Discovery of Novel Alkylated (bis)Urea and (bis)Thiourea Polyamine Analogues with Potent Antimalarial Activities

Abstract

A series of alkylated (bis)urea and (bis)thiourea polyamine analogues were synthesized and screened for antimalarial activity against chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro. All analogues showed growth inhibitory activity against P. falciparum at less than 3 μM, with the majority having effective IC(50) values in the 100-650 nM range. Analogues arrested parasitic growth within 24 h of exposure due to a block in nuclear division and therefore asexual development. Moreover, this effect appears to be cytotoxic and highly selective to malaria parasites (>7000-fold lower IC(50) against P. falciparum) and is not reversible by the exogenous addition of polyamines. With this first report of potent antimalarial activity of polyamine analogues containing 3-7-3 or 3-6-3 carbon backbones and substituted terminal urea- or thiourea moieties, we propose that these compounds represent a structurally novel class of antimalarial agents.

Full text

rXXXX American Chemical Society Adx.doi.org/10.1021/jm200463z |J. Med. Chem. XXXX, XXX, 000–000
ARTICLE
pubs.acs.org/jmc
Discovery of Novel Alkylated (bis)Urea and (bis)Thiourea Polyamine
Analogues with Potent Antimalarial Activities
Bianca K. Verlinden,
†
Jandeli Niemand,
†
Janette Snyman,
†
Shiv K. Sharma,
‡
Ross J. Beattie,
‡
Patrick M. Woster,
§
and Lyn-Marie Birkholtz*
,†
†
Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, PO Box x20, Pretoria, 0028,
South Africa
‡
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University,
Detroit, Michigan 48202, United States
§
Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425,
United States
’INTRODUCTION
Malaria remains one of the most deadly parasitic diseases, with
nearly 250 million new cases each year, resulting in approxi-
mately one million deaths (www.who.int). The spreading resis-
tance of Plasmodium falciparum to existing antimalarials including
chloroquine, antifolates, and artemisinin has resulted in a press-
ing need to discover new chemotherapeutic agents against this
disease.
1
One class of promising antiparasitic agents include
inhibitors of polyamine biosynthesis
2
as well as polyamine analogues,
3
with ample evidence indicating that these rapidly dividing cells
have an exquisite need for the presence of polyamines for a
myriad of cellular functions during cell growth and division.
4,5
The naturally occurring polyamines putrescine (1), spermi-
dine (2), and spermine (3) (Figure 1) interact with a variety of
cellular effector sites due to their highly cationic nature and
specific spatial orientation of positive charge and are therefore
able to stabilize DNA, RNA, and other acidic cellular con-
stituents.
4
Polyamine analogues are structurally similar to the
naturally occurring polyamines and act as either polyamine
antimetabolites that deplete intracellular polyamine pools or
polyamine mimetics that displace the natural polyamines from
their binding sites without substituting for their cellular functions.
6
Particularly, terminal alkylation of polyamines and polyamine
analogues results in a change of pK
a
of the amine groups of these
molecules, resulting in nonfunctional polyamine characteristics.
7,8
Moreover, these analogues may compete for polyamine uptake and,
in mammalian cells in particular, can induce polyamine catabolism.
3
The first generation of antiparasitic alkylpolyamines, typified
by the N,N0-bis(benzyl)-substituted polyamine analogue MDL
27695 (4, Figure 1), exhibited antitrypanosomal and antiplas-
modial activity in the μM range.
911
A bis[(2-phenyl)benzyl)]-
spermine analogue of 4known as BW-1 (5, Figure 1) was sub-
sequently shown to have inhibitory activity against various strains
of Trypanosoma and the microsporidial, Encephalitozoon cuniculi,
particularly by blocking polyamine uptake and inhibiting poly-
amine oxidase activity
9,11,12
and additionally being curative in a
rodent model of infection with the microsporidial organism.
9
Derivatives of 5include analogues of the substituted (bis)biguanide
known as 2d (6, Figure 1) that, in addition to depleting the
polyamine pool, inhibits trypanothione reductase (a spermidine
glutathione conjugate) activity in trypanosomes.
8
Compound 6
and its derivatives are highly active antiparasitic agents, with in
vitro IC
50
s against Trypanosoma brucei as low as 90 nM. Several
urea- and thiourea-based isosteres of 6have subsequently been
shown to be effective epigenetic modulators in mammalian cells
by influencing selective chromatin marks in tumor cell lines
through inhibition of lysine specific demethylase 1, thereby decreas-
ing cancerous cell growth.
13
On the basis of the success of terminally (bis)alkylated polyamine
analogues against other parasites, several analogues of 6, as well
as a new generation of (bis)urea and (bis)thiourea alkylated
Received: April 18, 2011
ABSTRACT: A series of alkylated (bis)urea and (bis)thiourea
polyamine analogues were synthesized and screened for anti-
malarial activity against chloroquine-sensitive and -resistant
strains of Plasmodium falciparum in vitro. All analogues showed
growth inhibitory activity against P. falciparum at less than
3μM, with the majority having effective IC
50
values in the
100650 nM range. Analogues arrested parasitic growth within
24 h of exposure due to a block in nuclear division and therefore asexual development. Moreover, this effect appears to be cytotoxic
and highly selective to malaria parasites (>7000-fold lower IC
50
against P. falciparum) and is not reversible by the exogenous
addition of polyamines. With this first report of potent antimalarial activity of polyamine analogues containing 373or363
carbon backbones and substituted terminal urea- or thiourea moieties, we propose that these compounds represent a structurally
novel class of antimalarial agents.

Bdx.doi.org/10.1021/jm200463z |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
isosteres of 6, were synthesized and evaluated for their ability to
inhibit the proliferation of malaria parasites. These compounds
contain a variety of carbon backbones, and terminal urea/
thiourea substituents that are symmetrically substituted aralkyl
substituents, and as such present a structurally novel class of
scaffolds, unrelated to any known antimalarials. This study reports
the antimalarial activity against drug sensitive and resistant
P. falciparum strains in vitro, their effects on the parasites’
DNA replication and polyamine-specific events.
’RESULTS AND DISCUSSION
Chemical Syntheses of Urea and Thiourea Polyamine
Analogues. To access a library of urea and thiourea analogues
isosteric to 6(720, Table 1; and 2539, Table 2) and anal-
ogous amidine analogues (2124, Table 1), we employed our
previously published synthesis
8,14
of precursor molecules 43,as
shown in Scheme 1. The appropriate diamine 40 (n=1,2,4,5)
was (bis)cyanoethylated (acrylonitrile, EtOH, reflux) to afford
the corresponding (bis)cyano intermediates 41. The central
nitrogens in 41 were then N-Boc protected ((Boc)
2
O, CH
2
Cl
2
/
aq NaHCO
3
)
15
to form 42, and the cyano groups were reduced
(Raney Ni) to yield the desired diamines 43.
