Bis(phenazine-1-carboxamides): Structure-activity relationships for a new class of dual topoisomerase I/II directed anticancer drugs

Article abstract of DOI:10.1021/jm990423f

Ring-substituted bis(phenazine-1-carboxamides), linked by a - (CH₂)₃NMe(CH₂)₃- chain, were prepared from the corresponding substituted phenazine-1-carboxylic acids by reaction of the intermediate imidazolides with bis(3-aminopropyl)methylamine. The compounds were evaluated for growth inhibitory activity in a panel of tumor cell lines, including P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia. The latter mutant lines are resistant to topoisomerase (topo) II targeted agents because of lower levels of the enzyme. Analogues with small, lipophilic substituents (e.g., Me, Cl) at the 9-position were the most potent inhibitors, superior to the corresponding dimeric bis(acridine-4-carboxamides) (bis-DACA analogues). Several of the compounds were preferentially (up to 2-fold) more cytotoxic toward the mutant Jurkat lines than the wild-type. To test whether this selectivity was related to topoisomerase action, the most potent of the compounds (9-methyl) was evaluated in a cell-free system. It poisoned topo I at drug concentrations of 0.25 and 0.5 μM and inhibited the catalytic activity of both topo I and topo II at concentrations of 1 and 5 μM, respectively. Results from the NCI human tumor cell line panel showed the compounds had preferential activity toward colon tumor lines (on average 9.5-fold more active in the HT29 line than in the cell line panel as a whole). Several analogues produced significant growth delays in the relatively refractory subcutaneous colon 38 tumor model in vivo. In particular the 9-methyl compound was substantially more potent in this tumor model than the clinical dual topo I/II poison DACA (total dose 90 versus 400 mg/kg) with comparable activity. The bis(phenazine-1-carboxamides) are a new and interesting class of dual topo I/II-directed anticancer drugs.

Full text of DOI:10.1021/jm990423f

1350
J . Med. Chem. 2000, 43, 1350-1358
Bis(p h en a zin e-1-ca r boxa m id es): Str u ctu r e-Activity Rela tion sh ip s for a New
Cla ss of Du a l Top oisom er a se I/II-Dir ected An tica n cer Dr u gs
J ulie A. Spicer, Swarna A. Gamage, Gordon W. Rewcastle, Graeme J . Finlay, David J . A. Bridewell,
Bruce C. Baguley, and William A. Denny*
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1000, New Zealand
Received August 20, 1999
Ring-substituted bis(phenazine-1-carboxamides), linked by a -(CH2)3NMe(CH2)3- chain, were
prepared from the corresponding substituted phenazine-1-carboxylic acids by reaction of the
intermediate imidazolides with bis(3-aminopropyl)methylamine. The compounds were evaluated
for growth inhibitory activity in a panel of tumor cell lines, including P388 leukemia, Lewis
lung carcinoma, and wild-type (J LC) and mutant (J LA and J LD) forms of human J urkat leukemia.
The latter mutant lines are resistant to topoisomerase (topo) II targeted agents because of
lower levels of the enzyme. Analogues with small, lipophilic substituents (e.g., Me, Cl) at the
9-position were the most potent inhibitors, superior to the corresponding dimeric bis(acridine-
4-carboxamides) (bis-DACA analogues). Several of the compounds were preferentially (up to
2-fold) more cytotoxic toward the mutant J urkat lines than the wild-type. To test whether this
selectivity was related to topoisomerase action, the most potent of the compounds (9-methyl)
was evaluated in a cell-free system. It poisoned topo I at drug concentrations of 0.25 and 0.5
µM and inhibited the catalytic activity of both topo I and topo II at concentrations of 1 and 5
µM, respectively. Results from the NCI human tumor cell line panel showed the compounds
had preferential activity toward colon tumor lines (on average 9.5-fold more active in the HT29
line than in the cell line panel as a whole). Several analogues produced significant growth
delays in the relatively refractory subcutaneous colon 38 tumor model in vivo. In particular,
the 9-methyl compound was substantially more potent in this tumor model than the clinical
dual topo I/II poison DACA (total dose 90 versus 400 mg/kg) with comparable activity. The
bis(phenazine-1-carboxamides) are a new and interesting class of dual topo I/II-directed
anticancer drugs.
There is current interest in dimeric analogues of
lipophilic, neutral, DNA mono-intercalating agents as
potential anticancer drugs. The bis(naphthalimide) LU
79953 (1) is a DNA bis-intercalator1 and very potent
cytotoxin, with broad-spectrum activity against a vari-
ety of human solid tumor cell lines (both in culture and
as xenografts in nude mice),2,3 and is in Phase I clinical
trial.4 The related bis(nitronaphthalimide) DMP 840 (2)
We have recently shown10 that, of a series of tricyclic
aromatic carboxamides, the bis(acridine-4-carboxamide)
(5) and the related bis(phenazine-1-carboxamide) (6: R
) H) showed the largest increase in potency over their
monomeric counterparts (3 and 4). More extensive
structure-activity relationship (SAR) studies of acri-
dine-substituted analogues of 5 showed that small
substituents (e.g., Me, Cl) at the 5-position provided
compounds of highest cytotoxicity, with IC50s as low as
2 nM against the Lewis lung carcinoma cell line.11
Several examples produced significant growth delays in
the relatively refractory subcutaneous colon 38 tumor
appears to be only a mono-intercalator5 but shows
curative activity against a variety of human solid tumor
xenografts in nude mice,6,7 and Phase I clinical trials
have been reported.8 The mode of action of these
compounds is still unclear,8,9 but they are known to be
topoisomerase inhibitors.4
* To whom correspondence should be addressed. Tel: 64-9-3737-
599. Fax: 64-9-3737-502. E-mail: b.denny@aukland.ac.nz.
10.1021/jm990423f CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

Bis(phenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1351
Sch em e 1a
Sch em e 3a
a
(i) CDI/DMF/45 °C, 30 min; (ii) [H2N(CH2)3]2NMe/THF/0-20
°C/4 h.
Sch em e 2a
a
(i) CuCl/Cu/N-ethylmorpholine/butane-2,3-diol/70 °C/18 h; (ii)
NaBH4/aq NaOH(or NaOEt/EtOH)/reflux/8 h.
required phenazine-1-carboxylic acids in moderate yields
(Scheme 3). 9-(Dimethylamino)phenazine-1-carboxylic
acid (7s) was prepared by reacting 9-fluorophenazine-
1-carboxylic acid (7p ) in 40% aqueous dimethylamine
in a bomb at 100 °C for 3 h.
a
(i) Pt-C/MeOH/50 psi/1 h; (ii) MeOH/reflux/15 min; (iii) DDQ/
benzene/reflux/15 h.
Resu lts a n d Discu ssion
model in vivo at substantially lower doses than the
corresponding monomer DACA (3).
The bis(phenazines) were evaluated in a panel of
cell lines in culture. They were used as the monohydro-
chloride salts, which were highly water-soluble. The
Because the acridine chromophore in the clinical
monomeric acridine-4-carboxamide DACA (3) undergoes
rapid oxidative metabolism to the acridone by enzymes
such as aldehyde oxidase,12 and because monomeric
phenazine-1-carboxamides are known13 to be effective
cytotoxins, we investigated bis(phenazine-1-carbox-
amide) analogues of 6 to determine substituent SAR and
to see whether analogues of superior activity to the bis-
(acridine-4-carboxamides) could be obtained.
murine P388 leukemia16
and murine Lewis lung carci-
noma17 lines were used to provide comparison with
previous data on related compounds. The three human
leukemia (J urkat) lines18,19 were used as examples of
human lines, and to provide insights into the mecha-
nism of cytotoxicity. J LC is the wild-type (sensitive) line,
while J LA and J LD, developed for resistance to amsa-
crine and doxorubicin, respectively, are 123-fold and
110-fold resistant, respectively, to the topo II inhibitor
amsacrine because of a reduced level of topo II enzyme.18
Table 1 provides the IC50 values for the P388, LL, and
J LC lines, together with ratios of IC50 values (J LA/J LC
and J LD/J LC) for the resistant J urkat lines. Ratios of
less than about 2-fold suggest a nontopo II mediated
mechanism of action. DACA (3), a dual topo I/II inhibi-
tor,20 has values of 2.3 and 2.5 for these ratios, while
the related monomeric phenazine (4) has values of 1.1
and 1.1.
