Polycationic sulfonamides for the sequestration of endotoxin

Article abstract of DOI:10.1021/jm061198m

Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality in the critically ill patient. We had previously shown that monoacylated polyamine compounds specifically bind to and neutralize the activity of LPS with high in vitro potency and afford complete protection in a murine model of endotoxic shock. Fatty acid amides of polyamines may be rapidly cleared from systemic circulation due to their susceptibility to nonspecific serum amidases and, thus, would be predicted to have a short duration of action. In a systematic effort to increase the likelihood of better bioavailability properties together with structural modifications that may result in gains in activity, we now report structure-activity relationships pertaining to endotoxin-binding and -neutralizing activities of homologated polyamine sulfonamides.

Full text of DOI:10.1021/jm061198m

J. Med. Chem. 2007, 50, 877-888
877
Polycationic Sulfonamides for the Sequestration of Endotoxin
Mark R. Burns,*,† Scott A. Jenkins,† Matthew R. Kimbrell,‡ Rajalakshmi Balakrishna,‡ Thuan B. Nguyen,‡
Benjamin G. Abbo,‡ and Sunil A. David*,‡
MediQuest Therapeutics, Inc., 22322 20th AVenue SE, Bothell, Washington 98021, and Department of Medicinal Chemistry,
UniVersity of Kansas, Lawrence, Kansas 66045
ReceiVed October 13, 2006
Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality
in the critically ill patient. We had previously shown that monoacylated polyamine compounds specifically
bind to and neutralize the activity of LPS with high in vitro potency and afford complete protection in a
murine model of endotoxic shock. Fatty acid amides of polyamines may be rapidly cleared from systemic
circulation due to their susceptibility to nonspecific serum amidases and, thus, would be predicted to have
a short duration of action. In a systematic effort to increase the likelihood of better bioavailability properties
together with structural modifications that may result in gains in activity, we now report structure-activity
relationships pertaining to endotoxin-binding and -neutralizing activities of homologated polyamine
sulfonamides.
Introduction
Endotoxins, or lipopolysaccharides (LPSa), are outer mem-
brane constituents of Gram-negative bacteria.1 A pivotal role
has been implicated for LPS in the pathogenesis of septic shock,
a syndrome of systemic toxicity that occurs as a consequence
of serious systemic infections (Gram-negative sepsis).2 Gram-
negative sepsis is the number one cause of deaths in the intensive
care unit,3 accounting for more than 200,000 fatalities in the
U.S. annually.4
The syndrome of multiple system organ failure, a hallmark
of septic shock, appears to be a consequence of a precipitous
activation of the innate immune response5,6 leading to the
uncontrolled production of a host of proinflammatory mediators,
including cytokines such as tumor necrosis factor-R (TNF-R),
interleukin-1â (IL-1â), and interleukin-6 (IL-6), primarily by
cells of the monocyte/macrophage lineage,7,8 as well as reactive
oxygen intermediates, eicosanoids, and proteases from neutro-
phils that, in concert, act to cause severe tissue damage.9 It has
been demonstrated by total synthesis10-13 that the toxicity of
LPS resides in its structurally highly conserved glycolipid
component called lipid A.14,15 Lipid A is composed of a
hydrophilic, bisphosphorylated diglucosamine backbone and a
hydrophobic domain of six (E. coli) or seven (Salmonella) acyl
chains in amide and ester linkages16-18 (Figure 1). The incidence
Figure 1. Structure of lipid A, the toxic moiety of bacterial li-
of sepsis has risen almost 3-fold from 1979 through 2000,19
and sepsis-associated mortality has essentially remained un-
changed at about 45%.20 The only available modality for the
therapy of sepsis is drotrecogin-alfa (activated recombinant
protein C),21 an antithrombotic agent whose efficacy has recently
been called into question.22-24 The urgent unmet need to develop
therapeutic options specifically targeting the pathophysiology
of sepsis has spurred a number of approaches, notable among
which are E5564, a synthetic lipid A analog25-28 that acts as
popolysaccharide.
an antagonist at the toll-like 4 receptor,29,30 the principal receptor
for LPS, and TAK-242,31,32 a small molecule inhibitor of
cytokine production. Both of these agents are currently in phase
III clinical trials.
One possible approach to therapeutically addressing 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.
Polymyxin B (PMB) is a membrane-active peptide antibiotic
known to sequester LPS and abrogate its toxicity. The pro-
nounced oto- and nephrotoxicity of PMB precludes its systemic
use and has led to the development of an extracorporeal
hemoperfusion cartridge based on PMB covalently immobilized
on a polystyrene-based fiber.33-35 Approved for clinical use in
Japan in late 2000, this provides a clinically validated proof-
of-concept of the therapeutic potential of sequestering circulating
* To whom correspondence should be addressed. Tel.: (425) 398-9580,
ext. 15 (M.R.B.); (785) 864-1610 (S.A.D.). Fax: (425) 398-9590 (M.R.B.);
(785) 864-1920 (S.A.D.). E-mail: markburns@mqti.com (M.R.B.);
sdavid@ku.edu (S.A.D.).
† MediQuest Therapeutics, Inc.
‡ University of Kansas.
a Abbreviations: LPS, lipopolysaccharide; PMB, polymyxin B; IL-1â,
interleukin-1â; IL-6, interleukin-6; TNF-R, tumor necrosis factor-R; NO,
nitric oxide
10.1021/jm061198m CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/26/2007

878 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Burns et al.
Scheme 1. Synthesis of Series 1 Compounds (SPM)a
LPS. A major goal in our laboratory has been to develop small-
molecule analogues of PMB that would sequester LPS with a
potency at least equivalent to that of PMB and, importantly, be
nontoxic and safe, so that it can be used parenterally for the
prophylaxis or therapy of Gram-negative sepsis.
We have, over the past decade, characterized the interactions
of lipid A with a number of classes of cationic amphipathic
molecules including proteins,36,37 peptides,38-42 pharmaceutical
compounds,43,44 and other synthetic polycationic amphiphiles.45-48
From these studies, we have determined that the pharmacophore
necessary for optimal recognition and neutralization of lipid A44
by small molecules requires two protonatable positive charges
so disposed that the distance between them is equivalent to the
distance between the two anionic phosphates on lipid A (∼14
Å), enabling ionic H-bonds between the phosphates on the lipid
A backbone and the positive charges on the compound. These
structural requisites are exemplified in the lipopolyamines, which
are of particular interest because they are active in vitro and
afford protection in animal models of Gram-negative sepsis,
are synthetically easily accessible, and, importantly, are non-
toxic, on account of their degradation to physiological substit-
uents (spermine and fatty acid).46,49-51 Fatty acid amides of
polyamines may be rapidly cleared from systemic circulation
due to their susceptibility to nonspecific serum amidases and,
thus, might be predicted to have a short duration of action. In
a systematic effort to increase the likelihood of better bioavail-
ability properties together with structural modifications that may
result in gains in activity, we have prepared and characterized
the homologated polyamine sulfonamides described in this
report.
a Reagents and conditions: (a) (i) F3CCO2Et, MeOH, -78 °C; (ii) Boc2O;
(iii) NaOH aq, 48%; (b) RSO2Cl, Et3N, THF, 57%; (c) HCl/CH3OH, 100%.
directly with Boc2O, and the resulting tetracarbamate 11 was
isolated in pure form by column chromatography in 61% yield.
This bis-cyano product was converted to the bis-amino product
12 by catalytic hydrogenation using Pd(OH)2 in CH3CO2H in
a 71% yield. Standard sulfonylation followed by acid depro-
tection gave the bis-sub-bis-HOMO-SPM Series 3 analogs
3A-E shown in Table 1.
Dialkylation of the primary amine of tri-Boc-spermine 6 or
the homologated tetra-Boc-carbamate intermediate 9 could be
accomplished using an excess of acrylonitrile at an elevated
temperature, as shown by Schemes 4 and 5, respectively. A
literature report showed that the addition of a catalytic amount
of cation exchange resin facilitated the production of the di-
adduct.58 Application of this technique to the present problem
helped to drive the conversion to the di-adducts 14 and 17,
which could be obtained following column chromatography in
90% and 46% yields, respectively. Hydrogenation using Pd-
(OH)2 in CH3CO2H again gave the desired product in good
yield. Sulfonylation, column chromatography, and HCl-mediated
Boc deprotection then gave the branched-SPM (analogs 4A-
E) and branched-HOMO-SPM (analogs 5A-E) compounds
shown in Table 1 (Series 4 and 5, respectively).
Results and Discussion
Synthesis of Polyamine Sulfonamides: Synthesis of the N1-
sulfonylated spermine analogs was accomplished most simply
by the direct sulfonylation of spermine in CH2Cl2 solution.52
This results in a mixture of mono- and bis-sulfonylated species
being generated through reaction with the primary amines of
spermine. Evidence supporting predominant primary versus
secondary amine reactivity is discussed below. Careful chro-
matography can provide pure products from this method, but
an improvement was enabled through the use of tri-Boc-
protected spermine 6. The synthesis of this useful intermediate53
has been described by Blagbrough and Geall54 and subsequently
modified by Wellendorph et al.55 Regiospecific sulfonylation
of 6 followed by column chromatography and subsequent HCl-
mediated deprotection was used to produce the spermine
sulfonamide analogs in Series 1 (SPM; Scheme 1; see Table 1
analogs 1A-E). The homologated spermine analogs (HOMO-
SPM) shown in Table 1 were produced via monoalkylation using
a Michael reaction between acrylonitrile and 6 (Scheme 2). Boc-
carbamate protection of the resulting secondary amine followed
by reduction of the nitrile using Pd(OH)2 in CH3CO2H gave
the desired homologated intermediate 956,57 in pure form
following column chromatography. Synthesis of the homolo-
gated spermine sulfonamides (HOMO-SPM) belonging to
Series 2 was then accomplished by sulfonylation of the primary
amine of this intermediate. Chromatography followed by acid-
mediated deprotection gave the desired molecules in pure form
(Table 1; analogs 2A-E).
The various analogs shown in Table 2 were synthesized by
direct sulfonylation of the modified polyamines using the direct
conditions given above. These analogs were purified by column
chromatography over silica gel using 80:18:2 CH2Cl2/MeOH/
concd NH4OH followed by conversion to their per-HCl salt
forms for characterization. Several examples such as 31 and 32
were synthesized using the corresponding acid chloride instead
of sulfonyl chloride. The special case of 22 deserves mentioning
here. We produced this analog via the regiospecific sulfonylation
of the secondary amino group of spermine through the use of
previously described N1, N14-bistrifluoroacetylspermine diTFA
salt.59 Workup, removal of TFA groups using NaOH in MeOH,
followed by chromatography gave 22 in pure form. Comparison
of this material and its primary regioisomer 1D (made either
by the direct or tri-Boc-spm sulfonylation methodologies) using
LC/MS demonstrated that they were distinct species (different
retention times, see Supporting Information). This analysis
furthermore showed that 1D synthesized by the direct spermine
sulfonylation route contained a minor amount of the secondary
sulfonamide impurity. This drop in the regiospecificity may be
unique for sulfonylation of spermine because we know of no
literature reference to loss of primary/secondary amine selectiv-
ity during carbonyl acylation reactions on spermine. Because
of this, all biological data presented in the following discussion
utilized materials synthesized using the regiospecific, Boc-
protected, intermediate routes for the compounds shown in Table
Utilization of the di-Boc-spermine 7 product obtained during
the synthesis tri-Boc-spermine intermediate 6 production for the
synthesis of the Series 3 analogs was accomplished by the route
depicted in Scheme 3. The bis-acylonitrile spermine adduct
formed from 7 and two equivalents of acrylonitrile was treated

Polyamine Sulfonamide Endotoxin-Sequestering Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 879
Scheme 2. Synthesis of Series 2 Compounds (HOMO-SPM)a
Scheme 4. Synthesis of Series 4 Compounds
(BRANCHED-SPM)a
a Reagents and conditions: (a) (i) CH2dCHCN, MeOH, 25 °C; (ii)
Boc2O, 66%; (b) Pd(OH)2, HOAc, H2, 71%; (c) RSO2Cl, Et3N, THF, 65%;
(d) HCl/CH3OH, 97%.
a Reagents and conditions: (a) CH2dCHCN (excess), MeOH, catalytic
Dowex 50W×400 (H+ form), reflux, 90%; (b) Pd(OH)2, HOAc, H2, 90%;
(c) RSO2Cl, Et3N, THF, 15%; (d) HCl/CH3OH, 75%.
Scheme 3. Synthesis of Series 3 Compounds
(Bis-Sub-Bis-HOMO-SPM)a
Scheme 5. Synthesis of Series 5 Compounds
(Branched-HOMO-SPM)a
a Reagents and conditions: (a) (i) CH2dCHCN (2 equiv), MeOH; (ii)
Boc2O, CH2Cl2, 61%; (b) Pd(OH)2, HOAc, H2, 71%; (c) RSO2Cl, Et3N,
THF, 23%; (d) HCl/CH3OH, 100%.
1. Analyses of these analogs by 1H-NMR, LC/MS, and
elemental analysis gave results consistent with their structures.
Affinity for Binding to Lipopolysaccharide and In Vitro
Endotoxin Neutralizing Activity: All analogs were evaluated
for binding affinity to LPS using a high-throughput fluorescent
dye displacement assay (ED50 values) together with two cell-
based assays measuring the inhibition of LPS-induced nitric
oxide (NO) and NFκB production. There was a progressive
increase in cell-based LPS-sequestering activities between C8
and C16 analogs for the monosulfonamide analogs in Series 1
and 2, although this trend is not paralleled in corresponding
enhancements in binding affinities (Table 1 and Figure 2). For
example, the SPM analog 1D (C16) showed high affinity to
LPS (ED50 value of 3.87 µM) and potent ability to inhibit the
cell-based LPS-induced cytokine production (NO IC50 value of
0.45 µM; NFκΒ IC50 value of 0.28 µM). Analog 2D (Series 2;
HOMO-SPM; C16) also showed similarly high LPS-neutral-
izing potency (NO IC50 value of 0.12 µM; NFκΒ IC50 value of
a Reagents and conditions: (a) CH2dCHCN (excess), MeOH, catalytic
Dowex 50W×400 (H+ form), reflux, 46%; (b) Pd(OH)2, HOAc, H2, 95%;
(c) RSO2Cl, Et3N, THF, 62%; (d) HCl/CH3OH, 96%.
0.20 µM) and an LPS affinity value of 4.71 µM. C8 chain length
containing analogs 1A and 2A had comparable ED50 values of
6.64 and 2.84 µM, respectively, whereas their NO inhibition
values (1.44 and 6.47 µM) and NFκB inhibition values (33.3
and 0.816 µM) were significantly diminished in comparison to
their more lipophilic counterparts (between a 3- and 54-fold
diminished NO inhibition and between a 3- and 166-fold
diminished NFκB inhibition). We ascribe the lack of significant
change in ED50 values (binding affinities) to the fact the
fluorescent displacement assay is very sensitive to electrostatic
interactions between ligand and LPS, but does not adequately

880 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Burns et al.
Table 1. Binding Affinity (BC Displacement; ED50) and Biological Activity (NO Inhibition in Murine J774 Cells; IC50; NFκâ Inhibition, IC50) of
Monosubstituted Spermine-Sulfonamide Analogs (SPM, Series 1), Monosubstituted Homologated Spermine-Sulfonamide Analogs (HOMO-SPM, Series
2), Bis-Substituted Bis-Homologated Spermine-Sulfonamide Analogs (Bis-Sub-Bis-HOMO-SPM, Series 3), Disubstituted Branched
Spermine-Sulfonamide Analogs (Branched-SPM, Series 4), Disubstituted Branched Homologated Spermine-Sulfonamide Analogs
(Branched-HOMO-SPM, Series 5
report hydrophobic interactions.60-62 Furthermore, the cell-based
cytokine inhibition data reflects a more complex, multistep
process.
apparent decrease in activity, as has been noted in other
lipopolyamines we examined earlier.51,63,64
To further define the molecular requirements of this inhibition,
a variety of analogs shown in Table 2 were produced. The
absolute requirement for a poly-charged species is demonstrated
by oxa- and carba-analogs 20 and 21. These nonpolyamine
analogs showed much weaker activities, suggesting that the
internal secondary amines (H-bond donor atoms) may contribute
to the enthalpy of binding via additional H-bonds with the
glycosidic backbone of lipid A (see Figure 1). This conjecture
is also supported by the observation that 22, which is substituted
on one of its internal secondary amines, shows greatly dimin-
ished activity compared to its primary amine-substituted re-
gioisomer 1D. Additional analogs containing the same number
of charges, but with differing spacing relative to spermine in
In contract to monosulfonamides discussed above, the bis-
sulfonamide analogs (Series 3), as well as branched-chain
compounds (Series 4 and 5), showed an apparent inverse
relationship between carbon lengths and binding affinity and
antiendotoxic activity. Maximal affinity and activity was
observed with C8 substitutions (3A, 4A, 5A) and decreased with
longer hydrophobic groups. This, however, is not inconsistent
with the results obtained with the monosulfonamides for the
compounds in Series 3-5 have two hydrophobic groups in
sulfonamide linkages, and the net carbon number for the C8
analogs is 16. The additional hydrophobicity gained with longer
appendages results in lower aqueous solubility, leading to an

Polyamine Sulfonamide Endotoxin-Sequestering Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 881
Table 2. Binding Affinity (BC Displacement; ED50) and Biological
Activity (NO Inhibition in Murine J774 Cells; IC50; NFκâ Inhibition,
IC50) of Miscellaneous Backbone-Modified Polyamine-Sulfonamide
Analogs (MISC-PA, Series 6)
attributable to an increased half-life of the sulfonamides versus
the carboxamides in these cell-based assays. The other observa-
tion was the marked dissociation between 25 and 26 (Table 2).
The N1,N1-bis(2-aminoethyl)ethane-1,2-diamine motif of 25
appears to correspond to greater affinity and activity than the
N1,N1-bis(3-aminopropyl)propane-1,3-diamine core structure of
26. We surmise that this may be a consequence of more
favorable multidentate ionic H-bonds65 formed with the lipid
A phosphates44 by 25.
Dissociation between NO and NFκB Inhibition Activities:
Although not readily apparent from a cursory inspection of
Figure 2, we have observed a rather pronounced dissociation
between NO inhibition on the one hand and NFκB attenuation
on the other. Plots of IC50 values of the compounds in the two
in vitro assays revealed a distinctly dimorphic distribution,
yielding two independent and divergent linear regression fits
(Figure 3, top panel). This was unexpected and had not been
observed before in several SAR studies on homologous se-
ries.50,60,66,67 Upon close examination of this data from these
two cell-based assays, we found that this was attributable to a
marked difference in SAR trends between the monosubstituted
(Series 1 and 2) compounds and the bis- and disubstituted/
branched (Series 3-5) compounds (Figure 3, bottom panels;
in these plots it is important to note that the symbol size
corresponds to the carbon number of the acyl groups). For the
Series 1 and 2 compounds, lengthening the acyl chain length
results in a dramatic decrease in the NO IC50 (∼10-5 M to
∼10-7 M), while changes in NFκB IC50 values are more subtle
(Figure 3, bottom left panel). The converse is true for Series
3-5 analogs in that increases in the acyl chain length result in
a pronounced increase in NFκB IC50 values, while the NO
inhibition constants are less affected. The presence of a
secondary target that is differentially affected by each of these
two classes of compounds is suggested by this data. We
hypothesize that this may be related to the different inhibitory
activities of these compounds on calmodulin, given that sper-
mine sulfonamides have been shown to antagonize calmodu-
lin68,69 and inducible NO synthase activity is Ca++/calmodulin-
dependent.70,71 The effect of these polyamine compounds on
calmodulin are currently being examined.
the polyamine backbone, maintain binding affinity and potency.
For instance, the norspermine backbone of 24 has a diamino-
propane central segment in contradistinction to the diaminobu-
tane fragment in spermine. Analog 27 contains an unsaturated
diaminobut-2-ene central core. Both of these compounds
displayed good activity.
Cytokine Inhibition in Human Blood Ex Vivo: It was
important to verify that the endotoxin-sequestering activity of
the most active polyamine compounds would be manifested in
the milieu of whole human blood, characterized not only by its
high ionic strength (∼300 mOsmoles), which attenuates elec-
trostatic interactions,44 but also by near-millimolar concentra-
tions of albumin. Albumin has been shown to bind both LPS36,37
as well as acylpolyamines.72 Furthermore, given the amphipathic
nature of both LPS and the polyamine analogs, it is conceivable
that substantial partitioning of both LPS and ligand into the
lipoprotein constituents could occur. For these reasons, and prior
to confirmatory experiments in an animal model, we compared
the effects of high-potency hits identified in the NFκB and NO
in vitro screening assays, relative to PMB as a reference
compound, in ex vivo cytokine release assays using whole
human blood. Gratifyingly, all analogs tested inhibited TNF-R,
IL-6, and IL-8 production in a dose-dependent manner with the
IC50 values considerably lower than that of PMB (Table 3),
demonstrating that these compounds were fully active under
physiological conditions.
Two observations were somewhat counterintuitive and are
worth noting. The branched carboxamide analogs 31 and 32
(Table 2), despite having good affinity for LPS in the binding
assays (1.32 µM and 1.00 µM, respectively), showed greatly
diminished biological activity in comparison to their sulfonamide
counterparts (4A and 5A); (NFκB IC50: 9.76 µM vs 0.669 µM
for carboxamide/sulfonamide branched-SPM pair, and 3.17 µM
vs 0.30 µM for carboxamide/sulfonamide branched-HOMO-
SPM pair). We do not yet understand the structural basis of
this apparent discrepancy but it suggests higher activity in
sulfonamide analogs. The observation that the sulfonamides
exhibit significantly higher activity in comparison to their
carboxamide counterparts is further supported by comparing to
some homologated analogs previously reported by this labora-
tory.51 The best in the sulfonamide series show low nanomolar
NO and cytokine-release inhibition activity (e.g., analog 2D:
0.12 and 0.20 µM), whereas the comparable carboxamide analog
(e.g., 4e 4.22 and 1.91 µM (in ref 51)) showed 10- to 35-fold
lower activities in these assays. These differences may also be
Protection against Endotoxin-Induced Lethality in Mice:
A well-established murine model of lethal septic shock46,50,51,64,73
was employed to evaluate the potencies of the three most active
test compounds. Cohorts of 10 CF-1 mice per group, sensitized

882 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Burns et al.
Figure 2. Binding affinities and inhibitory potencies in in vitro NO release and NFκB nuclear translocation assays for Series 1-5.
to the lethal effects of LPS with D-galactosamine, were
challenged with a supralethal dose of LPS (2× LD100 dose )
200 ng/animal) administered intraperitoneally (i.p.). This was
preceded by a subcutaneous (s.c.) injection of graded doses of
1D, 2D, or 5A given 1 h prior to LPS challenge. These
compounds respectively represent the best-in-class of the
spermine-sulfonamide Series 1 (SPM), Series 2 (HOMO-
SPM), and Series 5 (branched-HOMO-SPM) and specifically
contain example(s) of both the linear monosulfonamide and
branched bis-sulfonamide. As shown in Figure 4, i.p. admin-
istration of 1D and 2D afforded a clear, dose-dependent
protection from lethality with complete protection being evident
at a dose of 200 µg/mouse (8 mg/kg). Compound 5A displayed
toxic effects above doses of 50 µg/animal (2 mg/kg).
inhibits LPS-induced NO and NFκB production in cell-based
assays at 0.12 and 0.20 µM, respectively. This compound affords
dose-dependent protection in a mouse model of septic shock.
The questions that remain to be carefully addressed are the
following: First, what is the toxicity profile of polyamine
sulfonamides such as 2D? Polyamine analogs have been known
to exert cytostatic and antineoplastic activities74-78 mediated
by a variety of mechanisms including the depletion of intrac-
ellular polyamine pools,79 inhibition of ornithine decarboxylase,
and the upregulation of spermidine/spermine acetyltransferase
activities.79-82 It would, therefore, not be surprising to find some
of these activities with compounds such as 2D. Indeed, a
pharmaprofiling (CEREP screening) of 2D shows several off-
target effects primarily involving amine transporters (data not
shown). However, sequestration of LPS in the context of
managing Gram-negative sepsis would entail a short-term use,
quite unlike the chronic dosing required in antineoplastic
regimens. What the consequences of these off-target effects upon
short-term administration would be and whether they would be
tolerable in the setting of an acute, but frequently fatal syndrome,
are questions that are crucial in the additional preclinical
Concluding Remarks: A series of novel, homologated
spermine sulfonamides were examined for their ability to
sequester endotoxin and inhibit the production of downstream
cytokine production. A single long-chain (C16-C18) or two
moderate-length (C8-C10) aliphatic group(s) are essential for
optimal LPS binding and neutralization. Compound 2D, the
principal lead, sequesters LPS with an IC50 of 4.2 µM and

Polyamine Sulfonamide Endotoxin-Sequestering Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 883
Figure 4. Dose-response of select compounds (left) and time-course
of protection afforded by 2D (right) in a D-galactosamine-sensitized
mouse model of septic shock. Dose-response: Cohorts of five mice
each were challenged with a supralethal (twice the LD100 dose; 200
ng/animal) of LPS. Graded doses of compound were administered
concurrently by separate intraperitoneal injection. Time-course: A fully
protective dose of 2D (200 µg/mouse) was given at various times
preceding LPS challenge. Lethality was scored at 24 h post-LPS
challenge in both experiments.
carboxamide analogs48,50,53 have an intrinsic advantage over the
sulfonamide compounds presented in this work in being
degraded to nontoxic metabolites (fatty acid and polyamine).
Time-course experiments aimed at understanding the pharma-
codynamics of 2D (Figure 4) do indeed suggest a much longer
half-life than some carboxamide analogs that we have previously
examined.48,50,53 This observation needs to be formally examined
via pharmacokinetic experiments. Equally important, it remains
to be seen whether the potential gain in half-life with the
sulfonamides is offset by additional and perhaps unexpected
toxicity. Work addressing these issues is currently in progress.
Figure 3. Top: Dimorphic correlation between in vitro NO release
and NFκB nuclear translocation IC50 values for Series 1-5. The series
number is shown in the boxed symbol and the corresponding compound
numbers are listed alongside. Compounds in hatched rectangle were
insoluble and were, therefore, inactive in either assay. Compounds that
were highly active in both screens (in ellipse) were selected for in vivo
testing in a murine model of septic shock. Bottom: Inverse relationship
between NO and NFκB inhibition activities between monosubstituted
analogs in Series 1 and 2 (left panel), and disubstituted/branched
compounds in Series 3-5 (right panel). The symbol sizes correspond
to the total carbon number of the hydrophobic chain.
Experimental Section
Chemistry: General Experimental Methods. The sources of
all chemical reagents and starting materials were of the highest
grade available and were used without further purification. Drying
of organic layers was performed using MgSO4. LC/MS analyses
were performed using a Hewlett-Packard 1050 system. Detection
was by a Finnigan AQA operating in ESI+ mode (m/z range 140
to 1600 amu). Gradient elution from 2 to 7 min at 0.2 mL/min was
performed using 2% to 100% CH3CN in H2O (both with 0.05%
TFA) using a Waters XTerra MS C18 2.1 × 150 mm (3.5 µm)
Table 3. Secondary Screen IC50 Values for Inhibition of LPS (100
ng/mL)-Induced Cytokine Release in Whole Human Blood for
Polymyxin B (PMB; Reference Compound) and Compounds That Were
Active in Both NO Release and NFκB Translocation Assaysa
1
column. H NMR spectra were recorded at 300 MHz on a Bruker
1
AV300 spectrometer at the University of Washington, Seattle. H
cytokine inhibition in ex vivo whole
human blood: IC50 values (µM)
NMR signals were generally multiples, unless otherwise noted,
designated as s ) singlet, d ) doublet, t ) triplet, or m ) multiplet.
Chemical shifts are relative to external 3-(trimethylsilyl)-1-pro-
panesulfonic acid, sodium salt.
cmpd
TNF-R
IL-6
IL-8
PMB
1D
1E
2B
2C
22.3
22.1
18.0
32.9
30.1
14.2
11.0
15.0
8.17
8.80
29.4
12.1
5.92
7.77
25.2
15.2
15.4
22.4
15.3
15.1
12.5
Monosubstituted Spermine-Sulfonamide Analogs (SPM)s
Direct Route Synthetic Method. Synthesis of N1-Hexadecylsul-
fonyl-1,14-diamino-5,10-diazabuta-decane Tris(hydrochloride)
Salt 1D. To the clear solution of 200 mg (1 mmol, 10 equiv) of
spermine in 10 mL of dry CH2Cl2 was added a solution of 32 mg
(0.1 mmol, 1 equiv) of hexadecylsulfonyl chloride in 1 mL of dry
CH2Cl2 dropwise at 25 °C. After stirring for 16 h, the heterogeneous
mixture was washed with 5% Na2CO3 and brine, dried, and
evaporated to give the crude product as a mixture of mono-, di-,
and trisubstituted spermine sulfonamides. Column chromatography
was performed using 900 mg of silica gel, eluting with CH2Cl2
containing 5 to 10% MeOH and 1% NH4OH. Product-containing
fractions were combined and evaporated to give 1D as a pure oil
2D
5A
a See Figure 3.
evaluation of compounds such as 2D. Careful evaluation of acute
and chronic toxicity using escalating dose-regimens as well as
a more detailed evaluation of its efficacy in large-animal models
of sepsis are being planned. Second, as mentioned earlier,

884 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Burns et al.
in its free base form. This was converted to its trihydrochloride
salt form by treatment with and evaporation from MeOH saturated
with hydrogen chloride gas. 1H NMR (D2O, ppm): 3.07 (m, 12H),
2.08 (m, 2H), 1.88 (m, 2H), 1.74 (m, 4H), 1.26 (m, 30H), 0.82 (t,
3H). Elem anal. Calcd for C26H61Cl3N4O2S: C, 52.03; H, 10.24;
N, 9.33. Found: C, 51.82; H, 10.24; N, 9.37. LC/MS by ESI+ mode
analysis observed m/z 491 at 11.9 min retention time.
product as a colorless oil. This was purified over silica gel using
CHCl3/MeOH/concd NH4OH, 92:8:2 to give 2.0 g (71%) of 9 as a
colorless oil. This intermediate is also used for the synthesis of
branched HOMO-spermine analogs depicted in Table 1 (Series 4).
A 0.20 g (0.30 mmol) portion of this material was dissolved in 7
mL of dry CH2Cl2 and treated with 0.063 mL (1.5 equiv) of
triethylamine, followed by 0.15 g (1.5 equiv) of solid hexadecyl-
sulfonyl chloride at 25 °C. Following 16 h, the reaction was diluted
with CH2Cl2 and washed with ice-cold 0.1 N HCl, H2O, and then
brine. Drying and evaporation gave the crude product. Chroma-
tography over silica gel using hexane/EtOAc 2:1 gave 0.171 g
(65%) of 10D as a colorless oil. This was dissolved in 5 mL of
CH3OH and treated with 5 mL of 6 N HCl at 25 °C. The resulting
colorless solution was stirred for 8 h when evaporation gave 0.121
g (97%) of 2D as a white solid in its tetrahydrochloride salt form.
1H NMR (D2O, ppm): 3.05 (br s, 16H), 2.08 (br s, 4H), 1.90 (br
s, 2H), 1.73 (m, 6H), 1.22 (m, 28H), 0.78 (br s, 3H). Elem anal.
Calcd for C29H69Cl4N5O2S: C, 50.21; H, 10.02; N, 10.09. Found:
C, 50.17; H, 9.96; N, 10.11. LC/MS by ESI+ mode analysis
observed m/z 548 at 11.7 min retention time. Synthesis of 2A (C8),
2B (C10), 2C (C12), and 2E (C18) followed the same procedure
except for the use of the appropriate sulfonylchloride.
Monosubstituted Spermine-Sulfonamide Analogs (Series 1:
SPM)sAlternative Synthetic Route Method: Synthesis of N1-
Hexadecylsulfonyl-1,14-diamino-5,10-diazabutadecane Tris(hy-
drochloride) Salt 1D: A solution of 0.20 g (0.40 mmol) of tri-
Boc-spm 6 in 2 mL of dry CH2Cl2 was treated with 0.061 mL of
triethylamine (1.10 equiv) followed by 0.143 g of solid hexade-
cylsulfonyl chloride at 25 °C. The resulting solution was stirred
for 16 h when TLC analysis (hexane/EtOAc 2:1) showed the
reaction was complete. The reaction solution was diluted in CH2-
Cl2 and washed with ice-cold 0.1 N HCl, H2O, and then brine,
dried with MgSO4, and evaporated to give the crude product as an
off-white foam. Chromatography over silica gel using hexane/
EtOAc 2:1 gave 0.18 g (57% yield) of the tri-Boc intermediate
8D. This material was completely dissolved in 5 mL of CH3OH
and treated with 5 mL of 6 N HCl at 25 °C. After 16 h, the solvents
were evaporated to give 0.14 g (100%) of 1D as its trihydrochloride
salt as a white solid. Characterization matched that found above.
Synthesis of 1A (C8), 1B (C10), 1C (C12), and 1E (C18) followed
the same procedure except for the use of the appropriate alkane-
sulfonylchloride.
N1-Octanylsulfonyl-1,18-diamino-5,9,14-triazaoctadecane Te-
trahydrochloride Salt 2A: 1H NMR (D2O, ppm): 3.05 (m, 18H),
2.03 (m, 4H), 1.83 (m, 2H), 1.67 (m, 6H), 1.32 (m, 2H), 1.18 (m,
8H), 0.73 (t, 3H). Elem anal. Calcd for C21H53Cl4N5O2S: C, 43.37;
H, 9.19; N, 12.04. Found: C, 43.23; H, 9.14; N, 11.85. LC/MS by
ESI+ mode analysis observed m/z 426 at 10.8 min retention time.
N1-Decanylsulfonyl-1,18-diamino-5,9,14-triazaoctadecane Te-
trahydrochloride Salt 2B: 1H NMR (D2O, ppm): 3.02 (m, 18H),
2.02 (m, 4H), 1.81 (m, 2H), 1.68 (m, 6H), 1.31 (m, 2H), 1.22 (m,
12H), 0.74 (t, 3H). Elem anal. Calcd for C23H57Cl4N5O2S: C, 45.32;
H, 9.42; N, 11.49. Found: C, 45.32; H, 9.45; N, 11.23. LC/MS by
ESI+ mode analysis observed m/z 464 at 10.9 min retention time.
N1-Dodecanylsulfonyl-1,18-diamino-5,9,14-triazaoctadecane
N1-Octanylsulfonyl-1,14-diamino-5,10-diazabutadecane Tris-
1
(hydrochloride) Salt 1A (C8): H NMR (D2O, ppm): 3.02 (m,
14H), 1.98 (m, 2H), 1.83 (m, 2H), 1.68 (m, 6H), 1.31 (m, 2H),
1.18 (m, 8H), 0.72 (t, 3H). Elem anal. Calcd for C18H45Cl3N4O2S:
C, 44.30; H, 9.29; N, 11.48. Found: C, 44.19; H, 9.25; N, 11.27.
LC/MS by ESI+ mode analysis observed m/z 379 at 10.7 min
retention time.
N1-Decanylsulfonyl-1,14-diamino-5,10-diazabutadecane Tris-
1
1
(hydrochloride) Salt 1B (C10): H NMR (D2O, ppm): 3.04 (m,
Tetrahydrochloride Salt 2C: H NMR (D2O, ppm): 3.02 (m,
14H), 1.99 (m, 2H), 1.82 (m, 2H), 1.67 (m, 6H), 1.33 (m, 2H),
1.19 (m, 12H), 0.76 (t, 3H). Elem anal. Calcd for C20H49-
Cl3N4O2S: C, 46.55; H, 9.57; N, 10.86. Found: C, 46.46; H, 9.50;
N, 10.69. LC/MS by ESI+ mode analysis observed m/z 407 at 10.9
min retention time.
18H), 2.02 (m, 4H), 1.83 (m, 2H), 1.72 (m, 6H), 1.37 (m, 2H),
1.22 (m, 16H), 0.74 (t, 3H). Elem anal. Calcd for C25H61-
Cl4N5O2S: C, 47.09; H, 9.64; N, 10.98. Found: C, 47.19; H, 9.68;
N, 10.87. LC/MS by ESI+ mode analysis observed m/z 492 at 11.1
min retention time.
N1-Dodecanylsulfonyl-1,14-diamino-5,10-diazabutadecane Tris-
N1-Octadecanylsulfonyl-1,18-diamino-5,9,14-triazaoctade-
1
1
(hydrochloride) Salt 1C (C12): H NMR (D2O, ppm): 3.07 (m,
cane Tetrahydrochloride Salt 2E: H NMR (D2O, ppm): 3.08
14H), 2.04 (m, 2H), 1.86 (m, 2H), 1.72 (m, 6H), 1.34 (m, 2H),
1.18 (m, 16H), 0.78 (t, 3H). Elem anal. Calcd for C22H53-
Cl3N4O2S: C, 48.56; H, 9.82; N, 10.30. Found: C, 48.40; H, 9.79;
N, 10.31. LC/MS by ESI+ mode analysis observed m/z 435 at 11.2
min retention time.
(br s, 18H), 2.12 (br s, 4H), 1.91 (br s, 2H), 1.74 (m, 6H), 1.34 (m,
2H), 1.23 (m, 28H), 0.74 (t, 3H). Elem anal. Calcd for C31H73-
Cl4N5O2S‚0.5H2O: C, 50.95; H, 10.21; N, 9.58. Found: C, 51.08;
H, 10.05; N, 9.32. LC/MS by ESI+ mode analysis observed m/z
576 at 12.8 min retention time.
N1-Octadecanylsulfonyl-1,14-diamino-5,10-diazabutadecane
Synthesis of Bis-Substituted Bis-Homologated Spermine-
Sulfonamide Analogs (Series 3: Bis-Sub-Bis-HOMO-SPM)s
Synthesis of N1,N14-Bis[N-octanylsulfonyl-3-aminopropyl]-1,14-
diamino-5,10-diazabutadecane Tetrahydrochloride Salt 3A: To
a solution of 2.6 g (6.5 mmol) of N5,N10-Bis[t-butoxycarbonyl]-
1,14-diamino-5,10-diazabutadecane (N5,N10-bisBOC-spm) 7 in 35
mL of CH3OH was added 0.85 mL (2 equiv) of acrylonitrile at rt
for 16 h when TLC analysis (95:5:0.2 CH2Cl2/CH3OH/NH4OH)
showed complete consumption of starting material. The solvents
were evaporated and the residue was dissolved in 100 mL of CH2-
Cl2 and treated with 3 g (2.15 equiv) of Boc2O. After 8 h, the
solvents were evaporated and the residue was purified by column
chromatography over silica gel using 3:2 hexane/EtOAc to give
2.8 g (61%) of 11 as a clear oil. This material was dissolved in 40
mL of glacial AcOH and treated with 3 g of Pd(OH)2 and 50 psi
of H2 pressure. After 4.5 h, the catalyst was filtered off and the
filtrate was evaporated. The resulting residue was dissolved in
EtOAc and washed with 1 N NaOH and brine, dried, and evaporated
to give the crude product as an oil. This material was purified by
column chromatography over silica gel using 95:5:0.5 CHCl3/
MeOH/NH4OH to give 2.0 g (71%) of 12 as a clear oil. A 0.20 g
(0.28 mmol) portion of this product was dissolved in 5 mL dry
CH2Cl2 and treated with 0.12 mL (3 equiv) of triethylamine
1
Tris(hydrochloride) Salt 1E (C18): H NMR (D2O, ppm): 3.04
(m, 14H), 2.05 (m, 2H), 1.94 (m, 2H), 1.78 (m, 6H), 1.26 (m, 30H),
0.82 (t, 3H). Elem anal. Calcd for C28H65Cl3N4O2S: C, 53.53; H,
10.43; N, 8.92. Found: C, 53.39; H, 10.46; N, 8.75. LC/MS by
ESI+ mode analysis observed m/z 519 at 12.5 min retention time.
Monosubstituted Homologated Spermine-Sulfonamide Ana-
logs (Series 2: HOMO-SPM)sSynthesis of N1-Hexadecanyl-
sulfonyl-1,18-diamino-5,9,14-triazaoctadecane tetrahydrochlo-
ride salt 2D: To the solution of 2.6 g (6.5 mmol) of tri-Boc-spm
6 in 120 mL of dry CH3OH was added 0.85 mL of acrylonitrile.
Following stirring for 18 h, TLC analysis (CH2Cl2/MeOH/NH4OH
90:8:2) showed the reaction was nearly complete. The solvent was
evaporated and the oily residue was dissolved in 100 mL of CH2-
Cl2 and treated with 3.06 g (14 mmol, 2.15 equiv) of Boc2O. After
16 h, the solvents were evaporated and the oily residue was purified
by chromatography over silica gel (hexanes/EtOAc 3:2) to give
2.8 g (66%) of monoalkylated product 8 as a colorless oil. This
intermediate was dissolved in 30 mL of glacial acetic acid, and 3
g of Pd(OH)2 was added. This mixture was placed under 50 psi of
H2 pressure and shaken for 15 h. The catalyst was removed by
filtering over a pad of Celite, and the pad was washed with CH3-
OH and the combined filtrates were evaporated to give the crude

Polyamine Sulfonamide Endotoxin-Sequestering Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 885
followed by 0.164 mL (3 equiv) of octanylsulfonylchloride drop-
wise. The reaction was stirred under argon for 16 h when the
reaction was diluted with CH2Cl2, washed with 0.1 N HCl and brine,
dried, and evaporated. The crude residue was purified over silica
gel chromatography using 1:1 hex/EtOAc to give 69 mg (23%) of
pure 13A product. This was directly deprotected using 2 mL of
1:1 CH3OH/6 N HCl. This solution was allowed to stand for 16 h
when evaporation gave 52 mg (100%) of pure 3A as its tetrahy-
1.71 (m, 8H), 1.34 (m, 4H), 1.22 (m, 16H), 0.77 (t, 6H). LC/MS
by ESI+ mode analysis observed m/z 669 at 11.1 min retention
time.
N1-Di[N-decanylsulfonyl-3-aminopropyl]-1,14-diamino-5,10-
1
diazabutadecane Tetrahydrochloride Salt 4B: H NMR (D2O,
ppm): 3.10 (m, 24H), 2.08 (m, 4H), 1.96 (m, 4H), 1.73 (m, 8H),
1.36 (m, 4H), 1.22 (m, 24H), 0.81 (t, 6H). LC/MS by ESI+ mode
analysis observed m/z 726 at 11.9 min retention time.
1
N1-Di[N-dodecanylsulfonyl-3-aminopropyl]-1,14-diamino-5,-
10-diazabutadecane Tetrahydrochloride Salt 4C: 1H NMR (D2O,
ppm): 3.23 (m, 6H), 3.07 (m, 18H), 2.19 (t, 4H), 2.08 (m, 4H),
1.84 (m, 4H), 1.72 (m, 4H), 1.50 (m, 4H), 1.21 (m, 16H), 0.77 (t,
6H). LC/MS by ESI+ mode analysis observed m/z 669 at 11.1 min
retention time.
drochloride salt. H NMR (D2O, ppm): 3.14 (m, 6H), 3.08 (m,
18H), 2.09 (m, 4H), 1.89 (m, 4H), 1.68 (m, 8H), 1.35 (m, 4H),
1.22 (m, 16H), 0.75 (t, 6H). Elem anal. Calcd for C32H76-
Cl4N6O4S2: C, 47.16; H, 9.40; N, 10.31. Found: C, 46.99; H, 9.40;
N, 10.09. LC/MS by ESI+ mode analysis observed m/z 670 at 12.7
min retention time. Synthesis of 3B (C10), 3C (C12), 3D (C18), and
3E (C20) followed the same procedure except for the use of the
appropriate sulfonylchloride.
N1,N14-Bis[N-decanylsulfonyl-3-aminopropyl]-1,14-diamino-
5,10-diazabutadecane Tetrahydrochloride Salt 3B: 1H NMR
(D2O, ppm): 3.10 (m, 24H), 2.11 (m, 4H), 1.92 (m, 4H), 1.72 (m,
8H), 1.37 (m, 4H), 1.23 (m, 24H), 0.78 (t, 6H). Elem anal. Calcd
for C36H84Cl4N6O4S2‚H2O: C, 48.63; H, 9.75; N, 9.45. Found: C,
48.94; H, 9.57; N, 9.34. LC/MS by ESI+ mode analysis observed
m/z 726 at 13.4 min retention time.
N1,N14-Bis[N-dodecanylsulfonyl-3-aminopropyl]-1,14-diamino-
5,10-diazabutadecane Tetrahydrochloride Salt 3C: Elem anal.
Calcd for C40H92Cl4N6O4S2: C, 51.82; H, 10.00; N, 9.06. Found:
C, 51.68; H, 9.99; N, 8.99. LC/MS by ESI+ mode analysis observed
m/z 782 at 15.3 min retention time.
N1,N14-Bis[N-hexadecanylsulfonyl-3-aminopropyl]-1,14-di-
amino-5,10-diazabutadecane Tetrahydrochloride Salt 3D: Elem
anal. Calcd for C48H108Cl4N6O4S2: C, 55.47; H, 10.47; N, 8.09.
Found: C, 55.28; H, 10.54; N, 7.81.
N1-Di[N-octadecanylsulfonyl-3-aminopropyl]-1,14-diamino-
5,10-diazabutadecane Pentahydrochloride Salt 4E: 1H NMR
(D2O, ppm): 3.23 (m, 6H), 3.08 (m, 18H), 2.08 (m, 4H), 1.90 (m,
4H), 1.71 (m, 8H), 1.34 (m, 4H), 1.22 (m, 16H), 0.77 (t, 6H). LC/
MS by ESI+ mode analysis observed m/z 950 at 21.5 min retention
time.
Disubstituted Branched Homologated Spermine-Sulfonamide
Analogs (Series 5: branched-HOMO-SPM)sSynthesis of N1-
Bis[N-octanylsulfonyl-3-aminopropyl]-1,18-diamino-5,9,14-tri-
azaoctadecane Pentahydrochloride Salt 5A: To a solution of 1.0
g (1.5 mmol) of the tetraBocHOMO-spermine derivative 9 produced
in Series 2 (HOMO-SPM) above in 20 mL of dry CH3OH was
added 1.0 mL (10 equiv) of acrylonitrile. A catalytic amount of
Dowex 50W×400 cation-exchange resin was added (250 mg). The
resulting clear mixture was heated to reflux to give a mixture of
mono- and bis-addition products. Following filtration, the solvents
were evaporated and the residue was purified by column chroma-
tography using a 1:1 to 2:1 EtOAc/hexane solvent mixture to give
0.53 g (46%) of the bis-addition product 11 as a colorless oil. A
0.52 g portion of this oil was dissolved in 20 mL of glacial acetic
acid and treated with 0.5 g of Pd(OH)2 under 50 psi H2 pressure
for 3.5 h. The resulting mixture was filtered over a pad of celite
and evaporated to give 0.51 g (95%) of 12 as a clear oil. LC/MS
analysis of this product confirmed identity and purity. A 1.0 g
portion of this product was dissolved in 20 mL of dry CH2Cl2 and
treated with 0.54 mL of Et3N (3 equiv) followed by 0.75 mL (3
equiv) of octanylsulfonylchloride at rt under an argon atmosphere.
After stirring for 16 h, the resulting reaction solution was evaporated
and the crude residue was partitioned between 75 mL of EtOAc
and 50 mL of cold 0.1 N HCl. The organic layer was washed again
using cold 0.1 N HCl then dried and evaporated to give the crude,
oily product. This was purified by silica gel chromatography using
98:2:0.2 CH2Cl2/MeOH/NH4OH to give 0.90 g (62%) pure 13A
product. This material was dissolved in 15 mL of MeOH and treated
with 15 mL of 6 N HCl. The resulting solution was stirred for 16
h when the solvents were evaporated to give 0.70 g (96%) of desired
product 5A in its pentahydrochloride salt as a white solid. Elem
anal. Calcd for C35H84Cl5N7O4S2‚3/2H2O: C, 44.94; H, 9.37; N,
10.48. Found: C, 44.85; H, 9.33; N, 10.43. LC/MS by ESI+ mode
analysis observed m/z 727 at 12.8 min retention time. Synthesis of
5B (C10), 5C (C12), and 5D (C16) followed the same procedure
except for the use of the appropriate sulfonylchloride.
N1,N14-Bis[N-octadecanylsulfonyl-3-aminopropyl]-1,14-diamino-
5,10-diazabutadecane Tetrahydrochloride Salt 3E: LC/MS by
ESI+ mode analysis observed m/z 950 at 24.8 min retention time.
