Lysine-spermine conjugates: Hydrophobic polyamine amides as potent lipopolysaccharide sequestrants

Article abstract of DOI:10.1016/j.bmc.2005.01.038

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Thera

Full text of DOI:10.1016/j.bmc.2005.01.038

Bioorganic & Medicinal Chemistry 13 (2005) 2523–2536
Lysine–spermine conjugates: hydrophobic polyamine amides
as potent lipopolysaccharide sequestrants
Mark R. Burns,a Stewart J. Wood,b Kelly A. Miller,b Thuan Nguyen,b
Jens R. Cromerb and Sunil A. Davidb,*
aMediQuest, Inc., 4101 Stone Way North, Suite 220, Seattle, WA 98103, USA
bDepartment of Medicinal Chemistry, University of Kansas, Life Sciences Research Laboratories, 1501 Wakarusa Drive,
Lawrence, KS 66049, USA
Received 11 November 2004; revised 21 January 2005; accepted 21 January 2005
Abstract—Lipopolysaccharides (LPS), otherwise termed ꢀendotoxinsꢁ, are outer-membrane constituents of Gram-negative bacteria.
Lipopolysaccharides play a key role in the pathogenesis of ꢀSeptic Shockꢁ, a major cause of mortality in the critically ill patient.
Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist
at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS
and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically
viable strategy. In this paper, the interactions of a focused library of lysine–spermine conjugates with lipopolysaccharide (LPS) have
been characterized. Lysine–spermine conjugates with the e-amino terminus of the lysinyl moiety derivatized with long-chain
aliphatic hydrophobic substituents in acyl or alkyl linkage bind and neutralize bacterial lipopolysaccharides, and may be of use
in the prevention or treatment of endotoxic shock states.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
45%, both calling to attention the fact that aggressive
antimicrobial therapy alone is insufficient in preventing
mortality in patients with serious illnesses, and empha-
sizing an urgent, unmet need to develop therapeutic op-
tions specifically targeting the pathophysiology of sepsis.
Endotoxins, or lipopolysaccharides (LPS), the predomi-
nant structural component of the outer membrane of
Gram-negative bacteria,1,2 play a pivotal role in septic
shock, a syndrome of systemic toxicity, which occurs
frequently when the bodyꢁs defense mechanisms are
compromised or overwhelmed, or as a consequence of
antibiotic chemotherapy of serious systemic infections
(Gram-negative sepsis).3–5 Referred to as ꢀblood poison-
ingꢁ in lay terminology, Gram-negative sepsis is the thir-
teenth leading cause of overall mortality6 and the
number one cause of deaths in the intensive care unit,7
accounting for more than 200,000 fatalities in the US
annually.8 Despite tremendous strides in antimicrobial
chemotherapy, the incidence of sepsis has risen almost
threefold from 1979 through 20009 and sepsis-associated
mortality has essentially remained unchanged at about
The presence of LPS causes a widespread activation of
the innate immune response,10,11 leading to the uncon-
trolled production of numerous inflammatory media-
tors, including tumor necrosis factor-a (TNF-a),
interleukin-1b (IL-1b), and interleukin-6 (IL-6), primar-
ily by cells of the monocyte/macrophage lineage.12,13
The unregulated overproduction of these mediators, as
well as others, such as nitric oxide produced by the
endothelial cell,14,15 leads to a systemic inflammatory re-
sponse characterized by fever, hypotension, coagulopa-
thy, hemodynamic derangement, tissue hypoperfusion,
and multiple organ failure,16,17 culminating frequently
in death.
The therapy of septic shock remains primarily sup-
portive, and specific modalities aimed at limiting the
underlying pathophysiology are, unfortunately, as
yet unavailable. One possible approach to addressing
Keywords: Endotoxin; Lipopolysaccharide; Sepsis; Lysine–spermine
conjugates.
*
Corresponding author. Tel.: +1 785 330 4316; fax: +1 785 330
4332; e-mail: sdavid@ku.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.038

2524
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
therapeutically the problem of Gram-negative sepsis has
been to target LPS itself by the use of an agent that
would bind to, and sequester it. It has been shown by to-
tal synthesis18–21 that the toxicity of LPS resides in its
structurally highly conserved glycolipid component
called Lipid A.22,23 Lipid A is composed of a hydrophilic,
bis-phosphorylated diglucosamine backbone, and a
hydrophobic domain of 6 (E. coli) or 7 (Salmonella) acyl
chains in amide and ester linkages24–26 (Fig. 1). The an-
ionic and amphiphilic nature of lipid A (Fig. 1) enables
it to bind to numerous substances that are positively
charged and also possess amphipathic character. We
have, over the past decade, characterized the interactions
of lipid A with a number of classes of cationic amphi-
pathic molecules including proteins,27,28 peptides,29–33
pharmaceutical compounds,34,35 and other synthetic
polycationic amphiphiles.36–38 Importantly, from these
and currently ongoing studies, we have determined the
pharmacophore necessary for optimal recognition and
neutralization of lipid A35 by small molecules requires
two protonatable positive charges so disposed that the
distance between them are equivalent to the distance be-
protection in animal models of Gram-negative sepsis, are
synthetically easily accessible, and, importantly, are non-
toxic, on account of their degradation to physiological
substituents (spermine and fatty acid).37,44
Ongoing research in our laboratory seeks to systemati-
cally identify structural variations in the polyamine
backbone that would impart additional, enthalpically-
driven H-bond/van der Waals interactions. The lysine–
spermine derivatives described in this report exemplify
a group of compounds that incorporate stereogenic H-
bond donor/acceptor functionalities at one end of the
polyamine scaffold. Besides confirming the obligatory
requirement of a terminally-placed long-chain hydro-
phobic group for optimal endotoxin sequestration, we
find significant differences in both the binding affinity
and neutralization potency of L- and D-lysine conju-
gates, suggesting that an iterative substitution of the
polyamine backbone with H-bond donor/acceptor func-
tionalities with appropriate stereochemistry may yield
highly potent, yet nontoxic endotoxin neutralizers.
˚
tween the two anionic phosphates on lipid A (ꢀ14 A),
enabling ionic H-bonds between the phosphates on the
lipid A backbone and the positive charges on the com-
pound. In addition, appropriately-positioned pendant
hydrophobic functionalities are necessary to further en-
hance binding affinity and stabilize the resultant com-
plexes via hydrophobic interactions with the polyacyl
domain of lipid A (for a recent review, see Ref. 39). These
structural requisites were first identified in certain mem-
bers of a novel class of compounds, the lipopolyamines,
which were originally developed, and are currently being
used as DNA transfection (lipofection) reagents.40–43
Compounds of the conjugated spermine class are of par-
ticular interest because they are active in vivo and afford
2. Results and discussion
2.1. Chemistry
The method used for the synthesis of lysine–spermine
conjugates enabled selective functionalization of the e-
nitrogen atom of lysine and chromatographic purifica-
tion prior to exposure of the extremely polar amine
groups. Specifically, blockage of the polar amino groups
on the polyamine conjugates used Boc-carbamates,
allowing normal-phase SiO2 chromatography instead
of the more time-consuming ion-exchange method pre-
viously reported.45 Synthesis of these analogues, shown
in Scheme 1, began by coupling the free base of sperm-
OH
O
HO
O
O
O
Spermine
1
Boc-
L
-Lys(Cbz)-ONp
+
(HO)2P
O
O
2
O
O
O
P(OH)2
NH
NH
O
O
O
a,b
O
RHN
O
HO
O
HO
H
H
N
Boc
N
O
O
BocHN
N
NHBoc
Boc
O
R = Cbz, 3
R = H, 4
c
d or e, f
14
H
N
14
14
R
12
X
H
O
14
H
N
H
N
14
H2N
N
NH2
H
X = O or
X = H, H
Scheme 1. Synthesis of lipophilic lysine–spermine conjugates. Condi-
tions and reagents: (a) CH3OH; (b) Boc2O, THF/H2O, Na2CO3;
(c) H2, Pd/C, EtOH; (d) RCOCl, Et3N, CH2Cl2; (e) 1. RCHO,
CH(OCH3)3, Et3N, CH2Cl2, 2. NaBH4, CH3OH; (f) 3 N HCl/CH3OH.
Figure 1. Structure of lipid A, the toxic moiety of bacterial lipopoly-
saccharide. Numbers indicate acyl chain length.

