Influence of polyamine architecture on the transport and topoisomerase II inhibitory properties of polyamine DNA-intercalator conjugates

Article abstract of DOI:10.1021/jm010181v

An efficient five-step synthetic method was developed to access a series of spermine derivatives containing appended acridine, anthracene, and 7-chloroquinoline motifs. The derivatives were composed of a spermine fragment covalently tethered at its N4 and

Full text of DOI:10.1021/jm010181v

3
682
J . Med. Chem. 2001, 44, 3682-3691
In flu en ce of P olya m in e Ar ch itectu r e on th e Tr a n sp or t a n d Top oisom er a se II
In h ibitor y P r op er ties of P olya m in e DNA-In ter ca la tor Con ju ga tes
†
‡
†
†
,†
Lu Wang, Harry L. Price, J ane J uusola, Martin Kline, and Otto Phanstiel IV*
Center for Discovery of Drugs and Diagnostics, Department of Chemistry, University of Central Florida,
Orlando, Florida 32816-2366, and Department of Chemistry, Stetson University, Deland, Florida 32720
Received April 23, 2001
An efficient five-step synthetic method was developed to access a series of spermine derivatives
containing appended acridine, anthracene, and 7-chloroquinoline motifs. The derivatives were
composed of a spermine fragment covalently tethered at its N4 and N9 positions to an aromatic
nucleus via an aliphatic chain (e.g., 8: acridine -[C4 aliphatic tether]-spermine-[C4 aliphatic
tether]-acridine). The distance separating the spermine and aromatic nuclei was altered via
different tethers composed of four or five methylene units. These bis ligands (8, 9, 12, and 13)
were shown to inhibit human DNA topoisomerase II (topo II) activity at 5 µM. Enzymatic
activity was assessed as the ability to unknot (decatenate) and cleave kinetoplast DNA (kDNA).
Polyamine conjugation did not disrupt the ability of the acridine-spermine conjugates 8 and
9
to inhibit topo II activity as compared with the 9-aminoacridine and 9-(N-butyl)aminoacridine
controls (at 5 µM). The parent polyamines, spermine (5 µM) and spermidine (10 µM), had little
effect on topo II activity. In general, the bis-substituted spermine derivatives (8, 9, 12, and 13)
were more efficient topo II inhibitors at 5 µM than their monosubstituted spermidine
counterparts (22-25) at 10 µM. Within the bisintercalator spermine series, insertion of an
additional methylene unit (i.e., C5 tethers) increased potency 2-fold (8, bis-C4-acridine, 47 h
IC50 ) 40 µM; 9, bis-C5-acridine, IC50 ) 17 µM). Comparison of the bis- and monoacridine
spermine motifs (8 and 17) revealed a 4-fold increase in potency for the latter architecture (94
h IC50 for 8, 74 µM; for 17, 17 µM). In general the bisintercalators (8, 9, 12, and 13) behaved
as cytostatic agents, while the monosubstituted acridine and anthracene derivatives (22-25)
were cytotoxic. Anthracene-containing conjugates were generally more toxic than their acridine
counterparts in an L1210 (murine leukemia) cell assay. Of the conjugates tested the
(
monointercalator)-spermine motif (e.g., 17) had the highest affinity for the L1210 polyamine
transporter as revealed by spermidine protection experiments.
In tr od u ction
The selective delivery of drugs to targeted cell types
is one of the most challenging aspects of modern
chemotherapy. Indeed, many of the side effects of
current therapeutics result from the nonselective de-
livery of the toxic chemotherapeutic agent. Ideal drugs
should target only the affected cell type. Biosynthesized
in humans, spermidine [SPD, H2N(CH2)3NH(CH2)4NH2]
and spermine [SPM, H2N(CH2)3NH(CH2)4NH(CH2)3-
NH2] are ubiquitous polyamines, which are important
F igu r e 1. (a) Amsacrine (AMSA), a topoisomerase II inhibitor,
and (b) 9-aminoacridine.
1
of a flat, aromatic chromophore between the DNA base
pairs. Many of the clinically used DNA intercalating
agents are potent inhibitors of nucleic acid synthesis,
and this was originally considered to be their primary
mode of action. For example, amsacrine (AMSA, Figure
regulators of cell growth and differentiation. In vivo
they represent physiological sources of polycations use-
2
ful in the stabilization of DNA topologies. Many tumor
types have been shown to contain elevated polyamine
levels and an active polyamine transporter (PAT) for
6
importing exogenous polyamines.3 These range from
1) is a selective inhibitor of DNA synthesis and shows
a linear dose-response relationship for inhibition of
neuroblastoma, melanoma, human lymphocytic leuke-
mia, and colonic and lung tumor cell lines to murine
7
DNA polymerase I in a cell-free system.
3
Another major biological effect of DNA-intercalating
agents is the production of DNA damage. Many inter-
calating agents cause DNA strand breaks at very low
concentrations and usually involve disruption of a
L1210 cells. Recognizing the opportunity for selective
drug delivery, several anticancer therapies have tried
to utilize the PAT to convey cytotoxic and genotoxic
agents to rapidly proliferating cells.4
,5
8
topoisomerase enzyme. Topoisomerase type I (topo I)
DNA-intercalating ligands have the capacity to bind
tightly but reversibly to DNA by insertion (intercalation)
and type II (topo II) represent two classes of the known
mammalian DNA topoisomerases. To untwist densely
packed DNA, these enzymes generate transient breaks
within the DNA strands, allow for topological changes
†
University of Central Florida.
Stetson University.
‡
1
0.1021/jm010181v CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/28/2001

Polyamine DNA-Intercalator Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3683
F igu r e 2. Some naturally occurring polyamines.
to occur, and then reseal the break.9-12 Topo I and topo
II act by creating temporary single-strand and double-
strand breaks in DNA, respectively. Presently, two
equilibrating complexes (“cleavable” and “noncleavable”)
have been shown to exist between topoisomerase II and
1
0-12
F igu r e 3. General structures of conjugates predicated upon
spermine.
DNA.
The equilibrium is normally shifted toward
the noncleavable complex as the cleavable form results
in permanent DNA strand breaks.1
0,11
The noncleavable
We were interested in whether a DNA intercalator
with an appended polyamine side chain such as sper-
mine (SPM) would show enhanced DNA scission activ-
ity. The design of the conjugates (shown in Figure 3) is
predicated upon the well-known affinity of polyamines
for DNA and the established DNA binding modes of the
complex allows for the cleaved strands to untwist and
to be reannealed, which in turn allows for the local
separation of complementary polynucleotide strands and
ultimately the generation of relaxed supercoils (both of
which are important events for DNA replication and
RNA transcription). An anti-cancer strategy suggested
4
,5d
17
acridine
and anthracene nuclei. In 2000 we dem-
1
2
by Nelson et al. involves perturbing this equilibrium
toward the cleavable complex, an event that results in
permanent DNA strand breaks and cell death. Mol-
ecules that stimulate the formation of the cleavable
complex would be of clear value in terms of developing
new chemotherapeutics and further elucidating the
specific mechanisms involved in the DNA cleaving
onstrated the competitive uptake of spermidine-acri-
dine and spermidine-anthracene conjugates via the
polyamine transporter and their in vitro inhibition of
4
topo II. As part of our continuing efforts to define the
scope and limitations of PAT for drug delivery, several
spermine analogues were designed for comparison to our
4
earlier spermidine models. This paper describes the
1
2
process. However, mere intercalation of the topoi-
somerase inhibitor with DNA is not sufficient for
antineoplastic activity (as measured in L1210 murine
synthesis and biological evaluation of these SPM-DNA
intercalator conjugates.
