-Diamino-4-methylbenzoic acid was prepared from

4-methyl anthranilic acid according to the literature.¹¹This

was reacted with 5-methoxy[1,2]naphthoquinone as described

in the general method¹⁴to yield the desired title compound.

¹H NMR (DMSO-d₆) δ 2.97 (s, 3H), 4.06 (s, 3H), 7.50 (d, 1H),

7.85-7.90 (m, 1H), 7.97 (d, 1H), 8.02 (d, 1H), 8.45-8.50 (m,

2H), 8.57 (d, 1H).

1

solid in quantitative yield. H NMR (d₃-MeOD) δ 1.20 (t, 3H),

1.90 (m, 2H), 3.09 (s, 6H), 3.53 (m, 2H), 3.82 (m, 1H).

3N¹,N¹-Tr im et h yl-b u t a n e-1,2-d ia m in e Dih yd r och lo-

r id e. 2-Methyl-3-[N-(tert-butoxycarbonyl)amino]butanal was

prepared from ^DL-valine methyl ester hydrochloride as previ-

ously described.²⁶A mixture of 2-methyl-3-[N-(tert-butoxycar-

bonyl)amino]butanal (3.76 g, 18.7 mmol), dimethylamine

hydrochloride (3.04 g, 37.3 mmol), sodium acetate (2.45 g, 29.9

mmol), and sodium cyanoborohydride (1.76 g, 28.0 mmol) in

methanol (80 mL) was stirred at room temperature for 18 h.

The solvent was removed in vacuo, the residue was dissolved

in ethyl acetate (300 mL), washed with water (300 mL), and

dried (MgSO₄), the solvent was removed in vacuo, and the

product was purified on silica eluting with 90% dichlorometane

and 10% methanol to yield N¹,N¹-Dimethyl-2-(N-tert-butoxy-

carbonyl)-3-methyl-butane-1,2-diamine as a white solid (2.2

g, 51%). To N¹,N¹-Dimethyl-2-(N-tert-butoxycarbonyl)-3-meth-

yl-butane-1,2-diamine (2.2 g, 9.5 mmol) was added a 4.0 M

solution of hydrochloric acid in dioxane (10 mL) at room

temperature. After the mixture was stirred for 30 min, the

volatile components were removed in vacuo to yield the desired

title compound as a white solid (1.8 g, 93%). MH⁺131. ¹H NMR

(d₃-MeOD) δ 1.20 (d, 6H), 2.20 (m, 1H), 3.10 (s, 6H), 3.50 (dd,

1H), 3.60 (dd, 1H), 3.75 (m, 1H).

9-Ch lor o-4-m eth oxy-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic Acid . 5-Chloro-3-nitroanthranilic acid was prepared ac-

cording to the procedure described by Flippin.²⁴Hydrogenation

of this material in ethyl acetate using 10% palladium on carbon

at 50 psi using hydrogen for 2 h yielded 2,3-diamino-5-chloro-

benzoic acid. This was reacted with 5-methoxy[1,2]naphtho-

quinone as described in the general method to yield the desired

title compound. ¹H NMR (DMSO-d₆) δ 4.05 (s, 3H), 7.48 (d,

1H), 7.82-7.86 (m, 1H), 7.92 (d, 1H), 8.30 (d, 1H), 8.48 (d, 1H),

8.54 (d, 1H), 8.68 (d, 1H), 14.1 (br. s, 1H).

4-Meth oxy-10-m eth ylam in o-ben zo[a ]ph en azin e-11-car -

boxylic Acid Meth yl Ester . 2,3-Diamino-6-fluoro-benzoic

acid methyl ester (1.0 g, 5.43 mmol) was coupled with

5-methoxy[1,2]naphthoquinone (0.73 g, 3.88 mmol) as previ-

ously described in the general methods to give 10-fluoro-4-

methoxybenzo[a]phenazine-11-carboxylic acid methyl ester

(530 mg, 77%). The 10-fluoro-4-methoxybenzo[a]phenazine-11-

carboxylic acid methyl ester (0.1 g, 0.29 mmol) was dissolved

in a 2 M solution of methylamine in tetrahydrofuran (5 mL).

The reaction was stirred for 15 h at room temperature. The

solvent and methylamine were removed in vacuo to give the

1

crude product obtained (0.10 g, 97%). H NMR (CDCl₃) δ 4.05

(R)-3N¹,N¹-Tr im eth yl-bu ta n e-1, 2-d ia m in e d iyd r och lo-

r id e sa lt was prepared as described above by using D-valine

(s, 3H), 4.18 (s, 3H), 7.18 (d, 1H), 7.50 (d, 1H), 7.70 (t, 1H),

7.87 (d, 1H), 8.18 (d, 1H), 8.36 (br. s, 1H), 8.40 (d, 1H), 8.95

(d, 1H).

1

as the starting material. H NMR (d₃-MeOD) δ 1.20 (d, 6H),

2.20 (m, 1H), 3.10 (s, 6H), 3.50 (dd, 1H), 3.60 (dd, 1H), 3.75

(m, 1H).

10-Am in o-4-m eth oxy-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic Acid Meth yl Ester . 10-Fluoro-4-methoxybenzo[a]phen-

azine-11-carboxylic acid methyl ester (0.47 g, 1.40 mmol) was

dissolved in DMF (10 mL), sodium azide (0.90 g, 13.9 mmol)

was added, and the reaction was heated at 90 °C for 15 h.

Water (50 mL) and 2 M sodium hydroxide (25 mL) were then

added, and the resulting brown precipitate was filtered and

washed with water and diethyl ether to give the title product

(0.24 g, 51%). ¹H NMR (CDCl₃) δ 4.20 (s, 3H), 7.24 (d, 1H),

7.70 (t, 1H), 7.72 (d, 1H), 7.90 (d, 1H), 8.46 (d, 1H), 8.51 (d,

1H), 8.85 (d, 1H).

N¹,N¹-Dim eth yl-3-p h en yl-p r op a n e-1,2-d ia m in e Dih y-

d r och lor id e. 1-Phenyl-2-[N-(tert-butoxycarbonyl)amino]pro-

panal was prepared from ^DL-phenylalanine methyl ester

hydrochloride as previously described.²⁶A mixture of 1-phenyl-

2-[N-(tert-butoxycarbonyl)amino]propanal (1.39 g, 5.58 mmol),

dimethylamine hydrochloride (0.91 g, 11.2 mmol), sodium

acetate (731 mg, 8.9 mmol), and sodium cyanoborohydride (526

mg, 8.36 mmol) in methanol (30 mL) was stirred at room

temperature for 18 h. The solvent was removed in vacuo, and

730 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

the residue was dissolved in ethyl acetate (200 mL), washed

with water (200 mL), and dried (MgSO₄), the solvent was

removed in vacuo, and the product was purified on silica

eluting with 90% dichlorometane and 10% methanol to yield

N¹,N¹-dimethyl-2-(N-tert-butoxycarbonyl-3-phenyl-propane-

1,2-diamine as a white solid (912 mg, 59%). To N¹,N¹-dimethyl-

2-(N-tert-butoxycarbonyl-3-phenyl-propane-1,2-diamine (912

mg, 3.2 mmol) was added a 4.0 M solution of hydrochloric acid

in dioxane (10 mL) at room temperature. After the mixture

was stirred for 30 min, the volatile components were removed

in vacuo to yield the desired title compound as a white solid

(826 mg, 100%). ¹H NMR (d₃-MeOD) δ 3.00 (br s, 1H), 3.15

(m, 2H), 3.50 (dd, 1H), 4.15 (m, 1H), 7.40-7.50 (m, 5H).

the mixture stirred for 20 min. Concentration of the mixture

in vacuo and purification of the product on silica eluting with

90% dichlorometane and 10% methanol yielded the desired

title compound as a white solid (1.94 g, 27%). ¹H NMR (d₃-

MeOD) δ 3.10 (s, 6H), 3.54 (m, 1H), 3.66 (dd, 1H), 3.93

(m, 3H).

3(S)-Am in o-4-(d im eth yla m in o)-bu ta n -2(S)-ol Dih yd r o-

ch lor id e. Hydrogen chloride gas was bubbled through a

solution of ^D-threonine (20 g, 168 mmol) in methanol (50 mL).

The resulting solution was stirred for 5 h at room temperature

and then reduced in vacuo to yield ^D-threonine methyl ester

hydrochloride as a white solid in quantitative yield. Treatment

of ^D-threonine methyl ester (28.45 g, 168 mmol, with di-tert-

butyl dicarbonate (36.64, 168 mmol) in acetonitrile (275 mL)

and triethylamine (16.9 g, 168 mmol) yielded N-(tert-butoxy-

carbonyl)-^D-threonine methyl ester (32.7 g, 140 mmol). Treat-

ment of N-(tert-butoxycarbonyl)-^D-threonine methyl ester (32.7

g, 140 mmol) with tert-butyldimethylsilyl chloride (26.2 g, 174

mmol) in dichloromethane (170 mL) with imidazole (19,1 g,

281 mmol) yielded the corresponding TBDMS protected alcohol

(35.5 g, 102 mmol). Treatment of this compound (15 g, 43

mmol) in toluene (130 mL) with 1 M diisobutyl aluminum

hydride in toluene (52 mL, 52 mmol) at -78 °C for 4 h yielded

the corresponding aldehyde (12.1 g, 38 mmol).²⁷Reductive

amination was carried out as described previously to give the

dimethylamino derivative which was treated with 4.0 M HCl

in dioxane as described previously to yield a golden oil.

Trituration with ether gave the title compound as a white solid

(3.98 g, 30%). ¹H NMR (d₃-MeOD) δ 1.42 (d, 3H), 3.10 (s, 6H),

3.53 (dd, 1H), 3.68 (dd, 1H), 3.80 (m, 1H), 4.17 (dd, 1H).

(S)-N¹,N¹-Dim eth yl-p r op a n e-1,2-d ia m in e Diyd r och lo-

r id e. 2-(S)-[N-(tert-Butoxycarbonyl)amino]propanal was pre-

pared from ^L-alanine methyl ester hydrochloride according to

the literature procedure.²⁶A mixture of the 2-(S)-[N-(tert-

butoxycarbonyl)amino]propanal (1.58 g, 9.1 mmol), dimethyl-

amine hydrochloride (1.49 g, 18.3 mmol), sodium acetate (1.20

g, 14.6 mmol), and sodium cyanoborohydride (0.86 g, 13.7

mmol) in methanol (10 mL) was stirred at room temperature

for 18 h. The reaction mixture was dissolved in ethyl acetate

(40 mL), washed with water (30 mL), and dried (MgSO₄), and

the solvent was removed in vacuo to yield a viscous oil. This

was dissolved in dichloromethane (30 mL), extracted with citric

acid (10 mL), basified with 1 M sodium hydroxide, and re-

extracted with ethyl acetate (2 × 20 mL). The organic layer

was reduced in vacuo, and the product was purified on silica

eluting with 90% dichlorometane and 10% methanol to yield

(S)-N¹,N¹-dimethyl-propane-1,2-diamine as a white solid (537

mg 15%). To (S)-N¹,N¹-dimethyl-propane-1,2-diamine (502 mg,

2.5 mmol) was added a 4.0 M solution of hydrochloric acid in

dioxane (5.5 mL) at room temperature. After the mixture was

stirred for 30 min, the volatile components were removed in

vacuo to yield the desired title compound as a viscous oil (450

3-Am in o-4-(d im eth yla m in o)-bu ta n -1-ol Dih yd r och lo-

r id e. ^DL-Homoserine (6.0 g, 50.4 mmol) was treated with

methanol saturated with HCl gas (100 mL) and stirred at room

temperature for 18 h. The solvent was removed in vacuo to

yield a white solid which was treated with with di-tert-butyl

dicarbonate (11.5 g, 52.9 mmol) in acetonitrile (90 mL) and

triethylamine (7.7 mL, 55.4 mmol) to yield the N-tert-butoxy-

carbonyl protected derivative (4.52 g, 22.5 mmol). Treatment

of this compound (2.55 g, 12.7 mmol) in toluene (40 mL) with

1 M diisobutyl aluminum hydride in toluene (22.8 mL, 22.8

mmol) at -78 °C for 4 h yielded the corresponding lactol (2.0

g, 9.8 mmol).²⁷Reductive amination on the lactol (502 mg, 2.47

mmol) was carried out as described previously to yield 3-[N-

(tert-butoxycarbonyl)amino]-4-(dimethylamino)-butan-1-ol (140

mg, 0.6 mmol) which was treated with 4.0 M HCl in dioxane

as described above to yield the desired title compound as a

white solid (138 mg, 100%). ¹H NMR (d₃-MeOD) δ 2.10 (m,

2H), 3.10 (s, 6H), 3.59 (ddd, 2H), 3.92 (m, 2H), 4.05 (m, 1H).

1

mg, 100%). H NMR (d₃-MeOD) δ 1.20 (d, 3H), 3.27 (m, 1H),

3.44 (m, 1H), 3.57 (s, 6H), 3.64 (dd, 1H).

(R)-N¹,N¹-Dim eth yl-p r op a n e-1,2-d ia m in e Dih yd r och lo-

r id e. 2-(R)-[N-(tert-Butoxycarbonyl)amino]propanal was pre-

pared from ^D-alanine methyl ester hydrochloride according to

the procedure described in the literature.²⁶

A mixture of 2-(R)-[N-(tert-butoxycarbonyl)amino]propanal

(16.21 g, 94 mmol), dimethylamine hydrochloride (15.28 g,

187.6 mmol), sodium acetate (11.53 g, 140 mmol), and sodium

cyanoborohydride (8.24 g, 131 mmol) in methanol (250 mL)

was stirred at room temperature for 18 h maintaining pH at

6-7 by the dropwise addition of acetic acid. The reaction

mixture was dissolved in ethyl acetate (400 mL), washed with

water (200 mL), and dried (MgSO₄), and the solvent was

removed in vacuo to yield a viscous oil which was purified on

silica eluting with 90% dichlorometane and 10% methanol to

yield (R)-N¹,N¹-dimethyl-propane-1,2-diamine as a white solid

(10.81 g, 57%). To this compound (3.17 g, 15.7 mmol) was

added a 4.0 M solution of hydrochloric acid in dioxane (20 mL)

at room temperature. After the mixture was stirred for 1 h,

the volatile components were removed in vacuo to yield the

desired title compound as a viscous oil (4.28 g, 100%). ¹H NMR

(d₃-MeOD) δ 1.57 (d, 3H), 3.08 (s, 6H), 3.50 (dd, 1H), 3.62, (dd,

1H), 4.03, (q, 1H).

