Designing the polyamine pharmacophore: Influence of N-substituents on the transport behavior of polyamine conjugates

Article abstract of DOI:10.1021/jm701341k

N-Ethylated N-arylmethyl polyamine conjugates were synthesized and evaluated for their ability to target the polyamine transporter (PAT). To understand the effect of N-ethylation upon PAT selectivity, ethyl groups were appended onto a PAT-selective N¹-anthracenenylmethyl homospermidine derivative. 1b. Bioevaluation in L1210 murine leukemia cells and in two Chinese hamster ovary cell lines (PAT-active CHO and PAT-deficient CHO-MG) revealed a dramatic decrease in PAT targeting ability upon N¹ or N⁵ ethylation of the pharmacophore 1b. Experiments using the amine oxidase inhibitor, aminoguanidine (AG, 2 mM), revealed that the N⁹-ethyl and N⁹-methyl analogues were able to retain their PAT selectivity and cytotoxicity properties in the presence or absence of AG. In contrast, the lead compound 1b (containing a terminal NH₂ group) revealed a dramatic reduction in both its PAT-targeting ability and cytotoxicity in the absence of AG. An improved balance between these three properties of PAT-targeting, cytotoxicity and metabolic stability can be attained via N-methylation at the N⁹-position.

Full text of DOI:10.1021/jm701341k

J. Med. Chem. 2008, 51, 2551–2560
2551
Designing the Polyamine Pharmacophore: Influence of N-Substituents on the Transport
Behavior of Polyamine Conjugates
Navneet Kaur,† Jean-Guy Delcros,‡ Jennifer Archer,† Nathan Z. Weagraff,† Bénédicte Martin,‡ and Otto Phanstiel IV*,†,§
Groupe Cycle Cellulaire, CNRS UMR 6061 Génétique et DéVeloppement, IFR 97 Génomique Fonctionnelle et Santé, Faculté de Médecine,
UniVersité Rennes 1, 2 AV. du Pr Leon Bernard, CS 34317, F-35043 Rennes Cédex, France, Department of Chemistry,
Post Office Box 162366, UniVersity of Central Florida, Orlando, Florida 32816-2366, and Department of Medical Education, College of
Medicine, UniVersity of Central Florida, 12201 Research Parkway, Orlando, Florida 32816-2201
ReceiVed October 25, 2007
N-Ethylated N-arylmethyl polyamine conjugates were synthesized and evaluated for their ability to target
the polyamine transporter (PAT). To understand the effect of N-ethylation upon PAT selectivity, ethyl groups
were appended onto a PAT-selective N1-anthracenenylmethyl homospermidine derivative, 1b. Bioevaluation
in L1210 murine leukemia cells and in two Chinese hamster ovary cell lines (PAT-active CHO and PAT-
deficient CHO-MG) revealed a dramatic decrease in PAT targeting ability upon N1 or N5 ethylation of the
pharmacophore 1b. Experiments using the amine oxidase inhibitor, aminoguanidine (AG, 2 mM), revealed
that the N9-ethyl and N9-methyl analogues were able to retain their PAT selectivity and cytotoxicity properties
in the presence or absence of AG. In contrast, the lead compound 1b (containing a terminal NH2 group)
revealed a dramatic reduction in both its PAT-targeting ability and cytotoxicity in the absence of AG. An
improved balance between these three properties of PAT-targeting, cytotoxicity and metabolic stability can
be attained via N-methylation at the N9-position.
Introduction
amines (3–5) prefer a hoe shape, 1b has ready access to a shovel
shape.15 Furthermore, these shape preferences were observed
Polyamines are essential cellular growth factors.1 Tumor cells
have been shown to contain elevated polyamine levels and have
active polyamine transport systems to import exogenous
polyamines.1 Due to the enhanced cellular need for these amine
growth factors and an activated transport system for their import,
one can selectively deliver polyamine-drug conjugates to
particular cell types.1–8 The structural tolerances accommodated
by the polyamine transporter (PATa) allow for the import of
non-native polyamine conjugates.2–6
Prior work in our laboratories and others have revealed the
structural requirements associated with the delivery of polyamine
conjugates via the PAT in murine leukemia (L1210) and Chinese
hamster ovary (CHO) cells.1–11 In short, the methylene “spacer”
units (Figure 1, 1: x, y, and z), the size of the N1-substituent,
and the degree of N1-substitution all influence PAT-mediated
delivery.4,6–8 Earlier studies in our laboratory revealed that the
homospermidine conjugate 1b (e.g., a 4,4-triamine) had 150-
fold higher cytotoxicity in CHO cells than in a mutant PAT-
deficient CHO cell line (CHO-MG).4,5,12,13
Recently, we demonstrated that the presence of a secondary
nitrogen at the N1 position (e.g., 1b) is a critical structural
element for utilizing PAT for cellular entry.7,14 The presence
of a tertiary amine at the N1-position blocked the ability of both
the dihydromotuporamines 3 and 4 and the N1-ethyl-N1-
(anthracenylmethyl)-polyamine 5 to utilize the PAT for cellular
entry. Molecular modeling studies revealed that molecular shape
may explain this sensitivity. While both 1b and N1 tertiary
1
in solution by H NMR studies.15 Because N1-ethylation had
such a dramatic impact on PAT targeting, we were interested
if other positions along the polyamine chain had the same
sensitivity to further N-alkylation.
N-Alkylation has many effects on the biological properties
of polyamines. For example, N-alkylated polyamines have been
shown to have enhanced metabolic stability due to their ability
to avoid N-acetylation by spermidine (SPD)-spermine acetyl
transferase, SSAT, and subsequent degradation by polyamine
oxidase (PAO).10a,16 N-Alkylated polyamines also have en-
hanced stability in the culture medium, wherein the additional
alkyl group prevents their oxidation by serum amine oxidases.16e
In all of our previous experiments, aminoguanidine (AG, 2 mM)
was routinely added in order to inhibit the serum amine oxidase
activity.16e,f This was important because the polyamine drug
conjugates, which contain a primary amine, for example, 1b,
could act as substrates for these enzymes. Indeed, secondary
amine motifs at the terminal position have been shown to impart
enhanced metabolic stability and improve bioactivity.10a
Compounds 6 and 7 were synthesized to better understand
how additional N-alkylation of the 1b motif altered the ability
of these derivatives to enter cells via the polyamine transport
system. Specifically, these new derivatives of 1b provided an
opportunity to probe how N-ethylation at the N5 and N9 positions
perturbed PAT-mediated delivery. In previous work,3,10a both
dimethyl and diethyl SPD (12a and 12b) were shown to be
competitive antagonists for the PAT via competition experiments
with SPD. In particular, SPD rescue experiments revealed that
the N1,N8-dimethyl SPD (12a) was more sensitive to SPD rescue
than the related N1,N8-diethyl compound (12b).2,3 Because
N-methyl derivatives may have higher affinity for PAT,10a
compound 8 was also synthesized for comparison to 7.
Tetraamines were previously shown to have high affinity for
the PAT (e.g., the L1210 Ki value of 9: 51 nM) but lower PAT
selectivity than 1b.5 Tetraamine 10 was synthesized to explore
* To whom correspondence should be addressed. Phone: (407) 823-5410.
Fax: (407) 823-2252. E-mail: ophansti@mail.ucf.edu.
†
Department of Chemistry, University of Central Florida.
Department of Medical Education, University of Central Florida.
Groupe cycle cellulaire, Université Rennes 1.
§
‡
a Abbreviations: PAT, polyamine transporter; CHO, Chinese hamster
ovary; CHO-MG, Chinese hamster ovary cells polyamine transport deficient
mutant; L1210, mouse leukemia cells; HBBS, Hanks balanced salt solution;
PBS, Phosphate buffered saline.
10.1021/jm701341k CCC: $40.75
2008 American Chemical Society
Published on Web 03/26/2008

2552 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Kaur et al.
Figure 1. Polyamine Architectures 1–12.
how N1-ethylation of a tetraamine influenced PAT selectivity.
Previous results showed that N1-ethyl,N1-anthracen-9-ylmethyl-
triamine 5 had a complete loss of PAT selectivity when
compared to 1b. However, while tetraamine 10 contains this
same N1 tertiary center, it also retains the homospermidine motif
(e.g., the two secondary and one primary nitrogen motifs) needed
for PAT recognition. Indeed, large N1 “cargoes” (e.g., 2) have
been shown to be delivered to cells via the PAT. Tetraamine
11 was synthesized to test whether a large pyrenyl group could
also be delivered via a tetraamine motif and for comparisons
with 10.
