Synthesis and evaluation of unsymmetrical bis(arylcarboxamides) designed as topoisomerase-Targeted anticancer drugs

Article abstract of DOI:10.1016/S0968-0896(01)00249-8

Symmetric dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric and unsymmetri

Full text of DOI:10.1016/S0968-0896(01)00249-8

Bioorganic & Medicinal Chemistry 10 (2002) 19–29
Synthesis and Evaluation of Unsymmetrical Bis(arylcarboxamides)
Designed as Topoisomerase-Targeted Anticancer Drugs
Julie A. Spicer,* Swarna A. Gamage, Graeme J. Finlay and William A. Denny
Auckland Cancer Society Research Centre, Faculty of Medical & Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1000, New Zealand
Received 23 April 2001; accepted 26 June 2001
Abstract—Symmetrical dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been
shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric
and unsymmetric dimers of a variety of intercalating chromophores of varied DNA binding strength, including naphthalimides,
acridines, phenazines, oxanthrenes and 2-phenylquinolines. The unsymmetrical dimers were prepared by sequential coupling of the
chromophores to linkers with selectively protected primary terminal amines to ensure high yields and unequivocal product. Pro-
tection of the internal (secondary) amines as BOC derivatives was used to ensure complete structural specificity, and was also an aid
to the purification of these very polar compounds. The growth inhibitory abilities (as IC50 values) of the compounds in a range of
cell lines showed that the nature of the linker chain was important, and independent of the nature of the chromophore, with com-
pounds containing the dicationic linker [–(CH2)2NH(CH2)2NH(CH2)2–] being on average 30-fold more potent than the corre-
sponding compounds containing the monocationic linker [–(CH2)3NMe(CH2)3–]. However, the chromophores also play a role in
determining biological activity, with the cytotoxicities of symmetric and unsymmetric dicationic dimers correlating with the overall
DNA binding abilities of the chromophores. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
the side chain binding in the major groove,18,19 butother
reports,23 suggest that the related compound DMP-840
(1) is a monointercalator, despite having a sufficiently
long linker chain to span at least two base pairs. A series
of bis(imidazoacridanones) (e.g., 3) also appear notot
be bis-intercalating agents.9 While these appear to work
primarily by inhibition of topoisomerase enzymes, this
mechanism of action is also varied. Bis(naphthalimides) 1
and 2 are both reported to inhibit topo II.20,21 A series of
bis(acridinecarboxamides) (4) and bis(phenazinecarbox-
amides) (5) inhibit both topo I and topo II, but studies
with mutated cell lines suggest that topo I inhibition is
primarily responsible for their biological activity.13,14
Dimeric analogues of a number of classes of neutral
DNA-intercalating chomophores joined by cationic lin-
kers have recently been studied as potential anticancer
agents. The bis(naphthalimide) DMP-840 (1) shows
broad-spectrum activity against a variety of human
solid tumour cell lines in culture,1,2 and as xenografts in
nude mice,3,4 and is in clinical trial.5,6 Other examples
under study include bis(benzonaphthalimides),7 bis(imi-
dazoacridinones),8,9 bis(triazoloacridinones),10 bis(an-
thracyclines),11,12 bis(acridine-4-carboxamides),13 bis
(phenazine-1-carboxamides)14,15 and bis(indeno[1,2-
b]quinoline-6-carboxamides).16 A comparative study of
a variety of bis(chromophores) with their monomeric
counterparts showed there were variable but significant
gains in potency for the dimeric species.17
This background led us to speculation about whether
the two chromophores have different roles, with one
intercalating DNA and the other interacting with
topoisomerase enzymes, and thus to an interest in
unsymmetrical dimeric compounds. There has been lit-
tle work reported on such compounds, but a study23 of
unsymmetrical analogues of 1 showed one to have
comparable biological activity and improved solubility.
We report here on a study that looks at a wider series of
unsymmetrical dimeric compounds, including many
with quite different chromophores.
The mode of interaction with DNA of these compounds
is not entirely clear. The bis(naphthalimide) LU 79553
(Elinafide, 2) is reported to bis-intercalate DNA, with
*Corresponding author. Tel.: +64-9-373-7599; fax: +64-9-373-7502;
e-mail: j.spicer@auckland.ac.nz
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00249-8

20
J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
Results and Discussion
higher cytotoxicity. For each different chromophore,
the symmetric compounds were also prepared and eval-
uated, to allow comparisons.
Two series of compounds were prepared, using naph-
thalimide, acridine, phenazine, oxanthrene and 2-
phenylquinoline chromophores, joined by either a
[–(CH2)3NMe(CH2)3–] (6–13) or biscationic [–(CH2)2
NH(CH2)2NH(CH2)2–] (14–27) linker. In the case of the
acridines and the phenazines, the 5-methyl (or the
topologically-equivalent 9-methyl) analogues respec-
tively were also used, because this substituent provides
The unsymmetrical monocationic compounds 11–13
(Table 1) were prepared as described in Scheme 1. The
appropriate chromophore acids (as the N-imidazolides)
were reacted with the monoBOC-protected triamine 28,
then the resulting products (29, 31) were deprotected
with trifluoroacetic acid to give the free amines 30 and
Table 1. Growth inhibitory properties of bis(arylcarboxamides) Ar1-CONH-R-NHCO-Ar2
No.
Ar1
Ar2
mp
IC50 (nM)a
LLc
IC50 ratio
d
P388b
JLC
A/Ce
D/Cf
R=(CH2)3NMe(CH2)3
6
7
8
9
10
11
12
13
17
17
13
14
14
Naphthalimide
Acridine
5-Meacridine
Phenazine
9-Mephenazine
Acridine
Phenazine
Naphthalimide
Acridine
5-Meacridine
Phenazine
9-Mephenazine
Phenazine
9-Mephenazine
9-Mephenazine
12,400
130
23
520
30
1.8
107
1.6
82
670
110
11
0.7
0.7
0.4
0.5
0.6
0.8
0.5
0.4
1.0
0.8
0.7
0.7
0.6
0.9
0.8
0.7
520
15
133
214
14
173
5.7
276
36
6.6
171–173
120 (dec)
116–121
19
1.9
5-Meacridine
R=(CH2)2NH(CH2)2NH(CH2)2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Oxanthrene
Phenylquinoline
Naphthalimide
Acridine
Oxanthrene
Phenylquinoline
Naphthalimide
Acridine
152–154
171–172
22
2130
5000
180
920
330
24
1400
470
70
840
410
19
6.9
3.8
0.59
0.18
31
14
5.2
0.8
0.8
0.5
0.7
0.3
0.5
0.5
0.4
0.4
0.7
0.4
0.7
0.5
0.5
1.0
1.0
0.7
0.9
1.0
0.6
1.0
1.2
0.7
0.9
0.8
1.1
0.6
0.6
170–171
32
44
Phenazine
Phenazine
112
6.3
2.8
750
95
39
14
19
5-Meacridine
9-Mephenazine
Acridine
Oxanthrene
Phenylquinoline
Acridine
Phenazine
Naphthalimide
5-Meacridine
5-Meacridine
9-Mephenazine
Phenazine
9-Mephenazine
9-Mephenazine
9-Mephenazine
9-Mephenazine
9-Mephenazine
9-Mephenazine
169–170
15
21
185–187
268–270
150–155
175–177
207–210
148–153
75–179
1980
410
280
72
51
22
1.1
0.41
0.83
0.32
4.5
3.9
18
aIC50: concentration of drug (nM) to reduce cell number to 50% of control cultures (see text).
bP388 murine leukemia.
cLewis lung carcinoma.
dJurkathuman leukemia.
eA/C=JLA/JLC.
fA/D=JLD/JLC.
Scheme 1.

J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
21
Scheme 2.
32 which were then coupled with the appropriate acid
N-imidazolides to give 11–13. The use of a mono-
protected amine ensured high yields and unequivocal
product at all steps in the synthesis. A reported23 similar
strategy involved the preparation of a diamide and
reduction with BH3.THF to afford a monoprotected
chiral polyamine. Previous syntheses of unsymmetric
bis(naphthalides), however, have employed a large
excess of the unprotected linker and isolation of the
monoadduct,24 or the use of equimolar mixtures of each
chromophore, followed by separation of the resulting
statistical mixture of products.24
with trifluoroacetic acid and the resultant primary
amine 49 reacted with 9-methylphenazine imidazolide to
afford compound 27. A second, and preferred, method
(Schemes 2 and 3) was to prepare orthogonally pro-
tected amine 36 as follows; alkylation of ethylenedia-
mine with an excess of chloroacetonitrile2 giving 33,
protection of the secondary amines as BOC derivatives
(34), and reduction of the nitriles with Raney nickel28 to
afford 35. One of the amines was then selectively pro-
tected as the trifluoroacetamide26 to give 36. This amine
was reacted with 9-methylphenazine imidazolide, giving
key intermediate 37 which could be deprotected and
reacted with various chromophores to afford the BOC-
protected analogues of compounds 21–26. The advan-
tage of this procedure is not only does it afford specifi-
city (as for 27), but in this case, having a BOC-protected
compound as the penultimate step aids in purification of
these otherwise very polar and relatively insoluble bis-
cationic compounds.