Disubstituted Branched Spermine-Sulfonamide Analogs (Se-
ries 4: branched-SPM)sSynthesis of N1-Di[N-hexadecylsulfo-
nyl-3-aminopropyl]-1,14-diamino-5,10-diazabutadecane Te-
trahydrochloride Salt 4D: To a solution of 2.0 g (4.0 mmol) of
triBoc-spm 6 in 100 mL of dry CH3OH was added 2.7 mL (10
equiv) of acrylonitrile. A catalytic amount of Dowex 50W×400
cation-exchange resin was added (250 mg). The reaction was
refluxed, and after 18 h, TLC analysis (CH2Cl2/MeOH/NH4OH 90:
8:2) showed the reaction was nearly complete. Following filtration,
the solvent was evaporated and the bis-alkylated adduct was purified
by column chromatography using 1:1 hexane/EtOAc to give 2.2 g
(90%) product as an oil. A 1.1 g (1.81 mmol) portion of this material
was dissolved in 20 mL of glacial acetic acid and 1 g of Pd(OH)2
on carbon was added. The mixture was placed under 50 psi H2
pressure and shaken for 5 h. This was followed by filtration of the
mixture over a Celite pad, and the pad was washed twice each with
CH3OH and H2O. The filtrate was diluted in EtOAc and 1 N NaOH,
and the organic layer was removed, dried, and evaporated to give
1.0 g (90%) of 15 as a clear oil. A 0.167 g (0.32 mmol) portion of
this triamine was dissolved in 10 mL of dry CH2Cl2, and 0.136
mL (0.96 mmol, 3 equiv) of triethylamine was added. To the
resulting solution was added 0.171 mL (1.0 mmol, 3.1 equiv) of
hexadecylsulfonyl chloride at 25 °C. Following stirring for 18 h,
the solvents were evaporated and the residue was purified over silica
gel using CHCl3/CH3OH/concd NH4OH 98:1.8:0.2 to give 0.058
g (15%) of pure 16D product. This material was dissolved in 5
mL of CH3OH and treated with 5 mL of 6 N HCl at 25 °C for 16
h. Evaporation gave 0.038 g (75%) of 4D as a white solid. LC/MS
by ESI+ mode analysis observed m/z 894 at 18.3 min retention
time. Synthesis of 4A (C8), 4B (C10), 4C (C12), and 4E (C18)
followed the same procedure except for the use of the appropriate
sulfonylchloride.
N1-Di[N-decanylsulfonyl-3-aminopropyl]-1,18-diamino-5,9,-
14-triazaoctadecane Pentahydrochloride Salt 5B: 1H NMR (D2O,
ppm): 3.26 (m, 6H), 3.06 (m, 22H), 2.09 (m, 10H), 1.68 (m, 8H),
1.38 (m, 4H), 1.25 (m, 24H), 0.75 (t, 6H). Elem anal. Calcd for
C39H92Cl5N7O4S2‚H2O: C, 47.67; H, 9.64; N, 9.98. Found: C,
47.32; H, 9.59; N, 9.80. LC/MS by ESI+ mode analysis observed
m/z 783 at 11.6 min retention time.
N1-Di[N-dodecanylsulfonyl-3-aminopropyl]-1,18-diamino-
1
5,9,14-triazaoctadecane Pentahydrochloride Salt 5C: H NMR
(D2O, ppm): 3.10 (m, 28H), 2.09 (m, 10H), 1.70 (m, 8H), 1.40
(m, 4H), 1.25 (m, 32H), 0.75 (t, 6H). LC/MS by ESI+ mode analysis
observed m/z 839 at 14.6 min retention time.
N1-Di[N-octanylsulfonyl-3-aminopropyl]-1,14-diamino-5,10-
N1-Di[N-hexadecanylsulfonyl-3-aminopropyl]-1,18-diamino-
5,9,14-triazaoctadecane Pentahydrochloride Salt 5D: LC/MS by
ESI+ mode analysis observed m/z 951 at 20.1 min retention time.
1
diazabutadecane Tetrahydrochloride Salt 4A: H NMR (D2O,
ppm): 3.23 (m, 6H), 3.08 (m, 18H), 2.08 (m, 4H), 1.90 (m, 4H),

886 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Burns et al.
Synthesis of Miscellaneous Analogs. The various miscellaneous
analogs shown in Table 2 were synthesized by sulfonylation of the
modified polyamine under standard conditions. These analogs were
purified by column chromatography over silica gel using 80:18:2
CH2Cl2/MeOH/concd NH4OH. Several examples such as 31 and
32 were synthesized using the corresponding acid chloride instead
of sulfonyl chloride. Values (m/z; ESI+) and retention times (in
minutes) obtained by LC-MS were as follows: 20, 493/17.9; 21,
489/18.9; 22, 492/11.7; 23, 365/10.8; 24, 422/11.1; 25, 436/12.2;
26, 477/11.9; 27, 434/11.2 min; 28, 338/10.8; 29, 394/11.1; 30,
377/10.8; 31, 627/11.0; 32. 1H NMR (D2O, ppm): 3.12 (m, 20H),
2.18 (t, 4H), 2.05 (m, 4H), 1.84 (m, 4H), 1.70 (m, 4H), 1.51 (m,
4H), 1.17 (m, 16H), 0.75 (t, 6H). LC/MS by ESI+ mode analysis
observed m/z 570 at 11.0 min retention time.
made via direct and regiospecific routes compared to 22. LC/MS
chromatographs of analogs 20-32. This material is available free
of charge via the Internet at http://pubs.acs.org.
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In Vitro Screening Assays: The relative binding affinities of
the sulfonamide analogs with LPS were determined using an
automated, rapid-throughput fluorescence-based displacement assay,
employing BODIPY-TR cadaverine (BC; (5-(((4-(4,4-difluoro-5-
(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)-ami-
no)pentylamine hydrochloride) as the probe.50,51,60,64 Results are
reported as the effective concentration displacing 50% of the LPS-
bound probe (ED50). The inhibition of NO in murine macrophage
J774A.1 cells stimulated with 100 ng/mL LPS was performed using
an automated Griess assay, as described previously.50,51,60,64 The
inhibition of induction of NFκB (a key transcripitional activator of
the innate immune system was quantified using human embryonic
kidney 293 cells cotransfected with TLR4 (LPS receptor), CD14,
and MD2 (co-receptors), available from InvivoGen, Inc. (HEK-
Blue, San Diego, CA), as per protocols provided by the vendor).
Stable expression of secreted alkaline phosphatase (seAP) under
control of NFκB/AP-1 promoters is inducible by LPS, and
extracellular seAP in the supernatant is proportional to NFκB
induction. seAP was assayed spectrophotometrically using an
alkaline phosphatase-specific chromogen at 620 nm using a rapid-
throughput, automated protocol employing a Bio-Tek P2000 liquid
handler. Inhibition of LPS-induced cytokine production was quanti-
fied concurrently in ex vivo healthy human blood using a FAC-
SArray multiplexed (TNF-R, IL-1â, IL-6, IL-8, IL-10, and IL-
12p70) flow-cytometric bead array system (Becton-Dickinson-
Pharmingen, San Jose, CA), as described previously.51 Data for
the above-mentioned three in vitro biological assays are presented
as the concentration of compound inhibiting 50% of the response
(IC50). All experiments were performed in triplicate, and polymyxin
B was included as a reference compound, along with appropriate
negative and positive controls.
Mouse Lethality Experiments: Female, outbred, 9- to 11-week-
old CF-1 mice (Charles River, Wilmington, MA) weighing 22-28
g were used as described elsewhere.46,51 The animals were sensitized
to the lethal effects of LPS by D-galactosamine.83-85 The lethal dose
causing 100% mortality (LD100) for the batch of LPS used (E. coli
0111:B4) was first determined by administering D-galactosamine
(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 cohorts
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. In experiments designed to test
dose-response effects of the polyamine sulfonamides in affording
protection against LPS-induced lethality, mice received graded doses
of compound diluted in saline, i.p. in one flank, immediately before
a supralethal (200 ng) LPS challenge, which was administered as
a separate i.p. injection into the other flank. In experiments in which
the temporal window of protection was to be examined, a fixed
dose of 200 µg/mouse of compound was administered at various
times, before or after supralethal (200 ng/mouse) LPS challenge.
Lethality was determined at 24 h post LPS challenge.
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