Ch em istr y
The bis(phenazine-1-carboxamides) (6a -6w ) were
prepared by activation of the acids 7 with 1,1′-carbon-
yldiimidazole. The resulting N-imidazolides (8) were
isolated (this was necessary to obtain pure products) and
immediately reacted with a stoichiometric amount of
bis(3-aminopropyl)methylamine (Scheme 1). Most of
the substituted phenazine-1-carboxylic acids (7) re-
quired were known and were prepared by reported
methods.13,14
The parent unsubstituted bis compound 6a was a
moderately potent cytotoxin, showing similar IC50 val-
ues in the cell line panel to those determined previ-
ously10 for the related bis(acridine-4-carboxamide) 5
(e.g., IC50 J LC 137 and 110 nM, respectively). A com-
parison of data for 6a and the corresponding bis-
(phenazine-2-carboxamide) 20 (IC50 J LC 2760 nM)10
confirmed the requirement for the linker chain to be peri
to the ring nitrogen (on the same side as the carbox-
4-Chlorophenazine-1-carboxylic acid (13) was pre-
pared from 2,3-dinitro-4-chlorobenzoic acid (9).15 Reduc-
tion of this by hydrogenation over Pt/C and condensation
of the resulting diamine (10) with cyclohexane-1,2-dione
(11) gave the tetrahydro derivative (12), which was
dehydrogenated with DDQ to 13 (Scheme 2) in low yield.
However, reaction of this with bis(3-aminopropyl)-
methylamine as above gave none of the desired bis(4-
chlorophenazine) product (possibly due to concomitant
displacement of the relatively labile chloro group).
The 9-phenoxy-, 6,9-dimethyl-, and 6-chloro-9-meth-
ylphenazine-1-carboxylic acids (7r , 7t, and 7u ) were
prepared by Ullmann-type condensation of the requisite
amines (14-16) with 2-bromo-3-nitrobenzoic acid (17)
to give the diphenylamines 18-20, respectively. Reduc-
tive cyclization of these with NaBH4 in refluxing aque-
ous sodium hydroxide or sodium ethoxide gave the

1352 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Spicer et al.
Ta ble 1. Cytotoxicities of Bis[(phenazine-1-carboxamido)propyl]methylamines
IC50 (nM)a
IC50 ratios
d
e,f
e,g
no.
R
mp
ref 10
206-207
75-78
P388b
520
LLc
J LC
173
J LA/J LC
J LD/J LC
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
H
2-Cl
107
8370
48
12
26
46
151
72
83
64
0.5
0.6
0.7
0.6
0.7
0.8
0.9
0.8
0.8
0.9
0.6
0.4
0.5
0.6
0.6
0.6
0.7
1.0
0.9
0.5
0.4
0.8
0.6
0.7
0.7
0.8
0.9
0.8
0.9
1.0
0.9
0.9
0.9
1.0
0.8
0.6
0.8
0.6
0.9
0.6
1.0
1.1
0.9
0.8
0.8
1.5
0.8
0.8
1.2 × 104
472
204
375
584
7740
623
2960
600
690
97
2370
208
74
3-Me
3-Cl
169-170
218-220
228-230h
198-200
213-215h
173.5-175
225-229h
161-162
210-121.5
182-186
262-264h
169-171.5
183-186
220-222
nci
4-Me
6-Me
102
172
209
280
249
195
148
42
6-Cl
7-Me
7-Cl
7-OMe
8-Me
108
44
24
8-Cl
8-OMe
9-Me
75
15
39
30
5.7
14
1.6
8.8
120
160
1310
310
5.3
5.2
410
3.2
30
9-Cl
9-F
294
815
3450
2280
26
29
450
24
177
124
1250
695
21
9-OMe
9-OPh
9-NMe2
6,9-diMe
6-Cl, 9-Me
6,7-benzo
8,9-benzo
6s
6t
6u
6v
6w
5
nci
97-101
200-202
138-142
214-215.5
ref 11
9.8
290
9.0
130
110
a
IC50; concentration of drug (nM) to reduce cell number to 50% of control cultures (see text). The value is the average of at least two
independent determinations; the coefficients of variation were between 15 and 22%. Murine P388 leukemia. c Murine Lewis lung
b
carcinoma. J LC: wild-type human J urkat leukemia. e Ratios of IC50s in the cell lines shown. f J LA: amsacrine-resistant J urkat. J LD:
d
g
h
i
doxorubicin-resistant J urkat. HCl salt. Noncrystalline.
amide) as previously for both monomeric phenazine-1-
carboxamides13 and bis(acridine-4-carboxamides).11
The ring-substituted compounds 6b-6u contain the
same linker chain [(CH2)3NMe(CH2)3] used in the
previous bis(acridine-4-carboxamide) study,11 selected
in order to allow direct comparison of substituent
effects in the two series. Because neither large or
hydrophilic groups were beneficial in the bis(acridine-
4-carboxamide) series, these were not prepared here.
Although fewer compounds were therefore studied,
substitutions were again made at every available chro-
mophore ring position. Overall, the substituted bis-
(phenazine-1-carboxamides) were more cytotoxic toward
the mouse carcinoma line LL than the mouse leukemia
line P388 (eq 1), but they had very similar IC50 values
for the human and mouse leukemia lines (eq 2). These
general relationships are similar to those found previ-
ously11 for substituted bis(acridine-4-carboxamides).
respectively) were evaluated (note that the substituent
numbering is different between the acridine and phena-
zine chromophores). In the 2- and 4-positions, only one
analogue was made. The 2-Cl compound 6b was much
less cytotoxic than the parent, due presumably to the
substituent ortho to the carboxamide side chain inter-
fering with the conformation of the latter. This phe-
nomenon was true also in both the monophenazine13
and the bis(DACA)11 series, and no other 2-substituted
compounds were made. In the 4-series, the 4-Me com-
pound 6e was substantially more cytotoxic than the
parent (although not as active as other Me analogues),
but the corresponding 4-Cl analogue could not be
prepared (see above).
In terms of absolute potency, the remainder of the
specific substituent effects fell into two categories, very
similar to those seen in the bis(acridine-4-carbox-
amides).11 First, 9-substituents (especially small lipo-
philic ones) were the most beneficial, with the 9-Me
analogue 6n proving the most cytotoxic of all the
compounds made (25-fold more potent than 6a in J LC
cells), and the 6,9-diMe and 6-Cl, 9-Me analogues (6t
and 6u ) being the next active. In contrast, substituents
in the remaining positions (3-, 4-, 6-, 7-, and 8-) showed
no marked effects. Compounds 6t and 6u show that, in
6,9-disubstituted derivatives, the 9-substituent domi-
nates in terms of biological effects, with these com-
pounds showing similar potencies to their corresponding
9-monosubstituted analogues. Too few compounds were
made to study QSAR for local substituent steric and
log(IC50)LL
)
0.91((0.11) log(IC50)P388 - 0.59((0.30) (1)
n ) 21
r ) 0.88
s ) 0.35
F ) 67
log(IC50)J LC
)
0.75((0.07) log(IC50)P388 - 0.10((0.26) (2)
n ) 21 r ) 0.97 s ) 0.17 F ) 103
For each position, except 2- and 4-, at least Cl and
Me substituents (representative of small, lipophilic
electron-withdrawing and electron-donating groups,

Bis(phenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1353
Ta ble 2. Cytotoxicity of Selected Bis(phenazine-1-carboxamides) in the NCI Cell Line Panel
GI50 (nM)a
no.
sub.
