Derivatives of pentamidine designed to target the Leishmania lipophosphoglycan

Article abstract of DOI:10.1016/j.tetlet.2004.11.112

Compound 7, based on pentamidine, was designed to increase the parent compound's affinity for the Leishmania cell surface. The leishmanicidal activity of 7 is similar to that of the nonboronated diamine 4 and appears to be more effective at lower concentrations. The Leishmania lipophosphoglycan (LPG) is the most abundant cell surface glycoconjugate of a family of infectious protozoa. Pentamidine, a common drug used in the treatment of Leishmania infections, has been modified with boronic acids so that it might bind more selectively to the phosphodisaccharide repeating unit of the LPG. This could serve to target the drug to the protozoan surface and increase its efficacy in vivo.

Full text of DOI:10.1016/j.tetlet.2004.11.112

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 695–698
Derivatives of pentamidine designed to target the
Leishmania lipophosphoglycan
Kari L. Kramp,a Kristin DeWitt,b Jason W. Flora,a David C. Muddiman,a,
Kelli M. Sluntb,* and Todd A. Houstona,c,
*
aDepartment of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA
bDepartment of Chemistry, University of Mary Washington, Fredericksburg, VA 22401, USA
cSchool of Science and Eskitis Institute, Griffith University, Nathan, QLD 4111, Australia
Received 1 October 2004; revised 22 November 2004; accepted 22 November 2004
Abstract—The Leishmania lipophosphoglycan (LPG) is the most abundant cell surface glycoconjugate of a family of infectious pro-
tozoa. Pentamidine, a common drug used in the treatment of Leishmania infections, has been modified with boronic acids so that it
might bind more selectively to the phosphodisaccharide repeating unit of the LPG. This could serve to target the drug to the pro-
tozoan surface and increase its efficacy in vivo.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Leishmaniasis is a potentially fatal disease that afflicts
millions of people in tropical regions of the world.1
The Leishmania protozoa that cause the disease are typi-
cally transmitted by sandflies during a bloodmeal. The
parasite exists within the sandfly as an extracellular,
noninfectious promastigote, but during the process of
metacyclogenesis, the promastigotes cease dividing, de-
tach from the epithelial cells of the midgut and migrate
to the mouthparts of the insect.2 These virulent metacy-
clic promastigotes are taken up by the host macrophages
when the sandfly feeds and differentiation into virulent
amastigotes takes place.3 Liposomal amphoteracin B is
uptaken by macrophages and this is the only treatment
currently approved by the US FDA4 for use against vis-
ceral leishmaniasis.5 Other methods that have been
explored for selective drug delivery include liposomal
entrapped antimony,6,7 methotrexate-maleylated BSA
conjugates,8 pentamidine-loaded methactrylate nano-
particles,9 aphidicolin nanosuspension formulations,10
and 8-aminoquinoline analogs conjugated to N-acetyl-
mannosamine containing polymers.11 While these meth-
ods allow selective uptake by infected macrophages,
little attention has been paid to enhancing a drugÕs affi-
nity to the protozoa itself.
The lipophosphoglycan (LPG, Fig. 1),12 the most abun-
dant cell surface glycoconjugate on the promastigote,
has been implicated in the invasion of macrophages by
the protozoa and protects the organism from the hydro-
lytic environment of these immune cells.13,14 The unique
structure of the phosphodisaccharide repeating unit of
the LPG displays an array of unsubstituted cis-vicinal
diols and these offer an attractive target for reversible
binding to boronic acids. Boronic acids are known to
have a greater affinity for galactose and mannose than
for glucose at physiological pH;15 thus, drug molecules
modified with boronic acids should selectively bind to
the Leishmania cell surface. Oligomeric boronates
should have an even higher affinity for the protozoan
LPG if they are properly spaced to match the distance
between each galactose and mannose in the repeating
phosphodisaccharide. We have recently reported the
synthesis of such oligomers based on d-aminoboronic
acids with varying length spacer units.16 Here, we report
modification of antileishmanial compounds using the
same chemistry to further explore the potential method
of using boronates in drug targeting.17,18
Pentamidine (1),19 a drug that has long been used to
treat leishmaniasis,20 offers an excellent template on
which to append boronic acids to bind to the LPG. In
Keywords: Boronate drug targeting; Pentamidine; Leishmania.
