Synthesis and biological evaluation of N1-(anthracen-9-ylmethyl)triamines as molecular recognition elements for the polyamine transporter

Article abstract of DOI:10.1021/jm030028w

An efficient modular synthesis of N¹-substituted triamines containing different tether lengths between nitrogen centers was developed. A series of N¹-(9-anthracenylmethyl)triamines were evaluated for biological activity in L1210 (mur

Full text of DOI:10.1021/jm030028w

J . Med. Chem. 2003, 46, 2663-2671
2663
Syn th esis a n d Biologica l Eva lu a tion of N1-(An th r a cen -9-ylm eth yl)tr ia m in es a s
Molecu la r Recogn ition Elem en ts for th e P olya m in e Tr a n sp or ter
Chaojie Wang,†,‡ J ean-Guy Delcros,§ J ohn Biggerstaff,† and Otto Phanstiel, IV*,†
Groupe de Recherche en Therapeutique Anticance´reuse, Faculte´ de Me´decine, 2, Avenue du Professeur Le´on Bernard,
35043 Rennes, France, and Department of Chemistry, P.O. Box 162366, University of Central Florida,
Orlando, Florida 32816-2366
Received J anuary 16, 2003
An efficient modular synthesis of N1-substituted triamines containing different tether lengths
between nitrogen centers was developed. A series of N1-(9-anthracenylmethyl)triamines were
evaluated for biological activity in L1210 (murine leukemia), R-difluoromethylornithine (DFMO)-
treated L1210, Chinese hamster ovary (CHO), and CHO-MG cell lines. All triamines 8 had
increased potency in DFMO-treated L1210 cells. The 4,4- and 5,4-triamine systems had the
highest affinity for the polyamine transporter (PAT) with L1210 Ki values of 1.8 and 1.7 µM,
respectively. This trend was also reflected in the CHO studies. Surprisingly, the respective
4,4- and 5,4-triamine systems had 150-fold and 38-fold higher cytotoxicity in CHO cells
containing active polyamine transporters. Initial microscopy studies revealed the rapid
formation of vesicular structures within A375 melanoma cells treated with the N1-(9-
anthracenylmethyl)homospermidine (4,4-triamine) conjugate. In summary, the 4,4- and 5,4-
triamines were identified as selective vector motifs to ferry anthracene into cells via the PAT.
In tr od u ction
partially offset by scavenging polyamines from exog-
enous sources.7 In fact, many tumor types have been
shown to contain elevated polyamine levels and an
active PAT for importing exogenous polyamines. These
range from neuroblastoma, melanoma, human lympho-
cytic leukemia, colonic, and lung tumor cell lines to
murine L1210 cells.6 In short, targeting of cancerous cell
types would stem from their enhanced need for these
biological growth factors.
One of the major shortcomings of current cancer
therapies is the nonselective delivery of the antineoplas-
tic drug to both targeted tumor cells and healthy cells.
Enhanced selectivity of such drugs could diminish their
associated toxicity by reducing their uptake by healthy
cells. Moreover, selective delivery would increase drug
potency by lowering the effective dosage required to kill
the affected cell type. Vector systems, which have
enhanced affinity for cancer cells, would be an important
advance in cancer chemotherapy. This report illustrates
the development of conjugates between toxic agents and
polyamines as one such vector system (see Figure 1).
The long-range goal of this research is to harness the
polyamine transporter (PAT, a cell surface protein) for
selective drug delivery.1,2 The selection process would
occur during the molecular recognition events involved
in the import of exogenous polyamines by cancer
cells.3-5 In vivo, the polyamines exist as polycations
(because the nitrogens are protonated at physiological
pH) and are required for cell growth. Their alignment
of point charges is recognized by the PAT and has been
shown to facilitate their import.5 Rapidly dividing cells
require large amounts of polyamines in order to grow.
These can be internally biosynthesized and also im-
ported from exogenous sources. The high specific activity
of polyamine transport in tumor cells is thought to be
associated with the “inability of biosynthetic enzymes
to provide sufficient levels of polyamines to sustain the
rapid cell division.”6 These bioproduction constraints are
To date, the transport of polyamines into mammalian
cells is a measured1,2,5 yet poorly understood phenom-
ena. While significant work has also been accomplished
in E. coli,3,4,8,9 yeast,10 and other systems,11 the proteins
involved in mammalian polyamine transport have not
yet been isolated and characterized beyond a kinetic
description. Clearly, the lack of structural detail associ-
ated with the mammalian transporter is a glaring void
in the current knowledge base.
How can one design a polyamine vector to utilize a
transporter of unknown structure? Several authors have
developed structure-activity relationships to indirectly
address this issue.1,2,5,12 Thus far, small structural
changes have resulted in dramatic differences in the
transport behavior of polyamine analogues. For ex-
ample, a single methylene (CH2) unit change in the
tether separating the nitrogen centers has been shown
to significantly alter analogue uptake.5 This report
focuses on the unsymmetrical linear polyamines, which
are N-substituted at only one end of the polyamine
chain. While the terminally bis-alkylated polyamines
have yielded diverse biological activity ranging from
anticancer to antidiarrheal agents,5,11 their monosub-
stituted analogues have limited publications describing
their use in vector design.1,2,5,13-21 This, in part, may
stem from their less direct syntheses, which involve
several steps.5,19 In fact, only a few efficient syntheses
* To whom correspondence should be addressed. E-mail: ophansti@
mail.ucf.edu. Phone: (407) 823-5410. Fax: (407) 823-2252.
† University of Central Florida.
‡ C.W. is a visiting scholar from the Department of Chemistry,
Henan University, Kaifeng, 475001, P. R. China.
§ Groupe de Recherche en Therapeutique Anticance´reuse, Faculte´
de Me´decine.
10.1021/jm030028w CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/20/2003

2664 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
F igu r e 1. Sample conjugate system and the native polyamines.
Sch em e 1
of unsymmetrically substituted polyamines were pub-
lished prior to 1993.22
There are several caveats to using polyamines to
selectively deliver toxic “cargoes”. First, rapidly dividing
normal cell types (e.g., bone marrow, intestinal epithe-
lium, and hair follicles) may also be affected by this
strategy. Whether polyamine conjugates can discrimi-
nate between cancer cell types and these other prolific
cell lines remains to be seen. Nevertheless, other
authors have observed that transformed cells are more
sensitive than normal cells to R-difluoromethylornithine
(DFMO) pretreatment. DFMO is a known ornithine
decarboxylase (ODC) inhibitor. ODC is responsible for
putrescine biosynthesis, and its inhibition has been
shown to increase the uptake of extracellular poly-
amines.23 Most pertinent to this study is the observation
that when DFMO is dosed in vivo, it increased the
uptake of radiolabeled putrescine specifically into tumor
cells and not into other normal tissue, even rapidly
growing tissue.23 This suggests that DFMO treatment
may also increase the potency of the present polyamine
conjugates. Second, the “cargoes” to be delivered in this
study (e.g., anthracene derivatives) are relatively inert.