MCF7
NCI/ADR
CAK-1
SKOV-3
HT29
HCT-116
midb
6a
6b
6c
6d
6f
6h
6i
6j
H
2-Cl
3-Me
3-Cl
6-Me
7-Me
7-Cl
7-OMe
8-Me
8-Cl
8-OMe
9-Me
9-Cl
9-F
9-OMe
9-OPh
9-NMe2
6,9-diMe
6-Cl, 9-Me
120
4070
310
110
480
550
330
2000
330
<10c
<10
<10
35
460
47
3480
1820
<10
<10
1050
>105
5730
125
225
850
44
16
210
5750
725
180
740
600
560
4170
290
70
72
20
93
1320
575
3160
2510
34
3.4 × 104
8.8 × 104
1.2 × 104
1460
135
130
<10
270
235
80
285
470
18
63
455
35
170
1170
370
430
530
3020
240
62
82
<10
15
1730
1420
5360
2910
<10
<10
920
1.2 × 104
2660
1140
2.0 × 104
1570
240
1210
19
350
7450
2.1 × 104
1630
1.1 × 104
35
5840
1960
9660
93
90
37
6k
6l
1.1 × 104
325
<10
<10
<10
<10
225
<10
1770
430
<10
<10
1880
<10
<10
<10
70
400
35
1790
530
<10
40
6m
6n
6o
6p
6q
6r
6s
6t
6u
800
220
790
1.4 × 104
1560
1.1 × 104
1.0 × 104
74
50
2030
50
a
b
GI50: concentration of drug (nM) resulting in inhibition of cell growth to 50% of controls. Mid: the average GI50 value for the drug
over the whole cell line panel. c Lowest dose tested.
electronic effects at individual positions. While most
attention was focused on 9-substituents, even here only
six analogues were made. However, the results quali-
tatively support the observations made in the bis-
(acridine-4-carboxamide) series11 that large substituents
are disfavored. Overall, the substituted bis(phenazine-
1-carboxamides) showed very similar cytotoxicities to
the corresponding substituted bis(acridine-4-carbox-
amides).11 Thus, comparing IC50 values in the J LC cell
line for all pairs of methyl-, methoxy-, and chloro-
substituted bis(phenazines) and bis(acridines) (bisacri-
dine data from ref 11) gave eq 3, with a slope of unity
running essentially through the origin:
activity relationships could be observed; thus the 9-OPh
analogue 6r was 2-fold more potent in the NCI/ADR line
than in MCF7, but the 9-OMe compound 6q was 445-
fold less potent.
As expected, the relatively refractory solid tumor lines
CAK-1 (renal) and SKOV3 (ovarian) were resistant,
with GI50 for most of the compounds being greater than
the GI50(mid) values. Overall, and to an even greater
extent than the bis(DACA) compounds,11 the bis(phena-
zines) were preferentially active toward colon cell lines.
In the representative HT29 line, selectivities [measured
as GI50(mid)/GI50(HCT-116)] varied from 1.7-fold (for the
9-OPh analogue 6r ) to >40-fold for the 2-Cl and 7-OMe
analogues 6b and 6j, with an average of 9.5-fold (note
these are underestimated by taking IC50 values in Table
2 of <10 nM as 10 nM for the purpose of calculation).
Selectivities for HCT-116 were similar. Compounds 6b
and 6j in particular were relatively nonpotent in all but
the colon lines.
log(IC50)J LC(phenazines) ) 0.99((0.18)
log(IC50)J LC(acridines) - 0.01((0.28) (3)
n ) 17
r ) 0.87
s ) 0.27
F ) 45
Most of the compounds were also evaluated in the
NCI cell human line panel,21 and results for six of those
lines are shown in Table 2 as GI50 values (the concen-
tration of drug resulting in inhibition of cell growth to
50% of controls; equivalent to IC50), together with the
GI50(mid) value (the average GI50 value for the drug over
the whole 60-cell line panel). As shown previously11 with
bis(DACA) derivatives, a comparison of the GI50(mid)
values and IC50 values in the J LC line shows a good
correlation (eq 4), indicating that the latter is a useful
model for broadly ranking cytotoxicities.
The low IC50 ratios (J LA/J LC and J LD/J LC) for the
9-methyl analogue 6n (Table 1) indicated selective
toxicity toward cell lines expressing low levels of topo
II. To investigate the action of this compound on
topoisomerases, purified topo I and II were incubated
with increasing concentrations of drug in the presence
of supercoiled plasmid DNA, and the products were
subjected to electrophoresis in the presence of ethidium
bromide to separate closed and open circular DNA.
While no evidence for poisoning of topo II was obtained,
drug concentrations of 0.1 and 0.25 µM caused the
appearance of nicked circular DNA, indicative of poison-
ing of topo I (Figure 1). Higher drug concentrations (1
µM and 5 µM) were found to inhibit the relaxation of
plasmid DNA by both topo I and II, respectively (data
not shown). Compound 6v showed cytotoxic as well as
cytostatic activity in the H460 human lung line25 by
clonogenic assay, causing 90% and 99% inhibition of
survival at 9.0 and 15 µM, respectively.
log(GI50)mid ) 0.97((0.10)
log(IC50)J LC + 0.57((0.22) (4)
n ) 19
r ) 0.92
F ) 93
Again, similarly to the bis(DACA) compounds,11
a
comparison of results in the MCF7 and NCI/ADR lines
(the latter overexpresses P-glycoprotein) shows the bis-
(phenazines) are moderately affected by P-glycoprotein
mediated multidrug resistance. On average, the com-
pounds were 38-fold less active in the P-glycoprotein
overexpressing NCI/ADR line, but no obvious structure-
To assess the in vivo antitumor potential of this
series, the parent compound (6a ) and three of the more
potent analogues (6m , 6n , and 6o) were evaluated in
the relatively refractory subcutaneous colon 38 tumor

1354 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Spicer et al.
are broadly similar to those found previously for bis-
(DACA) compounds; small lipophilic substituents peri
to the ring nitrogen (in the 9-position) are the most
beneficial, with substituents in the remaining positions
showing no marked effects. Results from the NCI
human tumor panel showed the compounds were mod-
erately affected by P-glycoprotein mediated multidrug
resistance, but were highly preferentially active toward
colon cell lines. The 9-methyl analogue 6n is a dual topo
I/II inhibitor and a topo poison. Analogues showed
significant growth delays in a refractory subcutaneous
colon 38 tumor model in vivo. The bis(phenazine-1-
carboxamides) are an interesting class of dual topo I/II-
directed anticancer drugs.
F igu r e 1. Topo I cleavable complex formation assays. Gel
electrophoresis of supercoiled plasmid pBR322 DNA with no
additions (lane 1); reacted with topo I (15 units) in the absence
of drug (lane 2); topo I plus camptothecin at 100 µM (lane 3);
topo I plus compound 6n at concentrations of 2 nM (lane 4);
10 nM (lane 5); 50 nM (lane 6); 100 nM (lane 7); 250 nM (lane
8); relaxed marker pBR322 DNA (lane 9). Supercoiled and
relaxed DNA run at the bottom of the gel whereas nicked open
circles, which represent the formation of topoisomerase I
cleavable complexes, run at the top.
Exp er im en ta l Section
Ch em istr y. Analyses were carried out in the Microchemical
Laboratory, University of Otago, Dunedin, NZ. Melting points
were determined on an Electrothermal 2300 melting point
apparatus. NMR spectra were obtained on a Bruker DRX-400
spectrometer and are referenced to Me4Si for organic solutions
and 3-(trimethylsilyl)propanesulfonic acid, sodium salt for D2O
solutions. Thin-layer chromatography was carried out on
aluminum-backed silica gel (Merck 60 F254) or alumina plates.
Flash column chromatography was carried out on Merck silica
gel (230-400 mesh) or alumina. Petroleum ether refers to the
fraction boiling at 40-60 °C. Satisfactory high-resolution mass
spectral data were obtained for the bis(phenazines), using a
VG 7070 spectrometer at nominal 5000 resolution. All of the
bis(phenazines) were judged to be >98% pure by reverse-phase
HPLC analysis with diode array detection.
Ta ble 3. In Vivo Antitumor Activity of Selected
Bis(phenazine-1-carboxamides) against Subcutaneous Colon 38
Carcinoma
optimal dose
(mg/kg)
growth delay
(days)
no.
dosing schedule
6a
6m
6n
6n
6o
every 4 days × 3
100 × 3
6.0
8.0
7.6
12.3
7.0
single dose
100
single dose
30
every 4 days × 3
30 × 3
single dose
100 × 3
P r ep a r a tion of P h en a zin e Acid s. 4-Ch lor op h en a zin e-
1-ca r b oxylic Acid (13). A solution of 4-chloro-2,3-dinitro-
benzoic acid (9)15 (1.6 g, 6.48 mmol) in MeOH was hydrogen-
ated over Pt/C for 30 min, and the resulting solution of crude
diamino acid 10 was filtered directly into a solution of
cyclohexane-1,2-dione (11) (1.57 g, 6.48 mmol) in MeOH (20
mL). The reaction mixture was stirred and heated to reflux
for 15 min, then concentrated to half volume, cooled, and
diluted with water. The resulting precipitate was collected and
dissolved in CH2Cl2. Insoluble material was removed by
filtration, and the solution was dried (Na2SO4) and evaporated
to give a crude product (1.4 g). This was chromatographed on
silica gel in CH2Cl2 to give 4-chloro-6,7,8,9-tetrahydrophena-
zine-1-carboxylic acid (12) (0.5 g, 33%): 1H NMR (CDCl3) δ
2.12 (m, 4 H, 2 × CH2), 3.30 (m, 4 H, 2 × CH2), 7.94 (d, J )
8.1 Hz, 1 H, H-3), 8.60 (d, J ) 8.1 Hz, 1 H, H-2) 15.01 (s, 1 H,
CO2H). Anal. (C13H11ClN2O2) C, H, N.