1-Meth yl-3-(R)-a m in op yr r olid in e Dih yd r och lor id e. A

solution of 3-R-(-)-1-benzyl-3-aminopyrrolidine (847 mg, 4.8

mmol) in tert-butyl alcohol (10 mL) and 1.0 N sodium hydrox-

ide solution (4.8 mL) was treated dropwise with a solution of

di-tert-butyl dicarbonate (1.06 g, 4.8 mmol) in tert-butyl alcohol

(5 mL). After 1.5 h, the tert-butyl alcohol was removed in

vacuo, the residue was dissolved in ethyl acetate (50 mL),

washed with water (50 mL), and dried (MgSO₄), and the

solvent was removed in vacuo to yield the desired 3-N-tert-

butoxycarbonyl protected derivative as a colorless gum (1.3 g,

4.7 mmol). A solution of the 3-N-tert-butoxycarbonyl protected

derivative (800 mg, 2.9 mmol) in tetrahydrofuran (5 mL) and

ethanol (5 mL) was stirred over palladium hydroxide catalyst

under an atmosphere of hydrogen for 24 h. The reaction

mixture was then filtered through Celite and the solvent

removed in vacuo to yield the desired 3-N-tert-butoxycarbonyl-

3-(R)-aminopyrrolidine in quantitative yield. To a solution of

N-tert-butoxycarbonyl-3-(R)-aminopyrrolidine (437 mg, 2.35

mmol) in methanol (10 mL) was added 37% formaldehyde

solution in H₂O (0.52 mL) and sodium borohydride (271 mg,

7.0 mmol). The reaction mixture was stirred for 24 h at room

temperature and then reduced in vacuo. The residue was

dissolved in chloroform (10 mL), washed with brine (10 mL)

and NaHCO₃solution (10 mL), and dried (MgSO₄), and the

solvent was removed in vacuo to yield the desired 3-N-tert-

(S)-2-Am in o-3-(d im eth yla m in o)-p r op a n -1-ol Dih yd r o-

ch lor id e. N-[(tert-Butoxy)carbonyl]-O-(tert-butyldimethylsi-

lyl)-R-serine methyl ester was prepared according to the

literature from ^D-serine methyl ester hydrochloride.²⁷Treat-

ment of N-[(tert-butoxy)carbonyl]-O-(tert-butyldimethylsilyl)-

R-serine methyl ester with diisobutyl aluminum hydride in

toluene at -70 °C for 2 h yielded the correponding aldehyde.²⁷

A mixture of the crude aldehyde (4.43 g, 22 mmol), dimeth-

ylamine hydrochloride (2.26 g. 27.7 mmol), sodium cyanoboro-

hydride (1.31 g, 20.8 mmol), and sodium acetate (1.83 g, 22.2

mmol) was stirred in methanol (55 mL) for 24 h at room

temperature. Aqueous workup yielded the dimethylamino

derivative which was dissolved in dioxane, and to this was

added a 4.0 M solution of hydrochloric acid in dioxane and

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 731

butoxycarbonyl-3-(R)-amino-1-methylpyrrolidine as a gum (390

mg, 1.95 mmol), deprotection with 4.0 M HCl solution in

dioxane as described above yielded the desired title compound

(337 mg, 100%) which was used without further purification.

Meth od C: Rea ction of Am in es Dir ectly w ith th e

Su bstitu ted Ben zo[a ]-11-ca r boxylic Acid Meth yl Ester s.

4-Met h oxy-b en zo[a ]p h en a zin e-11-ca r b oxylic Acid (2-

(Dim eth yla m in o)-1-m eth yl-eth yl)-a m id e (4j′′). A mixture

of 4-methoxy-benzo[a]phenazine-11-carboxylic acid methyl

ester (350 mg, 1.1 mmol) and 1-(dimethylamino)-2-propyl-

amine (2 mL, 15.5 mmol) was heated to 110 °C for 4 h. The

reaction mixture was then cooled, and the excess amine was

removed in vacuo. The residue was then purified using flash

chromatography on silica (ethyl acetate and then 25% metha-

nol in ethyl acetate) to yield the title compound (164 mg, 38%)

N¹,N¹,N²,N²-Tetr a m eth yl-p r op a n e-1,2,3-tr ia m in e Tr i-

h yd r och lor id e. To ice cooled thionyl chloride (35 mL) was

added 1,3-bis(dimethylamino)-propan-2-ol (4.91 g, 33.58 mmol)

dropwise over 45 min with stirring. After the addition was

complete, the mixture was stirred for a further 4 h. Excess

thionyl chloride was removed under reduced pressure to give

the product as the hydrochloride salt as a cream solid (8.9 g).

The free base was obtained by treating a suspension of the

hydrochloride salt in toluene (18 mL) with sodium hydroxide

(2.4 equiv) in water (14 mL) for 30 min. The organic layer was

removed, dried over MgSO₄, and filtered, and the solvent was

removed in vacuo to yield 2-chloro-N,N,N¹,N¹-tetramethylpro-

pane-1,3-diamine as a yellow liquid. 2-Chloro-N,N,N¹,N¹-

tetramethylpropane-1,3-diamine (1.6 g, 9.71 mmol) as a solu-

tion in toluene (14 mL) was treated with potassium phthalimide

(1.98 g, 10.68 mmol). The stirred mixture was heated at reflux

for 18 h and cooled to ambient temperature, the solvent was

removed under reduced pressure to yield a beige solid, and

recrystallization from diethyl ether afforded a fawn solid (1.85

g, 6.7 mmol). This solid (0.953, 3.46 mmol) as a solution in

ethanol (10 mL) was treated with hydrazine hydrate (0.22 mL,

6.93 mmol), and the mixture was stirred at ambient temper-

ature for 18 h. The suspension was removed by filtration, the

filtrate acidified with 2 mL of 2 M hydrochloric acid, and the

solvent removed in vacuo to give the product as a cream solid

1

as a yellow solid. H NMR (DMSO-d₆) δ 1.53 (d, 3H, J ) 6.5

Hz), 2.37 (s, 6H), 2.55 (dd, 1H, J ) 12.2, 6.2 Hz), 2.84 (dd, 1H

J ) 12.2, 7.8 Hz), 4.10 (s, 3H), 4.55-4.65 (m, 1H), 7.28-7.30

(m, 1H), 7.75-7.80 (m, 1H), 7.95-8.02 (m, 2H), 8.42 (dd, 1H

J ) 8.4, 1.5 Hz), 8.57 (d, 1H, J ) 9.5 Hz), 8.81 (d, 1H, J ) 8.2

Hz), 9.02 (dd, 1H, J ) 7.3, 1.6 Hz), 10.9 (d, 1H, J ) 7.2 Hz).

MS (DCI/NH₃) m/e 389 (M + H)⁺.

The following compounds were prepared in an analogous

manner from the appropriate substituted benzo[a]-11-carbox-

ylic acid activated as the acyl imidazole and reacted with the

appropriate amine using general method A.

3-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4b) was prepared from

3-methoxy-benzo[a]phenazine-11-carboxylic acid (129 mg, 0.90

mmol) and N,N-dimethylethylenediamine (0.5 mL, 4.5 mmol)

to yield the title compound as a yellow solid (281 mg, 84%),

mp 172-173 °C. ¹H NMR (DMSO-d₆) δ 2.36 (s, 6H), 2.61 (t,

2H, J ) 5.5 Hz), 3.72 (q, 2H, J ) 5.5 Hz), 4.02 (s, 3H), 7.44

(dd, 1H, J ) 2.65, 8.9 Hz), 7.64 (d, 1H, J ) 2.6 Hz), 8.03-7.95

(m, 2H), 8.24 (d, 1H, J ) 9.3 Hz), 8.40 (dd, 1H, J ) 1.49, 8.2

Hz), 8.74 (dd, 1H, J ) 1.4, 7.6 Hz), 9.32 (d, 1H, J ) 8.9 Hz),

10.39 (t, 1H). MS (DCI/NH₃) m/e 375 (M + H)⁺. Anal.

(C₂₂H₂₂N₄O₂) C, H, N.

1

(795 mg, 90%). MH⁺172. H NMR (d₃-MeOD) δ 2.40 (s, 6H),

2.45 (s, 6H), 2.54 (m, 1H), 2.65 (m, 1H), 2.97 (m, 1H), 3.01 (t,

1H), 3.12 (m, 1H).

Gen er a l Meth od s for th e P r ep a r a tion of Su bstitu ted

Ben zo[a ]-11-ca r boxa m id es. Meth od A. Activa tion of 5

w ith 1,1′-Ca r bon yl Diim id a zole (CDI). 4-Meth oxy-ben zo-

[a ]p h en a zin e-11-ca r boxylic Acid (2-(Dim eth yla m in o)-

eth yl)-am ide (4c). A mixture of 4-methoxy-benzo[a]phenazine-

11-carboxylic acid (129 mg, 0.42 mmol) and CDI (138 mg, 0.85

mmol) was stirred in dry DMF (8 mL) at room temperature

for 4 h. To this mixture was added N,N-dimethylethylenedi-

amine (0.5 mL, 4.5 mmol), and the reaction mixture was

stirred at room temperature for a further 30 min. The volatile

components were then removed in vacuo, the residue was

dissolved in dichloromethane (20 mL), washed with water

(2 × 20 mL), and dried (MgSO₄), and the solvent was removed

in vacuo to provide crude product. This was purified using flash

chromatography eluting with 5% methanol in dichloromethane

to yield the title compound as a bright yellow solid (120 mg,

76%), mp 166-169 °C. ¹H NMR (DMSO-d₆) δ 2.36 (s, 6H), 2.61

(t, 2H, J ) 5.5 Hz), 3.72 (q, 2H, J ) 5.5 Hz), 4.02 (s, 3H), 7.44

(dd, 1H, J ) 2.65, 8.9 Hz), 7.64 (d, 1H, J ) 2.6 Hz), 7.95-8.03

(m, 2H), 8.24 (d, 1H, J ) 9.3 Hz), 8.40 (dd, 1H, J ) 1.49, 8.2

Hz), 8.74 (dd, 1H, J ) 1.4, 7.6 Hz), 9.32 (d, 1H, J ) 8.9 Hz),

10.39 (t, 1H). MS (DCI/NH₃) m/e 375 (M + H)⁺. Anal.

(C₂₂H₂₂N₄O₂) C, H, N.

2-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4a ) was prepared from

2-methoxy-benzo[a]phenazine-11-carboxylic acid (184 mg, 0.61

mmol) and N,N dimethylethylenediamine (0.5 mL, 4.5 mmol)

to give the title compound as a yellow solid 191 mg, 84%), mp

1

181-184 °C. H NMR (DMSO-d₆) δ 2.47 (s, 6H), 2.90 (t, 2H,

J ) 2.91 Hz), 3.79 (q, 2H, J ) 6.8 Hz, 4.06 (s, 3H), 7.56 (m,

1H), 7.81 (d, 1H J ) 9.20 Hz), 8.04 (m, 2H), 8.18 (d, 1H J )

9.24 Hz), 8.41(d, 1H J ) 7.48 Hz), 8.55 (m, 2H), 9.97 (br.t,

1H). MS (DCI/NH₃) m/e 375 (M + H)⁺.

4-Nitr o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-(d i-

m eth yla m in o)-eth yl)-a m id e (4i) was prepared from the

mixture of 4-nitro-benzo[a]phenazine-11-carboxylic acid, 3-ni-

tro-benzo[a]phenazine-11-carboxylic acid (69 mg, 0.21 mmol),

and N,N-dimethylethylenediamine (0.25 mL, 2.25 mmol) and

then purified using flash chromatography to give the title

1

compound as a yellow gum (26 mg, 36%). H NMR (CDCl₃) δ

2.50 (s, 6H), 2.75 (t, 2H), 3.95 (q, 2H), 7.95 (t, 1H), 8.10 (t,

1H), 8.30 (d, 1H), 8.45 (d, 1H), 8.50 (d, 1H), 8.70 (d, 1H), 9.10

(dd, 1H), 9.90 (d, 1H), 10.50 (br, 1H). MS (DCI/NH₃) m/e 390

(M + H)⁺.

Met h od B: Act iva t ion of 5 w it h Th ion yl Ch lor id e.

4-Met h oxy-b en zo[a ]p h en a zin e-11-ca r b oxylic Acid (2-

(Dim eth yla m in o)-1,1-d im eth yl-eth yl)-a m id e (4l′′). A mix-

ture of 4-methoxy-benzo[a]phenazine-11-carboxylic acid (1.0

g, 3.2 mmol) and thionyl chloride (10 mL) was heated to reflux

for 6 min. The thionyl chloride was then removed in vacuo.

The residue was dissolved in dry dichloromethane (10 mL),

the solution cooled to 0 °C, and 1-(dimethylamino)-2-methyl-

2-aminopropane (5 mL) was added. After being stirred for 2

h, the reaction mixture was diluted with dichloromethane (50

mL), washed with sodium bicarbonate solution (50 mL), and

dried (MgSO₄), and the solvent was removed in vacuo to

provide crude product which was purified on silica eluting with

95% dicloromethane and 5% methanol to give the title com-

pound as a yellow solid (945 mg, 74%), mp 158-162 °C. ¹H

NMR (DMSO-d₆) δ 1.56 (s, 6H), 2.30 (s, 6H), 2.78 (s, 2H), 4.07

(s, 3H), 7.48 (d, 1H), 7.86 (m, 1H), 7.98 (d, 1H), 8.07 (m, 1H),

8.43 (d, 1H), 8.50 (d, 1H), 8.56 (d, 1H), 8.95 (d, 1H), 9.67 (br,

1H). MS (DCI/NH₃) m/e 403 (M + H)⁺.

3-Nitr o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-(d i-

m eth yla m in o)-eth yl)-a m id e (4h ) was prepared from the

mixture of 4-nitro-benzo[a]phenazine-11-carboxylic acid, 3-ni-

tro-benzo[a]phenazine-11-carboxylic acid (69 mg, 0.21 mmol),

and N,N-dimethylethylenediamine (0.25 mL, 2.25 mmol) and

then purified using flash chromatography to give the title

compound as a yellow solid (7 mg, 9%), mp 245.5-246.5 °C.

¹H NMR (CDCl₃) δ 2.50 (s, 6H), 3.10 (t, 2H), 4.05 (q, 2H), 8.10

(dd, 1H), 8.15 (dd, 2H), 8.20 (d, 1H), 8.45 (d, 1H), 8.60 (dd,

1H), 9.0 (dd, 1H), 9.90 (d, 1H), 10.40 (br. s, 1H). MS (DCI/

NH₃) m/e 390 (M + H)⁺.

4-Ben zyloxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4c′) was prepared from

4-benzyloxy-benzo[a]phenazine-11-carboxylic acid (46 mg, 0.12

mmol) and N,N-dimethylethylenediamine (70 µL, 0.60 mmol)

to give the title compound as a yellow solid (20 mg, 37%), mp

1

172-173 °C. H NMR (CDCl₃) δ 2.42 (s, 6H), 2.75 (t, 2H J )

6.0 Hz), 3.89-3.93 (m, 2H), 5.35 (s, 2H), 7.33-7.49 (m, 4H),

732 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

7.55 (d, 2H, J ) 7.2 Hz), 7.73-7.77 (m, 1H), 7.95-8.0 (m, 2H),

8.40 (dd, 1H, J ) 8.6, 1.5 Hz), 8.63 (d, 1H, J ) 9.4 Hz), 9.00-

9.07 (m, 2H), 10.90 (br. s, 1H). MS (DCI/NH₃) m/e 451

(M + H)⁺.

