Dicationic bis(9-methylphenazine-1-carboxamides): Relationships between biological activity and linker chain structure for a series of potent topoisomerase targeted anticancer drugs

Article abstract of DOI:10.1021/jm0003283

Bis(9-methylphenazine-1-carboxamides) joined by a variety of dicationic (CH₂)_nNR(CH₂)_mNR- (CH₂)_n linkers of varying length (carboxamide N-N distances from 11.0 to 18.4 ?) and rigidity were prepared by reaction of 9-methylphenazine-1-carboxylic acid imidazolide with the appropriate polyamines. The compounds were evaluated for growth inhibitory properties in P388 leukemia, Lewis lung carcinoma, and wild-type (JL_C) and mutant (JL_A and JL_D) forms of human Jurkat leukemia with low levels of topoisomerase II (topo II). The compounds all had IC₅₀ ratios of <1 in the resistant Jurkat lines, consistent with topo II inhibition not being the primary mechanism of action. Analogues joined by an (CH₂)₂NR(CH₂)₂NR (CH₂)₂ linker were' extremely potent cytotoxins, with selectivity toward the human cell lines, but absolute potencies declined sharply from R = H through R = Me to R = Pr and Bu. In contrast, (CH₂)₂NR(CH₂)_3-NR (CH₂)₂ compounds showed reverse effects, with the R = Me analogue being more potent than the R = H one as well as being the most potent in the series [IC₅₀ in JL_C cells 0.08 nM, superior to that for the clinical bis(naphthalimide) LU 79553]. Overall, the IC₅₀s of analogues with linker chains (CH₂)_nNH(CH₂)_mNH (CH₂)_n were inversely proportional to linker length. Constraining the rigidity of the linker chain by incorporating a piperazine ring did not decrease potency significantly. A representative compound bound tightly to DNA with high selectivity for GC sites, compatible with recent work suggesting that compounds of this type place their side chains in the major groove, making specific contacts with guanine bases. Representative compounds were susceptible to transport mediated resistance, being much less effective in cells that overexpressed P-glycoprotein. Overall the results suggest these compounds have a similar mode of action, mediated primarily by poisoning of topo I (possibly with some involvement of topo II). The bis(9-methylphenazine-1-carboxamides) show very high in vitro growth inhibitory potencies compared to their monomeric analogues. Two compounds showed in vivo activity in murine colon 38 syngeneic and HT29 human colon tumor xenograft models using intraperitoneal dosing.

Full text of DOI:10.1021/jm0003283

J . Med. Chem. 2001, 44, 1407-1415
1407
Dica tion ic Bis(9-m eth ylp h en a zin e-1-ca r boxa m id es): Rela tion sh ip s betw een
Biologica l Activity a n d Lin k er Ch a in Str u ctu r e for a Ser ies of P oten t
Top oisom er a se Ta r geted An tica n cer Dr u gs
Swarna A. Gamage,† J ulie A. Spicer,† Graeme J . Finlay,† Alistair J . Stewart,# Peter Charlton,#
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, and Xenova Ltd., 240 Bath Road, Slough SL1 4EP, Berkshire, England
Received August 1, 2000
Bis(9-methylphenazine-1-carboxamides) joined by a variety of dicationic (CH2)nNR(CH2)mNR-
(CH2)n linkers of varying length (carboxamide N-N distances from 11.0 to 18.4 Å) and rigidity
were prepared by reaction of 9-methylphenazine-1-carboxylic acid imidazolide with the
appropriate polyamines. The compounds were evaluated for growth inhibitory properties in
P388 leukemia, Lewis lung carcinoma, and wild-type (J LC) and mutant (J LA and J LD) forms of
human J urkat leukemia with low levels of topoisomerase II (topo II). The compounds all had
IC50 ratios of <1 in the resistant J urkat lines, consistent with topo II inhibition not being the
primary mechanism of action. Analogues joined by an (CH2)2NR(CH2)2NR(CH2)2 linker were
extremely potent cytotoxins, with selectivity toward the human cell lines, but absolute potencies
declined sharply from R ) H through R ) Me to R ) Pr and Bu. In contrast, (CH2)2NR(CH2)3-
NR(CH2)2 compounds showed reverse effects, with the R ) Me analogue being more potent
than the R ) H one as well as being the most potent in the series [IC50 in J LC cells 0.08 nM;
superior to that for the clinical bis(naphthalimide) LU 79553]. Overall, the IC50s of analogues
with linker chains (CH2)nNH(CH2)mNH(CH2)n were inversely proportional to linker length.
Constraining the rigidity of the linker chain by incorporating a piperazine ring did not decrease
potency significantly. A representative compound bound tightly to DNA with high selectivity
for GC sites, compatible with recent work suggesting that compounds of this type place their
side chains in the major groove, making specific contacts with guanine bases. Representative
compounds were susceptible to transport mediated resistance, being much less effective in cells
that overexpressed P-glycoprotein. Overall the results suggest these compounds have a similar
mode of action, mediated primarily by poisoning of topo I (possibly with some involvement of
topo II). The bis(9-methylphenazine-1-carboxamides) show very high in vitro growth inhibitory
potencies compared to their monomeric analogues. Two compounds showed in vivo activity in
murine colon 38 syngeneic and HT29 human colon tumor xenograft models using intraperitoneal
dosing.
Compounds containing two neutral, relatively lipo-
philic DNA monointercalating chromophores linked by
a flexible chain are of current interest as potential
topoisomerase (topo) inhibitors and anticancer drugs.
Most such compounds have been symmetric, employing
chromophores such as naphthalimides,1-4 benzonaph-
thalimides,5 imidazoacridanones,6,7 anthracyclinones,8,9
acridinecarboxamides,10 and phenazinecarboxamides,11
but some unsymmetric compounds with dissimilar chro-
mophores have also been reported.12 Many of these bis
compounds are much more potent cytotoxins than the
corresponding monomers, and some show broad spec-
trum activity against a variety of human solid tumor
cell lines, both in culture and as xenografts in nude
mice. The bis(naphthalimide) (1: DMP 840) has under-
gone clinical trials4 and is reported to be a topo II
inhibitor in yeast.3 The related analog LU 79553 (2) is
highly effective against tumor xenografts in vivo13 and
is reported to bisintercalate DNA, with the side chain
binding in the major groove,14,15 and to inhibit topo II.15
Most of the structure-activity work with these com-
pounds has focused on alterations in the linker chain,
with relatively few changes in the terminal chro-
mophores. In contrast, our studies on bis(acridine-4-
carboxamides)10 3 and bis(phenazine-1-carboxamides)11
4 focused on evaluation of the role of chromophore
substituents and showed that structure-activity rela-
* Address correspondence to Dr. William A. Denny. Ph: (64 9) 373
7599 ext 6144. Fax: (64 9) 373 7502. E-mail: b.denny@auckland.ac.nz.
† Auckland Cancer Society Research Centre.
#
Xenova Ltd.
10.1021/jm0003283 CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/28/2001

1408 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Gamage et al.
Sch em e 1a
Sch em e 2a
a
(i) Raney Ni/H2/dry EtOH/NH3(g)/20 °C/5 d, or LiAlH4/THF/
reflux/4 h.
tionships (SAR) earlier derived for the corresponding
monomers16-18 applied to the bis compounds. Thus
analogues bearing small, lipophilic substituents (par-
ticularly methyl) at the unoccupied position peri to the
ring nitrogen [position 5 for bis(acridine-4-carbox-
amides) (e.g., 5) and position 9 for bis(phenazine-1-
carboxamides) (e.g., 6)] showed the highest cytotoxicity
in the monomeric series.17,18
a
(i) BOCNH(CH2)3CO2H/CDI/THF; (ii) HCl(g)/MeOH, then
Amberlite (OH-) resin; (iii) BH3-Me2S/THF; (iv) EtCO2CF3/
MeOH/N2/20 °C/15 h, then BOC2O/0-20 °C/3 h, then NH4OH/
20 °C/15 h; (v) 38, CDI/DMF/50-60 °C/15 h; (vi) HCl(g)/MeOH/
20 °C/5 d.