8,16
Compounds 43
(n= 1, 2, 4, 5) were then reacted with 2 equiv of appropriate
alkyl- or aryl-substituted isocyanates or isothiocyanates 44 in
anhydrous CH
2
Cl
2
, followed by acid removal of the N-Boc
protection groups (HCl in EtOAc)
15
to afford the desired urea
or thiourea products (720 and 2539). The amidine analo-
gues compounds 2124 were prepared (Scheme 2) by reacting
diamines 43 with 2 equiv of S-naphthylmethyl thioacetimidate
hydrobromide 46 (prepared by refluxing 2-bromomethyl-
naphthalene with thiacetamide in CHCl
3
according to literature
procedure
14
) using absolute ethanol, and the Boc protecting
groups were subsequently removed with HCl in EtOAc.
In Vitro Activity of Polyamine Analogues against P. falci-
parum.The first diverse library of isoteric (bis)urea and (bis)thiourea
alkylated polyamine analogues was tested for possible growth
inhibitory affect against intraerythrocytic P. falciparum in vitro
(Table 1). The majority of these compounds showed in vitro
inhibitory activity against both drug resistant (W2 chloroquine
resistant strain, HB3 antifolate resistant strain) and sensitive
P. falciparum (strain 3D7) at concentrations below 3 μM(Table1).
Compound 6, containing terminal (bis)diphenylpropylguanidine
moieties, is active against P. falciparum at 298 nM. Conversely, it
is clear that amidine substituted analogues that lacked terminal
alkyl groups were not active against the malaria parasite. Com-
pound 21, an amidine analogue containing a 333 carbon
backbone, which lacks any (bis)urea and (bis)thiourea substit-
uents, was the least effective compound (IC
50
= 147 μM,
Table 1). Of the 19 compounds tested, 15 have potent in vitro
antimalarial activity (<1 μM), with the six most active com-
pounds (6,9,13,15,16, and 20) displaying IC
50
values in the
range of 144405 nM against the 3D7 strain of P. falciparum
(Table 1). Moreover, all of these compounds had IC
50
values in
the nM range against drug resistant P. falciparum, with com-
pounds 13,15,16, and 20 being more active against chloroquine-
resistant P. falciparum (strain W2) displaying low resistance
factors (ranging from 0.19 to 0.61) compared to chloroquine
against this strain. This suggests that these analogues are mini-
mally affected by the resistance mechanisms of, e.g., chloroquine,
with a mechanistically distinct mode of action (Table 1).
Analysis of the antimalarial effects of this first series of (bis)urea
and (bis)thiourea alkylated polyamine analogues suggests that the
most potent compounds contain either a 373or343
carbon backbones, with the 373 carbon backbone delivering
the best activity against the parasite. Compounds with (bis)urea
substituents exhibited the most potent antimalarial activity, followed
by the (bis)thiourea substituted compounds. The diphenylpropyl
substituents proved more effective than the diphenylethyl substit-
uentsasterminalgroupsofthesecompounds.Analysisofthe
amidine alkylated polyamine analogues suggests that the 333
and 343 carbon backbones are not effective against the parasite
and that analogues with a 373 carbon backbone are active in the
nM range, particularly when they have terminal diphenylpropyl
substituents. On the basis of results observed with this first
evaluation of the antimalarial activity of polyamine analogues, the
selective design and synthesis of a second series of compounds,
predicted to have a higher antimalarial capacity, was attempted. This
second series of 15 compounds contained 363, 373, and
343 backbones but with a variety of terminal substituents.
Figure 1. The natural polyamines putrescine (1), spermidine (2), and spermine (3) and antimalarial polyamine analogues MDL 27695 (4), BW-1 (5),
and compound 6.

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Journal of Medicinal Chemistry ARTICLE
Table 1. In Vitro Antimalarial Activity of the Compounds 624, against P. falciparum Strains 3D7, HB3, and W2
a
Values are the means (SE of at least three independent experiments (ng3).
b
P. falciparum drug sensitive strain 3D7.
c
P. falciparum chloroquine
resistant strain HB3.
d
P. falciparum antifolate resistant strain W2.
e
Resistance index (RI) defined as the ratio of the IC
50
values of the resistance to
sensitive strains, W2/3D7.
f
Indicates IC
50
values in μM.
g
Control drug, chloroquine

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Journal of Medicinal Chemistry ARTICLE
Of the second series of analogues, 13 had potent antimalarial
activity with IC
50
values in the range of 88846 nM (Table 2).
The five most potent compounds (25,29,30,38, and 39), with
IC
50
values ranging between 88211 nM, were also active
against chloroquine resistant P. falciparum (W2 strain). These
compounds all have a 373or363 carbon architecture,
the majority with (bis)urea and terminal aralkyl substituents. Com-
pounds 34 and 35 were the least effective against the parasite,
with 34 not active in the μMrange,and35 with an IC
50
= 14.08 μM
(Table 2). Both of these contain 363 carbon backbones with
(bis)urea substituents, similar to the structures of the most
effective compounds, with the exception that they feature alkyl
rather than aralkyl substituent rings. This second generation of
synthesized (bis)urea and (bis)thiourea alkylated polyamine
analogues suggests that the most potent compounds contain
either a 363or373 carbon backbones, with the 363
Table 2. In Vitro Antimalarial Activity of the Second-Generation Compounds (2539), against P. falciparum Strains 3D7 and W2
a
Values are the means (SE of at least 3 independent experiments (ng3).
b
P. falciparum drug sensitive strain 3D7.
c
P. falciparum antifolate resistan
strain W2.
d
Resistance index (RI) defined as the ratio of the IC
50
values of the resistance to sensitive strains, W2/3D7.
e
Indicates IC
50
values in μM.
f
Control drug, chloroquine

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Journal of Medicinal Chemistry ARTICLE
carbon backbone delivering the best activity against the parasite.
Compounds with (bis)urea substituents exhibited the most potent
antimalarial activity, followed by the (bis)thiourea substituted
compounds. The selection of terminal substituents of these com-
pounds are of vital importance in enhancing their antimalarial
activity, compounds with terminal phenyl rings had the best
activity, followed by diphenylethyl substituents, benzyl rings, and
last diphenylmethyl substituents. Compounds with no terminal
aralkyl groups were the least effective against the parasite with
IC
50
values in the high μM range, thus demonstrating the impor-
tance of bulky terminal substituents for antimalarial activity.
Some terminally, symmetrically substituted polyamine analo-
gues target DNA and exert a cytotoxic effect through DNA
aggregation.
7
In most instances, the polyamine analogues
(compounds 9,13,15,16, and 25,29,30,38, and 39) elicited
a significant cytotoxic response in the parasite (P< 0.05), as
measured by a decrease in viable cell numbers (% parasitemia,
Figure 2). However, even through compound 20 was active
against the parasite, it was not able to decrease viable cell numbers in
P. falciparum over time, indicating a cytostatic action on in vitro
growth. During its asexual intraerythrocytic development,
P. falciparum replicates its DNA as it develops from single-
nucleated ring (1N) and trophozoite stages (1N) to multinucleated
schizont (2N or >2N) stages that result in up to 32 daughter
merozoites (mononucleated) being formed from a single parent
parasite.
