Novel alkylpolyamine analogues that possess both antitrypanosomal and antimicrosporidial activity

Article abstract of DOI:10.1016/S0960-894X(01)00315-8

A novel series of alkyl- or aralkyl-substituted polyamine analogues was synthesized containing a 3-7-3 polyamine backbone. These analogues were evaluated in vitro, and in one case in vivo, for activity as antitrypanosomal agents, and for activity against opportunistic infection caused by Microsporidia. Compound 21 inhibits trypanosomal growth with an IC₅₀ as low as 31 nM, while compound 24 shows promising activity in vitro against trypanosomes, and against Microsporidia in vitro and in vivo.

Full text of DOI:10.1016/S0960-894X(01)00315-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1613–1617
Novel Alkylpolyamine Analogues That Possess Both
Antitrypanosomal and Antimicrosporidial Activity
Yu Zou,a Zhiqian Wu,a Nilantha Sirisoma,a Patrick M. Woster,a,*
Robert A. Casero, Jr.,b Louis M. Weiss,c Donna Rattendi,d
Schennella Laned and Cyrus J. Bacchid
aDepartment of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, USA
bThe Johns Hopkins Oncology Center, 424 N. Bond St., Baltimore, MD 21231, USA
cAlbert Einstein College of Medicine, Bronx, NY 10461, USA
dHaskins Laboratories and Department of Biology, Pace University, New York, NY 10038, USA
Received 26 March 2001; accepted 3 May 2001
Abstract—A novel series of alkyl- or aralkyl-substituted polyamine analogues was synthesized containing a 3-7-3 polyamine back-
bone. These analogues were evaluated in vitro, and in one case in vivo, for activity as antitrypanosomal agents, and for activity
against opportunistic infection caused by Microsporidia. Compound 21 inhibits trypanosomal growth with an IC50 as low as 31 nM,
while compound 24 shows promising activity in vitro against trypanosomes, and against Microsporidia in vitro and in vivo. # 2001
Published by Elsevier Science Ltd.
There is no doubt that significant advancements in
anti-infective therapy have improved the quality of life
in developed nations. However, in underdeveloped
countries, there exist major infectious diseases that
account for a large portion of global morbidity. Some
of these diseases have the potential to become a threat
to those living in North America. Tuberculosis claims
an estimated 2 million lives each year, and drug-resis-
tant strains originally found in New York and Russia
are now being identified in other locations. Malaria,
African trypanosomiasis and Leishmaniasis accounted
for an additional 1,210,000 deaths in 1999.1 Also of
concern is the increasing incidence of opportunistic
infections such as those caused by Pneumocystis carinii
and strains of Microsporidia. Drug discovery efforts
against the diseases mentioned above are limited
because infected persons in underdeveloped areas can-
not afford even a single course of therapy, or because
the infected population represents too small of a drug
market. Clearly, there is a need for new antiinfective
agents that are potent, nontoxic and inexpensive to
manufacture. We recently described the synthesis and
evaluation of a small series of (bis)alkylated polyamines
that possess promising antitrypanosomal effects in
vitro.2 Analogues possessing a 3-3-3 carbon skeleton
(Fig. 1), such as BENSpm 1, CPENSpm 2, and
CHENSpm 32,3 have significant antitumor effects in
vitro and in vivo, but are inactive against cultured try-
panosomes. By contrast, analogues with a 3-7-3-carbon
skeleton, typified by MDL 27695 4,4 CHE-3-7-3 5,2,3
and bis-CH-3-7-3 62,3 are inactive as antitumor agents,
but possess promising antitrypanosomal effects in
vitro. We now report second generation compounds
in the 3-7-3 series, their biological evaluation as anti-
trypanosomal agents, and their activity against Micro-
sporidia in vitro and in vivo.