Sch em e 3a
Following this identification of 9-methylphenazine as
the optimal chromophore (in terms of cytotoxicity) for
bis analogues, in the present paper we explore structure-
activity relationships for bis(9-methylphenazine-1-car-
boxamides) 7-22, employing a variety of dicationic
linker chains where the length, rigidity, and charge
density on the chain are varied.
a
(i) CDI/DMF/50-60 °C/15 h; (ii) H2N-R-NH2/THF/20 °C/15 h.
Reaction of 9-methylphenazine-1-carboxylic acid (38),
6,9-dimethylphenzazine-1-carboxylic acid (39), or 6-chloro-
9-methylphenazine-1-carboxylic acid (40) with 1,1′-
carbonyldiimidazole (CDI) in DMF gave the correspond-
ing imidazolides. These were purified from excess
reagent by crystallization from CH2Cl2/petroleum ether,
then suspended in THF and treated with 0.5 equiv of
the appropriate diamine (Scheme 3).
Ch em istr y
Many of the linker chains for this study were com-
mercially available. The N,N′-dialkyl-N,N′-bis(2-ami-
noethyl)-1,X-diaminoalkanes (24, 26, 28, 30) were pre-
pared by formation of the corresponding bis(cyanomethyl)
analogues (23, 25, 27, 29, respectively) and subsequent
reduction of these with either LiAlH4 or Raney nickel
(Scheme 1).5,19 1,4-Bis(2-aminoethyl)piperazine (32) and
the known20 1,4-bis(aminobutyl)piperazine (34) were
similarly prepared via the corresponding dinitriles 31
and 33. N,N′-Bis(4-aminobutyl)-1,4-butanediamine (37)
for compound 20 was prepared from the corresponding
bisamides 35 and 36 according to the method of Brana
et al. (Scheme 2).5
For the preparation of compounds 18 and 20, it was
found desirable to protect the secondary amines of
linkers 41 and 37, as difficulties were encountered in
purification of the desired products. Thus the primary
amine groups were selectively protected by reaction with
ethyl trifluoroacetate, the remaining secondary amines

Dicationic Bis(9-methylphenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1409
Ta ble 1. Growth Inhibitory Properties of Bis(phenazine-1-carboxamides)
IC50 (nM)b
LLd
IC50 ratios
linka
(Å)
e
no.
fm
R
P388c
J LC
46
A/Cf
D/Cg
5h
6
6.4
18
15
21
33
170
250
7.4
1.6
740
2240
48
5.6
39
3.3
2.8
20
4.6
1.1
0.6
0.5
0.4
0.6
0.5
0.7
0.4
0.4
0.6
0.8
0.5
0.5
0.2
0.3
0.5
0.4
0.3
4.8
1.1
0.6
0.9
0.6
0.7
0.8
0.6
0.8
1.3
0.9
0.7
0.5
1.0
1.2
0.9
0.6
0.8
0.6
207
7i
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
C
(CH2)3NMe(CH2)3
9.8
11.0
11.0
11.0
11.0
12.2
12.2
13.5
10.3
12.5
15.2
14.7
16.0
18.4
11.0
11.0
12.3
5.6
0.18
5.3
16
46
0.24
0.08
1.7
8
9
(CH2)2NH(CH2)2NH(CH2)2
(CH2)2NMe(CH2)2NMe(CH2)2
(CH2)2NEt(CH2)2NEt(CH2)2
(CH2)2NPr(CH2)2NPr(CH2)2
(CH2)2NH(CH2)3NH(CH2)2
(CH2)2NMe(CH2)3NMe(CH2)2
(CH2)3NH(CH2)2NH(CH2)3
(CH2)2NpipN(CH2)2
10
11
12
13
14
15
16
17
18
19
20
21
22
2
48
120
1.2
0.3
35
12
11
(CH2)3NpipN(CH2)3
(CH2)4NpipN(CH2)4
0.46
0.34
31
112
290
3.9
10
1.3
1.1
1.3
8.7
96
(CH2)3NH(CH2)3NH(CH2)3
(CH2)3NH(CH2)4NH(CH2)3
(CH2)4NH(CH2)4NH(CH2)4
(CH2)2NH(CH2)2NH(CH2)2
(CH2)2NH(CH2)2NH(CH2)2
LU 79553
430
880
890
8.3
37
67
0.6
0.6
0.7
1.5
11
a
b
Length of linker chain (fully extended N-N distance between the carboxamides, see text). IC50; concentration of drug (nM) to reduce
cell number to 50% of control cultures (see text). c P388 murine leukemia. Lewis lung carcinoma. e J urkat human leukemia. f A/C )
J LA/J LC. D/C ) J LD/J LC. Data from ref 18. Data from ref 11.
d
g
h
i
were BOC-protected, and then the trifluoroacetamides
were cleaved with aqueous ammonia in a one-pot
procedure.21 The BOC-protected amines (42, 43) were
then reacted with the 9-methylphenazine imidazolide
as above, and the resulting BOC-protected intermedi-
ates (44, 45) were purified before subsequent cleavage
to the final products (Scheme 2).
mechanism. While the bis(naphthalimide) 2 is re-
ported15 as a topo II inhibitor, the J urkat ratios for this
compound in Table 1 (0.3 and 0.6) also suggest a similar
mechanism.
Compounds 7-20 in Table 1 explore structure-
activity relationships for the linker chain in bis(9-
methylphenazine-1-carboxamides). Linker chain lengths
were compared by computing the carboxamide N-N
distances (in the fully extended form) using the 3D
module from ACD Labs. With the possible exception of
7, all compounds had N-N distances compatible with
a possible bisintercalative mode of DNA binding, by
comparison with the known14,15 bisintercalator 2. This
has an N-N distance of 12.3 Å and, as a bis(imide),
probably a more rigid overall conformation than the bis-
(carboxamides). All of the bis compounds had IC50 ratios
in the resistant J urkat lines of <1, consistent with a
primarily non topo II mechanism of action.
As reported previously,11 the monocationic analogue
7 is a much more potent cytotoxin than the correspond-
ing monomer 6 (10-fold in LL, 37-fold in J LC). With the
shortest linker chain (an N-N distance of 9.8 Å), this
compound has the least flexibility and low cationic
charge density.
The dicationic compounds 8-11, with (CH2)2NR-
(CH2)2NR(CH2)2 linkers (separation distance 11.0 Å),
showed very clear SAR. The unmethylated compound
8 shows similar activity to 7 in the murine cell lines,
but a sharply enhanced potency (30-fold; IC50 0.18 nM)
in the human leukemia lines. Alkylation of the NH
groups in the linker with Me, Et, or Pr groups (com-
pounds 9-11) resulted in a steady loss of potency in all
cell lines as the size of the alkyl group was increased.
The closely related (CH2)2NR(CH2)3NR(CH2)2 ana-
logues 12 and 13, with an additional methylene unit
Str u ctu r e-Activity Rela tion sh ip s for Cellu la r
Gr ow th In h ibition
The structures of the bis compounds prepared by the
above methods are listed in Table 1, together with their
cytotoxicities (as IC50 values) in a panel of cell lines. The
selection of the cell line panel has been discussed
previously.10,18 P388 is a murine leukemia line,16 and
Lewis lung is a murine carcinoma22 line. The three
human leukemia (J urkat) lines23,24 provide some insight
into the mechanism of cytotoxicity. J LC is the wild-type
(sensitive) line, while J LA is 85-fold resistant to the topo
II inhibitor amsacrine because of a reduced level of topo
II enzyme. J LD is a doxorubicin-resistant line, also
primarily by virtue of lower topo II levels. IC50 values
are given for the P388, LLTC, and J LC lines, together
with ratios of IC50 values against J LC and the other two
J urkat lines (ratios J LA/J LC and J LD/J LC). Values of
these ratios of less than about 2-fold suggest a likely
non topo II mediated mechanism of action. The 5-me-
thylacridine-4-carboxamide 5 was the most potent com-
pound in the monomeric series of DACA analogues,16
but the ratios of 4.6 and 4.8 (Table 1 and ref 18) suggest
a predominantly topo II mechanism. In contrast, the
9-methylphenazine-1-carboxamide 6, while also among
the most potent monomeric phenazine analogues,17 has
ratios of only 1.1 and 1.1, respectively (Table 1), sug-
gestive of a primarily non topo II or mixed topo I/II

1410 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Gamage et al.