17,18
Polyamines have been shown to be important for
DNA replication and therefore implied life cycle development in
Plasmodia.
19
The effects of the polyamine analogues (compounds
9,13,15, and 16, and 25,29,30,38, and 39) on the ability of the
parasite to replicate its DNA were monitored. An untreated
parasite population contained 28% of parasites with single nuclei
(1N), whereas 38% and 34% were in multinucleated schizonts
forms of 2N or >2N, respectively (Figure 3), demonstrating
complete intraerytrocytic asexual development as expected after
72 h of development as measured by flow cytometry. Treatment
of P. falciparum with compound 20 produced similar cytometric
profiles to untreated parasites and was not able to prevent DNA rep-
lication, confirming its cytostatic nature. However, for compound 16,
Scheme 1
Scheme 2

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Journal of Medicinal Chemistry ARTICLE
a dramatic halt in schizogony and associated nuclear division was
observed with parasites containing 78% 1N rings/trophozoites,
20% 2N and 2% >2N schizonts after treatment with this
compound. Compounds 9,13,15,25,29, and 30 revealed
similar profiles to that of compound 16 (results not shown).
Compounds 16,38, and 39 had the greatest inhibitory affect on
DNA replication and nuclear division with the majority of the
parasites being confined to the ring stage (Figure 3).
Treatment of P. falciparum with polyamine biosynthesis
inhibitors like the substrate analogues α-difluoromethylornithine
and 3-aminooxy-1-aminopropane has been shown to be reversible,
and therefore the inhibitory effect is alleviated with the addition
of exogenous polyamines to the parasite.
20,21
To investigate the
influence of exogenous polyamines on P. falciparum growth
inhibition observed with the current series of polyamine analo-
gues, polyamine reversal studies were performed to determine if
treated parasites could recover when supplemented with exo-
genous putrescine. The inhibitory effect observed with the poly-
amine compounds could not be reversed with exogenous poly-
amines for any of the most potent compounds (Figure 4). The
decreased cell viability observed for compound 16 was again
visible over a 72 h time period, and this was already evident after the
first 24 h, during which the parasite needed to start nuclear division.
Therefore, the cytotoxic action of these polyamine analogues on
P. falciparum seems to be independent of changes in the polyamine
pool. This may be due to the analogues’ability to block the
intracellular binding sites of the natural polyamines, or to displace
intracellular polyamines from their binding sites.
10
Alternatively,
the mode of action of these polyamine analogues against P. falciparum
may be independent of the polyamine pathway. In trypanosomes,
polyamine analogues act as competitive inhibitors of enzymes not
directly related to polyamine biosynthesis.
22
It remains to be seen
if the polyamine analogues manifest their effect on P. falciparum
through targeting epigenetic control mechanisms, as has been ob-
served in mammalian cells.
13
Theparasiteseemstobeexquisitely
sensitive toward epigenetic regulatory mechanisms, particularly for
the control of expression of variant gene families.
23
P. falciparum appear to be highly sensitive to the polyamine
analogues described above, with the majority showing parasite
IC
50
values below 500 nM. To ensure that this effect was not
merely due to general toxicity of the compounds, in vitro cyto-
toxicity testing was performed in a sensitive mammalian cell line.
A subset of the compounds described in this manuscript, notably
14,15,and16, have been evaluated as potential antitumor agents in
the Calu-6 nonsmall cell human lung carcinoma cell line.
13
It is
Figure 2. Viable cell count of P. falciparum (3D7) treated with
compounds 16 or 20 (2 IC
50
). Parasites were treated for 72 h, after
which parasitemia was determined microscopically with Giemsa stains
(counting 100 parasites per slide 10,n= 3) and DNA levels were
quantified using SYBR Green I incorporation. Black bars, % parasitemia
at 0 h; gray bars, after 72 h. Results are the mean of three independent
experiments, performed in triplicate, (SE. Significance is indicated at
P< 0.05 (*) as determined with a Student-ttest.
Figure 3. Flow cytometric analysis of nuclear division of P. falciparum
treated with compounds 16,38, and 39 (2 IC
50
). Ring or trophozoite
stage parasites contain 1 nucleus (1N), followed by nuclear division in
late trophozoites (2N) and multinucleated schizonts (>2N), repre-
sented as the % parasites in each population. Flow cytometric measure-
ment of nuclear content was performed with SYBR Green I staining of
DNA tracked in the FITC channel. Data are represented as the mean of
three independent experiments, performed in duplicate, (SE.
Figure 4. Influence of exogenous addition of polyamines to P. falcipar-
um (3D7) treated with polyamine analogues. Parasites were treated with
compound 16 alone (2 IC
50
, gray circles), or supplemented with
1 mM putrescine after either 24 h (upward triangles) or 48 h (downward
triangles). Parasitemia was monitored for 72 h using SYBR Green I.
Untreated parasites are indicated with squares. Data are represented as
the mean of three independent experiments, performed in duplicate,
(SE. Error bars fall within symbols where not shown. Significance is
indicated at P< 0.05 (*) as determined with two-way ANOVA.

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Journal of Medicinal Chemistry ARTICLE
important to note that these compounds exert antitumor effects
through re-expression of aberrantly silenced tumor suppressor genes
and as such are not inherantly cytotoxic in mammalian cells when used
as single agents. Maximal cytotoxicity to tumor cells in vitro and in vivo
can only be achieved through synergistic effects with a traditional agents
such as 5-azacytidine. The GI
50
values in the Calu-6 cell line for
1416 alone range between 10 and 40 μM and thus they are not
generally cytotoxic agents. The most active compounds against
P. falciparum (compounds 9,13,15,16,and20,and25,29,30,
38,and39) showed remarkable selectivity toward the parasite
compared to a mammalian cell line (HepG2 human hepatocellular
liver carcinoma, Table 3), with the majority of the compounds
(particularly compounds 20,25,29,30,38,and39) showing >500-
fold selectivity toward the parasite. Remarkably, the most active
compound (30)is∼7000-fold more selective toward the parasite.
Comparatively, compounds 14,15,and16 were not the most
effective against P. falciparum and inhibited cell growth in Calu-6 cells
at μM concentrations (Table 3), but even these compounds showed
more than ∼10-fold selectivity against the malaria parasite.
13
’CONCLUSIONS
The results presented here indicate that the title compounds
do not show general cytotoxicity in mammalian cells and that P.
falciparum seems to show selective sensitivity to these polyamine
analogues. These results are encouraging in implicating this
series of polyamine analogues as highly selective antimalarial
agents. Moreover, the ability of polyamine analogues to cure
in vivo infections of malaria in the murine model of Plasmodia
berghei should provide clues as to the ultimate antimalarial
potential of this structurally distinct class of compounds, and
these studies are currently underway.
