New Benzophenone-derived bisphosphonium salts as leishmanicidal leads targeting mitochondria through inhibition of respiratory complex II

Article abstract of DOI:10.1021/jm901677h

A set. of benzophenone-derived bisphosphonium salts was synthesized and assayed for lethal activity on the human protozoan parasite Leishmania. A subset of them, mostly characterized by phosphonium substituents with an intermediate hydrophobicity, inhibit

Full text of DOI:10.1021/jm901677h

1
788 J. Med. Chem. 2010, 53, 1788–1798
DOI: 10.1021/jm901677h
New Benzophenone-Derived Bisphosphonium Salts as Leishmanicidal Leads Targeting Mitochondria
through Inhibition of Respiratory Complex II
†
,§
†,§
,†
,‡
J. Rom ꢀa n Luque-Ortega, Peter Reuther, Luis Rivas,* and Christophe Dardonville*
†
‡
Centro de Investigaciones Biol oꢀ gicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, and Instituto de Quı´mica M eꢀ dica, CSIC, Juan de la
Cierva 3, E-28006 Madrid, Spain. These authors contributed equally.
§
Received November 13, 2009
A set of benzophenone-derived bisphosphonium salts was synthesized and assayed for lethal activity on
the human protozoan parasite Leishmania. A subset of them, mostly characterized by phosphonium
substituents with an intermediate hydrophobicity, inhibited parasite proliferation at low micromolar
0
range ofconcentrations. Thebestof thissubset, 4,4 -bis((tri-n-pentylphosphonium)methyl)benzophenone
dibromide, showed a very scarce toxicity on mammalian cells. This compound targets complex II of the
respiratory chain of the parasite, based on (i) a dramatically swollen mitochondrion in treated parasites,
(
(
ii) fast decrease of cytoplasmic ATP, (iii) a decrease of the electrochemical mitochondrial potential, and
iv) inhibition of the oxygen consumption rate using succinate as substrate. Thus, this type of compounds
represents a new lead in the development of leishmanicidal drugs.
Introduction
Chart 1. Known Triphenylphosphonium Compounds with
Antischistosomal (I, III) or Antitrypanosomal Activity (II)
and Newly Synthesized Benzophenone-Derived Bisphospho-
nium Salts (IV)
Leishmaniasis encompasses the different clinical manifesta-
tionscaused by infection withthe protozoan speciesbelonging
to the genus Leishmania. It ranges from the cutaneous form, in
many cases self-healing, to the visceral leishmaniasis caused
by species of the donovani complex, mortal if untreated.
According to WHO estimations, 12 million people are in-
fected worldwide, with 2 million new cases per year, from
which 500 000 cases correspond to the visceral form (http://
www.who.int/leishmaniasis/burden/en/). Nowadays, chemo-
therapy is the only effective way to fight the disease. Until
recently, first-line drugs were constituted almost exclusively
by pentavalent organic antimonials; aside from their intrinsic
high toxicity, their efficacy is threatened by rampant resis-
1
tance. In fact, they are progressively being replaced by either
liposomal formulations of amphotericin B, despite their high
2
cost, or miltefosine, the first successful oral drug against the
3
visceral form of leishmaniasis. Nevertheless, the paucity of
8,9 10
rhodesiense, T. cruzi (II, Chart 1), and Leishmania mex-
new leads in the pipeline is a worrisome scenario, raising
concern about the near future. Thus, the search and develop-
11
icana. However, the literature about the pharmacological
4
applications of phosphonium derivatives is relatively scarce,
encompassing three major fields mainly. The first one consists
of phosphonium compounds, such as p-xylylene bis(triphe-
ment of new leads are an urgent priority.
During the screening of in-house made libraries of aromatic
and aliphatic dicationic derivatives against Trypanosoma
12
5
,6
brucei, the trypanocidal effect of the bisphosphonium deri-
nylphosphonium) or poly(methylene)bis(triphenylphospho-
nium salt (I and III, Chart 1), which showed marked antic-
holinesterase and anticholinergic activities on Schistosoma
0
vative 4,4 -bis((tri-n-pentylphosphonium)methyl)benzophenone
5
dibromide was discovered. This compound showed a good
trypanocidal activity in vitro with a low DNA-binding affi-
13
mansoni. In fact, the phosphonium analogue of choline is
7
nity, in contrast to the high affinity of its guanidine counter-
metabolized and incorporated into phospholipids in mamma-
14,15
lian cells without apparent toxicity.
part, also trypanocidal. Previously, other authors reported
for benzyltriphenylphosphonium derivatives cidal activities
on animal infections by trypanosomatids such as T. brucei
A second group of phosphonium compounds is character-
ized by a strong amphipathic character endowed by the
presence of at least one long alkyl chain substituent. They
16
were extensively used as biocides, in either monomeric or
17-19
*To whom correspondence should be addressed. For L.R.: phone,
polymeric form.
They work as cationic detergents, dis-
þ34 918373112; fax, þ34 915627518; e-mail, luis.rivas@cib.csic.es. For
C.D.: phone, þ34 912587490; fax, þ34 915644853; e-mail, dardonville@
iqm.csic.es.
rupting the cell membrane. Some specificity for this process is
achieved by profiting the higher anionic character of cell
r
pubs.acs.org/jmc
Published on Web 02/03/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1789
a
Scheme 1
phosphines were commercially available except tripentyl-
5
phosphine, which was prepared as previously reported.
The substitution pattern (R , R , and R ) of compounds
1
2
3
1
-19 is shown in Table 1.
Cytotoxicity against Leishmania Parasites. The reduction
of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide] by living cells constitutes a suitable parameter
to assess the viability in an extensive range of cell lines,
2
8
a
Reagents and conditions: (i) NBS, benzoyl peroxide cat., CCl
P, DMF or toluene, 110 °C.
4
,
including Leishmania. As such, the capability of bispho-
sphonium salts to inhibit MTT reduction by Leishmania
parasites was used to assess the potential leishmanicidal
activity of these compounds. The results were compiled in
Table 1. From the initial set of 19 bisphosphonium com-
pounds, 11 showed a LC50 on L. donovani promastigotes at
the low micromolar range. Under identical criteria, the same
subset became active on L. pifanoi axenic amastigotes, except
8
1 2 3
0 °C; (ii) R R R
membranes onprokaryotesandlowereukaryotes (reviewedin
ref 20).
Finally, some phosphonium derivatives act as lipophilic
ions with a delocalized charge and enhanced hydrophobicity.
These characteristics afford their transfer across biological
membranes despite their cationic nature, with a privileged
accumulation in organelles with a strong electrochemical
1
9, with a LC50 higher than 50 μM, the cutoff established in
the present study to select the active candidates (Table 1).
Interestingly, amastigotes, the pathological form of the
parasite in vertebrates, were more susceptible than promas-
tigotes, with the sole exception of compound 14 and the
aforementioned 19. The results underscore certain structural
features modulating the leishmanicidal activity. For com-
pounds with homoalkyl groups, the activity was restricted to
those with a narrow range of substituents, from five- to eight-
carbon length, suggesting an important role for hydropho-
bicity, tightly limited in values. Compound 7, with n-pentyl
substituents, showed the highest potency. The combination
of two alkyl groups plus an aromatic substituent was not
enough to recover activity for methyl (10) or ethyl (11)
substituents, but a better activity was achieved when two
phenyl groups (13, 14) or cyclohexyl substituents were pre-
sent (compound 12). The triphenylphosphonium (15) or
diphenyltolyl (17, 18) compounds showed activities very
close to the n-pentyl substituted compound. In particular,
the diphenyl-p-tolyl analogue (18) displayed the second best
activity of the whole series. Inclusion of a heterocycle was
well tolerated for 2-pyridyl (16) but impaired activity by
more than 1 log order for furanyl (19). As compound 7
turned out to be the most active molecule, on promastigotes
and amastigotes, its toxicity on mammalian cells was as-
sayed. The LC50 of 7 on mouse peritoneal macrophages was
2
1-23
potential, such as mitochondria.
Hence, the massive
accumulation of these lipophilic cations into mitochondria
may by itself lead to mitochondrial dysfunction, which in turn
will trigger the apoptotic mitochondrial pathway (reviewed in
ref 24). This is especially relevant in transformed cells, with a
a
25,26
higher ΔΨm than their nontransformed counterparts.
As far as Leishmania is concerned, the above-mentioned
properties of phosphonium derivatives are particularly rele-
vant if one considers the strong anionic character of the
2
7
parasite plasma membrane and the key role of mitochondria
in the bioenergetics of this parasite. This could in turn
represent a target for antileishmanialchemotherapy. Toassess
this hypothesis, we have synthesized and assayed a number
of bisphosphonium benzophenone derivatives bearing differ-
ent substituents on the phosphonium moieties (IV, Chart 1).