In the triethylenetetramine-linked series of compounds
14–27, symmetrical reference compounds 14, 15, 17–20
were prepared via reaction of two equivalents of the
appropriate acid N-imidazolide with one equivalent of
triethylenetetramine linker. Bisnaphthalimide 16 was
prepared simply by heating a solution of 1,8-naphthalic
anhydride and triethylenetetramine in absolute ethanol
atreflux. 25
The growth inhibitory abilities (as IC50 values) of the
compounds were evaluated in a panel of cell lines which
has been discussed previously.13 P388 is a murine leu-
kemia line, and Lewis lung is a murine carcinoma line.
The three human leukemia (Jurkat) lines29,30 provide
some insight into the mechanism of cytotoxicity. JLC is
the wild-type line, sensitive to topo II inhibitors, while
Two approaches were taken in order to prepare the
unsymmetrical compounds 21–27. A firstapproach
(Scheme 4) was to prepare26 triBOC-protected triethyl-
enetetramine 47, and to react this with 5-methylacridine
imidazolide. The BOC protection was then removed

22
J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
Scheme 4.
previously for the corresponding monomers,13,14,17
which in turn were correlated with their levels of DNA
binding.31 Thus the least potent of the symmetric dimers
(14, 15) are those with the weakest binding chromo-
phores, the bicyclic phenylquinoline, and the least
aromatic tricyclic oxanthrene. The known bis(naphtha-
limide)22 (16) and bis(phenazine)32 (18), as well as the
new bis(acridine) (17) were the next most potent sym-
metric dimers. It is known that methyl groups peri t o t he
ring nitrogen in monomeric acridine- and phenazine-
carboxamides substantially increase both DNA binding
and cytotoxic potency,33,34 and that this potency increase
is carried forward to the bis-compounds such as 19 and 20,
that are up to 60-fold more potent than the corresponding
unsubstituted analogues 17 and 18 (Table 1).
Scheme 3.
JLA is 85-fold resistant to the topo II inhibitor amsa-
crine because of a reduced level of the enzyme, and JLD
is a doxorubicin-resistant line, also primarily by virtue
of lower topo II levels. In Table 1, IC50 values are given
for the P388, LLTC and JLC lines, together with ratios
of IC50 values againstJL
and the other two Jurkat
C
lines (ratios JLA/JLC and JLD/JLC). Values of these
ratios of less than about 2-fold suggest a likely non-topo
II mediated mechanism of action.
To study the effect of the linker chain, a comparison
was made of the eight monocationic compounds (6–13)
with the corresponding dicationic ones (16–21, 25, 27).
Pairwise comparison of the IC50 values in the JLC line
gave eq (1):
Compounds 21–27 of Table 1 are the unsymmetrical
dimers studied. The acridine/phenazine dimer 21 was,
surprisingly, less effective than either of the corre-
sponding symmetric compounds 17 and 18. The
remainder of the compounds all contained a 9-methyl-
phenazine as one chromophore, while the others were a
group with varied DNA binding ability. The cytotoxic
potencies of the unsymmetrical dimers always fell
between those of the two corresponding symmetric
dimers (14–19 on one hand, and 20 on the other), with
almostidentical rank order.
LogJLCðmonocationicÞ ¼ 0:85ðÆ0:13ÞlogJLC
 ðdicationicÞ þ 1:47
ð1Þ
 ðÆ0:11Þ
n ¼ 8 r ¼ 0:94 F ¼ 42
Conclusions
This shows that compounds joined by the dicationic
linker are on average 30-fold more potent than the cor-
responding monocationic compounds in the human
leukemia line, with the two different linker chains con-
tributing quite consistently to the activity, independent
of the nature of the chromophore. However, the poten-
cies of these compounds in the Lewis lung line and P388
lines were less related, and essentially independent of the
nature of the linker chain. We have noted previously
that related dicationic compounds show much greater
potencies towards human- rather than murine-derived
tumor cell lines,14ꢀ16 and the same is true here.
Limited structural studies to date of symmetrical dimers
of lipophilic DNA-intercalating chromophores linked
by cation-containing chains have not provided a con-
sistent picture of their mode of interaction with DNA.
There has been speculation that the two chromophores
in these compounds may have independent roles,23 with
one intercalating DNA and the other interacting with
topoisomerase enzymes. Our present study, comparing
the biological activity of a series of symmetric and
asymmetric dimers of chromophores with varying DNA
binding abilities, show that their cytotoxicity in cell lines
appears to be related primarily to the DNA binding abil-
ities of the chromophores. This suggests the chromo-
phores probably do nothave differentroles, but
primarily contribute to the overall DNA binding of the
compounds.
To study the effects of differing chromophores, a larger
series of these were prepared with the dicationic linker
(14–27). The cytotoxic potencies of the symmetric com-
pounds (14–20) ranked well with those reported

J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
23
Experimental
CH2CH2CH2NHCOAr), 2.18 (s, 3H, NCH3), 2.46 (t,
J=6.7 Hz, 2H, CH2CH2CH2NHBOC), 2.56 (t, J=7.2
Hz, 2H, CH2CH2CH2NHCOAr), 3.20 (m, 2H,
CH2NHBOC), 3.74 (q, J=6.2 Hz, 2H, CH2NHCOAr),
5.45 (br s, 1H, NHBOC), 7.89–8.00 (m, 3H, 3ꢂArH),
8.22–8.26 (m, 1H, ArH), 8.28–8.32 (m, 1H, ArH), 8.40
(dd, J=8.7, 1.5 Hz, 1H, ArH), 9.02 (dd, J=7.1, 1.5 Hz,
1H, ArH), 11.03 (br s, 1H, CONH).
Chemistry
Analyses were carried outin hte Microchemical
Laboratory, University of Otago, Dunedin, NZ. Melt-
ing points were determined on an Electrothermal 2300
melting point apparatus. NMR spectra were obtained
on a Bruker DRX-400 spectrometer, and are referenced
t o M4eSi for organic solutions and 3-(trimethylsilyl)-
propanesulfonic acid, sodium saltfor D 2O solutions.
Thin-layer chromatography was carried out on alumi-
nium-backed silica gel plates (Merck 60 F254). 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. Mass spectra
were obtained on a Varian VG 7070 spectrometer at
nominal 5000 resolution.
To a solution of 29 (545 mg, 1.21 mmol) in CH2Cl2 (8
mL), was added trifluoroacetic acid (8 mL). This mix-
ture was stirred at room temperature for 2 h at which
pointhte reaciton was compleet by TLC. All solvenst
were removed under reduced pressure and the oily resi-
due partitioned between CH2Cl2 (100 mL) and 1 M
Na2CO3 (100 mL). The aqueous layer was extracted
with additional CH2Cl2 (4ꢂ100 mL) and all CH2Cl2
extracts were combined and dried with Na2SO4. The
solventwas removed under reduced pressure to give N-
{3-[(3-aminopropyl)(methyl)amino]propyl}-1-phenazine-
carboxamide (30) (392 mg, 92%) as an oil, which was
N-{3-[{3-[(4-Acridinylcarbonyl)amino]propyl}(methyl)-
amino]propyl}-1-phenazinecarboxamide (11) (Scheme 1).
tert-Butyl
3-[(3-aminopropyl)(methyl)amino]propyl-
carbamate (28) was prepared as reported previously.35
A solution of di-tert-butyldicarbonate (2.51 g, 11.5
mmol) in THF (15 mL) was added, over the course of
1.5 h, to a solution of N1-(3-aminopropyl)-N1-methyl-
1,3-propanediamine (5.00 g, 34.4 mmol) in THF (15
mL), which was maintained at 0 ꢁC (ice/water). The
reaction mixture was stirred for a further 18 h at room
temperature, then the solvent was removed under
reduced pressure and the resulting residue partitioned
between NaCl (satd) (100 mL) and CH2Cl2 (200 mL).
The CH2Cl2 layer was washed with a further portion
of NaCl solution (100 mL), then dried (Na2SO4), and
the solvent removed under reduced pressure to give 28
(2.58 g, 46%) as a viscous oil which was used directly:
1H NMR (CDCl3) d 1.44 [br s, 9H, C(CH3)3], 1.58–1.67
(m, 6H, 2ꢂCH2CH2CH2 and NH2), 2.22 (s, 3H,
NCH3), 2.34–2.40 (m, 4H, 2ꢂCH2NCH3), 2.74 (t,
J=6.9 Hz, 2H, CH2NH2), 3.12–3.21 (br m, 2H,
CH2NHBOC), 5.37 (br s, 1H, NHBOC).
used directly: H NMR (CDCl3) d 1.61–1.67 (m, 4H,
1
CH2CH2CH2NH2), 2.00 (quin, J=7.1 Hz, 2H,
CH2CH2CH2NHCOAr), 2.29 (s, 3H, NCH3), 2.46 (t,
J=7.2 Hz, 2H, CH2CH2CH2NH2), 2.59 (t, J=7.2 Hz,
2H, CH2CH2CH2NHCOAr), 2.75 (t, J=6.8 Hz, 2H,
CH2NH2), 3.72 (q, J=6.5 Hz, 2H, CH2NHCOAr), 7.89–
7.99 (m, 3H, ArH), 8.22–8.25 (m, 1H, ArH), 8.28–8.32
(m, 1H, ArH), 8.40 (dd, J=8.6, 1.5 Hz, 1H, ArH), 9.01
(dd, J=7.1, 1.5 Hz, 1H, ArH), 11.01 (br s, 1H, CONH).