4-Meth yl-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-(d i-

m eth ylam in o)-eth yl)-am ide (4p) was prepared from 4-meth-

yl-benzo[a]phenazine-11-carboxylic acid (100 mg, 0.35 mmol)

and N,N-dimethylethylenediamine (0.2 mL, 1.73 mmol) to give

the title compound as a yellow solid (26 mg, 26%), mp 166-

171 °C. ¹H NMR (CDCl₃) δ 2.44 (s, 6H), 2.60 (t, 2H, J ) 5.7

Hz), 2.75 (s, 3H), 3.91 (q, 2H, J ) 5.7 Hz), 7.69 (t, 1H, J ) 6.9

Hz), 7.73 (t, 1H, J ) 7.6 Hz), 7.98 (d, 1H, J ) 7.3 Hz), 8.02 (d,

1H, J ) 9.6 Hz), 8.31 (d, 1H, J ) 9.4 Hz), 8.42 (dd, 1H, J )

8.4, 1.5 Hz), 9.02 (dd, 1H, J ) 7.3, 1.5 Hz), 9.34 (d, 1H, J )

7.9 Hz), 10.88 (br. s, 1H). MS (DCI/NH₃) m/e 359 (M + H)⁺.

4-P r op-2-yn yloxy-ben zo[a ]ph en azin e-11-car boxylic acid

(2-(d im eth yla m in o)-eth yl)-a m id e (4d ′) was prepared from

4-prop-2-ynyloxy-benzo[a]phenazine-11-carboxylic acid (55 mg,

0.17 mmol) and N,N-dimethylethylenediamine (100 µL, 0.85

mmol) to give the title compound as a yellow solid (19 mg,

28%), mp 173-174 °C. ¹H NMR (CDCl₃) δ 2.37 (s, 6H), 2.55

(t, 1H, J ) 2.4 Hz), 2.70 (t, 2H, J ) 6.0 Hz), 3.82-3.87 (m,

2H), 4.95 (d, 2H J ) 2.4 Hz), 7.32 (d, 1H, J ) 8.3 Hz), 7.70-

7.74 (m, 1H), 7.90-7.95 (m, 2H), 8.35 (dd, 1H, J ) 8.50, 1.6

Hz), 8.50 (d, 1H, J ) 9.8 Hz), 8.95 (dd, 1H, J ) 7.30, 1.5 Hz),

9.01 (d, 1H, J ) 8.2 Hz), 10.80 (br. s, 1H). MS (DCI/NH3) m/e

399 (M + H)⁺.

3,4-Dim eth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid

(2-(d im eth yla m in o)-eth yl)-a m id e (4q′) was prepared from

3,4-dimethoxy-benzo[a]phenazine-11-carboxylic acid (45 mg,

0.13 mmol) and N,N-dimethylethylenediamine (44 µL, 0.40

mmol) to give the title compound as a yellow solid (21 mg,

39%), mp 155-159 °C. ¹H NMR (CDCl₃) δ 2.40 (s, 6H), 2.70

(t, 2H, J ) 6.0), 3.84 (q, 2H, J ) 6.0 Hz), 4.01 (s, 3H), 4.04 (s,

3H), 7.40 (d, 1H, J ) 9.0 Hz), 7.90-7.81 (m, 2H), 8.40-8.30

(m, 2H), 8.91 (d, 1H J ) 7.3 Hz), 9.11 (d, 1H, J ) 8.9 Hz),

10.82 (br. s, 1H). MS (DCI/NH3) m/e 405 (M + H)⁺.

4-F lu or o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-(d i-

m eth ylam in o)-eth yl)-am ide (4m ) was prepared from 4-fluoro-

benzo[a]phenazine-11-carboxylic acid (100 mg, 0.34 mmol) and

N,N-dimethylethylenediamine (0.2 mL, 1.71 mmol) to give the

title compound as a yellow solid (29 mg, 29%), mp 172-175

1

°C. H NMR (CDCl₃) δ 2.37 (s, 6H), 2.68 (t, 2H, J ) 5.8 Hz),

3.83 (q, 2H, J ) 5.5 Hz), 7.47 (m, 1H), 7.71 (m, 1H), 7.93 (dd,

1H, J ) 8.5, 7.2 Hz), 7.97 (d, 1H, J ) 9.5), 8.29 (d, 1H, J ) 9.5

Hz), 8.36 (dd, 1H, J ) 8.4, 1.5 Hz), 8.97 (dd, 1H, J ) 7.3, 1.5

Hz), 9.22 (d, 1H, J ) 8.1 Hz), 10.67 (br. s, 1H). MS (DCI) m/e

363 (M + H)⁺.

4-Meth ylsu lfan yl-ben zo[a ]ph en azin e-11-car boxylic acid

(2-(d im eth yla m in o)-eth yl)-a m id e (4j) was prepared from

4-methylsulfanyl-benzo[a]phenazine-11-carboxylic acid which

was prepared from 4-Fluoro-benzo[a]phenazine-11-carboxylic

acid (58 mg, 0.2 mmol) by treatment with sodium thiomethox-

ide (55 mg, 1 mmol) in DMSO (3 mL). The mixture was heated

at 130 °C for 2 h, quenched with acetic acid (2 mL) and water

(5 mL), and extracted with ethyl acetate (2 × 10 mL). The

organics were dried (MgSO₄) and reduced in vacuo to give the

product which was used crude in the next step. ¹H NMR

(DMSO-d₆) δ 2.70 (s, 3H), 7.91 (m, 2H), 8.11 (m, 2H), 8.45 (d,

1H), 8.52 (m, 2H), 9.00 (d, 1H). MS (DCI/NH3) m/e 321 (M +

H)⁺. 4-Methylsulfanyl-benzo[a]phenazine-11-carboxylic acid

(223 mg, 0.17 mmol) and N,N-dimethylethylenediamine (100

µL, 0.85 mmol) gave the title compound as a yellow glass (4.7

mg, 9%). ¹H NMR (CDCl₃) δ 2.43 (s, 6H), 2.66 (s, 3H), 2.75 (m,

2H), 3.90 (m, 2H), 7.76 (m, 2H), 7.99 (m, 1H), 8.05 (d, 1H, J )

9.7 Hz), 8.42 (dd, 1H, J ) 8.4, 1.5 Hz), 8.63 (d, 1H, J ) 9.7

Hz), 9.02 (dd, 1H, J ) 7.2, 1.5 Hz), 9.32 (m, 1H), 10.79 (br. s,

1H). MS (EI) m/e 390 (M⁺).

4-E t h oxy-b en zo[a ]p h en a zin e-11-ca r b oxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4a ′) was prepared from

4-ethoxy-benzo[a]phenazine-11-carboxylic acid (240 mg, 0.75

mmol) and N,N-dimethylethylenediamine (411 µL, 3.75 mmol)

to give the title compound as a yellow solid (56 mg, 19%), mp

1

151-152 °C. H NMR (DMSO-d₆) δ 1.52 (t, 3H, J ) 6.9 Hz),

2.35 (s, 6H), 2.65 (t, 2H, J ) 5.9 Hz), 3.72-3.75 (m, 2H), 4.30

(q, 2H, J ) 6.9 Hz), 7.43 (d, 1H, J ) 7.9 Hz), 7.80-7.84 (m,

1H), 7.92 (d, 1H, J ) 9.5 Hz), 8.05-8.10 (m, 1H), 8.42 (dd,

1H, J ) 8.6, 1.6 Hz), 8.45 (d, 1H, J ) 9.5 Hz), 8.72 (dd, 1H,

J ) 7.1, 1,5 Hz), 8.94 (d, 1H, J ) 8.2 Hz), 10.25 (t, 1H, J ) 5.0

Hz). MS (DCI/NH₃) m/e 389 (M + H)⁺. Anal. C₂₃H₂₄N₄O₂

C, H, N.

4-Isobu toxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4b′) was prepared from

4-isobutoxy-benzo[a]phenazine-11-carboxylic acid (10.8 mg,

0.031 mmol) and N,N-dimethylethylenediamine (17 µL, 0.16

mmol) to give the title compound as a yellow solid (9.6 mg,

74%), mp 152-154 °C. ¹H NMR (CDCl₃) δ 10.87 (br, 1H), 8.93

(m, 2H), 8.54 (d, 1H, J ) 9.5 Hz), 8.35 (dd, 1H, J ) 8.4, 1.5

Hz), 7.89 (m, 2H), 7.69 (t, 1H, J ) 8.0 Hz), 7.19 (m, 1H), 3.93

(d, 2H, J ) 6.4 Hz), 3.87 (m, 2H), 2.75 (m, 2H), 2.40 (s, 6H),

2.24 (m, 1H), 1.11 (d, 6H, J ) 6.8 Hz). MS (EI) m/e 416 (M⁺).

4-(2-Met h oxy-et h oxy)-b en zo[a ]p h en a zin e-11-ca r b ox-

ylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4i′) was pre-

pared from 4-(2-methoxy-ethoxy)-benzo[a]phenazine-11-car-

boxylic acid (45 mg, 0.13 mmol) and N,N-dimethylethylene-

diamine (70 µL, 0.64 mmol) to give the title compound as a

yellow solid (5 mg, 9%). ¹H NMR (CDCl₃) δ 2.69 (s, 6H), 2.92-

3.10 (m, 2H), 3.49 (s, 3H), 3.83-3.93 (t, 2H, J ) 4.4 Hz), 3.94-

4.10 (m, 2H), 4.28-4.40 (t, 2H, J ) 4.4 Hz), 7.22 (d, 1H, J )

8.0 Hz), 7.80 (t, 1H), 7.84-7.96 (m, 2H, J ) 4.7, 8.3 Hz), 8.37

(d, 1H, J ) 8.2 Hz), 8.54 (d, 1H, J ) 9.4 Hz), 8.82 (d, 1H, J )

7.4 Hz), 8.90 (d, 1H, J ) 6.9 Hz), 11.10 (br. s, 1H). MS (DCI/

NH₃) m/e 419 (M⁺).

4-Br om o-b en zo[a ]p h en a zin e-11-ca r b oxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4o) was prepared from

4-bromo-benzo[a]phenazine-11-carboxylic acid (52 mg, 0.15

mmol) and N,N-dimethylethylenediamine (81 µL, 0.75 mmol)

to give the title compound as a yellow solid (13 mg, 21%), mp

1

171-172 °C. H NMR (CDCl₃) δ 2.37 (s, 6H), 2.69 (br. t, 2H,

J ) 5.9 Hz), 3.84 (m, 2H), 7.61 (t, 1H, J ) 7.9 Hz), 7.94 (m,

1H), 8.02 (m, 2H), 8.36 (dd, 1H, J ) 8.4, 1.5 Hz), 8.47 (d, 1H,

J ) 9.8 Hz), 8.97 (dd, 1H, J ) 7.3, 1.6 Hz), 9.41 (d, 1H, J )

8.0 Hz), 10.61 (br. s, 1H). MS (DCI/NH₃) m/e 423:425, 1:1

(M + H)⁺. Anal. (C₂₁H₁₉BrN₄O) C, H, N.

4-Cya n o-b en zo[a ]p h en a zin e-11-ca r b oxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4q) was prepared from

4-cyano-benzo[a]phenazine-11-carboxylic acid (1.0 g, 3.34 mmol)

and N,N-dimethylethylenediamine (5 mL, 45 mmol) to give

the title compound as a yellow solid (200 mg, 16%), mp 172-

176 °C. ¹H NMR (DMSO-d₆) δ 2.35(s, 6H), 2.62 (t, 2H), 3.72

(q, 2H), 8.00 (m, 2H), 8.10-8.74 (dd, 1H), 8.24 (d, 1H), 8.35

(d, 1H), 8.41 (m, 2H), 9.62 (d, 1H), 9.99 (t, 1H). MS (DCI) m/e

370 (M + H)⁺.

[11-(2-Dim eth yla m in o-eth ylca r ba m oyl)-ben zo[a ]p h en -

a zin -4-yloxy]-a cetic a cid eth yl ester (4k ′) was prepared

from 4-ethoxycarbonylmethoxy-benzo[a]phenazine-11-carbox-

ylic acid (144 mg, 0.39 mmol) and N,N-dimethylethylenedi-

amine (210 µL, 1.95 mmol) to give the title compound as a

4-Meth a n esu lfon yl-ben zo[a ]p h en a zin e-11-ca r boxylic

a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4k ) was prepared

from 4-methanesulfonyl-benzo[a]phenazine-11-carboxylic acid

(120 mg, 0.34 mmol) and N,N-dimethylethylenediamine (186

µL, 1.7 mmol) to give the title compound as a yellow solid (85

1

yellow solid (34 mg, 22%), mp 167-168 °C. H NMR (CDCl₃)

δ 1.27 (t, 3H, J ) 7.1 Hz), 2.36 (s, 6H), 8.94 (dd, 1H, J ) 7.3,

1.5 Hz); 8.58 (d, 1H, J ) 9.5 Hz) 8.34 (dd, 1H, J ) 8.4, 1.5

Hz), 7.91 (m, 2H), 7.66 (t, 1H, J ) 8.1 Hz), 7.08 (d, 1H, J )

8.0 Hz), 4.83 (2H, s), 4.27 (q, 2H, J ) 7.1 Hz), 3.83 (m, 2H),

2.69 (m, 2H), 9.01 (d, 1H, J ) 8.2 Hz), 10.76 (br. s, 1H). MS

(DCI/NH₃) m/e 447 (M + H)⁺. Anal.(C₂₅H₂₆N₄O₄) C, H, N.

1

mg, 59%), mp 204 °C dec. H NMR (DMSO-d₆) δ 2.99 (s, 6H),

3.53 (br. t, 2H, J ) 6.3 Hz), 3.57 (s, 3H), 4.04 (br. m, 2H), 8.24

(m, 2H), 8.40 (d, 1H, J ) 9.8 Hz), 8.59 (m, 2H), 8.65 (m, 1H),

9.10 (d, 1H, J ) 9.8 Hz), 9.64 (d, 1H, J ) 8.0 Hz), 9.79 (br. m,

1H). MS (DCI/NH₃) m/e 423 (M + H)⁺.

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 733

4-Ch lor o-b en zo[a ]p h en a zin e-11-ca r b oxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4n ) was prepared from

4-chloro-benzo[a]phenazine-11-carboxylic acid (75 mg, 0.24

mmol) and N,N-dimethylethylenediamine (100 µL, 0.85 mmol)

(t, 2H, J ) 6.1 Hz), 3.82-3.87 (m, 2H), 4.06 (s, 3H), 4.10 (s,

3H), 6.78 (br s 1H), 7.19 (d, 1H, J ) 7.7 Hz), 7.63-7.70 (m,

2H), 7.85 (d, 1H, J ) 9.4 Hz), 8.28 (d, 1H, J ) 9.4 Hz), 8.45 (d,

1H, J ) 9.4 Hz), 8.95 (d, 1H, J ) 8.1 Hz). MS (DCI/NH₃) m/e

405 (M + H)⁺.

