SAR studies of diphenyl cationic trypanocides: Superior activity of phosphonium over ammonium salts

Article abstract of DOI:10.1021/ml500408d

In previous studies, we have shown that phosphonium salt diphenyl derivatives are attractive antitrypanosomal hit compounds with EC₅₀ values against Trypanosoma brucei in the nanomolar range. To evaluate the role of the cationic center on the t

Full text of DOI:10.1021/ml500408d

Letter
pubs.acs.org/acsmedchemlett
SAR Studies of Diphenyl Cationic Trypanocides: Superior Activity of
Phosphonium over Ammonium Salts
Christophe Dardonville,* Abdulsalam A. M. Alkhaldi, and Harry P. De Koning‡
,
†
‡,§
†
Instituto de Química Med
́
ica, IQM−CSIC, Juan de la Cierva 3, E−28006 Madrid, Spain
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow, United Kingdom
‡
§
Department of Biology, College of Science, Aljouf University, Sakaka, Saudi Arabia
ABSTRACT: In previous studies, we have shown that
phosphonium salt diphenyl derivatives are attractive anti-
trypanosomal hit compounds with EC50 values against
Trypanosoma brucei in the nanomolar range. To evaluate the
role of the cationic center on the trypanocidal activity and
extend the structure−activity relationship (SAR) of this series,
trialkylammonium, pyridinium, and quinolinium salt analogues
were synthesized and evaluated in vitro against T. b. brucei.
Similar SARs were observed with ammonium and phospho-
nium salts showing that charge dispersion and lipophilic
groups around the cationic center are crucial to obtain
submicromolar activities. The new compounds were equally
effective against wild type (T. b. brucei s427) and resistant strains (TbAT1-KO and TbB48) of trypanosomes indicating that the
P2 and high affinity pentamidine transporters (HAPT) are not essential to their trypanocidal action. Similarly to phosphonium
salt derivatives, diffusion seems to be the main route of entry into trypanosomes.
KEYWORDS: Trypanosoma brucei, ammonium salt, quinolinium salt, pyridinium salt, P2-transporter,
high affinity pentamidine transporter (HAPT)
arasitic protozoa of the genus Trypanosoma cause human
and animal trypanosomiases in Africa. Two subspecies of
Structure−activity relationship (SAR) studies revealed that
bulky substituents on the phosphorus atoms are required to
achieve submicromolar EC50 values within this series. In the
present work, we wanted to learn whether the nature of the
cationic center was important for the observed trypanocidal
activity of these hit compounds. Thus, new cationic analogues
of the hits with a nitrogen atom as cationic center (i.e.,
trialkylammonium, pyridinium, and quinolinium salts) were
synthesized and tested in vitro against wild type and resistant
lines of T. brucei.
P
T. brucei (T. b. gambiense and T. b. rhodesiense) are pathogenic
to man and cause human African trypanosomiasis (HAT) in
sub-Saharan Africa. In contrast, T. b. brucei, T. congolense, and T.
1
,2
vivax cause veterinary diseases in domestic and wild animals.
African trypanosomiasis is one of the most neglected tropical
diseases as shown by the lack of safe drugs to treat both (early
and late) stages of the illness. These drugs, which have been
used for decades, are subject to treatment failure (drug
resistance) and have severe drawbacks such as unacceptable
The ammonium (1a−c, 2a−b, 3b−c, and 3f), pyridinium
(1d−3d), and quinolinium (1e−3e) derivatives were synthe-
sized by nucleophilic substitution of the (bis)brominated
3
toxicity and parenteral administration.
Cationic drugs are an important class of trypanocides used
for the treatment of human and veterinary trypanosomiasis
6
precursors 1−3 with tertiary amines in refluxing acetonitrile
(
Scheme 1). The compounds were isolated and purified by
(
e.g., pentamidine, diminazene, homidium, and isomethami-
4
,5
crystallization.
dium). In previous studies we have shown that a new class of
cationic compounds, mono- and bisphosphonium salt deriva-
tives with a diphenyl scaffold, have submicromolar activities
The compounds were evaluated against the T. b. brucei s427
wild type strain and two drug resistant strains, TbAT1-KO and
1
0,11
6
,7
B48, using an alamarBlue-based assay.
TbAT1-KO is a
against T. brucei and Leishmania parasites. These compounds
display a good selectivity index relative to human cell lines, are
not cross-resistant with existing trypanocides, and require only
trypanosome strain lacking a functional P2-transporter and is
12
resistant to diminazene. The B48 strain is a mutant derived
13
6
from the TbAT1-KO strain with a nonfunctional high affinity
a short exposure time for trypanocidal activity. Like other
8
,9
dicationic trypanocides,
the bisphosphonium salts are
expected to accumulate in the mitochondria. Indeed, a
mitochondrial target for these compounds was demonstrated
in Leishmania.