With the above compounds in mind, we had certain expecta-
tions. For example, the bis-alkylated polyamine analogues have
been found to mimic the functions of the natural polyamines
and are more stable to intracellular polyamine metabolism and
degradation pathways (i.e., SSAT and PAO).10a,16 Related
studies with N1-(methylaryl)polyamines (e.g., 1b and 2) revealed
that large N1-substituents are not cleaved (e.g., no homosper-
midine is observed).6 Therefore, in terms of design, one should
be able to use either N-alkyl or larger alkylaryl substituents to
avoid degradation. As shown in this study, the N-methyl
substituents were superior, as they retained good PAT selectivity
and avoided degradation. Using these insights compounds 6, 7,
8, 10, and 11 were designed to probe the effect of additional
N-alkylation and terminal aminobutylation on compound 1b (the
most PAT-selective ligand). These studies are important as they
further define the structural tolerances of PAT and provide
insight into the design constraints for the polyamine vector and
placement of the cytotoxic “cargo”.
whether tetraamines were as sensitive as the triamines to N1
substitution and to the size of the N1-substituent.
The N5-ethyl derivative 6 was synthesized from commercially
available 1,4-diaminobutane 13 (shown in Scheme 1), which
was selectively converted to mono Boc-protected putrescine 14
in 90% yield.8 Amine 14 was then alkylated using 4-bromo-
butyronitrile to give secondary amine 15 in 65% yield.8
N-Alkylation of 15 using EtBr and K2CO3 generated nitrile 16
in 85% yield. Reduction of 16 by hydrogenation over Raney
Ni gave the primary amine 17, which upon reductive amination
with 9-anthraldehyde resulted in the secondary amine 18 in 72%
yield. Lastly, the Boc group was removed using 4 N HCl in
ethanol to provide the N5-ethyl derivative 6 in 95% yield.
Previously synthesized 1b4 was used in the synthesis of the
N9-ethyl derivative 7. The selective N-alkylation of the primary
amine in 1b was accomplished with the cesium method reported
by Jung.17 When CsOH·H2O (1 equiv) in DMF, 4 Å molecular
sieves, and ethyl bromide were used, the secondary amine 19
was synthesized in 21% yield (Scheme 2). Some of the N,N-
diethylated compound was also separated by column chroma-
tography. Further treatment of 19 with 4 N HCl resulted in
compound 7. To confirm the structure of compounds 7 and 19,
di-tert-butyldicarbonate was used in excess to Boc-protect all
the available amines of 7 to synthesize the tricarbamate 20. The
1H NMR spectrum of 20 showed the presence of three Boc groups
and the absence of a doublet of triplets at 3.03 ppm, which indicated
that a RCH2CH2NHBOC group was not present. This observation
confirmed that there is no NH carbamate available in compound
20. Both observations confirmed the regiochemistry of the N-ethyl
1
group in compounds 19 and 7. Indeed, a comparison of the H
Results and Discussion
NMR spectra of 5, 6, and 7 (see Supporting Information) showed
distinct differences and ruled out conclusively any misassignment
of the N-Et regiochemistry.
Synthesis. New conjugates 6-8, 10, and 11 were synthesized
to probe the role of N-ethylation at the N5 and N9 positions and

Designing the Polyamine Pharmacophore
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2553
Scheme 1a
a Reagents and conditions: (a) di-t-butyl dicarbonate/10% TEA/MeOH, 90%; (b) K2CO3/CH3CN/4-bromobutyronitrile, 65%; (c) K2CO3/CH3CN/C2H5Br,
85%; (d) Raney Ni/NH4OH/EtOH/NH3, 94%; (e) 9-anthraldehyde/25% MeOH/CH2Cl2; (f) 50% MeOH/CH2Cl2/NaBH4, 72%; (g) EtOH/4 N HCl, 95%.
Scheme 2a
As shown in Scheme 5, reductive amination of amine 31 with
9-anthraldehyde resulted in the secondary amine 32 in 72%
yield, which upon N-alkylation with EtBr and K2CO3 in
acetonitrile resulted in tertiary amine 33 in 81% yield. Lastly,
the Boc groups were removed using 4 N HCl in ethanol to
provide the N1-ethyl-N1-anthracenylmethyl tetraamine 10 in 96%
yield.
Finally, as shown in Scheme 6, reductive amination of amine
31 with 1-pyrenyl aldehyde resulted in the secondary amine 34
in 70% yield. Removal of the Boc groups with 4 N HCl gave
the N1-pyrenyl tetraamine 11 in 90% yield.
Biological Evaluation. Once synthesized, the conjugates were
screened for cytotoxicity in L1210, CHO, and CHO-MG cells.
L1210 (mouse leukemia) cells were selected to enable com-
parisons with the published IC50 and Ki values for a variety of
polyamine substrates.2–6 CHO cells were chosen along with a
mutant PAT deficient cell line (CHO-MG) to comment on
selective transport via the PAT.4–6,9 The results are shown in
Table 1.
L1210 IC50 and Ki Studies. The IC50 values listed in Table
1 represent the concentration of the polyamine conjugate
required to reduce the relative cell growth by 50%. The Ki values
a Reagents and conditions: (a) Na2CO3/CH2Cl2; (b) CsOH ·H2O/molecular
sieves/DMF/C2H5Br, 21%; (c) 4 N HCl/EtOH, 93%; (d) di-t-butyl dicar-
in Table 1 were determined for [14C]SPD uptake and reflect
the affinity of the polyamine derivative for the PAT on the cell
surface. With both parameters, one can determine whether high
affinity for the transporter (e.g., low Ki value) translated into
high cytotoxicity (e.g., low IC50 value) and so on.
bonate/10% TEA/MeOH, 94%.
As shown in Scheme 3, the synthesis of compound 8 utilized
the previously synthesized alcohol 21.7 The HCl salt of amine
22 was obtained using 4 N HCl in ethanol. Regioselective
reductive amination of amine 22 with 9-anthraldehyde in the
presence of TEA resulted in the desired secondary amine 23 in
95% yield. Reaction of 23 with di-tert-butyl dicarbonate
provided the alcohol 24 in 96% yield.5 Mesylation of alcohol
24 resulted in mesylate 25,5 which was reacted with excess
methylamine to obtain amine 26 in 73% yield. Lastly, removal
of the Boc groups with 4 N HCl provided the N9-methyl
derivative 8 in 95% yield.
Previous results8 showed that although the Ki values provide
relative affinity measures of polyamine derivatives toward the
PAT system, they were of minimal value in predicting their
cytotoxicity. This is likely due to the fact that polyamine
transport is a multistep process, which involves cell surface
interactions followed by uptake across the cell membrane.1,5
In addition, some polyamine derivatives have been shown to
have a high affinity for the PAT but are not transported into
the cells.11
For the synthesis of tetraamines 10 and 11, triBoc-amine 3118
was synthesized as depicted in Scheme 4. Secondary amine 15
(Scheme 4) was Boc-protected using di-tert-butyl dicarbonate
to provide nitrile 27 in 95% yield, which was then reduced to
amine 28 in 90% yield by hydrogenation over Raney Ni.8 Amine
28 was then alkylated using 4-bromo-butyronitrile to give
secondary amine 29 in 62% yield, which was further Boc-
protected to give nitrile 30 in 94% yield. Reduction of nitrile
30 by hydrogenation over Raney Ni gave the triBoc-amine 31
in 90% yield.
Nevertheless, sometimes correlations between Ki values and
cytotoxicity become apparent. For example, upon N1-alkylation
of 1b to provide the N1-Et derivative 5, the Ki value increased
(1b: 1.8 µM; 5: 24.4 µM). Indeed, this simple N1-alkylation
resulted in a nearly 14-fold increase in Ki value (1.8 vs 24.4
µM) and a 74-fold increase in L1210 IC50 value (0.3 vs 22.2
µM, i.e., a dramatic shift to lower cytotoxicity). Molecular
modeling studies suggested that this was related to changes in
molecular shape induced by the tertiary amine (N1-ethylation
in compound 1b).15

2554 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Kaur et al.