It is possible that other anticancer agents or “cargoes”,
which have more reactive chemical entities, may inter-
act with PAT or other cell surface receptors and dimin-
ish the anticipated selectivity. In this report, our goal
was not to develop new anticancer agents per se but to
use a stable model architecture (anthracene) in order
to understand which polyamine sequence was best able
to access the PAT.
The conjugates in this report are dualistic in design
and comprise an anthracene nucleus covalently bound
to a polyamine framework. In this manner, cells will
be challenged to import the same lethal cargo (an-
thracene) using a modified polyamine motif (for cellular
entry via the PAT).1,2 The anthracene component was
selected because of significant preliminary data, which
revealed its increased potency over an acridine analogue
in murine leukemia (L1210) cells.1,2 Moreover, the
anthracene provides a convenient UV “probe” for com-
pound identification and elicits a toxic response from
cells upon entry (presumably through DNA coordina-
tion).24 Early studies had revealed that branched
polyamines have moderate affinity for the PAT and can
provide access to cells.1,2 However, recent data sug-
gested that the linear polyamines were superior vectors
over their branched counterparts.12 In this manner, a
series of polyamine-anthracene conjugates were syn-
thesized and screened for uptake via the PAT.
can now comment on transport affinity and potency as
a function of polyamine-drug conjugate structure.
Syn th esis
As shown in Scheme 1, the reductive amination of 1
to 4 was achieved in two steps via in situ generation of
the imine 3. A homologous series of imines (3a -d ) were
prepared from 1 and different amino alcohols. Each
imine was then reduced to its respective amine 4 with
NaBH4 in good yield without purification. Solvent
removal by rotary evaporation at 40-50 °C facilitated
imine formation and provided satisfactory yields of the
2° amines, 4a -d , (68-81%) after the two-step process.
The 2° amines 4a -d were N-protected to form 5 using
excess di-tert-butyl dicarbonate, Boc2O. Interestingly,
compound 5b, which contained three methylene units,
was unstable, even when it was stored at low temper-
ature (0-5 °C) under nitrogen. This finding is in direct
contrast to 5a , 5c, and 5d , which were stable at room
temperature.
In the seemingly routine tosylation step, shown in
Scheme 2, we were unable to obtain the desired com-
pound 6a from the N-Boc protected 5a . Tosylate 6b was
isolated in lower yield (51%) but was unstable to
prolonged storage. In contrast, tosylates 6c and 6d were
prepared in higher yields (88%) and were relatively
stable. However, their respective colors and 1H NMR
spectra slowly changed during prolonged storage in the
refrigerator. Therefore, the tosylates 6 were best gener-
ated and used as soon as possible.
1
Interesting H NMR behavior was observed for 5a -
c. The N-Boc protection of 4 led to the expected
downfield shift of the “benzylic CH2” from δ ∼4.7 in 4
to δ ∼5.5 in 5 because of the inductive effects of the Boc
group. However, the non-benzylic N1-CH2 in 5a -c and
the other CH2 units were all shifted upfield (especially
the CH2-O group, e.g., δ 3.80 in 4b to δ 3.25 in 5b). A
similar chemical shift change had been observed with
a series of N-tosyl-N-anthracenylmethylpolyamine de-
By judicious choice of amine substrates (e.g., a
synthetic library) and proper biological experiments
(e.g., IC50 and Ki measurements) for selected cell types
(murine leukemia L1210, CHO, and CHO-MG cells), one

N1-(Anthracen-9-ylmethyl)triamines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2665
Sch em e 2
Sch em e 3
rivatives.25 These derivatives revealed a novel orienta-
tion wherein the polyamine “tail” was oriented over the
shielding cone of the anthracene ring presumably
because of the steric effects of the N-tosyl group. The
N-Boc derivatives 5a -c also seem to adopt this unusual
orientation, which results in the unexpected upfield
chemical shifts. However, this structural feature disap-
peared upon O-tosylation of 5 to form 6.
The tosylated compounds (6b-d ) were reacted with
excess putrescine or 1,3-diaminopropane to form the six
N1-Boc protected triamines 7b-g. These triamines were
isolated but were again unstable to prolonged storage
at low temperature. Therefore, they were consumed in
the next step immediately after purification. The N-Boc
groups of 7 were removed by 4 N HCl, and the six target
compounds 8b-g were formed in good yield. Impurities
in 8 were removed simply by washing the solids with
absolute ethanol.
As shown in Scheme 3, derivatives 4a -d were previ-
ously converted to their respective N,O-bistosylates,
9a -d .25 Displacement of the terminal tosylate by bu-
tanediamine provided the series 10a -d in good yield.25
Derivatives 10a -d represent triamine systems contain-
ing two large aromatic “cargoes” and have one of the
terminal amines sequestered as a sulfonamide. In
addition, the N-(anthracen-9-ylmethyl)-N-butylamine
HCl salt (11) shown in Scheme 3 was synthesized in
58% yield by reductive amination of 9-anthraldehyde
and butylamine followed by treatment with 4 N aqueous
HCl. Compound 11 was an important control because
it represented a charged anthracene moiety without an
attached polyamine vector. How these large structural
perturbations influence the cytotoxicity of the biocon-
jugate were evaluated via IC50 determinations.
the purposes of this study, the CHO-MG cell line
represents cells with very low PAT activity and provides
a model for alternative modes of entry or action. These
alternative modes of entry, which are non-PAT-based,
include passive diffusion or utilization of another trans-
porter. The alternative modes of action may include
interactions on the outer surface of the plasma mem-
brane or other membrane-receptor interactions. In
contrast, the parent CHO cell line represents a cell type
with high PAT activity.27,28 Comparison of efficacy in
these two lines provided an important screen to detect
selective conjugate delivery via the PAT. For example,
a conjugate with high utilization of the polyamine
transporter would be very toxic to CHO cells but less
so to CHO-MG cells.12,27 Therefore, IC50 determination
Biologica l Eva lu a tion
Three cell lines were chosen for bioassay. L1210
(mouse leukemia) cells were selected to enable compari-
sons with the IC50, and Ki values were determined
previously for a variety of polyamine substrates.5,26
Chinese hamster ovary (CHO) cells were chosen along
with a mutant cell line (CHO-MG) in order to comment
on how the synthetic conjugates gain access to cells.12
The CHO-MG cell line is polyamine-transport-deficient
and was isolated after chronic selection for growth
resistance to methylglyoxalbis(guanylhydrazone).27 For

2666 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
F igu r e 2. Polyamine biosynthetic pathway.