Chemistry
The syntheses leading to (bis)alkylated polyamines,
which are outlined in Scheme 1, are facile and straight-
forward, and as such are suitable for the efficient and
cost-effective production of a variety of active analo-
gues. The tetraamine 7 can be readily synthesized from
1,3-bis-[(amino)methyl]cyclohexane in three steps.2
Cyanoethylation5 followed by Raney nickel reduction5
of the cyano groups affords the corresponding tetra-
amine in high yield, which is then tetramesitylated (2-
mesitylenesulfonyl chloride, dichloromethane, 10%
*Corresponding author. Tel.:+1-313-577-1525; fax: +1-313-577-
2033; e-mail: woster@wizard.pharm.wayne.edu
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00315-8

1614
Y. Zou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1613–1617
aqueous NaOH)6 to afford the protected intermediate 7.
Bis alkylation of 7 (Method A, NaH, 2.2 equiv of R-X)2
affords the (bis)-alkylated intermediates, which are
deprotected (30% HBr in AcOH)7 to yield the pure
desired target compounds 8–12 as the tetra-
hydrobromide salts following recrystallization from
ethanol/water.
this stereochemistry was retained during the palla-
dium(0) coupling reaction.
The remaining target compounds were synthesized
using method C, outlined in Scheme 1. Bis alkylation of
20 (NaH, 1.5 or 2.2 equiv of R-X)2 afforded the (mono)-
or (bis)-alkylated intermediates, respectively, which
were deprotected (30% HBr in AcOH)7 to yield the pure
desired target compounds 21–25 as their tetra-
hydrobromide salts following recrystallization from
ethanol/water.
A second route (Scheme 1, method B) was employed for
the synthesis of analogues 16–19. The monoalkylated
1,3-diaminopropanes 13–155 were appended to the
appropriate cis-1-acetoxy-4-aminocycloalkane8 by pal-
ladium(0) catalyzed coupling as previously described,
affording the target compounds 16–19 in a single step.
These analogues were converted to the corresponding
trishydrobromide salts by anion exchange chromato-
graphy, and purified by recrystallization from ethanol/
water. The absolute stereochemistry of all three cis-1-
acetoxy-4-aminocycloalkanes used was determined by
Mosher amide analysis, as previously described,9 and
Each synthetic intermediate and target compound was
1
fully characterized by H and 13C NMR (Bruker QE
300), and by IR spectroscopy (Nicolet Magna 550). The
purity of all target compounds was assessed by either
combustion analysis (Atlantic Microlabs) or high reso-
lution mass spectrometry. The alkylpolyamine analo-
gues synthesized in this series to date are shown in
Figure 2.
Figure 1. Structures of the biologically active alkylpolyamines 1–6.
Scheme 1.

Y. Zou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1613–1617
1615
Antitrypanosomal Assay
containing medium at 3 and 6 days. On day 8, wells
were fixed overnight, stained with Giemsa, and counted
using an inverted microscope at 400Â magnification.
Five confluent fields (240 cells/field) were counted for
control as well as for drug treated wells. The percentage
of infected cells in the presence of the compound was
compared to the percentage of infected control cells.
IC50 values are given in mM.
Assays for inhibition of trypanosomal growth following
treatment with compounds of general structure 4 were
conducted as previously described.2,10 Activities of the
test compounds were compared to that of melarsen
oxide, a known trypanocidal agent, as a positive con-
trol. Trypanosomes were grown in modified IMDM
+20% horse serum in 24-well microplates at 37 ꢀC.
Wells were inoculated with 1Â105 trypanosomes, and
compounds were solubilized in the medium. One half
the volume of each well was changed daily. Cell counts
(Coulter counter) were made at 24 and 48 h. Control
cells grew to 5Â106/mL. IC50 values were determined
from semi-log plots. T.b. brucei Lab 110 EATRO is a
pentamidine and melarsen-sensitive strain and T.b. rho-
desiense KETRI 243 As-10-3 is a melarsen and penta-
midine resistant clone of a clinical isolate, KETRI 243.
KETRI 269 is also a melarsen-resistant clinically iso-
lated strain.