’EXPERIMENTAL SECTION
All reagents and dry solvents were purchased from Aldrich Chemical
Co. (Milwaukee, WI), Sigma Chemical Co. (St. Louis, MO), or Acros
Chemical (Chicago, IL) and were used without further purification
except as noted below. Triethylamine was distilled from potassium
hydroxide and stored in a nitrogen atmosphere. Methanol was distilled
from magnesium and iodine under a nitrogen atmosphere and stored
over molecular sieves. Methylene chloride was distilled from phosphorus
pentoxide, and chloroform was distilled from calcium sulfate. Tetrahy-
drofuran was purified by distillation from sodium and benzophenone.
Dimethyl formamide was dried by distillation from anhydrous calcium
sulfate and was stored under nitrogen. Preparative scale chromato-
graphic procedures were carried out using E. Merck silica gel 60,
230440 mesh. Thin layer chromatography was conducted on Merck
precoated silica gel 60 F-254. Ion exchange chromatography was
conducted on Dowex 1 8200 anion exchange resin. All
1
Hand
13
C
NMR spectra were recorded on a Varian Mercury 400 MHz spectro-
meter, and all chemical shifts are reported as δvalues referenced to TMS.
In all cases,
1
H NMR,
13
C NMR, and IR spectra were consistent with
assigned structures. Mass spectra were recorded on a Kratos MS 80 RFA
(EI and CI) or Kratos MS 50 TC (FAB) mass spectrometer. Prior to
biological testing, target molecules 739 were determined to be 95%
pure or greater by HPLC chromatography using an Agilent series 1100
high-performance liquid chromatograph fitted with a C18 reversed-
phase column. Compounds 720 were previously synthesized and their
1
H and
13
C NMR spectra have been reported.
13
General Procedure for the Synthesis of Urea and Thiourea
Analogues. Step A. In a 100 mL round-bottom flask, the centrally Boc
substituted diamino compounds 43 (0.5 mmol) were dissolved in 10 mL
of HPLC grade CH
2
Cl
2
and a solution of alkyl-, benzyl-, or phenyl-
substituted isocyanate or isothiocyanate, 44 (1.0 mmol, 2 equiv) in 5 mL
of CH
2
Cl
2
under cold condition was added. The flask was protected with
N
2
atmosphere, and the reaction mixture was allowed to stir at room
temperature for 1824 h, the progress for formation of product was
monitored by TLC using CH
2
Cl
2
/MeOH/NH
4
OH (94.4:5.0:0.5 or
89:10:1). After completion of the reaction, CH
2
Cl
2
was removed under
reduced pressure on a rotary evaporator to produce a viscous colorless
material. The product was transferred into the next step without further
purification.
Step B. The above crude product was dissolved in anhydrous
MeCO
2
Et (6.0 mL) and after 1 M HCl in MeCO
2
Et (6.0 mL) was
added, the reaction mixture becomes cloudy. The flask was protected
with N
2
atmosphere and the reaction mixture was allowed to stir at room
temperature for 2448 h, and the progress for formation of product was
monitored by TLC using CH
2
Cl
2
:MeOH:NH
4
OH (89:10:1 and
78:20:2). After completion of the reaction as confirmed by TLC, and
the nature of the product (white crystalline materials separated from the
solution), MeCO
2
Et was removed under reduced pressure on a rotary
evaporator to produce a white powder. The solid product was well
stirred with 20 mL of fresh MeCO
2
Et and the soluble part was decanted,
and the solid so obtained was vacuum-dried to give pure product as a
white solid.
1,14-Bis-{3-[1-(10,10-diphenylmethyl)thioureado]}-4,11-diazatetrade-
cane Hydrochloride, 25 (RJB-92-09C).
1
H NMR (DMSO-d
6
): δ8.83
(bs, 6H, NH), 8.26 (bs, 2H, NH), 7.28 (bs, 16H, ArH), 7.21 (bs, 4H,
ArH), 6.71 (b, 2H, CHPh
2
), 3.39 (bs, 4H, NCH
2
), 2.87 (bs, 4H,
NCH
2
), 2.82 (bs, 4H, NCH
2
), 1.85 (bs, 4H, CH
2
CH
2
), 1.58 (bs, 4H,
CH
2
CH
2
), 1.28 (bs, 4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ183.21
(CdS), 143.41, 129.07, 127.89, 127.57 (ArC), 61.28, 47.22, 45.23,
41.39, 26.38, 26.08, 25.85 (CH
2
).
1,14-Bis-{3-[1-(20,20-diphenylethyl)thioureado]}-4,11-diazatetrade-
cane Hydrochloride, 26 (RJB-92-09).
1
H NMR (DMSO-d
6
): δ9.00
(bs, 4H, NH), 7.80 (bs, 2H, NH), 7.54 (b, 2H, NH), 7.28
(bs, 16H, ArH), 7.17 (bs, 4H, ArH), 4.37 (b, 2H, CHPh
2
),
4.01 (b, 4H, NCH
2
), 3.43 (bs, 4H, NCH
2
), 2.79 (bs, 8H, NCH
2
),
1.80 (bs, 4H, CH
2
CH
2
), 1.60 (bs, 4H, CH
2
CH
2
), 1.30 (bs,
4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ183.00 (CdS), 143.40,
Table 3. Selectivity of Polyamine Analogues for Growth
Inhibition of P. falciparum (3D7) Compared to Selected
Mammalian Cells
compd P. falciparum
(IC
50
,μM)
Calu-6
(GI
50
,μM)
a
HepG2
(GI
50
,μM)
b
SI
c
(Calu-6/Pf)
SI
c
(HepG2/Pf)
90.144 (0.031 23.92 (0.4 166
13 0.253 (0.003 24.52 (1.4 97
14 1.316 (0.01 9.4 nd 7
15 0.329 (0.009 38.3 18.92 (1.39 38 58
16 0.405 (0.008 10.3 24.67 (0.3 25 61
20 0.355 (0.09 >200 >500
25 0.211 (0.007 >200 >500
29 0.106 (0.011 >200 >1500
30 0.088 (0.007 619.3 (25.8
d
7038
38 0.130 (0.002 >200 >1500
39 0.1 (0.003 33.8 (3.24 338
CQ 0.009 22.16 (0.39 2462
a
Data obtained from Sharma et al.
13
Calu-6 are human nonsmall cell
lung carcinoma cells.
b
Values are the means (SE of at least 2
independent experiments performed in duplicate. HepG2 are human
hepatocellular liver carcinoma cells.
c
Selectivity indices were determined
as the compound GI
50
mammalian cell/IC
50
P. falciparum.
d
Values are
the means (SE of 3 independent experiments performed in duplicate.

Hdx.doi.org/10.1021/jm200463z |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
129.17, 128.65, 127.08 (ArC), 50.46, 48.71, 47.22, 45.12, 41.22,
26.25, 26.14, 25.84 (CH
2
).
1,14-Bis-{3-[ 1-( 30,30-diphenylpropyl)thioureado]}-4,11-diazatetra-
decane Hydrochloride, 27 (RJB-92-11C).