1

to give the title compound as a yellow solid (19 mg, 21%). H

NMR (CDCl₃) δ 2.38 (s, 6H), 2.62-2.70 (t, 2H, J ) 8.0 Hz),

3.78-3.86 (m, 2H, J ) 5.4, 3.0 Hz), 7.69 (t, 1H, J ) 8.0 Hz),

7.72 (d, 1H, J ) 0.9, 6.8 Hz), 7.94 (t, 1H, J ) 1.3, 7.2 Hz), 8.04

(d, 1H, J ) 9.5 Hz), 8.37 (d, 1H, J ) 1.5, 6.9 Hz), 8.51 (d, 1H,

J ) 9.4 Hz), 8.98(d, 1H, J ) 1.5, 5.6 Hz), 9.99 (d, 1H, J ) 8.0

Hz), 10.60 (br. s, 1H). MS (DCI/NH₃) m/e 379 (M + H)⁺.

4-Acetyloxy-11-(2-dim eth ylam in o-eth ylcar bam oyl)-ben -

zo[a ]p h en a zin e (4r ) was prepared from 4-acetyloxy-benzo-

[a]phenazine-11-carboxylic acid (46 mg, 0.13 mmol) and N,N-

dimethylethylenediamine to give the title compound as a

4-Meth oxy-8-m eth yl-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4r ′) was pre-

pared from 4-methoxy-8-methyl-benzo[a]phenazine-11-carbox-

ylic acid (120 mg, 0.38 mmol) and N,N-dimethylethylenediamine

(0.21 mL, 1.9 mmol) to give the title compound as a yellow

1

solid (38 mg, 26%), mp 170-172 °C. H NMR (CDCl₃) δ 2.42

(s, 6H), 2.76 (t, 2H, J ) 6.0 Hz), 3.00 (s, 3H), 3.88-3.92 (m,

2H), 4.10 (s, 3H), 7.30 (d, 1H), 7.75-7.79 (m, 1H), 7.82 (d, 1H,

J ) 9.4 Hz), 8.03 (d, 1H, J ) 9.7 Hz), 8.55 (d, 1H, J ) 9.7 Hz),

8.90 (d, 1H, J ) 7.4 Hz), 9.03 (d, 1H, J ) 8.0 Hz), 10.95 (br. s,

1H). MS (DCI/NH₃) m/e 389 (M + H)⁺.

1

yellow solid. H NMR (CDCl₃) δ 2.38 (s, 6H), 2.70 (t, 2H, J )

5.8 Hz), 3.87 (q, 2H, J ) 5.8 Hz), 4.03 (s, 3H), 7.81 (t, 1H, J )

7.9 Hz), 7.96 (t, 1H, J ) 7.8), 8.07 (d, 1H, J ) 9.8 Hz), 8.35-

8.40 (m, 2H), 9.00 (d, 1H, J ) 7.2 Hz), 9.13 (d, 1H, J ) 9.8

Hz), 9.68 (d, 1H, J ) 8.1 Hz), 10.65 (br. s, 1H). MS (DCI/NH₃)

m/e 403 (M + H)⁺.

4-Meth ylsu lfa m oyl-ben zo[a ]p h en a zin e-11-ca r boxylic

a cid (2-(d im eth yla m in o)-eth yl)-a m id e; tr iflu or o-a ceta te

(4l) was prepared from the mixture of 4-methylsulfamoyl-

benzo[a]phenazine-11-carboxylic acid, 3-methylsulfamoyl-ben-

zo[a]phenazine-11-carboxylic acid, and N,N-dimethylethylene-

diamine. The two isomers were separated using preparative

HPLC to give the title compound as a yellow solid, mp 224-

226 °C. ¹H NMR (CDCl₃/d₄-MeOD) δ 2.67 (s, 3H), 3.05 (s, 6H),

3.62 (t, 2H), 4.25 (t, 2H), 8.00-8.10 (m, 3H), 8.45-8.50 (m,

2H), 8.88 (d, 1H), 8.99 (d, 1H), 9.35 (d, 1H). MS (DCI/NH₃)

m/e 438 (M + H)⁺.

2-Nitr o-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-(d i-

m eth yla m in o)-eth yl)-a m id e (4g) was prepared from 2-nitro-

benzo[a]phenazine-11-carboxylic acid and N,N-dimethyleth-

ylenediamine, to give the title compound as a yellow solid. ¹H

NMR (CDCl₃) δ 2.92 (d, 6H, J ) 5.0 Hz), 3.59 (q, 2H, 5.6 Hz),

4.26 (q, 2H, 6.1 Hz), 7.98 (dd, 1H, J ) 7.3, 8.5 Hz), 8.10-8.03

(m, 2H), 8.13 (d, 1H, J ) 9.3 Hz), 8.41 (dd, 1H, J ) 8.0, 1.4

Hz), 8.56 (dd, 1H, J ) 8.5, 2.3 Hz), 8.80 (dd, 1H, J ) 6.6, 1.4

Hz), 9.98 (d, 1H, J ) 2.2 Hz), 10.72 (br. t, 1H). MS (DCI/NH₃)

m/e 390 (M + H)⁺.

9-Ch lor o-4-m eth oxy-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4s′) was pre-

pared from 9-chloro-4-methoxy-benzo[a]phenazine-11-carbox-

ylic acid (245 mg, 0.72 mmol) and N,N-dimethylethylenediamine

(0.4 mL, 3.6 mmol) to give the title compound as a yellow solid

1

(67 mg, 23%), mp 188-190. H NMR (CDCl₃) δ 2.42 (s, 6H),

2.75 (t, 2H, J ) 5.9 Hz), 3.86-3.90 (m, 2H), 4.08 (s, 3H), 7.25

(d, 1H, J ) 7.8 Hz), 7.72-7.77 (m, 1H), 7.89 (d, 1H, J ) 9.3

Hz), 8.36 (d, 1H, J ) 2.5 Hz), 8.56 (d, 1H, J ) 9.6 Hz), 8.92 (d,

1H, J ) 2.5 Hz), 8.95 (d, 1H, J ) 8.1 Hz), 10.75 (br. s, 1H).

MS (DCI/NH₃) m/e 411/409 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (1-

d im eth yla m in om eth yl-p r op yl)-a m id e (4m ′′) was prepared

from 4-methoxy-benzo[a]phenazine-11-carboxylic acid (48 mg,

0.16 mmol) and N¹,N¹-dimethyl-butane-1,2-diamine dihydro-

chloride (61 mg, 0.32 mmol) in the presence of triethylamine

(67 µL, 0.96 mmol) to give the title compound as a yellow solid

(19 mg, 30%), mp 110-114 °C. ¹H NMR (CDCl₃) δ 1.11 (t, 3H,

J ) 7.5 Hz), 1.78-1.89 (m, 1H), 1.98-2.08 (m, 1H), 2.36 (s,

6H), 2.59 (dd, 1H, J ) 12.5, 6.1 Hz), 2.82 (dd, 1H, J ) 12.5,

7.6), 4.10 (s, 3H, OMe), 4.46-4.54 (m, 1H), 7.27 (d, 1H, J )

9.0 Hz), 7.76 (t, 1H, J ) 8.1 Hz), 7.94-8.01 (m, 2H), 8.41 (d,

1H, J ) 8.5 Hz), 8.57 (d, 1H, J ) 9.4 Hz), 8.79 (d, 1H, J ) 8.2

Hz), 9.01 (d, 1H, J ) 7.1 Hz), 10.92 (d, 1H, J ) 8.3). MS (DCI/

NH₃) m/e 403 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (1-

d im eth yla m in om eth yl-2-m eth yl-p r op yl)-a m id e (4n ′′) was

prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

(87 mg, 0.29 mmol) and 3N¹,N¹-trimethyl-butane-1,2-diamine

dihydrochloride salt (290 mg, 1.43 mmol) in the presence of

triethylamine (0.48 mL, 3.43 mmol) to give the title compound

as a yellow solid (77 mg, 64%). ¹H NMR (CDCl₃) δ 1.03 (d, 3H,

J ) 2.6 Hz), 1.04 (d, 3H, J ) 2.6 Hz), 2.16 (m, 1H), 2.28 (s,

6H), 2.59 (dd, 1H, J ) 12.6, 4.8 Hz), 2.83 (dd, 1H, J ) 12.6,

9.1 Hz), 4.01 (s, 3H), 4.49 (m, 1H), 7.18 (d, 1H, J ) 7.8 Hz),

7.68 (t, 1H, J ) 8.1), 7.90 (m, 2H), 8.33 (dd, 1H, J ) 8.6, 1.5

Hz), 8.48 (d, 1H, J ) 9.6 Hz), 8.71 (d, 1H, J ) 8.2 Hz), 8.95

(dd, 1H, J ) 7.3, 1.4 Hz), 10.90 (br. d, 1H). MS (DCI/NH₃) m/e

417 (M + H)⁺.

9-Br om o-4-m et h oxy-b en zo[a ]p h en a zin e-11-ca r b oxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4t′) was pre-

pared from 9-bromo-4-methoxy-benzo[a]phenazine-11-carbox-

ylic acid (24 mg, 0.064 mmol) and N,N-dimethylethylenediamine

(0.5 mL, 4.25 mmol) to give the title compound as a yellow

1

solid (24 mg, 86%), mp 161-163 °C. H NMR δ 2.33 (s, 6H),

2.74 (t, 2H), 3.85 (q, 2H), 4.09 (s, 3H), 7.10 (d, 1H), 7.90-8.04

(m, 3H), 8.38 (dd, 1H), 8.55 (t, 1H), 8.92 (dd, 1H), 10.73 (br.t,

1H). MS (DCI/NH₃) m/e 453/455 (M + H)⁺, 1:1.

The following compounds were prepared in an analogous

manner from the appropriate substituted benzo[a]-11-carbox-

ylic acid activated as the acid chloride and reacted with the

appropriate amine using general method B.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (1-

d im eth yla m in om eth yl-2-p h en yl-eth yl)-a m id e (4o′′) was

prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

(98 mg, 0.32 mmol) and N¹,N¹-dimethyl-3-phenyl-propane-1,2-

diamine dihydrochloride salt (81 mg, 0.32 mmol) in the

presence of triethylamine (100 µL, 7.14 mmol) to give the title

compound as a yellow solid (70 mg, 47%), mp 154-158 °C. ¹H

NMR (CDCl₃) δ 2.31 (s, 6H), 2.60 (dd, 1H, J ) 12.5, 6.1 Hz),

2.76 (dd, 1H, J ) 12.4, 8.3 Hz), 3.21 (dd, 1H, J ) 13.7, 5.5

Hz), 3.27 (dd, 1H, J ) 13.7, 5.9 Hz), 4.09 (s, 3H), 4.83 (m, 1H),

7.04 (t, 1H, J ) 7.3 Hz), 7.16 (t, 2H, J ) 7.5), 7.22 (d, 1H, J )

8.0 Hz), 7.32 (d, 2H, J ) 7.1), 7.55 (t, 1H, J ) 8.1 Hz), 7.96 (t,

1H, J ) 9.2 Hz), 7.99 (d, 1H, J ) 8.3 Hz), 8.24 (d, 1H, J ) 8.2

Hz), 8.42 (dd, 1H, J ) 8.5, 1.6 Hz), 8.55 (d, 1H, J ) 9.4 Hz),

9.02 (dd, 1H, J ) 7.2, 1.5 Hz), 11.0 (br. d, 1H). MS (DCI/NH3)

m/e 465 (M + H)⁺.

3-Dim et h yla m in o-2-[(4-m et h oxy-b en zo[a ]p h en a zin e-

11-ca r bon yl)-a m in o]-p r op ion ic a cid m eth yl ester (4r ′′)

was prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic

acid (1.9 g, 6.25 mmol) and 4-aza-^DL-leucine methyl ester

hydrochloride (1 g, 6.9 mmol) in the presence of pyridine (30

mL). The product was purified using preparative HPLC

(isocratic 60% water/40% MeCN) to yield the trifluoroacetate

salt of the desired compound as a yellow solid (165 mg, 5%),

1

mp 200 °C dec. H NMR (DMSO-d₆) δ 3.04(s, 6H), 3.90-3.70

(m, 2H), 3.96 (s, 3H), 4.10 (s, 3H), 5.61 (m, 1H), 7.34 (d, 1H,

J ) 7.95 Hz), 7.88 (t, 1H, J ) 8.15 Hz), 7.97 (m, 2H), 8.52 (dd,

1H, J ) 8.6, 1.5 Hz), 8.60 (d, 1H, J ) 9.52 Hz), 8.94 (m, 2H),

11.93 (br. d, 1H). MS (DCI/NH₃) m/e 433 (M + H)⁺.

4,10-Dim eth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid

(2-(d im eth yla m in o)-eth yl)-a m id e (4v′) was prepared from

4,10-dimethoxy-benzo[a]phenazine-11-carboxylic acid (628 mg,

1.88 mmol) and N,N-dimethylethylenediamine (1.03 mL, 9.4

mmol) to give the title compound as a yellow solid (116 mg,

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-1-(S)-m eth yl-eth yl)-a m id e ((S)-4j′′) was

prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

1

15%), mp 119 °C dec. H NMR (DMSO-d₆) 2.25 (s, 6H), 2.80

734 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

(362 mg, 1.19 mmol) and (S)-N¹,N¹-dimethyl-propane-1,2-

diamine dihydrochloride salt (209 mg, 1.19 mmol) in the

presence of triethylamine (0.35 mL, 2.5 mmol) to give the title

compound as a yellow glass (9 mg, 2%). ¹H NMR (CDCl₃) δ

1.53 (d, 3H, J ) 6.5 Hz), 2.37 (s, 6H), 2.55 (dd, 1H, J ) 12.2,

6.2 Hz), 2.84 (dd, 1H, J ) 12.2, 7.8 Hz), 4.10 (s, 3H), 4.55-

4.65 (m, 1H), 7.28-7.30 (m, 1H), 7.75-7.80 (m, 1H), 7.95-

8.02 (m, 2H), 8.42 (dd, 1H, J ) 8.4, 1.5 Hz), 8.57 (d, 1H, J )

9.5 Hz), 8.81 (d, 1h, J ) 8.2 Hz), 9.02 (dd, 1H, J ) 7.3, 1.6

Hz), 10.9 (d, 1H, J ) 7.2 Hz). MS (DCI/NH₃) m/e 389 (M +

H)⁺. This compound was 80% optically pure as determined by

NMR using the chiral shift reagent 2,2,2-trifluoro-1-(9-anthra-

nyl) ethanol and comparing to the racemate (4j′′) and the

>95% optically pure enantiomer ((R)-4j′′).