Ta ble 1. Biological Evaluation of Triamines 8b-g, N-Tosyl Derivatives 10a -d , and 11 in L1210 Cellsa
L1210
IC50 (µM)
L1210 + DFMO
IC50 (µM)
L1210/(L1210 + DFMO)
L1210
Ki (µM)
compd (tether)
IC50 ratio
8b (3,3)
8c (3,4)
8d (4,3)
8e (4,4)
8f (5,3)
8g (5,4)
10a (2,4)
10b (3,4)
10c (4,4)
10d (5,4)
11 (N-butyl)
1.8 ( 0.4
0.7 ( 0.3
0.4 ( 0.1
0.3 ( 0.04
1.3 ( 0.1
0.4 ( 0.1
3.3 ( 0.2
6.3 ( 0.5
7.4 ( 1.0
6.2 ( 0.3
14.6 ( 0.1
1.4 ( 0.3
0.3 ( 0.1
0.2 ( 0.02
0.15 ( 0.1
0.7 ( 0.1
0.3 ( 0.1
3.9 ( 0.9
7.7 ( 1.1
8.1 ( 1.6
6.9 ( 0.8
21.9 ( 3.6
1.3
2.3
2
33.4 ( 2.6
2.5 ( 0.3
6.2 ( 0.6
1.8 ( 0.1
5.0 ( 0.6
1.7 ( 0.2
ND
ND
ND
ND
62.3 ( 4.2
2
1.9
1.3
0.9
0.8
0.9
0.9
0.7
a
Individual L1210 IC50 values are listed in µM, and the IC50 ratio is dimensionless. ND ) not determined.
Ta ble 2. Biological Evaluation of Triamines 8b-g, N-Tosyl
in these two CHO lines provided a relative ranking of
delivery via the PAT. In short, highly selective, vectored
conjugates should give high CHO-MG/CHO IC50 ratios.
As mentioned previously, DFMO is a known ODC
inhibitor.29 ODC is the enzyme responsible for the
biosynthesis of putrescine from L-ornithine (see Figure
2). Putrescine (PUT) is then converted to the longer
polyamines, spermidine (SPD) and spermine (SPM), via
successive aminopropylation steps (Figure 2). Inhibition
of ODC blocks intracellular putrescine biosynthesis and
leads to a significant increase in polyamine uptake,
which allows the cell to acquire polyamines from exog-
enous sources. Therefore, cells that are treated with
DFMO should be more susceptible to the polyamine-
vectored conjugates and should provide lower IC50
values.11,12 IC50 values for L1210 cells with and without
DFMO treatment were determined. Conjugates, which
selectively target the PAT, should be more potent with
DFMO-treated cells and should provide L1210/(L1210
+ DFMO) IC50 ratios greater than 1.
All the derivatives listed in Table 1 (e.g., 8b-g, 10a -
d , and 11) were cytotoxic to L1210 cells and had IC50
values less than 15 µM. The DFMO-treated L1210 cells
were more susceptible to conjugates 8b-g than the
sulfonamides 10 or control 11. This finding suggests
that conjugates such as 8 use the PAT to enter cells.
DFMO treatment typically resulted in higher potency
for 8 and other previous polyamine conjugates.12 It
should be noted that in the present study, the DFMO
was coadministered with the polyamine conjugate and
therefore DFMO’s potency enhancement may not have
been maximal. Nevertheless, our findings are consistent
with those of other authors.12
Derivatives 10a -d , and 11 in the CHO-MG and CHO Cell
Lines
CHO-MG
IC50 (µM)
CHO
IC50 (µM)
CHO-MG/CHO
IC50 ratio
compd (tether)
8b (3,3)
8c (3,4)
8d (4,3)
8e (4,4)
8f (5,3)
8g (5,4)
10a (2,4)
10b (3,4)
10c (4,4)
10d (5,4)
11 (N-butyl)
3.4 ( 0.5
8.8 ( 1.2
9.5 ( 1.1
66.7 ( 4.1
10.1 ( 1.2
57.3 ( 2.9
7.1 ( 0.4
11.1 ( 1.3
10.6 ( 1.9
7.8 ( 1.9
11.2 ( 2.3
1.9 ( 0.4
2.5 ( 0.7
0.4 ( 0.1
0.45 ( 0.1
4.1 ( 0.5
1.5 ( 0.1
5.1 ( 0.6
10.2 ( 0.9
10.4 ( 1.6
7.1 ( 0.8
10.5 ( 2.0
1.8
3.5
24
148
2.5
38
1.4
1.1
1.0
1.1
1.1
62.3 µM) revealed the significantly higher PAT affinity
(lower Ki values) of conjugates containing a preferred
polyamine sequence. Although control 11 was able to
kill cells, it did so with lower potency than the vectored
systems 8e and 8g. This finding is significant because
it suggests that the 4,4- or 5,4-triamine motif selectively
vectored the anthracene cargo into cells via the PAT.
This selectivity became very apparent in the latter CHO
studies.
As shown in Table 2, biological evaluation of tri-
amines 8b-g, 10a -d , and 11 in the two CHO cell lines
revealed striking preferences by certain polyamine
architectures for the PAT. Perhaps most exciting was
the fact that the 4,4-triamine 8e displayed a nearly 150-
fold preference for the CHO over the CHO-MG cell line.
In contrast, the 3,3-triamine analogue 8b preferred this
line by only 1.8-fold. These findings demonstrate that
CHO and CHO-MG IC50 comparisons are an excellent
way to rank polyamine vectors and their transport.12
The effectiveness of the triamines 8e and 8g correlated
nicely with the L1210 IC50 and Ki results, wherein each
had enhanced potency with DFMO-treated cells and
high affinity for the polyamine transporter. Indeed, the
triamine systems (8e and 8g) seem to be well-behaved
systems, which give consistent data in all three cell
lines.
The Ki values in Table 1 reflect the affinity of the
polyamine derivative for the polyamine transporter on
the cell surface. Conjugates with an appended 4,4-
triamine (8e) or 5,4-triamine (8g) sequence had the
lowest Ki values (1.8 and 1.7 µM, respectively) and,
therefore, the highest affinity for the L1210 polyamine
transporter. A comparison of 8e and 8g with the
N-(anthracen-9-ylmethyl)butylamine control 11 (Ki )

N1-(Anthracen-9-ylmethyl)triamines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2667
Ta ble 3. L1210 IC50 and Ki Data5
importing large “cargoes” as long as they are tethered
at only one end.6,12 The 4,4-triamine 8e represents a
potential “home run” for these vectored systems in terms
of its potential 150-fold selectivity and observed low IC50
values in the CHO and L1210 cell lines (Tables 1 and
2). Comparisons between the CHO and CHO-MG cell
lines (i.e., respective IC50 values) provided a sensitive
screen for identifying architectures, which are more
specific for the PAT. Recall that in the proposed model,
the CHO cells represent cell types with active polyamine
transporters and the CHO-MG cell line represent cells
with low PAT activity. The observed 150-fold selectivity
in distinguishing between these two cell lines represents
a major advance in polyamine vector design. In short,
certain polyamine vectors can selectively deliver “large”
toxic agents to cells with highly active polyamine
transporters.
entry no.