Effect of polyamine analogues on mice infected with
Encephalitozoon cuniculi
CD8 knockout mice (C57B/6JDCD8)12 were infected
with 3Â107 or 1Â106 spores, respectively, by intraper-
itoneal injection 24 h before treatment.13 Animals were
treated with intraperitoneal drug for 5 days followed by
2 days without drug and then 5 more days of drug.
Animals that survived >28 days post-infection with no
histologic or PCR evidence of parasites were considered
cured. Tissues were obtained from mice at the end
of the observation periods, fixed and embedded per
standard protocols. Tissue sections were stained
with heamatoxylin and eosin (H&E) and tissue
chromotrope stain for microsporidia. These sections
were examined in a blinded fashion for pathology
and presence of organisms.
Antimicrosporidial Assays
Encephalitozoon cuniculi in vitro
RK-13 cells grown as a monolayer in Falcon 24-well
plates were infected with E. cuniculi (in controls 50–80%
of cells were infected).11 Drugs were added to duplicate
wells and incubated for 7 days, changing the drug
Results
The results of the in vitro screening of alkylpolyamine
analogues 2–6, 8–10, 16, 18, 19, 21, and 23–25 against
four strains of trypanosomes are shown in Table 1.
Consistent with our previous studies,2 the 3-3-3 analo-
gues 2 and 3 had no detectable activity against any of
the four strains of trypanosome tested. Analogues 16,
18, and 19, which contain a 1,3-diaminopropane back-
bone, were also devoid of activity. Marginal anti-
trypanosomal activity was observed in the case of
analogues 8–10. Although these analogues more closely
Table 1. In vitro IC50 values (mM) for growth inhibition of four
strains of Trypanosoma brucei by bis-substituted polyamine analgues
Compound
LAB110
K243
K269
K243 As-10-3
2
3
4
5
6
8
9
10
16
18
>100
>100
14.5
>100
>100
15.1
18.4
0.98
4.15
81.5
14.0
>100
>100
>100
0.04
0.38
0.19
>100
>100
12.3
21.0
0.69
5.1
>100
1.71
—
—
—
—
—
>100
>100
13.0
26.2
0.78
56
>100
2.15
—
—
—
0.165
0.79
0.20
5.5
18.0
0.125
4.05
73.0
3.25
>100
>100
>100
0.031
0.31
0.24
22
0.001
19
21
22
24
0.75
16.5
0.5
25
Melarsen oxide
18.5
0.04
—
Each data point represents the average of two determinations which
differed by less than 5% in all cases. >100 refers to the fact that there
was no appreciable inhibitory activity up to the highest concentration
tested.
Figure 2. Structures of ‘second generation’ alkylpolyamines evaluated
for antitrypanosomal and antimicrosporidial activity.

1616
Y. Zou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1613–1617
resemble the active 3-7-3 analogues than compounds 16,
18, and 19, they contain a ring in the central carbon
chain which restricts their rotation. As was described
previously,2 compounds 4, 5, and 6 have significant
activity against all four strains of trypanosomes, and are
equally effective against the arsenic-resistant strain
K243 As-10-3. Among the second generation analo-
gues, compounds 21, 23, and 24 showed activity that
was as much as an order of magnitude greater than
compound 6, the most active first generation analo-
gue. In particular, compound 21 showed activity
comparable to the currently used arsenical melarsen
oxide, with the added benefit that it is active against
the arsenic resistant K243 As-10-3 at a concentration
of 0.165 mM.
Discussion
Trypanosomes are hemoflagellates that live in the blood
and tissues of their human hosts. African trypanoso-
miasis is caused by Trypanosoma brucei rhodesiense or
Trypanosoma brucei gambiense, while South American
trypanosomiasis is caused by a distinct organism, Try-
panosoma cruzi. In these organisms, the polyamines
putrescine and spermidine are synthesized,2 but no
spermine is formed. Spermidine is instead used to pro-
duce trypanothione, a disulfide intermediate analogous
to glutathione, which protects the organism from oxi-
dative stress.14 Melaminophenyl arsenical compounds
such as melarsoprol and melarsen oxide are currently
the only therapy for late-stage trypanosomiasis. These
compounds enter trypanosomal cells via a recently
characterized adenine transporter,15 and react with try-
panothione to form an inactive, stable intermediate
called melT.16 Although arsenical drugs can be effective in
the treatment of late-stage trypanosomiasis, they produce
severe side effects, are extremely painful to administer, and
are lethal to the patient in 5% of cases. There is a clear
need to discover new antitrypanosomal agents that do not
produce the adverse effects associated with arsenicals. The
analogues described in Table 1, and in particular analogue
21, show promising in vitro activity against four major
strains of trypanosome, including one arsenical-resistant
line, K 243 As-10-3. It has recently been reported that a
series of substituted polyamines act as competitive inhibi-
tors of trypanothione reductase, the enzyme that synthe-
sizes trypanothione.17 Additional experiments are
required to determine whether compounds 2–25 exert
their antitrypanosomal effects by interacting with the
polyamine pathway in trypanosomes.
The results of in vitro testing against the microsporidial
organism E. cuniculi are summarized in Table 2. All of
the analogues evaluated (8–12, 16–19, 21, 23, 24, and
25) had some degree of activity in the micromolar
range. However, the most potent analogues were 16, 18,
19, and 24, and IC50 values were determined for these
compounds.
Because microsporidia must be grown on a feeder
monolayer layer of cells, a compound with promising
activity must produce potent activity against the organ-
ism without causing frank cytotoxicity to the mono-
layer. This requirement was satisfied by compounds 16,
18, and 24. However, analogue 24 had the most dra-
matic effect on E. cuniculi in vitro (IC50=0.47 mM,
Table 2), and as such was selected for further evaluation
in a murine model for microsporidial infection. Mice
that were left untreated died on day 21 (two mice), day
22 (two mice), and day 24 (one mouse). However, five of
five mice treated with compound 24 at either 1, or 5 mg/
kg/day survived past day 28, and as such were con-
sidered cured of the infection. In surviving animals, no
evidence of microsporidia was found in tissue samples,
either by microscopy or by PCR analysis.
Microsporidia are obligate intracellular, spore-forming
parasites that infect every major animal group. They
form small unicellular spores that contain a coiled polar
tube which facilitates transmission of infection to other
cells. Microsporidiosis is a common human infection,
but is self limited or asymptomatic in immunocompe-
tent hosts. However, microsporidial infection has
become a significant problem among immunocompro-
mised patients.18 The polyamines putrescine, spermidine
and spermine, are present in mature microsporidian
spores,19 and it has recently been reported that they
possess a eukaryotic-like polyamine metabolism.18 In
light of these facts, it is not surprising that polyamine
analogues such as those in Table 2 exhibit significant
antimicrosporidial activity. In addition, compounds 16,
18, and 24 were effective against the organism without
producing overt cytotoxicity in the host RK-13 cell
layer. The most potent of these analogues, compound
24 (IC50=0.47 mM) was further examined in a murine
model for microsporidiosis, and was found to be cura-
tive at two different dosage levels (1 mg/kg and 5 mg/
kg). Additional in vivo testing of analogue 24 is cur-
rently underway. Additional studies are required to
determine the mechanism by which alkylpolyamine
analogues exert their antitrypanosomal and anti-
microsporidial effects. These studies, as well as the
design and synthesis of additional analogues in the ser-
ies, is an ongoing concern in our laboratories.
Table 2. In vitro growth inhibition activity against Encephalitozoon
cuniculi produced by alkylpolyamine analogues
Toxicity
to compd
Activity
Monolayer
8
9
86% Inhibition (100 mM)
100% Inhibition (100 mM)
100% Inhibition (100 mM)
100% Inhibition (250 mM)
100% Inhibition (100 mM)
IC50=232 mM
Y
Y
Y
Y
Y
N
Y
N
Y
Y
Y
N
Y
10
11
12
16
17
18
19
21
23
24
25
76% Inhibition (100 mM)
IC50=36 mM
IC50=204 mM
47% Inhibition (50 mM)
87% Inhibition (500 mM)
IC50=0.47 mM
100% Inhibition (50 mM)
E. cuniculi was grown on RK-13 cells infected as a monolayer (ave 50–
80% infected cells). The percentage of infected cells was determined
after 7 days in the presence of the test compound, and compared to the
percentage of infected control cells. All values listed are derived from
three to five growth curves.

Y. Zou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1613–1617
1617
Acknowledgements
7. Roemmele, R. C.; Rappoport, H. J. Org. Chem. 1988, 53,
2367.
8. Sirisoma, N. S.; Woster, P. M. Tetrahedron Lett. 1998, 39,
1489.
9. Wu, Y. Q.; Woster, P. M. Bioorg. Med. Chem. 1993, 1, 349.
10. Guo, J. Q.; Wu, Y. Q.; Rattendi, D.; Bacchi, C. J.; Wos-
ter, P. M. J. Med. Chem. 1995, 38, 1770.
11. Coyle, C. M.; Kent, M.; Tanowitz, H. B.; Wittner, M.;
Weiss, L. M. J. Infect. Dis. 1998, 177, 515.
12. Kahn, I. A.; Schwartzman, J. D.; Kasper, L. H.; Moretto,
M. J. Immunol. 1999, 162, 6086.
These studies were supported by National Institutes of
Health Grants CA63552 (P.M.W.), CA85509 (P.M.W.),
and AI 41398 (L.M.W., C.J.B.). Additional support was
provided by a grant from the UNDP/World Health
Organization, Tropical Disease Research Grant
#960644 (P.M.W.), and a Pace University Scholarly
Research Award (C.J.B.). The expert technical assis-
tance of Mr. Benjamin Wei is gratefully acknowledged.
13. Didier, E. S.; Varner, P. W.; Didier, P. J.; Aldras, A. M.;
Millichamp, N. J.; Murphey-Corb, M.; Bohm, R.; Shadduck,
J. A. Folia Parasitol. 1994, 41, 1.
14. Yarlett, N.; Bacchi, C. J. Biochem. Soc. Trans. 1994, 22,
875.
15. Carter, N. S.; Berger, B. J.; Fairlamb, A. H. J. Biol. Chem.
1995, 270, 28153.
16. Scott, A. G.; Tait, A.; Turner, C. M. Exp. Parasitol. 1997,
86, 181.
References and Notes
1. Morel, C. M. Parisitol. Today 2000, 16, 503.
2. Bellevue, F. H.; Boahbedason, M. L.; Wu, R. H.; Casero,
R. A., Jr.; Rattendi, D.; Lane, S.; Bacchi, C. J.; Woster, P. M.
Bioorg. Med. Chem. Lett. 1996, 6, 2765.
3. Woster, P. M.; Casero, R. A. J. Med. Chem. 2001, 44, 1.
4. Edwards, M. L.; Stemerick, D. M.; Bitonti, A. J.; Dumont,
J. A.; McCann, P. P.; Bey, P.; Sjoerdsma, A. J. Med. Chem.
1991, 34, 569.
17. Li, Z.; Fennie, M. W.; Ganem, B.; Hancock, M. T.;
Kobaslija, M.; Rattendi, D.; Bacchi, C. J.; O’Sullivan, M. C.
Bioorg. Med. Chem. Lett. 2001, 11, 251.
5. Bergeron, R. J.; Garlich, J. R.; Stolowich, N. J. J. Org.
Chem. 1984, 49, 2997.
6. Yajima, H.; Takeyama, M.; Kanaki, J.; Nishimura, O.;
Fujino, M. Chem. Pharm. Bull. 1978, 26, 3752.
18. Bacchi, C. J.; Lane, S.; Weiss, L. M.; Yarlett, N.; Tak-
vorian, P.; Wittner, M. J. Euk. Microbiol. 2001, in press.
19. Coyle, C. M.; Bacchi, C. J.; Yarlett, N.; Tanowitz, H. B.;
Wittner, M.; Weiss, L. M. J. Euk. Microbiol. 1996, 43, 96S.