As the DNA-binding properties of some of these compounds
cannot fully account for their trypanocidal effects and likely
7
for their leishmanicidal activities, we set out to study alter-
native mechanisms. In tune with the aforementioned proper-
ties of this class of compounds, their effects on plasma
membrane integrity and mitochondrial functionality in
Leishmania were studied in detail. By complementary bioe-
nergetics and morphological approaches, we have defined the
impairment of mitochondrial functionalityas the basis forthe
leishmanicidal mechanism of bisphosphonium benzophe-
nones, exemplified by 7, the best within this series. This
compound severely impaired mitochondrial ATP synthesis
through inhibition of the respiratory complex II (succinate
dehydrogenase).
2
8.6 ( 4.8 μM. This represents a therapeutic index of 28,
defined as the ratio between the respective LC50 values for
mammalian cells and for amastigotes, the pathological form
in mammals. According to its highest leishmanicidal activity
and scarce toxicity on mammalian cells, compound 7 was
selected for the subsequent set of assays dealing with its
mechanism of action.
Results
Chemistry. The bisphosphonium salts 1-19 were synthe-
sized by reaction of 4,4 -bisbromomethylbenzophenone
with an excess of the corresponding phosphine in either an-
hydrous DMF or anhydrous toluene (Scheme 1). The
bisphosphonium salts were isolated with satisfactory yields
by crystallization from the reaction mixture. All the starting
Decrease of Intracellular ATP Levels. The maintenance of
an adequate intracellular ATP level is essential for cellular
homeostasis. The in vivo luminescence of 3-Luc promasti-
gotes affords a real-time monitorization of changes in the
cytoplasmic ATP pool. Accordingly, compound 7 caused a
drop of the luminescence of the 3-Luc strain in a concentra-
tion-dependent manner, reaching the end-point 10-15 min
after addition (Figure 1). Altogether, these results suggest a
decrease of the cytoplasmic ATP pool caused by 7.
Maintenance of Plasma Membrane Integrity. The drop of
intracellular ATP observed in the previous assay can be
caused either by inhibition of mitochondrial ATP synthesis,
essential in the bioenergetics of Leishmania, or by permea-
bilization of the parasite plasma membrane, leading to ATP
release or its futile hydrolysis by ionic pumps, in an attempt
0
2
9
a
m
Abbreviations: ΔΨ , mitochondrial membrane potential; BSA,
bovine seroalbumin; DMNPE-luciferin, D-luciferin 1-(4,5-dimethoxy-
2-nitrophenyl) ethyl ester; FCCP, carbonyl cyanide p-trifluoromethox-
yphenylhydrazone; HBSS-Glc, Hanks’ balanced salt solution plus 10
mM D-glucose; LC50, drug concentration required to inhibit 50% of
parasite proliferation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide; PI, propidium iodide; rhodamine 123, 2-(6-amino-
3-imino-3H-xanthen-9-yl)benzoic acid methyl ester, chloride; SD, stan-
dard deviation; SDS, sodium dodecyl sulfate.

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790 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Luque-Ortega et al.
m
Table 1. In Vitro Antileishmanial Activity and ΔΨ Variation Induced by Bisphosphonium Salts 1-19
LC50 (μM)
a
b
c
compd
R
1
R
2
R
3
L. donovani promastigote
L. pifanoi amastigote
% ΔΨ
m
miltefosine
12.50 ( 2.90
>50
>50
26.30 ( 3.40
>50
>50
75.00
1
2
3
4
5
6
7
8
9
Me
Et
Me
Et
Me
Et
n-Pr
i-Pr
n-Bu
i-Bu
n-Pr
i-Pr
n-Bu
i-Bu
n-Pr
i-Pr
n-Bu
i-Bu
>50
>50
>50
>50
>50
>50
>50
>50
n-C
c-C
5
H
11
11
17
n-C
c-C
5
H
11
11
17
n-C
c-C
5
H
11
11
17
1.09 ( 0.14
3.30 ( 1.19
5.66 ( 0.46
>50
0.97 ( 0.01
1.67 ( 0.06
1.37 ( 0.05
>50
48.00
42.00
10.80
6
H
6
H
6
H
n-C
Me
Et
8
H
n-C
Me
Et
8
H
n-C
Ph
Ph
Ph
Ph
Ph
Ph
8
H
10
11
12
13
14
15
16
17
18
19
>50
>50
c-C
Me
Et
6
H
11
c-C
Ph
Ph
Ph
Ph
Ph
Ph
6
H
11
2.10 ( 0.84
19.31 ( 0.64
6.90 ( 0.50
2.73 ( 0.99
3.03 ( 0.66
1.90 ( 0.01
1.20 ( 0.29
18.12 ( 1.20
1.45 ( 0.17
1.64 ( 0.5
14.01 ( 1.20
1.53 ( 0.03
2.58 ( 0.30
1.51 ( 0.05
1.38 ( 0.02
>50
83.12
100.00
100.00
67.25
Ph
Ph
Ph
Ph
2-pyridyl
o-tolyl
p-tolyl
2-furanyl
b
100.00
55.75
53.25
2-furanyl
2-furanyl
a
c
Promastigotes of L. donovani strain MHOM/SD/00/1S-2D. Amastigotes of L. pifanoi strain MHOM/VE/60/Ltrod. Percentage of rhodamine 123
accumulated in the promastigote mitochondrion after overnight incubation at the respective LC50, relative to untreated cells.
promastigotes by compound 7 was visualized by transmis-
sion electron microscopy. Electron micrographs showed an
extremely swollen mitochondrion for parasites treated with 7
at 5 μM (Figure 2). This organelle appeared as a huge,
electron translucent vacuole, occupying a large fraction of
the intracellular space. In fact, mitochondrion was only
identified by the presence of the kinetoplast, the mitochon-
drial network of concatenated DNA circles, characteristic of
trypanosomatids. Furthermore, the plasma membrane ap-
peared mostly intact, which confirmed the above results with
SYTOX green.
Effect on Mitochondrial Functionality. The cumulative
evidence of the results obtained by the two previous techni-
ques prompted us to get a deeper insight into mitochondrial
functionality. In the first step, we examined the variation of
ΔΨ , essential to drive the mitochondrial ATP synthesis,
m
assessed by rhodamine 123 accumulation in promastigotes,
and monitored by cytofluorometry. The ΔΨ -driven accu-
Figure 1. Variation of intracellular ATP level assessed by in vivo
luminescence of 3-Luc promastigotes after addition of compound 7.
m
7
mulation of rhodamine showed a consistent decreasing trend
after incubation with growing concentrations of 7 (Figure 3).
The same result was obtained when the incubation was
carried out for 4 h in HBSS, conditions used for luminescence
and cytotoxicity (data not shown). Rhodamine 123 accumu-
lation also decreased around 50% for most of the remaining
leishmanicidal benzophenone-derived bisphosphonium
compounds at their respective LC50 (Table 1), confirming
mitochondrial damage inflicted by these drugs. Neverthe-
less, compounds 13, 14, and 16 did not follow the same trend
(Table 1), suggesting alternative targets.
Promastigotes (2 ꢀ 10 cells/mL) were preloaded with 25 μM
DMNPE-luciferin. When the luminescence signal reached a pla-
teau, luminescence was considered as 100% and compound 7 was
added (t = 0). Variation in luminescence was normalized relative to
the control untreated parasites. Compound 7 concentrations are as
follows (μM): 0.6 (4), 1.0 (1), 2.5 (O), 8.0 (b).
to recover the initial ionic gradients. The latter alternative
was examined by monitoring the entrance of the vital dye
SYTOX green. Even at 25 μM, i.e., a 25-fold higher concen-
tration than its LC , compound 7 was unable to produce any
5
0
significant increase of SYTOX green fluorescence, ruling out
damage to the plasma membrane integrity (data not shown).
Morphological Alterations Induced by Compound 7. In a
further step, the morphological damage inflicted to the
In a step forward, we set out to define the molecular target
of 7 in the mitochondrial ATP synthesis. To afford free
access of specific substrates and inhibitors to the different
complexes of the respiratory chain, digitonin-permeabilized

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1791
m
Figure 3. VariationofΔΨ ofL. donovanipromastigotesmonitored
byrhodamine 123accumulation. Parasites treated with7overnightas
well as control cells were loaded with 0.3 μg/mL rhodamine 123, and
fluorescence level was measured by cytofluorometry. Compound 7
concentration is shown at the side of each trace. Full depolarized
parasites were obtained by incubation with 1 mM KCN.