1
H NMR (DMSO-d
6
): δ8.86
(bs, 4H, NH), 7.87 (bs, 4H, NH), 7.317.24 (m, 16H, ArH), 7.14
(t, 4H, J= 7.2 Hz, ArH), 4.01 (t, 2H, J= 7.2 Hz, CHPh
2
), 3.43 (b, 4H,
NCH
2
), 3.22 (b, 4H, NCH
2
), 2.83 (b, 8H, NCH
2
), 2.23 (b, 4H, NCH
2
),
1.82 (bs, 4H, CH
2
CH
2
), 1.52 (bs, 4H, CH
2
CH
2
), 1.29 (bs, 4H,
CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ145.37, 129.11, 128.30, 126.79
(ArC), 48.62, 47.22, 45.17, 41.00, 34.92, 26.36, 26.11, 25.86 (CH
2
).
1,14-Bis-{3-[ 1-( phenyl)thioureado]}-4,11-diazatetradecane Hydr-
ochloride 28 (RJB-92-06).
1
H NMR (DMSO-d
6
): δ10.09 (bs, 2H, NH),
8.90 (b, 4H, NH), 8.33 (bs, 2H, NH), 7.45 (d, 4H, J= 8.0 Hz, ArH),
7.28 (t, 4H, J= 8.0 Hz, ArH), 7.06 (t, 2H, J= 7.6 Hz, ArH), 3.54 (b,
4H, NHCH
2
), 2.84 (b, 8H, NCH
2
), 1.96 (bs, 4H, CH
2
CH
2
), 1.60 (bs,
4H, CH
2
CH
2
), 1.35 (bs, 4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
):
δ181.29 (CdS), 140.10, 129.16, 124.61, 123.38, 47.21, 45.25, 41.34,
28.74, 26.12, 25.88.
1,14-Bis-{3-[1-(benzyl)thioureado]}-4,11-diazatetradecane Hydro-
chloride 29 (RJB-92-11).
1
H NMR (DMSO-d
6
): δ8.96 (bs, 4H, NH),
8.25 (b, 2H, NH), 8.05 (bs, 2H, NH), 7.367.20 (m, 10H, ArH), 4.64
(s, 4H, ArCH
2
NH), 3.48 (bs, 4H, NHCH
2
), 2.82 (bs, 8H, NCH
2
), 1.85
(bs, 4H, CH
2
CH
2
), 1.60 (bs, 4H, CH
2
CH
2
), 1.30 (bs, 4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ182.00 (CdS), 140.01, 128.90, 127.91,
127.44, 47.23, 45.15, 41.30, 31.98, 26.34, 26.14, 25.85.
1,14-Bis-{3-[1-( phenyl)ureado]}-4,11-diazatetradecane Hydrochloride
30 (RJB-92-04).
1
HNMR(DMSO-d
6
): δ8.99 (s, 2H, NH), 8.88 (bs, 4H,
NH), 7.37 (d, 4H, J=8.0Hz,ArH), 7.19 (t, 4H, J=7.2Hz,ArH), 6.84
(t, 2H, J=7.2Hz,ArH), 6.66 (bs, 2H, NH), 3.14 (b, 4H, NHCH
2
), 2.85
(b, 8H, NCH
2
), 1.77 (t, 4H, J=6.8Hz,CH
2
CH
2
), 1.59 (bs, 4H, CH
2
CH
2
),
1.29 (bs, 4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ156.36 (CdO), 141.23,
129.28, 121.62, 118.20, 47.22, 45.28, 36.83, 27.29, 26.09, 25.86.
1,14-Bis-{3-[1-(benzyl)ureado]}-4,11-diazatetradecane Hydrochloride
31 (RJB-92-13).
1
H NMR (DMSO-d
6
): δ9.06 (bs, 4H, NH), 7.30
7.16 (m, 14H, ArH, and NH), 4.18 (s, 4H, ArCH
2
NH), 3.08 (b, 4H,
NHCH
2
), 2.79 (b, 8H, NCH
2
), 1.73 (b, 4H, CH
2
CH
2
), 1.58 (bs, 4H,
CH
2
CH
2
), 1.27 (bs, 4H, CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ159.33
(CdO), 141.50, 128.88, 127.60, 127.20, 47.16, 45.12, 43.56, 36.99,
27.44, 26.10, 25.82.
1,14-Bis-{3-[1-( ethyl)thioureado]}-4,11-diazatetradecane Hydro-
chloride 32 (RJB-92-08).
1
H NMR (DMSO-d
6
): δ9.92 (bs, 4H, NH),
7.79 (s, 2H, NH), 7.79 (s, 2H, NH), 3.42 (bs, 4H, NHCH
2
), 3.31 (bs,
4H, NCH
2
), 2.82 (bs, 8H, NCH
2
), 1.82 (b, 4H, CH
2
CH
2
), 1.59 (b, 4H,
CH
2
CH
2
), 1.30 (b, 4H, CH
2
CH
2
), 1.02 (t, 6H, J= 7.20 Hz, CH
3
).
13
C
NMR (DMSO-d
6
): δ45.13, 41.04, 39.00, 26.36, 26.11, 25.84, 15.11.
1,14-Bis-{3-[1-( n-propyl)thioureado]}-4,11-diazatetradecane Hy-
drochloride 33 (RJB-92-14C).
1
H NMR (DMSO-d
6
): δ8.93 (bs, 4H,
NH), 7.83 (bs, 2H, NH), 7.76 (bs, 2H, NH), 3.43 (bs, 4H, NHCH
2
),
3.25 (bs, 4H, NCH
2
), 2.83 (bs, 8H, NCH
2
), 1.82 (bs, 4H, CH
2
CH
2
),
1.59 (bs, 4H, CH
2
CH
2
), 1.44 (q, 4H, J= 7.2 Hz, CH
2
CH
3
), 1.29 (b, 4H,
CH
2
CH
2
), 0.82 (t, 6H, J= 7.20 Hz, CH
3
).
13
C NMR (DMSO-d
6
): δ
47.20, 46.00, 45.13, 41.03, 26.36, 26.12, 25.84, 22.70,12.08.
1,14-Bis-{3-[1-(ethyl)ureado]}-4,11-diazatetradecane Hydrochlor-
ide 34 (RJB-92-15C).
1
H NMR (DMSO-d
6
): δ9.09 (bs, 4H, NH), 7.27
(b, NH), 7.79 (s, 2H, NH), 3.042.95 (m, 8H, NHCH
2
), 2.80 (bs, 8H,
NCH
2
), 1.71 (b, 4H, CH
2
CH
2
), 1.60 (b, 4H, CH
2
CH
2
), 1.29 (b, 4H,
CH
2
CH
2
), 0.95 (t, 6H, J= 6.8 Hz, CH
3
).
13
C NMR (DMSO-d
6
): δ
159.22 (CdO), 47.15, 45.10, 37.00, 34.92, 27.34, 26.11, 25.81, 16.23.
1,14-Bis-{3-[1-( n-propyl)ureado]}-4,11-diazatetradecane Hydro-
chloride 35 (RJB-92-13C).