R1
R2
m
n
IC50 (µM, 96 h) Ki (µM)
1
2
3
4
5
6
7
propyl (Pr) Pr
1
1
1
1
1
2
2
1
1
2
2
2
2
2
60
100
33
28
55
6
125
33
26
3
8.5
67
5
Pr
Pr
Pr
H
H
Pr
H
Pr
Pr
H
Pr
Pr
0.6
The N-tosyl derivatives 10a -d and the control 11
demonstrated similar potency in both the CHO-MG and
CHO cell lines (with IC50 values ranging from 5.1 to 11.2
µM). These findings suggested that these particular
analogues are entering the cell via a non-PAT-mediated
pathway to elicit their toxic response. Nevertheless, the
vectored polyamine conjugates (8e and 8g) were sig-
Another outcome of this study was use of 8b-g as
new polyamine probes that have differential affinity for
the PAT and are fluorescent. In this regard, the
conjugates (8) could be tracked by deconvolution mi-
croscopy.
nificantly less potent in the CHO-MG cell line (IC50
)
66.7 and 57.3 µM, respectively) versus the control 11
(IC50 ) 11.2 µM). Therefore, certain polyamine archi-
tectures (like those found in 8e and 8g) can alter the
uptake characteristics of the appended anthracene.
Indeed, the proper polyamine sequence imparted high
selectivity for the PAT.
Decon volu tion Micr oscop y
Recently other authors have suggested two mecha-
nisms for polyamine transport into mammalian cells
(e.g., via receptor-mediated endocytosis or via a protein
channel).6,35 Of the two, the receptor-mediated endocy-
tosis pathway had the greatest experimental support.6,35
For example, Cullis reasoned that a rigid protein
channel with a fixed diameter would be sensitive to
steric effects at either end of the polyamine chain. An
envagination process, however, would be more tolerant
of bulky “cargoes” appended to the polyamine vector.
Indeed, Cullis concluded that the polyamine transporter
has a “surprising broad structural tolerance”.6
One of the limitations of the above biochemical assays
is that they are indirect measurements and do not
provide cellular localization information. Where do the
conjugates go once they enter the cell? Conjugate
localization information could help explain both the
mechanism of action of these systems and their trans-
port behavior. Deconvolution microscopy offered a unique
ability to address this question. By the synthesis of
conjugates with dramatically different transport proper-
ties (e.g., 8b vs 8e), this project generated important
probes for studying transport phenomena. The an-
thracene moiety offers a convenient UV “handle” to
monitor the location of the drug conjugate within the
cell. The UV spectra of these anthracene conjugates
suggest a consistent λmax near 364 nm and an emission
maxima near 417 nm. As shown in Figure 3, when A375
melanoma cells are incubated for 3 min with 10 µM 8e,
deconvolution microscopy revealed the rapid formation
of fluorescent vesicles (containing 8e) within the cell.
Control experiments with water or DMSO were “dark”
and gave no fluorescence with this filter set. These
results are exciting because they suggest that the
homologous series 8 could be used as fluorescent probes
to study polyamine uptake.
Another way of illustrating this selectivity enhance-
ment is to consider the differences in hydrophobicity
between the control 11 and the triamine derivatives
8b-g. Hydrophobic compounds (like 11) should be
expected to permeate across the cell membrane without
the use of a specific transport system. In contrast, the
appended polyamine motif in 8 should significantly
reduce the hydrophobicity of these conjugates. If one
assumes that each triamine motif (existing as a polyam-
monium tail) imparts similar water solubility to the
appended anthracene, then one would anticipate that
8b-g would all give the same biological data in Table
2. This is clearly not the case (e.g., 8b vs 8e). Therefore,
the data cannot be explained by a simple change in
hydrophobicity. Instead, the dramatic differences ob-
served in the CHO cell lines are more consistent with
the attachment of a selective molecular recognition
element for a particular cell surface receptor (i.e., the
PAT).
Other authors have shown differences in transport
affinity (Ki) and cytotoxicity (IC50) behavior for the N,N-
dialkylpolyamine and N-monoalkylpolyamine architec-
tures.5 In an elegant study by Bergeron et al., the
monopropylpolyamine derivatives gave lower Ki values
than their dialkylated counterparts (Table 3).5 However,
in some cases, this property did not always correlate
with higher potency (e.g., IC50 values for entries 1 and
2 in Table 3). Subtleties in tether length were also
apparent. For example, as shown in Table 3, the 3,4
triamine system (entry 4) had a lower Ki than its 4,3
counterpart (entry 5). A similar trend was observed in
Table 1 with 8c and 8d . Therefore, the polyamine
transporter is capable of distinguishing between iso-
meric systems that have the same overall length but a
different separation of point charges.5,30-34 Indeed, the
current study has identified some of the optimal motifs
in order to initiate transport via this pathway.
Our findings are consistent with those reported by the
Cullis and Poulin groups, which also observed the
formation of vesicles and suggested receptor-mediated
endocytosis as a mode of transport for both a N-
In terms of vector design, the monoalkylated polyamine
motif is more attractive because cells seem capable of

2668 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
F igu r e 3. Deconvolution microscopy images of an A375 cell treated with 8e (10 µM). This technique provides an image of the
entire cell by using software to overlay a series of planar images (or slices) to give a stacked translucent view of the entire cell
(leftmost image above). The lightest regions contain the highest concentration of the fluorescent agent 8e. The three representative
slices (second from left, second from right, and rightmost images) were chosen from a total of 32 images. A scale bar is shown in
micrometers.
1H NMR and 13C NMR spectra were recorded at 300 and 75
MHz, respectively. TLC solvent systems are based on volume
percent, and NH4OH refers to concentrated aqueous NH4OH.
Elemental analyses were performed by Atlantic Microlabs
(Norcross, GA). High-resolution mass spectrometry was per-
formed by Dr. David Powell at the University of Florida Mass
Spectrometry facility. The syntheses of N-tosyl derivatives 9
and 10 have been previously described.25
methylanthranilic acid (MANT)-polyamine conjugate6
and a spermidine-C2-BODIPY conjugate.35 Therefore,
the sequestration of 8e into vesicles (along with similar
findings by other investigators) supports an endocytotic
process.6,35 Whether other importation mechanisms are
in play remains to be seen.