*
˚
its extended form pentamidine spans over 18 A from
end to end—a distance greater than the maximal dis-
tance of separation between any two phosphates in the
Corresponding authors. Tel.: +61 7 3875 4115; fax: +61 7 3875
6572; e-mail: t.houston@griffith.edu.au
Present address: Mayo Clinic, Rochester, MN, USA.
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.11.112

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K. L. Kramp et al. / Tetrahedron Letters 46 (2005) 695–698
O
HO
HO
OH
O
HO
O
O
HO
OH
O
O
P
O
O
n (navg=16-32)
cap
repeating phosphodisaccharide
hexasaccharide core
lyso-alkyl-PI
Figure 1. General structure of Leishmania LPG.1
˚
repeat unit (<12 A). Due to the dication formed in aque-
O
n
O
ous solution and the flexible nature of the pentyl linker
in the drug, it should have some modest intrinsic affinity
for the cell surface LPG. Addition of one or two boronic
acids to both amidines would be expected to increase the
compoundÕs affinity for the LPG repeat. The reversible
nature of boronate–carbohydrate binding would still
allow the modified pentamidine to enter the cell while
increasing the effective concentration of the drug at the
cell surface. Pentamidine is a known DNA minor groove
binder and can inhibit topoisomerase.21 One question to
be addressed by this work is whether the substitution of
boronates on this core structure diminishes its effective-
ness to kill the protozoa or whether it makes the drug
more effective at lower concentrations by targeting it
to the cell surface (Fig. 2).
H
N
B
O
HO
H
N
H
B
O
O
O
O
O
O
O
O
O
O
O
O
P
P
O
O
OH
O
OH
HO
O
O
HO
O
OH
OH
Figure 2. Possible binding modes of 5–7 to LPG.
phenylboronate substitution had on the activity of the
parent diamines.
To simplify the synthesis of boronic acid derivatives, a
series of related diamines (2–4)22 were targeted for
study. The amidine group has been implicated in the
toxicity of 1 through its affinity for the imidazoline I2
receptor and diamines based on 2 have been identified
as novel DNA minor groove binding agents.23 Other
derivatives of the pentamidine core structure have
antiprotozoal activity against Leishmania and related
trypanosomes24,25 and alternative topoisomerase
inhibitors possess antileishmanial activity.26 Original
studies with pentamidine had shown that compounds
with either a three or five carbon linker had the best try-
panocidal activity.19 Each of these offered sufficient
spacing to allow both cationic ammonium species to
bind to two phosphates in the LPG repeat unit simulta-
neously. Even the three-carbon linker of 5 allows for
O
O
H2N
NH2
NH
NH
1
O
O
n
H2N
NH2
2: n = 1
3: n = 2
4: n = 3
O
O
˚
greater than 15 A separation between the amines in ex-
H
H
N
n
tended form. The diamines 2–422,23 and their boronic
acid derivatives 5–727 were readily synthesized through
the following sequence: (1) reaction of 4-hydroxybenzo-
nitrile with an appropriate dibromide, (2) nitrile reduc-
tion with LAH,22 and (3) double reductive amination
with o-formylphenylboronic acid.16,28 Initially, mass
spectral analysis of these compounds using FAB in
either positive or negative ion modes gave molecular
ion peaks much larger than expected. It became appa-
rent that the diboronates were extracting two molecules
of glycerol from the FAB matrix and this was confirmed
by observing an increase in 32 mass units when rerun-
ning the samples with a thioglycerol matrix. With these
compounds in hand, their lethality to growing protozoa
could then be tested in vitro to determine what effect
N
B(OH)2
B(OH)2
5: n = 1
6: n = 2
7: n = 3
H
N
H
N
N
H
B(OH)2
B(OH)2
8
As can be seen in Table 1,29 the diamine derivatives are
not as lethal as pentamidine to the protozoa (Leishmania

K. L. Kramp et al. / Tetrahedron Letters 46 (2005) 695–698
697
Table 1. Percent parasite fatality
120
100
Cmpd 2
80
60
40
20
0
Cmpd 4
Cmpd 5
Cmpd 7
Pent
3.13
6.25
12.5
25
50
100
Concentration (mM)
chagasi promastigotes); however, some interesting
trends are evident. The 4-carbon-linked compound 3
was less active (data not shown) than either 2 or 4,
which is consistent with the SARof other pentamidine
derivatives with the 5-carbon-linker (4) displaying best
activity.19 The boronate derivatives 5 and 7 of the 3-car-
bon-(2)23 and 5-carbon-linked (4)22 dibenzylic amines,
displayed very similar activities indicating the boronate
substitution does not drastically alter the desired biologi-
cal activity of these compounds.