Figure 4. Variation of oxygen consumption rates of L. donovani
promastigotes by 7. Bar scales for oxygen concentration (Y axis)
and time (X axis) are included. Oxygen consumption rates were
calculated from the corresponding slopes of the traces. Arrows
stand for the addition of the indicated substrates and inhibitors at
their respective final concentrations as stated: 10 μM FCCP, 60 μM
digitonin, 100 μM ADP, 0.1 mM TMPD plus 1.7 mM ascorbate,
Figure 2. Electron microscopy of L. donovani promastigotes trea-
ted with compound 7: (A) control parasites; (B) parasites incubated
for 4 h with 5 μM 7, a concentration that caused ∼80% inhibition of
proliferation. Mitochondrial kinetoplast is indicated by the arrow;
bar, 1 μm.
parasites were used. The integrity of the inner mitochondrial
membrane was preserved, as after ADP addition the re-
spiration increased recovering state 3, in which ADP is no
longer the limiting substrate for oxidative phosphoryla-
tion. At this state, oxygen consumption rate of digitonin-
2
mM malonate, 6.7 mM R-glycerophosphate (R-GP), and 1 μM
antimycin A: (A) intact parasites; (B, C) digitonin-permeabilized
promastigotes.
not revert this inhibition, ruling out any specific inhibition
-1
-8
permeabilized parasites was 9.4 nmol ꢀ min ꢀ 10 cells
of 7 on F
TMPD-ascorbate, an electron donor to cytochrome c,
F -ATPase (Figure 4A). In contrast, addition of
1 0
30
(Figure 4B). The addition of 16 μM compound 7 to digito-
nized parasites leads to a full inhibition of the oxygen
consumption rate using succinate as substrate (Figure 4B).
Addition of 10 μM FCCP, a mitochondrial uncoupler, did
restored the oxygen consumption rate at 50% of the initial
value (Figure 4B). This excludes cytochrome c oxidase
(complex IV) as the site of inhibition and circumscribes

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792 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Luque-Ortega et al.
1
Figure 5. Assessment by cytofluorometry of apoptosis in L. donovani promastigotes treated with 7. The presence of a sub-G peak,
characteristic of the apoptotic cell population, was identified by nucleic acid staining with PI, and its percentage (D) is indicated in each
histogram.
Scheme 2. Respiratory Chain in Leishmania with the Specific
Substrates and Inhibitors Used in This Study
Leishmania Killing Involves an Apoptotic Process. Since the
mitochondrion plays a pivotal role in the onset of apoptosis
in Leishmania, the induction of apoptosis by compound 7
was investigated.
a
Overnight incubation of promastigotes with different
concentrations of compound 7 led to an increase in the
sub-G peak region, accounting for fragmented chromatin
1
and hence for apoptosis (Figure 5). The percentage of
promastigotes with a PI fluorescence in the peak region
typical of apoptotic cells was 22% at the LC , while in
5
0
parasites treated with miltefosine, a typical apoptosis indu-
1
3
cer in Leishmania, the percentage was 25% at 15 μM, its
LC50 on promastigotes (Figure 5).
a
Sites of electron feeding or inhibition are indicated by solid or dotted
line, respectively. UQ, ubiquinone; Cyt C, cytochrome c.
Discussion
the potential target upstream of cytochrome c (Scheme 2).
The oxygen consumption rate inhibited by 7 was partially
The choice of phosphonium substituents with moderate
hydrophobicity led to compounds with an intermediate lipo-
philicity capable of traversing the plasma membrane and
(
33%) reinstated upon addition of 6.7 mM R-glyceropho-
30
24
sphate (data not shown), a substrate of complex I. Con-
sequently, this result ruled out complexes I and III as targets
for 7 and supports complex II as the more likely target
for this drug. To verify this hypothesis, the succinate-
dependent respiration was inhibited with malonate at the
level of complex II, and oxygen consumption was allowed
to proceed with R-glycerophosphate as the sole substrate
through complex I. Under these conditions, i.e., without a
functional complex II, compound 7 did not exert any
inhibitory effect on the R-glycerophosphate-dependent re-
spiration (Figure 4C), pointing at complex II as the main
target within oxidative phosphorylation.
accumulating in organelles driven by electrical potentials.
According to this hydrophobicity criteria for the substituents,
from the set of compounds tested, those with leishmanicidal
activity belong to this group. The substituents are medium
length alkyl chains (five to eight carbons), aryl groups, or a
combination of both. Nevertheless, the literature regarding
these compounds is mostly based on the presence of a single
phosphonium group in the molecule. Actually, the uptake of
dications may be impaired by the double charge present in the
molecule. In fact, the desolvatation process (i.e., energy
required to remove solvating water molecules) is the main
energy demand to move a cation from the aqueous medium to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1793
2
1
the hydrophobic lipid membrane. In practical terms, the
bisphosphonium cations require a higher threshold hydro-
phobicity than monophosphonium compounds to traverse
specific tumoricidal activity of phosphonium compounds
was based on their larger accumulation in mitochondria,
due to the significant higher ΔΨm in transformed versus
21
24,34-36
the membrane which for the set of compounds assayed was
provided by the benzophenone scaffold connecting the two
phosphonium moieties. As the entrance of the vital dye
SYTOX green into parasites is nil, even at concentrations
higher than those required for full lethality, plasma membrane
permeabilization was ruled out as a major cause of parasite
killing. Thereby, the lethal activity must involve intracellular
targets and thus uptake of compound 7 and other active
compounds by the parasite. Accordingly, the balance of
hydrophobicity with charge, required for the translocation
across the membrane, is finely tuned. In fact, changes in the
overall polarity of the whole molecule, achieved with a single
incorporation of a heterocycle with slightly higher polarity
than the phenyl substituent, impaired (2-pyridinyl, 16) or
almost abrogated (2-furanyl, 19) leishmanicidal activity. The
same happened when hydrophobicity was decreased for short
alkyl substituents (1-6, 10, 11). This exquisite modulation by
subtle changes in the hydrophobicity of phosphonium sub-
stituents has been employed to optimize the mitochondrial
uptake of MitoQ, consisting of an antioxidant ubiquinone
nontransformed cells.
lues reported are similar to those for mammalian cells
For Leishmania, the ΔΨm va-
37
(-150 mV in L. donovani promastigotes vs -140 to -180 mV
in mammalian cells ). Although plasma membrane potential
is higher in Leishmania (about -100 to -110 mV)
3
8
3
9-41
than in
4
2
many typical eukaryotic resting cells (-30 to -60 mV), the
only predicted effect will be a faster transfer of bisphospho-
nium compounds from the external medium into the intracel-
lular space. Nevertheless, the final mitochondrial accumula-
tion of 7, driven solely by ΔΨ , will be similar in both types of
m.
cells. Additional facts to discard an unspecific effect are
provided by the integrity of the inner mitochondrial mem-
brane throughout the time span of the experiment, as well as
preservation of the functionality of the rest of the components
within the respiratory chain. This is evidenced by the unal-
tered oxygen consumption rate obtained in the presence of 7,
when the system was artificially fed with substrates outside
complex II, i.e., R-glycerophosphate and TMPD-ascorbate.
Hence, a specific inhibition of compound 7 on the mito-
chondrial complex II is the most feasible scenario to account
for its leishmanicidal activity. In cancer cells, other lipophilic
cations such as rhodamine 123 or AA-1 (thiopyrilium) work
3
1,32
coupled to a phosphonium group.
The cellular uptake and accumulation for phosphonium
2
2
compounds are driven by electrical potential; hence, the
plasma membrane potential and in a further step the higher
ΔΨ of the mitochondrion will lead to a massive accumula-
25
by specific inhibition, in this case the F F -ATPase. Drugg-
1
0
abilityof complexIIinLeishmaniahas beenreported for other
43,44
43
also leading to apoptosis of the parasite, as
m
compounds
happens with 7.
2
3,24,33
tion of these compounds in this organelle.