Received: October 6, 2014
Accepted: December 10, 2014
7
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ml500408d | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis of Ammonium, Pyridinium, and
Quinolinium Salts
the cytotoxicity against human endothelial kidney (HEK) cells
was also determined, and the selectivity indices were
a
15
calculated (Table 1).
The trialkylammonium salts with long alkane chains (n-
C H > n-C H ≫ n-C H ) and the dibenzyl derivative 3f
6
13
4
9
2
5
were the most potent of the new series with EC values against
50
T. brucei in the low micromolar (1b, 2b, and 3b) to nanomolar
range (1c and 3c). The monocationic trihexylammonium salt
3c (EC50 (s427) = 23 nM) was two times less active than
pentamidine (EC50 (s427) = 10 nM) but 14 times more active
than diminazene in this assay. More importantly, this
compound was equally active against the TbAT1-KO and
B48 resistant strains (RF = 0.8 and 0.6, respectively) contrary
to pentamidine (RF = 0.6 and 32.9) and diminazene (RF = 2.3
and 3.1).
In contrast, the quinolinium salts (1e and 2e) displayed
micromolar range activities (>10 μM), whereas the pyridinium
salts (1d−3d) were mostly inactive (EC50 > 50 μM).
Importantly, all the compounds had similar activities against
wild type and resistant lines of T. brucei (i.e., RF ≈ 1), which
indicates that the P2 and HAPT transporters are not essential
for the uptake of these compounds by the parasite. Hence,
cross-resistance with existing trypanocides is not likely to
appear with these compounds. The compounds showed low
cytotoxicity against HEK cells, which turned into selectivity
indices >25 for 1e, 2b, 3b, and 3f and >100 for 1b, 1c, and 3c.
The comparison of these data with previous results obtained
a
Reagents and conditions: (i) R N (2.2 equiv for 1 and 2; 1.1 equiv for
3
3
1
), CH CN, 80 °C; (ii) pyridine or quinoline (2.2 equiv for 1 and 2;
3
.1 equiv for 3), CH CN, 80 °C; (iii) Bn NH (1.1 equiv), K CO (1.2
3
2
2
3
equiv), 60 °C then HCl /dioxane.
g
14
pentamidine transporter (HAPT). This strain is resistant to
diminazene, pentamidine, and melaminophenyl arsenicals.
13
6
with phosphonium salt analogues informed on the influence of
The resistance factor (RF) with respect to wild type
trypanosomes was calculated for both resistant strains. Finally,
P ↔ N replacement on the antitrypanosomal activity of these
series (Table 2). In most cases, phosphonium salts have EC50
a
Table 1. In Vitro Antitrypanosomal Activity (EC , μM) against Wild Type and Resistant Strains of T. b. brucei and
5
0
Cytotoxicity against HEK Cells (CC , μM)
5
0
a
b
c
12
Mean of three experiments ± SEM. T. b. brucei s427 trypomastigotes. T. b. brucei knockout strain lacking a functional P2-transporter and
d
e
13
resistant to diminazene aceturate. Resistance factor compared to WT. The B48 strain is a mutant derived from the TbAT1-KO strain with a
nonfunctional high affinity pentamidine transporter (HAPT). This strain is resistant to diminazene, pentamidine, and melaminophenyl arsenicals.
f
g
h
Human endothelial kidney cells. Selectivity index = [CC /EC (T.b.b.WT)]. Not determined.
50
50
B
dx.doi.org/10.1021/ml500408d | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 2. SAR Studies: Effect of P ↔ N Replacement on Antitrypanosomal Activity and Cytotoxicity
T. b. brucei WT EC50 (μM)
NR3 PR3
>100
HEK cells CC50 (μM)
a
a
cmpd
R
NR3
PR3
>300
1a
1b
1c
2a
2b
3b
3c
C H
36.5 ± 3.5
nd
2
5
b
b
n−C H
2.8 ± 0.2
0.59 ± 0.06
>300
>300
4
9
n-C H
0.102 ± 0.024
>100
0.037 ± 0.004
>100
21.8 ± 0.3
nd
22.1 ± 1.1
>300
6
13
C H
2
5
n-C H
11.5 ± 0.4
4.6 ± 0.6
0.48 ± 0.11
5.9 ± 0.7
>300
>300
4
9
b
b
n-C H
>300
>300
4
9
n-C H
0.023 ± 0.004
0.011 ± 0.004
15.4 ± 0.2
13.4 ± 0.3
6
13
a
b
Data taken from ref 6. EC50 value for R = i-C H (isobutyl).