4
,13
leukemia cells).
A series of substituted acridines have
Resu lts a n d Discu ssion
been evaluated for antineoplastic activity by Denny and
co-workers.13 After an extensive investigation, Denny
found that both DNA intercalation and an appropriately
placed side chain appeared to be an absolute require-
ment for antitumor activity with the acridine systems
studied.13
Syn th esis. The synthesis of each conjugate involves
a series of protection and deprotection steps similar to
those outlined by Cain and co-workers for the synthesis
1
8,19
of substituted acridines.
In each case, spermine was
conjugated at the N4 (and/or N9) position via an
aliphatic carbon tether to an acridine, anthracene, or
quinoline nucleus. The quinoline derivative allowed for
comparison between bicyclic and tricyclic aromatic
nuclei. The central attachment was predicated on the
findings by Porter in 1982, wherein spermidine could
be extensively derivatized at the central N4 position and
still be taken up by the polyamine transporter.16 The
N4,N9-alkylation step was designed to maintain the
basicity of the internal nitrogens, which was also shown
Virtually all cells contain substantial amounts of at
least one of the polyamines: putrescine, spermidine, or
spermine (Figure 2). Polyamines are a requirement for
the optimum growth and replication of various cell types
and are present in higher concentrations in rapidly
proliferating cells.1,14 The fact that polyamines can be
taken up by tissues from the circulation is known, since
the metabolism of labeled polyamines has been studied
in vivo. Tissues with a high demand for polyamines
1
6
to be critical to uptake.
(prostate, tumors, or normal but rapidly dividing cells)
take up exogenous polyamines in increased amounts via
a specific uptake system.3 More recently, studies have
indicated that polyammonium cations (PACs) have a
very high DNA affinity but are loosely bound and can
Each conjugate required the generation of a function-
alized spermine fragment with selectively protected
amino groups. As shown in Scheme 1, the selective
acylation of the primary amino groups of spermine
with 2-[t-(butoxycarbonyl)oxyimino]-2-phenylacetoni-
,15
“read” DNA very rapidly because of their otherwise
1
12
unconstrained motion. These properties make PACs and
related polycations ideal for drug delivery when the drug
trile (BOC-ON) gave N ,N -bis(t-butoxycarbonyl)sper-
2
0,21
4
9
mine 1 in 75% yield.
N ,N -Alkylation of 1 with
2
needs to reach specific sites in the DNA. Finally, Porter
either 4-bromobutyronitrile or 5-bromovaleronitrile
gave the corresponding nitriles 2 and 3 in 65% and
77% yield, respectively. Raney nickel reduction of 2 and
3 in the presence of ammonia under high pressure (75
psi) of H2 (g) gave the respective amines 4 (98%) and 5
(97%).
et al. have shown that a wide variety of N-4 substituents
1
6
on the spermidine molecule do not disrupt its uptake.
Therefore, the internal amine position was selected for
the introduction of substituents (with antitumor proper-
ties) onto the polyamine backbone.

3
684 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Wang et al.
Sch em e 1a
a
Reagents: (a) BOC-ON, THF, 0 °C; (b) Br-CH2(CH2)nCH2CN,
refluxing acetonitrile, solid K2CO3; (c) H2 gas (75 psi), NH4OH,
Raney Ni (50% slurry), absolute EtOH saturated with NH3 gas.
Sch em e 2a
F igu r e 4. Structures of other amine derivatives tested.
tected with 4 N HCl to give the HCl salts (8 and 9) in
6
0% and 70% yield, respectively.
In addition to the acridine derivatives, two an-
thracene-spermine conjugates (12 and 13) were syn-
thesized. As shown in Scheme 2, the respective amines
4
and 5 were condensed with 9-anthraldehyde and,
after reduction with NaBH4, afforded the tethered
amine adducts 10 (78%) and 11 (75%), respectively.23
The BOC protecting groups of 10 and 11 were then
deprotected with 4 N HCl to give the HCl salt of
the desired conjugates 12 (86%) and 13 (80%), respec-
tively.
Two monoacridine- and monoquinoline-spermine
conjugates (17 and 19, Figure 4) were also synthesized.
4
As shown in Scheme 3, N -alkylation of 1 with 1 equiv
of 4-bromobutyronitrile gave the corresponding nitrile
a
Reagents: (a) 9-chloroacridine, molten phenol, 50 °C; (b) 4 N
1
4 (73% yield). Raney nickel reduction of 14 in the
HCl, MeOH; (c) 9-anthraldehyde, 4 Å molecular sieves followed
by NaBH4.
presence of ammonia and H2 (g) gave the respective
amine 15 (95%). The respective amine 15 was split and
condensed either with 9-chloroacridine or 4,7-dichloro-
quinoline to afford the tethered amine adducts 16 (70%)
and 18 (54%), respectively. The BOC protecting groups
of 16 and 18 were then deprotected with 4 N HCl to
give the HCl salt of the desired conjugates 17 (72%) and
19 (72%), respectively.
The syntheses of the bisacridine conjugates 8 and 9
are shown in Scheme 2. The respective amines 4 and 5
were coupled to 9-chloroacridine by use of an excess of
molten phenol to give 6 and 7 in 79% and 73% yield,
respectively.4
,22
The BOC protecting groups on the
spermine-acridine conjugates 6 and 7 were then depro-

Polyamine DNA-Intercalator Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3685
Sch em e 3a
F igu r e 5. Topoisomerase II inhibition. Decatenation assay
results are displayed as a negative image of the electrophoresis
gel. All lanes contain 0.5 µg of kDNA and 2.0 units of human
topo II, with the exception of lane 1. The respective drugs and
conjugates were evaluated at 5 µM. Lane 1, negative control
(kDNA only); lane 2, positive control (topo II); lane 3, 9-ami-
noacridine; lane 4, conjugate 8; lane 5, conjugate 9; lane 6,
conjugate 12; lane 7, conjugate 13; lane 8, Berenil; lane 9,
spermine; lane 10, 9-(N-butyl)aminoacridine. Forms of DNA
are denoted as Roman numerals (I, II, and III, see text).
conjugates 22-25 derivatives (shown in Figure 6: 10
4
µM, panel A; 20 µM, panel B). For comparison, reac-
tions were also carried out with Berenil (Figure 4), a
3
4,35
known inhibitor of topo II,
9-aminoacridine, 9-(N-
butylamino)acridine, spermidine, and spermine. At 5
µM, spermine conjugates 8, 9, 12, and 13 (lanes 4-7 in
Figure 5) completely inhibit the activity of topo II,
resulting in the absence of lower molecular weight DNA
bands (i.e., the kDNA remains intact). Spermidine
conjugates 22-25 (10 µM, lanes 3-6 in Figure 6) clearly
interfere with the topo II-mediated decatenation of
kDNA but are less efficient inhibitors (as lower molec-
ular weight DNA bands are still generated). This is
consistent with the general observation that bisinter-
calator systems are typically more efficient DNA binding
agents than their monointercalator counterparts.30 The
results also parallel the avid binding characteristics
observed with these ligands with model oligonucleotides
a
Reagents: (a) Br-CH2CH2CH2CN, refluxing acetonitrile, solid
K2CO3; (b) H2 gas (75 psi), NH4OH, Raney Ni (50% slurry),
absolute EtOH saturated with NH3 gas; (c) 9-chloroacridine,
molten phenol, 50 °C; (d) 4,7-dichloroquinoline; (e) 4 N HCl, MeOH.