Although in
this work we did not attempt to define the intracellular
distribution of compound 7, from the experimental results it
was concluded beyond reasonable doubt that mitochondria
constitute an essential target for that compound. This is
demonstrated by morphological data, obtained from micro-
graphs where the mitochondrion of Leishmania appeared
dramatically swollen in parasites treated with 7. This result
was validated from a functional perspective; 7 provokes a
prominent decrease of cytoplasmic ATP level, as assessed in
real time by the rapid decay of luminescence of living 3-Luc
The uniqueness of the complex II of Leishmania as target
for 7 may be disputed on the basis of the following. (i) Its
dicationic character will presumably afford binding to either
nuclear or kinetoplast DNA, as described for its guanidine
homologue, but this possibility was ruled out on the grounds
7
of the poor affinity of 7 for DNA. (ii) Compound 7 is active
on the bloodstream forms of the African trypanosome
5
T. b. rhodesiense. It is well established that the bioenergetic
requirements of this form of parasite are fulfilled by glyco-
45
lysis rather than through oxidative phosphorylation, as
2
9
parasites. Once the leakage of intracellular ATP or its
enhanced hydrolysis associated with membrane permeabiliza-
tion was discarded as the reason for these effects, impairment
of ATP synthesis, essentially of mitochondrial origin in
Leishmania, came out as the most feasible target for com-
pound 7. In fact, this compound causes the decrease of ΔΨm,
which drives mitochondrial ATP synthesis, a trend also ob-
served for most of the compounds for which leishmanicidal
effect is associated with inhibition of rhodamine 123 accumu-
lation.
Leishmania does. Nevertheless, for this trypanosomal form
the maintenance of a rudimentary mitochondrion with a
residual ΔΨm is essential, although sustained by the electro-
46
genic activity of mitochondrial ATPase without the con-
course of cytochrome-containing complexes. Altogether these
data suggest the existence of additional targets aside from
complex II, at least for other trypanosomatids.
Conclusions
Finally, the mitochondrial dysfunction created by 7 was
unequivocally pinpointed to the inhibition of complex II,
according to the systematic dissection of the functionality of
the whole set of components involved in the oxidative phos-
phorylation process.
According to experimental data provided by other authors,
we surmise two major alternatives to account for the inhibi-
tion of complex II: either by an unspecific effect due to a
general mitochondrial dysfunction or by a specific effect of 7
on complex II.
Although the potential of phosphonium compounds
as leishmanicidal drugs has been previously described for
African and American trypanosomes and very succintly
mentioned for L. braziliensis and L. mexicana, this work
demonstrates the feasibility of benzophenone-derived bispho-
sphonium salts as new leishmanicidal lead compounds. A fine
modulation of the activity and specificity was achieved by
the adequate selection of substituent based on their hydro-
phobicity. Two additional facts stressed this assertion. First is
the higher susceptibility of amastigotes, as the pathological
form of the parasite in vertebrates, over promastigote,
and second, compound 7 acts over complex II of the mito-
chondrial respiratory chain. The importance of this target
for Leishmania is highlighted by the total dependence of
this parasite on mitochondrial ATP synthesis to fulfill its
Under the hypothesis of an unspecific origin for the inhibi-
tion of complex II by 7, this would likely be due to the
perturbation of the biophysical parameters of the inner
mitochondrial membrane after massive ΔΨ -driven insertion
m
of 7 as a lipophilic phosphonium compound. In fact, the

1
794 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Luque-Ortega et al.
0
bioenergetic requirements. Furthermore, in quantitative terms,
complex II is the main gate for the metabolic feeding of the
respiratory chain. All these items make highly attractive the
exploitation of bisphosphonium salts as new leishmanicidal
leads. Further progress in SAR studies is currently ongoing.
4,4 -Bis((triethylphosphonio)methyl)benzophenone Dibromide
(2). The reaction was carried out following the general proce-
dure using dry toluene instead of DMF and using triethylpho-
sphine (1 M in THF). The yellow precipitate was collected by
filtration and rinsed with toluene. Recrystallization from hot
DMF afforded 2 as a yellowish solid (58%): mp 284-288 °C
1
with previous softening (DMF). H NMR (300 MHz, DMSO) δ
.79 (d, J = 8.0, 4H, ArH), 7.58 (dd, J = 8.0, 2.0, 4H, ArH), 4.02
Experimental Section
7
(
d, J = 16.1, 4H, PhCH P), 2.26 (dq, J = 15.2, 7.6, 12H, CH
2
2
-
Chemistry. All dry solvents were purchased from Aldrich or
Fluka in Sure/Seal bottles. All reactions requiring anhydrous
conditions or an inert atmosphere were performed under a
13
CH
3
), 1.14 (dt, J = 18.3, 7.6, 18H, CH
3
). C NMR (75 MHz,
), 134.85 (d, J = 8.5,
C ), 130.76 (d, J = 6.7, CH), 130.68 (d, J = 3.9, CH), 24.92 (d,
q
DMSO) δ 195.05 (CO), 136.54 (d, J = 3.4, C
q
positive pressure of N . All reactions were monitored by
2
1
13
HPLC-MS. H and C NMR spectra were recorded on a
Bruker Advance 300 or Varian Inova 400 spectrometer.
J = 44.1, PhCH
CH
ESI-HRMS m/z 443.2634 (M-H) (C27
443.2627). HPLC g95% pure.
2
P), 11.13 (d, J = 48.1, CH ), 5.58 (d, J = 5.4,
2
þ
þ
2þ
3
). LRMS (ES ) m/z 443.25 [(M - H) ], 222.32 [M , 100%].
1
Chemical shifts of the H NMR spectra were internally refer-
enced to the residual proton resonance of the deuterated sol-
þ
H
41OP
2
requires
0
vents: CDCl
given in Hz. Chemical shifts of the C NMR spectra were
3
(7.24 ppm) and DMSO (δ 2.5 ppm). J values are
4,4 -Bis((tripropylphosphonio)methyl)benzophenone Dibromide
(3). The reaction was carried out following the general procedure
using dry toluene instead of DMF and using tri-n-propylpho-
sphine. The mixture was allowed to cool to room temperature
whereupona yellow stickyresidue was precipitatedonthe walls of
the tube. The supernatant was discarded and the precipitate
dissolved in a little DMF. Then 3 was obtained as a yellowish
solid (61%) following workup I: mp 90-95 °C with previous
1
3
referenced to the carbon resonance of the deuterated solvents:
CDCl (77.23 ppm) and DMSO (δ 39.51 ppm). Melting points
3
were determined in open capillary tubes with a SMP3-Stuart
Scientific apparatus and are uncorrected. The solvent used for
crystallization is indicated between parentheses. The purity of
the compounds was g95% as assessed by combustion analysis
or HPLC. Elemental analysis was performed on a Heraeus
CHN-O rapid analyzer. Analytical results were within (0.4%
of the theoretical values unless otherwise noted. Analytical
HPLC-MS was run with a Waters Sunfire C18 3.5 μm
1
softening (DMF/toluene). H NMR (300 MHz, DMSO) δ 7.80
(d, J = 8.1, 4H, ArH), 7.57 (dd, J = 8.1, 1.9, 4H, ArH), 4.01 (d,
J = 16.0, 4H, PhCH P), 2.24-2.17 (m, 12H, PCH CH ), 1.53
(dp, J = 14.9, 7.3, 12H, CH CH ), 1.01 (dt, J = 7.3, 1.2, 18H,
2
CH
3.4, C
(d, J = 5.0, CH), 25.93 (d, J = 43.9, PhCH P), 20.01 (d, J = 46.2,
2
2
2
3
1
3
(
4.6 mm ꢀ 50 mm) column on a Waters 2695 separation module
3
). C NMR (75MHz, DMSO) δ194.64(CO), 136.60 (d, J =
coupled with a Waters Micromass ZQ spectrometer using
electrospray ionization (ES ). The following HPLC conditions
q
), 134.92 (d, J = 8.6, C ), 130.74 (d, J = 2.8, CH), 130.64
q
þ
2
were used: column temperature = 30 °C, gradient time = 5 min,
PCH ), 15.64 (d, J = 16.7, CH
2
2
), 15.06 (d, J = 4.3, CH ). LRMS
3
þ
þ
2þ
H
2
O/CH
3
CN (90:10 f 0:100) (HCO
2
H 0.1%), flow rate = 1
(ES ) m/z 527.33 [(M - H) ], 264.35 [M , 100%]. Anal. Calcd
mL/min, UV detection with diode array (λ = 190-400 nm).
HRMS spectra were recorded on a QTOF 6520 Agilent Tech-
nologies mass spectrometer.
33 54 2 2 2
(C H Br OP 2H O): C, 54.70; H, 8.07. Found: C, 54.56;
3
H, 7.90.
0
4,4 -Bis((triisopropylphosphonio)methyl)benzophenone Dibro-
mide (4). The reaction was carried out following the general
procedure using dry toluene instead of DMF and using triiso-
propylphosphine (technical grade 90%). Compound 4 was
obtained as a yellowish solid (59%) following workup I and
General Procedure for the Synthesis of the Bisphosphonium
0
Salts 1-19. A Kimax tube was charged with 4,4 -bis(bromo-
methyl)benzophenone (100 mg, 0.272 mmol) and flushed with
5
argon. Anhydrous DMF (3 mL) was added followed by the
phosphine (1.1 mmol, 4 equiv). The tube was flushed with argon,
stopped, and the reaction mixture was stirred at 110 °C for 20 h.