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
2525
ine 1 with either the L- or D-stereoisomer of the ortho-
6
gonally-protected, active ester Boc-Lys(Cbz)-ONp 2.
Dropwise addition of the active ester to a solution of
spermine gave the statistical distribution of mono-, di-,
and un-substituted products. Reaction of the remaining
unsubstituted amino groups of spermine with an excess
of Boc2O produced the per-Boc mixture. The resulting
mixture could now be separated by standard silica gel
chromatography. The purified mono-acylated derivative
3 was then subjected to catalytic hydrogenation in order
to remove the Cbz-protecting group and gain the free
amino intermediate 4. It was found to be critical to
use ketone-free ethanol during this hydrogenation in or-
der to prevent formation of a higher Rf, alkylated side-
product. The amine 4 was then functionalized by stan-
dard acylation or reductive alkylation conditions to
produce the protected forms of the Lys–spermine
analogues. For the mono-alkyl derivatives, the imines
were pre-formed then reduced using NaBH4. In the case
of dialkylated analogues 30 and 37, excess aldehyde was
used in a reductive amination reaction with NaBH3CN.
In several cases unique functional groups were synthe-
sized using common reaction conditions or from com-
mercial sources. The derivatized intermediates were
purified using SiO2 chromatography, and the Boc-
groups removed using 3 N HCl in MeOH to afford the
Lys–spermine analogues in their HCl salt forms. Com-
7
P
P
P
8
P
P
11
14
17
27
25
37
28
26
PMB
P
P
120
60
P
P
P
P
P
P
P
10-7
10-6
10-5
10-4
10-3
Compound Concentration (M)
Figure 2. Binding affinity of compounds to LPS determined by the
BODIPY-Cadaverine displacement method. Polymyxin B (PMB) was
used as the reference compound. Alkyl compounds are depicted with
open symbols and dotted lines, and acyls with closed symbols and solid
lines. The Z0 factor of the HTS assay for quantifying ED50 (relative
binding affinity) is 0.82, and the CVs at 0% and 100% probe
displacement are 4.1% and 6.2%, respectively.51
1
pounds were characterized by TLC, H and 13C NMR,
and LC/MS and all spectra were consistent with struc-
tures assigned.
LPS-bound BC being relatively insensitive to hydropho-
bic substituents, and dominated by electrostatic interac-
tions.46 The consequence of this limitation is the lack of
discrimination between ligands that merely bind LPS,
and those that truly neutralize LPS activity. All promis-
ing leads are also therefore routinely screened in NO
inhibition assays. Chain lengths were critical determi-
nants for NO-inhibiting activity as shown by the alkyl
homologs C6 31 (>1000 lM), C7 29 (46 lM), D11-C16
27 (0.66 lM), as well as the acyl series from C8 17
(160 lM) and to C20 7 (1.2 lM).
2.2. Structure–activity relationships: binding affinity
and in vitro neutralization potency
We examined the relative binding affinities of the Lys–
spermine analogues with a recently-described46 high-
throughput fluorescence based displacement assay,
using BODIPY-TR cadaverine (BC), and are reported
as half-maximal effective displacement of probe (ED50)
in Figure 2, and Tables 1–3. Murine monocytes
(J774A.1 cells) produce measurable quantities of NO
on exposure to LPS and provide a model for the rapid
assessment of compounds to neutralize LPS activity.
Compounds that neutralize LPS inhibit NO production
in a dose-dependant manner from which 50% inhibitory
concentrations (IC50) can be determined, as shown in
Figure 3, and Tables 1–3. In all experiments, Polymyxin
B (PMB), a decapeptide antibiotic, known to bind and
(ii) Chain unsaturation. trans-Unsaturation of the acyl
chain was found to increased binding as shown by com-
paring D9-L-Lys-C16 15 (3.8 lM) with L-Lys-C16 14
(11 lM), and D11-L-Lys-C18 10 (4.2 lM) with L-Lys-
C18 9 (16 lM). Similarly for the alkyls, the cis-unsatu-
rated L-Lys-D11-C16 27 displayed a higher ED50 of
2.6 lM compared to its saturated counterpart, C16 26
(5.6 lM). However, this is not paralleled by an improve-
ment in LPS-neutralizing activity; for instance, the fully
saturated L-Lys-C16 14 (IC50: 6.4 lM), and the unsatu-
rated 15 (IC50: 8.8 lM) are equipotent. We surmise that
the observed enhanced affinity with the unsaturated ana-
logues may also be an artifactual consequence of the
probe displacement method. The unsaturated com-
pounds are, in general marginally more water soluble
than their saturated homologs, and thus may exhibit a
higher effective local concentration at the LPS-bulk sol-
vent interface.34 It is to be noted that in vitro bioassays,
as well as in animal models, the problem of differential
solubility is mitigated by the presence of physiological
concentrations of albumin, which serve to solubilize
neutralize LPS,29,47,48 was used as
compound.
a
reference
(i) Hydrocarbon chain length. Lysine–spermine ana-
logues with an unsubstituted e-amino lysine, 5 (L-Lys,
ED50: 40 lM), and 6 (D-Lys, ED50: 58 lM) showed poor
binding in the displacement assays, and negligible inhi-
bition of LPS-induced NO production. Substitution of
the e-amino group of lysine manifests in an increase in
affinity (Table 1), but no striking correlation between
hydrocarbon chain-length and affinity is evident (Fig.
4, inset). In contrast, increasing carbon chain length is
clearly correlated to the potency of inhibition of LPS
activity (Fig. 4). We have earlier shown that this appar-
ent discordance is attributable to the displacement of

2526
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
Table 1. Lysine–spermine long acyl chain homologs
R2
R1
N
H
N
H
N
H2N
N
H
NH2
*
O
D, L
IDR
R2
Note
Stereo
ED50 (lM)
NO IC50 (lM)
1
5
6
H
H
H
L
40.42
58.42
>1000
>1000
H
D
O
O
O
O
O
7
H
H
H
H
H
H
H
H
H
H
H
C20
L
D
L
L
L
L
D
L
L
L
L
6.46
1.21
8
C18
8.8
1.98
9
C18
16.39
4.2
18.14
NA
10
11
12
13
14
15
16
17
D11, C18
C17
6.71
5.93
9.94
10.74
3.82
5.63
12.97
4.49
O
O
C16
NA
S
O
C16
2.29
O
O
O
O
C16
6.41
D9, C16
C14
8.85
1.60
C8
162.24
both LPS and ligand.28 Unsaturation of the hydropho-
bic substituent, therefore, while not expected to result
in higher potency compounds, is a potentially useful
strategy that we are presently evaluating in enhancing
the solubility of other, less-soluble analogues.
(iv) Stereochemistry of Lys residue. Inverting the stereo-
chemistry of the a-carbon of lysine caused no apprecia-
ble effect on binding affinity for the stereoisomeric pair
D-Lys-C16 13 (10 lM) and L-Lys-C16 14 (11 lM), but a
distinct enhancement for longer chain D-Lys-C18
8
(8.8 lM), as compared to L-Lys-C18 9 (16 lM). This is
consistent with the higher potency for the D-analogues
in inhibition NO production. The D-stereoisomers are
expected to be less susceptible to proteolytic cleavage,
and the higher observed activity may be a consequence
of the longer persistence of the D-isomer in the assay sys-
tem. It is also possible that the lipid A moiety is a chiral,
and the mode of binding may be effected by the configu-
ration of asymmetric centers. Future isothermal titration
calorimetry experiments will help to clarify this issue.
(iii) Steric interactions. Although analogues with short
bulky substituents showed increased binding with
increasing chain carbon number, for instance, comp-
aring isobutyl 35 (101 lM) with 32 (9.6 lM) derived
from (S)-(ꢁ)-citronellal and the bis-alkylated meth-
ylcyclohexyl 37 (4.0 lM) (Table 3), none of these com-
pounds are potent LPS neutralizers, as are the di- and
tri-ether homologs 24 and 25 (IC50: >1000 mM) and
the polyethylene glycol polymer 23 (320 lM). The
biphenyls 38 and 21 and anthracene 22 all yielded rea-
sonably high LPS affinities (ED50: 3.7, 7.9, and
7.1 lM, respectively) but were poor inhibitors of LPS
bioactivity (IC50: >100 lM). These results emphasize
the obligatory requirement for long-chain aliphatic
hydrocarbon substituents for optimal biological
potency.
(vi) Alkylation versus acylation. Alkyl compounds bound
more strongly than their acyl equivalents; compare, for
example: alkyl C16 26 (5.6 lM) and acyl C16 14
(11 lM). This may be attributable to the loss of a pro-
tonatable positive charge on acylating the e-amino
group, leading to poorer solubility, as mentioned earlier.

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
Table 2. Lysine–spermine mixed acyl analogues
2527
R2
R1
N
H
N
H
N
H2N
N
H
NH2
*
O
D, L
IDR
18
R2
Note
Stereo
ED50 (lM)
NO IC50 (lM)
1
O
H
H
D
D
298.85
>1000
>1000
O
19
20
327.04
16.16
O
O
H
H
D
D
NA
21
22
7.86
7.09
124.30
H
L
108.10
323.54
O
O
23
24
25
H
H
H
Polymer
L
L
L
310.95
572.5
O
n
O
O
O
>1000
O
O
O
O
O
495.19
>1000
2.3. Comparison of IC50 and ED50
bind to the conserved lipid A portion, we expected that
there would be little variation in binding to a diverse
range of LPS from different bacteria. As shown in Figure
6, the highest affinity Lys–spermine analogues were
shown to consistently bind to LPS from different bacteria
in the 1–10 lM region and the relatively poor binders
bound to all the LPS in the 10–100 lM range. This clearly
shows that the Lys–spermine compounds bind to a vari-
ety of LPS structures, and thus may be clinically useful.
The graph of IC50 versus ED50 values display a linear
trend with a correlation coefficient of R = 0.60 (Fig. 5).
LPS binders with strong hydrophobic interactions
strayed from linearity due to the BC-LPS displacement
assay not accurately predicting hydrophobic interac-
tions, which have been shown to be crucial for LPS neu-
tralization. This is seen also for the aromatic and bulky
substituents, which are relatively bereft of biological
activity in contrast to their high binding affinities and
so appeared as a cluster in the upper left hand side of
the IC50 versus ED50 graph (Fig. 5).
2.5. Dose-dependent inhibition of proinflammatory cyto-
kines in human whole blood, determined by multiplexed
cytometric bead assay
2.4. Comparison of LPS from different Gram-negative
bacteria
Having verified that the Lys–spermine compounds were
active in inhibiting NO production in murine macro-
phages, we wished to independently confirm that they
would also inhibit LPS-induced inflammatory responses
in human cells. The activity of a subset of active Lys–
spermine compounds was examined for their ability to
inhibit TNF-a and IL-6 production in whole human
Although the structure of lipid A is highly conserved
among Gram-negative bacteria, the polysaccharide
domain is highly diverse among Gram-negative bacte-
ria.49,50 Since the Lys–spermine library was designed to

2528
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
Table 3. Lysine–spermine mixed alkyl analogues
R2
R1
N
H
N
H
N
H2N
N
H
NH2
*
O
D, L
IDR
R2
Note
Stereo
ED50 (lM)
NO IC50 (lM)
1
H
H
C16
L
L
5.56
2.59
NA
26
27
D11, C16
0.66
28
29
30
31
H
H
R1
H
C7
D
L
3.86
5.99
2.14
7.13
>100
46.43
38.83
>1000
C7
bis, C7
C6
L
D
32
33
H
H
D
L
9.55
>1000
>1000
12.07
34
35
H
H
D
D
10.93
838.41
72.88
100.58
36
H
D
16.08
>1000
37
38
R1
H
bis
L
4.04
3.71
>1000
105.12
D
blood, stimulated ex vivo with LPS. As shown in Figure
7, the rank-order of the inhibitory potencies in this assay
generally parallels NO inhibition activity, 8 being almost
as potent as polymyxin B, the reference compound.
gates such as DOSPER37 had shown the window of pro-
tection to be very short, a 15 min window of protection.
We wished to examine if 8, with its greater anticipated
hydrolytic stability, would afford a more extended
time-window of protection. 200 lg of 8 in a final volume
of 0.2 mL injections were administered intraperitoneally
at times of ꢁ6, ꢁ4, ꢁ2, 0, +1, and +2 relative to time-
zero, the time at which all mice were challenged with
200 ng/mouse LPS injections. Compound 8 provided
significant protection up to 6 h prior to LPS challenge
(Table 5). Based on these results, another time-course
experiment with subcutaneous, rather than i.p. injections
was undertaken with a much longer time window (ꢁ24,
ꢁ16, ꢁ12, ꢁ8, ꢁ4, 0, and +2 h relative to the time of
LPS administration). We wished to test if in this treat-
ment regime, which is characterized by a slow, gradual
systemic absorption from the site of injection, a longer
duration of protection would be observed. Lethality
was once again assessed 24 h following the final injection.
Two of the five mice in the ꢁ24 cohort survived, as did
three of the five in the ꢁ16, ꢁ12, and ꢁ8 cohorts (Table
6), indicating significant protection even when the com-
pound is administered 16 h ahead of LPS challenge.
These results indicate a significantly prolonged temporal
window of protection compared to DOSPER.37
2.6. Protective effects in a mouse model of endotoxic
shock
Based on the results of the displacement assays, NO, and
cytokine inhibition data, 8 was elected for detailed eval-
uation in animal experiments. We had previously deter-
mined that the LD100 (lethal dose—100%) dose was
100 ng per mouse (female, outbred, CF-1 mice, sensitized
with 800 mg/kg D-galactosamine). In all experiments
reported in this paper, a supralethal dose of 200 ng per
mouse, in a final volume of 0.2 mL saline was used.
The dose–response of protection afforded by 8 is de-
picted in Table 4. It is noteworthy that full protection
against the supralethal LPS challenge is observed at a
dose of 200 lg/mouse of 8. In these dose–response exper-
iments, mice received graded doses of compound diluted
in saline intraperitoneally (i.p.), in one flank, immedi-
ately before a supralethal (200 ng) LPS challenge, which
was administered as a separate i.p. injection into the
other flank. Previous studies with labile spermine conju-

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
2529
-1
-2
-3
-4
-5
-6
31
-4.6
-4.8
-5.0
-5.2
-5.4
-5.6
0.4
0.3
0.2
0.1
P
17
P
14
13
8
31
P
11
7
16
P
PMB
7
8
11
13
14
15
16
17
31
15
P
4
6
8
10
12
14
16
18
20
22
17
Carbon Length
29
P
15
14
11
13
16
P
8
7
P
P
P
P
P
P
P
P P
4
6
8
10
12
14
16
18
20
22
10-7
10-6
10-5
10-4
10-3
Carbon Length
Compound Concentration (M)
Figure 4. Correlation of NO inhibitory potency with carbon-length of
straight-chain acyl/alkyl analogues. Inset: Correlation between binding
affinity (BC Displacement) and carbon chain length. The poor
correlation is a consequence of the relative insensitivity of the probe
displacement method to discriminate between binders and true
neutralizers.
Figure 3. Nitric oxide (NO) inhibition in murine J774A.1 cells
stimulated for 14 h with 10 ng/mL E. coli 0111:B4 LPS, by polymyxin
B (reference compound), acyl (closed symbols) and alkyl (open
symbols) Lys–spermine analogues. Inhibition constant (IC50) values,
determined from the four-parameter logistic fits, are presented in
Tables 1–3. The CVs for the NO assay is 3.2%.
107
106
3. Conclusions
In conclusion, the interactions of a focused library of
lysine–spermine conjugates with Gram-negative bacte-
rial lipopolysaccharides have been characterized. Ly-
sine–spermine conjugates with the e-amino terminus of
the lysinyl moiety derivatized with long-chain aliphatic
hydrophobic substituents in acyl or alkyl linkage bind
to the lipid A moiety of LPS, and neutralize their toxic-
ity. The presence of long-chain aliphatic hydrophobic
functionalities is an obligatory requirement for biologi-
cal activity. The utilization of nontoxic and ubiquitous
building blocks (spermine, lysine, and long-chain fatty
acid) in the synthesis of these compounds would predict
low systemic toxicity, and are therefore excellent leads in
the development of novel therapeutic agents aimed at
the prevention or treatment of endotoxic shock states.
5
18
19
24
25
6
105
104
103
102
101
100
10-1
33
31
38
34
23
39
15
35
30
11
22
14
13
16
R = 0.60
103
100
101
102
ED (µM)
50
Figure 5. Correlation of binding affinity of the Lys–spermine ana-
logues (ED50) determined by BC fluorescent probe displacement, with
NO inhibition (IC50) in murine J774 cells.
4. Experimental
4.1. General synthetic methods
40 lm flash chromatography packing, respectively.
TLC analysis used the following solvent systems with
detection by ninhydrin staining: (a) hexane/ethyl ace-
tate/methanol 48:48:4; (b) 2-propanol/pyridine/glacial
acetic acid/H2O, 4:1:1:2; (c) CHCl3/MeOH/NH4OH
85:15:1. LC/MS analyzes were performed using a Gilson
The sources of all chemical reagents and starting mate-
rials were of the highest grade available and were used
without further purification. Thin-layer chromatogra-
phy analysis and column chromatography were per-
formed using Merck F254 silica gel plates and Baker

2530
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
Table 4. Dose-dependent protection of CF-1 mice challenged with a
supralethal dose 200 ng/mouse by compound 8 in cohorts of five
-4
P. aeruginosa
E. coli
10
animals
S. typhimurium
V. cholera
Amount of
compound used
(lg/mouse)
No of live
mice/no of
total mice tested
S. flexnerii
0
10
0/5
0/5
-5
10
50
1/5
100
200
4/5*
5/5*
Lethality was recorded at 24 h post-LPS injection. Ratios denote live/
total animals. Asterisks indicate statistical significance (P < 0.05;
Fisher one-tailed exact test).
-6
10
1
2
3
4
5
6
7
8
9
Primary-Screen Rank
Table 5. Time-course protection afforded by 8 in the D-galactosamine
sensitized CF-1 mouse lethality model
Figure 6. Lysine–spermine compounds bind to LPS isolated from
diverse Gram-negative bacteria. The rank-order of binding potency is
consistent.
Time of LPS
administration (h)
No of live mice/no
of total mice tested
ꢁ6
ꢁ4
ꢁ2
0
3/5
4/5*
4/5*
4/5*
0/5*
2/5
+1
+2
Animals were injected with 200 lg of 8 intraperitoneally at times noted
with respect to LPS challenge (200 ng/mouse). Lethality was recorded
at 24 h following LPS injection. Asterisks indicate statistical signifi-
cance (P < 0.05; Fisher one-tailed exact test).
2
P
P
Table 6. Time-course protection afforded by compound 8 in the
D-galactosamine sensitized CF-1 mouse lethality model
P
P
P
P
P
0
P
Time of LPS
administration (h)
No. of live mice/total
no. of mice tested
-7
-6
-5
-4
10
10
10
10
ꢁ24
ꢁ16
ꢁ12
ꢁ8
ꢁ4
0
2/5
3/5
3/5
3/5
5/5*
1/5
1/5
2.0
1.5
1.0
0.5
0.0
P
PM B
8
30
38
+2
Animals were injected with 200 lg of 8 subcutaneously at times noted
with respect to LPS challenge (200 ng/mouse). Lethality was recorded
at 24 h following LPS injection. Asterisks indicate statistical signifi-
cance (P < 0.05; Fisher one-tailed exact test).
P
P
P
P
P
P
P
P
Detection was by a Finnigan AQA operating in ESI+
mode (m/z range 140–1600 amu) together with an Agi-
lent 1100 series DAD detector (UV range 220–
320 nm). Gradient elution from 2 to 7 min. was per-
formed using 2–100% CH3CN in H2O (both with
0.05% heptafluorobutyric acid added as the volatile
ion-pairing reagent). 1H and 13C NMR spectra were
recorded at 500 and 125.8 MHz, respectively, on a
Brucker WM500 spectrometer at the University of
10-7
10-6
10-5
10-4
Figure 7. Inhibition by select Lys–spermine compounds of proinflam-
matory cytokines TNF-a and IL-6 in human blood stimulated with
10 ng/mL E. coli 0111:B4 LPS. Means of triplicates of a single
representative experiment are shown. The CV for the cytokine
measurements are 4.3%.
322 HPLC system coupled to a 215 liquid handler.
Retention of these polar molecules on C-18 reverse-
phase HPLC media was facilitated by the use of 0.05%
heptafluorobutyric acid as an ion-pairing reagent in
the mobile phase. This allowed analysis of the com-
pounds in their underivatived forms.
1
Washington, Seattle. H NMR signals were generally
multiples unless otherwise noted as s = singlet, d = dou-
blet, or t = triplet. Chemical shifts are relative to exter-
nal 3-(trimethylsilyl)-1-propanesulfonic acid, sodium
salt.

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
2531
4.2. Synthetic methods for precursor compounds
(5.5 mL, 3.0 equiv) and dry CH2Cl2 (100 mL) via syringe
under an atmosphere of argon. The resulting solution
was chilled to 0 ꢁC in an ice bath and palmitoyl chloride
(6.0 mL, 1.5 equiv) was added via syringe. After stirring
under an argon atmosphere overnight TLC analysis (c)
showed the expected product had formed. The solution
was diluted in CH2Cl2 (100 mL) and H2O (100 mL). The
organic layer was removed and the aqueous layer was
extracted twice more with CH2Cl2 (2 · 100 mL). The
combined organic layer was extracted with ice cold
0.1 N HCl (100 mL) then brine and dried over MgSO4,
filtered, and concentrated to give the crude oil. This
was purified via silica gel chromatography (column
dimensions 8 · 17 cm) using stepwise elution with hex-
anes/EtOAc 1:1 containing 0%, 2%, 3%, 4%, 5%, and
6% MeOH (500 mL each) to give the Boc-protected
product 13 as a clear oil (6.48 g, 51%). Removal of the
protecting groups was accomplished by treating a stirred
solution of the above product (6.48 g, 6.68 mmol) in
MeOH (50 mL) with 6 N HCl (50 mL) at room temper-
ature. After 3 h TLC analysis (b) showed the reaction
was complete. The solvents were evaporated to give
the desired product 14 in its 4HCl salt form as a white
solid (4.78 g, 100%). TLC analysis (b); Rf = 0.19. 1H
NMR (D2O, d): 3.93 (1H), 3.45 (1H), 3.03 (13H), 2.12
(2H), 2.02 (2H), 1.85 (4H), 1.75 (s, 4H), 1.43 (4H),
1.32 (2H), 1.11 (24H), 0.72 (t, 3H). 13C NMR (D2O,
ppm): 175.7, 169.8, 53.4, 46.7 (m), 45.2, 44.6, 38.8,
36.5, 36.1, 31.9, 30.4, 30.0 (m), 29.9 (m), 29.6 (m),
29.4, 28.2, 25.7, 25.5, 23.6, 22.8, 22.7, 22.3, 21.7, 13.8.
LC/MS (ret time, 7.2 min), calcd for C32H68N6O2 m/z
568, obsd 569 (MH+). Anal. Calcd for C32H72Cl4N6O2:
C, 53.77; H, 10.15; N, 11.76. Found: C, 53.51; H, 10.09;
N, 11.51.
4.2.1. Boc-L-Lys(Cbz)-N1-spermine-Boc3 (3). To a stirred
solution of spermine 1 (11.30 g, 1.4 equiv, free base
form) in MeOH (200 mL) was added dropwise over
1.5 h the active ester 2 (20.0 g, 40 mmol) in MeOH
(200 mL) at room temperature. After this dropwise
addition, TLC analysis (b) showed the expected mixture
of products had formed (di-substituted side-product
Rf = 0.76; mono-substituted desired product Rf = 0.50
and un-substituted spermine Rf = 0.08). If the optimal
ratio was not produced additional active ester in MeOH
was added dropwise. After stirring for 2 h, the solvent
was evaporated to give a yellow solid that was sus-
pended in THF (300 mL) and H2O (100 mL). A solution
of di-tert-butyl carbonate (43.5 g, 5.0 equiv) in tetrahy-
drofuran (50 mL) was added at room temperature.
The pH was adjusted periodically to ꢀ10 with a 10%
Na2CO3 solution.
A precipitate was noted after
10 min. After stirring for 18 h, TLC analysis (a) showed
the expected products had formed (elution order had in-
verted from that given above). Most of the THF was
evaporated in vacuo. The resulting mixture was dis-
solved in EtOAc (400 mL) and H2O (400 mL). The
organic layer was removed and the aqueous layer was
re-extracted with EtOAc (3 · 400 mL). The combined
organic layers were washed with ice-cold 0.1 N HCl
(2 · 250 mL) followed by brine. The organic layer was
dried over MgSO4, filtered, and concentrated to give a
crude oil, which was purified via silica gel chromatogra-
phy (column dimensions 8 · 17 cm) using stepwise elu-
tion with 1:1 hexanes/EtOAc containing 0%, 2%, 3%,
4%, and 5% MeOH (1 L each). The order of elution is
Boc4-spermine (25% yield (spermine can be recovered
after acid deprotection and conversion to the free base)),
the desired mono-substituted Boc-Lys(Cbz)-spermine-
Boc3 3 (19.4 g, 56% yield) and finally eluting last was
4.4. Representative mono-alkylation reaction
1
the di-substituted side-product. H NMR of the desired
product showed this to be a mixture of cis- and trans-
carbamate rotomers. It was used in the next reactions
without further characterization.
4.4.1. L-Lys(3,3-dimethyl-1-butane)-N1-spermine (18). To
0.58 g (0.82 mmol) of amine 4 in 5 mL of dry CH2Cl2
under argon was added 0.27 mL (3 equiv) of trimethyl-
orthoformate, 0.17 mL of Et3N (1.5 equiv), and
0.31 mL (3 equiv) of 3,3-dimethylbutyraldehyde. The
resulting solution was stirred at rt for 2 h when the sol-
vents were evaporated. The oily residue was dissolved in
5 mL of CH3OH and 70 mg (2 equiv) of NaBH4 was
added. After 2 h the solvent was evaporated and the res-
idue was partitioned between 0.01 N HCl and CH2Cl2
(50 mL each). The aqueous part was washed with an
additional portion of CH2Cl2 and the combined organic
layers were washed with brine, dried with MgSO4, and
evaporated to give 0.64 g crude oil. Column chromato-
graphy used CHCl3/MeOH/concd NH4OH 96:4:0.2 to
give 0.31 g (64% yield) pure protected product. This
was dissolved in 3 mL of CH3OH and treated with
3 mL of 6 N HCl at rt for 3 h. Evaporation gave
0.24 g (96% yield) of 18 as a white solid. TLC analysis
(b); Rf = 0.21. 1H NMR (D2O, d): 3.95 (1H), 3.31
(2H), 3.05 (14H), 2.04 (2H), 1.88 (4H), 1.71 (6H), 1.53
(2H), 1.41 (2H), 0.88 (9H). LC/MS (ret time, 6.1 min),
calcd for C22H50N6O m/z 414, obsd 415 (MH+). Anal.
Calcd for C22H55Cl5N6OÆ1/2H2O: C, 43.61; H, 9.31; N,
13.87. Found: C, 43.63; H, 9.37; N, 13.84.
4.2.2. Boc-L-Lys-N1-spermine-Boc3 (4). To a stirred solu-
tion of the orthogonally protected lysine–spermine con-
jugate 3 (19.4 g, 22.5 mmol) in EtOH (200 mL, must use
ketone and aldehyde free EtOH!) was added palladium
10 wt % on activated carbon (10.0 g) in a round-bottom
flask. The reaction flask was purged 3· with H2 then
placed under 5 psi H2 pressure. After stirring for 4.0 h
at room temperature, TLC analysis (c) showed the reac-
tion was complete. An extra amount of activated char-
coal was added to the mixture and the catalyst was
removed by filtering over a pad of Celite. The pad was
washed with EtOH (2 · 50 mL) and the combined
filtrates were evaporated to give 4 as white foam in
quantitative yield. Following evaluation by the above
TLC system this product was used directly in the next
steps.
4.3. Representative acylation reaction
4.3.1. L-Lys(palmitoyl)-N1-spermine (14). To the amine
precursor 4 (9.66 g, 13.22 mmol) was added Et3N

2532
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
4.5. Representative di-alkylation reaction
4.6.4. D-Lys-e-(stearoyl)-N1-spermine (8). TLC analysis
(b); Rf = 0.13. 1H NMR (D2O, d): 3.94 (1H), 3.47
(1H), 3.06 (13H), 2.13 (2H), 2.04 (2H), 1.87 (4H), 1.75
(4H), 1.47 (4H), 1.36 (2H), 1.16 (28H), 0.79 (3H). 13C
NMR (D2O, ppm): 175.9, 170.1, 53.4, 47.1 (m), 45.5,
44.7, 39.0, 36.8 (m), 36.0, 31.9, 30.6, 29.8 (m), 29.6,
29.3, 28.4, 25.9, 25.7, 23.8, 23.2, 23.1, 22.8, 13.8. LC/
MS (ret time, 7.4 min), calcd for C32H68N6O2 m/z 597,
obsd 598 (MH+).
4.5.1. L-Lys-e-(bis-(n-heptyl))-N1-spermine (30). A solu-
tion containing 0.22 g (0.30 mmol) of amine 4,
0.42 mL (3 mmol, 10 equiv) of n-heptanal, and 0.19 g
(3 mmol, 10 equiv) of NaBH3CN in 10 mL of CH3OH
was treated with glacial HOAc (five drops). The pH
was measured to be 4 by paper. Following overnight
stirring the solvent was evaporated and the residue
was partitioned between 1 N NaOH and CH2Cl2
(50 mL each). An additional CH2Cl2 wash of the aque-
ous layer was performed and the combined organic lay-
ers were washed with brine, dried over MgSO4, and
evaporated to give 0.33 g crude product. Column
chromatography used CHCl3/MeOH/concd NH4OH
(96:4:0.2) to give 0.20 g (71% yield) pure protected
product. This was dissolved in 1 mL of CH3OH and
treated with 1 mL of 6 N HCl at rt for 3 h. Evapora-
tion gave 0.11 g (73% yield) of 30 as a white solid.
4.6.5. L-Lys-e-(stearoyl)-N1-spermine (9). LC/MS (ret
time, 7.4 min), calcd for C34H72N6O2 m/z 597, obsd
598 (MH+).
4.6.6. L-Lys-e-(heptadecanoyl)-N1-spermine (11). TLC
1
analysis (b); Rf = 0.19. H NMR (D2O, d): 3.96 (1H),
3.47 (1H), 3.08 (13H), 2.14 (2H), 2.04 (2H), 1.87 (4H),
1.78 (4H), 1.50 (4H), 1.36 (2H), 1.22 (26H), 0.78 (3H).
13C NMR (D2O, ppm): 175.7, 169.9, 53.2, 47.2 (m),
45.4, 44.8, 39.0, 36.6 (m), 36.1, 31.9, 29.9 (m), 29.5,
29.3, 28.4, 25.8, 25.7, 23.8, 22.6, 22.5, 22.2, 22.0, 14.8.
LC/MS (ret time, 7.2 min), calcd for C33H70N6O2 m/z
583, obsd 584 (MH+).
1
TLC analysis (b), Rf = 0.21. H NMR (D2O, d): 3.93
(t, 1H), 3.28 (2H), 3.04 (16H), 2.02 (2H), 1.85 (4H),
1.71 (s, 4H), 1.62 (6H), 1.40 (4H), 1.25 (14H), 0.81
(t, 6H). 13C NMR (D2O, ppm): 175.7, 169.8, 53.4,
46.7 (m), 45.2, 44.6, 38.8, 36.5, 36.1, 31.9, 30.4, 30.0
(m), 29.9 (m), 29.6 (m), 29.4, 28.2, 25.7, 25.5, 23.6,
22.8, 22.7, 22.3, 21.7, 13.8. LC/MS (ret time,
7.3 min), calcd for C32H68N6O2 m/z 568, obsd 569
(MH+).
4.6.7.
L-Lys-e-(hexadecanesulfonamide)-N1-spermine
(12). 1H NMR (D2O, d): 4.04 (1H), 3.53 (1H), 3.30
(1H), 3.22 (2H), 3.17 (14H), 2.18 (2H), 2.00 (4H), 1.82
(6H), 1.67 (2H), 1.52 (4H), 1.34 (22H), 0.95 (t, 3H).
LC/MS (ret time, 7.3 min), calcd for C32H70N6O3S m/z
619, obsd 620 (MH+).
4.6. Representative individual analogues
4.6.1. L-Lys-N1-spermine (5).1 TLC analysis (b);
Rf = 0.04. LC/MS (ret time, 5.5 min), calcd for
C16H38N6O m/z 330, obsd 331 (MH+).
4.6.8. D-Lys-e-(palmitoyl)-N1-spermine (13). TLC analy-
sis (b); Rf = 0.21. H NMR (D2O, d): 3.94 (1H), 3.47
1
(1H), 3.06 (13H), 2.13 (2H), 2.04 (2H), 1.87 (4H), 1.75
(4H), 1.47 (4H), 1.36 (2H), 1.16 (24H), 0.78 (3H). 13C
NMR (D2O, ppm): 175.7, 169.8, 53.4, 47.2 (m), 45.6,
44.8, 39.0, 36.6 (m), 36.1, 31.9, 29.8 (m), 29.6, 29.3,
28.4, 25.9, 25.7, 23.8, 22.8, 23.1, 22.8, 22.1, 14.0. LC/
MS (ret time, 7.2 min), calcd for C32H68N6O2 m/z 569,
obsd 570 (MH+).
4.6.2. D-Lys-N1-spermine (6). Synthesis of analogue 6
used Boc-D-Lys(Boc)-ONp in place of the orthogonally
protected lysine derivative used for the synthesis of 14.
Coupling with spermine followed by protection of the
remaining amino groups as their Boc-carbamates gave
the protected intermediate following purification by col-
umn chromatography. Deprotection using 6 N HCl in
CH3OH gave the desired product 6. TLC analysis (b);
4.6.9. L-Lys(ene-D9-palmitoyl)-N1-spermine (15). LC/MS
(ret time, 7.3 min), calcd for C32H66N6O2 m/z 566, obsd
1
Rf = 0.04. H NMR (D2O, d): 3.92 (t, 1H), 3.29 (2H),
1
3.07 (10H), 2.93 (t, 2H), 2.04 (2H), 1.84 (4H), 1.72
(4H), 1.54 (2H), 1.34 (2H). 13C NMR (D2O, ppm):
168.7, 52.2, 45.8 (m), 44.2, 43.6, 40.0, 37.8, 35.4 (m),
29.0, 25.4, 24.6, 22.6, 21.8 (m), 20.2. LC/MS (ret time,
5.5 min), calcd for C16H38N6O m/z 330, obsd 331
(MH+). HRMS m/z calcd for C16H38N6O (M + H)
331.3185, found 331.3173. Anal. Calcd for
C16H43Cl5N6OÆ3/2H2O: C, 35.60; H, 8.59; N, 15.57.
Found: C, 35.58; H, 8.47; N, 15.41.
567 (MH+). H NMR (D2O, d): 5.29 (2H), 3.96 (1H),
3.49 (1H), 3.08 (14H), 2.16 (2H), 2.07 (2H), 1.87 (6H),
1.75 (4H), 1.47 (4H), 1.38 (2H), 1.16 (17H), 0.78
(3H). Anal. Calcd for C32H70Cl4N6O2Æ2H2O: C, 51.33;
H, 9.96; N, 11.22. Found: C, 51.30; H, 9.60; N,
11.45.
4.6.10. L-Lys-e-(myristoyl)-N1-spermine (16). TLC anal-
1
ysis (b); Rf = 0.22. H NMR (D2O, d): 3.92 (1H), 3.27
(2H), 3.03 (14H), 2.12 (2H), 2.07 (4H), 1.83 (4H), 1.66
(6H), 1.48 (4H), 1.20 (20H), 0.78 (3H). LC/MS (ret time,
7.0 min), calcd for C30H64N6O2 m/z 541, obsd 542
(MH+).
4.6.3. L-Lys-e-(eicosanoyl)-N1-spermine (7). TLC analy-
sis (b); Rf = 0.08. H NMR (D2O, d): 3.94 (1H), 3.48
1
(1H), 3.06 (13H), 2.15 (2H), 2.06 (2H), 1.88 (4H), 1.75
(4H), 1.47 (4H), 1.36 (2H), 1.16 (32H), 0.77 (3H). 13C
NMR (D2O, ppm): 175.5, 169.8, 53.4, 46.9 (m), 45.6,
44.8, 38.8, 36.8 (m), 36.0, 31.9, 30.4, 29.7 (m), 29.5,
29.3, 28.5, 25.9, 25.7, 23.8, 23.2, 23.1, 22.8, 21.7, 13.8.
LC/MS (ret time, 7.6 min), calcd for C36H76N6O2
m/z 625, obsd 626 (MH+).
4.6.11. L-Lys-e-(octanoyl)-N1-spermine (17). TLC analy-
sis (b); Rf = 0.20. LC/MS (ret time, 5.7 min), calcd for
C21H46N6O2 m/z 414, obsd 415 (MH+). Anal. Calcd
for C24H56Cl4N6O2ÆH2O: C, 46.45; H, 9.42; N, 13.54.
Found: C, 46.36; H, 9.39; N, 13.49.

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
2533
4.6.12. D-Lys-e-(isopropoyl)-N1-spermine (18). TLC
analysis (b); Rf = 0.24. H NMR (D2O, d): 3.90 (1H),
3.28 (3H), 3.05 (13H), 2.40 (1H), 2.02 (2H), 1.82 (4H),
1.71 (s, 2H), 1.47 (2H), 1.28 (2H), 0.99 (6H). 13C
NMR (D2O, ppm): 180.8, 175.8, 53.2, 47.0 (m), 45.2,
44.6, 38.6, 36.4 (m), 35.1, 30.4, 28.1, 25.7, 23.8, 29.4,
22.8 (m), 21.6, 18.9. LC/MS (ret time, 5.5 min), calcd
for C20H44N6O2 m/z 400, obsd 401 (MH+).
32.0, 30.6, 29.9 (m), 29.8, 29.5, 29.4, 29.1, 26.5, 25.9,
25.6, 25.5, 23.8, 22.9, 22.8, 21.7, 13.9. LC/MS (ret time,
7.2 min), calcd for C32H70N6O m/z 555, obsd 556
(MH+). Anal. Calcd for C32H75Cl5N6OÆ3/2H2O: C,
50.29; H, 10.29; N, 11.00. Found: C, 50.30; H, 10.05;
N, 10.67.
1
4.6.20. D-Lys-e-(3,3-dimethyl-1-butyl)-N1-spermine (34).
1
TLC analysis (b); Rf = 0.06. H NMR (D2O, d): 3.91
4.6.13. D-Lys-e-(2-norbornaneacetoyl)-N1-spermine (20).
(1H), 3.33 (1H), 3.23 (1H), 3.05 (14H), 2.01 (2H), 1.86
(4H), 1.71 (4H), 1.67 (2H), 1.50 (2H), 1.38 (2H), 0.85
(9H). LC/MS (ret time, 6.1 min), calcd for C22H50N6O
m/z 414, obsd 415 (MH+).
1
TLC analysis (b); Rf = 0.22. H NMR (D2O, d): 3.88
(1H), 3.24 (2H), 3.05 (13H), 2.02 (4H), 1.80 (4H), 1.68
(4H), 1.33 (8H), 0.98 (5H). LC/MS (ret time, 6.0 min),
calcd for C25H50N6O2 m/z 466, obsd 467 (MH+).
4.6.21. D-Lys-e-(3-methylpropyl)-N1-spermine (35). TLC
1
4.6.14.
D-Lys-e-(4-biphenycarboxamide)-N1-spermine
analysis (b); Rf = 0.06. H NMR (D2O, d): 3.90 (1H),
1
(21). TLC analysis (b); Rf = 0.13. H NMR (D2O, d):
7.77 (6H), 7.43 (3H), 3.87 (1H), 3.48 (2H), 3.16 (2H),
2.95 (10H), 1.94 (2H), 1.83 (2H), 1.72 (2H), 1.62 (6H),
1.34 (2H). LC/MS (ret time, 6.3 min), calcd for
C29H46N6O2 m/z 511, obsd 512 (MH+).
3.32 (1H), 3.24 (1H), 3.05 (10H), 2.82 (2H), 2.02 (2H),
1.82 (6H), 1.71 (6H), 1.37 (1H), 0.90 (6H). LC/MS (ret
time, 5.8 min), calcd for C20H46N6O m/z 387, obsd 388
(MH+).
4.6.22. L-Lys-e-(bis-(cyclohexyl))-N1-spermine (37). TLC
1
4.6.15.
L-Lys-e-(4-(1-pyrene)-butanoyl)-N1-spermine
analysis (b); Rf = 0.22. H NMR (D2O, d): 3.96 (1H),
(22). Synthesis of analogue 22 was by acylation with 1-
pyrenebutanoic acid succinimidyl ester from Molecular
Probes, Eugene, OR (cat. no. P-130). TLC analysis
3.30 (2H), 3.04 (18H), 2.06 (2H), 1.86 (4H), 1.72
(16H), 1.40 (2H), 1.18 (6H), 0.97 (4H). 13C NMR
(D2O, ppm): 169.8, 60.2, 54.1, 53.2, 47.0 (m), 45.3,
44.6, 36.6 (m), 32.9, 30.3 (m), 25.4, 25.0, 23.8, 22.8,
22.3, 21.7. LC/MS (ret time, 6.4 min), calcd for
C30H62N6O m/z 523, obsd 524 (MH+).
1
(b); Rf = 0.15. H NMR (D2O, d): 7.34 (d, 1H), 7.22
(3H), 7.08 (2H), 6.98 (2H), 6.88 (d, 1H), 3.74 (t, 1H),
3.18 (1H), 3.01 (4H), 2.93 (2H), 2.86 (1H), 2.77 (1H),
2.72 (2H), 2.65 (2H), 2.56 (1H), 2.40 (2H), 1.97 (2H),
1.73 (2H), 1.68 (2H), 1.54 (6H), 1.40 (2H), 0.98 (4H).
LC/MS (ret time, 6.6 min), calcd for C36H52N6O2 m/z
601, obsd 602 (MH+).
4.6.23. D-Lys-e-(4-phenylbenzyl)-N1-spermine (38). TLC
1
analysis (b); Rf = 0.11. H NMR (D2O, d): 7.55 (9H),
4.21 (s, 2H), 3.93 (1H), 3.32 (1H), 3.24 (1H), 3.04
(12H), 2.04 (2H), 1.80 (4H), 1.72 (6H), 1.40 (2H). 13C
NMR (D2O, ppm): 169.8, 141.6, 139.6, 130.5, 139.9,
129.3, 128.2, 127.6, 127.0, 53.1, 50.6, 47.1, 47.0, 46.5,
45.3, 44.6, 36.6 (m), 30.3, 25.5, 25.1, 23.8, 22.9 (m),
21.6. LC/MS (ret time, 6.3 min), calcd for C29H48N6O
m/z 497, obsd 498 (MH+).
4.6.16. L-Lys-e-(methylpolyethyleneglycolpropionyl)-N1-
spermine (23). The active ester used to acylate the e-
nitrogen atom was mPEG-SPA (mw 2000) from Nektar
Therapeutics (cat. no. 2M4MODO1). TLC analysis (b);
Rf = 0.24. 1H NMR (D2O, d): 3.80 (1H), 3.50 (large
OCH2 envelope), 3.42 (6H), 2.92 (15H), 2.34 (1H),
1.96 (1H), 1.75 (4H), 1.62 (4H), 1.41 (1H), 1.23 (1H).
LC/MS (ret time, 6.1 min), obsd an envelope of m/z cen-
tered at 650.
4.7. Rapid-throughput fluorescence displacement assay
for quantifying binding affinities to LPS
The BODIPY-TR-cadaverine (BC; 5-(((4-(4,4-difluoro-
5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl) phen-
oxy)acetyl)amino)pentylamine, hydrochloride; obtained
from Molecular probes, Inc., Eugene, OR) displacement
assay to quantify the affinities of binding of compounds
to LPS has been described in detail recently.46 This assay
was performed in a rapid-throughput format as follows.
The first column (16 wells) of a Corning Nonbinding
Surface 384-well flat-bottom black fluorescence micro-
plate contained 15 test compounds plus polymyxin B,
all at 5 mM, and were serially twofold diluted across
the remaining 23 columns, achieving a final dilution of
0.596 nM in a volume of 40 lL. Polymyxin B (PMB),
a peptide antibiotic known to bind and neutralize
LPS47 served as the positive control and reference com-
pound for every plate, enabling the quantitative assess-
ment of repeatability and reproducibility (CV and Z0
factors) for the assay. The Z0 factor of the HTS assay
for quantifying ED50 (relative binding affinity) is 0.82,
and the CVs at 0% and 100% probe displacement are
4.6.17. L-Lys-e-(2-[2-(2-methoxyethoxy)ethoxy]acetoyl)-
N1-spermine (24). TLC analysis (b); Rf = 0.11. 1H
NMR (D2O, d): 4.01 (3H), 3.91 (1H), 3.62 (6H), 3.31
(8H), 3.03 (12H), 2.06 (2H), 1.82 (6H), 1.53 (1H), 1.32
(1H). LC/MS (ret time, 5.4 min), calcd for
C23H50N6O5 m/z 490, obsd 491 (MH+).
4.6.18. L-Lys-e-(2-(2-methoxyethoxy)acetoyl)-N1-sperm-
ine (25). TLC analysis (b); Rf = 0.09. 1H NMR (D2O, d):
3.98 (3H), 3.92 (1H), 3.62 (6H), 3.31 (6H), 3.24 (6H),
3.03 (6H), 2.06 (2H), 1.87 (2H), 1.80 (4H), 1.53 (1H),
1.32 (1H). LC/MS (ret time, 5.7 min), calcd for
C21H46N6O4 m/z 446, obsd 447 (MH+).
4.6.19. L-Lys-e-(nhexadecyl)-N1-spermine (26). TLC
1
analysis (b); Rf = 0.11. H NMR (D2O, d): 3.97 (1H),
3.48 (1H), 3.04 (15H), 2.04 (2H), 1.91 (4H), 1.75 (8H),
1.48 (2H), 1.22 (26H), 0.91 (3H). 13C NMR (D2O,
ppm): 168.8, 53.4, 48.0, 47.3, 47.1 (m), 45.4, 44.7, 36.7,