Acridine-4-carboxylic acid (274 mg, 1.23 mmol) was
reacted with CDI (300 mg, 1.85 mmol) to form the imi-
dazolide which was isolated by precipitation from
CH2Cl2/petroleum ether as above. The imidazolide was
suspended in THF (15 mL), the suspension cooled to
0 ꢁC (ice/water), then a solution of 30 (392 mg, 1.12
mmol) in THF (10 mL) slowly added. The reaction
mixture was stirred for 2 h at 0 ꢁC, then 18 h at room
temperature. The solvent was removed under reduced
pressure and the residue was partitioned between
CH2Cl2 (100 mL) and 1 M Na2CO3 (100 mL). The
CH2Cl2 layer was dried with Na2SO4, the solvent
removed under reduced pressure to give an orange solid.
This was chromatographed on alumina, eluting with
CH2Cl2/MeOH (199:1), and then on silica gel, eluting
with CH2Cl2/MeOH/Et3N (395:4:1), to give 11 (560 mg,
83%): mp (CH2Cl2/n-hexane) 171–173 ꢁC; 1H NMR
(CDCl3) d 1.88 (quin, J=5.6 Hz, 2H, CH2CH2CH2),
2.02 (quin, J=6.0 Hz, CH2CH2CH2), 2.38 (s, 3H,
NCH3), 2.65–2.70 (m, 4H, 2ꢂCH2NCH3), 3.62–3.68 (m,
2H, CH2NHCO), 3.76 (q, J=6.5 Hz, 2H, CH2NHCO),
7.11 (t, J=7.7 Hz, 1H, ArH), 7.22–7.29 (m, 1H, ArH),
7.36 (d, J=8.3 Hz, 1H, ArH), 7.65 (ddd, J=8.3, 6.9, 1.5
Hz, 1H, ArH), 7.85–7.93 (m, 3H, 3ꢂArH), 8.00 (dd,
J=7.5, 0.9 Hz, 1H, ArH), 8.09–8.13 (m, 1H, ArH),
8.23–8.27 (m, 1H, ArH), 8.36 (dd, J=8.6, 1.5 Hz, 1H,
ArH), 8.42 (d, J=8.1 Hz, 1H, ArH), 8.53 (dd, J=8.0,
1.2 Hz, 1H, ArH), 8.88 (dd, J=7.2, 1.5 Hz, 1H, ArH),
9.15 (s, 1H, H-9), 11.03 [br s, 1H, NH (phenazine)],
12.55 [br s, 1H, NH (acridine)]. Analysis calcd for
Phenazine-1-carboxylic acid34 (494 mg, 2.24 mmol) was
reacted with CDI (544 mg, 3.36 mmol) in dry DMF (15
mL) at30 ꢁC for 2.5 h. The DMF was removed under
reduced pressure and the resulting yellow solid was dis-
solved in a mixture of petroleum ether and CH2Cl2 (40
mL, 3:1). Upon cooling, the imidazolide crystallised out
and this crude material was used in the following cou-
pling reaction. The crude imidazolide was suspended in
THF (20 mL), cooled to 0 ꢁC (ice/water), then a THF
(20 mL) solution of 28 (659 mg, 2.69 mmol) was added.
The reaction mixture was allowed to stir for a further 2 h
at0 ꢁC, and the solvent was removed under reduced
pressure. The resulting yellow oil partitioned between
CH2Cl2 (200 mL) and 1 M (Na2CO3) (200 mL), and the
CH2Cl2 layer was dried with Na2SO4, and evaporated
under reduced pressure. The residue was chromato-
graphed on alumina, eluting with CH2Cl2/MeOH
(399:1) to give tert-butyl 3-(methyl{3-[(1-phenazi-
nylcarbonyl)amino]propyl}amino)propylcarbamate (29)
1
(992 mg, 98%) as an oil, which was used directly: H
NMR (CDCl3) d 1.41 [br s, 9H, C(CH3)3], 1.44 (br s,
2H, CH2CH2CH2NHBOC), 1.95–2.04 (m, 2H,
.
C34H32N6O2 2H2O: C, 68.9; H, 6.1; N, 14.2. Found: C,
68.8; H, 5.8; N, 14.1%.

24
J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
9-Methyl-N-{3-[methyl(3-{[(5-methyl-4-acridinyl)carbonyl]-
amino}propyl)amino]propyl}-1-phenazinecarboxamide (13).
Activation and coupling of 9-methylphenazine-1-car-
boxylic acid34 with 28 as above gave tert-butyl 3-
[methyl(3-{[(9-methyl-1-phenazinyl)carbonyl]amino}pro-
pyl)amino]propylcarbamate (31) (89%) as an oil, which
J=8.6, 7.1 Hz, 1H, ArH), 7.94 (d, J=9.2 Hz, 1H, ArH),
8.00 (d, J=8.6 Hz, 1H, ArH), 8.08 (d, J=8.5 Hz, 1H,
ArH), 8.21 (dd, J=8.7, 1.5 Hz, 1H, ArH), 8.31 (dd,
J=8.7, 1.5 Hz, 1H, ArH), 8.89 (dd, J=7.1, 1.5 Hz, 1H,
ArH), 8.92 (dd, J=7.2, 1.5 Hz, 1H, ArH), 10.87 (br s,
2H, 2ꢂNH). HRMS (FAB+) calcd for C34H34N7O2
572.2774 (MH+), found 572.2762. Analysis calcd for
1
was used directly: H NMR (CDCl3) d 1.41 [br s, 9H,
.
C(CH3)3],
1.65
(quin,
J=6.6
Hz,
2H,
C34H33N7O2 H2O: C, 69.3; H, 6.0; N, 16.6. Found: C,
69.0; H, 5.7; N, 16.4%.
CH2CH2CH2NHBOC), 1.98 (quin, J=7.2 Hz, 2H,
CH2CH2CH2NHCOAr), 2.24 (s, 3H, NCH3), 2.42 (t,
J=6.7 Hz, 2H, CH2CH2CH2NHBOC), 2.53 (t, J=7.3
Hz, 2H, CH2CH2CH2NHCOAr), 2.94 (s, 3H, ArCH3),
3.12–3.23 (br s, 2H, CH2NHBOC), 3.73 (q, J=6.7 Hz,
2H, CH2NHCOAr), 5.39 (br s, 1H, NHBOC), 7.77 (dt,
J=6.5, 1.1 Hz, 1H, ArH), 7.81 (dd, J=8.5, 6.8 Hz, 1H,
ArH), 7.97 (dd, J=8.7, 7.2 Hz, 1H, ArH), 8.14 (d,
J=8.4 Hz, 1H, ArH), 8.39 (dd, J=8.6, 1.5 Hz, 1H,
ArH), 9.02 (dd, J=7.2, 1.5 Hz, 1H, ArH), 11.13 (br t,
J=5.2 Hz, 1H, CONH).
N-(2-{[2-({2-[(1-Oxanthrenylcarbonyl)amino]ethyl}amino)-
ethyl]amino}ethyl)-1-oxanthrenecarboxamide (14). 1-
Oxanthrenecarboxylic acid31 (935 mg, 4.11 mmol) was
reacted with CDI (1.00 g, 6.17 mmol) in dry DMF (10
mL) at room temperature for 2 h. This solution was
diluted with benzene (10 mL) then treated with lipo-
philic Sephadex resin to remove excess CDI.36 The
mixture was stirred at room temperature for 1 h, then
the resin was collected by filtration and the filtrate con-
centrated under reduced pressure. The resulting crude
imidꢁazolide was dissolved in THF (10 mL) and cooled
t o 0 C (ice/water). A solution of N,N0-bis(aminoethyl)-
ethanediamine (300 mg, 2.05 mmol) in THF (10 mL)
was then added and the entire mixture stirred at room
temperature for 48 h, at which point precipitation of the
producthad occurred. The reaciton mixutre was con-
centrated, diluted with water and the solid collected by
filtration to give 14 (995 mg, 86%): mp (CH2Cl2/n-hex-
Deprotection of 31 as above gave N-{3-[(3-aminopro-
pyl)(methyl)amino]propyl} - 9 - methyl - 1 - phenazinecar-
boxamide (32) (85%), as an oil which was used directly:
1H NMR (CDCl3) d 1.62 (quin, J=7.0 Hz, 2H,
CH2CH2CH2NH2), 1.98 (quin, J=7.3 Hz, 2H,
CH2CH2CH2NHCOAr), 2.26 (s, 3H, NCH3), 2.43 (t,
J=7.2 Hz, 2H, CH2CH2CH2NH2), 2.53 (t, J=7.3 Hz,
2H, CH2CH2CH2NHCOAr), 2.75 (m, 2H, CH2NH2),
2.93 (s, 3H, ArCH3), 3.73 (q, J=6.7 Hz, 2H,
CH2NHCOAr), 7.76 (dt, J=6.7, 1.3 Hz, 1H, ArH), 7.81
(dd, J=8.4, 6.9 Hz, 1H, ArH), 7.97 (dd, J=8.6, 7.1 Hz,
1H, ArH), 8.13 (dd, J=8.7, 1.0 Hz, 1H, ArH), 8.38 (dd,
J=8.7, 1.5 Hz, 1H, ArH), 9.00 (dd, J=7.1, 1.5 Hz, 1H,
ArH), 11.11 (br s, 1H, CONH).
ꢁ
1
ane) 152–154 C; H NMR (CDCl3) d 2.86 (br s, 4H,
2ꢂCH2), 2.90 (t, J=5.8 Hz, 4H, 2ꢂCH2), 3.55 (q,
J=5.6 Hz, 4H, 2ꢂCH2), 6.78–6.82 (m, 2H, 2ꢂArH),
6.83–6.97 (m, 10H, 10ꢂArH), 7.67 (dd, J=7.5, 2.2 Hz,
2H, 2ꢂArH), 7.80 (br s, 2H, 2ꢂCONH). HRMS
(FAB+) calcd for C32H31N4O6 567.2244 (MH+), found
.
567.2248. Analysis calcd for C32H30N4O6 0.5H2O: C,
66.8; H, 5.4; N, 9.7. Found: C, 66.9; H, 5.4; N, 9.7%.
Activation and coupling of 5-methylacridine-4-car-
boxylic acid33 with 32 as above gave 13 (66%): mp
(CH2Cl2/n-hexane) 116–121 ꢁC; 1H NMR (CDCl3) d
1.94–2.02 (m, 4H, 2ꢂCH2CH2CH2), 2.32 (s, 3H,
NCH3), 2.58–2.63 (m, 4H, 2ꢂCH2NCH3), 2.73 (s, 3H,
ArCH3), 2.80 (s, 3H, ArCH3), 3.66–3.74 (m, 4H,
2ꢂCH2NHCO), 7.31 (dd, J=8.4, 6.8 Hz, 1H, ArH),
7.49–7.53 (m, 2H, 2ꢂArH), 7.59 (dd, J=8.2, 7.2 Hz,
1H, ArH), 7.63–7.69 (m, 2H, ArH), 7.90 (dd, J=8.7, 7.2
Hz, 1H, ArH), 7.95–8.00 (m, 2H, 2ꢂArH), 8.28 (dd,
J=8.7, 1.5 Hz, 1H, ArH), 8.61 (s, 1H, H-9), 8.90–8.95
(m, 2H, 2ꢂArH), 10.87 [br s, 1H, NH (phenazine)] and
11.78 [br s, 1H, NH (acridine)]. HRMS (FAB+) calcd
for C36H37N6O2 585.2978 (MH+), found 585.2985.
2-Phenyl-N-[2-({2-[(2-{[(2-phenyl-8-quinolinyl)carbonyl]-
amino}ethyl)amino]ethyl}amino)ethyl]-8-quinolinecarbox-
amide (15). 2-Phenylquinoline-8-carboxylic acid37 was
activated with CDI to form the imidazolide which was
isolated using Sephadex resin as above. The imidazolide
was then reacted with N,N0-bis(aminoethyl)ethanedia-
mine, and the solid product after workup was purified
by column chromatography on silica gel, eluting with
CH2Cl2/MeOH (4:1), to give 15 (62%): mp (CH2Cl2/n-
hexane) 171–172 ꢁC; 1H NMR (CDCl3) d 2.64 (br s, 4H,
2ꢂCH2), 2.87 (t, J=6.2 Hz, 4H, 2ꢂCH2), 3.65 (q,
J=5.9 Hz, 4H, 2ꢂCH2), 7.42–7.53 (m, 6H, 6ꢂArH),
7.63 (t, J=7.7 Hz, 2H, 2ꢂArH), 7.83 (d, J=8.6 Hz, 2H,
2ꢂArH), 7.92 (dd, J=8.1, 1.5 Hz, 2H, 2ꢂArH), 7.96–
8.01 (m, 4H, 4ꢂArH), 8.26 (d, J=8.6 Hz, 2H, 2ꢂArH),
8.84 (dd, J=7.4, 1.5 Hz, 2H, 2ꢂArH), 11.45 (br s, 2H,
2ꢂCONH). HRMS (FAB+) calcd for C38H37N6O2
609.2978 (MH+), found 609.2982. Analysis calcd for
.
Analysis calcd for C36H36N6O2 1.5H2O: C, 70.7; H, 6.4;
N, 13.7. Found: C, 71.1; H, 6.4; N, 13.8%.
9-Methyl-N-{3-(methyl{3-[(1-phenazinylcarbonyl)amino]-
propyl}amino)propyl]-1-phenazinecarboxamide (12).
Activation and coupling of phenazine-1-carboxylic acid
with 32 as above gave 12 (77%): mp (CH2Cl2/n-hexane)
.
C38H36N6O2 0.75H2O: C, 73.3; H, 6.0; N, 13.5. Found:
C, 73.4; H, 6.2; N, 13.6%.
120 ꢁC (dec.); H NMR (CDCl3) d 1.96–2.07 (m, 4H,
1
2ꢂCH2CH2CH2), 2.36 (s, 3H, NCH3), 2.65–2.73 (m,
7H, 2ꢂCH2NCH3 and ArCH3), 3.68–3.78 (m, 4H,
2ꢂCH2NHCO), 7.47 (dt, J=6.9, 1.1 Hz, 1H, ArH),
7.57 (ddd, J=8.7, 6.6, 1.2 Hz, 1H, ArH), 7.63 (dd,
J=8.7, 6.8 Hz, 1H, ArH), 7.71 (ddd, J=8.7, 6.7, 1.5 Hz,
1H, ArH), 7.87 (dd, J=8.7, 7.2 Hz, 1H, ArH), 7.90 (dd,
N-(2-{[2-({2-[(4-Acridinylcarbonyl)amino]ethyl}amino)-
ethyl]amino}ethyl)-4-acridinecarboxamide (17). 4-Acri-
dinecarboxylic acid33 was activated with CDI to form
the imidazolide, which was isolated using Sephadex
resin as above. The imidazolide was then reacted

J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
25
with N,N0-bis(aminoethyl)ethanediamine and the solid
productafetr workup was chromaotgraphed on silica
gel, eluting with a gradient of MeOH in CH2Cl2 (98:2)
(100%) as a white solid which was used directly; 1H
NMR (CDCl3) d 1.46 [s, 18H, 2ꢂC(CH3)3], 1.57 (br s,
4H, 2ꢂNH2), 2.83 (br s, 4H, 2ꢂCH2), 3.30 (br m, 8H,
4ꢂCH2).
to give 17 (72%): mp (CH2Cl2/hexane) 170–171 ꢁC; H
1
NMR (CDCl3) d 2.91, (s, 8H, 4ꢂCH2), 3.53 (q, J=5.4
Hz, 4H, 2ꢂCH2), 7.53 (t, J=7.4 Hz, 2H, 2ꢂArH), 7.68
(dd, J=8.3, 7.1 Hz, 2H, 2ꢂArH), 7.81–7.85 (m, 2H,
2ꢂArH), 8.03 (d, J=8.3 Hz, 2H, 2ꢂArH), 8.22 (d,
J=8.9 Hz, 2H, 2ꢂArH), 8.26 (dd, J=8.5, 1.4 Hz, 2H,
2ꢂArH), 8.64 (dd, J=7.1, 1.6 Hz, 2H, H-3), 9.87 (s, 2H,
H-9), 11.56 (t, J=5.0 Hz, 2H, 2ꢂCONH). Analysis
The above diamine 35 was reacted with ethyl tri-
fluoroacetate according to a published method26 to give
N-aminoethyl-N,N0-bis(tert-butoxycarbonyl)-N0-[(N-tri-
fluoroacetamido)aminoethyl]ethylenediamine
(36)
(39%) as an oil: 1H NMR (CDCl3) d 1.46 [s, 18H,
2ꢂC(CH3)3], 1.95 (br s, 2H, NH2), 2.84–2.96 (m, 2H,
CH2), 3.20–3.48 (m, 10H, 5ꢂCH2), 7.99, 8.21, 8.51 (all br
s, total 1H, NH). Analysis calcd for C18H33F3N4O5; C,
48.9; H, 7.5; N, 12.7. Found: C, 48.9; H, 7.5; N, 12.4%.
.
calcd for C34H32N6O2 2H2O: C, 68.9; H, 6.1; N, 14.2.
Found: C, 68.4; H, 5.7; N, 14.0%.