4
9
against T. brucei from 2- to 24-times lower than the
corresponding ammonium salts, but the cytotoxicity of both
series against HEK cells is similar, which means that
phosphonium salts generally have higher therapeutic indices
than ammonium and quinolinium salts. In both series, more
lipophilic alkane substituents (i.e., alkyl chains with >4
methylene units) were favored and produced compounds
with submicromolar EC50 values.
dispersion and lipophilic groups around the cationic center are
crucial to obtain submicromolar activities. Nitrogen and
phosphorus cationic compounds are taken up into trypano-
somes independently from the known T. b. brucei drug
transporters. This indicates that the dicationic compounds
reported here should not be prone to cross-resistance with
clinically used trypanocidal drugs such as melarsoprol,
diminazene, and pentamidine, as extensive evidence from
laboratory models and clinical studies link resistance to these
Phosphonium salt derivatives based on the diphenyl scaffolds
1
2,13,14,17
1
−3 are promising compounds active in vitro against wild type
drugs to the P2 and HAPT1 transporters
the test strains utilized here.
absent from
and resistant lines of T. brucei. They also display a good
selectivity index versus human cell lines. In the present study,
6
we synthesized new ammonium, pyridinium, and quinolinium
salt derivatives using the same diphenyl scaffolds to check the
importance of the phosphorus atom in these series.
Replacement of the cationic center from phosphorus to
biososteric nitrogen atom led to compounds with disparate
activities. The trialkylammonium salt derivatives displayed
antiparasitic activities with the same order of magnitude as
the trialkylphosphonium salts, showing no improvement in
selectivity. We have shown earlier that in this series increased
activity is obtained with positive charge dispersion over a large
EXPERIMENTAL METHODS
■
Chemistry. All dry solvents were purchased from Aldrich or Fluka
in Sure/Seal bottles. All reactions requiring anhydrous conditions were
performed under argon atmosphere. The reactions were performed in
screw-capped Kimax tubes and monitored by HPLC−MS. Analytical
HPLC−MS was run with an Xbridge C18−3.5 μm (2.1 × 100 mm)
column on a Waters 2695 separation module coupled with a Waters
Micromass ZQ spectrometer using electrospray ionization (ESI ). All
compounds are >95% pure by HPLC otherwise noted. H and
NMR spectra were recorded on a Bruker Advance 300 spectrometer.
H NMR chemical shifts were internally referenced to the residual
+
1
13
C
1
6
surface. Hence, the 2- to 5-fold higher activity of the
phosphonium salt derivatives relative to the ammonium salts
proton resonance of the deuterated solvent for CD OD (δ = 3.49
3
H
ppm) or TMS (δ = 0 ppm) for CDCl . Peak multiplicity is given as
H
3
may possibly be related to the lower charge density of the
singlet (s), doublet (d), triplet (t), quadruplet (q), m (multiplet), and
br (broad peak). J values are given in Hz. C NMR spectra were
referenced to the chemical shift of the deuterated solvent: CD OD (δ
16
13
phosphorus atom that arises from a larger atomic radius. The
- to 7-fold higher activities of the quinolinium salts (i.e., charge
2
3
C
=
49.0 ppm) and CDCl (δC = 77.16 ppm). Peak assignments are
delocalized over two aromatic rings) compared with the
pyridinium salts reported here also support this view.
3
noted as follows: aromatic H (ArH), pyridine H (PyrH), and quinoline
H (QuinH). Melting points (uncorrected values) were determined in
open capillary tubes with a Mettler Toledo MP70 melting point
system or with a Reichert Jung Thermovar apparatus.
General Procedure for the Synthesis of Ammonium,
Pyridinium, and Quinolinium Salts. The reactions were carried
out under argon in Kimax tubes stopped with a screwcap. To a
6
As for phosphonium analogues the anti-T. brucei activity of
the trialkylammonium compounds requires a substantial level
of lipophilicity around the cationic N atom. It is also not
dependent on entry through the P2 and HAPT transporters.
These results, similar to a series of dicationic choline-derived
9
solution of linker 1−3 (1 equiv) in anhydrous CH CN (0.8 mL) was
compounds, indicate that diffusion is the most probable
3
added 2.2 equiv (1.1 equiv for linker 3) of the corresponding tertiary
amine. The tube was stopped up and heated at 80 °C for 75 min. The
product was purified by crystallization as specified in each case.