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-1(R)-m eth yl-eth yl)-a m id e ((R)-4j′′) was

prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

(207 mg, 0;68 mmol) and (R)-N¹,N¹-dimethyl-propane-1,2-

diamine diydrochloride salt (238 mg, 1.36 mmol) in the

presence of triethylamine (0.3 mL, 21 mmol) to give the title

(150 mg, 0.49 mmol) and 3-amino-4-(dimethylamino)-butan-

1-ol diydrochloride salt (122 mg, 0.58 mmol) in the presence

of triethylamine (1.5 mL, 10.8 mmol) to give the title compound

as a yellow solid (112 mg, 54%), mp 143-146 °C. ¹H NMR

(CDCl₃) δ 1.99 (m, 1H), 2.10 (m, 1H), 2.35 (s, 6H), 2.63 (dd,

1H, J ) 12.2, 2.1 Hz), 3.44 (dd, 1H, J ) 12.2, 6.0 Hz), 3.80-

3.67 (m, 2H), 4.02 (s, 3H), 4.59 (q, 1H, J ) 7.1 Hz), 7.19 (d,

1H, J ) 7.7 Hz), 7.69 (t, 1H, J ) 8.1 Hz), 7.94-7.86 (m, 2H),

8.35 (dd, 1H, J ) 8.5, 1.5 Hz), 8.48 (d, 1H, J ) 9.5 Hz), 8.53

(d, 1H, J ) 8.2 Hz), 8.92 (dd, 1H, J ) 7.2, 1.5 Hz), 11.05 (d,

1H). MS (DCI/NH₃) m/e 419 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

piper idin -1-yl-eth yl)-am ide (4d ′′) was prepared from 4-meth-

oxy-benzo[a]phenazine-11-carboxylic acid (250 mg, 0.82 mmol)

and 1-(2-aminoethyl) piperidine (0.59 mL, 4.1 mmol) to give

the title compound as a yellow solid (95 mg, 28%), mp 141-

143 °C. ¹H NMR (CDCl₃) δ 1.35 (m, 2H), 1.45 (m, 4H), 2.47

(m, 4H), 2.7 (t, 2H), 3.7 (m, 2H), 4.07 (s, 3H), 7.5 (d, 1H) 7.83

(m, 1H), 7.95 (d, 1H), 8.07 (m, 1H), 8.43 (m, 1H), 8.5 (m, 1H),

8.62 (m, 1H), 8.73 (m, 1H), 10.05 (br, 1H). MS (DCI/NH₃) m/e

414 (M + H)⁺.

compound as a yellow solid (73 mg, 28%), mp 116-118 °C;

1

[R]²³-120.0° (c ) 1.00, CHCl₃); H NMR (CDCl₃) δ 1.53 (d,

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

m or ph olin -4-yl-eth yl)-am ide (4f′′) was prepared from 4-meth-

oxy-benzo[a]phenazine-11-carboxylic acid (250 mg, 0.82 mmol)

and 4-(2-aminoethyl) morpholine (0.54 mL, 4.11 mmol) to give

the title compound as a yellow solid (187 mg, 54%), mp 201-

D

3H, J ) 6.5 Hz), 2.38 (s, 6H,), 2.51-2.60 (m, 1H), 2.82-2.90

(m, 1H), 4.11 (s, 3H), 4.56-4.65 (m, 1H), 7.29 (d, 1H, J ) 8.2

Hz), 7.78 (t, 1H, J ) 8.1 Hz), 7.96-8.02 (m, 2H), 8.43 (d, 1H,

J ) 8.4 Hz), 8.58 (d, 1H, J ) 9.5), 8.82 (d, 1H, J ) 8.4 Hz),

9.03 (d, 1H, J ) 7.3 Hz). MS (DCI/NH₃) m/e 389 (M + H)⁺.

This compound was >95%% optically pure as determined by

NMR using the chiral shift reagent 2,2,2-trifluoro-1-(9-anthra-

nyl)ethanol and comparing to the racemate (4j′′). Treatment

of the above compound with HCl in ether and recrystallization

from ether and aqueous ethanol gave the hydrated monohy-

1

202 °C. H NMR (CDCl₃) δ 2.62 (t, 4H), 2.90 (t, 2H), 3.65 (t,

4H), 3.93 (m, 2H), 4.20 (s, 3H), 7.25 (m, 1H), 7.70 (t, 1H), 8.00

(m, 2H), 8.45 (m, 1H), 8.60 (d, 1H), 8,75 (d, 1H), 9.0 (d, 1H),

10.9 (br, 1H). MS (DCI/NH₃) m/e 417 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

p yr r olid in -1-yl-et h yl)-a m id e (4h ′′) was prepared from

4-methoxy-benzo[a]phenazine-11-carboxylic acid (220 mg, 0.72

mmol) and 1-(aminoethyl)pyrrolidine (0.46 mL, 3.6 mmol) to

yield the title compound as a yellow solid (222 mg, 77%), mp

148-150 °C. ¹H NMR (CDCl₃) δ 1.82 (t, 4H), 2.75 (t, 4H), 3.20

(t, 2H), 3.90-3.99 (m, 2H), 4.11 (s, 3H), 7.26-7.31 (m, 1H),

7.78 (t, 1H), 7.95-7.98 (m, 1H), 7.98-8.03 (m, 1H) 8.4-8.46

(m, 1H), 8.55-8.61 (m, 1H), 8.78-8.83 (m, 1H) 8.98-9.30 (m,

1H). MS (DCI/NH₃) m/e 401 (M + H)⁺.

drochloride salt as a yellow solid, mp 235-236 °C; [R]²³

D

-127.4° (c ) 0.833, H₂O); MS (DCI/NH₃) m/e 389 (M + H)⁺.

Anal. (C₂₃H₂₅ClN₄O₂‚3(¹/₂)H₂O) C, H; calcd 6.5; found 5.8;

Cl; N.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-1(S)-h yd r oxym eth yl-eth yl)-a m id e ((S)-

4v′′) was prepared from 4-methoxy-benzo[a]phenazine-11-

carboxylic acid (709 mg, 2.33 mmol) and (S)-2-amino-3-

(dimethylamino)-propan-1-ol dihydrochloride salt (738 mg,

3.86 mmol) in the presence of triethylamine (10.8 mL, 77.4

mmol) to give the title compound as a yellow solid (165 mg,

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

dieth ylam in o-eth yl)-am ide (4c′′) was prepared from 4-meth-

oxy-benzo[a]phenazine-11-carboxylic acid (255 mg, 0.84 mmol)

and N,N-diethylethylenediamine (0.59 mL, 4.2 mmol) to give

the title compound as an orange solid (70 mg, 34%), mp 74 °C

1

18%), mp 88 °C dec. H NMR (CDCl₃) δ 2.39 (6H, s), 2.88 (dd,

1H, J ) 12.4, 7.4 Hz), 2.96 (dd, 1H, J ) 12.4, 6.0 Hz), 4.06 (s,

3H), 4.17 (m, 2H), 4.63 (m, 1H), 5.32 (br. s, 1H), 7.21 (d, 1H,

J ) 8.0 Hz), 7.72 (t, 1H, J ) 8.0 Hz), 7.86 (d, 1H, J ) 9.5 Hz),

7.90 (m, 1H), 8.35 (dd, 1H, J ) 8.6, 1.5 Hz), 8.41 (d, 1H, J )

9.5 Hz), 8.76 (d, 1H, J ) 8.2 Hz), 8.91 (dd, 1H, J ) 7.2, 1.5

Hz), 10.97 (br. s, 1H). MS (DCI/NH₃) m/e 405 (M + H)⁺. This

compound was >95%% optically pure as determined by NMR

using the chiral shift reagent 2,2,2-trifluoro-1-(9-anthranyl)

ethanol and comparing to the racemate (4p ′′).

1

dec. H NMR (DMSO-d₆) δ 0.99 (t, 6H), 2.65 (q, 4H), 2.79 (t,

2H), 3.65-3.72 (m, 2H), 4.07 (s, 3H), 7.49-7.53 (m, 1H), 7.8-

7.87 (m, 1H), 7.98 (d, 1H), 8.04-8.11 (m, 1H), 8.45 (m, 1H),

8.51 (m, 1H), 8.67 (m, 1H), 8.89 (m, 1H),10.15-10.25 (br, 1H).

MS (DCI/NH₃) m/e 403 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid {2-

[bis-(2-h yd r oxy-eth yl)-a m in o]-eth yl}-a m id e (4g′′) was pre-

pared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

(250 mg, 0.82 mmol) and N,N-bis(2-hydroxyethyl)ethylenedi-

amine (0.57 mL, 4.11 mmol) to give the title compound as a

yellow gum (131 mg, 37%). ¹H NMR (CDCl₃) δ 2.75 (t, 4H),

3.00 (t, 2H), 3.56 (t, 4H), 3.80-3.89 (m, 2H), 4.40 (s, 3H), 7.16-

7.22 (m, 1H), 7.69-7.75 (m, 1H), 7.85-7.89 (m, 1H), 7.89-

7.93 (m, 1H), 8.33-8.39 (m, 1H), 8.47-8.51 (m, 1H), 8.51-

8.55 (m, 1H), 8.89-8.95 (m, 1H), 11.29 (br, 1H). MS (DCI/NH₃)

m/e 435 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (1-

m eth yl-p yr r olid in -3-(R)-yl)-a m id e (4t′′) was prepared from

4-methoxy-benzo[a]phenazine-11-carboxylic acid (586 mg, 1.93

mmol) and 1-methyl-3-(R)-aminopyrrolidine dihydrochloride

salt (383 mg, 2.21 mmol) in the presence of triethylamine (0.92

mL, 6.5 mmol) to yield the title compound as a yellow solid

(138 mg, 19%), mp 164-167 °C. ¹H NMR (CDCl₃) δ 2.02-2.10

(m, 1H), 2.40-2.46 (m, 1H), 2.52-2.61 (m, 1H), 2.58 (s, 3H),

2.75-2.79 (m, 1H), 2.99-3.02 (m, 1H), 3.13-3.18 (m, 1H), 4.10

(s, 3H), 4.90-4.98 (m, 1H), 7.26 (d, 1H), 7.75-7.80 (m, 1H),

7.95-8.00 (m, 2H), 8.42(dd, 1H, J ) 8.4, 1.5 Hz), 8.58 (d, 1H,

J ) 9.6 Hz), 8.99-9.03 (m, 2H), 11.32 (d, 1H). MS (DCI/NH₃)

m/e 387 (M + H)⁺. Anal. (C₂₃H₂₂N₄O₂) C, H, N.

4-Meth oxy-ben zo[a ]ph en azin e-11-car boxylic acid (1(S)-

d im eth yla m in om eth yl-2(S)-h yd r oxy-p r op yl)-a m id e ((S)-

4u ′′) was prepared from 4-methoxy-benzo[a]phenazine-11-

carboxylic acid (500 mg, 1.65 mmol) and 3(S)-amino-4-

(dimethylamino)-butan-2(S)-ol dihydrochloride salt (1.1 g, 5.25

mmol) in the presence of triethylamine (4.57 mL, 32.9 mmol)

to give the title compound as an orange solid (161 mg, 23%),

1

mp 189-190 °C. H NMR (CDCl₃) δ 1.37 (d, 3H, J ) 6.4 Hz),

2.36 (s, 6H), 2.88 (dd, 1H, J ) 13.1, 4.8 Hz), 3.06 (dd, 1H, J )

13.1, 5.2 Hz), 3.48 (s, 1H), 4.06 (s, 3H), 4.43 (m, 2H), 7.35 (d,

1H, J ) 8.0 Hz), 7.75 (t, 1H, J ) 8.2 Hz), 7.99-7.91 (m, 2H),

8.42 (dd, 1H, J ) 8.6, 1.5 Hz), 8.53 (d, 1H, J ) 9.6 Hz), 9.02

(dd, 1H, J ) 7.3, 1.5 Hz), 9.27 (d, 1H, J ) 8.2 Hz), 11.21 (br.

s, 1H). MS (DCI/NH₃) m/e 419 (M + H)⁺. Anal. (C₂₄H₂₆N₄O₃)

C, H, N. This compound was >95% optically pure as deter-

mined by NMR using the chiral shift reagent 2,2,2-trifluoro-

1-(9-anthranyl) ethanol.

4-Meth oxy-ben zo[a ]ph en azin e-11-car boxylic acid [1-(di-

m eth yla m in o)-1-(2-h yd r oxyeth yl)]-eth yla m id e (4q′′) was

prepared from 4-methoxy-benzo[a]phenazine-11-carboxylic acid

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 735

(R)-4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid

(1-d im eth yla m in om eth yl-2-m eth yl-p r op yl)-a m id e ((R)-

4n ′′) was prepared from 4-methoxy-benzo[a]phenazine-11-

carboxylic acid (504 mg, 1.66 mmol) and (R)-3N¹,N¹-trimethyl-

butane-1, 2-diamine dihydrochloride (504 mg, 2.48 mmol) in

the presence of triethylamine (3.0 mL, 21.3 mmol) to give the

title compound as a yellow solid (350 mg, 51%). ¹H NMR

(CDCl₃) δ 1.10 (d, 3H, J ) 2.4 Hz), 1.12 (d, 3H, J ) 2.5 Hz),

2.22 (m, 1H), 2.34 (s, 6H), 2.64 (dd, 1H, J ) 12.6, 5.1 Hz), 2.89

(dd, 1H, J ) 12.6, 9.0 Hz), 4.09 (s, 3H,), 4.56 (m, 1H), 7.26 (d,

1H, J ) 7.7 Hz), 7.75 (t, 1H, J ) 8.1 Hz), 7.97 (m, 2H), 8.41

(dd, 1H, J ) 8.5, 1.5 Hz), 8.56 (d, 1H, J ) 9.5 Hz), 8.80 (d, 1H,

J ) 8.3 Hz), 9.03 (dd, 1H, J ) 7.3, 1.6 Hz), 11.0 (br d, 1H). MS

(DCI/NH₃) m/e 417 (M + H)⁺. This compound was >95%%

optically pure as determined by NMR using the chiral shift

reagent 2,2,2-trifluoro-1-(9-anthranyl) ethanol.

NMR (CDCl₃) δ 2.40 (s, 6H), 2.70 (t, 2H), 2.90 (s, 6H), 3.85 (q,

2H), 7.40 (d, 1H), 7.70 (t, 1H), 7.90 (dd, 2H), 8.40 (dd, 1H),

8.53 (d, 1H), 8.95 (dd, 1H), 9.10 (d, 1H), 10.85 (br, 1H). MS

(DCI/NH₃) m/e 388 (M + H)⁺.

4-Meth oxy-10-m eth ylam in o-ben zo[a ]ph en azin e-11-car -

boxylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4z′) was

prepared from 4-methoxy-10-methylamino-benzo[a]phenazine-

11-carboxylic acid methyl ester (104 mg, 0.30 mmol) and N,N-

dimethylethylenediamine (5 mL, 45 mmol) to yield the title

compound as an orange solid (32 mg, 27%), mp 310-312 °C.

¹H NMR (CDCl₃) δ 2.40 (s, 6H), 2.77 (t, 2H, J ) 6.5 Hz), 3.18

(d, 3H, J ) 5.1 Hz), 3.83 (q, 2H, J ) 6.2 Hz), 4.10 (s, 3H), 7.20

(d, 1H, J ) 7.8 Hz), 7.56 (d, 1H, J ) 9.6 Hz), 7.70 (t, 1H, J )

8.1 Hz), 7.90 (d, 1H, J ) 9.5 Hz), 8.12 (d, 1H, J ) 9.6 Hz),

8.40 (d, 1H, J ) 9.5 Hz), 8.80 (d, 1H, J ) 8.3 Hz), 11.12 (m,

1H), 11.92 (m, 1H). MS (DCI/NH₃) m/e 404 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2,3-

bis)-(d im eth yla m in o)-p r op yl)a m id e (4b′′) was prepared

from 4-methoxy-benzo[a]phenazine-11-carboxylic acid (210 mg,

0.69 mmol) and N¹,N¹,N²,N²-tetramethyl-propane-1,2,3-tri-

amine trihydrochloride (273 mg, 1.1 mmol) to yield the title

compound as a yellow solid (90 mg, 30%), mp 126.5-128 °C.