5-Methyl-N-[2-({2-[(2-{[(5-methyl-4-acridinyl)carbonyl]-
amino}ethyl)amino]ethyl}amino)ethyl]-4-acridinecarbox-
amide (19). 5-Methyl-4-acridinecarboxylic acid33 was
activated with CDI to form the imidazolide, which was
isolated using Sephadex resin as above. The imidazolide
was then reacted with N,N0-bis(aminoethyl)ethanedia-
mine and the solid product after workup was chroma-
tographed on silica gel, eluting with a gradient of
MeOH in CH2Cl2 (98:2) to give 19 (76%): mp 169–
9-Methyl-1-phenazinecarboxylic acid34 (647 mg, 2.72
mmol) was activated with CDI and the imidazolide iso-
lated by precipitation from CH2Cl2/petroleum ether as
above. The imidazolide was dissolved in THF (80 mL)
and a THF (20 mL) solution of the triprotected amine
36 (1.07 g, 2.28 mmol) was added to the stirred solution.
This mixture was stirred for a further 15 h at room
temperature. The solvent was then removed under
reduced pressure and the resulting oil dissolved in
CH2Cl2 (100 mL), then washed with 1 M Na2CO3 (100
mL). The CH2Cl2 layer was then dried with Na2SO4 and
evaporated down to afford a dark yellow oil which was
purified by chromatography on silica gel, eluting with
CH2Cl2/MeOH (199:1), to give N-1-{2-[N0-tert-butoxy-
carbonyl-N0-(2-{N-tert-butoxycarbonyl-N-[2-(N-trifluoro-
acetamido) aminoethyl]}aminoethyl)]aminoethyl} - 9 -
methyl-1-phenazinecarboxamide (37) (1.36 g, 88%) as a
170 ꢁC; H NMR (CDCl3) d 2.80 (s, 6H, 2ꢂArCH3),
1
2.85 (s, 4H, 2ꢂCH2), 2.99 (t, J=6.2 Hz, 4H, 2ꢂCH2),
3.74 (q, J=6.1 Hz, 4H, 2ꢂCH2), 7.39 (dd, J=8.4, 6.8
Hz, 2H, H-2), 7.57–7.61 (m, 4H, H-6, H-7), 7.75 (d,
J=8.7 Hz, 2H, H-8), 8.02 (dd, J=8.4, 1.5 Hz, 2H, H-1),
8.68 (s, 2H, H-9), 8.91 (dd, J=7.2, 1.5 Hz, 2H, H-3),
11.81 (t, J=5.5 Hz, 2H, 2ꢂCONH). Analysis calcd for
C36H36N6O2: C, 73.9; H, 6.2; N, 14.4. Found: C, 73.3;
H, 6.3; N, 14.4%.
foam: 1H NMR (CDCl3)
d 1.33–1.45 [m, 18H,
9-Methyl-N-(2-{[2-({2-[(1-oxanthrenylcarbonyl)amino]-
ethyl}amino)ethyl]amino}ethyl)-1-phenazinecarboxamide
(22) (Scheme 2). Ethylenediamine was alkylated with
an excess of chloroacetonitrile by a reported method27
followed by purification by filtration through a plug of
flash silica in CH2Cl2/MeOH (99:1) to give N,N0-bis
(cyanomethyl)ethylenediamine (33) (88%): mp (EtOAc/
n-hexane) 41–42 ꢁC (lit.38 bp 133–135 ꢁC/1 mm, mp not
2ꢂC(CH3)3], 2.89 (s, 3H, ArCH3), 3.27–3.63 (m, 10H,
5ꢂCH2), 3.82 (br s, 2H, CH2), 7.75–7.86 (m, 2H,
2ꢂArH), 7.99 (t, J=7.8 Hz, 1H, ArH), 8.14 (d, J=8.5 Hz,
1H, ArH), 8.23 [br s, 1H, NHC(O)CF3], 8.42 (d, J=8.0
Hz, 1H, ArH), 8.94–9.02 (m, 1H, ArH), 11.33-11.43 (m,
1H, CONH). Analysis calcd for C32H41N6O6F3: C, 58.0;
H, 6.2; N, 12.7. Found: C, 57.8; H, 6.1; N, 12.8.
1
previously recorded); H NMR (CDCl3) d 1.58 (s, 2H,
2ꢂNH), 2.90 (s, 4H, 2ꢂCH2), 3.63 (s, 4H, 2ꢂCH2).
The above monotrifluoroacetamide (37) (1.30 g, 1.96
mmol) was dissolved in a mixture of MeOH (60 mL)
and water (40 mL). K2CO3 (1.35 g, 9.82 mmol) was
added and the mixture heated at reflux for 2 h. The
reaction mixture was allowed to cool, concentrated
under reduced pressure, and saturated Na2CO3 (50 mL)
added. This aqueous mixture was extracted with CHCl3
(9ꢂ80 mL) then all CHCl3 fractions combined, dried
(Na2SO4), and the solvent removed under reduced
pressure to afford N-1-[2-(N0-tert-butoxycarbonyl-N0-{2-
[N-(aminoethyl)-N-tert- butoxycarbonyl]aminoethyl})-
The dinitrile 33 (3.00 g, 21.7 mmol) was treated with di-
tert-butyldicarbonate (19.0 g, 87.0 mmol) in a mixture
of THF (90 mL), water (10 mL) and triethylamine (10
mL). All solvents were removed under reduced pressure
and the resulting residue was partitioned between water
(100 mL) and EtOAc (2ꢂ100 mL). The combined
EtOAc layers were dried (Na2SO4), the solvent was
evaporated, and the resulting solid was chromato-
graphed on silica gel, eluting with EtOAc/petroleum
ether (1:1), to give N,N0-bis(tert-butoxycarbonyl)-N,N0-
bis(cyanomethyl)ethylenediamine (34) (6.59 g, 90%):
aminoethyl]-9-methyl-1-phenazinecarboxamide
(38)
(1.11 g, 100%) as a yellow oil which was used directly:
1H NMR (CDCl3) d 1.41 [s, 18H, 2ꢂC(CH3)3], 2.73–
2.95 (m, 5H, CH2 and ArCH3), 3.18–3.46 (m, 6H,
3ꢂCH2), 3.51–3.64 (br s, 2H, CH2), 3.78–3.89 (m, 2H,
CH2), 7.74–7.84 (m, 2H, 2ꢂArH), 7.92–8.00 (m, 1H,
ArH), 8.14 (d, J=8.3 Hz, 1H, ArH), 8.40 (br s, 1H, ArH),
8.95–9.03 (m, 1H, ArH), 11.15–11.41 (m, 1H, CONH).
mp (EtOAc/n-hexane) 112–113 ꢁC; H NMR (CDCl3) d
1
1.50 [s, 18H, 2ꢂC(CH3)3], 3.52 (s, 4H, 2ꢂCH2), 4.13 (br
s, 4H, 2ꢂCH2). Analysis calcd for C16H26N4O4: C, 56.8;
H, 7.7; N, 16.6. Found: C, 56.5; H, 7.8; N, 16.7%.
Hydrogenation of 34 using W-7 Raney nickel was carried
t o giveN,N0-bis(amino-
28
outusing a reporetd mehtod,
ethyl)-N,N0-bis(tert-butoxycarbonyl)ethylenediamine (35)
1-Oxanthrenecarboxylic acid31 (99 mg, 0.43 mmol) was
activated with CDI (105 mg, 0.65 mmol) in DMF (5 mL)

26
J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
as above. The imidazolide was dissolved in THF (10
mL) and a THF solution of 38 (245 mg, 0.43 mmol)
added slowly to the cooled, (ice/water) stirred solution.
The reaction mixture was stirred at room temperature
for 4 days, the solvent was removed under reduced
pressure, and the residue was partitioned between
CH2Cl2 (100 mL) and 1 M Na2CO3 (50 mL). The
CH2Cl2 layer was dried with Na2SO4 and evaporated,
and the residue was chromatographed on alumina,
eluting with CH2Cl2/MeOH (199:1), to give N-1-{[2-(N0-
tert-butoxycarbonyl-N0-{[2-(N-tert-butoxycarbonyl-N-{[2-
(oxanthrene - 1 - carbonyl)amino]ethyl})amino]ethyl})-
10H, 5ꢂCH2), 3.69–3.83 (m, 2H, CH2), 7.44–7.66 (m,
4H, 4ꢂArH), 7.73 (br m, 1H, ArH), 7.79 (t, J=7.7 Hz,
1H, ArH), 7.89 (d, J=8.6 Hz, 1H, ArH), 7.91–8.05 (m,
4H, 4ꢂArH), 8.12 (d, J=8.6 Hz, 1H, ArH), 8.27–8.41
(m, 2H, 2ꢂArH), 8.77–8.87 (m, 1H, ArH), 8.92–9.00
(m, 1H, ArH), 11.07-11.32 (m, 1H, CONH), 11.49-11.66
(m, 1H, CONH). Analysis calcd for C46H51N7O2: C,
69.2; H, 6.4; N, 12.3. Found: C, 69.2; H, 6.7; N, 12.3%.