Different workup procedures were used depending on whether
the product precipitated from the reaction mixture or not. For
workup I, the mixture was allowed to cool to room temperature
and the precipitated product was collected by filtration, rinsed
1
recrystallization from DMF: mp 277-278 °C (DMF). H NMR
(300 MHz, DMSO) δ 7.78 (d, J = 8.1, 4H, ArH), 7.61 (dd, J =
8.1, 1.7, 4H, ArH), 4.20 (d, J = 14.9, 4H, CH P), 3.05-2.78 (m,
6H, CH), 1.31 (dd, J = 15.9, 7.2, 36H, CH
2
1
3
3
). C NMR (75
), 135.55 (d,
MHz, DMSO) δ 194.78 (CO), 136.43 (d, J = 2.8, C
q
J = 8.0, C ), 131.07 (d, J = 4.6, CH), 130.75 (CH), 22.04 (d, J =
40.4), 20.50 (d, J = 41.0), 16.63 (d, J = 3.2, CH
q
þ
successively with toluene and Et
2
O, and dried under vacuum.
3
). LRMS (ES )
þ
1
For workup II, the reaction mixture was transferred to a flask.
Then toluene (10 mL) was added to precipitate the product. The
flask was stored in the refrigerator overnight. The supernatant
m/z 527.27 [(M - H) ]. Anal. Calcd (C33
H
2
54Br OP
2
/
3
H
2
O
/ C H NO): C, 56.80; H, 7.99, Br, 22.23. Found: C, 56.64; H,
3
3
1
3
3
7
8.38; Br, 22.08.
0
was removed and the precipitate rinsed with toluene. Et
(
spatula. The solid was collected, rinsed with Et O, and dried
2
O
10 mL) was added, and the precipitate was triturated with a
4,4 -Bis((tributylphosphonio)methyl)benzophenone Dibromide
(5). The reaction was carried out following the general procedure
with tri-n-butylphosphine. Compound 5 was obtained as a beige
2
under vacuum.
The synthesis of compounds 7 and 15
solid (87%) following workup II: mp 154.2-157.7 °C with
5
47,48
1
was reported
before. We report here for the first time the detailed H and
previous softening (Et
2
O). H NMR (300 MHz, DMSO) δ 7.79
1
(d, J = 7.9, 4H, ArH), 7.57 (d, J = 7.9, 4H, ArH), 4.01 (d, J =
16.0, 4H, PhCH P), 2.19 (br m, 12H, PCH CH ), 1.41 (br m,
1
3
C NMR spectroscopic data for 15.
0
2
2
2
13
4,4 -Bis((trimethylphosphonio)methyl)benzophenone Dibromide
1). The reaction was carried out following the general procedure
24H, CH
2
CH
2
), 0.90 (t, J = 6.8, 18H, CH
3
). C NMR (75 MHz,
), 134.98 (d, J =8.4,
C ), 130.67 (CH), 130.61 (CH), 25.90 (d, J = 43.8, PhCH P),
(
with trimethylphosphine (1 M in toluene). The product was
DMSO) δ195.12(CO), 136.64 (d, J = 3.4, C
q
q
2
obtained as a colorless solid (82%) following workup I: mp
2
23.70 (d, J = 15.9, CH ), 22.92 (d, J = 4.5, CH ), 17.79 (d, J =
2 2
1
þ
þ
08-213 °C with previous softening (DMF/toluene). H NMR
46.8, PCH ), 13.60 (CH
2
3
). LRMS (ES ) m/z 611.35 [(M - H) ],
2þ
þ
(
300 MHz, DMSO) δ 7.81 (d, J = 7.8, 4H, ArH), 7.52 (dd, J =
306.38 [M , 100%]. ESI-HRMS m/z 611.4507 (M - H)
(C H OP requires 611.4505). HPLC g95% pure.
8
1
1
.2, 2.3, 4H, ArH), 3.97 (d, J = 17.5, 4H, PhCH P), 1.86 (d, J =
2
39 65
2
13
0
4.7, 18H, CH
3
). C NMR (75 MHz, DMSO) δ 195.21 (CO),
), 135.01 (d, J = 9.1, C ), 130.77 (d, J = 3.2,
4,4 -Bis((triisobutylphosphonio)methyl)benzophenone Dibromide
(6). The reaction was carried out following the general procedure
with triisobutylphosphine. Compound 6 was obtained as an off-
white solid (56%) following workup II: mp 79-85 °C (DMF/
36.59 (d, J = 3.7, C
q
q
CH), 130.49 (d, J = 5.2, CH), 29.84 (d, J = 48.2, CH ), 7.43 (d,
2
þ
þ
J = 54.0, CH ). LRMS (ES ) m/z 359.11 [(M - H) ], 180.23
3
2þ
þ
1
[ , 100%]. ESI-HRMS m/z 359.1696 (M - H) (C21
requires 359.1688). HPLC g95% pure.
M
H29OP
2
toluene). H NMR (300 MHz, DMSO) δ 7.80 (d, J = 8.0, 4H,
ArH), 7.60 (dd, J = 8.0, 1.8, 4H, ArH), 4.09 (d, J=15.6, 4H,

Article
PhCH
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1795
0
2
P), 2.26 (dd, J = 13.1, 6.1, 12H, PCH CH), 2.12 (m, J =
2
4,4 -Bis((dicyclohexylphenylphosphonio)methyl)benzophenone
Dibromide (12). The reaction was carried out following the
general procedure with dicyclohexylphenylphosphine. Workup
13
6.1, 6H, CH), 1.02 (d, J = 6.6, 36H, CH3). C NMR (75 MHz,
q
DMSO) δ 195.13 (CO), 136.63 (d, J = 3.3, C ), 134.91 (d, J = 8.7,
C
q
), 131.00 (d, J= 4.8, CH), 130.64 (d, J = 2.3, CH), 28.00 (d, J =
3.0, CH P), 27.62 (d, J = 42.8, CH P), 24.88 (d, J = 8.6, CH ),
3.03 (d, J = 4.6, CH ). LRMS (ES ) m/z 611.37 [(M - H) ],
II followed by recrystallization from EtOH/Et O afforded 12 as a
2
1
4
2
3
2
2
3
yellowish solid (33%): mp >300 °C (dec). H NMR (400 MHz,
þ
þ
þ
2
CDCl ) δ 8.10(t, J = 8.7, 4H, ArH), 7.67 (m, 6H, ArH), 7.12 (brs,
3
2þ
06.33 [M , 100%]. ESI-HRMS m/z 611.4514 (M - H)
8H, ArH), 5.33 (d, J = 15.2, 4H, PhCH P), 3.09 (m, 4H, PCH),
2
1
3
(
C
39
H
65OP
2
requires 611.4505). HPLC 92% pure.
,4 -Bis((tricyclohexylphosphonio)methyl)benzophenone Dibro-
2.25-1.00 (m, 40H, CH2). C NMR (101 MHz, CDCl ) δ 194.27
3
0
4
(
CO), 135.70 (d, J = 3.2), 135.12 (d, J = 8.7), 134.32 (d, J = 2.3),
mide (8). The reaction was carried out following the general
procedure with tricyclohexylphosphine. The mixture was con-
centrated to ∼1 mL, resulting in a yellowish oil that was diluted
1
5
2
34.18 (d, J = 7.6), 130.83 (d, J = 4.8), 130.24, 130.10 (d, J =
.9), 115.35 (d, J = 73.7, C ), 31.28 (d, J = 42.3, PCH ), 26.73,
q
2
þ
6.55, 25.74, 24.82 (d, J = 40.7, PCH ). LRMS (ES ) m/z 755.36
2
þ
2þ
2
with toluene (10 mL). Addition of Et O (10 mL) caused pre-
[
(
(M - H) ], 378.21 [M , 100%]. ESI-HRMS m/z 755.4508
þ
cipitation of an oily residue. The flask was stored in the refrig-
erator overnight. The supernatant was removed, and the oily
precipitate was rinsed with toluene. Et O (10 mL) was added to
2
M - H) (C51
H
65OP
2
requires 755.4505). HPLC g 95% pure.