Scheme 3a
a Reagents and conditions: (a) EtOH/4 N HCl, 97%; (b) 9-anthraldehyde/TEA/25% MeOH/CH2Cl2; (c) 50% MeOH/CH2Cl2/NaBH4 95%; (d) di-t-butyl
dicarbonate/10% TEA/MeOH, 96%; (e) methane sulfonyl chloride/TEA, 94%; (f) methylamine/CH3CN, 73%.
Scheme 4a
a Reagents and conditions: (a) di-t-butyl dicarbonate/10% TEA/MeOH, 95%; (b) Raney Ni/NH4OH/EtOH/NEt3, 90%; (c) K2CO3/CH3CN/4-
bromobutyronitrile, 62%; (d) Raney Ni/NH4OH/EtOH/NEt3, 90%.
Scheme 5a
Scheme 6a
a Reagents and conditions: (a) 31/25% MeOH/CH2Cl2; (b) 50% MeOH/
CH2Cl2/NaBH4 72%; (c) K2CO3/CH3CN/C2H5Br, 81%; (d) EtOH/4 N HCl,
96%.
a Reagents conditions: (a) 25% MeOH/CH2Cl2; (b) 50% MeOH/CH2Cl2/
NaBH4 70%; (c) EtOH/4 N HCl, 90%.
In the present investigation, when the position of the N1-
ethyl group was changed to N5- and N9-ethyl (i.e., compounds
6 and 7), the Ki values approached that of compound 1b. As
the position of the tertiary nitrogen center moves away from
the anthracene unit, the Ki value decreases (5: 24.4; 6: 5.5; 7:
3.5 µM), implying an increased affinity for PAT in this series.
To explore how N9-substituents affected PAT-binding affinity,
the Ki values of compounds 7 (N9-Et) and 8 (N9-Me) were also
determined. As listed in Table 1, the Ki values for the
N9-derivatives (7: 3.5 µM and 8: 8.2 µM) were similar and yet
significantly lower than the N1-derivative 5 (24.4 µM). These
data suggest that in terms of additional N-alkylation of 1b
polyamine conjugates containing N5- and N9-alkyl substituents
have higher PAT affinity than those with additional N1-
substituents (e.g., 5).
In the case of tetraamines 95 and 10, the Ki values were
significantly lower, which was consistent with previous results
with other tetraamines,5 and reflected their higher binding
affinity. N1-Ethylation of tetraamine 9 resulted in nearly a 10-
fold increase in Ki value for compound 10 (9: 0.05 vs 10: 0.46
µM), which is consistent with the above trend observed with
triamines 1b and 5 (1b: 1.8 vs 5: 24.4 µM). This observation
indicated that the presence of the tertiary amine at the N1
position also lowers the PAT binding affinity of tetraamines.
The presence of the larger pyrenyl cargo in compound 11 also
increased the Ki value to 0.14 µM (vs 9: 0.051 µM). This
suggested that the size of the N1-substituent can also alter the
affinity of the polyamine conjugate for the PAT, a finding that
is consistent with previous results with N1-substituted
polyamines.8

Designing the Polyamine Pharmacophore
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2555
Table 1. Biological Evaluation of Polyamine Derivatives in L1210, CHO, and CHO-MG Cells in the Presence of AGa
cmpd (tether)
L1210 IC50 in µM
L1210 Ki values (µM)
ref
CHO-MG IC50 in µM
CHO IC50 in µM
IC50 ratiob
1b: Antmethyl (4,4)
0.30 ((0.04)
22.2 ((1.2)
0.88 ((0.02)
1.01 ((0.04)
0.40 ((0.05)
7.5 ((0.3)
1.8 ((0.1)
24.4 ((1.5)
5.5 ((1.5)
4
7
66.7 ((4.1)
21.9 ((0.9)
7.0 ((0.2)
57.9 ((2.3)
60.0 ((2.6)
33.2 ((1.7)
55.7 ((5.1)
25.3 ((2.9)
1.2 ((0.1)
0.45 ((0.10)
22.2 ((0.7)
3.9 ((0.5)
9.8 ((0.3)
4.9 ((0.2)
10.6 ((3.1)
17.1 ((1.9)
2.4 ((0.1)
1.1 ((0.1)
148
1
5: N1-Ethyl-N1-antmethyl (4,4)
6: N5-Ethyl-N1-antmethyl (4,4)
7: N9-Ethyl-N1-antmethyl (4,4)
8: N9-Methyl-N1-antmethyl (4,4)
9: Antmethyl (4,4,4)
1.8
5.9
12.2
3.1
3.3
10.5
1.1
3.5 ((0.2)
8.2 ((0.6)
0.05 ((0.06)
0.46 ((0.10)
0.14 ((0.02)
4.3 ((0.2)
5
8
10: N1-Ethyl-N1-antmethyl (4,4,4)
11: Pyr-methyl (4,4,4)
3.4 ((0.3)
1.2 ((0.1)
1.5 ((0.1)
12c: Bis-N1,N9-antmethyl (4,4)
a Definitions used in Table 1, column 1: Ant ) Anthracen-9-yl, column 4: ref denotes the reference number in which the data was originally reported.
A blank in the ref column denotes new data. Cells were incubated for 48 h at 37 °C with the respective conjugate. b The ratio denotes the (CHO-MG/CHO)
IC50 ratio, a measure of PAT selectivity.
In the L1210 experiments, the higher Ki value of 5 correlated
with its lower cytotoxicity (IC50 of 5: 22.2 µM). Indeed, the
IC50 value increased significantly from the parent system, 1b
(0.3 µM). This revealed that at least for these compounds both
the PAT binding affinity and the conjugate’s cytotoxicity were
sensitive to the degree of alkylation at the N1 position. As the
position of the tertiary amine was moved from N1 (5) to N5 (6)
and N9 (7), the cytotoxicity increased (IC50 value: 5: 22.2 µM;
6: 0.88 µM; 7: 1.01 µM). In terms of N9-substituent effects,
the small N9-alkyl groups of 7 and 8 did not significantly alter
the cytotoxicity profile seen with the parent 1b (e.g., IC50 1b:
0.30 µM; 7: 1.01 µM; IC50 8: 0.40 µM). Indeed, a SPD rescue
experiment, wherein the competitive antagonist SPD is added,
showed significant rescue of cells from compound 8. Specifi-
cally, a significant increase in the IC50 value was observed in
the presence of added SPD (IC50 8: 0.4 µM; IC50 8+SPD: 1.38
µM). Similar SPD protection was previously observed with 12a
and 12b.2,3 This result suggests that 8 is able to access cells
via the same PAT in L1210 cells, as utilized by the native SPD.
In general, the SPD protection assays were performed to
determine whether uptake of the selected polyamine analogues
is mediated, in part or in whole, by the polyamine transport
apparatus. To answer this question, competition assays were
performed in the absence and presence of SPD. To maximize
the protective effects of SPD, an excess of SPD (200 µM) was
used during these experiments. Use of excess SPD provided a
high level of competition with the selected polyamine derivatives
in terms of using the polyamine transport system for cellular
entry. Note: a similar SPD protection effect was observed in
CHO cells for compounds 1b, 7, and 8.
In the case of the tetraamines 10 and 11, the opposite trend
was observed. A modest increase in cytotoxicity was observed
upon N-ethylation (IC50 value decreased from 7.5 µM (9) to
3.4 µM (10)). This suggests that a motif with two secondary
nitrogens and one primary nitrogen is sufficient for cellular entry.
Interestingly, this premise could also explain the increase in
cytotoxicity of the N1-Et tetraamine 10 (3.4 µM) over its N1-Et
triamine 5 (IC50: 22.2 µM) counterpart. As will be demonstrated
in the following CHO studies, this is due to the enhanced PAT
targeting abilities of 10.
In summary, the L1210 Ki and IC50 values allowed compari-
sons to published polyamine systems and revealed that, at least
in the series 5-7, the higher affinity for PAT translated into a
more cytotoxic compound.8 The CHO and CHO-MG IC50
comparisons were even more informative.
(dNH)NH2]) using a single-step selection after mutagenesis with
ethylmethanesulfonate.12,13
For the purposes of this study, the CHO-MG cell line
represents cells with no PAT activity and provided a model for
alternative modes of entry or action, which are independent of
PAT. These alternative modes of entry include passive diffusion
or utilization of another transporter. The alternative modes of
action may also include interactions on the outer surface of the
plasma membrane or other membrane receptor interactions.
In contrast, the parent CHO cell line represents a cell type
with high PAT activity.12,13 Comparison of conjugate cytotox-
icity in these two CHO cell lines provided an important screen
to detect selective conjugate delivery via the PAT. For example,
a conjugate with high utilization of the PAT would be very
toxic to CHO cells, but less so to CHO-MG cells.4–6,9 In short,
highly selective PAT ligands should give high (CHO-MG/CHO)
IC50 ratios.
As reported earlier,8 dramatic differences in cytotoxicity were
observed with 1b (CHO-MG/CHO IC50 ratio: 148), a highly
PAT-selective substrate. The CHO-MG/CHO IC50 ratios listed
in Table 1 suggested that PAT targeting is influenced by the
degree of substitution of nitrogen at the N1 position of the
polyamine vector. A direct correlation was observed between
cytotoxicity and polyamine conjugate uptake.8 Therefore, the
relative toxicities observed in CHO and CHO-MG cells
represent a measure of differential uptake via PAT and provide
a measure of PAT selectivity.8
When this CHO screen was used, N1-alkylation (5) gave a
complete loss of PAT selectivity (IC50 ratio CHO-MG/CHO )
1). As expected, cytotoxicity was also effected, wherein
compound 5 (CHO IC50 of 5: 22.2 µM) was 49 times less
cytotoxic then compound 1b (0.45 µM). Thus, the presence of
the tertiary amine at the N1 position of the homospermidine
vector resulted in a loss of ability to use PAT and reduced
cytotoxicity. When the position of the tertiary nitrogen was
moved away from the N1 position (5) to the N5 (6) and N9 (7)
positions, it also resulted in a decrease in cytotoxicity when
compared to the parent compound 1b.
The CHO-MG/CHO IC50 ratios revealed that PAT selectivity
was very sensitive to alkylation at N1 and N5, with CHO-MG/
CHO ratios of 1 and 1.8, respectively. Interestingly, N9-
ethylation of 1b provided compound 7, which was 5.9 times
more toxic to CHO than CHO-MG cells. Although compound
1b was more selective in using PAT, the tertiary N9-amine, 7,
could also be accommodated by PAT, albeit to a lesser degree
of selectivity. However, previous results showed that very large
N9-substituents (e.g., compound 12c) resulted in the complete
loss of PAT selectivity (Table 1). Pursuing this insight,
compound 8 with a methyl group at the N9 position was
evaluated in the CHO cell lines. The smaller N9-methyl
substituent resulted in increased PAT selectivity (ratio IC50
CHO and CHO-MG Studies. CHO cells were chosen along
with a mutant cell line (CHO-MG) to comment on how the
synthetic conjugates gain access to cells.4–6,9 The CHO-MG cell
line is polyamine-transport deficient and was isolated after
selection for growth resistance to methylglyoxalbis(guanylhy-
drazone), MGBG, (CH3C[dN-NHC(dNH)NH2]CH[dN-NHC-

2556 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Kaur et al.
In contrast, the PAT selectivities of N9-alkylated compounds 7
and 8 were retained in the absence of AG, suggesting that these
compounds are not the substrates for serum amine oxidases and
are still able to utilize the PAT. In the absence of AG,
compounds 7 and 1b have the same PAT selectivity (ratio IC50
CHO-MG/CHO: 4). However, in the absence of AG, the N9-
methyl analogue 8 has a higher selectivity (ratio IC50 ratio: 11.5)
than either its N-ethyl derivative 7 or the parent system, 1b.
Table 2. Biological Evaluation of Polyamine Derivatives in CHO and
CHO-MG Cells in the Absence of AG (IC50 Values in µM)a
CHO-MG
IC50
w/o AG
IC50
IC50
CHO IC50
w/o AG
cmpd (tether)
ratiob
ratiob
w/o AG with AG
1b: Antmethyl(4,4)
7: N9-Ethyl-
7.1 ((0.3)
1.7 ((0.1)
4.2
3.9
148
5.9
51.2 ((1.8) 13.2 ((0.5)
N1-antmethyl (4,4)
8: N9-Methyl-
56.2 ((2.0)
4.9 ((0.1)
11.5
12.3
N1-antmethyl (4,4)
Conclusions
a Definitions used in Table 1, column 1: ant ) anthracen-9-yl; cells were
incubated for 48 h with the respective conjugate. b The ratio denotes the
(CHO-MG/CHO) IC50 ratio, a measure of PAT selectivity.
A series of polyamine conjugates were synthesized to probe
the sensitivity of the PAT to small structural changes in its
substrate and to provide insight into potential “points of
attachment” onto the polyamine pharmacophore. A direct
correlation was found between PAT selectivity and the degree
of alkylation at the N1, N5, and N9 positions. N-Alkylation with
small substituents at the terminal N9 position still retained
modest PAT selectivity. This PAT selectivity was maintained
even in the absence of AG, whereas the lead compound, 1b,
showed a dramatic reduction in PAT selectivity in the absence
of AG. Large N1-cargoes could be accommodated (e.g., 11) by
PAT as long as the proper polyamine motif was in place. These
insights will be applied to the future design of polyamine-drug
conjugates, which are metabolically stable and still retain PAT
selectivity (e.g., 8).
CHO-MG/CHO for 8: 12.2 vs 7: 5.9). The increased PAT
selectivity of N-methyl analogue 8 over its N-ethyl counterpart
7 is likely due to steric effects, wherein the smaller substituent
at the N9 position is better accommodated by PAT. Indeed, the
ability to target PAT increased as one reduced the size of the
N9-substituent within the series 12c, 7, 8, and 1b.
Conclusively, the presence of the tertiary center at N1 and
N5 positions either obliterated or significantly diminished the
ability of conjugates to utilize the PAT for cellular entry,
whereas N9-methylation retained significant PAT selectivity.
Interestingly, N1-alkylation in the case of tetraamines 9 and
10 did not affect the PAT selectivity. Although the PAT
selectivity was reduced relative to triamine 1b, the CHO-MG/
CHO IC50 ratios for tetraamines 9 and 10 were 3.1 and 3.3,
respectively. This suggested that the presence of two secondary
amines and one primary amine is a sufficient motif for cellular
entry via PAT. Indeed, the truncated motif 5 had no PAT
selectivity, whereas tetraamine 10 (with the intact motif) showed
3-fold selectivity. The presence of the larger cargo (pyrenyl) in
tetraamine 11 resulted in a 10.5-fold increase in PAT selectivity,
which suggests that very large N1 cargoes could also be
accommodated by the PAT as long as the proper polyamine
motif was present.
Experimental Section
Materials. Silica gel (32–63 µm) and chemical reagents were
purchased from commercial sources and used without further
1
purification. All solvents were distilled prior to use. H and 13C
NMR spectra were recorded at 300 and 75 MHz, respectively. TLC
solvent systems are based on volume % and NH4OH refers to
concentrated aqueous NH4OH. Elemental analyses were performed
by Atlantic Microlabs (Norcross, GA).
Biological Studies. Murine leukemia (L1210), CHO, and CHO-
MG cells were grown in RPMI medium supplemented with 10%
fetal calf serum, glutamine (2 mM), penicillin (100 U/mL), and
streptomycin (50 µg/mL). 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 (AG, 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. Cells in early to
mid log-phase were used.
IC50 Determinations. Cell growth was assayed in sterile 96-
well microtiter plates (Becton-Dickinson, Oxnard, CA, U.S.A.).
L1210 cells were seeded at 5e4 cells/mL of medium (100 µL/well).
CHO and CHO-MG cells were plated at 2e3 cells/mL. Drug
solutions (10 µL per well) of appropriate concentration were added
at the time of seeding for L1210 cells and after an overnight
incubation for the CHO cell lines. 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 for absorbance (540
nm) measurements.19
Prior work with extended tethers in the N1 position (1c and
1d) revealed decreases in PAT selectivity as the N1-tether was
extended from methylene (1b) to ethylene (1c) and propylene
(1d). The current finding with 11 is significant because it implies
that larger cargoes can be accommodated if the correct motif is
present.18 The nature of this amine message will be explored
in further work. The present findings have direct applications
in the future design of PAT selective drug conjugates.
N-Alkylated polyamines have been shown to have enhanced
metabolic stability due to their ability to avoid degradation by
serum amine oxidases (present in the culture medium) and by
the intracellular polyamine oxidase (PAO).10a,16 Aminoguani-
dine16e,f (AG) is a known inhibitor of the serum amine oxidases
and is routinely added (at 2 mM) during our cell culture
experiments to avoid polyamine drug degradation by the serum
oxidase. We speculated that, in the absence of AG, the
polyamine conjugates could be degraded by the serum oxidases
and converted to other metabolites, which could affect the
measured cytotoxicity and PAT selectivity of the conjugates.
Prior experiments revealed that polyamine metabolic stability
could be modulated via steric effects.10a Therefore, even though
the N9-alkylated polyamine conjugates 7 and 8 showed lower
PAT selectivity than the lead compound 1b, it was possible
that they were more metabolically stable. To test this hypothesis,
we determined the cytotoxicity of 1b, 7, and 8 in the presence
and absence of AG (Table 2).
Ki Procedure. The ability of the conjugates to interact with the
polyamine transport system was determined by measuring competi-
tion by the conjugates against radiolabeled SPD uptake in L1210
cells. This procedure was used to obtain the data listed in Table 1.
Initially, the Km value of SPD transport was determined as
previously described.20
The ability of conjugates to compete for [14C]SPD uptake were
determined in L1210 cells by a 10-min uptake assay in the presence
of increasing concentrations of competitor using 1 µM [14C]SPD
as substrate. Ki values for inhibition of SPD uptake were determined
using the Cheng-Prusoff equation21 from the IC50 value derived
by iterative curve fitting of the sigmoidal equation describing the
As expected, the PAT selectivity of lead compound 1b was
lowered from 148 to 4 (Table 2) in the absence of AG, which
clearly suggests that 1b is a substrate for serum amine oxidases.

Designing the Polyamine Pharmacophore
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2557
velocity of SPD uptake in the presence of the respective
competitor.11,22 L1210 cells were grown and maintained according
to established procedures10d and were washed twice in HBSS prior
to the transport assay.
(4-Amino-butyl)-carbamic Acid tert-Butyl Ester, 14.8 1,4-
Diaminobutane (13; 30 g, 341 mmol) was dissolved in a solution
of triethylamine and methanol (10% TEA in MeOH, 250 mL). A
solution of di-tert-butyl dicarbonate (25 g, 115 mmol) in methanol
(50 mL) was added dropwise to this mixture with vigorous stirring
at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, then
warmed slowly to rt and stirred overnight. The methanol and TEA
were removed in vacuo to yield an oily residue that was dissolved
in CH2Cl2 (200 mL) and washed with a solution 10% aq sodium
carbonate (3 × 80 mL). The CH2Cl2 layer was dried over anhydrous
sodium sulfate, filtered, the solvent was removed in vacuo, and
the oily residue was purified by flash column chromatography to
give the product 148 as a clear oil (90%), Rf ) 0.38 (NH4OH/
N1-{4-[(Anthracen-9-ylmethyl)-amino]-butyl}-N1-ethyl-butane-
1,4-diamine, Hydrochloride Salt, 6. A solution of Boc-protected
18 (300 mg, 0.63 mmole) was dissolved in absolute ethanol (13
mL) and stirred at 0 °C for 10 min. A 4 N HCl solution (22 mL)
was added to the reaction mixture dropwise and stirred at 0 °C for
20 min and then at room temperature overnight. The solution was
concentrated in vacuo to give 6 as a yellow solid in 95% yield. 1H
NMR (D2O) δ 8.69 (s, 1H), 8.26 (d, 2H), 8.16 (d, 2H), 7.73 (m,
2H), 7.63 (m, 2H), 5.25 (s, 2H), 3.30 (t, 2H), 3.20 (q, 2H), 3.16
(m, 4H), 3.03 (t, 2H), 1.75 (m, 8H), 1.26 (t, 3H); 13C NMR (D2O)
δ 131.3, 130.9, 130.7, 129.9, 128.2, 126.0, 122.9, 121.2, 52.0, 51.8,
48.3, 47.4, 46.9, 43.3, 39.2, 24.4, 23.2, 21.1, 18.2, 8.5. HRMS
(FAB) calcd for C25H35N3 ·3HCl (M + H - 3HCl)+, 378.2909;
found, 378.2916.
1
MeOH/CHCl3, 1:10:89); H NMR (300 MHz, CDCl3) δ 4.68 (br
s, 1H, NHCO), 3.13 (m, 2H), 2.70 (t, 2H), 1.62–1.33 (m, 13H, 2
× CH2, 3 × CH3).
[4-(3-Cyano-propylamino)-butyl]-carbamic Acid tert-Butyl
Ester, 15.8 4-Bromo-butyronitrile (11.8 g, 80 mmol) was dissolved
in anhydrous acetonitrile and was added to the stirring mixture of
compound 14 (17 g, 90 mmol) and anhydrous K2CO3 (22 g, 159
mmol). The mixture was then stirred overnight at 75 °C under a
N2 atmosphere. After confirmation of the disappearance of the nitrile
by TLC, the solution was concentrated under reduced pressure. The
residue was dissolved in CH2Cl2 (100 mL) and washed three times
with 10% aqueous sodium carbonate. The organic layer was
separated, dried with anhydrous Na2SO4, filtered, and concentrated
under vacuum. Flash column chromatography of the residue gave
15 as a light yellow oil. Yield 65%; Rf ) 0.35 (MeOH/NH4OH/
CHCl3, 5:0.5:94.5); 1H NMR (CDCl3) δ 4.92 (bt, 1H), 3.12 (q,
2H), 2.73 (t, 2H), 2.61 (t, 2H), 2.45 (t, 2H), 1.81 (q, 2H), 1.39–1.6
(m, 13H).
N-Anthracen-9-ylmethyl-N′-(4-ethylamino-butyl)-butane-1,4-
diamine, Hydrochloride Salt, 7. A solution of 19 (49 mg, 0.13
mmol) was dissolved in absolute ethanol (6 mL) and stirred at 0
°C for 10 min. A 4 N HCl solution (11 mL) was added to the
reaction mixture dropwise and stirred at 0 °C for 20 min and then
at room temperature overnight. The solution was concentrated in
Vacuo to give 7 as a yellow solid in 93% yield. 1H NMR (D2O) δ
8.64 (s, 1H), 8.22 (d, 2H), 8.13 (d, 2H), 7.71 (m, 2H), 7.61 (m,
2H), 5.19 (s, 2H), 3.28 (t, 2H), 3.09 (m, 8H), 1.77 (m, 8H), 1.30
(t, 3H); 13C NMR (D2O) δ 133.7, 133.3, 133.1, 132.2, 130.5, 128.3,
125.3, 123.5, 49.9, 49.6, 49.1, 45.7, 25.7, 13.4. HRMS (FAB) calcd
for C25H35N3 ·3HCl (M + H - 3HCl)+, 378.2909; found, 378.2906.
N-Anthracen-9-ylmethyl-N′-(4-methylamino-butyl)-butane-
1,4-diamine, Hydrochloride Salt, 8. A solution of Boc-protected
26 (180 mg, 0.32 mmole) was dissolved in absolute ethanol (13
mL) and stirred at 0 °C for 10 min. A 4 N HCl solution (22 mL)
was added to the reaction mixture dropwise and stirred at 0 °C for
20 min and then at room temperature overnight. The solution was
concentrated in vacuo to give 8 as a yellow solid in 95% yield. 1H
NMR (300 MHz, D2O) δ 8.32 (s, 1H), 7.99 (d, 2H), 7.93 (d, 2H),
7.60 (m, 2H), 7.50 (m, 2H), 4.87 (s, 2H), 3.16 (t, 2H), 3.02 (m,
6H), 2.70 (s, 3H), 1.72 (m, 8H); 13C NMR (D2O) δ 130.7, 130.4,
130.1, 129.5, 127.7, 125.5, 122.5, 120.4, 48.4, 47.2, 47.1, 47.0,
42.8, 32.9, 23.1, 23.0, 22.9. HRMS (FAB) calcd for C24H33N3 ·3HCl
(M + H - 3HCl), 364.2747; found, 364.2715.
N-(4-Amino-butyl)-N′-[4-(anthracen-9-ylmethyl-ethyl-amino)-
butyl]-butane-1,4-diamine, Hydrochloride Salt, 10. A solution
of 33 (140 mg, 0.18 mmol) was dissolved in absolute ethanol (10
mL) and stirred at 0 °C for 10 min. A 4 N HCl solution (20 mL)
was added to the reaction mixture dropwise and stirred at 0 °C for
20 min and then at room temperature overnight. The solution was
concentrated in vacuo to give 10 as a yellow solid in 96% yield.
1H NMR (D2O) δ 8.68 (s, 1H), 8.17 (m, 4H), 7.75 (m, 2H), 7.64
(m, 2H), 5.25 (s, 2H), 3.09–3.00 (m, 12H), 2.82 (m, 2H), 1.78 (m,
10H), 1.44 (m, 5H); 13C NMR (D2O) δ 131.3, 131.0, 129.8, 128.2,
125.7, 122.7, 119.6, 107.1, 49.6, 49.5, 47.2, 47.1, 47.0, 46.7, 39.0,
24.2, 23.1, 23.0, 22.9, 20.9, 8.8. HRMS (FAB) calcd for
C29H44N4 ·4HCl (M + H - 4HCl)+, 449.3639; found, 449.3629.
N-[4-(4-Amino-butylamino)-butyl]-N′-(4,6-dihydro-pyren-1-
ylmethyl)-butane-1,4-diamine, Hydrochloride Salt, 11. A solution
of 34 (200 mg, 0.27 mmol) was dissolved in absolute ethanol (10
mL) and stirred at 0 °C for 10 min. A 4 N HCl solution (20 mL)
was added to the reaction mixture dropwise and stirred at 0 °C for
20 min and then at room temperature overnight. The solution was
concentrated in vacuo to give 11 as a yellow solid in 90% yield.
1H NMR (D2O) δ 8.25 (m, 2H), 8.15 (m, 2H), 7.95 (m, 3H), 7.90
(m, 2H), 4.64 (s, 2H), 3.1 - 2.90 (m, 12H), 1.85–1.60 (m, 12H);
13C NMR (D2O) δ 131.8, 130.7, 130.0, 128.8, 128.6, 128.3, 127.2,
126.7, 126.0, 125.9, 124.9, 123.6, 123.3, 123.0, 121.4, 64.5, 57.7,
49.1, 48.2, 47.2, 47.1, 46.7, 39.1, 24.2, 23.1, 17.1. HRMS (FAB)
calcd for C29H40N4 ·4HCl (M + H - 4HCl)+, 445.3326; found,
445.3321.
{4-[(3-Cyano-propyl)-ethyl-amino]-butyl}-carbamic Acid tert-
Butyl Ester, 16. Bromoethane (385 mg, 3.53 mmol) was dissolved
in anhydrous acetonitrile and was added to a stirred mixture of
compound 15 (300 mg, 1.18 mmol) and anhydrous K2CO3 (487
mg, 3.53 mmol). The mixture was then stirred overnight at 75 °C
under a N2 atmosphere. After confirmation of the disappearance of
15 by TLC, the solution was concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 (100 mL) and washed three
times with aqueous Na2CO3. The organic layer was separated, dried
with anhydrous Na2SO4, filtered, and concentrated under vacuum.
Flash column chromatography of the residue gave 16 as a light
yellow oil. Yield 85%; Rf ) 0.35 (MeOH/CH2Cl2, 3:97); 1H NMR
(CDCl3) δ 4.94 (b, 1H), 3.10 (q, 2H), 2.37–2.52 (m, 8H), 1.76 (q,
2H), 1.44 (m, 13H), 1.00 (t, 3H); 13C NMR (CDCl3) δ 155.9, 119.9,
78.9, 53.3, 51.6, 47.3, 40.6, 28.6, 28.2, 24.7, 23.7, 14.9, 11.8.
{4-[(4-Amino-butyl)-ethyl-amino]-butyl}-carbamic Acid tert-
Butyl Ester, 17. The nitrile 16 (290 mg, 1.02 mmol) was dissolved
in ethanol (30 mL). NH4OH (3 mL) and Raney nickel (1.5 g) were
added and ammonia gas was bubbled through the solution for 10
min at 0 °C. The suspension was hydrogenated at 50 psi for 24 h.
Air was bubbled through the solution, and the Raney nickel was
removed by filtering through a sintered glass funnel keeping the
Raney nickel residue moist at all times. The ethanol and NH4OH
were removed in vacuo, and the oily residue dissolved in CH2Cl2
and was washed with a 10% aqueous sodium carbonate solution
(2 × 50 mL). The organic layer was dried over anhydrous sodium
sulfate and filtered, and the solvent was removed in vacuo to give
the product 17 as a light yellow oil (94% yield) that was used in
the next step without further purification; 1H NMR (300 MHz,
CDCl3) δ 5.20 (br s, 1H), 3.35 (m, 2H), 2.15–2.80 (m, 8H),
0.90–1.70 (m, 21H); 13C NMR (CDCl3) δ 156.1, 78.9, 53.4, 47.5,
42.4, 40.8, 32.1, 28.7, 28.4, 24.9, 24.5, 11.8.
[4-({4-[(Anthracen-9-ylmethyl)-amino]-butyl}-ethyl-amino)-
butyl]-carbamic Acid tert-Butyl Ester, 18. To a stirred solution
of 17 (275 mg, 0.96 mmol) in 25% MeOH/CH2Cl2 (20 mL) was
added a solution of 9-anthraldehyde (164 mg, 0.80 mmol) in 25%
MeOH/CH2Cl2 (15 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete
(monitored by disappearance of the 1H NMR (CDCl3) signal at
11.40 ppm). The solvent was removed in vacuo, the solid residue

2558 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Kaur et al.
was dissolved in 50% MeOH/CH2Cl2 (40 mL), and the solution
was cooled to 0 °C. NaBH4 (2.40 mmol) was added in small
portions to the solution, and the mixture was stirred at rt overnight.
The solvent was removed in vacuo, the solid residue was dissolved
in CH2Cl2 (50 mL) and washed with 10% aq Na2CO3 solution (3
× 30 mL). The CH2Cl2 layer was separated, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo to give an oily residue.
The oil was purified by flash column chromatography to yield the
product 18 as a pale yellow viscous oil (72%), Rf ) 0.3 (MeOH/
NH4OH/CH2Cl2, 6:0.5:93.5); 1H NMR (CDCl3) δ 8.30 (d, 2H), 8.27
(s, 1H), 7.93 (d, 2H), 7.48 (m, 2H), 7.40 (m, 2H), 4.66 (s, 2H),
3.03 (q, 2H), 2.85 (t, 2H), 2.44 (q, 2H), 2.35 (m, 4H), 1.40–1.54
(m, 17H), 0.96 (t, 3H); 13C NMR (CDCl3) δ 156.0, 131.7, 131.5,
130.2, 129.1, 127.1, 126.0, 124.9, 124.1, 78.8, 53.3, 53.2, 50.5,
47.3, 45.9, 40.6, 28.6, 28.3, 24.7, 24.7, 11.6. HRMS (FAB) m/z
calcd for C30H44N3O2 (M + H)+, 478.3434; found, 478.3442.
N-Anthracen-9-ylmethyl-N′-(4-ethylamino-butyl)-butane-1,4-
diamine, 19. A saturated sodium carbonate solution (20 mL) was
added to a vigorously stirred solution of 1b (500 mg, 1.09 mmol)
in CH2Cl2 (50 mL). The organic layer was separated and was
washed twice with saturated sodium carbonate. The combined
organic layers were dried with anhydrous Na2SO4, filtered, and
concentrated to give the free amine of 1b as a pale yellow thick
oil in 98% yield.
CH2Cl2 (15 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete
(monitored by NMR). The solvent was removed in vacuo, the solid
residue was dissolved in 50% MeOH/CH2Cl2 (40 mL), and the
solution was cooled to 0 °C. NaBH4 (4.50 mmol) was added in
small portions to the solution, and the mixture was stirred at rt
overnight. The solvent was removed in vacuo, and the solid residue
was dissolved in CH2Cl2 (50 mL) and washed with 10% aq Na2CO3
solution (3 × 30 mL). The CH2Cl2 layer was separated, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to give an
oily residue, 23 (95%), which was used in the next step without
1
further purification. H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H),
8.26 (d, 2H), 7.92 (d, 2H), 7.48 (m, 2H), 7.40 (m, 2H), 4.63 (s,
2H), 3.48 (t, 2H), 2.80 (t, 2H), 2.51 (t, 4H), 1.46–1.58 (m, 8H);
13C NMR (CDCl3) δ 131.7, 131.4, 130.1, 129.1, 127.0, 126.0,
124.8, 124.0, 62.4, 50.4, 49.6, 49.4, 45.8, 32.5, 28.7, 28.0, 27.7.