2534
M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
4.1% and 6.2%, respectively.51 Robotic liquid handling
was performed on a Precision 2000 automated micro-
plate pipetting system, programmed using the Precision
Power software, Bio-Tek Instruments Inc., VT, USA.
Stock solutions of LPS (5 mg/mL; E. coli 0111:B4; pro-
cured from Sigma) and BC (500 lM) were prepared in
Tris buffer (pH 7.4, 50 mM). One milliliter each of the
LPS and BC stocks were mixed and diluted in Tris buffer
to a final volume of 100 mL, yielding final concentra-
tions of 50 lg/mL of LPS and 5 lM BC. Forty microli-
ters of this BC:LPS mixture was added to each well of
the plate using the Precision 2000. Fluorescence mea-
surements were made at 25 ꢁC on a Fluoromax-3 with
Micromax Microwell 384-well plate reader, using Data-
Max software, Jobin Yvon Inc., NJ. The BC excitation
wavelength was 580 nm, emission spectra were taken at
620 nm with both emission and excitation monochroma-
tor bandpasses set at 5 nm. The fluorescence of BC is
quenched upon binding to LPS, and the displacement
of BC by the compounds results in de-quenching (inten-
sity enhancement) of BC fluorescence. Effective displace-
ments (ED50) were computed at the midpoint of the
fluorescence signal versus compound concentration dis-
placement curve, determined using an automated four-
parameter sigmoidal fit utility of the Origin plotting
software (Origin Lab Corp., MA), as described in the
preceding paper. Z0 factors52 computed using the equa-
4.9. Multiplexed cytokine assay ex vivo in human blood
One hundred microliters aliquots of fresh whole blood,
anticoagulated with EDTA, obtained by venipuncture
from healthy human volunteers with informed consent
and as per guidelines approved by the Human Subjects
Experimentation Committee, were exposed to an equal
volume of 50 ng/mL of E. coli 0111:B4 LPS, with graded
concentrations of test compounds diluted in saline for
4 h in a 96-well microtiter plate. The effect of the com-
pounds on modulating cytokine production examined
using a FACSArray multiplexed flow-cytometric bead
array (CBA) system (Becton–Dickinson–Pharmingen,
San Jose, CA). The system uses a sandwich ELISA-
on-a-bead principle,55,56 and is comprised of 6 popula-
tions of microbeads that are spectrally unique in terms
of their intrinsic fluorescence emission intensities (de-
tected in the FL3 channel of a standard flow cytometer).
Each bead population is coated with a distinct capture
antibody to detect six different cytokines concurrently
from biological samples (the human inflammation
CBA kit includes TNF-a, IL-1b, IL-6, IL-8, IL-10,
and IL-12p70). The beads are incubated with 30 lL of
sample, and the cytokines of interest are first captured
on the bead. After washing the beads, a mixture of opti-
mally paired second antibodies conjugated to phycoery-
thrin is added, which then forms a fluorescent ternary
complex with the immobilized cytokine, the intensity
(measured in the FL2 channel) of which is proportional
to the cytokine concentration on the bead. The assay
was performed according to protocols provided by the
vendor. Standard curves were generated using recombi-
nant cytokines provided in the kit. The data were ana-
lyzed in the CBA software suite, which is integral to
the FACSArray system.
tion: 1 ꢁ [3(SD+ SD )/(A ꢁ A0)] where SDand SD , A
and A0 are standard deviations for the signal and noise,
and means of signal and noise, respectively, yielded a Z0
factor of 0.821 and an inter-plate CVs of 5.2%.
0
0
4.8. Nitric oxide assay
Nitric oxide production was measured as total nitrite in
murine macrophage J774A.1 cells using the Griess re-
agent system.53,54 Murine macrophage J774A.1 cells
were grown in RPMI-1640 cell-culture medium contain-
ing L-glutamine and sodium bicarbonate and supple-
mented with 10% fetal bovine serum, 1% L-glutamine–
penicillin–streptomycin solution, and 200 lg/mL L-argi-
nine at 37 ꢁC in a 5% CO2 atmosphere. Cells were plated
at ꢀ2 · 106/mL in a volume of 40 lL/well, in 384 well,
flat-bottomed, cell culture treated microtiter plates until
confluency and subsequently stimulated with 100 ng/mL
lipopolysaccharide (LPS). Concurrent to LPS stimula-
tion, serially diluted concentrations of test compounds
were added to the cell medium and left to incubate over-
night for 16 h. Polymyxin B was used as reference com-
pound in each plate. Positive- (LPS stimulation only)
and negative-controls (J774.A1 medium only) were in-
cluded in each experiment. Nitrite concentrations were
measured adding 30 lL of supernatant to equal volumes
of Griess reagents (50 lL/well; 0.1% NEDsolution in
ddH2O and 1% sulfanilamide, 5% phosphoric acid solu-
tion in ddH2O) and incubating for 15 min at room
temperature in the dark. Absorbance at 535 nm was
measured using a Molecular Devices Spectramax M2
multifunction plate reader (Sunnyvale, CA). Nitrite
concentrations were interpolated from standard curves
obtained from serially diluted sodium nitrite
standards.
4.10. Mouse lethality experiments
Female, outbred, 9–11-week-old CF-1 mice (Charles
River, Wilmington, MA) weighing 22–28 g were used
as described elsewhere.37 Upon arrival, the mice were al-
lowed to acclimatize for a week prior to experimenta-
tion, housed 5 per cage in a controlled environment at
the AALAC-accredited University of Kansas Animal
Care Facility, and allowed access to mouse chow and
water ad libitum. The animals were sensitized to the
55,57,58
lethal effects of LPS by D-galactosamine.
The
lethal dose causing 100% mortality (LD100) dose of the
batch of LPS used (E. coli 0111:B4 procured from Sig-
ma) was first determined by administering D-galactos-
amine (800 mg/kg) and LPS (0, 10, 20, 50, 100, 200 ng/
mouse) as a single injection intraperitoneally (i.p.) in
freshly prepared saline to batches of five animals in a
volume of 0.2 mL. The expected dose–response profile
was observed in two independent experiments with all
five mice receiving 100 ng succumbing within 24 h,
establishing the LD100 dose to be 100 ng/mouse. Exper-
iments designed to test dose–response effects of the acyl-
spermines in affording protection against LPS-induced
lethality, mice received graded doses of compound di-
luted in saline, i.p., in one flank, immediately before a
supralethal (200 ng) LPS challenge, which was adminis-
tered as a separate i.p. injection into the other flank. In

M. R. Burns et al. / Bioorg. Med. Chem. 13 (2005) 2523–2536
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was to be examined, a fixed dose of 200 lg/mouse of
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or after supralethal (200 ng/mouse) LPS challenge.
Lethality was determined at 24 h post LPS challenge.
18. Galanos, C.; Luderitz, O.; Rietschel, E. T.; Westphal, O.;
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