A mixture of 12 (10 mg, 0.038 mmol) and DDQ (20 mg, 0.088
mmol) in benzene (5 mL) was refluxed overnight. Solvents
were removed under reduced pressure, and the residue was
chromatographed on silica gel, eluting with CH2Cl2/MeOH
(99:1) to give 13 (1.2 mg, 12%): 1H NMR (CDCl3) δ 8.03-8.11
(m, 2 H, H-7,8), 7.72 (d, J ) 8.2 Hz, 1 H, H-3), 8.32 (dd, J )
8.4, 1.8 Hz, 1 H, H-6 or H-9), 8.49 (dd, J ) 7.6, 1.6 Hz, 1 H,
H-6 or H-9), 8.91 (d, J ) 7.7 Hz, 1 H, H-2), 15.35 (s, 1 H, CO2H).
HRMS (FAB+) calcd for C13H835ClN2O2 258.0919 (MH+); found,
258.0914.
F igu r e 2. Growth of colon 38 adenocarcinoma in the absence
of drug treatment (b) and following administration of com-
pound 6n (commencing at day 0 on the graph, or 8 days
after tumor inoculation). The schedules tested in this experi-
ment were single dose (30 mg/kg; O), repeated daily dose
(10 mg/kg × 7; 3) and intermittent dose (30 mg/kg every 4
days × 3; 2).
9-P h en oxyp h en a zin e-1-ca r boxylic Acid (7r ). A mixture
of 2-aminodiphenyl ether (14) (15 g, 80 mmol), 2-bromo-3-
nitrobenzoic acid (17) (10 g, 40 mmol), CuCl (1 g) in N-
ethylmorpholine (15 mL), and butane-2,3-diol (25 mL) were
stirred at 80 °C for 4 h. The cooled mixture was diluted with
0.01 N aqueous NaOH, clarified with charcoal, and filtered
through Celite. The cloudy solution was seeded with 2-amino-
diphenyl ether to precipitate unreacted starting material, and
the resulting clear solution was acidified with dilute HCl to
give N-(2-phenoxyphenyl)-3-nitroanthranilic acid (18) (13 g,
91%): mp (benzene/petroleum ether) 153-155 °C; 1H NMR
(CDCl3) δ 6.95-7.08 (m, 8 H, ArH), 7.09-7.30 (m, 2 H, ArH),
8.11 (dd, J ) 8.1, 1.7 Hz, 1 H, ArH), 8.22 (dd, J ) 7.8, 1.6 Hz,
1 H, ArH), 9.69 (br s, 1 H, NH). Anal. (C19H14N2O5) C, H, N.
model. The data are shown in Table 3 and Figure 2. The
most active analogue was 6n , which showed a growth
delay of 12.3 days (at 30 mg/kg using a 4 days × 3 dosing
schedule). This is comparable to the activity of the
clinical agent DACA in this model, which showed a
growth delay of 12.5 days (at 200 mg/kg using a
schedule of 200 mg/kg × 2)22 but at a considerably lower
total dose (90 versus 400 mg/kg).
Con clu sion s
The bis(phenazine-1-carboxamides) are potent cyto-
toxins in cell culture. Structure-activity relationships

Bis(phenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1355
A solution of 18 (10 g, 2.85 mmol) and NaBH4 (10 g) in 2 N
aqueous NaOH (600 mL) was heated under reflux for 48 h.
The cooled mixture was filtered, and the solid was dissolved
in hot water. Acidification with concentrated HCl gave a
mixture of the desired product and the amine resulting from
direct reduction. The dried mixture was suspended in boiling
MeOH, and sufficient Et3N was added to just give a homo-
geneous solution. Acidification with AcOH then precipitated
7r (0.93 g, 10%): mp 232-234 °C; 1H NMR [(CD3)2SO] δ 7.22-
7.25 (m, 3 H, ArH), 7.45-7.50 (m, 3 H, ArH), 8.02 (dd, J )
8.8, 7.6 Hz, 1 H, ArH), 8.15-8.18 (m, 2 H, ArH), 8.59 (dd, J )
8.8, 1.4 Hz, 1 H, ArH), 8.68 (dd, J ) 7.0, 1.2 Hz, 1 H, ArH),
15.02 (br s, 1 H, COOH). Anal. (C19H12N2O3) C, H, N. The
filtrate from the original reaction mixture was acidified with
concentrated HCl to give phenazine-1-carboxylic acid (resulting
from displacement of the phenoxy group during ring closure).
9-(Dim eth yla m in o)p h en a zin e-1-ca r boxylic Acid (7s).
A solution of 9-fluorophenazine-1-carboxylic acid14 (7p ) (200
mg, 0.8 mmol) in 40% aqueous Me2NH (20 mL) was heated at
100 °C in a bomb for 3 h. The resulting intensely purple
solution was diluted with water and then neutralized with
AcOH. The aqueous solution was then extracted with CHCl3
(3 × 50 mL) until all color was removed. The organic layer
was further washed with water (1 × 150 mL) and then dried
over Na2SO4, and the solvent was removed under reduced
pressure. The resulting purple solid was dissolved in a minimal
amount of CH2Cl2, and petroleum ether was added until
crystallization occurred, giving 7s as dark purple needles (210
mg, 95%): mp 186-187.5 °C; 1H NMR (CDCl3) δ 3.16 [s, 6 H,
N(CH3)2], 7.26 (dd, J ) 6.8, 1.8 Hz, 1 H, H-6 or H-8), 7.81-
7.88 (m, 2 H, H-7 and H-8 or H-6), 8.01 (dd, J ) 8.7, 7.0 Hz,
1 H, H-3), 8.48 (dd, J ) 8.7, 1.2 Hz, 1 H, H-4), 8.91 (dd, J )
7.0, 1.3 Hz, 1 H, H-2). Anal. (C15H13N3O2) C, H, N.
Bis[3-(3-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6c): Exa m p le of Gen er a l Meth od of Sch em e 1.
A suspension of 3-methylphenazine-1-carboxylic acid (7c)13
(1.02 g, 4.29 mmol) in DMF (10 mL) was treated with 1,1′-
carbonyldiimidazole (1.39 g, 8.57 mmol), and the mixture was
stirred at 45 °C for 30 min. After cooling, the mixture was
diluted with CH2Cl2/petroleum ether (1:1) to complete precipi-
tation of the crude imidazolide (8c), which was collected,
washed with petroleum ether, dried, and immediately added
to an ice-cold solution of bis(3-aminopropyl)methylamine (0.31
g, 2.15 mmol) in THF (50 mL). The mixture was stirred at 20
°C for 4 h, then volatiles were removed under reduced
pressure, and the residue was partitioned between CH2Cl2 and
aqueous 1 M Na2CO3. The organic layer was washed with
water, dried, and evaporated, and the residue was chromato-
graphed on alumina-90. Elution with CH2Cl2/MeOH (20:1),
followed by crystallization from CH2Cl2/petroleum ether, gave
bis[(3-methylphenazine-1-carboxamido)propyl]methylamine (6c)
(1.05 g, 84% from the acid): mp (CH2Cl2/n-hexane) 75-78 °C;
1H NMR (CDCl3) δ 2.03 (quin, J ) 7.0 Hz, 4 H, 2 × CH2CH2-
CH2), 2.37 (s, 3 H, NCH3), 2.67 (d, J ) 0.9 Hz, 6 H, 2 × CH3),
2.73 [t, J ) 7.2 Hz, 4 H, CH2N(CH3)CH2], 3.73 (q, J ) 6.3 Hz,
4 H, 2 × CH2NH), 7.62-7.70 (m, 4 H, 2 × H-7 and 2 × H-8),
7.98-8.03 (m, 6 H, 2 × H-6, 2 × H-9 and 2 × H-2 or 2 × H-4),
8.73 (d, J ) 2.1 Hz, 2 H, 2 × H-4 or 2 × H-2), 10.88 (br
t, J ) 5.2 Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd
for C35H36N7O2 586.2930 (MH+); found, 586.2937. Anal.
(C35H35N7O2‚H2O) C, H, N.