1
H NMR (DMSO-d
6
): δ9.07 (bs, 4H,
NH), 7.77 (b, 4H, NH), 3.04 (t, 4H, J= 6.0 Hz, NHCH
2
), 2.90
(t, 4H, J=7.2Hz,NCH
2
), 2.79 (b, 8H, NCH
2
), 1.71 (q, 4H, J=6.8
Hz, CH
2
CH
2
), 1.60 (b, 4H, CH
2
CH
2
), 1.361.28 (m, 8H,
CH
2
CH
2
), 0.79 (t, 6H, J=7.6Hz,CH
3
).
13
C NMR (DMSO-d
6
):
δ159.36 (CdO), 47.14, 45.10, 41.87, 36.96, 27.38, 26.10, 25.80,
23.77, 12.02.
1,12-Bis-{3-[1-(10,10-diphenylmethyl)ureado]}-4,9-diazadodecane
Hydrochloride, 36 (SKS-96-02).
1
H NMR (DMSO-d
6
): δ8.95 (b, 4H,
NH), 8.19 (b, 2H, NH), 7.397.18 (m, 20H, ArH), 6.48 (b, 2H, NH),
5.86 (d, 2H, J= 8.4 Hz, CHPh
2
), 3.07 (t, 4H, J= 5.6 Hz, NCH
2
), 2.75 (b,
8H, NCH
2
), 1.79 (b, 4H, CH
2
CH
2
), 1.58 (b, 4H, CH
2
CH
2
).
13
C NMR
(DMSO-d
6
): δ158.64 (CdO), 144.45, 129.04, 127.59, 127.37 (ArC),
57.78, 46.55, 45.09, 36.86, 27.47, 23.20 (CH
2
).
1,12-Bis-{3-[ 1-( 20,20-diphenylethyl)ureado]}-4,9-diazadodecane Hydro-
chloride, 37 (SKS-96-01).
1
HNMR(DMSO-d
6
): δ9.11 (bs, 4H, NH),
7.347.15 (m, 20H, ArH), 4.10 (t, 2H, J= 6.0 Hz, CHPh
2
), 3.64 (m, 4H,
NCH
2
), 3.01 (bs, 4H, NCH
2
), 2.81 (bs, 4H, NCH
2
), 2.75 (bs, 4H, NCH
2
),
1.68 (bs, 8H, CH
2
CH
2
).
13
CNMR(DMSO-d
6
): δ159.05 (CdO), 143.77,
129.10, 128.58, 126.92 (ArC), 51.72, 46.51, 45.06, 44.53, 36.83, 27.39, 23.25
(CH
2
).
1,15-Bis-{3-[1-(10,10-diphenylmethyl)ureado]}-4,12-diazapentade-
cane Hydrochloride, 38 (SKS-96-02C).
1
H NMR (DMSO-d
6
): δ8.93
(bs, 4H, NH), 7.287.14 (m, 20H, ArH), 5.88 (bs, 2H, CHPh
2
), 3.08
(bs, 4H, NCH
2
), 2.77 (bs, 4H, NCH
2
), 2.69 (bs, 4H, NCH
2
), 1.71 (bs,
4H, CH
2
CH
2
), 1.53 (bs, 4H, CH
2
CH
2
), 1.20 (b, 6H, CH
2
CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ159.30 (CdO), 144.02, 129.60, 127.38,
127.60 (ArC), 57.70, 42.26, 45.07, 36.81, 28.56, 27.47, 26.37, 25.88
(CH
2
).
1,15-Bis-{3-[1-( 20,20-diphenylethyl)ureado]}-4,12-diazapentadecane
Hydrochloride, 39 (SKS-96-01C).
1
H NMR (DMSO-d
6
): δ9.06 (bs, 4H,
NH), 7.317.14 (m, 20H, ArH), 4.10 (t, 2H, J= 7.6 Hz, CHPh
2
), 3.64
(m, 4H, NCH
2
), 3.01 (t, 4H, J= 5.6 Hz, NCH
2
), 2.74 (bs, 8H, NCH
2
),
1.68 (m, 4H, CH
2
CH
2
), 1.64 (m, 4H, CH
2
CH
2
), 1.16 (bs, 6H,
CH
2
CH
2
CH
2
).
13
C NMR (DMSO-d
6
): δ159.11 (CdO), 143.75,
129.11, 128.58, 126.94 (ArC), 51.71, 47.26, 45.06, 44.55, 36.82, 28.62,
27.39, 26.42, 25.91 (CH
2
).
S-2-Naphthylmethyl Thioacetimidate Hydrobromide 46 (SKS-84-
31). To a stirred solution of thioacetamide (1.127 g, 15 mmol) in
anhydrous CHCl
3
(40 mL) was added 2-bromomethylnaphthalene 45
(3.40 g, 15 mmol) by cooling the reaction flask. The reaction mixture
was allowed to stir at room temperature for 5 min and then heated to
reflux for 2 h, cooled to back to room temperature, and placed in an ice
bath. The resulting solid was filtered off, washed with 50 mL CHCl
3
, and
dried in vacuum for 3 h to afford 46 (3.83 g, 86%) as a white solid,
1
H
NMR (DMSO-d
6
): δ8.00 (s, 1H ArH), 7.96 (m, 3H, ArH), 7.56
(m, 3H, ArH), 3.80 (s, 2H, SCH
2
), 2.62 (s, 3H, CH
3
).
13
C
NMR (DMSO-d
6
): δ193.48 (CdS), 133.48, 133.15, 131.42, 129.48,
128.83, 128.40, 128.37, 127.51, 127.42, 127.34 (ArC), 36.74 (CH
2
),
24.86 (CH
3
).
General Procedure for the Synthesis of Amidine Analo-
gues. The diamino compound 43 (0.50 mmol) was dissolved in 12 mL
of absolute ethanol, and a solution of S-2-naphthylmethyl thioacetimi-
date hydrobromide 46 (310 mg, 1.0 mmol, 2 equiv) was added under
cold condition. The flask was protected with N
2
atmosphere, the
resulting suspension that eventually becomes homogeneous was allowed
to stir at room temperature for 4872 h, and the progress for formation
of product was monitored by TLC using CH
2
Cl
2
/MeOH/NH
4
OH
(89:10:1). After completion of the reaction, the ethanol was removed
under reduced pressure on a rotary evaporator to produce a viscous
colorless material, which was purified by stirring the mixture with dry
ether, discarded ether soluble part, and insoluble material was again
stirred with fresh ether (25 mL). The white solid so obtained was dried
in vacuum.
1,11-Bis-(acetamidinyl)-4,8-di( tert-butyloxycarbonyl)-4,8-diazoun-
decane Hydrobromide 47a (SKS-84-40).
1
H NMR (DMSO-d
6
): δ9.38
(bs, 1H, NH), 9.06 (bs, 1H, NH), 8.55 (bs, 1H, NH), 8.27 (bs, 1H, NH),
7.19 (t, 2H, NH), 3.203.05 (m, 12H, NCH
2
), 2.11 (s, 6H, CH
3
),
1.721.60 (m, 6H, CH
2
), 1.33 (s, 18H, C[CH
3
]
3
).

Idx.doi.org/10.1021/jm200463z |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
1,12-Bis-(acetamidinyl)-4,9-di( tert-butyloxycarbonyl)-4,9-diazodo-
decane Hydrobromide 47b (SKS-84-31C).
1
H NMR (DMSO-d
6
):
δ9.18 (b, 1H, NH), 9.04 (b, 1H, NH), 8.50 (b, 1H, NH), 7.91 (b,
1H, NH), 7.08 (t, 2H, NH), 3.1o (b, 12H, NCH
2
), 2.11 (s, 6H, CH
3
),
1.71 (m, 4H, CH
2
), 1.421.30 (bs, 22H, CH
2
and C[CH
3
]
3
).
1
H NMR
(CD
3
OD): δ3.30 (t, 4H, J= 7.2 Hz, NCH
2
), 3.24 (bs, 8H, NCH
2
), 2.24
(s, 6H, CH
3
), 1.90 (m, 4H, CH
2
), 1.53 (m, 4H, CH
2
), 1.45 (s, 18H,
C[CH
3
]
3
).
1,14-Bis-(acetamidinyl)-4,11-di( tert-butyloxycarbonyl)-4,11-diazo-
tetradecane Hydrobromide 47c (SKS-99-05C).
1
H NMR (CD
3
OD):
δ3.303.19 (m, 12H, NCH
2
), 2.24 (s, 6H, CH
3
), 1.87 (b, 4H, CH
2
),
1.55 (b, 4H, CH
2
), 1.45 (s, 18H, C[CH
3
]
3
), 1.32 (b, 4H, CH
2
).
1,15-Bis-(acetamidinyl)-4,12-di( tert-butyloxycarbonyl)-4,12-diazo-
pentadecane Hydrobromide 47d (SKS-84-33C).
1
H NMR (CD
3
OD):
δ3.283.19 (m, 12H, NCH
2
), 2.22 (s, 6H, CH
3
), 1.86 (m, 4H, CH
2
),
1.54 (m, 4H, CH
2
), 1.45 (s, 18H, C[CH
3
]
3
), 1.29 (m, 6H, CH
2
).
Cleavage of Boc Group. The compound 47ad(0.430.45
mmol) was stirred with 6 mL of anhydrous MeCO
2
Et for 5 min, and 1 M
HCl in MeCO
2
Et (5 mL) was added. The flask was protected with N
2
atmosphere, and the reaction mixture was allowed to stir at room
temperature for 1824 h, the progress for formation of product was
monitored by TLC (CH
2
Cl
2
:MeOH:NH
4
OH 78:20:2). After comple-
tion of the reaction, MeCO
2
Et was removed under reduced pressure on
a rotary evaporator to produce a white powder. The solid product was
well stirred with 20 mL of fresh MeCO
2
Et, the decanted the soluble part
was decanted, and the solid so obtained was vacuum-dried to give pure
product 2124 as a white solid.
1,11-Bis-(acetamidinyl)-4,8-diazoundecane Hydrochloride 21 (SKS-
84-40C).
1
H NMR (DMSO-d
6
): δ9.75 (s, 2H, NH), 9.29 (s, 2H,
NH), 9.23 (s, 4H, NH), 8.79 (s, 2H, NH), 2.98 (b, 12H, NCH
2
), 2.13 (s,
6H, CH
3
), 2.07 (b, 2H, CH
2
), 1.89 (b, 4H, CH
2
).
13
C NMR (DMSO-
d
6
): δ164.79 (dCH), 44.83, 44.61, 39.54 (NCH
2
), 24.73, 22.85(CH
2
),
19.27 (CH
3
).
1
H NMR (D
2
O): δ3.35 (t, 4H, J= 6.4 Hz, NCH
2
),
3.183.11 (m, 8H, NCH
2
), 2.19 (s, 6H, CH
3
), 2.122.09 (m, 2H,
CH
2
), 2.082.01 (m, 2H, CH
2
).
13
C NMR (D
2
O): δ164.80 (dCH),
45.35, 44.83, 39.37 (NCH
2
), 29.86, 24.07(CH
2
), 18.70 (CH
3
).
1,12-Bis-(acetamidinyl)-4,9-diazododecane Hydrochloride 22 (SKS-
84-35).
1
H NMR (DMSO-d
6
): δ9.88 (s, 2H, NH), 9.10 (s, 6H, NH),
8.81 (s, 2H, NH), 3.34 (m, 4H, NCH
2
), 2.94 (b, 4H, NCH
2
), 2.87 (b, 4H,
NCH
2
), 2.14 (s, 6H, CH
3
), 1.90 (m, 4H, CH
2
), 1.71 (b, 4H, CH
2
).
13
C
NMR (DMSO-d
6
): δ164.79 (dCH), 46.62, 44.72, 39.84 (NCH
2
), 24.71,
23.21 (CH
2
), 19.26 (CH
3
);
1
HNMR(D
2
O): δ3.34 (t, 4H, J=7.2Hz,
NCH
2
), 3.133.06 (m, 8H, NCH
2
), 2.20 (s, 6H, CH
3
), 2.02 (m, 4H,
CH
2
), 1.76 (m, 4H, CH
2
).
13
CNMR(D
2
O): δ47.26, 45.19, 39.40
(NCH
2
), 24.09, 23.07 (CH
2
), 18.69 (CH
3
). HR-MS m/z285.4
1,14-Bis-(acetamidinyl)-4,11-diazotetradecane Hydrochloride, 23
(SKS-99-06).
1
H NMR (DMSO-d
6
): δ9.79 (s, 2H, NH), 9.27 (s, 2H,
NH), 9.13 (s, 4H, NH), 8.82 (s, 2H, NH), 3.33 (b, 4H, NCH
2
), 2.93
(b, 4H, NCH
2
), 2.83 (b, 4H, NCH
2
), 2.14 (s, 6H, CH
3
), 1.90 (b, 4H,
CH
2
), 1.63 (b, 4H, CH
2
) 1.30 (b, 4H, CH
2
).
13
C NMR (DMSO-d
6
):
δ164.77 (dCH), 47.28, 44.77, 39.98 (NCH
2
), 26.11, 25.74, 24.73-
(CH
2
), 19.22 (CH
3
).
1
H NMR (D
2
O): δ3.21 (t, 4H, J= 6.4 Hz,
NCH
2
), 2.96 (t, 4H, J= 7.2 Hz, NCH
2
), 2.90 (t, 4H, J= 7.6 Hz, NCH
2
),
2.06 (s, 6H, CH
3
), 1.89 (m, 4H, CH
2
), 1.54 (b, 4H, CH
2
) 1.25 (b, 4H,
CH
2
).
13
C NMR (D
2
O): δ165.30 (dCH), 47.81, 44.97, 39.36
(NCH
2
), 25.45, 25.34, 23.99(CH
2
), 19.10 (CH
3
).
1,15-Bis-(acetamidinyl)-4,12-diazopentadecane Hydrochloride 24
(SKS-84-34).
1
H NMR (DMSO-d
6
): δ9.78 (s, 2H, NH), 9.25 (s, 2H,
NH), 9.12 (s, 4H, NH), 8.81 (s, 2H, NH), 3.33 (m, 4H, NCH
2
), 2.93 (b,
4H, NCH
2
), 2.82 (b, 4H, NCH
2
), 2.14 (s, 6H, CH
3
), 1.89 (m, 4H,
CH
2
), 1.62 (b, 4H, CH
2
) 1.27 (b, 6H, CH
2
).
13
C NMR (DMSO-d
6
):
δ164.77 (dCH), 47.35, 44.75, 39.84 (NCH
2
), 28.58, 26.39, 25.86,
24.75 (CH
2
), 19.26 (CH
3
).
1
H NMR (D
2
O): δ3.34 (t, 4H, J= 6.4 Hz,
NCH
2
), 3.09 (t, 4H, J= 8.0 Hz, NCH
2
), 3.03 (t, 4H, J= 8.0 Hz, NCH
2
),
2.20 (s, 6H, CH
3
), 2.04 (m, 4H, CH
2
), 1.66 (m, 4H, CH
2
), 1.35 (b, 6H,
CH
2
).
13
C NMR (D
2
O): δ48.05, 45.05, 39.46 (NCH
2
), 27.97, 25.73,
24.09 (CH
2
), 18.71 (CH
3
).
In Vitro Cultivation of P. falciparum.P. falciparum 3D7 (chloroquine
sensitive), W2 (chloroquine resistant), and HB3 (antifolate resistant) strains
were used to assess the in vitro antimalarial efficacy of the polyamine analogues.
The parasites were maintained in O
+
human red blood cells suspended at 5%
hematocrit in RPMI-1640 culture medium containing 23.81 mM sodium
bicarbonate, 0.024 mg/mL gentamycin, 25 mM HEPES, 0.2% glucose, 0.2 mM
hypoxanthine, and 5 g/L Albumax II. The parasites were incubated with
moderate shaking at 60 rpm at 37 °Cinanatmosphereof5%CO
2
,5%O
2
,and
90% N
2
.
24
Cultures were synchronized to >95% in the ring stage with 10%
(w/v) D-sorbitol treatment.
In Vitro Assessment of Antimalarial Activity. In vitro activity
against erythrocytic stages of P. falciparum (strains 3D7 and W2) was
determined using the Malaria SYBR Green I-based fluorescence assay
(MSF)
25
based on the DNA binding properties of this dye. Compounds
were dissolved in a nonlethal DMSO concentration (<0.013%),
26
serially diluted and added to the ring stage P. falciparum (1% parasitemia,
2% hematocrit) and incubated at 37 °C, static for 96 h. Subsequently,
equal volumes (100 μL) of the parasite suspension were added to SYBR
Green I lysis buffer (0.2 μL/mL of 10000SYBR Green I (Invitrogen),
20 mM Tris, pH 7.5, 5 mM EDTA, 0.008% (w/v) saponin, 0.08% (v/v)
Triton X-100) and incubated in the dark for 1 h at room temperature.
Fluorescence was read with a Fluoroskan Ascent FL microplate reader at
an excitation of 485 nm and emission of 538 nm. The data was
represented as percentage of untreated control to determine cell
proliferation. Nonlinear regression curves were generated using Sigma
Plot 11.0, from which the 50% inhibitory concentrations (IC
50
) could be
determined. Each compound was tested in duplicate for at least three
independent biological replicates.
Determination of Parasite DNA Replication. The effect of the
compounds on P. falciparum DNA replication and nuclear division was
determined using flow cytometry. Parasites (2% parasitemia, 2% hemat-
ocrit) were treated with test compounds (2 IC
50
), and 50 μL samples
were isolated at set time intervals following drug exposure. Parasites
were fixed with of 1 mL of 0.025% glutaraldehyde for 45 min and kept at
4°C until use. Fixed cells were washed twice with 1PBS, resuspended
in 20 μL PBS, and stained with 20 μL 1:1000 SYBR Green I for 30 min in
the dark at room temperature. DNA fluorescence was measured with a
BD FACS Aria I flow cytometer (Becton Dickinson) analyzing 10
6
cells
for each sample with fluorescence emission collected at an excitation
wavelength of 488 nm with 502 nm long band-pass and 530 nm band-
pass emission filters. BD FACS Diva Software (6.1.1) and FlowJo v9.1
(Tree Star) were used to analyze the data.
Determination of Polyamine Reversal of Inhibition. The
ability of exogenous putrescine to rescue polyamine analogue treated
parasites was determined using polyamine reversal studies that were set
up in the format similar to that of the MSF assay. P. falciparum 3D7
cultures were treated in early ring stages with test compounds at 2 
IC
50
. After 24 and 48 h, the cultures were supplemented with 1 mM
putrescine and incubated for a further 24 or 48 h, after which parasitemia
was determined as described for the MSF assay. Statistical analysis was
performed with GraphPad InStat 3.10, all data given are the mean of at
least three independent biological repeats.
Cytotoxicity Determinations in Mammalian Cells. Human
hepatocellular liver carcinoma cells (HepG2, kind gift by Duncan
Cromarty, University of Pretoria) were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10% heat
inactivated fetal bovine serum and 1% penicillin/streptomycin at
37 °C (5% CO
2
, 90% humidity). Cytotoxicity was measured using the
lactate dehydrogenase assay (LDH). Cells (100000) were seeded in 96-
well plates and grown for 24 h at 37 °C, after which cells were treated
with various concentrations of the compounds. After 48 h exposure, cells

Jdx.doi.org/10.1021/jm200463z |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry ARTICLE
were pelleted at 250gfor 10 min and LDH activity was measured in the
supernatant (10 μL) by adding 100 μL LDH reaction mix (BioVision)
and incubating for 30 min at room temperature. Colorimetric detection
of NADH levels (as a measurement of LDH-mediated oxidation of
lactate indicating LDH activity) occurred at 450 nm. Experiments were
performed in duplicate for at least two independent biological repeats.
’AUTHOR INFORMATION
Corresponding Author
*Phone: +27 12 420 2479. Fax: +27 12 362 5302. E-mail:
lbirkholtz@up.ac.za.
’ACKNOWLEDGMENT
We thank Wayne Barnes at the Flow Cytometry and Cell
Sorting Unit in the Department of Biochemistry, University of
Pretoria, for technical assistance and Bridgette Cummings for
proofreading the manuscript. This work was supported by the
South African Malaria Initiative (www.sami.org.za), the South
African National Research Foundation (NRF Grant FA2007050-
300003) and the University of Pretoria (L.M.B.) and National
Institutes of Health grant 7RO1-CA149095 (P.M.W.). B.V. was
supported by grants from TATA Africa and the South African
Malaria Initiative. Any opinion, findings and conclusions or
recommendations expressed in this paper are those of the
author(s) and therefore the NRF does not accept any liability
in regard hereto.
’ABBREVIATIONS USED
MSF, malaria SYBR Green I-based fluorescence assay
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