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.tetlet.
2004.11.112.
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Importantly, the activities of the boronate derivatives
appeared to decrease less rapidly than their respective
diamines at lower concentration and this may be due
to increased affinity for LPG that in turn increases its
effective concentration around the organism. The results
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any cytotoxic effects against the protozoa at concentra-
tions used here.
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tency versus Leishmania protozoa at low millimolar con-
centrations. While aromatic boronic acids are somewhat
toxic, this adverse property should be reduced by the
presence of an amine that can form an intramolecular
Lewis acid–base complex. Not only would such a com-
plex buffer the Lewis acidity of the boronate, but since
it is charge neutral, it would also facilitate the com-
poundÕs passage through biological membranes.30–32
We are currently synthesizing more complex boronate
derivatives in attempts to increase their potency and will
study their binding properties to the repeat oligosaccha-
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We wish to thank Virginia Commonwealth University
for support through the Mary Kapp Fund and Grant-
In-Aid Program. Funding from the Jeffress Memorial
Trust is also gratefully acknowledged. We also thank
Professor Richard D. Pearson (U. Virginia) for supply-
ing the Leishmania protozoa.
19. Ashley, J. N.; Barber, H. J.; Ewins, A. J.; Newbery, G.;
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(C38H46B2N2O8–H+, diglycerol-boronate adduct) calcd
679.36, found 679.19.
Compound 7: 1H NMR(300 MHz, CD 3OD) d 1.70 (m,
2H), 1.85 (m, 4H), 3.85 (s, 4H), 3.90 (s, 4H), 4.00 (t,
J = 6.2 Hz, 4H), 6.95 (d, J = 8.8 Hz, 4H), 7.05 (m, 2H),
7.15 (m, 4H), 7.35 (d, J = 8.8 Hz, 4H), 7.45 (m, 4H) ppm.
13C NMR(300 MHz, CD 3OD) d 22.77, 29.02, 50.48,
52.90, 67.80, 114.68, 126.56, 126.7, 127.15, 130.96, 141.2,
159.50 ppm. FAB-MS (C39H48B2N2O8–H+, diglycerol-
boronate adduct) calcd 693.37, found 693.23.
24. Donkor, I. O.; Huang, T. L.; Tao, B.; Rattendi, D.; Lane,
S.; Vargas, M.; Goldberg, B.; Bacchi, C. J. Med. Chem.
2003, 46, 1041–1048.
25. Stephens, C. E.; Brun, R.; Salem, M. M.; Werbovetz, K.
A.; Tanious, F.; Wilson, W. D.; Boykin, D. W. Bioorg.
Med. Chem. Lett. 2003, 13, 2065–2069.
28. Gray, C. W., Jr.; Houston, T. A. J. Org. Chem. 2002, 67,
5426–5428.