Bis[3-(2-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6b). From similar reaction of 2-chlorophenazine-1-
carboxylic acid13 (7b) (45% yield): mp (CH2Cl2/n-hexane) 206-
1
207 °C; H NMR (CDCl3) δ 1.83 (quin, J ) 6.0 Hz, 4 H, 2 ×
CH2CH2CH2), 2.17 (s, 3 H, NCH3), 2.72 [t, J ) 6.1 Hz, 4 H,
CH2N(CH3)CH2], 3.67 (q, J ) 6.0 Hz, 2 H, 2 × CH2NH), 7.03
(br t, J ) 5.9 Hz, 2 H, 2 × CONH), 7.47 (d, J ) 9.4 Hz, 2 H,
2 × H-3 or 2 × H-4), 7.60-7.68 (m, 4 H, 2 × H-7 and 2 × H-8),
7.89 (d, J ) 9.3 Hz, 2 H, 2 × H-4 or 2 × H-3), 7.91-7.97 (m,
4 H, 2 × H-6 and 2 × H-9). HRMS (FAB+) m/z calcd
for C33H3035Cl2N7O2 626.1838 (MH+); found, 626.1854. Anal.
(C33H29Cl2N7O2) C, H, N.
6-Ch lor o-9-m eth ylp h en a zin e-1-ca r boxylic Acid (7u ). A
mixture of 5-chloro-2-methylaniline (16) (8.63 g, 61.0 mmol),
2-bromo-3-nitrobenzoic acid (17) (10.0 g, 41.0 mmol), CuCl (0.5
g), copper powder (0.1 g) in butane-2,3-diol (25 mL), and
N-ethylmorpholine (15 mL) was stirred and heated for 18 h
at 70 °C. The reaction mixture was diluted with 0.5 M NH4-
OH (500 mL), then filtered through Celite. The orange filtrate
was then slowly added to a stirred solution of 2 N HCl, and
the resulting yellow precipitate was collected by filtration,
dried, and recrystallized to give 2-[(5-chloro-2-methylphenyl)-
amino]-3-nitrobenzoic acid (20) (6.97 g, 55%): mp (EtOAc/n-
Bis[3-(3-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6d ). From similar reaction of 3-chlorophenazine-
1-carboxylic acid13 (7d ) (76% yield): mp (CH2Cl2/n-hexane)
1
169-170 °C; H NMR (CDCl3) δ 2.02 (quin, J ) 6.9 Hz, 4 H,
1
hexane) 228-230 °C; H NMR (CDCl3) δ 2.35 (s, 3 H, CH3),
2 × CH2CH2CH2), 2.36 (s, 3 H, NCH3), 2.72 [t, J ) 7.3 Hz, 4
H, CH2N(CH3)CH2], 3.71 (q, J ) 6.3 Hz, 4 H, 2 × CH2NH),
7.69-7.76 (m, 4 H, 2 × H-7 and 2 × H-8), 7.94-8.00 (m, 4 H,
2 × H-6 and 2 × H-9), 8.20 (d, J ) 2.5 Hz, 2 H, 2 × H-4), 8.74
(d, J ) 2.5 Hz, 2 H, 2 × H-2), 10.65 (br t, J ) 5.2 Hz, 2 H, 2
× CONH). HRMS (FAB+) m/z calcd for C33H3035Cl2N7O2
626.1838 (MH+); found, 626.1824. Anal. C33H29Cl2N7O2‚H2O)
C, H, N.
6.79 (d, J ) 2.1 Hz, 1 H, H-6′), 6.96-7.00 (m, 2 H, H-4′ and
H-5), 7.15 (d, J ) 8.0 Hz, 1 H, H-3′), 8.07 (dd, J ) 8.1, 1.8 Hz,
1 H, H-4 or H-6), 8.24 (dd, J ) 7.9, 1.7 Hz, 1 H, H-6 or H-4),
9.51 (s, 1 H, NH). Anal. (C14H11ClN2O2) C, H, N.
A solution of 20 (3.59 g, 11.7 mmol) and NaBH4 (2.62 g, 68.8
mmol) in 2 M NaOH was heated at reflux for 8 h. The reaction
mixture was then cooled and acidified with AcOH, and the
resulting precipitate was collected and recrystallized to give
7u (1.42 g, 45%): mp (acetone) 255-257 °C; 1H NMR [(CD3)2-
SO] δ 2.86 (s, 3 H, CH3), 7.90 (dd, J ) 7.4, 1.1 Hz, 1 H, ArH),
8.11-8.18 (m, 2 H, ArH), 8.57-8.61 (m, 2 H, ArH), 14.52 (br
s, 1 H, COOH). Anal. (C14H9ClN2O2) C, H, N, Cl.
6,9-Dim eth ylp h en a zin e-1-ca r boxylic Acid (7t). Reaction
of 2-bromo-3-nitrobenzoic acid (17) and 2,5-dimethylaniline
(15) as above gave 2-[(2,5-dimethylphenyl)amino]-3-nitro-
benzoic acid (19) (65%): mp (benzene/acetone) 215-217 °C;
1H NMR [(CD3)2SO] δ 2.10 (s, 3 H, CH3), 2.23 (s, 3 H, CH3),
6.53 (s, 1H, H-6′), 6.79 (d, J ) 7.4 Hz, 1 H, H-4′), 7.02 (t, J )
8.0 Hz, 1 H, H-5), 7.11 (d, J ) 7.7 Hz, 1 H, H-3′), 8.03 (dd,
J ) 8.1, 1.4 Hz, 1 H, H-6), 8.22 (dd, J ) 7.7, 1.5 Hz, 1 H, H-4),
9.84 (br s, 1 H, NH), 13.8 (br s, 1 H, CO2H). Anal. (C15H14N2O4)
C, H, N.
Bis[3-(4-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6e). From similar reaction of 4-methylphenazine-
1-carboxylic acid13 (7e) (78% yield): mp (CH2Cl2/n-hexane)
1
218-220 °C; H NMR (CDCl3) δ 2.04 (quin, J ) 7.0 Hz, 2 H,
2 × CH2CH2CH2), 2.38 (s, 3 H, NCH3), 2.75 [t, J ) 7.3 Hz, 4
H, CH2N(CH3)CH2], 2.90 (s, 6 H, 2 × CH3), 3.71 (q, J ) 6.3
Hz, 4 H, 2 × CH2NH), 7.58 (ddd, J ) 8.6, 6.7, 1.3 Hz, 2 H,
ArH), 7.65 (ddd, J ) 8.6, 6.6, 1.4 Hz, 2 H, ArH), 7.70 (dd, J )
7.2, 1.0 Hz, 2 H, ArH), 7.94 (dd, J ) 8.6, 0.9 Hz, 2 H, ArH),
8.00 (d, J ) 8.7 Hz, 2 H, ArH), 8.77 (d, J ) 7.3 Hz, 2 H, ArH),
10.88 (br s, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd
for
C
35H36N7O2 586.2930 (MH+); found, 586.2922. Anal.
(C35H35N7O2‚2.5H2O) C, H, N.
Bis[3-(6-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6f). From similar reaction of 6-methylphenazine-
1-carboxylic acid13 (7f) (47% yield): mp (HCl salt from MeOH/
Reductive ring closure of 19 with NaOC2H5/EtOH/NaBH4
under the above conditions gave 7t (64%): mp (MeOH) 246-
247 °C; 1H NMR [(CD3)2SO] δ 2.78 (s, 3 H, CH3), 2.83 (s, 3 H,
CH3), 7.80 (d, J ) 7.0 Hz, 1 H, H-7 or H-8), 7.83 (d, J ) 7.0
Hz, 1 H, H-7 or H-8), 8.12 (dd, J ) 8.5, 7.2 Hz, 1 H, H-3), 8.56
(d, J ) 8.7 Hz, 1 H, H-4), 8.66 (d, J ) 7.0 Hz, 1 H, H-2), 15.24
(br s, 1 H, CO2H). Anal. (C15H12N2O2) C, H, N.
1
EtOAc) 228-230 oC; H NMR (CDCl3) δ 2.06 (quin, J ) 6.9
Hz, 4 H, 2 × CH2CH2CH2), 2.39 (s, 3 H, NCH3), 2.79 (s, 6 H,
2 × CH3), 2.81 [t, J ) 7.0 Hz, 4 H, CH2N(CH3)CH2], 3.75 (q,
J ) 6.1 Hz, 4 H, 2 × CH2NH), 7.42 (t, J ) 7.8 Hz, 2 H, 2 ×
H-8), 7.61 (d, J ) 8.8 Hz, 2 H, ArH), 7.87 (dd, J ) 8.5, 7.1 Hz,
4 H, 2 × H-3 and ArH), 8.27 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 ×

1356 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Spicer et al.
H-4), 8.88 (dd, J ) 7.0, 1.5 Hz, 2 H, 2 × H-2), 10.93 (br s, 2 H,
2 × CONH). Anal. (C35H35N7O2‚0.5H2O) C, H, N.