,4 -Bis((diphenylmethylphosphonio)methyl)benzophenone Di-
0
4
bromide (13). The reaction was carried out following the general
procedure with methyldiphenylphosphine. Compound 13 was
obtained as an off-white solid (71%) following workup II: mp
the oily precipitate which was triturated with a spatula to afford 8
as a colorless solid (75%): mp 151-156 °C with previous softening
1
1
(
J = 7.7, 4H, ArH), 7.57 (d, J = 7.7, 4H, ArH), 4.17 (d, J = 14.7,
DMF/toluene/Et O). H NMR (300 MHz, DMSO) δ 7.79 (d,
2
110-113 °C (dec). H NMR (300 MHz, DMSO) δ 7.97 (dd, J =
12.8, 7.3, 8H, ArH), 7.86 (dd, J = 8.0, 6.2, 4H, ArH), 7.74 (td,
J = 7.6, 3.4, 8H, ArH), 7.61 (d, J = 7.9, 4H, ArH), 7.38-7.26 (m,
13
4
H, PhCH P), 2.58 (m, 6H, PCH), 2.15-1.14 (m, 60H, CH ). C
NMR (75 MHz, DMSO) δ 195.09 (CO), 136.64 (C ), 135.79 (d,
2
2
q
4H, ArH), 4.82 (d, J = 16.9, 4H, PhCH P), 2.63 (d, J = 14.1, 6H,
2
1
3
J = 7.9, C ), 131.03 (d, J = 4.5, CH), 130.72 (CH), 29.89 (d, J =
CH3). C NMR (75MHz, DMSO) δ195.03(CO), 136.60 (d, J =
3.8), 135.02 (d, J = 2.5), 133.85 (d, J = 8.3), 133.06 (d, J = 10.1,
CH), 130.93 (d, J = 5.3), 130.43 (d, J = 3.0), 130.16 (d, J = 12.4,
CH), 120.07 (d, J = 84.6, C ), 29.05 (d, J = 47.4, PCH ), 4.86 (d,
q
3
3
1
7
9.4, PCH ), 26.41 (CH ), 26.24 (CH ), 25.31 (CH ), 21.80 (d, J =
2 2 2 2
þ
þ
2þ
,
9.9, PCH). LRMS (ES ) m/z 767.44 [(M - H) ], 383.99 [M
þ
00%]. ESI-HRMS m/z 767.5451 (M - H) (C H OP requires
51
77
2
q
2
þ
þ
67.5444). HPLC g95% pure.
0
J = 55.6, CH
3
). LRMS (ES ) m/z 607.13 [(M - H) ]. Anal.
4,4 -Bis((trioctylphosphonio)methyl)benzophenone Dibromide
9). The reaction was carried out following the general procedure
Calcd (C H Br OP 2C H NO): C, 62.79; H, 5.39, Br, 18.99.
1
Found: C, 62.70; H, 5.83; Br, 18.74. HPLC g95% pure.
0
4
38
2
2
3
3
7
(
with tri-n-octylphosphine (technical grade 90%). Compound 9
was isolated as a yellowish hygroscopic solid (33%) following the
4,4 -Bis((diphenylethylphosphonio)methyl)benzophenone Dibro-
mide (14). The reaction was carried out following the general
procedure with ethyldiphenylphosphine. Compound 14 was ob-
tained as an off-white solid (67%) following workup II: mp
1
same workup as described for 8. H NMR (300 MHz, CDCl ) δ
3
7
.57 (m, 4H, ArH), 7.45 (d, J = 7.6, 4H, ArH), 4.66 (d, J = 16.1,
1
4
H, PhCH P), 2.31 (br m, 12H, PCH CH ), 1.7-1.1 (m, 72H,
2
2
2
118-123 °C with previous softening (DMF/toluene). H NMR
13
(
CH ) ), 0.79(t, J = 5.5, 18H, CH ). C NMR(75MHz, CDCl3)
(300 MHz, DMSO) δ 8.05-7.65 (m, 20H, ArH), 7.54 (d, J = 7.8,
2
6
3
δ 194.53, 136.43, 134.37 (d, J = 9.1), 130.72, 130.64, 31.77, 30.99
2
4H, ArH), 7.17 (m, 4H, ArH), 4.90 (d, J = 16.2, 4H, PhCH P),
13
C
(
(
4
d, J = 14.8), 29.05 (d, J = 6.6), 22.70, 22.02 (d, J = 4.7), 19.71
d, J = 24.1), 18.94, 14.18. LRMS (ES ) m/z 947.86 [(M - H) ],
3.02 (m, 4H, PCH
NMR (75 MHz, DMSO) δ 195.01 (CO), 136.56 (d, J = 3.7),
135.20 (d, J = 2.1), 133.77 (d, J = 9.5, CH), 130.76 (d, J = 5.2),
130.34 (CH), 130.18 (CH), 117.91 (d, J = 82.7, C
45.7, PCH ), 12.64 (d, J = 50.6, PCH ), 5.97 (d, J = 4.7, CH
2
2
CH ), 1.14 (td, J = 7.4, 21, 6H, CH
3
3
).
þ
þ
2þ
2þ
73.96 [M , 100%]. ESI-HRMS m/z 473.4093 (M - H)
(
C
63
H
112OP
2
requires 473.4094). HPLC g95% pure.
,4 -Bis((dimethylphenylphosphonio)methyl)benzophenone Di-
q
), 27.11 (d, J =
).
0
4
2
þ
3
þ
2þ
bromide (10). The reaction was carried out following the general
procedure with dimethylphenylphosphine. Compound 10 was
obtained as an off-white solid (79%) following workup II: mp
LRMS (ES ) m/z 635.18 [(M - H) ], 318.26 [M , 100%]. ESI-
þ
HRMS m/z 635.2628 (M - H) (C H OP requires 635.2627).
43
41
2
HPLC 92% pure.
1
0
7,48
9
5-100 °C (dec). H NMR (300 MHz, DMSO) δ 8.05-7.89 (m,
H, ArH), 7.82 (dd, J = 7.6, 5.7, 2H, ArH), 7.76-7.61 (m, 8H,
4,4 -Bis((triphenylphosphonio)methyl)benzophenone Dibromide
4
4
ArH), 7.31 (dd, J = 8.3, 2.4, 4H, ArH), 4.25 (d, J = 17.0, 4H,
(15).
The reaction was carried out following the general
procedure with triphenylphosphine. Compound 15 was obtained
as an off-white solid (66%) following workup II: mp >200 °C
(dec). H NMR (300 MHz, DMSO) δ 8.30-7.61 (m, 30H, ArH),
7.52 (d, J = 7.8, 4H, ArH), 7.14 (d, J = 7.7, 4H, ArH), 5.33 (d,
J = 16.1, 4H, CH
136.81 (d, J = 3.7, C
J = 8.4), 131.33 (d, J = 4.9), 130.56 (d, J = 12.5), 130.27 (d, J =
2.7), 117.93 (d, J = 85.9, C ), 28.43 (d, J = 46.1, CH ). LRMS
(ES ) m/z 731.21 [(M - H) ], 366.3 [M , 100%]. HPLC g95%
pure.
1
3
PhCH
DMSO) δ195.15 (CO), 136.56 (d, J = 3.9, C
C ), 134.51 (d, J = 9.1), 132.31 (d, J = 10.0, CH), 130.58 (m,
2
P), 2.25 (d, J = 14.5, 12H, CH
3
). C NMR (75 MHz,
1
q
), 134.62(d, J = 2.6,
q
1
3
CH), 129.79 (d, J = 12.4, CH), 120.75 (d, J = 84.2, C ), 30.83 (d,
q
2
). C NMR (75 MHz, DMSO) δ 195.08 (CO),
), 135.60, 134.44 (d, J = 10.0), 133.47 (d,
þ
J = 46.5, CH ), 6.42 (d, J = 54.5, CH
3
3
). LRMS (ES ) m/z 483.13
(M - H) ], 242.23 [M , 100%]. ESI-HRMS m/z 483.2000
q
þ
2þ
[
(
þ
M - H) (C H OP requires 483.2001). HPLC 94% pure.