Ta ble 2. Growth Inhibitory Properties of Selected
Bis(phenazine-1-carboxamides) in the NCI Cell Line Panel
Ta ble 3. Growth Inhibitory Properties of Selected
Bis(phenazine-1-carboxamides) in Other Tumor Cell Lines
GI50 (nM)a
HT-29 MOLT4 U937
AuxB1
D3F
midb
a
a
a
a
a
no.
IC50
IC50
IC50
IC50
ratiob IC50
ratiob
no. MCF7 NCI-ADR CAK-1 SKOV3 HT29 HCT-116
7
8
<10c
<10
19
15
<0.1
8030
906
<10
<10
220
<10
<0.1 <10
8890
13600
<10
<10
<10
<10
20
13
0.29
524
1995
7
8
21
2
1.5
0.35
6.9
0.57
3.7
4.5
31.3
47.1
30.3
2.9
4.4
6.5
0.57
0.91
0.77
0.08
0.15
0.15
0.0062
0.067
0.037
0.047
0.52
0.34
13 <10
14
19
<0.1
<0.1
24
74
1000
8280
27
366
21
335
a
IC50; concentration of drug (nM) to reduce cell number to 50%
a
GI50: concentration of drug (nM) resulting in inhibition of cell
b
of control cultures (see text). Ratio IC50[CHrC5]/IC50[AuxB1].
Ratio IC50[D3F-C10]/IC50[D3F].
b
growth to 50% of controls. mid: the average GI50 value for the
drug over the whole cell line panel. c Lowest dose tested.
controls; equivalent to IC50), together with the GI50(mid)
value (the average GI50 value for the drug over the
whole 60-cell line panel). The individual cell line results
are of limited value since many were not titrated down,
but the overall GI50(mid) values correlate reasonably
well with the J LC results in Table 1. For the less active
compounds 14 and 19, a comparison of results in the
MCF7 and NCI/ADR lines (the latter overexpresses
P-glycoprotein) shows they are significantly affected by
P-glycoprotein mediated multidrug resistance. Four
compounds (2, 7, 8, 21) were also evaluated in HT-29
human colon and MOLT4 and U937 human leukemia
lines (Table 3). All showed similar patterns of activity,
with the MOLT4 line proving most sensitive.
between the cationic centers (separation distance 12.2
Å), showed quite different behavior. The unmethylated
compound 12 showed similar cytotoxicities to 8 in the
cell line panel, but in this case methylation of the NH
groups to give 13 led to significant increases in potency,
making it the most effective compound in all of the cell
lines (e.g., an IC50 of 0.08 nM in J LC, 10-fold superior
to that of the clinical agent 2). Similar SAR have been
reported previously, with related pairs of both bis-
(naphthalimides)2 and bis(indeno[1,2-b]quinoline-6-car-
boxamides),25 but the reasons for this quite consistent
behavior are not clear.
Compounds 14, 18, and 19 have progressively longer
linker chains, (CH2)3NH(CH2)nNH(CH2)3, where n is
2-4. Analogues 14 and 18, separated by 13.5 and 14.7
Å, respectively) showed comparable activity (albeit less
than 8 or 12), whereas 19 (16.0 Å) was much less
effective. Compound 20, with the longest linker chain
[(CH2)4NH(CH2)4NH(CH2)4; 18.4 Å], was the least active
of all the compounds studied. In fact, for the subset of
compounds with linker chains (CH2)xNH(CH2)nNH-
(CH2)x, there is an excellent inverse correlation between
potency and linker length (e.g., eq 1).
Mech a n ism of Action Stu d ies
The binding constants of 7 and 8 to DNA (to the
copolymers poly‚[dA-dT] and poly‚[dG-dC]) were deter-
mined at 0.01 ionic strength by the ethidium displace-
ment method.28 The monocationic compound 7 bound
equally well to both copolymers, with binding constants
of 8 × 107 and 7 × 107 M-1. The dicationic compound 8
showed a similar level of binding to poly‚[dA-dT] (9 ×
107 M-1) but bound much more strongly to poly‚[dG-
dC] (K ) 1.6 × 109 M-1). This high GC-selectivity is
consistent with reported binding models for both acridi-
necarboxamides29 and the bis(naphthalimide) 2,15 where
the side chains lie in the major groove and make specific
contacts with guanine bases.
log IC50 (J LC) ) 0.36 ((0.07)[length] - 4.92 ((1.1)
(1)
n ) 5, r ) 0.985, F ) 94.7
A similar trend was seen with bis(indeno[1,2-b]quino-
line-6-carboxamides), where extending the linker chain
from (CH2)2NH(CH2)3NH(CH2)2 to (CH2)3NH(CH2)3NH-
(CH2)3 led to a 200-fold drop in potency.25
To further evaluate mechanism of action, selected
compounds were evaluated in a series of cell lines, along
with the bis(naphthalimide) 2 (Table 3). The CHrC5 line
is derived from the AuxB1 Chinese hamster line and
has highly amplified expression of the P-glycoprotein
drug efflux pump.30 The DC3F-C10 line is derived from
the D3F Chinese hamster line and has a point mutation
in the topo I enzyme, leading to a >60-fold resistance
to camptothecin.31 The CHrC5/AuxB1 ratios show that
all the compounds are susceptible to the P-glycoprotein
mediated resistance (although it should be noted that
this cell line has a high level of overexpression). The
low DC3F-C10/D3F ratios are consistent with previous
data that all of these compounds, because they bind
DNA rather than topo I protein, are not affected by this
point mutation.32
Compounds 15-17 constrain the linker chain by
incorporating a piperazine ring. Analogue 15, with the
shortest and most rigid of all the dicationic linkers, has
relatively low potency. There are no strictly comparable
linear chain compounds to compare 16 and 17 to, but
they are among the most potent derivatives in the set.
These results suggest that, as shown previously with
bis(benzonaphthalimides),5 constraining the linker chain
is acceptable, provided it has some minimum length and
overall flexibility. On the other hand, too long and
flexible a chain is disadvantageous, presumably because
of unfavorable entropic effects on DNA binding.26 Com-
pounds 21 and 22 are analogues of 8 with an additional
substituent at the 5-position. As shown previously with
monocationic bis(phenazine-1-carboxamides),11 while
these substituents are acceptable they do not signifi-
cantly affect cytotoxicity.
These compounds are expected to be efficient DNA
intercalating agents, and this is demonstrated for 8 in
Figure 1, where using topo I to relax the supercoiled
DNA before addition of 8 indicated that the compound
can induce drug-dependent supercoils in the DNA,
typical behavior for a DNA intercalator.33,34
Selected compounds were also evaluated in the NCI
cell human line panel,27 and results for six of those lines
are shown in Table 2 as GI50 values (the concentration
of drug resulting in inhibition of cell growth to 50% of

Dicationic Bis(9-methylphenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1411
toneal route, with a growth delay of 7.8 days at 20 mg/
kg (Table 4). Compound 8 was relatively inactive
against HT29 by this route, with a growth delay of only
2.4 days at 13.3 mg/kg. However, 8 has been reported35
to show good activity in this model (22-day growth
delay) when given intravenously (15 mg/kg, q4d × 3)
and to induce complete tumor regression in the majority
of animals in the H69 SCLC model.