35Cl2N7O2 626.1838 (MH+); found, 626.1860. Anal. (C33H29
Cl2N7O2) C, H, N, Cl.
-
Bis[3-(6-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6g). From similar reaction of 6-chlorophenazine-1-
carboxylic acid13 (7g) (56% yield): mp (CH2Cl2/MeOH) 198-
Bis[3-(8-methoxyphenazine-1-carboxamido)propyl]meth-
yla m in e (6m ). From similar reaction of 8-methoxyphenazine-
1-carboxylic acid13,14 (7m ) (99% yield): mp (CH2Cl2/hexane)
1
1
200 °C; H NMR (CDCl3) δ 2.03 (quin, J ) 7.0 Hz, 4 H, 2 ×
182-186 °C; H NMR (CDCl3) δ 1.92 (quin, J ) 6.4 Hz, 2 ×
CH2CH2CH2), 2.37 (s, 3 H, NCH3), 2.73 [t, J ) 7.2 Hz, 4 H,
CH2N(CH3)CH2], 3.72 (q, J ) 6.2 Hz, 4 H, CH2NH), 7.62 (dd,
J ) 8.7, 7.2 Hz, 2 H, 2 × H-8), 7.74 (dd, J ) 7.2, 1.2 Hz, 2 H,
2 × H-7 or 2 × H-9), 7.91 (dd, J ) 8.8, 1.2 Hz, 2 H, 2 × H-9 or
2 × H-7), 7.93 (dd, J ) 8.7, 7.1 Hz, 2 H, 2 × H-3), 8.39 (dd,
J ) 8.7, 1.6 Hz, 2 H, 2 × H-4), 8.88 (dd, J ) 7.1, 1.6 Hz, 2 H,
2 × H-2), 10.59 (br t, J ) 5.1 Hz, 2 H, 2 × CONH). HRMS
(FAB+) m/z calcd for C33H30Cl2N7O2 626.1838 (MH+); found,
618.1840. Anal. (C33H29Cl2N7O2) C, H, N, Cl.
CH2CH2CH2), 2.30 (s, 3 H, NCH3), 2.71 [m, 4 H, CH2N(CH3)-
CH2], 3.60 (q, J ) 6.1 Hz, 4 H, 2 × CH2NH), 3.85 (s, 6 H, 2 ×
OCH3), 7.06 (s, 2 H, 2 × H-9), 7.19 (dd, J ) 9.4, 2.4 Hz, 2 H,
2 × H-7), 7.69 (d, J ) 9.4 Hz, 2 H, 2 × H-6), 7.80 (dd, J ) 8.7,
7.2 Hz, 2 H, 2 × H-3), 8.11 (dd, J ) 8.5, 1.4 Hz, 2 H, 2 × H-4),
8.48 (J ) 7.1, 1.5 Hz, 2 H, 2 × H-2), 10.39 (t, J ) 5.4 Hz, 2 H,
2 × CONH). Anal. (C35H35N7O4) C, H, N.
Bis[3-(9-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6n ). From similar reaction of 9-methylphenazine-
1-carboxylic acid13 (7n ) (82% yield): mp (HCl salt from
MeOH/EtOAc) 262-264 °C; 1H NMR (CDCl3) δ 1.99 (quin,
J ) 7.3 Hz, 4 H, 2 × CH2CH2CH2), 2.32 (s, 3 H, NCH3), 2.60
[t, J ) 7.4 Hz, 4 H, CH2N(CH3)CH2], 2.79 (s, 6 H, 2 × CH3),
3.70 (q, J ) 6.7 Hz, 4 H, 2 × CH2NH), 7.56 (d, J ) 6.7 Hz,
2 H, 2 × H-8), 7.65 (dd, J ) 8.7, 7.2 Hz, 2 H, 2 × H-7),
7.89 (dd, J ) 8.7, 7.2 Hz, 2 H, 2 × H-3), 7.97 (d, J ) 8.6 Hz,
2 H, 2 × H-6), 8.27 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 × H-4),
8.93 (dd, J ) 7.2, 1.5 Hz, 2 H, 2 × H-2), 10.94 (br s, 2 H, 2 ×
CONH). Anal. (C35H35N7O2.HCl) C, N, Cl; H, found 6.4, calcd
5.9%.
Bis[3-(7-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6h ). From similar reaction of 7-methylphenazine-
1-carboxylic acid (7h )13 (63% yield): mp (HCl salt from MeOH/
1
EtOAc) 213-215 °C; H NMR (CDCl3) δ 2.06 (quin, J ) 6.9
Hz, 4 H, 2 × CH2CH2CH2), 2.38 (s, 3 H, NCH3), 2.44 (s, 6 H,
2 × CH3), 2.79 [t, J ) 7.4 Hz, 4 H, CH2N(CH3)CH2], 3.75 (q,
J ) 6.2 Hz, 4 H, 2 × CH2NH), 7.40 (dd, J ) 8.9, 1.6 Hz, 2 H,
2 × H-8), 7.62 (br s, 2 H, 2 × H-6), 7.77 (d, J ) 8.9 Hz, 2 H, 2
× H-9), 7.86 (dd, J ) 8.5, 7.1 Hz, 2 H, 2 × H-3), 8.22 (dd, J )
8.6, 1.5 Hz, 2 H, 2 × H-4), 8.86 (dd, J ) 7.2, 1.5 Hz, 2 H, 2 ×
H-2), 10.85 (t, J ) 4.9 Hz, 2 H, 2 × CONH). Anal. (C32H35N7O2‚
2HCl‚2H2O) C, N, Cl: H; found 6.6; requires 6.0%.
Bis[3-(9-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6o). From similar reaction of 9-chlorophenazine-1-
carboxylic acid13 (7o) (86% yield): mp (CH2Cl2/MeOH) 169-
Bis[3-(7-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6i). From similar reaction of 7-chlorophenazine-1-
carboxylic acid (7i)13 (71% yield): mp (CH2Cl2/MeOH) 173-
1
171.5 °C; H NMR (CDCl3) δ 2.01 (quin, J ) 7.3 Hz, 4 H, 2 ×
1
175 °C; H NMR (CDCl3) δ 2.02 (quin, J ) 6.9 Hz, 4 H, 2 ×
CH2CH2CH2), 2.32 (s, 3 H, NCH3), 2.62 [t, J ) 7.4 Hz, 4 H,
CH2N(CH3)CH2], 3.70 (q, J ) 6.2 Hz, 4 H, 2 × CH2NH), 7.64
(dd, J ) 8.8, 7.4 Hz, 2 H, 2 × H-7), 7.80 (dd, J ) 7.2, 1.0 Hz,
2 H, 2 × H-6 or 2 × H-8), 7.95 (dd, J ) 8.7, 7.2 Hz, 2 H, H-3),
8.01 (dd, J ) 8.7, 1.0 Hz, 2 H, 2 × H-8 or 2 × H-6), 8.27 (dd,
J ) 8.7, 1.5 Hz, 2 H, 2 × H-4), 8.99 (dd, J ) 7.2, 1.5 Hz, 2 H,
H-2), 10.94 (br t, J ) 5.0 Hz, 2 H, 2 × CONH). HRMS (FAB+)
m/z calcd for C33H30Cl2N7O2 626.1838 (MH+); found, 618.1848.
Anal. (C33H29Cl2N7O2) C, H, N.
CH2CH2CH2), 2.37 (s, 3 H, NCH3), 2.73 [t, J ) 7.2 Hz, 4 H,
CH2N(CH3)CH2], 3.73 (q, J ) 6.2 Hz, 4 H, 2 × CH2NH), 7.54
(dd, J ) 9.3, 2.4 Hz, 2 H, H-8), 7.84 (d, J ) 9.3 Hz, 2 H, 2 ×
H-9), 7.90 (d, J ) 2.5 Hz, 2 H, 2 × H-6), 7.92 (dd, J ) 8.7, 7.1
Hz, 2 H, 2 × H-3), 8.20 (dd, J ) 8.7, 1.6 Hz, 2 H, 2 × H-4),
8.88 (dd, J ) 7.1, 1.5 Hz, 2 H, 2 × H-2), 10.54 (br t, J ) 5.1
Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd for C33H30
Cl2N7O4 626.1838 (MH+); found, 618.1844. Anal. (C33H29
Cl2N7O2) C, H, N, Cl.