¹H NMR (CDCl₃) δ 2.25 (s, 6H), 2.32-2.38 (m, 1H), 2.52 (s,

6H), 2.56-2.62 (m, 1H), 3.00-3.09 (m, 1H), 3.65-3.74 (m, 1H),

4.01-4.09 (m, 1H), 4.11 (s, 3H), 7.28 (d, 1H, J ) 7.7 Hz), 7.78

(t, 1H, J ) 8.0 Hz), 7.96-8.01 (m, 2H), 8.42 (d, 1H, J ) 8.5

Hz), 8.58 (d, 1H, J ) 9.6 Hz), 8.98-9.03 (m, 2H). MS (DCI/

NH₃) m/e 432 (M + H)⁺.

10-Am in o-4-m eth oxy-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4y′) was pre-

pared from 10-amino-4-methoxy-benzo[a]phenazine-11-car-

boxylic acid methyl ester (100 mg, 0.30 mmol) and N,N-

dimethylethylenediamine (3 mL, 27 mmol) to give the title

compound as a red solid (16 mg, 14%), mp 170-172 °C. ¹H

NMR (CDCl₃) δ 2.42 (s, 6H), 2.70 (t, 2H J ) 6.4 Hz), 3.87 (q,

2H, J ) 6.1 Hz), 4.09 (s, 3H), 7.20 (d, 1H, J ) 7.7 Hz), 7.23 (d,

1H, J ) 9.3 Hz), 7.72 (t, 1H, J ) 8.1 Hz), 7.91 (d, 1H, J ) 9.5

Hz), 8.01 (d, 1H, J ) 9.3 Hz), 8.43 (d, 1H, J ) 9.3 Hz), 8.80

(d, 1H, J ) 8.2 Hz), 11.75 (m, 1H). MS (DCI/NH₃) m/e 390

(M + H)⁺.

The following compounds were prepared in an analogous

manner from the appropriate substituted benzo[a]-11-carbox-

ylic acid methyl ester and the appropriate amine using general

method C.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

m eth yla m in o-eth yl)-a m id e (4i′′) was prepared from 4-meth-

oxy-benzo[a]phenazine-11-carboxylic acid methyl ester (95.5

mg, 0.3 mmol) and N-methylethylenediamine (1 mL, 11 mmol)

to give the title compound as a yellow solid (34 mg, 32%), mp

104-106 °C. ¹H NMR (CDCl₃) δ 2.64 (s, 3H), 3.12 (t, 2H), 3.90-

3.96 (m, 2H), 4.11 (s, 3H), 7.29 (s, 1H), 7.63-7.71 (m, 1H),

7.92-7.95 (m, 1H), 7.95-8.01 (m, 1H), 8.38-8.45 (m, 1H),

8.55-8.61 (m, 1H), 8.89-8.95 (m, 1H), 8.95-9.02 (m, 1H),

11.08 (br, 1H). MS (DCI/NH₃) m/e 361 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (3-

(d im eth yla m in o)-p r op yl)-a m id e (4a ′′) was prepared from

4-methoxy-benzo[a]phenazine-11-carboxylic acid methyl ester

(50 mg, 0.16 mmol) and 3-(dimethylamino) propylamine (1.0

mL, 8.1 mmol) to give the title compound as a yellow solid (37

1

mg, 64%), mp 97-99 °C. H NMR (DMSO-d₆) 1.92 (t, 2H, J )

7.0 Hz), 2.2 (s, 6H), 3.62 (q, 2H, J ) 6.7 Hz), 4.07 (s, 3H), 7.3

(d, 1H, J ) 8.0 Hz), 7.85 (t, 1H, J ) 8.1 Hz), 7.98 (d, 1H, J )

9.5 Hz), 8.05 (t, 1H, J ) 7.3 Hz), 8.42 (d, 1H, J ) 8.6 Hz), 8.49

(d, 1H, J ) 9.6 Hz), 8.52 (d, 1H, J ) 6.9 Hz), 8.72 (d, 1H, J )

8.1 Hz), 9.95 (br, 1H). MS (DCI/NH₃) m/e 389 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (3-

a m in o-2-h yd r oxy-p r op yl)-a m id e (4e′′) was prepared from

4-methoxy-benzo[a]phenazine-11-carboxylic acid methyl ester

(50 mg, 0.16 mmol) and 1,3-diamino-2-hydroxypropane (500

mg, 5.6 mmol) to yield the title compound as a beige solid (24

mg, 40%), mp 199 °C dec. ¹H NMR (CDCl₃) δ 2.81 (dd, 1H,

J ) 12.6, 7.5 Hz), 2.97 (dd, 1H, J ) 12.6, 3.7 Hz), 3.70-3.75

(m, 1H), 3.90-4.00 (m, 2H), 4.01 (s, 3H), 7.15 (d, 1H, J ) 8.0

Hz), 7.65-7.71 (m, 1H), 7.81 (d, 1H, J ) 9.5 Hz), 7.85-7.90

(m, 1H), 8.31 (d, 1H, J ) 7.5 Hz), 8.42 (d, 1H, J ) 9.6 Hz),

8.80 (d, 1H, J ) 8.2 Hz), 8.88 (d, 1H, J ) 6.1 Hz), 11.2 (br,

1H). MS (DCI/NH₃) m/e 377 (M + H)⁺.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-p r op yl)-a m id e (4k ′′) was prepared from

4-methoxy-benzo[a]phenazine-11-carboxylic acid methyl ester

(100 mg, 0.31 mmol) and N²,N²-dimethyl-propane-1,2-diamine

(0.5 mL, 3.7 mmol) to give the title compound as a yellow solid

(30 mg, 25%). ¹H NMR (CDCl₃) δ 1.18 (d, 3H, J ) 6.5 Hz),

2.45 (s, 6H), 3.08 (m, 1H), 3.70 (m, 1H), 3.93 (m, 1H), 4.05 (s,

3H), 7.23 (m, 1H), 7.75 (m, 1H), 7.95 (m, 2H), 8.40 (m, 1H),

8.52 (m, 1H), 8.97 (m, 2H), 10.76 (br s, 1H). MS (DCI/NH₃)

m/e 389 (M + H)⁺.

In t er con ver sion of Su bst it u t ed Ben zo[a ]-11-ca r b ox-

am ides. 4-Hydr oxy-ben zo[a ]ph en azin e-11-car boxylic Acid

(2-(Dim eth ylam in o)-eth yl)-am ide Hydr obr om ide Salt (4f).

To a solution of 4-methoxy-benzo[a]phenazine-11-carboxylic

acid (2-(dimethylamino)-ethyl)-amide (727 mg, 1.94 mmol) in

dry dichloromethane (15 mL) cooled to -5 °C was added a 1.0

M solution of boron tribromide in dichloromethane (13.6 mL,

13.6 mmol). After being stirred for 4 h, the reaction mixture

was poured onto ice/water (50 mL), yielding a precipitate which

was collected by filtration. This was triturated from a hot

methanol/ethyl acetate mixture to yield the title compound as

1

a beige solid (505 mg, 72%), mp 220 °C dec. H NMR (DMSO-

d₆) 2.91 (s, 6H), 3.45 (t, 2H), 3.97-4.02 (m, 2H), 7.37 (d, 1H),

7.72-7.78 (m, 1H), 7.92(d, 1H), 8.08-8.12(m, 1H), 8.45-8.51

(m, 2H), 8.54-8.61 (m, 2H), 9.42 (br, 1H), 10.13 (t, 1H), 10.67

(s, 1H). MS (EI) m/e 360 (M⁺). Anal. (C₂₁H₂₀N₄O₂) C, H, N.

3-Hyd r oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(dim eth ylam in o)-eth yl)-am ide (4e). To a solution of 3-meth-

oxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-

ethyl)-amide (170 mg, 0.45 mmol) in dry DMF (3 mL) was

added sodium thioethoxide (380 mg, 4.5 mmol). The reaction

mixture was then heated to reflux under argon for 3 h, cooled

to room temperature, and acidified with 1 M HCl. The volatile

components were removed in vacuo. The residue was purified

using column chromatography (20% methanol in dichlo-

romethane) to yield the title compound as a red solid (142 mg,

1

88%), mp 230 °C dec. H NMR (DMSO-d₆) δ 2.50 (s, 6H), 2.58

(br, 2H), 3.88 (br, 2H), 7.41 (m, 2H), 7.89 (d, 1H, J ) 9.4 Hz),

8.03 (m, 1H), 8.13 (d, 1H, J ) 9.4 Hz), 8.42 (dd, 1H, J ) 1.51,

8.4 Hz), 8.66 (m, 1H), 9.14 (br, 1H), 10.40 (br, 1H), 10.58 (s,

1H). MS (DCI/NH₃) m/e 361 (M + H)⁺. Anal. (C₂₁H₂₀N₄O₂) C,

H, N.

2-Hyd r oxy-ben zo[a ]p h en a zin e-11-ca r boxylic a cid (2-

(d im eth yla m in o)-eth yl)-a m id e (4d ) was prepared in an

analogous manner from 2-methoxy-benzo[a]phenazine-11-car-

boxylic acid (2-(dimethylamino)-ethyl)-amide (200 mg, 0.54

4-Dim et h yla m in o-b en zo[a ]p h en a zin e-11-ca r b oxylic

a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4w ) was prepared

from the mixture of 4-(dimethylamino)-benzo[a]phenazine-11-

carboxylic acid methyl ester and 3-(dimethylamino)-benzo[a]-

phenazine-11-carboxylic acid methyl ester (289 mg, 0.87 mmol)

and N,N-dimethylethylenediamine (3 mL, 27 mmol), followed

by purification on silica eluting with 10% methanol in dichlo-

romethane to remove the minor isomer, to give the title

compound as a yellow solid (75 mg, 22%), mp 114-115 °C. ¹H

736 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

mmol) to give the title compound as a yellow solid (151 mg,

78%), mp 172-174 °C. ¹H NMR (DMSO-d₆) δ 2.89 (s, 6H), 3.46

(t, 2H, J ) 6.0 Hz), 4.07 (q, 2H, J ) 6.0 Hz), 7.40 (m, 1H),

7.81 (d, 1H, J ) 9.2 Hz), 8.02 (d, 1H, J ) 8.5 Hz), 8.10 (m,

1H), 8.18 (d, 1H, J ) 9.2 Hz), 8.49 (m, 2H), 8.56 (m, 1H), 10.13

(br.t, 1H), 10.56 (s, 1H). MS (DCI/NH₃) m/e 361 (M + H)⁺.

4-Cyan om eth oxy-ben zo[a ]ph en azin e-11-car boxylic acid

(2-(d im eth yla m in o)-eth yl)-a m id e (4e′). To a suspension of

4-hydroxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethyl-

amino)-ethyl)-amide hydrobromide salt (230 mg, 0.52 mmol)

in dry DMF (3 mL) were added potassium tert-butoxide (175

mg, 1.56 mmol) and then bromoacetonitrile (47 µL, 0.68 mmol).

The reaction mixture was heated to 100 °C for 1 h and then

cooled to room temperature, diluted with ethyl acetate (30 mL),

washed with sodium carbonate solution (30 mL) and brine (30

mL), and dried (MgSO₄), and the solvent was removed in vacuo

to the crude product. This was purified using flash chroma-

tography on silica eluting with 25% MeOH in ethyl acetate to

yield the title compound as a yellow solid (74 mg, 36%), mp

4.83 (s, 2H), 7.12 (d, 1H, J ) 7.8 Hz), 7.71-7.76 (m, 1H), 7.95-

8.02 (m, 2H), 8.45 (d, 1H, J ) 8.4, 1.4 Hz), 8.63 (d, 1H, J ) 9.6

Hz), 9.03 (dd, 1H, J ) 7.2, 1.6 Hz), 9.15 (d, 1H, J ) 8.1 Hz),

10.80 (br, 1H). MS (DCI/NH₃) m/e 417 (M + H)⁺.

Eth yl-ca r ba m ic a cid 11-(2-(d im eth yla m in o)-eth ylca r -

ba m oyl)-ben zo[a ]p h en a zin -4-yl ester (4h ′). A mixture of

4-hydroxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethyl-

amino)-ethyl)-amide hydrobromide salt (540 mg, 1.2 mmol),

triethylamine (0.51 mL, 3.6 mmol), and ethyl isocyanate (0.29

mL, 3.6 mmol) was stirred in dry DMF (3 mL). The product

slowly precipitated from the reaction mixture and was collected

by filtration and washed with ether to yield the title compound

as a yellow solid (210 mg, 41%), mp 302 °C dec. ¹H NMR

(DMSO-d₆) δ 0.9 (t, 3H, J ) 7.4 Hz), 2.4 (s, 6H), 2.75 (br, 2H),

3.37 (t, 2H, J ) 6.4 Hz), 5.3 (br, 2H), 7.57 (d, 1H, J ) 7.2 Hz),

7.75 (t, 1H, J ) 7.4 Hz), 7.91 (t, 2H, J ) 7.1 Hz), 8.13 (d, 1H,

J ) 9.6 Hz), 8.3 (d, 1H, J ) 8.4 Hz), 8.95 (d, 1H, J ) 6.7 Hz),

9.21 (d, 1H, J ) 7.6 Hz), 10.76 (br, 1H). MS (DCI/NH₃) m/e

432 (M + H)⁺.

Acetic a cid 11-(2-(d im eth yla m in o)-eth ylca r ba m oyl)-

ben zo[a ]p h en a zin -4-yl ester (4g′). A mixture of 4-hydroxy-

benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-

ethyl)-amide (46 mg, 0.13 mmol), triethylamine (71 µL, 0.52

mmol), and acetyl chloride (20 µL, o.29 mmol) in dichlo-

romethane (2 mL) was stirred at room temperature for 2 h.

The volatile components were removed in vacuo, and the

residue was purified using column chromatography on silica

eluting with 10% methanol in dichloromethane to yield the

title compound as a yellow gum (27 mg, 52%). ¹H NMR

(DMSO-d₆) δ 2.37 (s, 6H), 2.52 (s, 3H), 2.65 (t, 2H, J ) 6.0

Hz), 3.72 (q, 2H, J ) 6.0 Hz), 7.74 (d, 1H, J ) 7.0 Hz), 7.99 (t,

1H, J ) 8.0 Hz), 8.08 (d, 1H, J ) 9.6 Hz), 8.12 (t, 1H, J ) 7.8

Hz), 8.31 (d, 1H, J ) 9.6 Hz), 8.47 (d, 1H, J ) 8.0 Hz), 8.75 (d,

1H, J ) 7.0 Hz), 9.38 (d, 1H, J ) 8.0 Hz). MS (DCI/NH₃) m/e

403 (M + H)⁺.