Future studies will relate PAT affinity and transport
properties to a particular cellular compartmentalization/
uptake process. An advantage of the present systems
such as 8 is that the UV probe is also the toxic entity
and not a UV prosthetic like MANT. Moreover, the N4-
spermidine-MANT conjugate used by Cullis had a Ki
value of 23.4 µM, which is over a log unit higher than
the chlorambucil-polyamine conjugates they were mod-
eling.6,20 In the present case, the conjugate 8 itself is
both the UV probe and the active drug. We are now
poised to compare several members of the series in a
time-lapse microscopy study, which will be the focus of
a future report.
Biologica l Stu d ies. Murine leukemia cells (L1210), CHO,
and CHO-MG cells were grown in RPMI medium (Eurobio,
Les Ulis, France) supplemented with 10% fetal calf serum, 2
mM glutamine (2 mM), penicillin (100U/mL), and streptomycin
(50 µg/mL) (BioMerieux, MArcyl’Etoile, France). L-Proline (2
µg/mL) was added to the culture medium for CHO-MG cells.
Cells were grown at 37 °C under a humidified 5% CO2
atmosphere. Aminoguanidine (2 mM) was added to the culture
medium to prevent oxidation of the drugs by the enzyme
(bovine serum amine oxidase) present in calf serum. Trypan
blue staining was used to determine cell viability before the
initiation of a cytotoxicity experiment. Typically, samples
contained less than 5% trypan blue positive cells (dead). For
example, L1210 cells in early to mid log phase were used.
IC50 Deter m in a tion s. Cell growth was assayed in sterile
96-well microtiter plates (Becton-Dickinson, Oxnard, CA).
L1210 cells were seeded at 5 × 104 cells/mL of medium (100
µL/well). Single CHO and CHO-MG cells harvested by
trypsinization were plated at 2 × 103 cells/mL. Drug solutions
(5 µL per well) of appropriate concentration were added at the
time of seeding for L1210 cells and after an overnight incuba-
tion for CHO and CHO-MG cells. In some experiments, DFMO
(5 mM) was added in the culture medium at the time of drug
addition. After exposure to the drug for 48 h, cell growth was
determined by measuring formazan formation from 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium using a Titertek
Multiskan MCC/340 microplate reader (Labsystems, Cergy-
Pontoise, France) for absorbance (540 nm) measurements.37
Ki P r oced u r e. The ability of the conjugates to interact with
the polyamine transport system was determined by measuring
competition by the conjugates against radiolabeled spermidine
uptake in L1210 cells. Initially, the Km value of spermidine
transport was determined in a reaction volume of 600 µL of
Hanks’ balanced salt solution (HBSS) containing 3 × 106 cells/
mL in the presence of 0.5, 1, 2, 4, 6, and 8 µM [14C]-spermidine.
The cell suspensions were incubated at 37 °C for 10 min. The
reaction was stopped by adding 3 mL of ice-cold phosphate-
buffered saline (PBS). The tubes were centrifuged, and the
supernatant was removed. The cell pellets were then washed
twice with ice-cold PBS, and the supernatant was removed.
The pellet was broken by sonication in 500 µL of 0.1% Triton
in distilled water. Two hundred microliters of the cell lysate
was transferred in a 5 mL scintillation vial containing 3 mL
of Pico-Fluor 15. The respective radioactivity of each sample
was measured using a scintillation counter. The Km value of
spermidine transport was determined by Lineweaver-Burke
analysis as described.38
Con clu sion s
An efficient modular synthesis of N1-substituted tri-
amines containing different tether lengths between
nitrogen centers was developed. This new methodology
should contribute to the limited synthetic approaches
used to access linear polyamines.36 Triamine conjugates
such as 8 had increased potency in DFMO-treated
L1210 cells, whereas the related N1-tosyl derivatives 10
and control 11 did not. All polyamine vectors do not
impart the same selectivity. Indeed, only a few of the
screened triamines demonstrated the desired vectoring
behavior. The 4,4- and 5,4-triamine systems had the
highest affinity for the PAT with L1210 Ki values of 1.8
and 1.7 µM, respectively. A survey of different polyamine
vectors revealed that the respective 4,4- and 5,4-
triamine systems had 150-fold and 38-fold higher cyto-
toxicity in CHO cells containing active polyamine
transporters. Initial microscopy studies revealed the
rapid formation of vesicular structures within A375
melanoma cells treated with the N1-(9-anthracenyl-
methyl)homospermidine conjugate 8e. In summary, the
4,4- and 5,4-triamines were identified as selective vector
motifs for the PAT. Future work will also probe the
influence of these conjugates on intracellular polyamine
pools and enzymes.
Exp er im en ta l Section
Ma t er ia ls. Silica gel (32-63 µm) and chemical reagents
were purchased from commercial sources and used without
further purification. All solvents were distilled prior to use.
The ability of conjugates to compete for [14C]-spermidine
uptake was determined in L1210 cells by a 10 min uptake

N1-(Anthracen-9-ylmethyl)triamines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2669
assay in the presence of increasing concentrations of competi-
tor, using 1 µM [14C]-spermidine as substrate. Ki values for
inhibition of spermidine uptake were determined using the
Cheng-Prusoff equation39 from the IC50 value derived by
iterative curve fitting of the sigmoidal equation describing the
velocity of spermidine uptake in the presence of the respective
competitor.40,41 Cell lines (murine leukemic L1210 cells) were
grown and maintained according to established procedures.7,42
Cells were washed twice in HBSS prior to the transport assay.
Gen er a l P r oced u r e for th e Syn th esis of N-Boc-N-
(An th r a cen -9-ylm eth yl)a m in o Alcoh ols, 5. The solution of
N-(anthracen-9-ylmethyl)amino alcohol 4 (5 mmol) in 20 mL
of pyridine/methanol (1:5 v/v) was stirred at 0 °C for 10 min.
A solution of di-tert-butyl dicarbonate (7.5 mmol) in methanol
(5 mL) was added dropwise over 10 min. The temperature was
allowed to rise to room temperature and the reaction mixture
was stirred overnight. The mixture was evaporated to dryness
under reduced pressure. The residue was dissolved in meth-
ylene chloride and washed with deionized water several times.
The organic layer was separated, dried over anhydrous Na2-
SO4, filtered, and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel.
After numerous syntheses of this type, we found that it was
not necessary to purify compounds 5 and 6 by column chro-
matography. In fact, these could be used directly in subsequent
steps to provide satisfactory yields and purities of the target
compounds 8. Therefore, as long as adducts 4 and 7 were pure,
one could avoid column chromatography on the other inter-
mediates 5 and 6.