-
-
Bis[3-(9-flu or op h en a zin e-1-ca r boxa m ido)p r op yl]m eth -
yla m in e (6p ). From similar reaction of 9-fluorophenazine-1-
carboxylic acid14 (7p ) (87% yield): mp (CH2Cl2/MeOH) 186-
Bis[3-(7-methoxyphenazine-1-carboxamido)propyl]meth-
yla m in e (6j). From similar reaction of 7-methoxyphenazine-
1-carboxylic acid13 (7j) (60% yield): mp (HCl salt from MeOH/
EtOAc) 225-229 °C; 1H NMR (free base in CDCl3) δ 2.03 (quin,
J ) 6.9 Hz, 4 H, 2 × CH2CH2CH2), 2.37 (s, 3 H, NCH3), 2.74
[t, J ) 7.2 Hz, 4 H, CH2N(CH3)CH2], 3.73 (q, J ) 6.3 Hz, 4 H,
2 × CH2NH), 3.93 (s, 6 H, 2 × OCH3), 7.10 (d, J ) 2.7 Hz, 2
H, 2 × H-6), 7.28 (dd, J ) 9.1, 3.1 Hz, 2 H, ArH), 7.78 (d, J )
9.5 Hz, 2 H, 2 × H-9), 7.83 (dd, J ) 8.7, 1.5 Hz, 2 H, ArH),
8.17 (dd, J ) 8.6, 1.5 Hz, 2 H, ArH), 8.81 (J ) 7.2, 1.5 Hz, 2
1
187 °C; H NMR (CDCl3) δ 2.02 (quin, J ) 7.1 Hz, 4 H, 2 ×
CH2CH2CH2), 2.36 (s, 3 H, NCH3), 2.72 [t, J ) 7.4 Hz, 4 H,
CH2N(CH3)CH2], 3.73 (q, J ) 6.1 Hz, 4 H, 2 × CH2NH), 7.30-
7.35 (m, 2 H, 2 × H-7 or 2 × H-8), 7.54-7.60 (m, 2 H, 2 × H-8
or 2 × H-7), 7.84 (d, J ) 9.0 Hz, 2 H, 2 × H-6), 7.94 (dd, J )
8.7, 7.0 Hz, 2 H, 2 × H-3), 8.25 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 ×
H-4), 8.95 (dd, J ) 7.0, 1.5 Hz, 2 H, H-2), 10.94 (br t, J ) 5.0
Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd for C33H30F2N7O2
594.2429 (MH+); found, 594.2403. Anal. (C33H29F2N7O2‚
0.5H2O) C, H, N.
H, ArH), 10.77 (t, J ) 4.6 Hz, 2 H, 2 × CONH). Anal. (C35H35
-
N7O4.2HCl‚3H2O) C, H, N.
Bis[3-(8-m eth ylph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6k ). From similar reaction of 8-methylphenazine-
1-carboxylic acid13,14 (7k ) (76% yield): mp (CH2Cl2/hexane)
Bis[3-(9-methoxyphenazine-1-carboxamido)propyl]meth-
yla m in e (6q). From similar reaction of 9-methoxyphenazine-
1-carboxylic acid13,14 (7q) (86% yield): mp (CH2Cl2/MeOH)
1
1
161-162 °C; H NMR (CDCl3) δ 2.16 (quin, J ) 6.6 Hz, 4 H,
220-222 °C; H NMR (CDCl3) δ 2.02 (quin, J ) 7.2 Hz, 4 H,
2 × CH2CH2CH2), 2.52 (s, 9 H, NCH3 and 2 × CH3), 2.93 [br
s, 4 H, CH2N(CH3)CH2], 3.76 (q, J ) 6.3 Hz, 4 H, 2 × CH2-
NH), 7.41 (d, J ) 8.6 Hz, 2 H, 2 × H-6), 7.77 (br s, 2 H, 2 ×
H-9), 7.86 (dd, J ) 8.5, 7.1 Hz, 4 H, 2 × H-3 and 2 × H-7),
8.26 (dd, J ) 8.6, 1.5 Hz, 2 H, H-4), 8.87 (dd, J ) 7.7, 1.5 Hz,
2 H, H-2), 11.00 (br s, 2 H, 2 × CONH). Anal. (C35H35N7O2‚
0.5H2O) C, H, N.
CH2CH2CH2), 2.39 (s, 3 H, NCH3), 2.73 [t, J ) 7.6 Hz, 4 H,
CH2N(CH3)CH2], 3.66 (q, J ) 6.0 Hz, 4 H, CH2NH), 3.90 (s, 6
H, 2 × OCH3), 6.60 (dd, J ) 6.7, 1.9 Hz, 2 H, 2 × H-6 or 2 ×
H-8), 7.32-7.38 (m, 4 H, 2 × H-7 and 2 × H-8 or 2 × H-6),
7.84 (dd, J ) 8.7, 7.2 Hz, 2 H, 2 × H-3), 8.11 (dd, J ) 8.7, 1.5
Hz, 2 H, 2 × H-4), 8.83 (dd, J ) 7.1, 1.5 Hz, 2 H, 2 × H-2),
11.12 (br t, J ) 4.7 Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z
calcd for C35H36N7O4 618.2829 (MH+); found, 618.2847. Anal.
(C35H35N7O4) C, H, N.
Bis[3-(8-ch lor oph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6l). From similar reaction of 8-chlorophenazine-1-
carboxylic acid13,14 (7l) (85% yield): mp (CH2Cl2/n-hexane)
Bis[3-(9-phenoxyphenazine-1-carboxamido)propyl]meth-
1
210-212 °C; H NMR (CDCl3) δ 2.04 (quin, J ) 7.0 Hz, 4 H,
yla m in e (6r ). From similar reaction of 9-phenoxyphenazine-
1
2 × CH2CH2CH2), 2.39 (s, 3 H, NCH3), 2.73 [t, J ) 7.2 Hz, 4
H, CH2N(CH3)CH2], 3.74 (q, J ) 6.3 Hz, 4 H, 2 × CH2NH),
7.56 (dd, J ) 9.2, 2.4 Hz, 2 H, 2 × H-7), 7.92 (dd, J ) 8.7, 7.2
Hz, 2 H, 2 × H-3), 7.98 (d, J ) 9.2, 2 H, 2 × H-6), 8.03 (d, J )
2.2 Hz, 2 H, 2 × H-9), 8.26 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 × H-4),
8.92 (dd, J ) 7.2, 1.5 Hz, 2 H, 2 × H-2), 10.64 (br t, J ) 5.2
1-carboxylic acid (7r ) (51% yield): as an orange oil; H NMR
(CDCl3) δ 1.71 (quin, J ) 7.3 Hz, 4 H, 2 × CH2CH2CH2), 1.97
(s, 3 H, NCH3), 2.31 [t, J ) 7.3 Hz, 4 H, CH2N(CH3)CH2], 3.43
(q, J ) 6.4 Hz, 4 H, 2 × CH2NH), 7.11-7.14 (m, 6 H, 2 × H-2′,
2 × H-6′ and 2 × H-6 or 2 × H-8), 7.18 (t, J ) 7.5 Hz, 2 H,
2 × H-4′), 7.39 (t, J ) 7.5 Hz, 4 H, 2 × H-3′ and 2 × H-5′),
7.69 (dd, J ) 8.7, 7.6 Hz, 2 H, 2 × H-7), 7.89 (m, 4 H, 2 × H-3
Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd for C33H30
-

Bis(phenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1357
and 2 × H-8 or 2 × H-6), 8.26 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 ×
H-4), 8.90 (dd, J ) 7.1, 1.5 Hz, 2 H, 2 × H-2), 10.98 (br t, J )
5.2 Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd for
C45H40N7O4 742.3142 (MH+); found, 742.3147.
were performed in duplicate, and coefficients of variation were
18% (P388), 22% (LL), 16% (J LC), 16% (J LA), and 16%(J LD).
Top oisom er a se Assa ys. Topo I assays were carried out25
in a reaction (30 µL) containing 50 mM Tris HCl (pH 7.5), 50
mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, 30 µg/
mL bovine serum albumin, and 0.125 µg pBR322 supercoiled
plasmid DNA, together with calf thymus topo I (5 units for
relaxation assays and 15 units for cleavable complex formation
assays). Topo II assays were carried out in a reaction (20 µL)
in relaxation and cleavage buffers supplied by the manufac-
turer (TopoGEN, Inc), pRYG supercoiled plasmid DNA (0.125
µg), and purified human topo II (10 units for cleavage assays
and 4 units for relaxation assays). Reactions were assembled
on ice with drug and topoisomerase added last, incubated at
37 °C for 30 min, and terminated by adding pre-warmed 1%
sodium dodecyl sulfate, followed by proteinase K treatment
(50 µg/mL) for an additional 30 min. Relaxation assay samples
were loaded onto 1% agarose gels in 40 mM Tris acetate buffer
(pH 8.0) containing 1 mM EDTA and were electrophoresed at
1.4 V/cm for 14-16 h.
In Vivo Colon 38 Tu m or Assa y. Colon 38 tumors were
grown subcutaneously from 1 mm3 fragments implanted in one
flank of mice (anesthetized with pentobarbitone 90 mg/kg).
When tumors reached a diameter of approximately 4 mm (7-8
days), mice were divided into control and drug treatment
groups (5 mice/group), with similar average tumor volumes
in each group. Drugs were administered as solutions of the
hydrochloride salts in distilled water and were injected in a
volume of 0.01 mL/g body weight in two equal injections
administered 1 h apart. The mice were monitored closely, and
tumor diameters were measured with callipers three times a
week. Tumor volumes were calculated as 0.52 × a2 × b, where
a and b are the minor and major tumor axes, and data were
plotted on a semilogarithmic graph as mean tumor volumes
(( SEM) versus time after treatment. The growth delay was
calculated as the time taken for tumors to reach a mean
volume 4-fold higher than their pretreatment volume.
Bis[3-(9-d im et h yla m in o)p h en a zin e-1-ca r b oxa m id o)-
p r op yl]m et h yla m in e (6s). From similar reaction of 9-(di-
methylamino)phenazine-1-carboxylic acid (7s) (78% yield) as
a red-purple oil: 1H NMR (CDCl3) δ 1.95 (quin, J ) 7.2 Hz, 4
H, 2 × CH2CH2CH2), 2.29 (s, 3 H, NCH3), 2.57 [t, J ) 7.3 Hz,
4 H, CH2N(CH3)CH2], 3.05 [s, 12 H, 2 × N(CH3)2], 3.68 (q, J )
6.5 Hz, 4 H, 2 × CH2NH), 7.07 (dd, J ) 7.2, 1.3 Hz, 2 H, 2 ×
H-6 or 2 × H-8), 7.65 (dd, J ) 8.7, 7.3 Hz, 2 H, 2 × H-7), 7.70
(dd, J ) 8.7, 1.3 Hz, 2 H, 2 × H-8 or 2 × H-6), 7.90 (dd, J )
8.6, 7.1 Hz, 2 H, 2 × H-3), 8.27 (dd, J ) 8.6, 1.4 Hz, 2 H, 2 ×
H-4), 8.87 (dd, J ) 7.1, 1.4 Hz, 2 H, 2 × H-2), 10.99 (br t, J )
5.1 Hz, 2 H, 2 × CONH). HRMS (FAB+) m/z calcd for
C
37H42N9O2 644.3461 (MH+); found, 644.3485.
Bis[3-(6,9-d im eth ylp h en a zin e-1-ca r boxa m id o)p r op yl]-
m eth yla m in e (6t). From similar reaction of 6,9-dimeth-
ylphenazine-1-carboxylic acid (7t) (53% yield): mp (CH2Cl2/
n-hexane) 97-101 °C; 1H NMR (CDCl3) δ 2.02-2.08 (br m,
4 H, 2 × CH2CH2CH2), 2.34 (s, 3 H, NCH3), 2.60-2.68 [br m,
4 H, CH2N(CH3)CH2], 2.68 (s, 6 H, 2 × ArCH3), 2.78 (s, 6 H,
2 × ArCH3), 3.70 (q, J ) 6.6 Hz, 4 H, 2 × CH2NH), 7.32-7.40
(m, 4 H, 2 × H-7 and 2 × H-8), 7.86 (dd, J ) 8.6, 7.2 Hz, 2 H,
2 × H-3), 8.28 (dd, J ) 8.7, 1.5 Hz, 2 H, 2 × H-4), 8.90 (dd,
J ) 7.1, 1.5 Hz, 2 H, 2 × H-2), 11.00 (br s, 2 H, 2 × CONH).
HRMS (FAB+) m/z calcd for C37H40N7O2 614.3243 (MH+);
found, 614.3237. Anal. (C37H39N7O2‚0.5H2O) C, H, N.
Bis[3-(6-ch lor o-9-m et h ylp h en a zin e-1-ca r b oxa m id o)-
p r op yl]m eth yla m in e (6u ). From similar reaction of 6-chloro-
9-methylphenazine-1-carboxylic acid (7u ) (84% yield): mp
(CH2Cl2/n-hexane) 200-202 °C; 1H NMR (CDCl3) δ 1.97 (quin,
J ) 7.2 Hz, 4 H, 2 × CH2CH2CH2), 2.31 (s, 3 H, NCH3), 2.59
[t, J ) 7.1 Hz, 4 H, CH2N(CH3)CH2], 2.76 (s, 6 H, 2 × CH3),
3.69 (q, J ) 6.7 Hz, 4 H, 2 × CH2NH), 7.50 (dd, J ) 7.6, 1.0
Hz, 2 H, 2 × H-8), 7.78 (d, J ) 7.5 Hz, 2 H, 2 × H-7), 7.93 (dd,
J ) 8.7, 7.2 Hz, 2 H, 2 × H-3), 8.41 (dd, J ) 8.7, 1.5 Hz, 2 H,
2 × H-2), 8.94 (dd, J ) 7.1, 1.5 Hz, 2 H, 2 × H-4), 10.72 (br s,
2 H, 2 × CONH). HRMS (FAB+) m/z calcd for C35H34Cl2N7O2
654.2151 (MH+); found, 654.2159. Anal. (C35H33Cl2N7O2‚
0.5H2O) C, H, N.
Ack n ow led gm en t. This work was supported by
Xenova Ltd, Slough, U.K., and the Auckland Division
of the Cancer Society of New Zealand.
Bis[3-(6,7-ben zoph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6v). From similar reaction of 6,7-benzophenazine-
1-carboxylic acid13 (7v) (66% yield): mp (CH2Cl2/hexane) 138-
Refer en ces
1
142 °C; H NMR (CDCl3) δ 2.08 (quin, J ) 6.8 Hz, 4 H, 2 ×
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CH2CH2CH2), 2.41 (s, 3 H, NCH3), 2.84 [t, J ) 7.3 Hz, 4 H,
CH2N(CH3)CH2], 3.78 (q, J ) 6.1 Hz, 4 H, 2 × CH2NH), 7.48
(d, J ) 9.2 Hz, 2 H, ArH), 7.59-7.76 (m, 10 H, ArH), 8.01 (dd,
J ) 8.4, 1.5 Hz, 2 H, ArH), 8.73 (dd, J ) 7.3, 1.5 Hz, 2 H,
ArH), 9.02 (d, J ) 7.7 Hz, 2 H, ArH), 10.84 (br t, J ) 5.0 Hz,
2 H, 2 × CONH). Anal. (C41H35N7O2) C, H, N.
Bis[3-(8,9-ben zoph en azin e-1-car boxam ido)pr opyl]m eth -
yla m in e (6w ). From similar reaction of 8,9-benzophenazine-
1-carboxylic acid13 (7w ) (77% yield): mp (CH2Cl2/hexane) 214-
1
215.5 °C; H NMR (CDCl3) δ 2.10 (quin, J ) 7.1 Hz, 4 H, 2 ×
CH2CH2CH2), 2.43 (s, 3 H, NCH3), 2.80 [t, J ) 7.4 Hz, 4 H,
CH2N(CH3)CH2], 3.70 (q, J ) 6.3 Hz, 4 H, 2 × CH2NH), 7.18-
7.31 (m, 2 H, ArH), 7.36 (t, J ) 7.2 Hz, 2 H, ArH), 7.48-7.57
(m, 6 H, ArH), 7.92 (dd, J ) 8.5, 7.2 Hz, 2 H, ArH), 8.28 (dd,
J ) 8.5, 1.5 Hz, 2 H, ArH), 8.61 (d, J ) 8.0 Hz, 2 H, ArH),
8.87 (dd, J ) 7.3, 1.5 Hz, 2 H, ArH), 10.52 (br s, 2 H, 2 ×
CONH). Anal. (C41H35N7O2‚0.5H2O) C, H, N.
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assays were performed by culturing cells at 4.5 × 103 (P388),
103 (LL), and 3.75 × 103 (J urkat lines) per well in microculture
plates (150 mL per well) for 3 (P388) or 4 days in the presence
of drug. Cell growth was determined by [3H]TdR uptake
(P388)23 or the sulforhodamine assay.24 Independent assays

1358 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
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