[4-(Anthracen-9-ylmethyl-tert-butoxycarbonyl-amino)-butyl]-
(4-hydroxy-butyl)-carbamic Acid tert-Butyl Ester, 24. The
synthesis of compound 24 has been reported previously.5
Methanesulfonic Acid 4-{[4-(Anthracen-9-ylmethyl-tert-bu-
toxycarbonyl-amino)-butyl]-tert-butoxycarbonyl-amino}-butyl
Ester, 25. The synthesis of compound 25 has been reported
previously.5
[4-(Anthracen-9-ylmethyl-tert-butoxycarbonyl-amino)-butyl]-
(4-methylamino-butyl)-carbamic Acid tert-Butyl Ester, 26. Com-
pound 25 (425 mg, 0.68 mmol) was dissolved in acetonitrile (10
mL) and added to a stirring solution of methylamine (4.77 mL of
2.0 M in MeOH, 9.55 mmol) in acetonitrile (20 mL). The mixture
was then stirred at 75 °C overnight under a N2 atmosphere. TLC
showed the absence of starting material 25 after 24 h and the
solution was then concentrated under reduced pressure. The residue
was dissolved in CH2Cl2 (30 mL) and washed three times with
aqueous sodium carbonate. The organic layer was separated, dried
with anhydrous Na2SO4, filtered, and concentrated under vacuum.
Flash column chromatography of the residue gave 26 as a yellow
oil (73%). 26: Rf ) 0.3 (MeOH/NH4OH/CH2Cl2, 8:1:91); 1H NMR
(300 MHz, CDCl3) δ 8.40 (s, 1H), 8.36 (d, 2H), 7.98 (d, 2H), 7.48
(m, 4H), 5.50 (s, 2H), 2.93–2.79 (m, 6H), 2.53 (t, 2H), 2.40 (s,
3H), 1.76–1.43 (m, 8H), 1.41–1.28 (m, 14H), 1.11 (m, 4H); 13C
NMR (CDCl3) δ 155.3, 131.3, 131.3, 129.2, 128.2, 126.3, 125.0,
124.1, 79.8, 79.0, 53.6, 51.9, 46.7, 44.5, 41.2, 36.6, 28.7, 28.6, 27.2,
26.1, 25.7. HRMS (FAB) m/z calcd for C34H49N3O4 (M + Na)+,
586.3615; found, 586.3608.
(4-tert-Butoxycarbonylamino-butyl)-[4-(3-cyano-propylamino)-
butyl]-carbamic Acid tert-Butyl Ester, 29. To a solution of the
amine 28 (1.32 g, 3.68 mmol) in anhydrous acetonitrile (20 mL)
was added potassium carbonate (1.7 g), and the suspension was
stirred at rt for 10 min. A solution of 4-bromobutyronitrile (0.54 g,
3.68 mmol) in acetonitrile (10 mL) was added, and the resulting
mixture was stirred at 50 °C under nitrogen for 24 h. TLC (1:10:
89 NH4OH/MeOH/CHCl3) showed the reaction was 95% complete.
The mixture was filtered and the volatiles were removed in vacuo
to give a semisolid residue. The residue was redissolved in CH2Cl2
(70 mL) and washed with saturated aq Na2CO3 (50 mL). The
organic layer was separated, dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The oily residue was purified by flash
column chromatography (MeOH/NH4OH/CH2Cl2, 7:1:92) to give
the product 29 as an oil in 62% yield. 1H NMR (300 MHz, CDCl3)
δ 4.60 (br s, 1H, NHCO), 3.15 (m, 6H, 3 × CH2), 2.74 (t, 2H,
CH2), 2.62 (t, 2H, CH2), 2.46 (t, 2H, CH2), 1.83 (quin, 2H, CH2),
1.55–1.39 (m, 26H, 4 × CH2, 6 × CH3); 13C NMR (CDCl3) δ
156.0, 155.6, 119.8, 79.3, 79.2, 53.6, 49.5, 48.1, 46.9, 40.4, 28.7,
28.6, 27.6, 27.3, 26.1, 25.9, 15.2.
To the stirred solution of the free amine of 1b (368 mg, 1.05
mmol) in DMF (6 mL), flame dried 4 Å powdered molecular sieves
(500 mg) and CsOH ·H2O (177 mg, 1.05 mmol) were added. The
solution was stirred for 30 min, bromoethane (77 mg, 0.96 mmol)
dissolved in DMF (3 mL) was added, and the reaction was stirred
overnight at rt. The molecular sieves were filtered off, DMF was
removed in vacuo, and the residue was dissolved in CH2Cl2 (50
mL) and washed with 10% aq Na2CO3 solution (3 × 30 mL). The
CH2Cl2 layer was separated, dried over anhydrous Na2SO4, filtered,
and removed in vacuo to give an oily residue. The oil was purified
by flash column chromatography (MeOH/NH4OH/CH2Cl2, 12:1:
1
87) to yield the product 19 as a pale yellow oil (21%). H NMR
(CDCl3) δ 8.37 (s, 1H), 8.30 (d, 2H), 7.98 (d, 2H), 7.52 (m, 2H),
7.44 (m, 2H), 4.70 (s, 2H), 2.87 (m, 2H), 2.57 (m, 8H), 1.45–1.57
(m, 8H), 1.09 (t, 3H); 13C NMR (CDCl3) δ 131.9, 131.6, 130.3,
129.2, 127.2, 126.2, 125.0, 124.2, 50.7, 50.0, 49.9, 46.0, 44.3, 28.2,
28.2, 28.1, 15.5.
Anthracen-9-ylmethyl-(4-{tert-butoxycarbonyl-[4-(tert-butoxy-
carbonyl-ethyl-amino)-butyl]-amino}-butyl)-carbamic Acid tert-
Butyl Ester, 20. Compound 7 (5 mg, 0.01 mmol) was dissolved
in a solution of triethylamine and methanol (10% TEA in MeOH,
10 mL). A solution of di-tert-butyl dicarbonate (11.20 mg, 0.05
mmol) in methanol (3 mL) was added dropwise to this solution
with vigorous stirring at 0 °C. The reaction was stirred at 0 °C for
30 min and then warmed slowly to rt. The solution was stirred
overnight. The methanol and TEA were removed in vacuo to yield
an oily residue that was dissolved in CH2Cl2 (30 mL) and washed
with a solution of 10% aq sodium carbonate (3 × 30 mL). The
CH2Cl2 layer was dried over anhydrous sodium sulfate, separated,
filtered, and concentrated in vacuo to provide a thick oil 20 (94%).
1H NMR (300 MHz, CDCl3) δ 8.45 (s, 1H), 8.38 (d, 2H), 8.02 (d,
2H), 7.51 (m, 4H), 5.52 (s, 2H), 3.15 (m, 4H), 2.60–2.97 (m, 6H),
1.05–1.59 (m, 38H).
4-(4-Amino-butylamino)-butan-1-ol, Hydrochloride Salt, 22.
A solution of Boc-derivative 21 (600 mg, 1.67 mmol) was dissolved
in absolute ethanol (20 mL) and stirred at 0 °C for 10 min. A 4 N
HCl solution (35 mL) was added to the reaction mixture dropwise
and stirred at 0 °C for 20 min and then at room temperature
overnight. The solution was concentrated in vacuo to give 22 as a
white solid in 97% yield. 1H NMR (300 MHz, D2O) δ 3.61 (t, 2H,
OCH2), 3.05 (m, 6H, NCH2), 1.75 (m, 6H, CH2), 1.61 (m, 2H,
CH2); 13C NMR (300 MHz, D2O): δ 60.9, 47.5, 46.9, 38.9, 28.4,
24.1, 22.9, 22.5.
(4-tert-Butoxycarbonylamino-butyl)-{4-[tert-butoxycarbonyl-
(3-cyano-propyl)-amino]-butyl}-carbamic Acid tert-Butyl Ester,
30. A solution of di-tert-butyl dicarbonate (0.92 g, 0.42 mmol) in
methanol (10 mL) was added dropwise to a vigorously stirred
solution of 29 (0.12 g, 0.28 mmol) in 10% TEA/MeOH at 0 °C.
The mixture was stirred at rt overnight. TLC (NH4OH/MeOH/
CHCl3, 1:10:89) showed the tert-butoxycarbonylation was complete.
The solvent was removed in vacuo to yield an oily residue that
4-{4-[(Anthracen-9-ylmethyl)-amino]-butylamino}-butan-1-
ol, 23. To a stirred solution of 22 (386 mg, 1.66 mmol) and TEA
(418 mg, 4.14 mmol) in 25% MeOH/CH2Cl2 (20 mL) was added
a solution of 9-anthraldehyde (310 mg, 1.50 mmol) in 25% MeOH/

Designing the Polyamine Pharmacophore
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2559
was then dissolved in dichloromethane (100 mL) and washed with
a solution of saturated aqueous Na2CO3 (3 × 30 mL). The organic
layer was dried over anhydrous Na2SO4, separated, filtered, and
concentrated in vacuo to give an oily residue, which was purified
by flash column chromatography (CHCl3/MeOH 99:1) to give 30
155.6, 131.4, 131.4, 131.0, 129.0, 127.2, 125.4, 125.3, 124.8, 79.3,
79.1, 52.7, 50.9, 47.5, 47.0, 46.8, 40.5, 28.7, 28.7, 27.6, 26.2, 25.8,
24.6, 12.2. HRMS (FAB) m/z calcd for C44H68N4O6 (M + H)+,
749.5212; found, 749.5210.
{4-[tert-Butoxycarbonyl-(4-tert-butoxycarbonylamino-butyl)-
amino]-butyl}-{4-[(4,6-dihydro-pyren-1-ylmethyl)-amino]-butyl}-
carbamic Acid tert-Butyl Ester, 34. To a stirred solution of 31
(150 mg, 0.28 mmol) in 25% MeOH/CH2Cl2 (20 mL) was added
a solution of 1-pyrenecarboxaldehyde (71 mg, 0.31 mmol) in 25%
MeOH/CH2Cl2 (15 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete (as
1
as an oil (94% yield), Rf ) 0.5 (CHCl3/MeOH, 98:2); H NMR
(CDCl3) δ 4.63 (br s, 1H, NHCO), 3.28 (t, 2H, CH2), 3.14 (m, 8H,
4 × CH2), 2.36 (t, 2H, CH2), 1.88 (quin, 2H, CH2), 1.58–1.35 (m,
35H, 4 × CH2, 9 × CH3); 13C NMR δ 155.9, 155.5, 79.9, 79.3,
79.0, 46.7, 45.8, 40.3, 28.6, 28.5, 27.5, 26.0, 25.7, 24.6, 14.9.
(4-Amino-butyl)-{4-[tert-butoxycarbonyl-(4-tert-butoxycarbo-
nylamino-butyl)-amino]-butyl}-carbamic Acid tert-Butyl Ester,
31. The nitrile 30 (500 mg, 0.95 mmol) was dissolved in ethanol
(30 mL). NH4OH (3 mL) and Raney nickel (1.5 g) were added
and ammonia gas was bubbled through the solution for 10 min at
0 °C. The suspension was hydrogenated at 50 psi for 24 h. Air
was bubbled through the solution and the Raney nickel was removed
by filtering through a sintered glass funnel, keeping the Raney nickel
residue moist at all times. The ethanol and NH4OH were removed
in vacuo, and the oily residue was dissolved in CH2Cl2 and washed
with 10% aq sodium carbonate (2 × 50 mL). The organic layer
was separated, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to give the product 31 as a light yellow oil
in 90% yield. The oil was used in the next step without further
1
monitored by H NMR). The solvent was removed in vacuo, the
solid residue was dissolved in 50% MeOH/CH2Cl2 (40 mL), and
the solution was cooled to 0 °C. NaBH4 (2.40 mmol) was added in
small portions to the solution, and the mixture was stirred at rt
overnight. The volatiles were removed in vacuo, and the solid
residue was dissolved in CH2Cl2 (50 mL) and washed with 10%
aq Na2CO3 solution (3 × 30 mL). The CH2Cl2 layer was separated,
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo
to give an oily residue. The oil was purified by flash column
chromatography (MeOH/NH4OH/CH2Cl2, 5:0.5:94.5) to yield the
product 34 as a pale yellow oil (70%), Rf ) 0.3 (5% MeOH/0.5%
1
NH4OH/CH2Cl2; H NMR (CDCl3) δ 8.32 (d, 1H), 8.07 (m, 4H),
7.96 (m, 4H), 4.43 (s, 2H), 3.06 (m, 10H), 2.78 (m, 2H), 1.20–1.63
(m, 39H); 13C NMR (CDCl3) δ 156.0, 155.5, 133.9, 131.3, 130.8,
130.6, 129.1, 127.6, 127.5, 127.1, 125.9, 125.1, 125.1, 125.0, 125.0,
124.9, 124.7, 123.2, 79.3, 79.2, 52.0, 50.0, 47.1, 46.8, 40.4, 28.7,
28.6, 27.7, 26.3, 25.8. HRMS (FAB) m/z calcd for C44H64N4O6 (M
+ H)+, 745.4899; found, 745.4872.
1
purification; Rf ) 0.5 (NH4OH/MeOH/CHCl3, 1:10:89). H NMR
(CDCl3) δ 4.65 (s, 1H, NHCO), 3.15 (m, 10H, 5 × CH2), 2.70 (t,
2H, CH2NH2), 1.58–1.39 (m, 39H, 6 × CH2, 9 × CH3); 13C NMR
(CDCl3) δ 155.9, 155.4, 79.2, 79.1, 47.0, 46.7, 40.3, 28.6, 28.5,
27.5, 26.1, 25.7. HRMS (FAB) calcd for C27H55N4O6 (M + 1),
531.4122; found (M + 1), 531.4111.
Acknowledgment. The authors thank Dr. David Powell at
the University of Florida mass spectrometry facility for his kind
assistance in acquiring the mass spectra of these compounds.
O.P. acknowledges the Broad Medical Research Program of the
Eli and Edythe L. Broad Foundation for their partial support of
this research and thank Dr. W. F. Flintoff (University of Western
Ontario) for his gracious donation of CHO-MG cell line.12
[4-({4-[(Anthracen-9-ylmethyl)-amino]-butyl}-tert-butoxycar-
bonyl-amino)-butyl]-(4-tert-butoxycarbonylamino-butyl)-car-
bamic Acid tert-Butyl Ester, 32. To a stirred solution of 31 (204
mg, 0.38 mmol) in 25% MeOH/CH2Cl2 (20 mL) was added a
solution of 9-anthraldehyde (87.2 mg, 0.42 mmol) in 25% MeOH/
CH2Cl2 (15 mL) under N2. The mixture was stirred at room
temperature overnight until the imine formation was complete (as
1
monitored by H NMR). The solvent was removed in vacuo. The
solid residue was dissolved in 50% MeOH/CH2Cl2 (40 mL) and
the solution was cooled to 0 °C. NaBH4 (2.40 mmol) was added in
small portions to the solution, and the mixture was stirred at rt
overnight. The solvent was removed in vacuo, and the solid residue
was dissolved in CH2Cl2 (50 mL) and washed with 10% aq Na2CO3
solution (3 × 30 mL). The CH2Cl2 layer was separated, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to give an
oily residue. The oil was purified by flash column chromatography
(MeOH/NH4OH/CH2Cl2, 6:0.5:93.5) to yield 32 as a pale yellow
viscous oil (72%), Rf ) 0.3 (MeOH/NH4OH/CH2Cl2, 6:0.5:93.5;
1H NMR (CDCl3) δ 8.35 (s, 1H), 8.29 (d, 2H), 7.96 (d, 2H), 7.44
(m, 4H), 5.24 (s, 2H), 3.12 (m, 10H), 2.87 (m, 2H), 1.24–1.55 (m,
39H); 13C NMR (CDCl3) δ 155.9, 155.5, 131.7, 131.5, 130.2, 129.1,
127.1, 126.1, 124.9, 124.1, 79.2, 79.2, 50.4, 47.0, 46.7, 45.9, 40.4,
28.7, 28.6, 27.6, 26.2, 25.8. HRMS (FAB) m/z calcd for C42H64N4O6
(M + H)+, 721.4893; found, 721.4904.
Supporting Information Available: Elemental analyses for
1
compounds 5-8, 10, 11, 18, 26, and 32-34 and the H and 13C
spectra of compounds 6-8, 10, 11, 16-20, 22-24, 26, and 29-34
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
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