Decon volu tion Micr oscop y. The microscope system in-
cluded an Applied Precision (Issaquah, WA) Deltavision
system equipped with a Nikon Eclipse TE200 inverted micro-
scope. Image deconvolution was performed using Applied
Precision SoftWorX software. Detached melanoma A375 cells
were prepared by 3 min incubation with trypsin. The cells were
then washed, centrifuged and resuspended in phosphate
buffered saline. The cells were then washed repetitively with
fresh media, and the cells were fixed onto a microscope slide
and imaged by the deconvolution microscope. This technique
provides an image of the entire cell by using software to
overlay a series of planar images (or slices) to give a stacked
translucent view of the entire cell. The lightest regions contain
the highest concentration of the fluorescent agent 8e.
An th r a cen -9-ylm eth yl(2-h yd r oxyeth yl)ca r ba m ic a cid
ter t-bu tyl ester , 5a : pale-yellow solid; mp 131-132 °C; yield
1
84%; Rf ) 0.21 (acetone/hexane 1:4); H NMR (CDCl3) δ 8.43
(s, 1H), 8.38 (br s, 2H), 8.01 (d, 2H), 7.43 (m, 4H), 5.50 (br s,
2H), 3.30 (t, 2H), 3.00 (br s, 3H, including -OH), 1.52 (br s,
9H). Anal. (C22H25NO3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N-(An th r a cen -
9-ylm eth yl)a m in o Alcoh ols, 4. To a stirred solution of amino
alcohol 2 (12 mmol) in 25% MeOH/CH2Cl2 (10 mL) was added
a solution of 9-anthraldehyde 1 (10 mmol) in 25% MeOH/CH2-
Cl2 (10 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete
(monitored by TLC). The solvent was evaporated under
vacuum to give the crude imine 3 as a bright-green solid, which
was used for reduction without further purification.
An th r acen -9-ylm eth yl(3-h ydr oxypr opyl)car bam ic acid
ter t-bu tyl ester , 5b: unstable pale-yellow solid; yield 90%,
Rf ) 0.23 (acetone/hexane 1:4); 1H NMR (CDCl3) δ 8.42 (s, 1H),
8.36 (d, 2H), 8.02 (d, 2H), 7.52 (m, 4H), 5.50 (br s, 2H), 3.25
(br s, 2H), 3.10 (br s, 2H), 1.62 (m, 11H). HRMS (FAB) calcd
for C23H28NO3 (M + H)+: 366.2069. Found: 366.2067.
An th r a cen -9-ylm eth yl(4-h yd r oxybu tyl)ca r ba m ic a cid
ter t-bu tyl ester , 5c: pale-yellow solid; mp 113-114 °C; yield
88%; Rf ) 0.13 (acetone/hexane 12:88); 1H NMR (CDCl3) δ 8.42
(s, 1H), 8.39 (d, 2H), 8.01 (d, 2H), 7.52 (m, 4H), 5.50 (br s, 2H),
3.25 (t, 2H), 2.80 (br s, 2H), 1.60 (br s, 9H), 1.20 (m, 4H). 13C
NMR δ 155.88, 131.52, 131.48, 129.45, 128.99, 126.53, 125.22,
124.32, 80.08, 62.35, 44.54, 41.28, 30.05, 28.94 (3C), 24.99.
Anal. (C24H29NO3) C, H, N. HRMS (FAB) calcd for C24H29NO3-
Na (M + Na)+: 402.2045. Found: 402.2070.
NaBH4 (30 mmol) was added in small portions to a solution
of imine 1:1 CH3OH/CH2Cl2 (20 mL) at 0 °C. The mixture was
stirred at room temperature overnight and then concentrated
under vacuum. The residue was dissolved in CH2Cl2 (50 mL)
and washed three times with aqueous Na2CO3 (pH 10, 50 mL).
The organic layer was separated, dried over anhydrous Na2-
SO4, filtered, and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel.
2-[(An t h r a cen -9-ylm et h yl)a m in o]et h a n ol, 4a : bright-
yellow solid; mp 116-118 °C; yield 77%; Rf ) 0.48, methanol/
chloroform, 1:9; 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.28
(d, 2H), 8.00 (d, 2H), 7.48 (m, 4H), 4.68 (s, 2H), 3.64 (t, 2H),
3.00 (t, 2H), 2.1 (br s, 2H); 13C NMR δ 131.75, 131.44, 130.45,
129.47, 127.63, 126.48, 125.23, 124.18, 61.12, 51.65, 45.41.
An th r a cen -9-ylm eth yl(5-h yd r oxyp en tyl)ca r ba m ic a cid
ter t-bu tyl ester , 5d : pale-yellow solid; mp 125-126 °C; yield
1
84%; Rf ) 0.28 (acetone/hexane 1:4); H NMR (CDCl3) δ 8.44
(s, 1H), 8.41 (d, 2H), 8.05 (d, 2H), 7.55 (m, 4H), 5.56 (br s, 2H),
3.38 (t, 2H), 2.82 (br s, 2H), 1.64 (br s, 9H), 1.25 (m, 4H), 0.98
(m, 2H); 13C NMR δ 155.97, 131.56, 131.53, 129.43, 128.34,
126.51, 125.23, 124.43, 79.94, 62.85, 44.84, 41.41, 32.33, 28.96
(3C), 28.43, 23.14. Anal. (C25H31NO3) C, H, N. HRMS (FAB)
calcd for C25H32NO3 (M + H)+: 394.2382. Found: 394.2385.
Gen er a l P r oced u r e for th e Tosyla tion of 5 To Give 6.
A solution of the N-Boc protected (anthracen-9-ylmethyl)amino
alcohol 5 (5 mmol) in 20 mL of dry pyridine was stirred at 0
°C for 10 min. p-Toluenesulfonyl chloride (TsCl, 7.5 mmol) was
added in small portions over 30 min. The mixture was stirred
for an additional hour, and the reaction flask was placed in a
refrigerator (0-5 °C) overnight. The mixture was poured into
200 mL of ice/water, and a hemisolid (or viscous liquid)
typically precipitated (or separated). After the upper layer was
decanted off, the residue was dissolved in methylene chloride
and washed several times with deionized water. The organic
layer was separated, dried over anhydrous Na2SO4, filtered,
and concentrated under vacuum. The residue was purified by
flash chromatography on silica gel to give 6.
Anal. (C17H17NO) C, H, N. HRMS (FAB) m/z calcd for C17H18
-
NO (M + H)+: 252.1388. Found: 252.1381.
3-[(An th r acen -9-ylm eth yl)am in o]pr opan -1-ol, 4b: bright-
yellow solid; mp 82-83 °C; yield 80%; Rf ) 0.51, methanol/
chloroform, 1:9; 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.28
(d, 2H), 8.00 (d, 2H), 7.46 (m, 4H), 4.70 (s, 2H), 3.80 (t, 2H),
3.10 (t, 2H), 2.90 (br s, 2H), 1.78 (m, 2H); 13C NMR δ 131.72,
130.98, 130.48, 129.48, 127.72, 126.56, 125.25, 124.06, 64.65,
50.83, 46.02, 31.14. Anal. (C18H19NO) C, H, N. HRMS (FAB)
m/z calcd for C18H20NO (M + H)+: 266.1545. Found: 266.1526.