31
33
2
q
þ
2
0
þ
2þ
4
,4 -Bis((diethylphenylphosphonio)methyl)benzophenone Dibro-
mide (11). The reaction was carried out following the general
procedure with diethylphenylphosphine. Compound 11 was ob-
tained as an off-white solid (60%) following workup II: mp
0
4,4 -Bis((diphenyl-2-pyridylphosphonio)methyl)benzophenone
Dibromide (16). The reaction was carried out following the
general procedure with diphenyl-2-pyridylphosphine. Com-
pound 16 was obtained as a beige solid (69%) following workup
1
6
8
(
9-76 °C (DMF/toluene). H NMR (300 MHz, DMSO) δ
.04-7.78 (m, 6H, ArH), 7.72 (dt, J = 7.4, 3.7, 4H, ArH), 7.64
d, J = 8.0, 4H, ArH), 7.29 (dd, J = 8.2, 2.1, 4H, ArH), 4.37 (d,
1
II: mp 175-178 °C (dec). H NMR (300 MHz, DMSO) δ 9.03 (d,
J = 16.0, 4H, PhCH
J = 19.2, 7.5, 12H, CH
CO), 136.51 (d, J = 3.6, C
J = 8.5), 132.99 (d, J = 8.7), 130.60 (d, J = 5.6, CH), 130.52
CH), 130.13 (d, J = 11.7, CH), 117.22 (d, J = 79.4, C ), 26.74(d,
2
P), 2.81-2.59 (m, 8H, CH
3
2
CH
). C NMR (75 MHz, DMSO) δ 195.01
), 134.88 (d, J = 2.6, C ), 134.27 (d,
3
), 1.16 (dt,
J = 4.5, 2H, ArH, 6-Hpyr), 8.21 (dt, J = 7.8, 6.1, 2H, ArH),
8.13-7.98 (m, 2H, ArH), 7.92 (dd, J = 14.6, 6.1, 6H, ArH),
7.83-7.64 (m, 16H, ArH), 7.46 (d, J = 7.9, 4H, ArH), 7.20 - 7.04
1
3
(
q
q
1
3
(m, 4H, ArH), 5.29 (d, J = 16.4, 4H, CH ). C NMR (75 MHz,
DMSO) δ 195.01 (CO), 152.44(d, J = 19.5), 144.04(d, J =116.4,
2
(
q
J = 44.7, CH P), 11.55 (d, J = 49.3, CH
2
2
P), 5.76 (d, J = 5.0,
CH ). LRMS (ES ) m/z 539.2 [(M - H) ], 270.29 [M , 100%].
q
C ), 138.84 (d, J = 9.9), 136.65 (d, J = 3.7), 135.61 (d, J = 2.7),
134.62 (d, J = 9.8, CH), 133.46 (d, J = 8.4), 132.17 (d, J = 23.8),
131.30 (d, J = 5.5), 130.43 (d, J = 12.5, CH), 130.12 (d, J = 2.7),
þ
þ
2þ
3
þ
ESI-HRMS m/z 539.2635 (M - H) (C H OP requires
3
5
41
2
5
39.2627). HPLC 92% pure.
q
128.99 (d, J = 3.2), 117.26 (d, J = 84.9, C ), 27.93 (d, J = 46.4,

1
796 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Luque-Ortega et al.
þ
þ
2þ
CH
2
). LRMS (ES ) m/z 733.12 [(M - H) ], 366.89 [M , 100%].
for the rest of the experiments unless otherwise stated. Aliquots
(120 μL) of this cellular suspension were incubated with the drugs
for 4 h, at either 25 or 32 °C for promastigotes or amastigotes,
respectively. Afterward, 20 μL aliquots of this suspension were
taken, added to 1 mL of HBSS-Glc, and centrifuged to remove
unbound reagent. Washed parasites were resuspended in 100 μL
of their respective growth medium devoid of phenol red, trans-
ferred into a new 96-well microplate, and allowed to proliferate
(48 h, 25 °C for promastigotes; 96 h, 32 °C for amastigotes).
Finally, an amount of 100 μL of MTT solution (1 mg/mL) in
HBSS-Glc was added and substrate reduction allowed to proceed
for 2 h at 25 or 32 °C for promastigotes or amastigotes, respec-
tively. Precipitated formazan was solubilized by addition of
100 μL of 10% (w/v) SDS solution and read in a 450 Bio-Rad
Anal. Calcd (C H Br N OP ): C, 65.79; H, 4.51, N, 3.13.
Found: C, 65.98; H, 4.80; N, 3.20.
4
9
40
2
2
2
0
4
,4 -Bis((diphenyl-o-tolylphosphonio)methyl)benzophenone
Dibromide (17). The reaction was carried out following the
general procedure with diphenyl(o-tolyl)phosphine. Compound
1
7 was obtained as an off-white solid (55%) following workup II:
1
mp 255.9-262.7 °C with previous softening (DMF/toluene). H
NMR (300 MHz, CDCl ) δ 8.17 (br, 2H, ArH), 7.9-7.45 (m,
6H, ArH), 7.22 (m, 8 H, ArH), 5.60 (d, J = 15.0, 4H, CH ), 1.99
s, 6H, CH3). C NMR (75 MHz, CDCl ) δ 195.58 (CO), 143.50
d, J = 8.4), 137.01 (d, J = 3.7), 136.81 (d, J = 10.9), 136.0 (d,
J = 2.2), 135.45 (d, J = 2.4), 134.53 (d, J = 9.8), 133.82 (d, J =
3
2
(
(
2
13
3
1
1
1.0), 133.19 (d, J = 8.2), 132.14 (d, J = 5.4), 130.75 (d, J =
2.6), 130.53 (d, J = 2.4), 128.65, 117.93 (d, J = 84.7, C ), 116.55
29
q
ELISA microplate reader equipped with a 600 nm filter. To
6
(
CH
d, J = 83.2, C ), 32.24 (d, J = 47.6, CH ), 23.29 (d, J = 4.4,
measure the short-term effect, an amount of 100 μL (1.6 ꢀ 10
q
2
þ
þ
2þ
3
). LRMS (ES ) m/z 759.35 [(M - H) ], 380.1 [M , 100%].
promastigotes) of the remaining parasites was washed with 1 mL of
HBSS-Glc, resuspended, and assayed for MTT reduction as above.
The induction of apoptosis by 7 was monitored by analysis of
þ
ESI-HRMS m/z 759.2936 (M - H) (C53
H
45OP
2
requires
7
59.2940). HPLC 92% pure.
0
4
,4 -Bis((diphenyl-p-tolylphosphonio)methyl)benzophenone
the sub-G peak in parasites incubated overnight with different
1
Dibromide (18). The reaction was carried out following the
general procedure with diphenyl(p-tolyl)phosphine. Compound
concentrations of 7. After the incubation, parasites were stained
with 50 μg/mL propidium iodide (PI) and individual cellular
fluorescence was measured in a Beckman Coulter FC500 MPL
cytofluorometer. As positive control for apoptosis induction,
parasites were treated in identical conditions with 15 μM
1
8 was obtained as an off-white solid (50%) following workup
1
II: mp 296.9-298.5 °C (DMF/toluene). H NMR (300 MHz,
CDCl ) δ 8.0-7.0 (m, 36H, ArH), 5.73 (br, 4H, CH ), 2.44 (s, 6H,
) δ 194.92 (CO), 146.47 (d, J =
3
2
13
31
CH
3
). C NMR (75 MHz, CDCl
3
miltefosine (hexadecylphosphocholine).
BALB/c peritoneal macrophages were resuspended in their
2
.5), 135.0 (br), 134.86, 134.68 (d, J = 10.2, CH), 133.13 (d, J =
.5), 132.29 (d, J = 5.0), 131.23 (d, J = 13.0), 130.36 (d, J = 12.5,
6
culture medium at a final density of 10 cells/mL, plated in a 96-
8
CH), 118.29 (d, J = 86.0, C
2
J = 46.1, CH ), 22.22 (CH
q
), 114.08 (d, J = 88.4, C ), 31.24 (d,
q
well culture microplate (100 μL/well), and incubated with
compound 7 for 24 h at 37 °C. Cytotoxicity was assessed using
the colorimetric MTT reduction assay and expressed as the
percentage of the value obtained for control cells.
þ
3
). LRMS (ES ) m/z 759.21 [(M -
þ
2þ
þ
H) ], 380.24 [M , 100%]. ESI-HRMS m/z 759.2929 (M - H)
(
C
53
H
45OP
2
requires 759.2940). HPLC g95% pure.
,4 -Bis((tri-2-furanylphosphonio)methyl)benzophenone Dibro-
0
4
Changes in Intracellular ATP Levels. The in vivo variation of
intracellular ATP levels in L. donovani promastigotes was
measured in 3-Luc parasites expressing a cytoplasmic form of
mide (19). The reaction was carried out following the general
procedurewithtri-2-furylphosphine. Compound19was obtained
as a beige solid (76%) following workup II: mp 214.5-223.7 °C
2
9
Photinus pyralis luciferase. Briefly, the substrate for luciferase,
DMNPE-luciferin, was prepared as a 5 mM stock solution in
DMSO. It was added to the 3-Luc promastigote suspension (2 ꢀ
1
with previous softening (DMF/toluene). H NMR (300 MHz,
CDCl
6
3
) δ 8.03 (br m, 14H, ArH), 7.43 (m, 6H, ArH), 6.78 (br s,
13
H, ArH, 4-Hfuranyl), 5.55 (br m, 4H, CH2). C NMR (75 MHz,
7
10 cells/mL) in HBSS-Glc at a final concentration of 25 μM,
CDCl
.3, C
3
) δ 195.21 (CO), 154.06 (d, J = 8.6, CH), 137.14 (d, J =
), 133.33 (d, J = 22.0, CH), 131.86(d, J = 6.3, CH), 131.28
and aliquots of this suspension were immediately distributed
into a 96-well microplate (100 μL/well). Compound 7 was added
once the luminescence reached a plateau. This point was con-
sidered as time zero and its luminescence taken as 100%.