Con clu sion s
The dicationic bis(9-methylphenazine-1-carboxam-
ides) are extremely potent cytotoxins in cell culture,
particularly in human cell lines, with generally greater
activity toward mutant lines underexpressing topo II.
There was a negative correlation between absolute IC50
values and linker length. This is similar to that observed
for dicationic bis(indeno[1,2-b]quinoline-6-carboxam-
ides)25 but not for dicationic bis(benzonaphthalimides),5
where in vitro potency did not alter significantly with
chain length. The highly GC-selective DNA binding of
8 is consistent with the reported binding model for the
related bis(naphthalimide) 2,15 with the linker chain
lying in the major groove, making specific contacts with
guanines.
F igu r e 1. Agarose gel of 8 with supercoiled and topo I relaxed
DNA. Lane 1: supercoiled DNA. Lane 2: relaxed DNA. Lane
3: relaxed DNA + 1.0 µM compound 8. Lane 4: relaxed DNA
+ 0.1 µM compound 8.
The compounds all showed higher activity toward
mutant J urkat leukemia lines that underexpress topo
II, and they also inhibited the action of purified topo I.
This is consistent with their primary mode of action
being via poisoning of topo I. However, other studies
on the stabilization of cleavable complex by 8 have
shown that this compound can poison both topo I and
II in a purified enzyme system.36 Studies with selected
examples (including 8) showed they had much lower
effectiveness in cells with artificially elevated P-glyco-
protein.
Two representative compounds (8 and 21) showed
significant but modest antitumor effects in vivo against
synegeneic colon 38 and HT29 human tumor xenografts
when given intraperitoneally, indicating that their very
high in vitro growth inhibitory potencies (compared to
their monomeric analogues) do not necessarily translate
into comparable increases in in vivo effectiveness.
However, 8 does show good activity against HT29
tumors when given intravenously.35
F igu r e 2. Effect of 8 (A) and 21 (B) on the growth of colon 38
adenocarcinoma. Panel A: Mice with subcutaneously im-
planted tumors of approximately 4 mm in diameter were
treated with 8 at doses of 20 mg/kg (4) or 13.3 mg/kg (3) on
an intermittent dose schedule (every 4 days × 3). Symbols (b)
show untreated tumor controls. Panel B: Mice were treated
as above with 21 at 30 mg/kg.
Ta ble 4. In Vivo Activity of Compounds 8 and 21
growth delay
no.
tumor
dose
(days)
8
colon 38
colon 38
colon 38
HT29
colon 38
colon 38
colon 38
colon 38
HT29
20
7.0
4.0
2.8
2.4
7.5
4.4
2.4
2.8
7.8
Exp er im en ta l Section
13.3
8.9
13.3
30
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.
21
20
13.3
8.9
20
In Vivo Stu d ies
Compounds 8 and 21 were evaluated both in the
relatively refractory subcutaneous syngeneic colon 38
tumor model in C57/Bl mice. Both compounds were
active in this model (growth delays of 7 days at 20 mg/
kg for 8, and 7.5 days at 30 mg/kg for 21 (Figure 2 and
Table 4)) when given intraperitoneally. This compares
with a growth delay of 12 days at 30 mg/kg previously
reported11 for the less cytotoxic analogue 7 in this model.
Compound 21 was also active against HT29 human
colon tumor xenografts in nude mice by the intraperi-
P r ep a r a tion of Lin k er Ch a in s. N,N′-Dim eth yl-N,N′-
b is(2-a m in oet h yl)-1,2-et h a n ed ia m in e (24). A solution of
N,N′-dimethyl-N,N′-bis(cyanomethyl)-1,2-ethanediamine37 (23)
(5.0 g, 100 mmol) in absolute EtOH saturated with dry
ammonia was hydrogenated over Raney nickel for 5 days at
20 °C (additional Raney nickel was added as required). The

1412 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Gamage et al.
catalyst was removed by filtration through a pad of Celite, and
evaporation of solvents gave essentially pure 24 (4.60 g, 88%)
that was used directly: 1H NMR (CDCl3) δ 2.24 (s, 6 H, 2 ×
CH3), 2.43 (br s, 4 H, 2 × CH2), 2.49 (s, 2 H, 2 × CH2), 2.73 (br
s, 2 H, 2 × CH2).
and CDI (5.89 g, 36.4 mmol) in dry THF (100 mL) was stirred
for 15 h at room temperature. A solution of 1,4-diaminobutane
(1.47 g, 16.6 mmol) in dry THF (50 mL) was slowly added to
the above solution of imidazolide. This mixture was allowed
to stir at room temperature for 2 days. The solvent was
removed under reduced pressure and the residue partitioned
between 1 M Na2CO3 (200 mL) and CHCl3 (200 mL). The
CHCl3 layer was then washed with water (300 mL) and 0.5 M
HCl solution (200 mL) and then dried (Na2SO4). The CHCl3
was removed under reduced pressure to give a white solid
which was purified by chromatography on silica gel (2-3%
MeOH in CH2Cl2 as eluant), giving N,N′-bis(4-tert-butyloxy-
carbonylamino-1-oxobutyl)-1,4-butanediamine (35) (2.72 g,
N ,N ′-Die t h yl-N ,N ′-b is(2-a m in oe t h yl)-1,2-e t h a n e d i-
a m in e (26). Sodium m-bisulfite (11.5 g, 60.5 mmol) was
dissolved in water (25 mL) and 40% aqueous formaldehyde
(7.75 mL). The solution was boiled for 10 min and cooled to
room temperature, and then N,N′-diethyl-1,2-ethanediamine
(6.6 g, 56.8 mmol) was added with vigorous stirring. After the
mixture was stirred at room temperature for 4 h, a solution
of NaCN (6.25 g, 127.5 mmol) in water (12.5 mL) was added,
and the mixture was stirred overnight at room temperature.
The product was extracted into CH2Cl2 and dried (Na2SO4).
Evaporation of solvents gave a yellow oil that was chromato-
graphed on alumina, eluting with CH2Cl2, to give N,N′-bis-
(cyanomethyl)-N,N′-diethyl-1,2-diaminoethane (25) (5.63 g,
51%) as an oil: 1H NMR (CDCl3) δ 1.11 (t, J ) 7.2 Hz, 6 H, 2
× CH2CH3), 2.60 (q, J ) 7.2 Hz, 4 H, 2 × CH2CH3), 2.67 [s, 4
H, N(CH2)2N], 3.69 (s, 4 H, 2 × CH2CN).
1
38%): mp (EtOAc) 118-119 °C; H NMR [(CH3)2SO] δ 1.39-
1.54 (m, 22 H, 6 × CH3 and 2 × CH2), 1.56 (quin, J ) 7.3 Hz,
4 H, 2 × CH2), 2.02 (t, J ) 7.5 Hz, 4 H, 2 × CH2), 2.88 (q, J )
6.6 Hz, 2 H, 2 × CH2), 2.97-3.04 (br m, 4 H, 2 × CH2), 6.78 [t,
J ) 5.4 Hz, 2 H, 2 × CONH (amide)], 7.75 [t, J ) 5.5 Hz, 2 H,
2 × CONH (carbamate)]. Anal. (C22H42N4O6) C, H, N.
Diamide 35 (2.09 g, 4.56 mmol) in MeOH (50 mL) saturated
with HCl(g) was stirred overnight at room temperature, and
the solvent was then removed under reduced pressure. The
residue was converted from the HCl salt to the free base by
elution through a short column of Amberlite (OH-) resin with
EtOH, to give N,N′-bis(4-amino-1-oxobutyl)-1,4-butanediamine
(36) as a white solid (1.18 g, 100%): mp (EtOH) 120-124 °C;
1H NMR (D2O) δ 1.51 (br s, 4 H, 2 × CH2), 1.94 (quin, J ) 7.6
Hz, 4 H, 2 × CH2), 2.36 (t, J ) 7.5 Hz, 4 H, 2 × CH2), 3.01 (t,
J ) 7.8 Hz, 4 H, 2 × CH2), 3.19 (br s, 4 H, 2 × CH2). Anal.
(C12H26N4O2‚0.5H2O) C, H, N.
A solution of 25 (0.522 g, 2.69 mmol) in THF (20 mL) was
added dropwise to a suspension of LiAlH4 (0.47 g, 12.38 mmol)
in THF (20 mL). The reaction mixture was heated under reflux
for 4 h and then cooled in ice, and water (0.47 mL), 15%
aqueous NaOH (0.47 mL), and water (1.41 mL) were added
sequentially. The resulting granular precipitate was filtered
and washed with Et2O. The combined filtrates were evaporated
under reduced pressure, and the residue was chromatographed
on silica gel (eluting with MeOH/CH2Cl2/NH4OH, 30:60:10) to
give 26 (0.45 g, 85%) as an oil (lit.38 oil): 1H NMR (CDCl3) δ
1.02 (q, J ) 7.2 Hz, 6 H, 2 × CH2CH3), 1.96 (br s, 4 H, 2 ×
NH2), 2.48-2.57 (m, 8 H, 4 × CH2), 2.52 [s, 4 H, N(CH2)2N],
2.73 (t, J ) 6.1 Hz, 4 H, 2 × NCH2CH2NH2).
Reduction of 36 with BH3‚Me2S gave the known39 polyamine
N,N′-bis(4-aminobutyl)butane-1,4-diamine (37) as a crude oil.
This oil was purified by derivatization as described for
compound 42, giving N,N′-bis[(4- tert-butyloxycarbonylamino)-
butyl]-1,4-butanediamine (43) as a transparent oil, 56% over
two steps from the amide; 1H NMR (CDCl3) δ 1.45 [s, 18 H, 2
× C(CH3)3], 1.39-1.54 (br m, 12 H, 6 × CH2 and 2 × NH2),
2.70 (t, J ) 7.0 Hz, 4 H, 2 × CH2NH2), 3.17 [br s, 8 H, 2 ×
CH2N(BOC)CH2].
N,N′-Di-(n -p r op yl)-N,N′-bis(2-a m in oeth yl)-1,2-eth a n e-
d ia m in e (28). Similar reaction of N,N′-di-n-propyl-1,2-
ethanediamine gave N,N′-bis(cyanomethyl)-N,N′-di-n-pro-
pyl-1,2-ethanediamine (27) (8.3 g, 66%) as an oil: 1H NMR
(CDCl3) δ 0.92 (t, J ) 7.4 Hz, 6 H, 2 × CH3), 1.49 (sext, J )
7.4 Hz, 4 H, 2 × CH2), 2.49 (t, J ) 7.4 Hz, 4 H, 2 × CH2), 2.66
(s, 4 H, 2 × CH2), 3.67 (s, 4 H, 2 × CH2). HRMS (EI) m/z calcd
for C12H22N4 (M+), 222.1845; found, 222.1850.
Reduction of 27 with LiAlH4 as above gave 28 (1.96 g,78%)
as an oil: 1H NMR (CDCl3) δ 0.87 (t, J ) 7.2 Hz, 6 H, 2 ×
CH3), 1.47 (sext, J ) 7.4 Hz, 4 H, 2 × CH2), 2.39 (t, J ) 7.5
Hz, 4 H, 2 × CH2), 2.50 (t, J ) 7.0 Hz, 4 H, 2 × CH2), 2.52 (s,
4 H, 2 × CH2), 2.73 (t, J ) 6.0 Hz, 4 H, 2 × CH2), 3.12 (s, 8 H,
2 × CH2 and 2 × NH2). HRMS (FAB) m/z calcd for C12H30N4
(MH+), 231.2549; found, 231.2551.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
1,2-eth a n ed ia m in e (8): Gen er a l Meth od of Sch em e 3. A
mixture of 9-methylphenazine-1-carboxylic acid17 (38) (3.59 g,
15.1 mmol) and CDI (3.66 g, 22.6 mmol) in DMF (20 mL) was
stirred and heated to 50-60 °C for 2 h. Most of the DMF was
removed under reduced pressure, and CH2Cl2 (20 mL) was
added to dissolve the crude imidazolide. Addition of CH2Cl2/
light petroleum (1:4, 200 mL) and cooling in ice gave crystalline
imidazolide, which was collected, washed with cold CH2Cl2/
light petroleum (1:4, 100 mL), and dried (3.34 g, 77%). This
was dissolved in THF (30 mL), cooled in ice/salt, and treated
with a solution of N,N′-(2-aminoethyl)-1,2-diaminoethane (0.85
g, 5.8 mmol) in THF (5 mL). The mixture was allowed to slowly
warm to room temperature and stirred overnight. Most of the
THF was evaporated, then water (200 mL) was added, and
the mixture was stirred for 1 h. The resulting precipitate was
filtered, washed with water, dried (vacuum; P2O5), then
dissolved in MeOH/AcOH, clarified with charcoal/Celite, fil-
tered, and basified with Et3N to give 8 (2.5 g, 77%): mp
N,N′-Bis[(3- ter t-bu tyloxyca r bon yla m in o)p r op yl]-1,3-
p r op a n ed ia m in e (42). To a solution of commercial N,N′-bis-
(3-aminopropyl)-1,3-propanediamine (41) (1.00 g, 5.32 mmol)
in MeOH (20 mL) under N2 was added ethyl trifluoroacetate
(2.64 g, 18.6 mmol). This mixture was stirred for 15 h at room
temperature at which point the reaction was complete by TLC.
This solution was then cooled to 0 °C (ice/water) and then
BOC2O (4.64 g, 21.3 mmol) added, and the reaction was
allowed to warm to room temperature with stirring. After 3 h
at this temperature, concentrated aqueous NH3 was added
until the pH reached 11, and then the solution was stirred for
15 h at room temperature. The mixture was then concentrated,
diluted with CHCl3 (300 mL), and dried with Na2SO4. All solids
were removed by filtration and washed thoroughly with CHCl3,
and the organic fractions were combined. The CHCl3 was then
removed under reduced pressure to yield an oil which was
purified by chromatography on silica (eluting with 1% NH4-
OH in MeOH/CH2Cl2 20:80) to give N,N′-bis[(3- tert-butyloxy-
carbonylamino)propyl]-1,3-propanediamine (42) as a pale yel-
low oil (1.71 g, 83%): 1H NMR (CDCl3) δ 1.45 (br s, 18 H, 6 ×
CH3), 1.52-1.88 (m, 12 H, 4 × CH2 and 2 × NH2), 2.69 (br t,
J ) 6.6 Hz, 2 H, 1 × CH2), 3.11-3.31 (br m, 8 H, 4 × CH2).
1
(MeOH) 243-245 °C; H NMR (CF3CO2D) δ 3.15 (s, 6 H, 2 ×
ArCH3), 3.90 (t, J ) 4.6 Hz, 4 H, 2 × CH2NHCH2), 4.06 (s, 4
H, NHCH2CH2NH), 4.33 (t, J ) 4.6 Hz, 4 H, 2 × CONHCH2),
8.30 (d, J ) 5.0 Hz, 2 H, ArH), 8.42-8.48 (m, 4 H, ArH), 8.55
(t, J ) 8.1 Hz, 2 H, ArH), 8.81 (d, J ) 8.9 Hz, 2 H, ArH), 9.09
(d, J ) 7.2 Hz, 2 H, ArH). Anal. (C34H34N8O2) C, H, N.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
N,N′-d im eth yl-1,2-eth a n ed ia m in e (9). Similar reaction of
38 and 24, followed by chromatography of the product on silica
gel (eluting with a gradient of 1-6% MeOH in CH2Cl2), gave
9 (1.10 g, 78%): mp (CH2Cl2/hexane) 124-125 °C; 1H NMR
(CDCl3) δ 2.33 (s, 6 H, 2 × NCH3), 2.63 (br s, 4 H, CH3NCH2
CH2NCH3), 2.74 (t, J ) 6.5 Hz, 4 H, CONHCH2CH2), 2.83 (s,
6 H, 2 × ArCH3), 3.73 (q, J ) 6.2 Hz, 4 H, 2 × CONHCH2),
7.61 (d, J ) 6.7 Hz, 2 H, H-8), 7.68 (dd, J ) 8.7, 6.8 Hz, 2 H,
H-7), 7.89 (dd, J ) 8.7, 7.2 Hz, 2 H, H-3), 7.98 (d, J ) 8.7 Hz,
N,N′-Bis[(4-ter t-bu tyloxyca r bon yla m in o)bu tyl]-1,4-bu -
ta n ed ia m in e (43). According to the method of Brana et al.,5
a mixture of N-BOC-4-aminobutyric acid (7.11 g, 33.1 mmol)

Dicationic Bis(9-methylphenazine-1-carboxamides)
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1413
(1% MeOH in CH2Cl2 as eluant) to give 15 (77%): mp (CH2-
2 H, H-6), 8.26 (dd, J ) 8.7, 1.5 Hz, 2 H, H-4), 8.89 (dd, J )
7.0, 1.5 Hz, 2 H, H-2), 10.85 (t, J ) 5.2 Hz, 2 H, 2 × CONH).
Anal. (C36H38N8O2‚2HCl‚0.5H2O) C, H, N, Cl.
1
Cl2/n-hexane) 279-281 °C; H NMR (CDCl3) δ 2.58-2.65 (br
s, 8 H, 4 × CH2pip), 2.77 (t, J ) 6.6, 4 H, 2 × CH2Npip), 2.95
(s, 6 H, 2 × ArCH3), 3.84 (q, J ) 6.2 Hz, 4 H, 2 × CH2NHCO),
7.74 (d, J ) 6.7 Hz, 2 H, ArH), 7.80 (dd, J ) 8.6, 6.8 Hz, 2 H,
ArH), 7.96 (dd, J ) 8.6, 7.2 Hz, 2 H, ArH), 8.13 (d, J ) 8.7 Hz,
2 H, ArH), 8.39 (dd, J ) 8.7, 1.5 Hz, 2 H, ArH), 8.99 (dd, J )
7.2, 1.5 Hz, 2 H, ArH), 11.03 (br t, J ) 5.0 Hz, 2 H, 2 × CONH),
HRMS (FAB+) m/z calcd for C36H37N8O2, 613.3039 (MH+);
found, 613.3080. Anal. (C36H36N8O2) C, H, N.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
N,N′-d ieth yl-1,2-eth a n ed ia m in e (10). Similar reaction of 38
and 26 gave 10 (78%) as an oil: 1H NMR (CDCl3) δ 0.99 (t, J
) 7.1 Hz, 6 H, 2 × CH2CH3), 2.64 (q, J ) 7.1 Hz, 4 H, 2 ×
CH2CH3), 2.71 [s, 4 H, N(CH2)2N], 2.82 (t, J ) 6.8 Hz, 4 H, 2
× CONHCH2CH2), 2.84 (s, 6 H, 2 × ArCH3), 3.72 (q, J ) 6.4
Hz, 4 H, 2 × CH2), 7.63 (d, J ) 6.7 Hz, 2 H, H-8), 7.70 (dd, J
) 8.7, 6.6 Hz, 2 H, H-7), 7.88 (dd, J ) 8.6, 7.2 Hz, 2 H, H-3),
8.01 (d, J ) 8.5 Hz, 2 H, H-4), 8.28 (dd, J ) 8.7, 1.7 Hz, 2 H,
H-6), 8.92 (dd, J ) 7.2, 1.5 Hz, 2 H, H-2), 10.91 (t, J ) 5.6 Hz,
2 H, 2 × CONH). HRMS (FAB) C38H43N8O2 (MH+) requires
643.3509; found, 643.3517.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
N,N′-d i-n -p r op yl-1,2-eth a n ed ia m in e (11). Similar reaction
of 38 and 28 gave 11 (97%) as an oil: 1H NMR (CDCl3) δ 0.78
(t, J ) 7.4 Hz, 6 H, 2 × CH2CH3), 1.41 (sext, J ) 7.4 Hz, 4 H,
2 × CH2CH3), 2.49 (t, J ) 7.5 Hz, 4 H, 2 × CH2CH2CH3), 2.70
(br s, 4 H, 2 × CH2), 2.82 (t, J ) 6.5 Hz, 4 H, 2 × CH2), 2.86
(s, 6 H, 2 × ArCH3), 3.71 (q, J ) 6.3 Hz, 4 H, 2 × CH2), 7.64
(d, J ) 6.8 Hz, 2 H, H-8), 7.71 (dd, J ) 8.7, 6.8 Hz, 2 H, H-7),
7.89 (dd, J ) 8.6, 7.2 Hz, 2 H, H-3), 8.03 (d, J ) 8.4 Hz, 2 H,
H-6), 8.30 (dd, J ) 8.7, 1.5 Hz, 2 H, H-4), 8.93 (dd, J ) 7.1, 1.6
Hz, 2 H, H-2), 10.92 (br s, 2 H, 2 × CONH). HRMS (FAB+)
m/z calcd for C40H47N8O2 (MH+), 671.3822; found, 671.3801.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
1,3-p r op a n ed ia m in e (12). Similar reaction of 38 and N,N′-
bis(2-aminoethyl)-1,3-propanediamine gave 12 (47%): mp
(CH2Cl2/MeOH) 194-195 °C; 1H NMR (CDCl3) δ 1.73 (quin, J
) 6.9 Hz, 2 H, CH2CH2CH2), 2.79 (t, J ) 6.9 Hz, 4 H, 2 ×
CH2), 2.88 (s, 6 H, 2 × ArCH3), 2.97 (t, J ) 6.2 Hz, 4 H, 2 ×
CH2), 3.75 (q, J ) 6.0 Hz, 4 H, 2 × CH2), 7.64-7.69 (m, 2 H,
ArH), 7.72 (dd, J ) 8.6, 6.8 Hz, 2 H, ArH), 7.93 (dd, J ) 8.7,
7.2 Hz, 2 H, ArH), 8.04 (dd, J ) 8.7, 0.9 Hz, 2 H, ArH), 8.33
(dd, J ) 8.7, 1.5 Hz, 2 H, ArH), 8.96 (dd, J ) 7.2, 1.5 Hz, 2 H,
ArH) and 11.06 (br t, J ) 5.3 Hz, 2 H, 2 × CONH); HRMS
(FAB+) m/z calcd for C35H37N8O2 601.3039 (MH+), found
601.3043. Anal. (C35H36N8O2‚0.5H2O) C, H: N; Found 19.0;
Calculated 18.4%.
N,N′-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
N,N′-d im eth yl-1,3-p r op a n ed ia m in e (13). Reaction of N,N′-
bis(cyanomethyl)-1,3-propanediamine (29) with Raney nickel
in EtOH in the presence of ammonia gas gave the known40
amine N,N′-bis(2-aminoethyl)- N,N′-dimethyl-1,3-propanedi-
amine (30) in 49% yield. This amine was reacted directly with
38 as above, followed by chromatography on silica (1-2%
MeOH in CH2Cl2 as eluant) to give 13 as a yellow foam (87%):
1H NMR (CDCl3) δ 1.60-1.65 (m, 2 H, CH2CH2CH2 - obscured
by H2O), 2.18 (s, 6 H, 2 × NCH3), 2.40 (t, J ) 7.3 Hz, 4 H, 2
× CH2), 2.57 (t, J ) 6.5 Hz, 4 H, 2 × CH2), 2.83 (s, 6 H, 2 ×
ArCH3), 3.69 (q, J ) 6.1 Hz, 2 × CH2), 7.61-7.64 (m, 2 H,
ArH), 7.66 (dd, J ) 8.6, 6.8 Hz, 2 H, ArH), 7.90 (dd, J ) 8.7,
7.2 Hz, 2 H, ArH), 7.97 (d, J ) 7.3 Hz, 2 H, ArH), 8.28 (dd, J
) 8.7, 1.5 Hz, 2 H, ArH), 8.95 (dd, J ) 7.2, 1.5 Hz, 2 H, ArH),
10.82 (br t, J ) 4.9 Hz, 2 H, 2 × CONH). Anal. (C37H40N8O2)
C, H, N.