Finally, to compare the activities of these conjugates
to known active spermidine analogues, spermidine
derivatives (20 and 21) were synthesized by the method
of Bergeron (65% and 63%, respectively).2 The related
spermidine derivatives (22-25) were prepared by our
3
0
in vitro.
Equally important results were obtained by carrying
out the decatenation reactions in the presence of the
minor-groove binder Berenil (i.e., diminazene aceturate,
lane 8 in Figure 5 and lane 7 in Figure 6) and the known
DNA intercalator 9-aminoacridine (lane 3 in Figure 5
4
4
published methods and are shown in Figure 4.
Biologica l Eva lu a tion . There has been recent inter-
est in tethering polyamines to existing cancer drugs
and bioactive agents to augment their activity and
3
6
and lane 8 in Figure 6). 9-(N-Butylamino)acridine
(lane 10 in Figure 5 and lane 9 in Figure 6), which
represents a molecular fragment of conjugates 8, 9, 22,
and 23 without the polyamine moiety, had similar
activity as 9-aminoacridine at 5 µM. When tested at 5
µM, the derivatives 8, 9, 12, and 13 possess similar
potency as Berenil. Other control reactions, which
included unmodified spermine (lane 9 in Figure 5) and
unmodified spermidine (lane 10 in Figure 6), had little
effect on topo II activity. Evaluation of these compounds
provided both verification of inhibition and insight into
the contribution that each molecular fragment (i.e.,
intercalator and polyamine) made to the observed
inhibitory activity.
4
,5,25-30
specificity.
Conjugates, which incorporated either
spermidine (22-25)4
,30b
or spermine (8, 9, 12, and 13)
30a
were evaluated for their biological activity. Enzymatic
activity was assessed as the ability to unknot (decat-
enate) and cleave Crithidia fasciculata kinetoplast DNA
3
1,32
33
(
kDNA).
A simple electrophoretic assay allowed for
the visualization of the reaction products (see Experi-
mental Section). Due to its high molecular weight,
catenated DNA (kDNA) has limited migratory ability
and remains near the top well (form I), but the lower
molecular weight decatenated forms [i.e., closed-circular
(
form II) and linear DNA (form III)] migrate further
down the gel. A potent topo II inhibitor is expected to
completely abrogate the decatenation process and to
leave (after electrophoresis) significant amounts of
kDNA remaining in the top well.
Decatenation reactions were carried out in the pres-
ence of the bisintercalator conjugates (8, 9, 12, and 13)
at 5 µM (shown in Figure 5) and monointercalator
It is important to recognize that the in vitro topo II
measurements remove the “transport-into-the-cell” hurdle
and measure direct interactions between the conjugates,
kDNA, and topo II. By design the polyamine appendage
was expected (a) to play a significant role in conjugate
uptake and (b) to show dramatic differences both in the
concentration of conjugate required to reduce viable cell

3
686 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Wang et al.
4
F igu r e 6. Topoisomerase II inhibition. Decatenation assay results are displayed as a negative image of the electrophoresis gel.
All lanes contain 0.5 µg of kDNA and 2.0 units of human topo II, with the exception of lane 1. The respective drugs and conjugates
were evaluated at 10 µM (panel A) and 20 µM (panel B). Lane 1, negative control (kDNA only); lane 2, positive control (topo II);
lane 3, conjugate 22; lane 4, conjugate 23; lane 5, conjugate 24; lane 6, conjugate 25; lane 7, Berenil; lane 8, 9-aminoacridine;
lane 9, 9-(N-butyl)aminoacridine; lane 10, spermidine. Forms of DNA are denoted as Roman numerals (I, II, and III, see text).
Ta ble 1. IC50 Values
than the related spermine (SPM) derivatives (see Table
: 12 vs 24 and 13 vs 25). [Note: 9-(N-butylamino)-
1
IC50 (µM)
acridine exhibits the highest activity among all of the
derivatives tested, which may be due to multiple modes
of activity.] Among the acridine conjugates tested (8, 9,
sample
47 h
94 h
74
23
61
25
17
8
9
1
1
1
1
2
2
2
2
9
(SPM bisC4 acridine)
(SPM bisC5 acridine)
40
17
44
18
23
1
7, 22, and 23), spermine monoacridine conjugate 17
2 (SPM bisC4 anthracene)
3 (SPM bisC5 anthracene)
7 (SPM monoC4 acridine)
9 (SPM monoC4 quinoline)
2 (SPD C4 acridine)
showed the greatest bioactivity at 94 h (IC50 ) 17 µM),
whereas the related spermidine acridine conjugate (22)
had a higher IC50 (55 µM). In this regard, the spermine
framework serves as a better vector than the spermidine
>100
>100
75
55
3 (SPD C5 acridine)
>100
13
>100
6
4
4 (SPD C4 anthracene)
5 (SPD C5 anthracene)
-(N-butyl)aminoacridine
motif. These results support our earlier premise that
7
2
5
2
conjugates which more closely resemble the parent
polyamines are more likely to be recognized by the
polyamine transporter. Spermine monoquinoline con-
jugate 19 exhibited a much higher IC50 value (>100 µM)
than any of the other conjugates. This is likely related
to the putative mode of action (topo II inhibition).
number by 50% (IC50) and in the transport kinetic (Ki)
measurements with cancer cell types.
IC50 Mea su r em en ts. A second set of experiments
was performed to determine the respective cytotoxicity
of 8, 9, 12 and 13 toward murine leukemia (L1210) cells
grown in tissue culture. The results of these toxicity
P AT Stu d ies. The bis systems are potent inhibitors
of topo II (by gel assay), and cytostatic agents. In
contrast, the spermidine monointercalator systems are
less efficient topo II inhibitors and yet are cytotoxic.
Therefore, the IC50 data cannot be rationalized by the
observed topo II inhibitory activity. Instead, one must
consider the conjugate’s ability to enter the cell. To
relate potency to uptake by the polyamine transporter,
several spermidine protection experiments were con-
ducted.
3
7
studies yielded IC50 values (Table 1). The reported
mean IC50 value for the parent, AMSA, is 0.26 µM for
six human cancer cell lines with varying levels of topo
II R and â isoforms.38 All tested compounds exhibited
concentration dependent toxicity. Time-dependent be-
havior was also observed. Our goal was not to generate
a highly toxic agent with little inherent selectivity [e.g.,
9
-(N-butylamino)acridine, 94 h IC50 ) 2 µM). Instead,
we were interested in how changes in the appended
polyamine architecture perturbed the overall efficacy of
the conjugate. Interestingly, the bis systems (8, 9, 12,
and 13) showed lower cytotoxicity at longer time (i.e.,
the IC50 is higher at 94 h than at 47 h). In direct
contrast, the positive control with an agent of known
toxicity (10 µM, methotrexate) and the monoalkylated
spermidine (22) and spermine (17) derivatives always
showed higher toxicity at 94 h than at 47 h (i.e., the
IC50 value at 94 h is lower than that at 47 h). These
observations are consistent with the bisintercalators
acting as temporary cytostatic agents. In general, the
bis systems containing the C5 tether length (9 and 13)
had higher activities (i.e., lower IC50 values) than the
corresponding conjugates containing the C4 tether
length (8 and 12). This tether trend was also evident in
the monosubstituted spermidine-anthracene conju-
gates (24 and 25). For the anthracene conjugates, the
spermidine (SPD) derivatives showed higher activity
Are the polyamine-DNA intercalator conjugates rec-
ognized and transported by the polyamine uptake
apparatus of L1210 cells? Several polyamine derivatives
were examined. Two controls, dimethylspermidine (20)
and diethylspermidine (21) have been shown to gain
4
,24
entry into the cell via the PAT.