Changes in luminescence were recorded for 80 min using a
luminescence optics setting. The measurements were averaged
every 4 s. In vitro inhibition of luciferase (EC 1.13.12.7) by the
4
q
(
d, J=9.9, C ), 131.05 (d, J=11.9, CH), 130.1 (d, J=125.2, C ),
q
q
þ
1
14.26 (d, J=10.0, CH), 32.54 (d, J=53.4, CH ). LRMS (ES )
2
þ
2þ
m/z 671.08 [(M - H) ], 336.0 [M , 100%]. ESI-HRMS
m/z 671.1379 (M - H) (C39
g 95% pure.
Cultured Cells. Promastigotes from the Leishmania donovani
strain MHOM/SD/00/1S-2D, its derived isogenic strain, 3-Luc,
which expresses a C-terminal mutated form of luciferase
þ
29 7 2
H O P requires 671.1383). HPLC
2
9
compound was tested as described using a commercial purified
enzyme (Roche Applied Sciences, Madrid, Spain).
Plasma Membrane Permeabilization. It was assessed by the
entrance of the vital dye SYTOX green (MW 600), according to
the procedure described previously. Briefly, compound 7 was
(
cytoplasmic luciferase), and L. pifanoi axenic amastigotes
2
9,49
50
(
MHOM/VE/60/Ltrod) were grown as described.
Peritoneal macrophages from BALB/c mice were obtained by
7
added to L. donovani promastigotes in HBSS-Glc (2 ꢀ 10
previous elicitation of the mouse by a single intraperitoneal
injection of 1 mL of 3% sodium thioglycolate, 4 days priors to
macrophage harvesting by peritoneal lavage.
Unless otherwise stated, reagents were purchased from Sig-
ma-Espa n~ a (Madrid, Spain). Propidium iodide, SYTOX Green,
rhodamine 123 [2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic
acid methyl ester, chloride], and DMNPE-luciferin [D-luciferin
1-(4, 5-dimethoxy-2-nitrophenyl) ethyl ester] were purchased
from Invitrogen (Barcelona, Spain).
Fluorescence and luminescence measurements were recorded
in a Polarstar Galaxy microplate reader (BMG Labotechnolo-
gies, Offenburg, Germany) fitted with the corresponding optical
setting, unless otherwise stated.
promastigotes/mL) preincubated with 1 μM SYTOX green.
Entrance of the dye into the intracellular compartment is only
afforded through injured membranes, with lesions large enough
to allow dye passage. The increase of fluorescence, due to
binding of the dye to intracellular nucleic acids, was monitored
using 485 and 520 nm filters for excitation and emission
wavelengths, respectively. Full permeation was considered as
that achieved by 0.1% Triton X-100 (TX-100).
Electron Microscopy. Promastigotes were incubated with
compound 7 according to the standard procedure, washed twice
in PBS, and fixed in 5% (w/v) glutaraldehyde in the same
medium. Afterward, parasites were included with 2.5% (w/v)
OsO for 1 h, gradually dehydrated in ethanol (30%, 50%, 70%,
4
Cytotoxic Activity Measurements. Promastigotes and amas-
tigotes were harvested at the late exponential phase, washed
twice in Hanks’ buffer, supplemented with 10 mM D-glucose
90%, and 100% (v/v); 30 min each) and propylene oxide (1 h),
embedded in Epon 812 resin, and observed in a Jeol-1230
5
0
electron microscope as described.
Effects on Mitochondrial Activity. Variation of the mitochon-
drial membrane potential (ΔΨ ) of promastigotes was measured
(
pH 7.2) (HBSS-Glc) at 4 °C, and resuspended in the same buffer
7
at 2 ꢀ 10 cells/mL. These standard conditions were maintained
m

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1797
3
9
by rhodamine 123 accumulation. To this end, promastigotes
were incubated in their growth medium with the leishmanicidal
benzophenone-derived bisphosphonium compounds at their res-
pective LC50 overnight, and dye incorporation was measured in a
Beckman Coulter FC500 MPL cytofluorometer (excitation and
emission wavelengths of 488 and 525 nm, respectively). Parasites
either untreated or fully depolarized by incubation with 1 mM
KCN were used as controls. Following the same protocol, a more
extensive range of concentrations was used to study the effect of
compound 7 on rhodamine 123 accumulation.
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drugs for antileishmanial activity in golden hamsters infected with
Leishmania donovani. Int. J. Parasitol. 1977, 7, 443–447.
(
12) Levi-Schaffer, F.; Tarrab-Hazdai, R.; Meshulam, H.; Arnon, R.
Effect of phosphonium salts and phosphoranes on the acetylcho-
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Effects of phosphonium compounds on Schistosoma mansoni.
J. Med. Chem. 1980, 23, 862–865.
3
1
(
14) Sim, E.; Cullis, P. R. P nuclear magnetic resonance studies of cell
membranes labelled with phosphonium phosphatidylcholine.
FEBS Lett. 1977, 79, 340–344.
Oxygen consumption rates were measured in a Clark oxygen
electrode (Hansatech, KingsLynn, U.K.) at 25 °C, using 1 mL of
a promastigote suspension (10 cells/mL) in respiration buffer
(
15) Sim, E.; Pasternak, C. A. The metabolism of the phosphonium
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8
supplemented with 5 mM succinate and 1 mg/mL fatty acid free
3
7
BSA as described. Cells were permeabilized with 60 μM
digitonin, which allows a selective permeation of the plasma
membrane but not of the inner mitochondrial membrane.
Afterward, 100 μM ADP was added to restore state 3 of
respiration, and once a steady rate was reached, compound 7
was added, followed by a selective set of substrates and inhibi-
tors specific for the different complexes of the respiratory chain,
at concentrations stated in the corresponding figures.
(
16) Kanazawa, A.; Ikeda, T.; Endo, T. Synthesis and antimicrobial
activity of dimethyl- and trimethyl-substituted phosphonium
salts with alkyl chains of various lengths. Antimicrob. Agents
Chemother. 1994, 38, 945–952.
5
1
(17) Kanazawa, A.; Ikeda, T.; Endo, T. Novel polycationic biocides;
synthesis and antibacterial activity of polymeric phosphonium
salts. J. Polym. Sci., Part A: Polym. Chem 1993, 31, 335–343.
(
18) Kenawy, E. R.; Abdel-Hay, F. I.; Abou El-Magd, A.; Mahmoud,
Y. Biologically active polymers: VII. Synthesis and antimicrobial
activity of some crosslinked copolymers with quaternary ammo-
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Statistical Analysis. Data represent the mean of triplicate
samples ( SD. LC50 values were calculated from the sigmoidal
inhibition curves using the SigmaPlot 11.0 software.
(
19) Popa, A.; Davidescu, C. M.; Trif, R.; Ilia, G.; Iliescu, S.; Dehelean,
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bacterial activity of quaternary phosphonium salts grafted on “4
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Acknowledgment. This work was supported by grants
from the Spanish Ministerio de Educaci oꢀ n y Ciencia to C.D.
Grant SAF2006-04698) and to L.R. (Grant PET2006-0139-
1), Fondo de Investigaciones Sanitarias (Grants PI061125,
(
0
(
20) Tashiro, T. Antibacterial and bacterium adsorbing macromole-
cules. Macromol. Mater. Eng. 2001, 286, 63–87.
(
21) Ross, M. F.; Da Ros, T.; Blaikie, F. H.; Prime, T. A.; Porteous,
C. M.; Severina, I. I.; Skulachev, V. P.; Kjaergaard, H. G.; Smith,
R. A.; Murphy, M. P. Accumulation of lipophilic dications by
mitochondria and cells. Biochem. J. 2006, 400, 199–208.
PS09/0192, and RD 06/0021/0006), and regional government
of Madrid (Grant S-BIO-0260/2006) to L.R. P.R. is recipient
of a Studienstiftung des Deutschen Volkes fellowship. We
gratefully acknowledge Dr. H. de Koning for his comments
and suggestions on the manuscript, and the IQM Mass
Spectrometry Service Centre for HPLC-MS analysis.
(22) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cocheme,
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in mitochondrial bioenergetics and free radical biology. Biochem-
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