N1,N4-Bis[3-(9-m eth ylph en azin e-1-car boxam ido)pr opyl]-
p ip er a zin e (16). Similar reaction of 38 and 1,4-bis(3-amino-
propyl)piperazine gave a crude product that was dissolved in
MeOH/AcOH, clarified with charcoal/Celite, filtered, and bas-
ified with Et3N to give 16 (45%): mp (MeOH) 252-253 °C; 1H
NMR (CDCl3) δ 1.98 (quin, J ) 7.3 Hz, 4 H, 2 × CH2CH2-
CH2), 2.52 (t, J ) 7.4 Hz, 12 H, CH2Npip & pipCH2), 2.92 (s,
6 H, 2 × ArCH3), 3.72 (q, J ) 6.5 Hz, 4 H, 2 × CH2NHCO),
7.75-7.82 (m, 4 H, ArH), 7.96 (dd, J ) 8.7, 7.2 Hz, 2 H, H-3),
8.13 (dd, J ) 8.4, 0.8 Hz, 2 H, ArH), 8.39 (dd, J ) 8.7, 1.5 Hz,
2 H, ArH), 9.00 (dd, J ) 7.2, 1.5 Hz, 2 H, H-2), 11.11 (t, J )
5.3 Hz, 2 H, 2 × CONH). Anal. (C38H40N8O2‚0.5H2O) C, H, N.
N1,N4-Bis[4-(9-m eth ylp h en a zin e-1-ca r boxa m id o)bu tyl]-
p ip er a zin e (17). Similar reaction of 38 and 1,4-bis(4-ami-
nobutyl)piperazine20 gave 17 (68%): mp (CH2Cl2/hexane) 181-
1
182 °C; H NMR (CDCl3) δ 1.69 (quin, J ) 7.8 Hz, 4 H, 2 ×
CH2), 1.82 (quin, J ) 7.3 Hz, 4 H, 2 × CH2), 2.42 (t, J ) 7.5
Hz, 4 H, 2 × CH2), 2.43 (br s, 8 H, 4 × CH2pip), 3.70 (q, J )
6.6 Hz, 4 H, 2 × CH2NHCO), 7.73-7.81 (m, 4 H, ArH), 7.95
(dd, J ) 8.6, 7.0 Hz, 2 H, H-3), 8.11 (d, J ) 7.7 Hz, 2 H, ArH),
8.37 (dd, J ) 8.7, 1.3 Hz, 2 H, ArH), 8.99 (dd, J ) 7.1, 1.5 Hz,
2 H, H-2), 11.05 (br t, J ) 5.2 Hz, 2 H, 2 × CONH). Anal.
(C40H44N8O2) C, H, N.
N,N′-Bis[3-(9-m eth ylph en azin e-1-car boxam ido)pr opyl]-
1,3-p r op a n ed ia m in e (18): Sch em e 2. Similar reaction of 42
(407 mg, 1.05 mmol) with 38 as above, followed by chroma-
tography on silica gel (CH2Cl2/MeOH 99:1 as eluant), gave the
BOC-protected bis-phenazine (44) as a crude yellow oil (570
mg, 78%): 1H NMR (CDCl3) δ 1.43 (br s, 18 H, 6 × CH3), 1.80
(br s, 2 H, CH2), 2.02 (quin, J ) 7.3 Hz, 4 H, 2 × CH2), 2.89 (s,
6 H, 2 × ArCH3), 3.23 (br s, 4 H, 2 × CH2), 3.37 (t, J ) 7.0 Hz,
4 H, 2 × CH2), 3.66 (q, J ) 6.8 Hz, 4 H, 2 × CH2), 7.70-7.78
(m, 4 H, ArH), 7.93 (t, J ) 7.7 Hz, 2 H, ArH), 8.07 (d, J ) 8.3
Hz, 2 H, ArH), 8.34 (d, J ) 8.4 Hz, 2 H, ArH), 8.94 (br d, J )
5.8 Hz, 2 H, ArH), 11.03 (br s, 2 H, 2 × CONH).
A solution of 44 (570 mg, 0.68 mmol) in MeOH (10 mL)
saturated with HCl(g) was stirred at room temperature for 5
days, when the reaction was complete by TLC. The solvent
was removed under reduced pressure, and the resulting solid
was then eluted through an Amberlite (OH-) resin column to
convert the material to the free base. The elution solvent
(EtOH) was removed under reduced pressure, and the residue
was purified by chromatography on silica (1% NH4OH and 20%
MeOH in CH2Cl2 as eluant) to give 18 (341 mg, 79%): mp (CH2-
Cl2/n-hexane) 155-160 °C; 1H NMR (400 MHz, CDCl3) δ 1.69-
1.79 (m, 2 H, CH2), 2.02 (quin, J ) 7.1 Hz, 4 H, 2 × CH2), 2.76
(t, J ) 6.7 Hz, 4 H, 2 × CH2), 2.84 (t, J ) 7.0 Hz, 4 H, 2 ×
CH2), 2.90 (s, 6 H, ArCH3), 3.77 (q, J ) 6.7 Hz, 4 H, 2 × CH2),
7.67-7.70 (m, 2 H, ArH), 7.73 (dd, J ) 8.4, 6.8 Hz, 2 H, ArH),
7.92 (dd, J ) 8.6, 7.2 Hz, 2 H, ArH), 8.06 (d, J ) 8.5 Hz, 2 H,
ArH), 8.33 (dd, J ) 8.5, 1.5 Hz, 2 H, ArH), 8.96 (dd, J ) 7.1,
1.4 Hz, 2 H, ArH), 11.07 (br t, J ) 5.3 Hz, 2 H, 2 × CONH).
HRMS (FAB+) m/z calcd for C37H41N8O2, 629.3352 (MH+);
found, 629.3391. Anal. (C37H40N8O2‚H2O) C, H, N.
N,N′-Bis[3-(9-m et h ylp h en a zin e-1-ca r b oxa m id o)p r o-
p yl]-1,2-eth a n ed ia m in e (14). Similar reaction of 38 and
N,N′-bis(3-aminopropyl)-1,2-ethanediamine gave 14 as a gum,
which was converted to the dihydrochloride salt (10%): mp
(MeOH) 276 °C;1H NMR (HCl salt; D2O) δ 2.07 (quin, J ) 6.7
Hz, 4 H, 2 × CH2), 2.82 (s, 6 H, 2 × ArCH3), 3.17 (m, 4 H, 2 ×
CH2), 3.31 (br s, 4 H, 2 × CH2), 3.65 (t, J ) 6.6 Hz, 4 H, 2 ×
CH2), 7.87 (m, 4 H, ArH), 7.96-8.00 (m, 4 H, ArH), 8.27-8.30
(m, 2 H, ArH), 8.60 (d, J ) 7.2 Hz, 2 H, ArH). HRMS (FAB+)
m/z calcd for C36H38N8O2, 615.3196 (MH+); found, 615.3196.