Deprotection of 40 with HCl(g) in MeOH as above,
followed by column chromatography on silica gel, elut-
ing with CH2Cl2/MeOH (4:1), gave 23 (91%): mp
(CH2Cl2/n-hexane) 150–155 ꢁC; 1H NMR (CDCl3) d
2.70-2.74 (m, 2H, CH2), 2.77–2.82 (m, 5H, CH2 and
ArCH3), 2.89 (t, J=6.0 Hz, 2H, CH2), 2.99 (t, J=6.0
Hz, 2H, CH2), 3.66 (q, J=5.8 Hz, 2H, CH2), 3.73 (q,
J=5.8 Hz, 2H, CH2), 7.43–7.57 (m, 4H, 4ꢂArH), 7.61
(d, J=6.7 Hz, 1H, ArH), 7.67–7.72 (m, 2H, 2ꢂArH),
7.80 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.91–7.98 (m, 3H,
3ꢂArH), 8.02 (d, J=8.8 Hz, 1H, ArH), 8.10 (d, J=8.6
Hz, 1H, ArH), 8.35 (dd, J=8.6, 1.5 Hz, 1H, ArH), 8.75
(dd, J=7.3, 1.4 Hz, 1H, ArH), 8.96 (dd, J=7.1, 1.4 Hz,
1H, ArH), 10.91 [br s, 1H, CONH (quinoline)], 11.48
[br s, 1H, CONH (phenazine)]. HRMS (FAB+) calcd
for C36H36N7O2 598.2930 (MH+), found 598.2938.
amino]ethyl}-9-methyl-1-phenazinecarboxamide
(39)
1
(284 mg, 82%) as a foam: H NMR (CDCl3) d 1.33–
1.39 [br s, 18H, 2ꢂC(CH3)3], 2.87 (s, 3H, ArCH3), 3.32–
3.64 (m, 10H, 5ꢂCH2), 3.78–3.87 (m, 2H, CH2), 6.77–
6.95 (m, 5H, 5ꢂArH), 7.11 [br s, 1H, CONH (oxan-
threne)], 7.58–7.83 (m, 4H, 4ꢂArH), 7.95 (t, J=7.4 Hz,
1H, ArH), 8.12 (d, J=8.3 Hz, 1H, ArH), 8.48 (br s, 1H,
ArH), 8.93–9.00 (m, 1H, ArH), 11.26–11.36 [m, 1H,
CONH
(phenazine)].
.
Analysis
calcd
for
C43H48N6O8 0.5H2O: C, 65.8; H. 6.3; N, 10.7. Found:
C, 65.9; H, 6.5; N, 10.8%.
Deprotection of 39 was carried outby dissolving hte
starting material (284 mg, 0.36 mmol) in MeOH (20
mL) containing HCl(g). This mixture was stirred for 2
days at room temperature, concentrated to half the ori-
ginal volume under reduced pressure, then filtered
through a short column of Amberlite (OHꢀ) resin elut-
ing with MeOH in order to convert the HCl salt of the
desired product 22 to the free base. A yellow foam was
obtained which was purified by chromatography on
silica gel, eluting with CH2Cl2/MeOH (4:1), to give 22
.
Analysis calcd for C36H35N7O2 2H2O: C, 68.2; H, 6.2;
N, 15.5. Found: C, 68.5; H, 6.0; N, 15.3%.
N-(2-{[2-({2-[(4-acridinylcarbonyl)amino]ethyl}amino)-
ethyl]amino}ethyl)-9-methyl-1-phenazinecarboxamide (24).
Acridine-4-carboxylic acid33 was activated with CDI to
the imidazolide which was isolated by precipitation
from CH2Cl2/petroleum ether then reacted with 38 as
above. Purification of the product by chromatography
on alumina, eluting with CH2Cl2/MeOH (199:1), gave N
-1-{2-[(N0 -tert-butoxycarbonyl-N0 -{2-[(N-tert-butoxy-
carbonyl-N-{2-[(acridinyl-4-carbonyl)amino]ethyl})ami-
no]ethyl})amino]ethyl}-9-methylphenazine-1-carboxamide
(41) (97%) as a foam: 1H NMR (CDCl3) d 1.24-1.39 [br
s, 18H, 2ꢂC(CH3)3], 2.80–2.86 (m, 3H, ArCH3), 3.36–
3.64 (m, 8H, 4ꢂCH2), 3.75–3.87 (m, 4H, 2ꢂCH2),
7.48–7.83 (m, 5H, 5ꢂArH), 7.86–7.99 (m, 2H,
2ꢂArH), 8.03–8.13 (m, 2H, 2ꢂArH), 8.18–8.26 (br s,
1H, ArH), 8.27–8.37 (br s, 1H, ArH), 8.76–8.83 (br s,
1H, ArH), 8.87–8.97 (m, 2H, 2ꢂArH), 11.04-11.30 (m,
1H, CONH), 11.78-11.92 (br s, 1H, CONH). Analysis
(164 mg, 78%): mp (CH2Cl2/n-hexane) 268–270 ꢁC; H
1
NMR (CDCl3) d 2.82-2.94 (m, 9H, 3ꢂCH2 and
ArCH3), 3.04 (t, J=5.8 Hz, 2H, CH2), 3.51 (q, J=5.5
Hz, 2H, CH2), 3.81 (q, J=5.8 Hz, 2H, CH2), 6.53 (dd,
J=7.9, 1.4 Hz, 1H, ArH), 6.72 (td, J=7.7, 1.8 Hz, 1H,
ArH), 6.77–6.87 (m, 3H, 3ꢂArH ), 6.92 (t, J=7.8 Hz,
1H, ArH), 7.63 (dd, J=8.0, 1.6 Hz, 1H, ArH), 7.65–
7.75 [m, 3H, 2ꢂArH and CONH (oxanthrene)], 7.87
(dd, J=8.6, 7.1 Hz, 1H, ArH), 8.05 (d, J=8.5 Hz, 1H,
ArH), 8.27 (dd, J=8.6, 1.6 Hz, 1H, ArH), 8.91 (dd,
J=7.2, 1.4 Hz, 1H, ArH), 11.16 [s, 1H, CONH (phena-
zine)]. HRMS (FAB+) calcd for C33H33N6O4 577.2564
(MH+), found 577.2579. Analysis calcd for
.
calcd for C44H49N7O6 0.5H2O: C, 67.7; H, 6.4; N, 12.6.
Found: C, 67.7; H, 6.1; N, 12.6%.
.
C33H32N6O4 H2O: C, 66.7; H, 5.8; N, 14.1. Found: C,
66.7; H, 5.6; N, 14.0%.
Deprotection of 41 with HCl(g) in MeOH as above,
followed by chromatography on alumina, eluting with
CH2Cl2/MeOH (99:1), gave 24 (96%): mp (CH2Cl2/n-
9-Methyl-N-[2-({2-[(2-{[(2-phenyl-8-quinolinyl)carbonyl]-
amino}ethyl)amino]ethyl}amino)ethyl]-1-phenazinecarbox-
amide (23). 2-Phenylquinoline-8-carboxylic acid37 was
activated with CDI to the imidazolide which was iso-
lated by precipitation from CH2Cl2/petroleum ether
then reacted with 38 as detailed above. Purification of
the product by column chromatography on silica gel,
eluting with CH2Cl2/MeOH (199:1), gave N-1-{[2-(N0-
tert-butoxycarbonyl-N0-{[2-(N-tert-butoxycarbonyl-N-{[2-
(2 - phenylquinoline - 8 - carbonyl)amino]ethyl})amino]-
ethyl})amino]ethyl}-9-methylphenazine-1-carboxamide
(40) (75%) as a foam: 1H NMR (CDCl3) d 1.23–1.34 [br
s, 18H, 2ꢂC(CH3)3], 2.86 (s, 3H, ArCH3), 3.29–3.59 (m,
hexane) 175–177 ꢁC; H NMR (CDCl3) d 2.73 (s, 3H,
1
ArCH3), 2.95–3.01 (m, 4H, 2ꢂCH2), 3.04 (sextet, J=2.8
Hz, 2ꢂCH2), 3.75 (sextet, J=5.9 Hz, 4H, 2ꢂCH2),
7.27–7.32 (m, 1H, ArH), 7.50–7.61 (m, 4H, 4ꢂArH),
7.70 (ddd, J=8.8, 6.6, 1.2 Hz, 1H, ArH), 7.81–7.87 (m,
2H, 2ꢂArH), 7.93 (dd, J=8.4, 1.5 Hz, 1H, ArH), 8.10
(d, J=8.6 Hz, 1H, ArH), 8.15 (dd, J=8.7, 1.5 Hz, 1H,
ArH), 8.41 [s, 1H, ArH (acridine H-9)], 8.81 (dd, J=7.1,
1.5 Hz, 1H, ArH), 8.84 (dd, J=7.1, 1.5 Hz, 1H, ArH),
10.86 [br t, J=5.5 Hz, 1H, CONH (phenazine)], 11.77–
11.83 [br s, 1H, CONH (acridine)]. Analysis calcd for

J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
27
.
C34H33N7O2 0.5H2O: C, 70.3; H, 5.9; N, 16.9. Found:
C, 70.5; H, 6.0; N, 17.0%.
Deprotection of 43 with HCl(g) in MeOH as above,
followed by column chromatography on silica gel, elut-
ing with CH2Cl2/MeOH (9:1), gave 26 (96%): mp
(CH2Cl2/n-hexane) 148–153 ꢁC; 1H NMR (CDCl3) d
2.81-2.93 (m, 7H, 2ꢂCH2 and ArCH3), 2.99 (t, J=6.5
Hz, 2H, CH2), 3.03 (t, J=6.1 Hz, 2H, CH2), 3.79 (q,
J=5.9 Hz, 2H, CH2), 4.27 (t, J=6.5 Hz, 2H, CH2), 7.67
(dd, J=8.1, 7.3 Hz, 2H, 2ꢂArH), 7.71–7.80 (m, 2H,
2ꢂArH), 7.94 (dd, J=8.6, 7.3 Hz, 1H, ArH), 8.06–8.12
(m, 3H, 3ꢂArH), 8.36 (dd, J=8.7, 1.3 Hz, 1H, ArH),
8.52 (dd, J=7.1, 1.0 Hz, 2H, 2ꢂArH), 8.97 (dd, J=7.1,
1.6 Hz, 1H, ArH), 11.09 (br s, 1H, CONH). HRMS
(FAB+) calcd for C32H31N6O3 547.2458 (MH+), found
9-Methyl-N-(2-{[2-({2-[(1-phenazinylcarbonyl)amino]-
ethyl}amino)ethyl]amino}ethyl)-1-phenazinecarboxamide
(25). Phenazine-1-carboxylic acid34 was activated with
CDI to the imidazolide which was isolated by pre-
cipitation from CH2Cl2/petroleum ether then reacted
with 38 as above. Purification of the product by chro-
matography on alumina, eluting with CH2Cl2/MeOH
(199:1), gave N-1-{2-[(N0-tert-butoxycarbonyl-N0-{2-
[(N-tert-butoxycarbonyl-N-{2-[(phenazinyl-1-carbonyl)-
amino]ethyl})amino]ethyl})amino]ethyl}-9-methylphena-
zine-1-carboxamide (42) (98%) as a foam: 1H NMR
(CDCl3) d 1.28–1.38 [br s, 18H, 2ꢂC(CH3)3], 2.80–
2.91 (m, 3H, ArCH3), 3.37–3.64 (m, 8H, 4ꢂCH2),
3.76–3.86 (m, 4H, 2ꢂCH2), 7.65–7.96 (m, 6H,
6ꢂArH), 8.03–8.12 (br s, 1H, ArH), 8.15–8.24 (m, 2H,
2ꢂArH), 8.29–8.39 (br s, 2H, 2ꢂArH), 8.89–8.98 (br
s, 2H, 2ꢂArH), 10.92–11.03 (m, 1H, CONH), 11.10–
11.34 (m, 1H, CONH). Analysis calcd for
.
547.2474. Analysis calcd for C32H30N6O3 2.5H2O: C,
65.0; H, 6.0; N, 14.2. Found: C, 65.2; H, 5.2; N, 14.3%.
N-(2-{[2-({2-[(4-Acridinylcarbonyl)amino]ethyl}amino)-
ethyl]amino}ethyl)-1-phenazinecarboxamide (21) (Scheme
3). 1-Phenazinecarboxylic acid34 was activated with
CDI and the imidazolide which was isolated by pre-
cipitation from CH2Cl2/petroleum ether then reacted
with 36 as detailed above. The solid obtained after
workup was purified by chromatography on alumina,
eluting with CH2Cl2 /MeOH (199:1), to give N-1-{2-[N-
tert-butoxycarbonyl-N0 (2-{N-tert-butoxycarbonyl-N-[2-
(N - trifluoroacetamido)aminoethyl]aminoethyl)] - ami-
noethyl}phenazine-1-carboxamide (44) (91%) as an oil
.
C43H48N8O6 H2O: C, 65.3; H, 6.4; N, 14.2. Found: C,
65.1; H, 6.4; N, 14.2%.
Deprotection of 42 with HCl(g) in MeOH as above,
followed by column chromatography on alumina, elut-
ing with CH2Cl2/MeOH (98:2), gave 25 (83%): mp
(CH2Cl2/n-hexane) 207–210 ꢁC; 1H NMR (CDCl3) d
2.75 (s, 3H, ArCH3), 2.96–2.98 (br s, 6H, 3ꢂCH2), 3.00
(t, J=5.8 Hz, 2H, CH2), 3.06 (t, J=5.7 Hz, 2H, CH2),
3.71 (q, J=5.5 Hz, 2H, CH2NHCO), 3.77 (q, J=5.7 Hz,
2H, CH2NHCO), 7.54–758 (m, 1H, ArH), 7.60–7.67 (m,
2H, 2ꢂArH), 7.78 (ddd, J=8.7, 6.6, 1.2 Hz, 1H, ArH),
7.83 (dd, J=8.6, 7.2 Hz, 1H, ArH), 7.86–7.91 (m, 3H,
3ꢂArH), 8.12 (d, J=8.7 Hz, 1H, ArH), 8.15 (dd,
J=8.7, 1.5 Hz, 1H, ArH), 8.21 (dd, J=8.7, 1.5 Hz, 1H,
ArH), 8.83 (dt, J=7.1, 1.5 Hz, 2H, 2ꢂArH), 10.91–
11.00 (m, 2H, 2ꢂCONH). Analysis calcd for
1
which was used directly: H NMR (CDCl3) d 1.39 [br s,
9H, C(CH3)3], 1.43 [br s, 9H, C(CH3)3], 3.31–3.68 (m,
10H, 5ꢂCH2), 3.82–3.89 (m, 2H, CH2), 7.90–8.44 [m,
7H, 6ꢂArH, NHC(O)CF3], 8.99 (br s, 1H, ArH), 11.13
(br s, 1H, CONH).
The above trifluoroacetamide 44 was deprotected as
above, using a solution of K2CO3 in MeOH/water at
reflux for 2 h to give the corresponding amine N-1-[2-
(N0-tert-butoxycarbonyl-N0-{2-[N-(aminoethyl)-N-tert-
butoxycarbonyl]-aminoethyl})aminoethyl]phenazine-1-
carboxamide (45) (100%) as an oil which was used
.
C33H32N8O2 0.5H2O: C, 68.1; H, 5.7; N, 19.3. Found:
1
C, 68.3; H, 6.0; N, 19.3%.
directly: H NMR (CDCl3) d 1.38 [br s, 9H, C(CH3)3],
1.43 [br s, 9H, C(CH3)3], 2.78 (t, J=6.5 Hz, 2H, NH2),
3.18–3.70 (m, 10H, 5ꢂCH2), 3.81–3.90 (m, 2H, CH2),
7.89–8.00 (m, 3H, ArH), 8.21–8.43 (m, 3H, 3ꢂArH),
9.00 (br s, 1H, ArH), 11.08 (br s, 1H, CONH).
N-{2-[(2-{[2-(1,3-Dioxo-1H-benzo[de]isoquinolin-2-(3H)-
yl)ethyl]amino}ethyl)amino]ethyl}-9-methyl-1-phenazine-
carboxamide (26). 1,8-Naphthalic anhydride (193 mg,
0.98 mmol) was added to a solution of 38 (544 mg, 0.98
mmol) in absolute EtOH (30 mL), and the mixture was
heated at reflux for 5.5 h, at which point the reaction
was complete by TLC. The solvent was removed under
reduced pressure, and the residue was chromatographed
on alumina, eluting with CH2Cl2/MeOH (199:1), to give
N-1-[(2-{N0-tert-butoxycarbonyl-N0-[(2-{N-tert-butoxy-
carbonyl-N-[2-(1,8-naphthalimido)ethyl]}amino)ethyl]}-
Acridine-4-carboxylic acid33 was activated with CDI
and the imidazolide isolated by precipitation from
CH2Cl2/petroleum ether as above. The imidazolide was
then reacted with a solution of 45 in THF. After
workup, the resulting crude product was purified by
chromatography on alumina, eluting with CH2Cl2/
MeOH (199:1), to give N-1-{[2-(N0-tert-butoxycarbonyl-
N0-{[2-(N-tert-butoxycarbonyl-N-{[2-(acridinyl-4-carbo-
nyl)amino]ethyl})amino]ethyl})-amino]ethyl}phenazine-
1-carboxamide (46) (89%), as a foam: 1H NMR
(CDCl3) d 1.42 [br s, 9H, C(CH3)3], 1.46 [br s, 9H,
C(CH3)3], 3.37–3.69 (m, 10H, 5ꢂCH2), 3.81–3.89 (m,
2H, CH2), 7.09–7.15 (m, 1H, ArH), 7.25 (t, J=7.4 Hz,
1H, ArH), 7.36 (d, J=8.3 Hz, 1H, ArH), 7.64 (t, J=7.2
Hz, 1H, ArH), 7.83–7.97 (m, 4H, 4ꢂArH), 8.11–8.27
(m, 3H, 3ꢂArH), 8.31–8.45 (m, 2H, 2ꢂArH), 8.53–8.61
(br s, 1H, ArH), 8.89–8.99 (br s, 1H, ArH), 11.10 [s, 1H,
CONH (phenazine)], 12.52 [s, 1H, CONH (acridine)].
amino)ethyl]-9-methylphenazine-1-carboxamide
(43)
(616 mg, 85%) as a foam: 1H NMR (CDCl3) d 1.05-1.15
[m, 9H, C(CH3)3], 1.26–1.33 [m, 9H, C(CH3)3], 2.87–
2.96 (m, 3H, ArCH3), 3.32–3.64 (m, 8H, 4ꢂCH2), 3.82
(br s, 2H, CH2), 4.31 (t, J=5.8 Hz, 2H, CH2), 7.67–7.82
(m, 4H, 4ꢂArH), 7.91–7.98 (m, 1H, ArH), 8.10–8.21
(m, 3H, 3ꢂArH), 8.34–8.41 (br s, 1H, ArH), 8.49–8.57
(m, 2H, 2ꢂArH), 8.93–9.01 (m, 1H, ArH), 11.15–11.37
(m,
1H,
CONH).
Analysis
calcd
for
.
C42H46N6O7 0.5H2O: C, 66.7; H, 6.3; N, 11.1. Found:
C, 66.6; H, 6.7; N, 11.2%.

28
J. A. Spicer et al. / Bioorg. Med. Chem. 10 (2002) 19–29
.
Analysis calcd for C43H47N7O6 1.5H2O: C, 65.8; H, 6.4;
N, 12.5. Found: C, 65.7; H, 6.1; N, 12.4.
CHCl3 (11ꢂ100 mL) and the combined extracts were
dried (Na2SO4) and evaporated to give N-1-[2-(N-{2-[N-
(2-aminoethyl)]aminoethyl})aminoethyl]-5-methylacri-
dine-4-carboxamide (49) (533 mg, 98%) as an oil which
Deprotection of 46 with HCl(g)/MeOH was carried out
as above gave 21 (96%) without further purification
after workup: mp (CH2Cl2/MeOH) 185–187 ꢁC; 1H
NMR (CDCl3) d 2.83–2.96 (m, 6H, 3ꢂCH2), 3.07 (t,
J=5.9 Hz, 2H, CH2), 3.46 (q, J=5.3 Hz, 2H, CH2),
3.80 (q, J=5.8 Hz, 2H, CH2), 7.01 (t, J=7.8 Hz, 1H,
ArH), 7.25 (ddd, J=8.1, 7.2, 0.9 Hz, 1H, ArH), 7.32 (d,
J=8.2 Hz, 1H, ArH), 7.45 (br s, 1H, ArH), 7.65 (ddd,
J=8.3, 7.0, 1.5 Hz, 1H, ArH), 7.79–7.90 (m, 4H,
4ꢂArH), 8.12–8.16 (m, 2H, 2ꢂArH), 8.26 (dd, J=8.7,
1.5 Hz, 1H, ArH), 8.39 (dd, J=8.1, 1.3 Hz, 1H, ArH),
8.43 (dd, J=8.1, 1.4 Hz, 1H, ArH), 8.89 (dd, J=7.1, 1.5
Hz, 1H, ArH), 11.11 [s, 1H, CONH (phenazine)], 12.20
[s, 1H, CONH (acridine)]. Analysis calcd for
1
was used directly: H NMR (CDCl3) d 2.58–2.68 (m,
2H, CH2), 2.70–2.76 (m, 4H, 2ꢂCH2), 2.82–2.86 (m,
2H, CH2), 2.93 (s, 3H, ArCH3), 3.03–3.07 (m, 2H,
CH2), 3.82–3.88 (m, 2H, CH2), 7.49–7.55 (m, 1H, ArH),
7.65–7.75 (m, 2H, 2ꢂArH), 7.89–7.94 (m, 1H, ArH),
8.13–8.18 (m, 1H, ArH), 8.88 (br d, J=5.3 Hz, 1H,
ArH), 8.98 (td, J=7.1, 1.5 Hz, 1H, ArH), 12.04 (br s,
1H, NH).
9-Methylphenazine-1-carboxylic acid (1.00 g, 4.20
mmol) was reacted with CDI (1.02 g, 6.30 mmol) to
form the imidazolide which was isolated by precipita-
tion from CH2Cl2/petroleum ether as above. The
resulting solid was suspended in THF (20 mL), cooled
.
C33H31N7O2 2H2O: C, 66.8; H, 5.9; N, 16.5. Found: C,
67.0; H, 5.7; N, 16.4%.
ꢁ
t o 0 C in ice/water, then treated slowly with a solution
of 49 (529 mg, 1.48 mmol) in THF (20 mL). The reac-
tion mixture was stirred for 2 h at 0 ꢁC, then 18 h at
room temperature. Solvent was removed under reduced
pressure and the residue was partitioned between
CH2Cl2 (100 mL) and 1 M Na2CO3 (100 mL). The
CH2Cl2 layer was dried (Na2SO4) and evaporated, and
the residue was chromatographed on alumina, eluting
with CH2Cl2/MeOH (49:1), to give 27 (533 mg, 61%):
mp (CH2Cl2/n-hexane) 175–179 ꢁC; 1H NMR (CDCl3) d
2.77 (s, 3H, ArCH3), 2.79 (s, 3H, ArCH3), 2.82–2.88 (m,
4H, 2ꢂCH2), 2.97 (t, J=6.0 Hz, 2H, CH2), 3.02 (t,
J=6.0 Hz, 2H, CH2), 3.70 (q, J=5.9 Hz, 2H, CH2),
3.79 (q, J=6.0 Hz, 2H, CH2), 7.31 (dd, J=8.4, 6.8 Hz,
1H, ArH), 7.51–7.70 (m, 5H, 5ꢂArH), 7.87 (dd, J=8.5,
7.1 Hz, 1H, ArH), 7.91–7.96 (m, 2H, 2ꢂArH), 8.26 (dd,
J=8.7, 1.5 Hz, 1H, ArH), 8.57 [s, 1H, H-9 (acridine)],
8.85–8.91 (m, 2H, 2ꢂArH), 10.83 [br t, J=5.1 Hz, 1H,
NH (phenazine)], 11.83 [br t, J=5.4 Hz, 1H, NH (acri-
dine)]. HRMS (FAB+) calcd for C35H36N7O2 586.2930
(MH+), found 586.2938. Analysis calcd for
9-Methyl-N-[2-({2-[(2-{[(5-methyl-4-acridinyl)carbonyl]-
amino}ethyl)amino]ethyl}amino)ethyl]-1-phenazinecarbox-
amide
(27)
(Scheme
4).
N,N0-bis(aminoethyl)
ethanediamine was reacted with di-tert-butyldicarbo-
nate according to a reported method.26 Purification of
the product by chromatography on silica gel, eluting
with CH2Cl2/MeOH (4:1), gave N-aminoethyl-N,N0-
bis(tert-butoxycarbonyl)-N-[(N-tert-butoxycarbonyl)-
aminoethyl]ethylenediamine (47) (59%) as a viscous oil
1
which was used directly; H NMR (CDCl3) d 1.39–1.50
[m, 29H, 3ꢂC(CH3)3, NH2], 3.00–3.62 (m, 12H,
6ꢂCH2), 4.45 (br s, 1H, NHBOC). HRMS (EI+) calcd
for C21H42N4O6 446.3104 (M+), found 446.3095.
5-Methylacridine-4-carboxylic acid (1.00 g, 4.23 mmol)
was reacted with CDI (1.02 g, 6.33 mmol) as above to
form the imidazolide which was isolated by precipita-
tion from CH2Cl2/petroleum ether as above. This was
suspended in THF (80 mL) at room temperature, and a
solution of 47 (2.04 g, 4.65 mmol) in THF (20 mL) was
slowly added. The reaction mixture was then stirred for
.
C35H35N7O2 1.5H2O: C, 68.6; H, 6.3; N, 16.0. Found:
C, 68.9; H, 6.0; N, 15.9%.
ꢁ
18 h at20 C, then the THF was removed under reduced
pressure and the resulting solid was partitioned between
CH2Cl2 (200 mL) and 1 M Na2CO3 (200 mL). The
CH2Cl2 layer was dried (Na2SO4) and evaporated, and
the residue was chromatographed on alumina, eluting
with CH2Cl2/MeOH (4:1), to give N-1-(-2-[N0tert-
butoxycarbonyl N0-(2-{N-tert-butoxycarbonyl-N-[2-(N-
tert-butoxycarbonyl)-aminoethyl]}aminoethyl)-5-methyl-
acridine-4-carboxamide (48) (1.41 g, 51%) as a foam
Acknowledgements
This work was supported by the Auckland Division of
the Cancer Society of New Zealand, and by Xenova
Ltd, Slough, UK.
1
which was used directly: H NMR (CDCl3) d 1.46 [m,
References and Notes
30H, 3ꢂC(CH3)3, ArCH3], 3.13–3.65 (m, 12H, 6ꢂCH2),
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7.74 (m, 2H, 2ꢂArH), 7.88–7.94 (m, 1H, ArH), 8.12–
8.19 (m, 1H, ArH), 8.85–8.90 (m, 1H, ArH), 8.93–9.00
(m, 1H, ArH), 12.23 (br s, 1H, NH).
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