29. Compounds 2–7 were tested for leishmanicidal activity
against the L. chagasi strain as follows. A 10 mM stock
solution for each of the eight trials (six compounds in
DMSO, pentamidine (1) in DMSO, and a DMSO
control) were prepared. Each drug was diluted to a
concentration of 400 lM with modified HOMEM buffer
system. Addition of 40 lL DMSO in 460 lL HOMEM
served as a solvent control. Initially, 100 lL of HOMEM
was placed in all 12 rows of 8 columns of an 8 · 12 sterile
cell well plate (FALCON 3872). Duplicate serial dilu-
tions of each drug were made as follows: 100 lL of the
400 lM drug solution was pipetted into wells 1 and 2
(rows 1 and 2), 100 lL of the solution in well 1 was
pipetted into well 3, and 100 lL of the solution in well 2
was pipetted into well 4. This procedure was repeated
until 100 lL was removed from wells 11 and 12 at the
end. To each well 100 lL of L. chagasi promastigotes at
approximately 5 · 106 parasites/mL was added, and the
cell well plate was placed in an incubator at 25 ꢁC
overnight. The parasites were counted after 24 and 48 h
incubation time by pipetting 10 lL of solution from each
well under the cover slip of a hemacytometer with a 5 · 5
major grid divided into 4 · 4 minor grids. The number of
parasites in two major grids was counted (viewed at 400·
magnification), averaged, and converted to parasites/mg
by multiplying by 25 · 104. The average parasite counts
were converted into percent parasite fatality as shown in
Table 1.
26. Slunt, K. M.; Grace, J. M.; MacDonald, T. L.; Pearson,
R . D.Antimicrob. Agents Chemother. 1996, 40, 706–709.
27. Synthesis of diboronates 5–7: The same general procedure
was carried out throughout the series.28 Compounds 222,23
(225 mg, 0.78 mmol), 322 (210 mg, 0.71 mmol), 422
(200 mg, 0.64 mmol) were each dissolved in reagent grade
MeOH (5 mL) along with o-formylphenylboronic acid
(234 mg, 1.56 mmol; 212 mg, 1.42 mmol; 191.5 mg,
1.28 mmol; respectively) and these were allowed to stir at
rt for 3–4 h. NaBH4 (74 mg, 67 mg, 60.5 mg, respectively)
was added and the reactions were stirred for an additional
2 h, then concentrated under vacuum. CH2Cl2 (10 mL)
was added and the mixture was filtered. Dropwise addition
of hexanes to the filtrate provided the products 5 (380 mg,
87% yield), 6 (200 mg, 48%), and 7 (225 mg, 62%) as white
powders.
Compound 5: 1H NMR(300 MHz, CD 3OD) d 2.24 (q, J =
6.2 Hz, 2H), 3.85 (s, 4H), 3.89 (s, 4H), 4.18 (t, J = 6.0 Hz,
4H), 7.00 (d, J = 8.8 Hz, 4H), 7.12 (m, 2H), 7.20 (m, 4H),
7.35 (d, J = 8.8 Hz, 4H), 7.50 (m, 2H) ppm. 13C NMR
(300 MHz, CD3OD) d 29.72, 51.03, 52.56, 64.42, 114.59,
126.35, 127.0, 128.0, 130.87, 141.5, 159.22 ppm. FAB-MS
(C37H44B2N2O8–H+, diglycerol-boronate adduct) calcd
665.34, found 665.22.
Compound 6: 1H NMR(300 MHz, CD 3OD) d 1.97 (m,
4H), 3.86 (s, 4H), 3.92 (s, 4H), 4.10 (m, 4H), 6.98 (d,
J = 8.8 Hz, 4H), 7.08 (m, 2H), 7.16 (m, 4H), 7.35 (d,
J = 8.8 Hz, 4H), 7.45 (m, 4H) ppm. 13C NMR(300 MHz,
CD3OD) d 26.09, 50.49, 52.97, 67.60, 114.58, 126.43,
126.78, 127.02, 130.92, 141.3, 159.36 ppm. FAB-MS
30. Gardiner, S. J.; Smith, B. D.; Duggan, P. J.; Karpa, M. J.;
Griffin, G. J. Tetrahedron 1999, 55, 2857–2864.
31. Draffin, S. P.; Duggan, P. J.; Duggan, S. A. M. Org. Lett.
2001, 3, 917–920.
32. Smith, B. D.; Davis, J. P.; Draffin, S. P.; Duggan, P. J.
Supramol. Chem. 2004, 16, 87–90.