N1,N4-Bis[2-(9-m eth ylp h en a zin e-1-ca r boxa m id o)eth yl]-
p ip er a zin e (15). Reaction of N,N′-bis(cyanomethyl)pipera-
zine41 (31) with Raney nickel in EtOH in the presence of
ammonia gas gave the known42 amine 1,4-bis(2-aminoethyl)-
piperazine (33) in 73% yield. This amine was reacted directly
with 38 as above, followed by chromatography on alumina
N,N′-Bis[3-(9-m eth ylph en azin e-1-car boxam ido)pr opyl]-
1,4-bu ta n ed ia m in e (19). Similar reaction of 38 and N,N′-
bis(3-aminopropyl)butanediamine gave 19 (73%): mp (CH2Cl2/
1
hexane) 86-90.5 °C; H NMR (CDCl3) δ 1.53 (quin, J ) 3.2
Hz, 4 H, 2 × CH2), 1.97 (quin, J ) 7.0 Hz, 4 H, 2 × CH2), 2.62
(t, J ) 6.2 Hz, 4 H, 2 × CH2), 2.79 (t, J ) 7.0 Hz, 4 H, 2 ×
CH2), 2.88 (s, 6 H, 2 × ArCH3), 3.74 (q, J ) 6.6 Hz, 4 H, 2 ×
CH2), 7.71-7.78 (m, 4 H, ArH), 7.93 (dd, J ) 8.7, 7.2 Hz, 2 H,
H-3), 8.08 (d, J ) 7.9, 0.8 Hz, 2 H, ArH), 8.34 (dd, J ) 8.7,

1414 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Gamage et al.
1.5 Hz, 2 H, H-4), 8.96 (dd, J ) 7.1, 1.5 Hz, 2 H, H-2), 11.05 (t,
J ) 5.2 Hz, 2 H, 2 × CONH). Anal. (C38H42N8O2‚1.5H2O)
C, H, N.
were obtained and cultured as described.32 Growth inhibition
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)44 or the sulforhodamine assay.45 Independent assays
were performed in duplicate.
N,N′-Bis[4-(9-m eth ylp h en a zin e-1-ca r boxa m id o)bu tyl]-
bu ta n ed ia m in e (20). Similar reaction of 43 with 38 as above,
followed by chromatography on silica gel (4% MeOH in CH2-
Cl2 as eluant) gave the BOC-protected bis-phenazine 45 as a
yellow foam (80%): 1H NMR (CDCl3) δ 1.34-1.49 [m, 22 H, 2
× C(CH3 )3 and 2 × CH2], 1.67-1.82 (m, 8 H, 4 × CH2), 2.90
(s, 6 H, 2 × ArCH3), 3.10-3.28 (br m, 8 H, 4 × CH2), 3.70 (q,
J ) 6.4 Hz, 4 H, 2 × CH2), 7.72-7.81 (m, 4 H, ArH), 7.95 (t,
J ) 7.9 Hz, 2 H, ArH), 8.11 (d, J ) 8.2 Hz, 2 H, ArH), 8.36 (d,
J ) 8.1 Hz, 2 H, ArH), 8.99 (d, J ) 6.3 Hz, 2 H, ArH), 11.09
(br s, 2 H, 2 × CONH). Anal. (C50H62N8O6‚3H2O) C, H, N.
Deprotection of 45 was carried out with MeOH/HCl(g) as
above, giving 20 as a pale yellow solid (64%): mp (CH2Cl2/n-
hexane) 122-126 °C; 1H NMR (CDCl3) δ 1.43-1.72 (m, 8 H, 4
× CH2), 1.82 (quin, J ) 7.4 Hz, 4 H, 2 × CH2), 2.55-2.62 (m,
4 H, 2 × CH2), 2.68 (t, J ) 7.2 Hz, 4 H, 2 × CH2), 2.95 (s, 6 H,
2 × ArCH3), 3.69 (q, J ) 6.6 Hz, 4 H, 2 × CH2NHCO), 7.71-
7.82 (m, 4 H, ArH), 7.96 (dd, J ) 8.6, 7.1 Hz, 2 H, ArH), 8.10
(d, J ) 8.2 Hz, 2 H, ArH), 8.36 (dd, J ) 8.6, 1.5 Hz, 2 H, ArH),
8.99 (dd, J ) 7.1, 1.6 Hz, 2 H, ArH), 11.08 (br s, 2 H, 2 ×
CONH), HRMS (FAB+) m/z calcd for C40H47N8O2, 671.3822
(MH+); found, 671.3815. Anal. (C40H46N8O2‚0.5H2O) C, H, N.
N,N′-Bis[2-(6,9-d im eth ylp h en a zin e-1-ca r boxa m id o)et-
h yl]-1,2-eth a n ed ia m in e (21). Similar reaction of 6,9-dim-
ethylphenazine-1-carboxylic acid11 (39) and N,N′-(2-aminoethyl)-
1,2-diaminethane gave 21 (99%): mp (dihydrochloride salt
from MeOH) 299 °C (dec); 1H NMR (CF3CO2D) δ 3.06 (s, 6 H,
2 × ArCH3), 3.09 (s, 6 H, 2 × ArCH3), 3.87 (br s, 4 H, 2 ×
CH2), 3.91 (br s, 4 H, 2 × CH2), 4.27 (br s, 4 H, 2 × CH2), 8.20
(d, J ) 7.3 Hz, 2 H, H-7 or H-8), 8.24 (d, J ) 7.3 Hz, 2 H, H-7
or H-8), 8.43 (t, J ) 8.1 Hz, 2 H, H-3), 8.96 (d, J ) 8.8 Hz, 2
H, H-4), 9.02 (d, J ) 7.3 Hz, 2 H, H-2). Anal. (C36H40Cl2N8O2)
C, H, N.
In Vivo Tu m or Assa ys. Colon 38 tumors were grown
subcutaneously from 1 mm3 fragments implanted in one flank
of C57/Bl mice (anesthetized with pentobarbitone 90 mg/kg).
HT-29 cells were grown in culture, and a suspension (5 × 106
cells) was implanted in each flank of nude mice. When tumors
reached a diameter of approximately 4-6 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 intraperitoneally in
a volume of 0.01 mL/g body weight, using an intermittent (q4d
× 3) schedule. 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.
Ack n ow led gm en t. This work was supported by
Xenova plc (U.K.) and the Auckland Division of the
Cancer Society of New Zealand. The authors thank Dr.
Li Zhuang, Ms. Elaine Marshall, Ms. Debra Greenhalgh,
and Ms. Tristan Gregory for help with the biological
assays.
Refer en ces
N,N′-Bis[2-(6-ch lor o-9-m eth ylp h en a zin e-1-ca r boxa m i-
do)eth yl]-1,2-eth an ediam in e (22). Similar reaction of 6-chlo-
ro-9-methylphenazine-1-carboxylic acid11 (40) and N,N′-(2-
aminoethyl)-1,1-diaminoethane gave 22 as a yellow solid
(6%): mp (HCl salt from MeOH/EtOAc) 301 °C (dec); 1H NMR
(CF3CO2D) δ 3.87 (br s, 4 H, 2 × CH2NH), 4.04 (br s, 4 H, 2 ×
CH2NH), 4.09 (s, 6 H, 2 × ArCH3), 4.29 (br s, 4 H, 2 ×
CONHCH2), 8.25 (br d, J ) 7.9 Hz, 2 H, ArH), 8.44 (br d, J )
7.7 Hz, 2 H, ArH), 8.53 (br s, 2 H, ArH), 9.03 (br d, J ) 8.7
Hz, 2 H, ArH) and 9.09 (br s, 2 H, ArH); HRMS (FAB+) m/z
calcd for C34H3335Cl2N8O2, 655.2104 (MH+); found, 655.2075.
Ca lcu la tion of Lin k er Ch a in Len gth s. These were
computed (between the carboxamide nitrogens) for the fully
extended forms of the linkers, using the 3D module of ACD
Labs, 133 Richmond Street West, Suite 605, Toronto, Ontario,
Canada M5H 2L3.
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