Both of these
derivatives are toxic to L1210 cells, and kinetic assays
have revealed that these SPD analogues inhibited PAT-
mediated transport of SPD via a competitive mecha-
2
4
nism. In terms of their respective affinity for PAT, the
Ki values for inhibition of SPD uptake for dimethyl 20
and diethyl 21 have been reported to be 5 and 19 µM,
2
4
respectively. Based on these Ki values, the approxi-
mate Km values for 20 and 21 transport via the PAT
2
4
are 2.5 and 9.5 µM, respectively. The cellular response
to 20 and 21 is death, which results from polyamine
2
4
depletion. Since the mode of SPD inhibition is com-
petitive and an excess of SPD is present in the system,
it is expected that transport of both 20 and 21 will be

Polyamine DNA-Intercalator Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3687
F igu r e 7. Demonstration of spermidine-mediated protection
against dimethylspermidine (20, upper panel) and diethyl-
spermidine (21, lower panel). See Experimental Section for
description of experimental conditions. (4) No spermidine; (O)
2
5 µM spermidine. Response is equivalent to cell death as
determined by the MTS/PMS colorimetric assay. A decreased
response (i.e., less cell death) is consistent with reduced drug
uptake. Data represent the average of two independent trials,
each performed in triplicate.
reduced by more than 90%, which in turn will signifi-
cantly reduce cell death. Therefore, inhibition of PAT-
mediated transport of 20 and 21 by exogenous spermi-
dine will be observed as an increase in cell viability.
In general, the spermidine (SPD) protection assays
were performed to determine whether uptake of selected
polyamine analogues is mediated, in part or in whole,
by the polyamine transport apparatus (PAT). To answer
this question, competition assays were performed in the
absence and presence of SPD. To maximize the protec-
tive effects of spermidine (SPD), an excess of SPD (25
µM) was used during these experiments. Use of a 25Km
excess of SPD ensures that SPD is transported into the
cell at nearly Vmax and, hence, provides a high level of
competition with the selected polyamine derivatives for
the PAT protein.
F igu r e 8. Spermidine protection experiments to assess the
ability of different polyamine conjugates to gain entry into the
cell via the PAT apparatus. (A) SPM monoC4 acridine (17);
(B) SPD C4 anthracence (24); (C) SPM bisC4 acridine (8); (D)
SPM bisC4 anthracene (12). (4) No spermidine; (O) 25 µM
spermidine. Response is equivalent to cell death as determined
by the MTS/PMS colorimetric assay. A decreased response (i.e.,
less cell death) is consistent with reduced drug uptake. Data
represent the average of two independent trials, each per-
formed in triplicate.
considers our topo II (and IC50) findings, which suggest
that if these analogues were transported efficiently into
the cell they would kill it. The second possibility, and
the most reasonable, is that 8 and 12 display a low
affinity for PAT, and a second process is responsible for
transport of these analogues. Analogous behavior has
been observed for in vitro model systems designed to
study the effect of uptake blockers on cellular responses
The results of experiments with 8, 12, 17, 20, 21, and
2
4 are shown in Figures 7 and 8. The graphs clearly
indicate that, over a 48-h period, the selected assay
format provides sufficient sensitivity to reveal changes
in cell viability. Note: the low concentration range of
3
9
2
0 and 21 used in these experiments was intended to
to agonists. In this regard, the low toxicity observed
with the bis ligands (8 and 12, Table 1) may be due to
their poor transport into the cell by PAT. Therefore, the
bisintercalating systems (e.g., 8 and 12) do not represent
optimum vector architectures, which target the PAT.
Spermidine protection experiments with the monoint-
ercalators 17 and 24 revealed significant cell rescue
(Figure 8A,B) upon addition of exogenous spermidine
(25 µM). This was especially evident for 17 (Figure 8A).
These observations are compelling as they suggest that
conjugates such as 17 are using the polyamine trans-
porter (PAT) to gain entry into L1210 cells. Although
17 is transported more readily into the cell, it has a
higher 94 h IC50 (17 µM) than 24 (6 µM). This finding
was rationalized by the observed greater toxicity of
produce a low level of toxicity and, hence, amplify any
observed SPD-mediated protection.
A high degree of SPD protection was observed for the
terminally alkylated spermidine models 20 and 21
(
Figure 7). This is consistent with their low Ki values
4
,24
and high affinity for PAT.
Moderate SPD protection
was observed with the monointercalators 17 and 24
Figure 8, panels A and B, respectively). Little, if any,
SPD protection was observed with the bisintercalators
(
8
and 12 (Figure 8, panels C and D, respectively).
There are several possible interpretations of these
results. The first possibility is that 8 and 12 exhibit an
even greater affinity for the transport apparatus than
either 20, 21, or SPD. This seems unlikely when one

3
688 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Wang et al.
anthracene SPD analogues (Table 1, 24 and 25) over
their acridine counterparts (22 and 23). These experi-
ments elegantly delineate the properties required for
optimal drug efficacy as a balance between ease of
transport and conjugate toxicity. Moreover, our findings
support spermidine’s antagonistic character in these
studies and bolster the premise that certain conjugates
can compete with spermidine for the transporter, while
others are excluded due to their structure. In summary,
the selectivity of the PAT can be used to deliver specific
architectures into cancer cells.
ments. Typically, samples contained less than 5% trypan blue
positive cells (dead). In these experiments, L1210 cells in early
to mid log phase were used.
Sp er m id in e P r otection Assa ys. L1210 cells were seeded
into 96-well plates and grown in medium containing the
polyamine oxidase inhibitor aminoguanidine (250 µM) and
increasing concentrations of drug, in the presence and absence
of a saturating concentration of spermidine (25 µM, ap-
proximate K
m
) 1.0 µM). Cells were grown for 48 h. Relative
cell viability was determined by the colorimetric MTS/PMS
assay.4
0,41
After a 2 h incubation, the absorbance at 490 nm
2
was recorded with a Wallac Victor plate reader operating in
absorbance mode. The relative viability was determined from
the following ratio: 1] }0 .0 × {[A490(cells+drug) - A490(drug blank)]/
Con clu sion s
[A
490(cells only)
-
A
490(blank)
1
N ,N12-Bis(t-bu toxyca r bon yl)sp er m in e (1).20,21 Spermine
We have demonstrated that polyamine homologation
can alter the topo II inhibitory activity, the delivery
(9.87 g, 49 mmol) was dissolved in THF (150 mL) and cooled
4
to 0 °C. BOC-ON (24.04 g, 98 mmol) in THF (300 mL) was
added dropwise and the reaction mixture was stirred for 2 h
at 0 °C, resulting in the complete conversion of spermine as
characteristics, cytotoxicity time dependence, and ef-
ficacy of the conjugate employed. In terms of vector
design, the bis-substituted analogues behaved as tem-
porary cytostatic agents, whereas the monosubstituted
polyamine derivatives were cytotoxic. In this regard,
longer tether lengths and further aminoalkylation of the
polyamine framework may lead to more effective vec-
tors. Future work will continue to illustrate the versa-
tility of polyamines and their ability to function as
modulators of drug delivery.
monitored by TLC (R
reaction mixture was then warmed to room temperature and
partitioned between a saturated aqueous Na CO solution and
dichloromethane. The organic layer was separated, dried over
anhydrous Na SO , filtered, and concentrated in a vacuum to
afford the crude product. The crude product was subjected to
flash chromatography on silica gel and eluted with 5% NH
OH/CH OH to give di-BOC amine 1 (14.67 g, 75%). 1: R
f
) 0.4, 40% NH
4 3
OH/CH OH). The
2
3
2
4
4
-
3
f
)
1
0
3
.30 in 5% NH
4 3 3
OH/CH OH; H NMR (CDCl ) δ 5.19 (br s, 2H),
.20 (q, 4H), 2.60 (m, 8H), 1.60 (m, 8H), 1.40 (s, 18H); Anal.