4- [(An th r acen -9-ylm eth yl)am in o]bu tan -1-ol, 4c: bright-
yellow solid; mp 87-88 °C; yield 81%; Rf) 0.51, methanol/
chloroform, 1:9; 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.26
(d, 2H), 8.00 (d, 2H), 7.49 (m, 4H), 4.68 (s, 2H), 3.50 (t, 2H),
2.90 (t, 2H), 1.65 (br s, 4H); 13C NMR δ 131.74, 130.73, 130.48,
129.51, 127.82, 126.64, 125.30, 123.96, 62.89, 50.58, 45.72,
32.72, 29.13. Anal. (C19H21NO) C, H, N. HRMS (FAB) m/z calcd
for C19H22NO (M + H)+: 280.1701. Found: 280.1679.
5- [(Anthracen-9-ylmethyl)amino]pentan-1-ol, 4d: bright-
yellow solid; mp 76-77 °C; yield 68%; Rf ) 0.26, methanol/
chloroform (5:95); 1H NMR (CDCl3) δ 8.38 (s, 1H), 8.27 (d, 2H),
7.98 (d, 2H), 7.45 (m, 4H), 4.65 (s, 2H), 3.50 (t, 2H), 2.82 (t,
2H), 1.78 (br s, 2H), 1.50 (m, 4H), 1.40 (m, 2H); 13C NMR
(CDCl3) δ 131.89, 131.77, 130.48, 129.44, 127.46, 126.40,
125.19, 124.29, 62.68, 50.60, 45.96, 32.68, 29.91, 23.67. Anal.
(C20H23NO) C, H, N. HRMS (FAB) m/z calcd for C20H24NO
(M + H)+: 294.1858. Found: 294.1835.
Tolu en e-4-su lfon ic a cid 3-(a n th r a cen -9-ylm eth yl-ter t-
bu toxyca r bon yla m in o)p r op yl ester , 6b: unstable bright-
yellow viscous liquid, yield 51%; Rf ) 0.21 (acetone/hexane 1:4);
1H NMR (CDCl3) δ 8.42 (s, 1H), 8.39 (d, 2H), 8.05 (d, 2H), 7.58
(m, 6H), 7.21 (d, 2H), 5.46 (s, 2H), 3.63 (t, 2H), 2.82 (t, 2H),
2.41 (s, 3H), 1.64 (br s, 11H). HRMS (FAB) calcd for C25H26
-
NO3S (M + 2H - Boc)+: 420.1633. Found: 420.1642.
Tolu en e-4-su lfon ic a cid 4-(a n th r a cen -9-ylm eth yl-ter t-
bu toxyca r bon yla m in o)bu tyl ester , 6c: pale-yellow viscous
liquid; yield 88%; Rf ) 0.38, acetone/hexane 1:3; 1H NMR

2670 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Wang et al.
(CDCl3) δ 8.42 (s, 1H), 8.37 (d, 2H), 8.00 (d, 2H), 7.62 (d, 2H),
N-(4-Am in obu tyl)-N-a n th r a cen -9-ylm eth ylbu ta n e-1,4-
d ia m in e tr ih yd r och lor id e sa lt, 8e: bright-yellow solid,
yield 91%; H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.42 (d,
7.48 (m, 4H), 7.22 (d, 2H), 5.46 (s, 2H), 3.63 (t, 2H), 2.77 (br s,
1
2H), 2.40 (s, 3H), 1.48 (br s, 9H), 1.20 (br s, 4H). Anal. (C31H35
-
NO5S‚0.5H2O) C, H, N. HRMS calcd for C31H35NO5S M+:
2H), 8.20 (d, 2H), 7.70 (t, 2H), 7.61 (t, 2H), 5.23 (s, 2H), 3.30
(t, 2H), 2.93 (m, 4H), 2.82 (t, 2H), 1.78-1.60 (m, 8H); 13C NMR
(D2O) δ 130.62, 130.32, 130.01, 129.45, 127.67, 125.48, 122.48,
120.37, 47.17, 47.11, 47.00, 42.76, 39.03, 24.19, 23.14, 23.02
(2C). Anal. (C23H34Cl3N3‚0.8H2O) C, H, N. HRMS (FAB) calcd
for C23H32N3 (M + H - 3HCl)+: 350.2590. Found: 350.2611.
N-(3-Am in op r op yl)-N-a n t h r a cen -9-ylm et h ylp en t a n e-
1,5-d ia m in e tr ih yd r och lor id e sa lt, 8f: bright-yellow solid,
yield 86%; 1H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.42 (d,
2H), 8.20 (d, 2H), 7.72 (t, 2H), 7.64 (t, 2H), 5.22 (s, 2H), 3.28
(t, 2H), 2.99 (m, 6H), 2.00 (m, 2H), 1.80 (m, 2H), 1.72 (m, 2H),
1.42 (m, 2H); 13C NMR (D2O) δ 130.54, 130.23, 129.91, 129.39,
127.60, 125.42, 122.42, 120.30, 47.62, 47.54, 44.69, 42.56,
36.80, 25.28, 25.22, 24.01, 23.20. Anal. (C23H34Cl3N3‚0.2H2O)
C, H, N. HRMS (FAB) calcd for C23H34Cl2N3 (M + H - HCl)+:
422.2130. Found: 422.2106.
N-(4-Am in obu tyl)-N-a n th r a cen -9-ylm eth ylp en ta n e-1,5-
d ia m in e tr ih yd r och lor id e sa lt, 8g: bright-yellow solid,
yield 88%; 1H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.42 (d,
2H), 8.20 (d, 2H), 7.73 (t, 2H), 7.64 (t, 2H), 5.23 (s, 2H), 3.26
(t, 2H), 2.93 (br s, 4H), 2.82 (t, 2H), 2.00 (m, 2H), 1.80 (m,
2H), 1.72 (br s, 4H), 1.42 (m, 2H); 13C NMR (D2O) δ 130.49,
130.21, 129.89, 129.38, 127.61, 125.41, 122.42, 120.23, 47.51,
47.46, 47.07, 42.50, 39.06, 25.29, 25.19, 24.22, 23.20, 23.04.