1

188-191 °C. H NMR (CDCl₃) δ 2.36 (s, 6H), 2.68 (t, 2H, J )

5.9 Hz), 3.81-3.84 (m, 2H), 5.02 (s, 2H), 7.30 (d, 1H, J ) 8.1

Hz), 7.71-7.75 (m, 1H), 7.92-7.98 (m, 2H), 8.36-8.41 (m, 2H),

8.96 (dd, 1H, J ) 7.2, 1.5 Hz), 9.15 (d, 1H, J ) 8.3 Hz), 10.70

(br, 1H). MS (DCI/NH₃) m/e 400 (M + H)⁺. Anal. (C₂₃H₂₁N₅O₂)

C, H, N.

The following compounds were prepared in an analogous

manner using 4-hydroxy-benzo[a]phenazine-11-carboxylic acid

(2-(dimethylamino)-ethyl)-amide hydrobromide salt and the

appropriate alkylating reagent.

4-(2-Mor ph olin -4-yl-eth oxy)-ben zo[a ]ph en azin e-11-car -

boxylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4p ′) was

prepared using N-(2-chloroethyl)morpholine hydrochloride to

yield the title compound as a yellow solid (51%), mp 135-137

°C. ¹H NMR (CDCl₃) δ 2.45 (s, 6H), 2.68-2.71 (m, 4H), 2.75

(t, 2H J ) 6.0 Hz), 3.01 (t, 2H, J ) 5.6 Hz), 3.75-3.79 (m,

4H), 3.88-3.92 (m, 2H), 4.36 (t, 2H, J ) 5.6 Hz), 7.25-7.27

(m, 1H), 7.71-7.75 (m, 1H), 7.95-8.01 (m, 2H), 8.41 (dd, 1H,

J ) 8.5, 1.5 Hz), 8.55 (d, 1H, J ) 9.5 Hz), 9.01-9.05 (m, 2H),

10.8 (br, 1H). MS (DCI/NH₃) m/e 474 (M + H)⁺.

[11-(2-Dim eth yla m in o-eth ylca r ba m oyl)-ben zo[a ]p h en -

a zin -4-yloxy]-a cetic a cid tr iflu or oa ceta te sa lt (4n ′). [11-

(2-Dimethylamino-ethylcarbamoyl)-benzo[a]phenazin-4-yloxy]-

acetic acid tert-butyl ester was prepared using tert-butyl

1

4-(3-Cya n o-p r op oxy)-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4f′) was pre-

pared using 4-bromobutyronitrile to give the title compound

bromoacetate (15%). H NMR (CDCl₃) δ 1.52 (s, 9H), 2.75 (t,

2H), 3.90 (q, 2H), 4.80 (s, 2H) 7.13 (d, 1H) 7.72 (t, 1H), 7.95

(d, 1H), 7. 98 (d, 1H), 8.41 (dd, 1H), 8.65 (d, 1H), 9.00 (dd,

1H), 9.07 (d, 1H). To a solution of [11-(2-(dimethylamino)-

ethylcarbamoyl)-benzo[a]phenazin-4-yloxy]-acetic acid tert-

butyl ester (18 mg, 0.04 mmol) in dry dichloromethane (1 mL)

was added trifluoroacetic acid (1 mL). After the mixture was

stirred for 4 h, the solvent was removed in vacuo to yield crude

product. Trituration with ether yielded the title compound as

a yellow solid (10 mg, 47%), mp 192-194 °C. ¹H NMR (DMSO-

d₆) δ 2.70 (s, 6H), 3.18 (t, 2H), 3.89-3.95 (m, 2H), 5.00 (s, 2H),

7.42 (d, 1H, J ) 8.0 Hz), 7.81-7.86 (m, 1H), 8.00 (d, 1H, J )

8.0 Hz), 8.08-8.12 (m, 1H), 8.46 (d, 1H, J ) 7.8 Hz), 8.55-

8.62 (m, 2H), 8.85 (d, 1H, J ) 8.1 Hz), 10.1 (br, 1H). MS (DCI/

NH₃) m/e 419 (M + H)⁺.

1

as a yellow solid (37%), mp 168-170 °C. H NMR (CDCl₃) δ

2.35-2.38 (m, 2H), 2.45 (s, 6H), 2.73-2.75 (m, 4H), 3.89-3.93

(m, 2H), 4.37 (d, 2H, J ) 5.7 Hz), 7.28-7.30 (m, 1H), 7.78-

7.80 (m, 1H), 7.98-8.01 (m, 2H), 8.45 (dd, 1H, J ) 8.4, 1.5

Hz), 8.56 (d, 1H, J ) 9.7 Hz), 9.05 (dd, 1H, J ) 7.2, 1.5 Hz),

9.08 (d, 1H, J ) 8.2 Hz), 10.8 (br, 1H). MS (DCI/NH₃) m/e 428

(M + H)⁺.

4-(3-Dim eth yla m in o-p r op oxy)-ben zo[a ]p h en a zin e-11-

ca r boxylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4o′)

was prepared using 3-dimethylaminopropyl chloride hydro-

chloride to give the title compound as a yellow solid (44%),

1

mp 145-147 °C. H NMR (CDCl₃) δ 2.15-2.21 (m, 2H), 2.34

(s, 6H), 2.44 (s, 6H), 2.63 (t, 2H, J ) 7.2 Hz), 2.79 (t, 2H, J )

6.0 Hz), 3.90-3.95 (m, 2H), 4.30 (t, 2H, J ) 6.3 Hz), 7.25-

7.27 (m, 1H), 7.71-7.74 (m, 1H), 7.95-8.01 (m, 2H), 8.40 (dd,

1H, J ) 8.40, 1.5 Hz), 8.55 (d, 1H, J ) 9.5 Hz), 8.99-9.03 (m,

2H), 10.89 (br, 1H). MS (DCI/NH₃) m/e 446 (M + H)⁺.

11-(2-Dim eth yla m in o-eth ylca r ba m oyl)-ben zo[a ]p h en -

a zin e-4-ca r boxylic a cid tr iflu or oa ceta te sa lt (4s). 11-(2-

Dimethylamino-ethylcarbamoyl)-benzo[a]phenazine-4-carbox-

ylic acid methyl ester (200 mg, 0.5 mmol) was sonicated in a

mixture of methanol (4 mL) and ammonium hydroxide (20

mL). The suspension was then heated to 50 °C for 92 h. All

volatiles were then removed in vacuo to yield crude product,

which was purified using preparative HPLC on reverse phase

(C₁₈) eluting with water 80% acetonitrile 20% to yield the title

4-Ca r ba m oylm et h oxy-b en zo[a ]p h en a zin e-11-ca r b ox-

ylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4m ′) was pre-

pared using 2-bromoacetamide to give the title compound as

1

a yellow solid (5%), mp 228 °C dec. H NMR (CDCl₃and two

1

compound as a yellow solid (20 mg, 8%), mp 227-229 °C. H

drops of MeOH-d₄) δ 2.32 (s, 6H), 2.68 (t, 2H, J ) 6.2 Hz),

3.78 (t, 2H, J ) 6.2 Hz), 4.68 (s, 2H), 7.20 (d, 1H, J ) 7.8 Hz),

7.70-7.72 (m, 1H), 7.90-7.95 (m,2H), 8.35 (dd, 1H, J ) 1.5,

8.5 Hz), 8.53 (d, 1H, J ) 9.8 Hz), 8.86 (dd, 1H, J ) 7.2, 1.5

Hz), 8.95 (d, 1H, J ) 8.3 Hz). MS (DCI/NH₃) m/e 418 (M +

H)⁺.

4-(2-Oxo-p r op oxy)-b en zo[a ]p h en a zin e-11-ca r b oxylic

a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4l′) was prepared

using chloroacetone to give the title compound as orange

crystals (25%), mp 182-183 °C. ¹H NMR (CDCl₃) δ 2.42 (s,

6H), 2.45 (s, 3H), 2.75 (t, 2H, J ) 5.9 Hz), 3.88-3.91 (m, 2H),

NMR (DMSO-d₆) δ 2.94 (d, 6H, J ) 3.7 Hz), 3.49 (m, 2H), 4.00

(q, 2H, J ) 6.2 Hz), 8.05 (t, 1H, J ) 7.9 Hz), 8.12-8.22 (m,

2H), 8.48-8.58 (m, 3H), 9.17 (d, 1H, J ) 9.8 Hz), 9.41 (d, 1H,

J ) 8.2 Hz), 9.82 (t, 1H). MS (DCI/NH₃) m/e 389 (M + H)⁺.

4-H yd r oxym et h yl-b en zo[a ]p h en a zin e-11-ca r b oxylic

a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4t). To a solution

of 11-(2-(dimethylamino)-ethylcarbamoyl)-benzo[a]phenazine-

4-carboxylic acid methyl ester (317 mg, 0.78 mmol) in tetrahy-

drofuran (18 mL) and 2-propanol (10 mL) at 0 °C was added

lithium borohydride (2.0 M solution in tetrahydrofuran, 1.97

Novel Angular Benzophenazines

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 737

mL, 1.97 mmol). The reaction mixture was stirred at room

temperature overnight and then quenched with ammonium

chloride solution (10 mL). The reaction mixture was extracted

with ethyl acetate (20 mL), dried (MgSO₄), and concentrated

in vacuo. The residue was purified using flash chromatography

on silica eluting with 10% MeOH in dichloromethane to yield

the title compound as a yellow solid (98 mg, 31%), mp 175-

176 °C. ¹H NMR (CDCl₃) δ 2.50 (br s, 6H), 2.85 (br m, 2H),

3.83 (m, 2H), 5.09 (s, 2H), 7.69 (m, 3H), 7.86 (m, 1H), 8.24 (m,

2H), 8.80 (dd, 1H, J ) 7.1, 1.5 Hz), 8.92 (dd, 1H, dd, J ) 7.1,

1.5 Hz) 10.64 (1H, br). MS (CI/NH₃) m/e 375 (M + H)⁺. Anal.

(C₂₂H₂₂N₄O₂‚3(¹/₂)H₂O) C, H, N.

4-(2-Hyd r oxy-eth oxy)-ben zo[a ]p h en a zin e-11-ca r boxyl-

ic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4j′). 4-[2-(tert-

Butyl-dimethyl-silanyloxy)-ethoxy]-benzo[a]phenazine-11-car-

boxylic acid (2-(dimethylamino)-ethyl)-amide was prepared

from 4-[2-(tert-butyl-dimethyl-silanyloxy)-ethoxy]-benzo[a]-

phenazine-11-carboxylic acid (358 mg, 0.8 mmol) and N,N-

dimethylethylenediamine (439 µL, 4.0 mmol). The intermedi-

ate was purified on silica eluting with 5% methanol in

dichloromethane to give (125 mg, 30%).

give the title compound as a yellow solid (10 mg, 16%), mp

204 °C dec. ¹H NMR (MeOH-d₄) δ 3.18 (s, 6H), 3.24 (s, 3H),

3.70 (t, 2H, J ) 6.0 Hz), 4.22 (t, 2H, J ) 6.0 Hz), 8.02-7.92

(m, 3H), 8.09 (dd, 1H, J ) 7.5, 8.2 Hz), 8.42 (d, 1H, J ) 8.5

Hz), 8.65 (d, 1H, J ) 9.7 Hz), 8.79 (d, 1H, J ) 6.2 Hz), 8.98 (d,

1H, J ) 7.9 Hz). MS (DCI/NH₃) m/e 438 (M + H)⁺.

4-Bis-(Meth a n esu lfon yla m in o)-ben zo[a ]p h en a zin e-11-

ca r b oxylic a cid (2-(d im et h yla m in o)-et h yl)-a m id e (4z)

was prepared in a similar manner from 4-amino-benzo[a]-

phenazine-11-carboxylic acid (2-(dimethylamino)-ethyl)-amide

(19.4 mg, 0.054 mmol) using excess methanesulfonyl chloride

(40 µL) and triethylamine (0.1 mL, 0.71 mmol) to give the title

compound as a yellow solid (13 mg, 47%), mp 226-227 °C. ¹H

NMR CDCl₃δ 2.45 (s, 6H), 2.75 (t, 2H), 3.60 (s, 6H), 3.90 (q,

2H), 7.85 (d, 1H), 7.95 (t, 1H), 8.05 (t, 1H), 8.20 (d, 1H), 8.35

(d, 1H), 8.45 (d, 1H), 9.08 (d, 1H), 9.75 (d, 1H), 10.65 (br, 1H).

MS (DCI/NH₃) m/e 516 (M + H)⁺.

4-(Cyan om eth yl-am in o)-ben zo[a ]ph en azin e-11-car box-

ylic Acid (2-(d im eth yla m in o)-eth yl)-a m id e (4v). To a

solution of 4-amino-benzo[a]phenazine-11-carboxylic acid (2-

(dimethylamino)-ethyl)-amide (69 mg, 0.19 mmol) in methanol

(10 mL) was added formaldehyde (37% solution, 1.0 mL),

potassium cyanide (102 mg, 1.47 mmol), and 2 N HCl (1.0 mL).

The reaction mixture was heated to 50 °C for 3 h. The reaction

mixture was then cooled to room temperature, diluted with

water (15 mL) and sodium bicarbonate solution (15 mL),

extracted into dichloromethane (2 × 15 mL), and dried

(MgSO₄), and the solvent was removed in vacuo to yield crude

product. This was purified using flash chromatography eluting

with 10% methanol in dichloromethane to yield the title

To a solution of 4-[2-(tert-butyl-dimethyl-silanyloxy)-ethoxy]-

benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-

ethyl)-amide (125 mg, 0.24 mmol) in tetrahydrofuran (5 mL)

was added a 1.0 M solution of tetrabutylammonium fluoride

(1.2 mL, 1.2 mmol). After being stirred for 1.5 h, the reaction

mixture was diluted with ethyl acetate (20 mL), washed with

water (20 mL), and dried (MgSO₄), and the solvent was

removed in vacuo to yield crude product which was purified

using flash chromatography eluting with 5% methanol in

dichloromethane to yield the title compound as an orange solid

(24 mg, 25%). ¹H NMR (CDCl₃) δ 2.35 (s, 6H). 2.68 (t, 2H, J )

6.0 Hz), 3.83 (q, 2H J ) 5.7 Hz), 4.27 (m, 2H), 4.11 (m, 2H),

7.11 (d, 1H, J ) 7.9 Hz), 7.57 (t, 1H, J ) 8.1 Hz), 7.85 (d, 1H,

J ) 9.6 Hz), 7.91 (m, 1H), 8.32 (dd, 1H, J ) 8.3, 1.5 Hz), 8.47

(d, 1H, J ) 9.6 Hz), 8.80 (d, 1H, J ) 8.3 Hz), 8.93 (dd, 1H,

1

compound as a violet solid (13 mg, 17%), mp 224-225 °C. H

NMR (CDCl₃) δ 2.45 (s, 6H), 2.80 (t, 2H), 3.10 (s, 2H), 3.90 (q,

2H, 7.03 (d, 1H,), 7.75 (t, 1H), 7.95 (m, 2H), 8.12 (d, 1H), 8.40

(d, 1H), 8.80 (d, 1H), 9.05 (d, 1H), 10.95 (br, 1H). MS (DCI/

NH₃) m/e 416 (M NH₄)⁺.

3-Dim et h yla m in o-2-[(4-m et h oxy-b en zo[a ]p h en a zin e-

11-ca r bon yl)-a m in o]-p r op ion ic Acid Hyd r och lor id e (4s′).

A mixture of 3-(dimethylamino)-2-[(4-methoxy-benzo[a]phen-

azine-11-carbonyl)-amino]-propionic acid methyl ester (150 mg,

0.35 mmol) and 1 M HCl (50 mL) was heated to reflux for 1 h.

After cooling to room temperature, the volatile components

were removed in vacuo to yield the title compound as a red

solid (158 mg, 100%). ¹H NMR (DMSO-d₆) δ 2.95 (br s, 6H),

3.80-3.60 (m, 2H), 4.10 (s, 3H), 5.39 (m, 1H), 7.54 (d, 1H, J )

8.00 Hz), 7.86 (t, 1H, J ) 8.19 Hz), 8.03 (d, 1H, J ) 9.49 Hz),

8.14 (t, 1H, J ) 7.25 Hz), 8.55 (m, 2H), 8.69 (d, 1H, J ) 7.06

Hz), 9.01 (d, 1H, J ) 8.19 Hz), 10.74 (br d, 1H). MS (DCI/

NH₃) m/e 419 (M + H)⁺.

4,10-Dih yd r oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid

(2-(Dim eth yla m in o)-eth yl)-a m id e (4w ′). To a cold solution

of 4,10-dimethoxy-benzo[a]phenazine-11-carboxylic acid (2-

(dimethylamino)-ethyl)-amide (96 mg, 0.24 mmol) in dichlo-

romethane (2 mL) was added a 1.0 M solution of boron

tribromide in dichloromethane (2.14 mL, 2.14 mmol). The

reaction mixture was stirred at room temperature for 16 h and

then ice (5 g) was added with sodium carbonate (5 mL) and

sodium chloride (5 mL). The organics were extracted into

dichloromethane (2 × 10 mL) and dried (MgSO₄), and the

solvent was removed in vacuo to yield an orange compound

which was recrystallized from dichloromethane, methanol, and

hexane to yield the title compound as an orange solid (6 mg,

8%), mp 257 °C (dec). ¹H NMR (DMSO-d₆) δ 2.33 (s, 6H), 2.67

(t, 2H, J ) 5.7 Hz), 3.75-3.80 (m, 2H), 7.32 (d, 1H, J ) 7.3

Hz), 7.64 (d, 1H, J ) 9.4 Hz), 7.70-7.74 (m, 1H), 7.85 (d, 1H,

J ) 9.4 Hz), 8.3 (d, 1H, J ) 9.4 Hz), 8.40 (d, 1H, J ) 9.4 Hz),

8.78 (d, 1H, J ) 8.1 Hz), 10.55 (br, 1H), 11.4 (br, 1H). MS (DCI/

NH₃) m/e 377 (M + H)⁺. Anal. (C₂₁H₂₀N₄O₃) C, H, N.

J

) 7.3, 1.5 Hz), 10.69 (br, 1H). MS (CI/NH₃) m/e 405

(M + H)⁺.

4-Am in o-b en zo[a ]p h en a zin e-11-ca r b oxylic Acid (2-

(Dim eth yla m in o)-eth yl)-a m id e (4u ). A mixture of 4-nitro-

benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-

ethyl)-amide (176 mg, 0.45 mmol), indium (154 mg, 0.75

mmol), and saturated ammonium chloride solution (5 mL) in

ethanol (20 mL) was heated to reflux for 3 h. The reaction

mixture was cooled to room temperature, quenched with water

(10 mL), and then filtered through Celite. The filtrate was

concentrated in vacuo, the residue was treated with saturated

sodium bicarbonate solution (10 mL), extracted into chloroform

(2 × 10 mL), and dried (MgSO₄), and the solvent was removed

in vacuo to yield the title compound as a red solid (163 mg,

1

100%), mp 243-244 °C. H NMR (CDCl₃) δ 2.50 (s, 6H), 2.90

(t, 2H), 3.95 (m, 2H), 7.10 (d, 1H) 7.65 (t, 1H), 7.90 (m, 2H),

8.05 (d, 1H), 8.35 (d, 1H), 8.70 (d, 1H), 8.95 (d, 1H), 11.0 (br,

1H). MS (DCI/NH₃) m/e 360 (M + H)⁺.

4-Acetyla m in o-ben zo[a ]p h en a zin e-11-ca r boxylic Acid

(2-(Dim eth yla m in o)-eth yl)-a m id e (4x). To a solution of

4-amino-benzo[a]phenazine-11-carboxylic acid (2-(dimethyl-

amino)-ethyl)-amide (20 mg, 0.056 mmol) in tetrahydrofuran

(5 mL) was added pyridine (0.1 mL) and acetyl chloride (20

µL). After being stirred for 1 h, the reaction mixture was

quenched with sodium bicarbonate solution (10 mL), extracted

with ethyl acetate (20 mL), separated, and dried (MgSO₄). The

solvent was removed in vacuo, and the residue was triturated

with ether to yield the title compound as a yellow solid (10

mg, 45%), mp 233 234 °C. ¹H NMR (CDCl₃) δ 2.35 (s, 6H),

2.40 (s, 3H), 2.65 (t, 2H), 3.80 (q, 2H), 7.45 (t, 1H), 7.70 (m,

2H), 7.90 (m, 2H), 8.20 (s, 1H), 8.25 (d, 1H), 8.70 (d, 1H), 8.80

(d, 1H), 10.10 (br, 1H). MS (DCI/NH₃) m/e 402 (M + H)⁺.

10-Hyd r oxy-4-m eth oxy-ben zo[a ]p h en a zin e-11-ca r box-

ylic Acid (2-(Dim eth yla m in o)-eth yl)-a m id e (4x′). To a cold

solution of 4,10-dimethoxy-benzo[a]phenazine-11-carboxylic

acid (2-(dimethylamino)-ethyl)-amide (300 mg, 0.74 mmol) in

dichloromethane (25 mL) was added a 1.0 M solution of boron

tribromide in dichloromethane (1.63 mL, 1.63 mmol). The

4-Met h a n esu lfon yla m in o-b en zo[a ]p h en a zin e-11-ca r -

boxylic a cid (2-(d im eth yla m in o)-eth yl)-a m id e (4y) was

prepared in an analogous manner from 4-amino-benzo[a]-

phenazine-11-carboxylic acid (2-(dimethylamino)-ethyl)-amide

(52 mg, 0.14 mmol) and methanesulfonyl chloride (10 µL) to

738 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3

Vicker et al.

reaction mixture was stirred at room temperature for 6 h, and

then ice (5 g) was added with sodium carbonate (5 mL) and

sodium chloride (5 mL). The organics were extracted into

dichloromethane (2 × 10 mL) and dried (MgSO₄), and the

solvent was removed in vacuo to yield a yellow solid which

was purified using flash chromatography on silica eluting with

ethyl acetate and 15% methanol to yield the title compound

as a yellow solid (61 mg, 21%), mp 181-182 °C. ¹H NMR

(CDCl₃) δ 2.42 (s, 6H), 2.78 (t, 2H J ) 6.0 Hz), 3.86-3.90 (m,

2H), 4.10 (s, 3H), 7.25 (d, 1H J ) 7.8 Hz), 7.57 (d, 1H J ) 9.4

Hz), 7.70-7.75 (m, 1H), 7.95 (d, 1H J ) 9.4 Hz), 8.22 (d, 1H

J ) 9.4 Hz), 8.51 (d, 1H J ) 9.6 Hz), 8.81 (d, 1H J ) 8.2 Hz),

11.65 (br, 1H), 16.17 (s, 1H). MS (DCI/NH₃) m/e 391 (M + H)⁺.

20 and 50 mg/kg or by oral gavage at 50 mg/kg. At various

times (2 min-24 h) after dosing, blood samples were obtained

from anaesthetized animals by cardiac puncture. The blood

samples were collected using heparinized syringes and cen-

trifuged to prepare plasma which was analyzed using HPLC.³⁰

Xen ogr a ft Stu d ies. Xenograft studies were performed

using H69 SCLC (7 × 10⁶) cells harvested from in vitro

incubations in PBS, with inoculation performed subcutane-

ously into the right flank of female CD1 nude mice (20-28 g).

Tumor volumes were estimated as described previously.³¹

When tumors had reached an average size of between 0.3 and

0.7 cm, the animals were randomized into groups of 7-8 by

tumor volume. The efficacy of XR11576 was evaluated follow-

ing iv administration and compared with the clinical candidate

TAS-103 (given iv).

4,9-Dim eth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid

(2-(Dim eth ylam in o)-eth yl)-am ide (4u ′). A mixture of 9-chlo-

ro-4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimeth-

ylamino)-ethyl)-amide (85 mg, 0.21 mmol) and a 25% solution

of sodium methoxide in methanol (4 mL) was heated to reflux

for 6 h. The reaction mixture was then cooled to room

temperature, diluted with ethyl acetate (15 mL), washed with

water (15 mL), and dried (MgSO₄), and the solvent was

removed in vacuo to yield a yellow solid which was purified

using flash chromatography on silica eluting with ethyl acetate

and 15% methanol to yield the desired title compound as a

Refer en ces

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65, 635-692.

(2) Hande, K. R. Biochim. Biophys. Acta 1998, 1400, 173-184.

(3) Dancey, J .; Eisenhauer, E. A. Current perspectives on camp-

tothecins in cancer treatment. Br. J . Cancer 1996, 74, 327-338.

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S. T. In vivo resistance towards anthracyclines, etoposide, and

cis-diamine-dichloroplatinum (II). Cancer Res. 1982, 42, 4719-

4725.

1

yellow solid (42 mg, 50%), mp 184-185 °C. H NMR (CDCl₃)

δ 2.42 (s, 6H), 2.75 (t, 2H, J ) 6.0 Hz), 3.85-3.90 (m, 2H),

4.07 (s, 3H), 4.10 (s, 3H),7.22 (d, 1H J ) 8.0 Hz), 7.64 (d, 1H

J ) 3.0 Hz), 7.72-7.77 (m, 1H), 7.91 (d, 1H J ) 9.4 Hz), 8.55

(d, 1H J ) 9.6 Hz), 8.70 (d, 1H J ) 3.0 Hz), 8.99 (d, 1H J )

8.0 Hz), 10.92 (br, 1H). MS (ESI +ve) m/e 405 (M +H)⁺.

(5) Hendricks, C. B.; Rowinsky, E. K.; Grochow, L. B., Donehower

R. C.; Kaufmann, S. H. Effect of P-glycoprotein expression on

the accumulation and cytotoxicity of Topotecan (SK & F 104864),

a new camptothecin analogue. Cancer Res. 1992, 52, 2268-2278.

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C.; Gondrad, L.; Saucier, J . M.; Bisagni, E.; Lavalle, F. Intopli-

cine (RP 64075) and its derivatives, a new class of antitumor

agents inhibiting both topoisomerase I and II activities. Cancer

Res. 1993, 53, 5987-5993.

(7) Finlay, G. J .; Riou, J . F.; Baguley, B. C. From amsacrine to

DACA (N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide): se-

lectivity for topoisomerases I and II among acridine derivatives.

Eur. J . Cancer 1996, 32A, 708-714. Dangerfield, W.; Mistry,

P.; Stewart, A. J .; Kofler, B.; Liddle, C.; Baker, M.; Bootle, D.;

Laurie, D.; Spencer, M.; Charlton, P. In vitro and in vivo

characterization of XR11576, a novel dual inhibitor of topo-

isomerase I and II. Proc. 92nd AACR. 2001, 42, 101.

(8) Utsugi, T.; Aoyagi, K.; Asao, T.; Okazaki, S.; Aoyagi, Y.; Sano,

M.; Wierzba, K.; Yamada, Y. Antitumor activity of a novel

quinoline derivative, TAS-103, with inhibitory effects on topo-

isomerases I and II. J pn. J . Cancer Res. 1997, 88, 992-1002.

(9) Fortune, J . M.; Velea, L.; Graves, D. E.; Utsugi, T.; Yamada, Y.;

Oscheroff, N. DNA Topoisomerases as Targets for the Anticancer

Drug TAS-103: DNA Interactions and Topoisomerase Catalytic

Inhibition. Biochemistry 1999, 38, 15580-15586.

4-Meth oxy-ben zo[a ]p h en a zin e-11-ca r boxylic Acid (2-

(Dim eth yla m in o)-1-h yd r oxym eth yl-eth yl)-a m id e (4p ′′). A

mixture of 3-(dimethylamino)-2-[(4-methoxy-benzo[a]phen-

azine-11-carbonyl)-amino]-propionic acid methyl ester (335 mg,

0.66 mmol) and lithium borohydride (72 mg, 3.3 mmol) in

tetrahydrofuran (10 mL) and 2-propanol (10 mL) was stirred

at room temperature for 18 h. A further 5 equiv of lithium

borohydride were added portionwise, and the mixture was

stirred for 18 h. The reaction was quenched with ammonium

chloride solution (5 mL), extracted with ethyl acetate (2 × 20

mL), and dried (MgSO₄), and the solvent was removed in vacuo

to yield a brown gum. Purification using flash chromatography

on silica eluting with ethyl acetate and 15% methanol and

trituration with ether yielded the desired title compound as

an orange powder (50 mg, 19%), mp 168-170 °C. ¹H NMR

(CDCl₃) δ 2.35, (s, 6H), 2.87 (m, 2H), 4.05 (m, 5H), 4.55 (m,

1H), 7.20 (s, 1H), 7.71 (t, 1H, J ) 8.14), 7.88 (m, 2H), 8.35 (dd,

1H, J ) 1.48, 8.47 Hz), 8.50 (1H, d, J ) 9.55 Hz), 8.85 (1H, d,

J ) 8.17 Hz), 8.91 (1H, dd, J ) 1.47 Hz, 7.29 Hz), 11.05, (1H,

d). MS (DCI/NH₃) m/e 405 (M + H)⁺.

(10) Spicer, J . A.; Gamage, S. A.; Atwell, G. J .; Finlay, G. J .; Baguley,

B. C.; Denny,.W. A. Structure-Activity Relationships for Acri-

dine Analogues of the Mixed Topoisomerase I/II Inhibitor N-[2-

(Dimethylamino)ethyl]acridine-4-carboxamide. J . Med. Chem.

1997, 40, 1919-1929.

Biology. Cytotoxicity Assa ys. The cytotoxicity of com-

pounds was measured using the H69 parental (H69/P) human

small cell lung carcinoma cell line and the drug resistant

human small cell lung carcinoma cell line H69/LX4 which

overexpresses P-glycoprotein (Pgp). The cytotoxicity of selected

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J M010329A

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Novel angular benzophenazines: Dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents

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