Exp er im en ta l Section
20 42 4 4
(C H N O ) C, H, N.
N ,N -Bis(3-cya n op r op yl)-N ,N12-bis(t-bu toxyca r bon yl-
4
9
1
Ma ter ia ls a n d Meth od s. Silica gel 60 (70-230 mesh) was
purchased from EM Science, Darmstadt, Germany. All other
reagents were purchased from the Acros Chemical Company
)
sp er m in e (2). Amine 1 (4.78 g, 12 mmol) and 4-bromobuty-
ronitrile (8.80 g, 60 mmol) were dissolved in acetonitrile (10
mL). Potassium carbonate (27.2 g, 28 mmol) was then added.
The mixture was allowed to reflux with constant stirring. TLC
1
and used without further purification. H NMR spectra were
recorded at 200 MHz.
(
4 3 f
15% NH OH/CH OH, R ) 0.45) was used to monitor the
Top o II Assa y. The protocol for assaying topoisomerase II
3
1,32
consumption of the starting material. After 24 h the reaction
mixture was concentrated, redissolved in dichloromethane, and
activity is well established.
were performed in buffer A containing 50 mM Tris-HCl (pH
.0), 120 mM KCl, 10 mM MgCl , 30 µg/mL bovine serum
Briefly, decatenation assays
washed with aqueous Na
rated, dried over anhydrous Na
trated. The crude residue was subjected to flash chromatog-
raphy and eluted with 10% CH OH/EtOAc to give 2 (4.14 g,
2
CO
3
. The organic layer was sepa-
8
2
2
SO , filtered, and concen-
4
albumin, 0.5 mM dithiothreitol, and 0.5 mM ATP. Reaction
mixtures contained 0.5 µg of catenated kinetoplast DNA, four
units of human topoiosomerase II, and the appropriate
polyamine conjugate (8, 9, 12, or 13) at 5 µM (Figure 5) and
conjugates 22-25 (see Figure 6: 10 µM, panel A; 20 µM, panel
B). Controls were run in parallel and contained no inhibitor
and no enzyme. Berenil, 9-aminoacridine, and 9-(N-butyl)-
aminoacridine were also evaluated at 10 µM (and 20 µM).
Reaction mixtures were incubated at 37 °C for 30 min.
3
1
6
5
5%). 2: R
f
) 0.29 in 10% CH
3 3
OH/EtOAc; H NMR (CDCl ) δ
.10 (br s, 2H), 3.09 (q, 4H), 2.35 (m, 16H), 1.62 (m, 12H), 1.40
(
s, 18H); Anal. (C28
H
52
N
6
O
4
) C, H, N.
N ,N -Bis(4-cya n obu tyl)-N ,N12-bis(t-bu toxyca r bon yl)-
4
9
1
sp er m in e (3). Amine 1 (1.20 g, 2.98 mmol) and 5-bromovale-
ronitrile (1.45 g, 8.95 mmol) were dissolved in acetonitrile (10
mL). Potassium carbonate (2.31 g, 16.8 mmol) was then added.
The mixture was refluxed with constant stirring. TLC (15%
NH OH/CH OH, R ) 0.45) was used to monitor the consump-
4 3 f
tion of the starting amine. After 24 h the mixture was
concentrated, redissolved in dichloromethane, and washed
2
Decatenation reactions were quenched with 20 mM Na EDTA
and 100 µg/mL proteinase K. Reaction products were separated
by electrophoresis at 2.5 V/cm through a 1.5% agarose gel. The
gel contained 0.5 µg/mL ethidium bromide and was submerged
in buffer B [1× TAE buffer: 40 mM Tris-HCl, 25 mM sodium
acetate, and 1 mM EDTA (pH 8.5)]. The reaction products were
visualized under ultraviolet light and photographed.
with aqueous Na
over anhydrous Na
residue was subjected to flash chromatography and eluted with
2
CO
3
. The organic layer was separated, dried
2
SO
4
, filtered, and concentrated. The crude
IC50 Deter m in a tion s a n d Meth od s for Tr a n sp or t Re-
la ted Stu d ies. Murine leukemia cells (L1210) were obtained
from the American Type Culture Collection (ATCC). All
reagents and materials used specifically for the growth and
maintenance of this cell line were obtained from Sigma
Chemical Co. and Fisher Scientific, Inc. Cells were grown and
maintained in RPMI-1640 medium, supplemented with 10%
horse serum and 1% antibiotic/antimycotic cocktail (100 units
of penicillin, 100 µg of streptomycin, and 0.25 µg of ampho-
1
3
0% CH
0% CH
3
OH/EtOAc to give 3 (1.33 g, 77%). 3: R
3 3
OH/CHCl ; H NMR (CDCl ) δ 5.50 (br s, 2H), 3.10
f
) 0.30 in
1
3
(q, 4H), 2.45 (m, 16H), 1.62 (m, 16H), 1.40 (s, 18H); high-
resolution mass spectrum (HRMS) fast atom bombardment
(
1
FAB) theory for (C30
H
56
N
6
O
4
) M + 1 ) 565.4441, found M +
) 565.4428.
N ,N -Bis(4-a m in obu tyl)-N ,N12-bis(t-bu toxyca r bon yl)-
4
9
1
sp er m in e (4). Nitrile 2 (2.69 g, 5 mmol) was dissolved in
absolute EtOH in a Parr shaker bottle. Concentrated NH OH
(4.7 mL) was added to the solution, followed by the addition
of Raney nickel catalyst (50% slurry, 3.34 g). NH gas was then
tericin B). Cells were grown at 37 °C in a 5% CO
2
atmosphere.
4
For these studies, cells were plated out into sterile 96-well
microtiter plates and grown for the specified period of time in
the absence and presence of increasing concentrations of drug.
A positive control containing methotrexate was included in all
trials. Cell viability was determined by a colorimetric assay
based on the NAD(P)H-dependent production of Formazan.4
Trypan blue staining was used to determine cell viability
before the initiation of the cytotoxicity and transport experi-
3
passed through the solution at 0 °C for 20 min. The reaction
mixture was placed in a Parr shaker and briefly evacuated,
and H
disappearance of starting material was monitored by TLC
(20% CH OH/EtOAc, R ) 0.40). The reaction took 78 h to
complete. The suspension was filtered and the recovered
2
gas was introduced at a pressure of 75 psi. The
0,41
3
f

Polyamine DNA-Intercalator Conjugates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3689
catalyst was washed with absolute EtOH. The filtrate was
OH). The mixture was concentrated under reduced pressure
concentrated and redissolved in CH
washed with aqueous Na CO (pH ) 10), separated, dried over
anhydrous Na SO , filtered, and concentrated. The product
was purified by flash chromatography and eluted with 16%
NH OH/CH OH to give the desired amine 4 (2.7 g, 98%). 4:
) 0.30 in 16% NH
s, 2H), 3.56 (br s, 4H), 3.12 (br m, 4H), 2.70 (br s, 4H), 2.40
br m, 12H), 1.60 (br m, 16H), 1.40 (s, 18H); HRMS (FAB)
theory for (C28
45.4763; Anal. (C28
2
Cl
2
. The CH
2
Cl
2
phase was
and the product was recrystallized with CH
1) to give the hygroscopic salt 8 (0.40 g, 60%). 8: H NMR (D
δ 7.71 (d, 4H), 7.50 (t, 4H), 7.15 (t, 4H), 7.00 (d, 4H), 3.70 (br
m, 4H), 3.45 (br m, 12H), 3.24 (t, 4H), 2.30 (br m, 4H), 2.00
3
OH and Et
2
O (1:
O)
1
2
3
2
2
4
4
3
(br m, 12H); HRMS (FAB) theory for (C44
699.4863, found M + 1 ) 699.4807; Anal. (C44
C, H, N.
H
H
58
N
N
8
) M + 1 )
6
1
R
f
4
OH/CH
3
OH; H NMR (CDCl
3
) δ 5.75 (br
64
8
Cl ‚3.3H O)
2
(
4
9
N ,N -Bis[5-(9-am in oacr idin yl)pen tyl]sper m in e Hexah y-
d r och lor id e (9). The acridine derivative 9 (0.70 g, 0.76 mmol)
was dissolved in dioxane (17 mL) at 0 °C, followed by the
addition of 4 N HCl (17 mL). The mixture was stirred for 4 h
at room temperature. The disappearance of the starting
60 6 4
H N O ) M + 1 ) 545.4754, found M + 1 )
5
H
60
N
6
O
4
‚H
2
O) C, H, N.
N ,N -Bis(5-a m in op en tyl)-N ,N12-bis(t-bu toxyca r bon yl-
4
9
1
)
sp er m in e (5). Nitrile 3 (1.33 g, 2.35 mmol) was dissolved in
absolute EtOH in a Parr shaker bottle. Concentrated NH OH
2.4 mL) was added to the solution, followed by the addition
4
material was monitored by TLC (R
OH). The mixture was concentrated under reduced pressure
f
) 0.35, 1% NH
4 3
OH/CH -
(
of Raney nickel catalyst (50% slurry, 1.67 g). NH
passed through the solution at 0 °C for 20 min. The reaction
mixture was placed in a Parr shaker and briefly evacuated,
3
gas was then
and the product was recrystallized with CH
3
OH and Et
2
2
O to
O) δ
give the hygroscopic salt 9 (0.38 g, 70%). 9: 1H NMR (D
7.55 (m, 8H), 7.50 (t, 4H), 7.15 (q, 4H), 7.00 (d, 4H), 3.50 (br
m, 4H), 3.35 (br m, 16H), 2.20 (br m, 4H), 1.85 (br m, 12H),
1.50 (br m, 4H); HRMS (FAB) theory for (C46
and H
disappearance of starting material was monitored by TLC
30% CH OH/CHCl , R
2
gas was introduced at a pressure of 75 psi. The
H
H
62
N
8
) M + 1 )
(
3
3
f
) 0.30). The reaction took 78 h to
727.5176, found M + 1 ) 727.5192; Anal. (C46
68
N
8
Cl ·4H O)
6
2
complete. The suspension was filtered and the recovered
catalyst was washed with absolute EtOH. The filtrate was
C, H, N.
N ,N -Bis[4-[9-(am in om eth yl)an th r acen yl]bu tyl]-N ,N12-
4
9
1
concentrated and redissolved in CH
washed with aqueous Na CO (pH ) 10), separated, dried over
anhydrous Na SO , filtered, and concentrated. The product
was purified by flash chromatography on silica gel with 16%
NH OH/CH OH to give the desired amine 5 (1.31 g, 97%). 5:
) 0.30 in 20% NH
s, 2H), 3.15 (q, 4H), 2.40 (br m, 12H), 2.05 (br s, 4H), 1.60 (br
2 2 2 2
Cl . The CH Cl phase was
bis(t-bu toxycar bon yl)sper m in e (10). 9-Anthraldehyde (0.43
g, 2 mmol) was dissolved in 25% CH OH/CH Cl (3 mL). The
3 2 2
diamine 4 (0.56 g, 1 mmol) was added to the solution dropwise
at room temperature. The mixture was stirred for 24 h. The
2
3
2
4
4
3
disappearance of 4 was monitored by TLC (20% NH
OH, R ) 0.32). After the imination was complete, the crude
product was concentrated and redissolved in a premixed
solvent (50% CH OH/CH Cl ) followed by the addition of
NaBH (0.19 g, 5 mmol). After being stirred for 12 h, the
mixture was concentrated, redissolved in CH Cl , and washed
with aqueous Na CO (pH ) 10). The organic layer was
separated, dried over anhydrous Na SO , filtered, and con-
centrated. The crude residue was subjected to flash chroma-
tography and eluted with 1% NH OH/CH OH to furnish the
desired anthracene derivative 10 (0.74 g, 78%). 10: R ) 0.30
) δ 8.40 (s, 2H), 8.00
3
OH/CH -
4
1
R
f
4 3 3
OH/CH OH; H NMR (CDCl ) δ 5.65 (br
f
m, 20H), 1.40 (s, 18H); HRMS (FAB) theory for (C30
64 6 4
H N O )
3
2
2
M + 1 ) 573.5067, found M + 1 ) 573.5068.
4
N ,N -Bis[4-(9-a m in oa cr id in yl)b u t yl]-N ,N12-b is(t-b u -
toxyca r bon yl)sp er m in e (6). 9-Chloroacridine (0.40 g, 2
mmol) and phenol (1.72 g, 18 mmol) were heated to 50 °C for
4
9
1
2
2
2
3
2
4
2
0 min. Amine 4 (0.50 g, 1 mmol) dissolved in hot phenol was
added to the stirred mixture. The reaction mixture was heated
to 100 °C. The disappearance of starting amine was monitored
4
3
f
1
by TLC (20% NH
was complete, the mixture was concentrated and dissolved in
CH Cl and washed with aqueous Na CO (pH ) 10). The
organic layer was separated, dried over anhydrous Na SO
filtered, concentrated, purified by flash chromatography, and
eluted with 0.5% NH OH/CH OH to give the desired acridine
derivative 6 (0.64 g, 79%). 6: R ) 0.40 in 0.5% NH OH/CH
) δ 8.09 (m, 8H), 7.62 (m, 4H), 7.32 (m,
H), 5.65 (br s, 2H), 5.35 (br s, 1H), 5.25 (br s, 1H), 3.78 (q,
H), 3.10 (q, 4H), 2.30 (m, 12H), 1.92 (s, 4H), 1.75 (m, 4H),
.50 (m, 4H), 1.40 (s, 18H); HRMS (FAB) theory for (C54
4
OH/CH
3
OH, R
f
) 0.32). After the reaction
4 3 3
in 1% NH OH/CH OH; H NMR (CDCl
(d, 4H), 7.50 (m, 8H), 5.30 (br s, 2H), 4.75 (s, 4H), 3.05 (q, 4H),
2.85 (t, 4H), 2.30 (br m, 12H), 1.60 (m, 16H), 1.40 (s, 18H);
2
2
2
3
2
4
,
HRMS theory for (C58
H
80
N
6
O
4
) M + 1 ) 925.6319, found M +
) C, H, N.
N ,N -Bis[5-[9-(a m in om e t h yl)a n t h r a ce n yl]p e n t yl]-
1 ) 925.6322; Anal. (C58
80 6 4
H N O
4
9
4
3
-
1
12
f
4
3
N ,N -bis(t-bu toxyca r bon yl)sp er m in e (11). 9-Anthralde-
hyde (0.36 g, 1.75 mmol) was dissolved in 25% CH OH/CH Cl
2.5 mL). The diamine 5 (0.50 g, 0.8 mmol) was added to the
1
OH; H NMR (CDCl
3
3
2
2
4
4
1
(
solution dropwise at room temperature. The mixture was
stirred for 24 h. The disappearance of 5 was monitored by TLC
H
74
N
8
O
4
)
M + 1 ) 899.5911, found M + 1 ) 899.5938.
(
20% NH
complete, the crude was concentrated and redissolved in a
premixed solvent (50% CH OH/CH Cl ) followed by the addi-
tion of NaBH (0.20 g, 5.26 mmol). After being stirred for 12
h, the mixture was concentrated, redissolved in CH Cl , and
washed with aqueous Na CO (pH ) 10). The organic layer
was separated, dried over anhydrous Na SO , filtered, and
concentrated. The crude residue was subjected to flash chro-
matography and was eluted with 2% NH OH/CH OH to give
the desired anthracene derivative 11 (0.55 g, 75%). 11: R
4 3 f
OH/CH OH, R ) 0.25). After the imination was
N ,N -Bis[5-(9-a m in oa cr id in yl)p en tyl]-N ,N12-bis(t-bu -
toxyca r bon yl)sp er m in e (7). 9-Chloroacridine (0.70 g, 3.3
mmol) and phenol (2.58 g, 27 mmol) were heated to 50 °C for
4
9
1
3
2
2
4
2
0 min. Amine 5 (0.92 g, 1.6 mmol) dissolved in hot phenol
2
2
was added to the stirred mixture. The reaction mixture was
heated to 100 °C. The disappearance of starting material was
2
3
2
4
monitored by TLC (20% NH
reaction was complete, the mixture was concentrated, dis-
solved in CH Cl , and washed with aqueous Na CO (pH )
0). The organic layer was separated, dried over anhydrous
Na SO , filtered, concentrated, purified by flash chromatog-
raphy, and eluted with 1% NH OH/CH OH to give the desired
acridine derivative 7 (1.08 g, 73%). 7: R ) 0.35 in 1% NH
) δ 8.09 (t, 8H), 7.62 (t, 4H), 7.32
t, 4H), 5.50 (br s, 2H), 3.80 (t, 4H), 3.15 (q, 4H), 2.30 (br m,
4 3 f
OH/CH OH, R ) 0.25). After the
4
3
2
2
2
3
f
)
1
1
4 3 3
0.30 in 2% NH OH/CH OH; H NMR (CDCl ) δ 8.35 (s, 2H),
2
4
8.30 (d, 4H), 8.00 (d, 4H), 7.50 (m, 8H), 5.60 (br s, 2H), 4.70
(s, 4H), 3.15 (q, 4H), 2.85 (t, 4H), 2.30 (br m, 12H), 1.60 (br m,
4
3
f
4
-
20H), 1.40 (s, 18H); HRMS theory for (C60
H
84
N
H
6
O
4
) M + 1 )
1
OH/CH
3
OH; H NMR (CDCl
3
953.6632, found M + 1 ) 953.6581; Anal. (C60
84
N
6
O
4
‚2H O)
2
(
C, H, N.
1
2H), 1.75 (br m, 4H), 1.50 (m, 16H), 1.40 (s, 18H); HRMS
) M + 1 ) 927.6224, found M +
4
9
N ,N -B is [4-[9-(a m in o m e t h y l)a n t h r a c e n y l]b u t y l]-
sp er m in e Hexa h yd r och lor id e (12). The anthracene deriva-
tive 10 (0.74 g, 0.8 mmol) was dissolved in dioxane (10 mL) at
0 °C, followed by addition of 4 N HCl (10 mL). The mixture
was stirred for 4 h at room temperature. The disappearance
(FAB) theory for (C56
H
78
N
8
O
4
1
) 927.6214.
4
9
N ,N -Bis[4-(9-am in oacr idin yl)bu tyl]sper m in e Hexah y-
d r och lor id e (8). The acridine derivative 6 (0.64 g, 0.7 mmol)
was dissolved in 1,4-dioxane (10 mL) at 0 °C, followed by the
addition of 4 N HCl (10 mL). The mixture was stirred for 4 h
at room temperature. The disappearance of the starting
4 3 f
of 10 was monitored by TLC (1% NH OH/CH OH, R ) 0.30).
The solution was concentrated and the resulting solid was
recrystallized with ethanol to give 12 as a hygroscopic solid
1
material was monitored by TLC (R
f
) 0.5, 1% NH
4
OH/CH
3
-
(0.65 g, 86%). 12: H NMR (D
2
O) δ 8.36 (s, 2H), 8.15 (d, 4H),

3
690 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Wang et al.
7
4
.95 (d, 4H), 7.67 (t, 4H), 7.55 (t, 4H), 5.02 (s, 4H), 3.30 (br m,
H), 3.10 (br m, 16H), 2.10 (br m, 4H), 1.80 (br m, 12H); HRMS
addition of 4 N HCl (1.5 mL). The mixture was stirred at room
temperature. The disappearance of the starting material was
theory for (C48
H
64
N
6
) M + 1 ) 725.5271, found M + 1 )
Cl ‚2H O) C, H, N.
N ,N -Bis[5-[9-(a m in om e t h yl)a n t h r a ce n yl]p e n t yl]-
monitored by TLC (R
the mixture was concentrated under reduced pressure and the
product was recrystallized with CH OH and Et O to give the
f
) 0.24, 2% NH
4 3
OH/CH OH). After 4 h,
7
25.5274; Anal. (C48
H
70
N
6
6
2
4
9
3
2
1
hygroscopic salt 17 (0.084 g, 72%). 17: H NMR (CD
8
2
3
OD) δ
.50 (d, 2H), 7.90 (t, 2H), 7.75 (d, 2H), 7.50 (t, 2H), 4.20 (br m,
H), 3.20 (br m, 4H), 3.20 (br m, 12H), 2.00 (br m, 12H); Anal.
sp er m in e Hexa h yd r och lor id e (13). The anthracene deriva-
tive 11 (0.55 g, 0.60 mmol) was dissolved in dioxane (7 mL) at
0
°C, followed by addition of 4 N HCl (7 mL). The mixture
was stirred for 4 h at room temperature. The disappearance
of 11 was monitored by TLC (2% NH OH/CH OH, R
(C
27
H
47
N
6
Cl
5
2
‚3.5H O) C, H, N.
N -[4-(7-Ch lor oq u in olin yl)b u t yl]-N ,N12-b is(t-b u t oxy-
4
1
4
3
f
) 0.30).
The reaction mixture was concentrated and the resulting solid
was recrystallized with ethanol to give 13 as a hygroscopic
solid (0.36 g, 80%). 13: H NMR (D
ca r bon yl)sp er m in e (18). 4,7-Dichloroquinoline (0.24 g, 1.26
mmol) and phenol (0.12 g, 1.25 mmol) were heated to 100 °C
for 20 min. Amine 15 (0.40 g, 0.84 mmol) dissolved in hot
phenol was added to the stirred mixture. The reaction mixture
was under 100 °C overnight. The reaction mixture was
1
2
O) δ 8.15 (d, 4H), 7.90 (s,
2
2
H), 7.65 (m, 8H), 7.50 (m, 4H), 4.80 (s, 4H), 3.30 (br m, 20H),
.30 (br m, 4H), 1.90 (br m, 12H), 1.50 (br m, 4H); HRMS
) M + 1 ) 753.5584, found M + 1 )
Cl ‚4H O) C, H, N.
N -(3-Cya n op r op yl)-N ,N12-bis(t-bu toxyca r bon yl)sp er -
theory for (C50
7
H
68
N
6
dissolved in CH
10). The organic layer was separated, dried over anhydrous
Na SO , filtered, concentrated, purified by flash chromatog-
2 2 2 3
Cl and washed with aqueous Na CO (pH )
53.5561; Anal. (C50
H
74
N
6
6
2
4
1
2
4
raphy, and eluted with 2% NH
4%). 18: R ) 0.20 in 2% NH
δ 8.50 (d, 1H), 7.95 (s, 1H), 7.80 (d, 1H), 7.35 (d, 1H), 6.40 (d,
4
OH/CH
3
OH to give 18 (0.29 g,
m in e (14). Amine 1 (4.23 g, 10.5 mmol) and 4-bromobuty-
ronitrile (1.55 g, 10.5 mmol) were dissolved in acetonitrile (10
mL). Potassium carbonate (1.74 g, 12.6 mmol) was then added.
The mixture was refluxed with constant stirring. TLC (15%
NH OH/CH OH, R ) 0.45) was used to monitor the consump-
4 3 f
tion of the starting amine. After 24 h the reaction mixture was
concentrated, redissolved in dichloromethane, and washed
1
5
f
4
OH/CH
3 3
OH; H NMR (CD OD)
1
1
H), 3.30 (t, 2H), 3.20 (m, 4H), 2.50 (m, 10H), 1.80 (br m, 12H),
.40 (s, 18H); Anal. (C33
H
55
O
4
N
6
2
Cl‚0.7H O) C, H, N.
4
N -[4-(7-Ch lor oq u in olin yl)b u t yl]sp er m in e P en t a h y-
d r och lor id e (19). Quinoline derivative 18 (0.27 g, 0.42 mmol)
was dissolved in dioxane (5 mL) at 0 °C, followed by the
addition of 4 N HCl (5 mL). The mixture was stirred at room
temperature. The disappearance of the starting material was
with aqueous Na
over anhydrous Na
residue was subjected to flash chromatography and eluted with
% NH OH/CH OH to give 14 (2.54 g, 73% after correction
for the recovery of unreacted starting material). 14: R ) 0.20
) δ 5.40 (br s, 2H), 3.15
m, 4H), 2.50 (br m, 12H), 1.70 (br m, 10H), 1.40 (s, 18H); Anal.
‚0.5H O) C, H, N.
N -(4-Am in ob u t yl)-N ,N12-b is(t-b u t oxyca r b on yl)sp er -
m in e (15). Nitrile 14 (2.54 g, 5.4 mmol) was dissolved in
absolute EtOH in a Parr shaker bottle. Concentrated NH OH
5 mL) was added to the solution, followed by the addition of
2
CO
3
. The organic layer was separated, dried
2
SO
4
, filtered, and concentrated. The crude
1
4
3
monitored by TLC (R
the mixture was concentrated under reduced pressure and the
product was recrystallized with CH OH and Et O to give the
hygroscopic salt 19 (0.084 g, 72%). 19: H NMR (D O) δ 8.18
f
) 0.20, 2% NH
4 3
OH/CH OH). After 4 h,
f
1
in 1% NH
4
OH/CH
3
OH; H NMR (CDCl
3
3
2
1
(
(
2
C
24
H
4
47
N
5
O
4
2
(d, 1H), 7.95 (d, 1H), 7.65 (d, 1H), 7.45 (dd, 1H), 6.70 (d, 1H),
3.55 (m, 2H), 3.10 (br m, 14H), 2.05 (br m, 4H), 1.75 (m, 8H);
1
Anal. (C23
H
44
N
6
Cl
6
2
‚4H O) C, H, N.
1
8
N ,N -Dim eth ylsp er m id in e Tr ih yd r och lor id e (20). De-
4
(
rivative 20 was synthesized by published methods in 65%
overall yield from spermidine.24 20: H NMR (D
1
O) δ 3.12-
Raney nickel catalyst (50% slurry, 3.50 g). NH
passed through the solution at 0 °C for 20 min. The reaction
mixture was placed in a Parr shaker and briefly evacuated,
3
gas was then
2
2.90 (m, 8H), 2.66 (s, 3H), 2.65 (s, 3H), 2.12-1.95 (m, 2H),
1.75-1.68 (m, 4H); 13C NMR 51.1, 49.9, 48.6, 47.3, 35.6, 35.5,
and H
disappearance of starting material was monitored by TLC (1%
NH OH/CH OH, R ) 0.20). The reaction was complete after
8 h. The suspension was filtered and the recovered catalyst
was washed with absolute EtOH. The filtrate was concentrated
and redissolved in CH Cl . The CH Cl phase was washed with
aqueous Na CO (pH ) 10), separated, dried over anhydrous
Na SO , filtered, and concentrated. The product was purified
by flash chromatography and eluted with 16% NH OH/CH
OH to give the desired amine 15 (2.4 g, 95%). 15: R ) 0.25 in
) δ 5.60 (br s, 1H), 5.20
br s, 1H), 3.20 (m, 4H), 2.70 (br m, 4H), 2.40 (br m, 8H), 1.60
br m, 12H), 1.40 (s, 18H); HRMS theory for (C24 ) M
1 ) 474.4019, found M + 1 ) 474.4001.
2
gas was introduced at a pressure of 75 psi. The
9 26 3 3
25.6, 25.5, 25.4; Anal. (C H N Cl ) C, H, N.
1
8
N ,N -Dieth ylsp er m id in e Tr ih yd r och lor id e (21). De-
4
3
f
rivative 21 was synthesized by published methods in 63%
overall yield from spermidine.24 21: H NMR (D
1
O) δ 3.19-
7
2
3.04 (m, 12H), 2.18-2.06 (m, 2H), 1.81-1.73 (m, 4H), 1.34-
1.24 (m, 6H); Anal. (C11 Cl ) C, H, N.
2
2
2
2
H
30
N
3
3
2
3
2
4
Ack n ow led gm en t. This research was supported by
an award from Research Corporation. We gratefully
acknowledge the additional financial support by the
University of Central Florida Sponsored Research In-
House Award, the Elsa U. Pardee Foundation, and the
Florida Hospital Gala Endowed Program for Oncologic
Research. The 200 MHz NMR spectrometer was pur-
chased by a grant from the National Science Foundation
4
3
-
f
1
1
4 3 3
0% NH OH/CH OH; H NMR (CDCl
(
(
51 5 4
H N O
+
4
N -[4-(9-Am in oa cr id in yl)bu tyl]-N ,N12-bis(t-bu toxyca r -
1
bon yl)sp er m in e (16). 9-Chloroacridine (0.06 g, 0.26 mmol)
and phenol (0.20 g, 2.09 mmol) were heated to 50 °C for 20
min. Amine 15 (0.15 g, 0.32 mmol) dissolved in hot phenol was
added to the stirred mixture. The reaction mixture was heated
to 100 °C. The disappearance of the starting amine was
(CHE-8608881). All mass spectral data were generously
provided by Dr. David H. Powell at the University of
Florida.
monitored by TLC (10% NH
min, the reaction mixture was concentrated and dissolved in
CH Cl and washed with aqueous Na CO (pH ) 10). The
organic layer was separated, dried over anhydrous Na SO
filtered, concentrated, purified by flash chromatography, and
eluted with 2% NH OH/CH OH to give the acridine derivative
6 (0.12 g, 70%). 16: R ) 0.24 in 2% NH
CDCl + 25% CD OD) δ 8.40 (d, 2H), 7.90 (d, 2H), 7.80 (t,
H), 7.40 (t, 2H), 4.10 (t, 2H), 3.10 (m, 4H), 2.76 (q, 4H), 2.50
4 3 f
OH/CH OH, R ) 0.20). After 20
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2
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3
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J M010181V