533.2236. Found: 533.2236.
Tolu en e-4-su lfon ic a cid 5-(a n th r a cen -9-ylm eth yl-ter t-
bu toxyca r bon yla m in o)p en tyl ester , 6d : pale-yellow vis-
cous liquid; yield 88%; Rf ) 0.25, acetone/hexane 1:4; 1H NMR
(CDCl3) δ 8.42 (s, 1H), 8.36 (d, 2H), 8.00 (d, 2H), 7.63 (d, 2H),
7.46 (m, 4H), 7.22 (d, 2H), 5.47 (s, 2H), 3.73 (br s, 2H), 2.76
(br s, 2H), 2.40 (s, 3H), 1.48 (br s, 9H), 1.24 (br s, 2H), 1.20 (br
s, 2H), 0.93 (br s, 2H). Anal. (C32H37NO5S‚0.5H2O) C, H, N.
HRMS (FAB) calcd for C32H38NO5S (M + H)+: 548.2471.
Found: 548.2501.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e N1-Boc
P r otected N1-(An th r a cen -9-ylm eth yl)tr ia m in es, 7. The
tosylated products 6 (1 mmol) and 1,4-diaminobutane or 1,3-
diaminepropane (10 mmol) were dissolved in acetonitrile (10
mL), and then the mixture was stirred at 75 °C under N2
overnight. After a check for the disappearance of the tosylate
by TLC, the solution was concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 (20 mL) and washed three
times with saturated aqueous sodium carbonate. The organic
layer was separated, dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel. The purified
products 7 were used immediately for next step (BOC depro-
tection). The isolated yields ranged between 59% and 75%.
Anal. (C24H36Cl3N3) C, H, N. HRMS (FAB) calcd for C24H36
-
Cl2N3 (M + H - HCl)+: 436.2286. Found: 436.2289.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e N1-
(An th r a cen -9-ylm eth yl)tr ia m in es, 8. The N1-Boc protected
triamine 7 (0.5 mmol) was dissolved in ethanol (5 mL), and
the mixture was stirred at 0 °C for 10 min. A 4 N aqueous
HCl (8 mL) solution was added dropwise at 0 °C. The mixture
was stirred at room temperature overnight. The solution was
then concentrated under reduced pressure (while maintaining
the water bath on the rotary evaporator below 60 °C), and a
bright-yellow solid precipitated. The solids were washed
several times with absolute ethanol and provided the pure
N-(An t h r a cen -9-ylm et h yl)b u t yla m in e, Mon oh yd r o-
ch lor id e, 11. Compound 11 was synthesized in 58% yield by
reductive amination of anthraldehyde and butylamine followed
by treatment with 4 N aqueous HCl. These two procedures
are described in the above general procedures for 4 and 8,
respectively.
11: yellow solid; yield 58%; Rf ) 0.5, methanol/chloroform,
1:20, + 1 drop of NH4OH; 1H NMR (300 MHz, DMSO-d6) δ
9.1 (br s, 2H, NH2 salt), 8.78 (s, 1H), 8.51 (d, 2H), 8.18 (d, 2H),
7.64 (m, 4H), 5.2 (br s, 2H), 3.2 (s, 2H), 1.73 (t, 2H), 1.36 (q,
2H), 0.92 (q, 3H); 13C NMR (CDCl3) δ 131.6, 131.26, 130.55,
129.46, 128.05, 125.72, 123.89, 120.86, 46.05, 41.92, 28.48,
20.32, 13.79.
1
target compounds 8b-g as HCl salts. The H NMR spectra of
polyamine conjugates were measured in 0.5 mL of DMSO-d6
and three drops of D2O. The use of DMSO-d6/D2O mixtures
resulted in better spectral resolution (compared to using pure
D2O as solvent). The 13C NMR spectra of the triamines were
measured in D2O to avoid the interference of DMSO carbon
signals.
Ack n ow led gm en t. The authors thank the China
Scholarship Council for partial support of C.W. This
work was supported in part by the Elsa U. Pardee
Foundation and the Florida Hospital Gala Endowed
Program for Oncologic Research. The authors are also
grateful to Ms. Fanta Konate for the synthesis of control
11.
N-(3-Am in op r op yl)-N-a n t h r a cen -9-ylm et h ylp r op a n e-
1,3-d ia m in e tr ih yd r och lor id e sa lt, 8b: bright-yellow solid,
yield 98%; 1H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.40 (d,
2H), 8.22 (d, 2H), 7.75 (t, 2H), 7.62 (t, 2H), 5.23 (s, 2H), 3.40
(t, 2H), 3.05 (m, 4H), 2.96 (t, 2H), 2.18 (m, 2H), 2.00 (m, 2H);
13C NMR δ 130.63, 130.45, 130.06, 129.47, 127.72, 125.50,
122.48, 120.06, 44.91, 44.82, 44.75, 43.08, 36.74, 23.98, 22.93.
Anal. (C21H30Cl3N3) C, H, N. HRMS (FAB) calcd for C21H30
Cl2N3 (M + H - HCl)+: 394.1817. Found: 394.1806.
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N1-{3-[(An t h r a cen -9-ylm et h yl)a m in o]p r op yl}b u t a n e-
1,4-d ia m in e tr ih yd r och lor id e sa lt, 8c: bright-yellow solid,
1
yield 98%; H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.42 (d,
2H), 8.20 (d, 2H), 7.72 (t, 2H), 7.62 (t, 2H), 5.23 (s, 2H), 3.42
(t, 2H), 3.05 (t, 2H), 2.98 (t, 2H), 2.82 (t, 2H), 2.20 (br s, 2H),
1.71 (br s, 4H); 13C NMR (D2O) δ 130.57, 130.41, 130.01,
129.45, 127.71, 125.48, 122.46, 119.98, 47.27, 44.76, 44.66,
43.01, 39.03, 24.16, 23.00, 22.92. Anal. (C22H32Cl3N3‚0.6H2O)
C, H, N. HRMS (FAB) calcd for C22H32Cl2N3 (M + H - HCl)+:
408.1973. Found: 408.1950.
N-(3-Am in opr opyl)-N-an th r acen -9-ylm eth ylbu tan e-1,4-
d ia m in e tr ih yd r och lor id e sa lt, 8d : bright-yellow solid,
yield 95%; 1H NMR (DMSO-d6 + D2O) δ 8.80 (s, 1H), 8.42 (d,
2H), 8.20 (d, 2H), 7.73 (t, 2H), 7.64 (t, 2H), 5.23 (s, 2H), 3.30
(t, 2H), 2.99 (m, 6H), 2.00 (m, 2H), 1.80 (m, 4H); 13C NMR
(D2O) δ 130.65, 130.37, 130.04, 129.48, 127.70, 125.51, 122.49,
120.30, 47.18 (2C), 44.78, 42.79, 36.81, 24.04, 23.14, 22.99.
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Anal. (C22H32Cl3N3) C, H, N. HRMS (FAB) calcd for C22H32
-
Cl2N3 (M + H - HCl)+: 408.1973. Found: 408.1958.

N1-(Anthracen-9-ylmethyl)triamines
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