Representative examples (1c, 1d, and 1e) are given below. The
synthesis and characterization of 1a, 1b, 2a, 2b, 2d, 2e, and 3b−3f will
be reported elsewhere.
mechanism of uptake into trypanosomes, although the presence
of an unknown transporter for lipophilic cations cannot be
excluded. However, it is likely that the lipophilic ammonium
and phosphonium compounds would have to pass both the
plasma membrane and the mitochondrial membrane, as such
compounds are generally believed to accumulate, and act, in the
N,N′-((Oxybis(4,1-phenylene))bis(methylene))bis(N,N-dihex-
ylhexan-1-aminium) Bromide (1c). The reaction was carried out by
following the general procedure starting from 4,4′-oxybis-
7
mitochondria (De Koning, manuscript in preparation).
In summary, modulation of the cationic center (from
phosphorus to nitrogen) of the diphenylether and benzophe-
none class of trypanocides resulted in 2- to 5-times lower
activities against wild type and resistant T. b. brucei lines.
Nevertheless, two new potent and selective trypanocidal
compounds were obtained, 1c and 3c, which displayed
nanomolar EC50 and SI > 100. Similar SARs were observed
with ammonium and phosphonium salts showing that charge
(
(
(bromomethyl)benzene) (54.6 mg, 0.153 mmol) and trihexylamine
114 μL, 0.337 mmol). Then, diethyl ether (4 mL) was added to the
cold reaction mixture, and the tube was allowed to stand at 4 °C
overnight. The supernatant was removed and the sticky precipitate was
covered with Et O and triturated with a spatula until formation of a
2
white solid. The product was recrystallized from CH Cl /Et O, rinsed
2
2
2
with Et O, and dried under vacuum. Off-white amorphous solid (29
2
1
mg, 21%); HPLC > 90% pure; H NMR (300 MHz, CDCl ) δ 7.58
3
C
dx.doi.org/10.1021/ml500408d | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(
d, J = 8.15, 4H, ArH), 6.89 (d, J = 8.15, 4H, ArH), 4.98 (s, 4H,
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(
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1
1,18
9,10,15
exactly as described.
AUTHOR INFORMATION
■
(
̈
13) Bridges, D. J.; Gould, M. K.; Nerima, B.; Maser, P.; Burchmore,
Corresponding Author
R. J. S.; De Koning, H. P. Loss of the high-affinity pentamidine
transporter is responsible for high levels of cross-resistance between
arsenical and diamidine drugs in African trypanosomes. Mol.
Pharmacol. 2007, 71, 1098−1108.
*
Tel: +34 912587490. Fax: +34 915644853. E-mail:
dardonville@iqm.csic.es.
Author Contributions
The manuscript was written through contributions of all
authors.
(
14) Munday, J. C.; Eze, A. A.; Baker, N.; Glover, L.; Clucas, C.;
Aguinaga Andres, D.; Natto, M. J.; Teka, I. A.; McDonald, J.; Lee, R.
S.; Graf, F. E.; Ludin, P.; Burchmore, R. J. S.; Turner, C. M. R.; Tait,
A.; MacLeod, A.; Maser, P.; Barrett, M. P.; Horn, D.; De Koning, H. P.
́
Funding
̈
Support of the Government of Saudi Arabia (Studenship to
A.A.M.A., Aljouf University, Saudi Arabia) is acknowledged.
This work was supported by grants from the Spanish Ministerio
de Ciencia e Innovacion
Notes
Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter
for pentamidine and melaminophenyl arsenic drugs and the main
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́
(Grant SAF2009−10399) to C.D.
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Gould, M. K.; Suksamrarn, A. Curcuminoid analogs with potent
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phosphonium based ionic liquids: a comparison of common anions.
Phys. Chem. Chem. Phys. 2014, 16, 15278−15288.
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̈
P. P.; Buscher, P.; De Koning, H. P.; Horn, D.; Maser, P. Aquaporin 2
mutations in Trypanosoma brucei gambiense field isolates correlate with
The authors declare no competing financial interest.
ABBREVIATIONS
■
(
BBB, blood−brain barrier; CNS, central nervous system; CSF,
cerebrospinal fluid; HAPT, high affinity pentamidine trans-
porter; HAT, human African trypanosomiasis; HEK cells,
human endothelial kidney cells; LAPT, low affinity pentamidine
transporter; RF, resistance factor; WT, wild type
(
D
dx.doi.org/10.1021/ml500408d | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
decreased susceptibility to pentamidine and melarsoprol. PLoS Negl.
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(
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E
dx.doi.org/10.1021/ml500408d | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX