Synthesis and structure-activity relationships of new quinolone-type molecules against trypanosoma brucei

Article abstract of DOI:10.1021/jm101439s

Human African trypanosomiasis (HAT) or sleeping sickness is caused by two subspecies of Trypanosoma brucei, Trypanosoma brucei gambiense, and Trypanosoma brucei rhodesiense and is one of Africa's old plagues. It causes a huge number of infections and cases of death per year because, apart from limited access to health services, only inefficient chemotherapy is available. Since it was reported that quinolones such as ciprofloxacin show antitrypanosomal activity, a novel quinolone-type library was synthesized and tested. The biological evaluation illustrated that 4-quinolones with a benzylamide function in position 3 and cyclic or acyclic amines in position 7 exhibit high antitrypanosomal activity. Structure-activity relationships (SAR) are established to identify essential structural elements. This analysis led to lead structure 29, which exhibits promising in vitro activity against T. b. brucei (IC₅₀ = 47 nM) and T. b. rhodesiense (IC₅₀ = 9 nM) combined with low cytotoxicity against macrophages J774.1. Screening for morphological changes of trypanosomes treated with compounds 19 and 29 suggested differences in the morphology of mitochondria of treated cells compared to those of untreated cells. Segregation of the kinetoplast is hampered in trypanosomes treated with these compounds; however, topoisomerase II is probably not the main drug target.

Full text of DOI:10.1021/jm101439s

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of New Quinolone-
Type Molecules against Trypanosoma brucei
Georg Hiltensperger,† Nicola G. Jones,‡ Sabine Niedermeier,† August Stich,§ Marcel Kaiser,∥,⊥
Jamin Jung,‡ Sebastian Puhl,† Alexander Damme,# Holger Braunschweig,# Lorenz Meinel,†
Markus Engstler,‡ and Ulrike Holzgrabe*,†
†Institut fur Pharmazie and Lebensmittelchemie, Universitat Wuerzburg, Am Hubland, 97074 Wurzburg, Germany
̈
̈
̈
‡Department of Cell and Developmental Biology, Universitat Wuerzburg, Am Hubland, 97074 Wurzburg, Germany
̈
̈
§Medical Mission Institute, Salvatorstrasse 7, 97067 Wurzburg, Germany
̈
∥Swiss Tropical and Public Health Institute, Socinstrasse 57, 4051 Basel, Switzerland
⊥Swiss University of Basel, Petersplatz 1, 4051 Basel, Switzerland
#Institute of Inorganic Chemistry, Universitat Wuerzburg, Am Hubland, 97074 Wurzburg, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Human African trypanosomiasis (HAT) or sleeping sickness is
caused by two subspecies of Trypanosoma brucei, Trypanosoma brucei gambiense, and
Trypanosoma brucei rhodesiense and is one of Africa’s old plagues. It causes a huge
number of infections and cases of death per year because, apart from limited access
to health services, only inefficient chemotherapy is available. Since it was reported
that quinolones such as ciprofloxacin show antitrypanosomal activity, a novel
quinolone-type library was synthesized and tested. The biological evaluation
illustrated that 4-quinolones with a benzylamide function in position 3 and cyclic or
acyclic amines in position 7 exhibit high antitrypanosomal activity. Structure−
activity relationships (SAR) are established to identify essential structural elements. This analysis led to lead structure 29, which
exhibits promising in vitro activity against T. b. brucei (IC50 = 47 nM) and T. b. rhodesiense (IC50 = 9 nM) combined with low
cytotoxicity against macrophages J774.1. Screening for morphological changes of trypanosomes treated with compounds 19 and
29 suggested differences in the morphology of mitochondria of treated cells compared to those of untreated cells. Segregation of
the kinetoplast is hampered in trypanosomes treated with these compounds; however, topoisomerase II is probably not the main
drug target.
mainly affecting the Democratic Republic of Congo, Angola,
Southern Sudan, and Uganda.4 In 1997, surveillance in sub-
Saharan Africa had been reinforced. Current estimations show
that about 60 million people are at risk in sub-Saharan Africa
with 50000−70000 cases occurring annually.5
INTRODUCTION
■
Human African trypanosomiasis (HAT), also known as
sleeping sickness, is caused by infection with one of two
subspecies of Trypanosoma brucei. Trypanosoma brucei
rhodesiense is found in Eastern and Southern Africa, whereas
Trypanosoma brucei gambiense occurs in Western and Central
Africa and is responsible for over 90% of all reported cases of
infection.1
In the last 120 years, Africa saw three severe sleeping sickness
epidemics. In 1896, the first one mainly affected Uganda and
Congo and caused approximately 500000 deaths.2,3 The
ensuing drug development, supported by colonial adminis-
trations, helped to combat the second severe outbreak in 1920.2
Another important approach toward the containment of HAT
was the elimination of the parasite reservoir so that within 11
years the prevalence of sleeping sickness decreased from 60% in
some areas to 0.2−4.1%. By the mid 1960s, most of the
endangered countries became independent. The upcoming
political instability had a disastrous effect on the health services,
furthermore, the control of HAT was no longer a priority and
led to the third and most recent epidemic in the 20th century,
Both subspecies are transmitted by the bite of infected tsetse
flies. T. b. gambiense HAT is primarily a chronic human disease.
T. b. rhodesiense HAT is primarily zoonotic with a huge animal
reservoir and causes the acute form of sleeping sickness. Both
forms of HAT show two clinical stages. The first corresponds
to the multiplication of the parasites in the blood and lymphatic
system. After crossing the blood−brain barrier, the trypano-
somes attack the central nervous system and neurological
symptoms appear. Without medical treatment, coma and finally
death results. The medical treatment is based on only five
commercially available drugs: suramine and pentamidine are
used in treatment of the first stage, and melarsoprol and
eflornithine, which both are able to cross the blood−brain
Received: November 9, 2010
Published: March 1, 2012
© 2012 American Chemical Society
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Figure 1. Structures of commercially available drugs.
barrier, are employed in the therapy of the second stage. A fifth
drug, nifurtimox, was recently introduced as a partner in
combination therapies for T. b. gambiense HAT (Figure 1).6 All
treatment regimens were established during the last 20 years,
and some suffer from severe, sometimes life-threatening side-
effects, e.g., melarsoprol as an arsenic containing drug, is lethal
in 4−6% of treated patients. Moreover, drug resistance is
spread to an extent that medical treatment of HAT is rapidly
losing its effectiveness in many African regions.7,8
Consequently, new active compounds are urgently needed.
Parasitic targets such as cytochrome-independent oxidase, fatty
acid biosynthesis, and purine salvage provide a basis for new
drug design.7,9,10 Another interesting target is the kinetoplast
DNA (kDNA) replication machinery in which the top-
oisomerase is responsible for the unique structure of
kDNA.11 Recently, it was reported that topoisomerase II
inhibitors like nalidixic acid, ciprofloxacin, gatifloxacin, and
moxifloxacin showed activity against the topoisomerases II of
trypanosomes (Figure 2).8,12 Even though structural variations
mainly with regard to the basic heterocycles in position 7 and
condensed rings (from N1 to C8 and N1 to C2) were
performed, the antitrypanosomal activity stayed in the low
micromolar range. Furthermore, ciprofloxacin derivatives
exhibited antitrypanosomal potency but seemed to lack in
vivo activity.13−16
The initial finding that the amidation of the carboxyl function
resulted in antitrypanosomal activity in the low micromolar
concentration range prompted us to synthesize and test a
library of quinolone-type molecules mainly characterized by a
benzylamide function in position 3 and an amine heterocycle in
position 7. A subsequent SAR analysis indicated the importance
of the amide group.
Screening to elucidate the target of these newly synthesized
compounds in trypanosomes was performed using a range of
organelle marker cell lines and fluorescence microscopy.
Initially observed changes in mitochondrion morphology
were followed up by a cell cycle analysis, which revealed that
kinetoplast segregation was affected. Because compound 29
exhibited promising in vitro activity against T. b. brucei and T. b.
rhodesiense, it was also tested in vivo in the T. b. rhodesiense
(STIB900) acute mouse model.
RESULTS AND DISCUSSION
■
Chemistry. The syntheses of 4-oxo-quinoline skeletons
were accomplished according to the Gould−Jacobs procedure,
starting off with the condensation of 3-chloro-4-fluoroaniline 1a
and 3-(trifluoromethyl)-aniline 1b, respectively, with diethyl 2-
(ethoxymethylene)-malonate (EMME). Subsequent cyclization
in boiling diphenyl ether resulted in ethyl 7-chloro-6-fluoro-4-
hydroxyquinoline-3-carboxylate 3a and ethyl 7-(trifluorometh-
yl)-4-hydroxyquinoline-3-carboxylate 3b.17−19 In both cases
regioisomers arose, where ethyl 5-chloro-6-fluoro-4-hydrox-
yquinoline-3-carboxylate 3a′ and ethyl 5-(trifluoromethyl)-4-
hydroxyquinoline-3-carboxylate 3b′ were obtained in low
amounts (<5%). Hence these isomer mixtures were carried
on to the next step without separation. The alkylation of
position 1 was achieved with corresponding alkyl halides in the
presence of potassium carbonate.20 After separation of the
regioisomers using normal phase column chromatography on
silica gel and hydrolysis of the ethyl ester, the corresponding 4-
oxo-1,4-dihydroquinoline-3-carboxylic acids 4a−c were isolated
in good yields.19,20 7-Chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid 4d was commercially
available. Ethyl 7-(trifluoromethyl)-4-oxo-1,4-dihydroquino-
line-3-carboxylic acid 4b was amidated in position 3 using i-
Figure 2. Structures of several 4-quinolones exhibiting antitrypanoso-
mal activity.
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a
Scheme 1
a
Reagents and conditions: (i) EMME, toluene, reflux; (ii) diphenyl ether, reflux; (iii) (1) alkyl halide, K2CO3, KI, DMF, 80 °C, (2) KOH (2M),
reflux; (iv) H2NR3, i-butyl chloroformiate, NMM, DMFabs, 0 °C/rt; (v) piperidine/piperidine-4-carboxamide, DMF, 90 °C; (vi) morpholine/N-
benzylmethylamine, MW (500 W), 120 °C; (vii) BF3·OEt2, CH2Cl2abs, reflux; (viii) (1) DMF, 60 °C, (2) KOH (2M), reflux; (ix) BBr3, CH2Cl2abs, 0
°C/rt.
butyl chloroformiate, N-methylmorpholine (NMM), and
corresponding amines to give compounds 5 and 6.19 The 7-
chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
derivatives 4a, 4c, and 4d were used for the chloride
substitution in position 7 with different cyclic and acyclic
amines. The substitution of either 4a, 4c, or 4d with piperidine
or piperidine-4-carboxamide was carried out in DMF at 80 °C
to give intermediates 7−10.19 This method did not work for
compounds 11 and 12. Hence, compounds 11 and 12 were
obtained by dissolving 4c in morpholine or N-benzylmethyl-
amine and heating under microwave irradiation (120 °C/500
W/4 h). Compound 13 was prepared via the borate complex of
4c.21 The boron ester was accomplished using boron trifluoride
diethyl etherate and served as internal Lewis acid, which
activates position 7 for nucleophilic aromatic substitution. The
chloride substitution was achieved in DMF containing a huge
amount of free dimethyl amine. Subsequent hydrolysis led to
intermediate 13. For amidation in position 3, the carboxylic
acids were activated using i-butyl chloroformiate and NMM.
Subsequently, the in situ generated mixed anhydride was
converted with different aryl amines to give the corresponding
amides 14−23 and 25−29. Demethylation of compound 19
with BBr3 resulted in compound 24 (Scheme 1).
fluorobenzene (Table 1). Nitration and reduction with iron and
methanol was accomplished according to Austin et al., yielding
the aniline 30.23 Subsequent condensation with EMME and
cyclization in boiling diphenyl ether resulted in ethyl 7-bromo-
6-fluoro-4-hydroxyquinoline-3-carboxylate 31, which was alky-
lated in position 1 with brombutane in the presence of
potassium carbonate and purified using normal phase column
chromatography.20,24 The Suzuki reaction was performed with
phenylboronic acid in the presence of Pd(OAc)2 and
triphenylphosphine under basic conditions.25 After ester
hydrolysis, the amidation of position 3 resulted in compound
34 (Scheme 2).
Structure−Activity Relationships. Since the antitrypano-
somal activity of ciprofloxacin and corresponding derivatives
was reported,8,12−16 we synthesized a quinolone-type library
and tested the intermediates of the synthetic pathway as well as
the 4-quinolone-3-carboxamides against Trypanosoma brucei.
The antitrypanosomal activity was evaluated in Trypanosoma
brucei brucei according to Raz et al., and cytotoxicity was
̈
measured in macrophages J774.1 using the AlamarBlue-based
assay.26−28 These data are displayed in Table 2. The most active
compound is the morpholino-substituted substance 29 having
an IC50 (T. b. brucei) of 0.047 μM and an IC50 value of 0.009
μM against the human pathogenic subspecies T. b. rhodesiense
(STIB900).
To introduce a phenyl residue in position 7, the Suzuki
coupling reaction was employed. Often, the oxidative addition
is the rate-determining step in the catalytic process and is
improved using aryl-bromides instead of aryl-chlorides.22
Therefore, the ethyl 7-bromo-4-quinolone-3-carboxylate inter-
mediate 32 was synthesized starting off with 1-bromo-2-
The intermediate 4-hydroxy-quinoline esters 3a−b (data not
shown) and 4-quinolone carboxylic acids 4a−d did not show
any activity. After amidation of 4b in position 3, resulting in
compound 5, the IC50 value decreased to 7.31 μM. Comparing
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Table 1. Substitution Pattern of Intermediates and 4-Quinolone-3-carboxamide Derivatives 4−34
a
Scheme 2
a
Reagents and conditions: (i) (1) HNO3, H2SO4, 0 °C/rt, (2) Fe, MeOH, reflux; (ii) (1) EMME, toluene, reflux, (2) diphenyl ether, reflux; (iii)
brombutane, K2CO3, KI, DMF, 80 °C; (iv) (1) phenylboronic acid, Pd(OAc)2, PPh3, K2CO3, DMF/H2O (9:1), 60 °C, (2) KOH (2M), reflux; (v)
benzylamine, i-butyl chloroformiate, NMM, DMFabs, 0 °C/rt.
benzylamides (5) with phenylamides (6) revealed the
amidation in position 3 to be essential for high activity, but
only for benzylamide and not for phenylamide derivatives,
indicating the need of flexible aromatic moieties in position 3.
The replacement of the chloride atom with cyclic and acyclic
amines in position 7, giving the 4-quinolone derivatives 7−13,
results in inactive or low activity. However, again the
subsequent amidation of 4-quinolone-3-carboxylic acids 7−13
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be pivotal. This finding is supported by the fact that most
fluoroquinolone acid derivatives reported in the literature were
found to exhibit at most a weak activity.12−16 Moreover, no in
vivo effectivity was found for these acids in mice.15
Table 2. Antitrypanosomal Activity, Cytotoxicity, and
Selectivity Index of 4-Quinolone-3-carboxamide Derivatives
a
and Intermediates
T. brucei brucei IC50 [μM]
3. The Influence of the Phenyl Substitution of the
Benzylamides in Position 3. The comparison of the
benzylamide, compounds 19 and 22−25 exhibited, that either
a nitro or methoxy group induces a high activity in comparison
to a hydroxy group (24) and a combination of methoxy and
nitro group (23). This result indicated that nitro and methoxy
groups, which can be regarded as H-bond acceptors, led to
higher trypanocidal activities than H-bond donors like the
hydroxy group. An additional H-bond acceptor on the aromatic
moiety decreased the activity (23). This might be due to steric
hindrance.
4. The Influence of Cyclic and Acyclic Amines in Position
7. To examine the influence of different amines, we have
chosen the n-butyl substituent in position 1 and the
benzylamide in position 3 and varied the amine substituent in
position 7. Whereas compounds 22 and 27 carrying a N-
benzylmethylamine and a piperidine-4-carboxamide, respec-
tively, showed low activity, the piperidino- and N,N-
dimethylamino substituted compounds 26 and 28, respectively,
are active in the submicromolar range of concentration. They
were even surpassed by the morpholino-substituted derivative
29 showing an IC50 value of 47 nM. Because smaller
substituents like piperidine, morpholine, and N,N-dimethyl-
amine showed higher activity, steric hindrance might play a
pivotal role. Morpholine comprises a H-bond acceptor, which
might enable interactions at this position.
cytotoxicity J774.1
selectivity
index (SI)
compd
48 h (sdv)
72 h (sdv)
IC50 [μM]
4a
4b
4c
5
>40
>40
>100
>100
>100
>100
>100
>100
>100
>100
93.0
>2.50
>2.50
>2.50
>13.7
>2.50
>2.50
>2.50
>2.50
6.39
>40
>40
>40
>40
7.31 (2.29)
>40
6.37 (1.25)
>40
6
7
>40
>40
8
>40
>40
9
>40
>40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
14.6 (0.51)
19.2 (2.36)
17.2 (1.00)
>40
18.1 (0.6)
22.2 (2.82)
23.0 (8.15)
>40
>100
43.2
>5.21
2.51
>100
>100
56.9
>2.50
>11.2
12.4
8.89 (4.17)
4.60 (0.53)
1.03 (0.13)
2.75 (0.31)
2.28 (0.51)
0.78 (0.14)
1.07 (0.16)
0.75 (0.23)
1.09 (0.47)
3.07 (0.59)
3.09 (0.56)
0.54 (0.14)
0.67 (0.11)
5.40 (0.55)
0.68 (0.16)
0.047 (0.00)
12.1 (2.15)
4.84 (0.76)
1.84 (0.32)
2.56 (1.12)
3.12 (1.19)
1.45 (0.61)
1.91 (0.65)
1.25 (0.20)
1.98 (0.80)
4.14 (0.48)
5.12 (0.14)
0.58 (0.14)
1.04 (0.22)
10.77 (1.37)
0.92 (0.01)
0.05 (0.01)
41.4
40.2
>100
41.4
>36.4
18.2
40.8
52.3
51.2
47.9
43.7
58.3
47.0
43.1
>100
41.6
>32.6
13.5
>100
47.8
>185.2
71.3
By means of the Suzuki reaction, a phenyl residue could be
realized in position 7. The resulting compound 34 showed an
IC50 value above 40 μM and emphasizes the need of saturated
N−C coupled substituents in this position.
86.0
15.9
>100
57.0
>147.1
1140
b
b
0.009
6333
Physicochemical Properties and Formulation. The pKa
of lead compound 29 was determined UV metrically at 4.53
0.18 (n = 6) and corroborated potentiometrically (pKa of 4.63).
The lipophilicity of the most active compounds 16, 19, 20, 21,
22, 25, 26, 28, and 29 was determined using RP HPLC and the
correlation between the capacity factor k′ and the logP values of
reference compounds.29 The logP values range from 2.4 to 4.0,
and the molecular weight of the most active compounds is in
the range of 395.5 g/mol and 523.6 g/mol. Additionally, all
compounds comprise less than 10 H-bond acceptors and less
than 5 H-bond donors, respectively. These properties correlate
with Lipinski’s Rule of Five and predict a good oral
bioavailability.30
34
>40
>40
>100
>2.50
a
All compounds are tested in duplicate to calculate mean value and
standard deviation (sdv). For comparison: pentamidine diisethionate
IC50 = 0.0029 μM; suramine Na IC50 = 0.31 μM; eflornitine HCl IC50
b
= 22.9 μM; melarsoprol IC50 = 0.0026 μM. Activity of compound 29
against T. b. rhodesiense and corresponding selectivity index.
exhibited an enhancement in activity. This finding clearly
demonstrated that a combination of benzylamides in position 3
and cyclic and acyclic amines in position 7 are necessary for
high antitrypanosomal activity. In the following, structure−
activity relationships of 7-amino-substituted 4-quinolone-3-
carboxamides will be discussed in detail:
The solubility of compound 29 in buffered aqueous media
has been assessed in preliminary tests, ranking the compound
as “poorly water-soluble” or slightly soluble or less according to
USP or EP pharmacopeial terminology. Drug solubility in water
is typically a function of (i) entropy of mixing, (ii) drug−drug
interactions associated with lattice energy in cases the drug is
crystalline, and (iii) the difference of adhesive interactions
(drug−water) and the sum of cohesive interactions (water−
water and drug−drug). The crystal structure of 26 (Figure 3),
one representative of this group of compounds, indicates such
high crystal forces. Compound 29 has a sharp melting point
determined at 164−165 °C (n = 3), further supporting the
assumption of rather strong crystal forces and arguably pointing
to the development of a formulation, within which compound
29 is presented in amorphous form or dissolved state.
1. The Influence of Alkyl Substituents in Position 1.
Comparison of derivatives 14−16, 17−19, and 20−22 with
either cyclopropyl-, n-propyl-, or n-butyl-residues in position 1
revealed n-butyl-substituted 4-quinolones of compounds 13−
15 and 16−18 to be advantageous, e.g., N-butyl compound 19
exhibited an IC50 value of 0.78 μM while the N-cyclopropyl
compound 17 showed an IC50 value of 2.75 μM. However, the
comparison of compounds 20−22 revealed the n-propyl-
substituted compound 21 to have the lowest IC50 value.
Nevertheless, the longer alkyl chains in position 1 the higher is
the activity.
2. Carboxylic Acid versus Aromatic Amide Function in
Position 3. Comparison of the acid derivatives with the amide
compounds revealed the benzylamide residue in position 3 to
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oral dose of 50 mg/kg given on four consecutive days. In the
absence of pharmacokinetic data, we cannot distinguish if this is
due to missing in vivo efficacy or related to the bioavailability of
the compound when delivered in strongly solubilized form, as
done in this study. Future studies will detail the interaction of
the compound with the different elements constituting the
micelles in the administered microemulsion, and appropriate
dissolution and pharmacokinetic studies should detail this
aspect. Strong solubilization has been demonstrated to
constitute an important parameter affecting the drug
bioavailability and, hence, bioactivity.33 As discussed before,
the formulation is not yet suitable for parenteral use.
The target of the quinolone-3-carboxylic acids was shown to
be the topoisomerase II of the trypanosome.8,14,15 However,
because the carboxylate function is essential for the interaction
of the drugs with the topoisomerase, it is quite likely that the
benzylamides address a different target.
To narrow down the target of these antitrypanosomal
compounds, 19 and 29 were subjected to a fluorescence
microscopy based screen. Recombinant marker cell lines, in
which organelles are fluorescently labeled by GFP or YFP
fusion to an organelle specific resident protein, were employed
to study the effect of the two compounds on the
mitochondrion, Golgi, ER, endosomes, and lysosome of the
cell. No significant changes in the morphology of the Golgi, ER,
endosomes, or lysosome could be observed. However,
Figure 3. Distinctive layers of compound 26. Two molecules in a
plane communicate via weak intermolecular H-bond interaction of the
aromatic proton at C5 and the carbonyl oxygen in position 4 (about
2.2−2.6 Å) as well as the fluoride atom at C6 and the amide proton
(about 3.1 Å). The distance between the aromatic skeleton of two
molecules is about 3.6 Å, indicating a possible interaction of π-
electronic systems.
For in vivo experiments, common aqueous DMSO mixtures
for dissolving 29 could not be used. Even the addition of 10%
of typical used surface-formulation ingredients like Tween 80 or
Cremophor RH40 did not substantially improve the water
solubility of compound 29. Thus, a preliminary oral
formulation consisting of Cremophor RH40 (35%), Capmul
MCM C8 (45%), triethylcitrate (10%), and ethanol (10%) was
developed which dissolves a concentration of 10 mg/mL of 29
and forms stable emulsions in aqueous solutions (stable >24 h).
The developed preconcentrate is suitable for oral use, and
similar formulations have been deployed.31 However, responses
to parenteral administration require further characterization,
particularly hemodynamic response in vitro and in vivo
responses (acid−base balance, blood gases, plasma electrolytes,
and arterial blood pressure or heart rate) due to ascending
doses of the vehicle should be characterized prior to using the
formulation.32
incubation with both compounds 19 (0.54
0.04 μM) and
29 (0.023 0.004 μM) at IC50 concentrations for 42 h
suggested a change in the morphology of the mitochondrion
compared to control cells. In untreated BSF T. brucei, the
mitochondrion is an elongated, tubular structure extending the
length of the cell. In cells treated with both compound 19 and
29, mitochondrion morphology appeared altered, showing
sheet-like patches in the cell-spanning mitochondrion. Figure 4
shows the observed phenotype with representative cells.
As it is known that ciprofloxacin acts as a topoisomerase II
inhibitor and compounds 19 and 29 are quinolone-type
structures, we further focused on the kinetoplast. A cell cycle
analysis of chemical treated cells showed that compounds 19
and more so 29 revealed an increased percentage of 1K1N cells
displaying segregating kinetoplasts, 1Kseg1N, suggesting that
both compounds interfere with correct segregation of the
kinetoplast (Figure 5). The insert in Figure 5 shows a cell
displaying a stark segregation defect. This phenotype was not
evident in ciprofloxacin treated cells. To further investigate the
In Vivo Activity and Target Search. An aqueous 1:1
dilution of the formulation, containing 10 mg/mL of 29, was
administered orally to T. b. rhodesiense (STIB900) infected
NMRI mice. Compound 29 did not show in vivo efficacy at an
Figure 4. Fluorescence microscopy images of a T. b. brucei mitochondrion marker cell line treated with DMSO (A), compound 19 (B), and
compound 29 (C). The mitochondrion, M, is shown in green. The cell surface (AMCA stained) and the nucleus, N, and kinetoplast, K, (DAPI
stained) are shown in blue. Whereas control cells (A) show the typical elongated, tubular structure of the mitochondrion of BSF trypanosomes, cells
treated with compounds 19 or 29 (B, C) show sheet-like patches in the tubular structure.
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Figure 5. Cell cycle analysis of a T. b. brucei mitochondrion marker cell
line before and after treatment for 42 h with DMSO, ciprofloxacin,
compound 19, and compound 29 (n = 300 each). Treatment with
compounds 19 and 29 lead to a shift in ratio of 1K1N to 1Kseg1N cells
from 2:1 to 3:2 and 1:1, respectively. The inset shows cells that have
been stained with DAPI to show the larger nucleus and smaller
kinetoplast. The upper figure (DMSO) shows two typical cells,
whereas the lower figure (comp) depicts a stark example of two cells
displaying a segregation defect in their kinetoplasts.
Figure 6. Knockdown of mitochondrial T. brucei topoisomerase II. (A)
Knockdown of TbTopoIImt in BSF trypanosomes affects cell growth
beginning 72 h after induction of RNAi with tetracycline (Tet). Three
independent clones were analyzed, and cell numbers in the growth
curves are shown as the average of these and their standard deviation.
The inset shows a Northern blot analysis of one clone to confirm
knockdown of topoisomerase II (TopoII) mRNA 24 h after induction
of RNAi. Tubulin (Tub) mRNA served as a loading control. (B)
Images illustrating the shrinkage of the kinetoplast over time after
RNAi induction. At 0 h, kinetoplasts are clearly visible (indicated by a
white arrowhead). kDNA loss of DAPI stained cells is first detected
after 48 h of induction (indicated by gray arrowheads). By 72 h post
induction, the majority of cells are dyskinetoplastid.
possibility that compounds 19 and 29 target the mitochondrial
topoisomerase II, TbTopoIImt, of T. brucei, we knocked down
TbTopoIImt in BSF cells by RNA interference. The function of
this TbTopoIImt in trypanosomes has been studied previously
in insect stage trypanosomes employing RNA interference.34,35
These studies showed that knockdown of TbTopoIImt lead to a
shrinkage and eventual loss of the kinetoplast. Knockdown of
TbTopoIImt in BSF trypanosomes produced a comparable
phenotype, where the kinetoplast was lost over time (Figure 6).
However, a segregation defect could not be detected, not even
transiently. This formally excludes the possibility that
compounds 19 and 29 target TbTopoIImt alone but suggests
these compounds also affect other proteins involved in T. brucei
kinetoplast segregation. This is in line with previous findings
that only β-keto-carboxylates are able to closely bind to the
topoisomerase II36 which, in turn, the amides cannot, indicating
that the topoisomerase is likely not the target of the quinolone-
amides.
treated with these compounds showed a defect in kinetoplast
segregation.
Compound 29 was selected for evaluation in a murine model
of human African trypanosomiasis. Therefore, a preliminary
formulation was developed, but oral administration proofed
ineffective against T. b. rhodesiense (STIB900) infected NMRI
mice. Nevertheless, the outstanding in vitro activity of 29
aganist T. b. brucei and T. b. rhodesiense suggests that this
compound serves as a promising lead structure for further
optimization. Thus, structural modification of 29 for increasing
the water solubility, as well as developing a more appropriate
parenteral formulation, will be the future goal.
CONCLUSION
■
The biological evaluation of the 4-quinolone-3-carboxamide
library revealed several promising lead compounds with
antitrypanosomal activity in the nanomolar concentration
range and low cytotoxicity. The SAR analysis illustrated the
need of alkyl chains in position 1, benzylamides in position 3,
and amine heterocycles in position 7 and led to compound 29
with an IC50 (T. b. brucei) of 47 nM and an IC50 value against T.
b. rhodesiense of 9 nM. Moreover, because the most active
compounds show a cytotoxicity higher than 40 μM, the gap
between trypanocidal activity and cytotoxicity augments with
increasing antiinfective activity, resulting in an SI value of 6333
for compound 29. Furthermore, the main advantage of this
group of compounds is the simple route of synthesis using the
well-known Gould−Jacobs procedure, which gives good overall
yields.
EXPERIMENTAL SECTION
■
Melting points were determined with a Stuart melting point apparatus
SMP11 (Bibby Scientific, UK) and not corrected. IR spectra were
obtained using a Biorad PharmalyzIR FT-IR spectrometer (Digilab,
Krefeld, Germany). TLC was performed on silica gel 60 F254
1
aluminum sheets (Merck, Darmstadt, Germany). H (400.132 MHz)
and 13C (100.613 MHz) NMR spectra were recorded on a Bruker AV
400 instrument (Bruker Biospin, Ettlingen, Germany). As internal
standard, the signals of the deuterated solvents were used (DMSO-d6:
1H 2.5 ppm, 13C 39.52 ppm; CDCl3: H 7.26 ppm, 13C 77.16 ppm).
1
Abbrevations for data quoted are: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; b, broad; dd, doublet of doublets; dt, doublet of
triplets; tt, triplet of triplets; tq, triplet of quartets. Coupling constants
(J) are given in Hz. Chemicals were of analytical grade and purchased
from Aldrich (Steinheim, Germany) and Merck (Darmstadt,
Germany).
For identifying the target of these compounds, the
antitrypanosomal substances 19 and 29 were subjected to a
fluorescence microscopy based screen. Incubation with both
compounds appeared to affect the morphology of the
mitochondrion. Further analysis revealed that trypanosomes
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Journal of Medicinal Chemistry
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The purities of new compounds was determined using micro-
analysis (C, H, N) and HPLC and agreed with the theoretical value
within 0.4% and ≥95%, respectively. Alternatively, HPLC analyses
were performed on an Agilent 1100 HPLC system with UV detector,
using a C18 reversed-phase Inertsil ODS-2 column (150 × 4.6 mL, 5
μm) eluting with a mixture of methanol/phosphate buffer (Method 1,
70% methanol:30% phosphate buffer. Method 2: gradient, 30%
methanol going up to 80% methanol in 5 min, 80% methanol going
down to 30% methanol in 5 min, 30% methanol for 2 min. Flow rate
1.0 mL/min, λ = 254.4 nm.).
3.3), 66.6 (2C), 51.4, 49.9 (2C), 30.5, 19.6, 13.3. Anal.
(C18H21FN2O4) Calcd: C 62.1, H 6.08, N 8.04. Found: C 62.0, H
6.24, N 7.80.
General Procedure for the Syntheses of 1-Butyl-7-(4-
carbamoylpiperidin-1-yl)-6-fluoro-N-(4-methoxybenzyl)-4-
oxo-1,4-dihydroquinoline-3-carboxamide (19) and N-Benzyl-
1-butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihydroquinoline-3-
carboxamide (29). 4-Oxo-quinolone-3-carboxylic acid (1 equiv) and
NMM (5 equiv) were dissolved in abs DMF (10 mL) at rt under Ar
atmosphere. After cooling to 0 °C, 4 equiv of i-butyl chloroformiate
were added and the mixture was stirred for 1 h. Then 4 equiv of the
corresponding amine were added and the mixture was stirred for
additional 2 h at rt. The solvent was removed in vacuo, and 20 mL of
water were added. The aqueous solution was extracted three times
with ethyl acetate (150 mL). The combined organic layers were dried
over sodium sulfate. The solvent was reduced in vacuo, and the residue
was purified by normal phase column chromatography on silica gel.
1-Butyl-7-(4-carbamoylpiperidin-1-yl)-6-fluoro-N-(4-me-
thoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (19).
Yield 46%; mp 195−196 °C. eluent: CHCl3/MeOH = 20:1 (Rf =
0.43). IR [cm−1]: 3193, 2931, 2868, 2833, 1653, 1488, 1233, 1031,
Syntheses of compounds 3a, 3b, 4a, 4b, 5, 6, 9, 30, and 31 were
performed according to refs 18−20, 23, and 24.
Synthesis of 1-Butyl-7-chloro-6-fluoro-4-oxo-1,4-dihydro-
quinoline-3-carboxylic Acid (4c). 3a (1 equiv) and potassium
carbonate (5 equiv) were suspended in DMF (15 mL) at rt. After 15
min of stirring, a catalytic amount of potassium iodide and 6 equiv of
1-bromobutane were added and the mixture was stirred at 80 °C for
24 h. After cooling to rt, the solvent was removed in vacuo and the
residue was purified by column chromatography on silica gel (eluent:
CHCl3/MeOH = 15:1, Rf = 0.66). The resulting ethyl 1-butyl-7-
chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate was sus-
pended in 20 mL of aqueous sodium hydroxide solution (2M) and
refluxed for 4 h. After cooling to rt, the solution was acidified by means
of concentrated hydrochloride acid (pH = 4). The precipitate was
collected, washed with a mixture of water and acetone (1:1), and dried
in vacuo to give compounds 4c.
1
3
803. H NMR (DMSO-d6, δ [ppm], J [Hz]): 10.3 (t, J = 5.7, 1H),
8.76 (s, 1H), 7.83 (d, 3JH,F = 13.4, 1H), 7.31 (s, 1H), 7.27 (d, 3J = 8.6,
4
3
2H), 7.07 (d, JH,F = 7.1, 1H), 6.90 (d, J = 8.6, 2H), 6.82 (s, 1H),
4.47−4.46 (m, 4H), 3.73 (s, 3H), 3.67−3.64 (m, 2H), 2.91−2.85 (m,
3
3
2H), 2.35−2.29 (m, 1H), 1.87−1.69 (m, 6H), 1.32 (tq, J = 7.2, J =
7.3, 2H), 0.92 (t, J = 7.3, 3H). 13C NMR (DMSO-d6, δ [ppm], J
3
Yield 87%; mp 233−236 °C. IR [cm−1]: 3048, 2959, 2872, 1717,
4
1
1
[Hz]): 176.0, 174.1 (d, JC,F = 2.2), 164.0, 158.3, 152.6 (d, JC,F
248.1), 147.7, 144.9 (d, JC,F = 11.0), 136.7, 131.5, 128.8 (2C), 121.2
=
1610, 1458, 1032, 897, 808. H NMR (DMSO-d6, δ [ppm], J [Hz]):
2
14.7 (s, 1H), 9.03 (s, 1H), 8.40 (d, 4JH,F = 5.3, 1H), 8.13 (d, 3JH,F = 8.6,
1H), 4.56 (t, 3J = 7.0, 2H), 1.74 (tt, 3J = 7.0, 3J = 7.0, 2H), 1.34 (tq, 3J
3
2
(d, JC,F = 6.6), 113.8 (2C), 111.3 (d, JC,F = 22.7), 110.0, 105.7 (d,
3JC,F = 2.2), 55.1, 52.8, 49.5 (2C), 41.6, 41.0, 30.3, 28.3 (2C), 19.2,
3
3
= 7.0, J = 7.0, 2H), 0.90 (t, J = 7.0, 3H). 13C NMR (DMSO-d6, δ
4
1
13.5. HPLC purity (method 1): 98.29%.
[ppm], J [Hz]): 176.5 (d, JC,F = 2.2), 165.5, 154.9 (d, JC,F = 249.6),
150.0, 136.4 (d, JC,F = 2.2), 127.4 (d, JC,F = 20.5), 123.3 (d, JC,F
6.5), 121.1, 111.9 (d, JC,F = 22.7), 107.7, 53.8, 30.8, 19.0, 13.5. Anal.
(C14H13ClFNO3) Calcd: C 56.5, H 4.40, N 4.70. Found: C 56.3, H
4.67, N 4.48.
4
2
3
N-Benzyl-1-butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihydro-
quinoline-3-carboxamide (29). Yield 58%; mp 164−165 °C.
Eluent: ethyl acetate (Rf = 0.57). IR [cm−1]: 3039, 2957, 2855,
=
2
1
1650, 1488, 1258, 1117, 929, 732. H NMR (DMSO-d6, δ [ppm], J
3
3
[Hz]): 10.4 (t, J = 5.9, 1H), 8.79 (s, 1H), 7.86 (d, JH,F = 13.6, 1H),
Synthesis of 1-Butyl-7-(4-carbamoylpiperidin-1-yl)-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (8). 4c (1 equiv)
and piperidine-4-carboxamide (8 equiv) were dissolved in DMF (20
mL) and stirred at 80 °C for 24 h. The solvent was removed in vacuo
and the residue resuspended in water (20 mL). After acidification with
concentrated hydrochloride acid (pH = 4), the mixture was stirred for
15 min at rt. The solid was filtered, washed several times with water,
and dried in vacuo to give compound 8.
4
3
7.35−7.24 (m, 5H), 7.08 (d, JH,F = 7.3, 1H), 4.55 (d, J = 5.9, 2H),
3
4.47 (t, J = 7.1, 2H), 3.80−3.78 (m, 4H), 3.26−2.23 (m, 4H), 1.76
(tt, 3J = 7.1, 3J = 7.0, 2H), 1.33 (tq, 3J = 7.0, 3J = 7.4, 2H), 0.92 (t, 3J =
7.4, 3H). 13C NMR (DMSO-d6, δ [ppm], J [Hz]): 174.0 (d, JC,F
=
4
1
2
2.2), 164.1, 152.4 (d, JC,F = 247.1), 147.7, 144.3 (d, JC,F = 10.6),
139.3, 136.5, 128.3 (2C), 127.3 (2C), 126.8, 121.4 (d, JC,F = 6.8),
111.4 (d, JC,F = 22.7), 110.0, 105.5 (d, JC,F = 2.8), 65.8 (2C), 52.8,
49.8 (2C), 42.0, 30.2, 19.1, 13.4. Anal. (C25H28FN3O3) Calcd: C 68.6,
H 6.45, N 9.60. Found: C 68.7, H 6.85, N 9.36.
3
2
3
Yield 52%; mp 226−228 °C. IR [cm−1]: 3196, 3049, 2951, 2872,
1
1716, 1625, 1472, 936, 808. H NMR (DMSO-d6, δ [ppm], J [Hz]):
15.3 (s, 1H), 8.90 (s, 1H), 7.85 (d, 3JH,F = 13.1, 1H), 7.32 (s, 1H), 7.12
(d, 4JH,F = 6.0, 1H), 6.83 (s, 1H), 4.54 (t, 3J = 7.0, 2H), 3.72−3.70 (m,
2H), 2.97−2.92 (m, 2H), 2.35−2.33 (m, 1H), 1.85−1.76 (m, 6H),
Trypanosome Assay According to References 26 and 27.
Parasite Culture. Trypomastigote forms of T. brucei brucei
laboratory strain TC 221 were cultured in Baltz medium
according to standard conditions.
3
3
3
1.33 (tq, J = 7.0, J = 7.0, 2H), 0.92 (t, J = 7.0, 3H). 13C NMR
4
In Vitro Cytotoxicity Assays. The test compounds were dissolved in
DMSO or NaOH (0.1 M). A defined number of parasites (104
trypanosomes per mL) were exposed in test chambers of 96-well plates
to various concentrations of the test substances in a final volume of
200 μL. Positive (trypanosomes in culture medium) and negative
controls (test substance without trypanosomes) were run with each
plate. The plates were then incubated at 37 °C in an atmosphere of 5%
CO2 for a total time period of 72 h. A reading was done at 48 h. The
effect of test substances was quantified in IC50 values by linear
interpolation of two different measurements. The activity of the test
substances was measured by light absorption in an MR 700 microplate
reader at a wavelength of 550 nm with a reference wavelength of 630
nm, using the AlamarBlue.
Macrophage Assay According to Reference 28. The macro-
phage cell line J774.1 was maintained in complete Click RPMI
medium. For the experimental procedures, cells were detached from
the flasks with a rubber police, washed twice with PBS, and suspended
at 2 × 106 cells per mL in complete Click RPMI medium. J774.1
macrophages were plated in complete RPMI medium (200 μL)
without phenol red in 96-well plates in the absence or presence of
(DMSO-d6, δ [ppm], J [Hz]): 176.0 (d, JC,F = 2.0), 175.8, 166.1,
152.9 (d, 1JC,F = 249.5), 148.7, 145.7 (d, 2JC,F = 10.3), 137.5, 118.9 (d,
3JC,F = 6.6), 111.0 (d, JC,F = 23.7), 106.8, 105.8 (d, JC,F = 3.7), 53.3,
2
3
49.4 (2C), 40.9, 30.3, 28.2 (2C), 19.1, 13.4. HPLC purity (method 2):
94.64%.
Synthesis of 1-Butyl-6-fluoro-7-morpholino-4-oxo-1,4-dihy-
droquinoline-3-carboxylic Acid (11). 4c (1 equiv) was dissolved in
morpholine (10 mL) and heated under microwave irradiation (500W/
120 °C) for 4 h. After cooling to rt, 20 mL of water were added and
the solution was acidified with concentrated hydrochloride acid (pH =
4). The precipitated solid was filtered and washed with water to give
compound 11 after drying in vacuo.
Yield 68%; mp 242−243 °C. IR [cm−1]: 3019, 2968, 2172, 1720,
1626, 1460, 1257, 942, 804. 1H NMR (CDCl3, δ [ppm], J [Hz]): 14.8
3
4
(s, 1H), 8.61 (s, 1H), 8.04 (d, JH,F = 13.1, 1H), 6.86 (d, JH,F = 6.8,
3
1H), 4.22 (t, J = 7.1, 2H), 3.92−3.90 (m, 4H), 3.29−3.27 (m, 4H),
1.88 (tt, 3J = 7.1, 3J = 7.5, 2H), 1.43 (tq, 3J = 7.5, 3J = 7.2, 2H), 0.99 (t,
3J = 7.2, 3H). 13C NMR (CDCl3, δ [ppm], J [Hz]): 176.7 (d, JC,F
=
4
2.5), 166.8, 153.2 (d, 1JC,F = 252.2), 147.5, 145.3 (d, 2JC,F = 9.9), 136.9,
3
2
3
120.8 (d, JC,F = 8.7), 112.7 (d, JC,F = 23.0), 108.0, 103.7 (d, JC,F
=
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various concentrations of the compounds and incubated for 24 h at 37
°C, 5% CO2, 95% humidity. Following the addition of AlamarBlue (20
μL), the plates were further incubated at similar conditions. The plates
were then read 24 and 48 h later. Control experiments to examine the
effect of cell density, incubation time, and DMSO concentration were
performed. Absorbance in the absence of compounds was set as 100%
of growth control.
mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose, pH 7.4) and
chemically fixed overnight in 2% formaldehyde at 4 °C or cell surface
stained prior to fixing. For staining, cells were washed 3× with TDB
and adjusted to a cell density of approximately 1 × 108 cells per mL.
Cell surface labeling was performed by incubation with 1 mM sulfo-
NHS-AMCA for 15 min on ice. After washing twice with TDB, cells
were chemically fixed as described above. Before data acquisition, the
nucleus and kinetoplast were stained by addition of DAPI.
Data Acquisition and Analysis. Image acquisition was performed
with a Till Photonics iMic Microscope and the LA Live Acquisition
software package. Three-dimensional images were acquired with 100×
magnification and 150 nm × 100 nm z-step size. The acquired raw
data was imported into the Huygens Essential Software (version 3.4,
Scientific Volume Imaging, BV, Hilversum, The Netherlands) for
digital deconvolution using the maximum likelihood algorithm and
100 iterations. Images of cells shown in Figures 4−6 were prepared by
maximum intensity z-projection of the deconvolved stacks using
ImageJ 1.42q (National Institutes of Health, USA).41
LogP Determination According to Reference 29 and pKa
Determination. LogP Determination by Means of RP HPLC. The
partition coefficients of all compounds were determined by RP-
chromatography using methanol/phosphate buffer 70:30 as an
eluent. The chromatographic systems were calibrated with
solutes for which an experimental octanol/water partition
coefficient was available.37 The capacity factors of the reference
substances were correlated against with experimental octanol/
water logP values, and the obtained correlation equation was
used to calculate logP values of the tested compounds.
Liquid chromatography was conducted on an Agilent 1100 series
system with a C18 reversed-phase Inertsil ODS-2 (MZ-Analytical,
Mainz, Germany) (150 × 4.6 mL, 5 μm) column. The methanol
(LiChrosolv, Merck, Germany)/buffer (70/30) mobile phase was used
at a flow rate of 1.5 mL/min. The phosphate buffer (10 mM) was
prepared by dissolving 1.361 g of potassium dihydrogen phosphate in
Millipore water and adjusting the solution to pH 7.4 with aqueous
sodium hydroxide solution (0.1 M). Then 0.02% N,N-dimethylhexyl-
amine was added to minimize the peak tailing. The following aromatic
compounds were applied for calibration: 2-phenylethanol, benzene,
chlorobenzene, toluene, ethylbenzene, cumene, biphenyl, and
anthracene.
First, 4 mg of the analytes were dissolved in 1 mL of DMSO (ACS
spectrophotometric grade, Sigma-Aldrich, Germany). Then 10 μL of
the DMSO solution were diluted with 990 μL of the mobile phase to a
concentration of 4 μg/mL. Experiments were run in duplicate, and
peak maxima were determined at 254.4 nm. Capacity ratios were
determined as k′ = (tr − to)/to, where tr is the average retention time of
the analyte and to is the average retention time of the solvent. A linear
regression was performed for the logk′/logP data of the reference
compounds (y = 2.3417x + 1.8415; r2 = 0.9803), and the regression
equation was used to calculate the logP of the compounds.
pKa was determined by UV assay using a Sirius T3 (Sirus; Forest
Row, UK). The UV-based approach is particularly suitable for poorly
water-soluble compounds with chromophores within five bond lengths
of the ionizable group. For the experiment, 3 μL of a 10 mM stock of
compound was used for each experiment (n = 6) and diluted with 1.5
mL of 0.15 KCl solution as starting solution and as described
before.38−40 This starting solution was titrated to pH 3 using 0.5 M
HCl and titrated through a pH range of 3−9 using 0.5 M KOH.
Absorbance was read over λ = 200−700 nm. The UV metric data was
confirmed potentiometrically and as described before.40 In brief, about
0.54 mg of compound was dissolved in 1150 μL of DMF. This
solution was filled to 1.5 mL using 0.15 M KCl. The solution was
titrated to pH 2 using 0.5 M HCl and titrated over a pH range of 2−12
thereafter and using 0.5 M KOH. Potentiometric changes were
recorded by a pH electrode. Addition of DMF was required for the
potentiometric approach due to solubility restrictions of compound
29.
Microscopy and Image Analysis. IC50 Determination. IC50
values for ciprofloxacin and compounds 19 and 29 in the
mitochondrion marker cell line were determined for 42 h of
incubation. Each culture was set up in triplicate, and IC50 values
were determined as described above with the exception of cell
densities being determined by counting with a hemocytometer.
Sample Preparation. Organelle marker cell lines for the
mitochondrion, Golgi, ER, endosomes, and lysosome were based on
bloodstream form T. b. brucei 13.90 and cultured in HMI-9 at 37 °C
and 5% CO2. Following incubation with the respective chemical,
dissolved in DMSO, for 42 h at IC50 concentrations or with 1%
DMSO as a control, 5 × 106 to 1 × 107 cells were harvested at 1400g
and 4 °C for 10 min. Cells were either washed twice in trypanosome
dilution buffer (TDB; 5 mM KCl, 80 mM NaCl, 1 mM MgSO4, 20
Knockdown of the Mitochondrial Topoisomerase II. A 500 bp
fragment of the topoisomerase II gene42 was amplified by PCR from
Lister 427 genomic DNA. This fragment was cloned into a p2T7−177
RNAi expression vector43 containing a puromycin cassette for
selection.
First, 3 × 107 cells of the mitochondrion marker cell line were
harvested by centrifugation at 1500g and resuspended in 100 μL of
AMAXA Basic Parasite Solution 1. Then, after addition of 10 μg of the
NotI linearized RNAi vector, transfection was performed with an
AMAXA nucleofector II using program X-001. Immediately after
transfection, cells were transferred to 30 mL of preincubated HMI-9
medium containing 5 μg/mL hygromycin, 2.5 μg/mL G418, and 2.5
μg/mL phleomycin. Serial dilutions of 1:10, 1:100, and 1:1000 were
distributed to 24-well plates. Eight hours post transfection, puromycin
was added to a final concentration of 0.3 μg/mL. Growth of
transformed cultures was first observed 5 days post transfection and
cultures derived from the 1:100 dilution expanded for further analysis.
RNAi was induced by addition of 1 μg/mL tetracycline. Knockdown of
topoisomerase II mRNA was confirmed by Northern blot analysis of
RNA samples prepared from cells prior to and 24 h after induction of
RNAi against topoisomerase II. Total RNA was prepared using the
RNeasy kit (QIAGEN) and RNA samples were denatured with glyoxal
and electrophoresis was performed on a vertical agarose gel. Blotting
occurred by upward capillary transfer. The blot was probed using the
DIG labeling sytem (Roche). DIG labeled DNA probes were
generated for topoisomerase II (primers used for PCR amplification:
5′gcggaggcacacaagtat and 5′acgaggaaaactagaata, yielding a 1025 bp
fragment) and for α-tubulin (primers used for PCR amplification:
5′cacttctatttatttatc and 5′gtcatcatggaggcgggc, yielding a 926 bp
fragment). Hybridization was carried out overnight at 50 °C, and
subsequent washes were performed at 42 °C. Topoisomerase II and
tubulin mRNA, respectively, was detected after incubation with the
chemiluminescence reagent CSPD (Roche).
In Vivo Activity against T. brucei rhodesiense.44 Experiments
in the T. b. rhodesiense (STIB900) acute mouse model were performed
as preciously described,44 with minor modifications. Briefly, female
NMRI mice were infected interperitoneally (ip) with 104 T. b.
rhodesiense bloodstream forms. Groups of four mice were treated
perorally (po) with the compound, dissolved in a aqueous 1:1 dilution
of the formulation (Cremophor RH40, Capmul MCM C8,
triethylcitrate, ethanol), on days 3−6 post infection. A control group
remained untreated. The parasitemia of all animals was checked every
second day up to day 6 postinfection. Death of animals was recorded
to calculate the mean survival time. Surviving and aparasitemic mice
were considered cured at 60 days and then euthanized. All protocols
and procedures used in the current study were reviewed and approved
by the local veterinary authorities of the Canton Basel-Stadt,
Switzerland.
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Journal of Medicinal Chemistry
Article
(10) Lee, S. H.; Stephens, J. L.; Paul, K. S.; Englund, P. T. Fatty acid
synthesis by elongases in trypanosomes. Cell 2006, 126, 691−699.
(11) Bodley, A. L.; Chakraborty, A. K.; Xie, S.; Burri, C.; Shapiro, T.
A. An unusual type IB topoisomerase from African trypanosomes. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 7539−7544.
(12) Nenortas, E.; Burri, C.; Shapiro, T. A. Antitrypanosomal activity
of fluoroquinolones. Antimicrob. Agents Chemother. 1999, 43, 2066−
2068.
ASSOCIATED CONTENT
* Supporting Information
Further syntheses and spectroscopic data of all compounds,
microanalysis, HPLC purities, logP values, and crystallographic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 0931-31-85461. E-mail: holzgrab@pharmazie.uni-
wuerzburg.de.
(13) Bringmann, G.; Hoerr, V.; Holzgrabe, U.; Stich, A.
Antitrypanosomal naphthylisoquinoline alkaloids and related com-
pounds. Pharmazie 2003, 58, 343−346.
■
(14) Nenortas, E.; Kulikowicz, T.; Burri, C.; Shapiro, T. A.
Antitrypanosomal activities of fluoroquinolones with pyrrolidinyl
substitutions. Antimicrob. Agents Chemother. 2003, 47, 3015−3017.
(15) Keiser, J.; Burri, C. Evaluation of quinolone derivatives for
antitrypanosomal activity. Trop. Med. Int. Health 2001, 6, 369−389.
(16) Ma, X.; Zhou, W.; Brun, R. Synthesis, in vitro antitrypanosomal
and antibacterial activity of phenoxy, phenylthio or benzyloxy
substituted quinolones. Bioorg. Med. Chem. Lett. 2009, 19, 986−989.
(17) Gould, R. G.; Jacobs, W. A. The synthesis of certain substituted
quinolines and 5, 6-benzoquinolines. J. Med. Chem. Soc. 1939, 61,
2890−2895.
(18) Shindikar, A. V.; Viswanathan, C. L. Novel fluoroquinolones:
design, synthesis and in vivo activity in mice against Mycobacterium
tuberculosis H37Rv. Bioorg. Med. Chem. Lett. 2005, 15, 1803−1806.
(19) Niedermeier, S.; Singethan, K.; Rohrer, S. G.; Matz, M.;
Kossner, M.; Diederich, S.; Maisner, A.; Schmitz, J.; Hiltensperger, G.;
Baumann, K.; Holzgrabe, U.; Schneider-Schaulies, J. A small-molecule
inhibitor of Nipah virus envelope protein-mediated membrane fusion.
J. Med. Chem. 2009, 52, 4257−4265.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Elena Katzowitsch and Tobias Olschlager (Institute
for Molecular Infection Biology of the University of Wurzburg)
for the cytotoxicity screening, Jennifer Rath and Antje Fuss
(Medical Mission Institute, Wurzburg) for testing the
compounds against T. brucei brucei, and the Deutsche
Forschungsgemeinschaft (SFB 630) for financial support.
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ABBREVIATIONS USED
■
BSF, bloodstream form; DAPI, 4′,6-diamidino-2-phenylindole;
EMME, diethyl 2-(ethoxymethylene)-malonate; ER, endoplas-
mic reticulum; GFP, green fluorescent protein; HAT, human
African trypanosomiasis; NMM, N-methylmorpholine; TDB,
trypanosome dilution buffer; sulfo-NHS-AMCA, sulfosuccini-
midyl-7-amino-4-methylcoumarin-3-acetate; YFP, yellow fluo-
rescent protein
(20) Koga, H.; Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T.
Structure−activity relationships of antibacterial 6,7- and 7,8-
disubstituted 1-a1kyl-1,4-dihydro-4-oxoquinoline-3-carboxylic acids. J.
Med. Chem. 1980, 23, 1358−1363.
(21) Heravi, M. M.; Jaddi, Z. S.; Oskooie, H. A.; Khaleghi, S.;
Ghassemzadeh, M. Regioselective synthesis of quinolone antibacterials
via borate complex of quinolone carboxylic acid. J. Chem. Res. 2005,
578−579.
REFERENCES
■
(1) Cecchi, G.; Paone, M.; Franco, J. R.; Fevre, E. M.; Diarra, A.;
Ruiz, J. A.; Mattioli, R. C.; Simarro, P. P. Towards the Atlas of human
African trypanosomiasis. Int. J. Health Geogr. 2009, 8, 10.1186/1476-
072X-8-15.
(2) African Trypanosomiasis (Sleeping Sickness); World Health
Organization Fact Sheet;World Health Organization: Geneva, 2006.
(3) Hide, G. History of sleeping sickness in East Africa. Clin.
Microbiol. Rev. 1999, 12, 112−125.
(4) RaadtP.The history of sleeping sickness. Fourth International
Cours on African Trypanosomoses, Tunis, Oct 11−28, 2005, World
Health Organization: Geneva, 2005.
(22) Miyaura, M.; Suzuki, A. Palladium-catalyzed cross-coupling
reactions of organoboron compounds. Chem. Rev. 1995, 95, 2457−
2483.
(23) Austin, W. B.; Bilow, N.; Kelleghan, W. J.; Lau, K. S. Y. Facile
synthesis of ethynylated benzoic acid derivatives and aromatic
compounds via ethynyltrimethylsilane. J. Org. Chem. 1981, 46,
2280−2286.
(24) Gilligan, P. J.; Witty, M. J.; McGuirk, P. R. Substituted
dihydroquinolone carboxylic acids, and antibacterial compositions
containing them. Patent EP/184384/19860611, 1986.
̀
(5) Fevre, E. M.; Wissmann, B.; Welburn, S. C.; Lutumba, P. The
burden of human African trypanosomiasis. PLoS Neglected Trop. Dis.
2008, 2 DOI: 10.1371/journal.pntd.0000333.
(25) Vice, S.; Bara, T.; Bauer, A.; Fort, J.; Josien, H.; McCombie, S.;
Miller, M.; Nazareno, A. P.; Tagat, J. Concise formation of 4-benzyl
piperidines and related derivatives using a Suzuki protocol. J. Org.
Chem. 2001, 66, 2487−2492.
(6) Priotto, G.; Kasparian, S.; Mutombo, W.; Ngouama, D.;
Ghorashian, S.; Arnold, U.; Ghabri, S.; Baudin, E.; Buard, V.;
Kazadi-Kyanza, S.; Ilunga, M.; Mutangala, W.; Pohlig, G.; Schmid,
C.; Karunakara, U.; Torreele, E.; Kande, V. Nifurtimox−eflornithine
combination therapy for second-stage African Trypanosoma brucei
gambiense trypanosomiasis: a multicentre, randomised, phase III, non-
inferiority trial. Lancet 2009, 374, 56−64.
(7) Cavalli, A.; Bolognesi, M. L. Neglected tropical diseases: multi-
target-directed ligands in the search for novel lead candidates against
trypanosoma and leishmania. J. Med. Chem. 2009, 52, 7339−7359.
(8) (a) Cavalcanti, D. P.; Fragoso, S. P.; Goldenberg, S.; Souza, W.;
Motta, M. C. M. The effect of topoisomerase II inhibitors on the
kinetoplast ultrastructure. Parasitol. Res. 2004, 94, 439−448.
(b) Kulikowicz, T.; Shapiro, T. A. Distinct genes encode type II
topoisomerases for the nucleus and mitochondrion in the protozoan
parasite Trypanosoma brucei. J. Biol. Chem. 2006, 281, 3048−3056.
(9) Sen, N.; Majumder, H. K. Mitochondrion of protozoan parasite
emerges as potent therapeutic target: exciting drugs are on the
horizon. Curr. Pharm. Dis. 2008, 14, 839−846.
(26) Raz, B.; Iten, M.; Grether-Buhler, Y.; Kaminsky, R.; Brun, R.
̈
̈
The AlamarBlue assay to determine drug sensitivity of African
trypanosomes (T. brucei rhodesiense and T. brucei gambiense) in vitro.
Acta Trop. 1997, 68, 139−147.
(27) Baltz, T.; Baltz, D.; Giroud, C.; Crocket, J. Cultivation in a semi-
defined medium of animal infective forms of Trypanosoma brucei, T.
equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985,
4, 1273−1277.
(28) Ahmed, S. A.; Gogal, R. M.; Walsh, J. E. A new rapid and simple
non-radioactive assay to monitor and determine the proliferation of
lymphocytes: an alternative to [3H]thymidine incorporation assay. J.
Immunol. Methods 1994, 170, 211−224.
(29) Alptuzun, V.; Prinz, M.; Horr, V.; Scheiber, J.; Radacki, K.;
̈
̈
̈
Fallarero, A.; Vuorela, P.; Engels, B.; Braunschweig, H.; Erciyas, E.;
Holzgrabe, U. Interaction of (benzylidene-hydrazono)-1,4-dihydropyr-
idines with β-amyloid, acetylcholine, and butyrylcholine esterases.
Bioorg. Med. Chem. 2010, 18, 2049−2059.
2547
dx.doi.org/10.1021/jm101439s | J. Med. Chem. 2012, 55, 2538−2548

Journal of Medicinal Chemistry
Article
(30) Lipinski, C. A.; Lombard, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3−26.
(31) Tenjarla, S. Microemulsions: an overview and pharmaceutical
applications. Crit. Rev. Ther. Drug Carrier Syst. 1999, 16, 461−521.
(32) Date, A. A.; Nagarsnker, M. S. Parenteral microemulsions: an
overview. Int. J. Pharm. 2008, 355, 19−30.
(33) Benita, S. Submicron Emulsion in Drug Targeting and Delivery;
Harwood Academic Publishers: Amsterdam, 1998.
(34) Wang, Z.; Englund, P. T. RNA interference of a trypanosome
topoisomerase II causes progressive loss of mitochondrial DNA.
EMBO J. 2001, 20, 4674−4683.
(35) Lindsay, M. E.; Gluenz, E.; Gull, K.; Englund, P. T. A new
function of Trypanosoma brucei mitochondrial topoisomerase II is to
maintain kinetoplast DNA network topology. Mol. Microbiol. 2008, 70,
1465−1476.
(36) Wohlkonig, A.; Chan, P. F.; Fosberry, A. P.; Homes, P.; Huang,
J.; Kranz, M.; Leydon, V. R.; Miles, T. J.; Pearson, N. D.; Perera, R. L.;
Shillings, A. J.; Gwynn, M. N.; Bax, B. D. Structural basis of quinolone
inhibition of type IIA topoisomerases and target-mediated resistance.
Nature Struct. Mol. Biol. 2010, 17, 1152−1153.
(37) Hansch, C.; Leo, A.; Hoekman, D. Exploring QSAR: Volume 2:
Hydrophobic, Electronic and Steric Constants; American Chemical
Society: Waschington DC, 1995.
(38) Clarke, J.; Cunliffe, A. E. Rapid spectrophotometric measure-
ment of ionisation constants in aqueous solution. Chem. Ind. (London)
1973, 281.
(39) Albert, A.; Serjeant, E. P. Ionisation constants of acids and bases.
Wiley Inc.: New York, 1962.
(40) Box, K.; Comer, J. E.; Gravestock, T.; Stuart, M. New ideas
about the solubility of drugs. Chem. Biodiversity 2009, 6, 1767−1788.
(41) Rasband, W. S. ImageJ; National Institutes of Health: Bethesda,
MD, 1997−2008; http://rsb.info.nih.gov/ij/.
(42) Strauss, P. R.; Wang, J. C. The TOP2 gene of Trypanosoma
brucei: a single-copy gene that shares extensive homology with other
TOP2 genes encoding eukaryotic DNA topoisomerase II. Mol.
Biochem. Parasitol. 1990, 38, 141−150.
(43) Wickstead, B.; Ersfeld, K.; Gull, K. Targeting of a tetracycline-
inducible expression system to the transcriptionally silent mini-
chromosomes of Trypanosoma brucei. Mol. Biochem. Parasitol. 2002,
125, 211−216.
(44) Kaiser, M.; Bray, M. A.; Cal, M.; Bourdin Trunz, B.; Torreele,
E.; Brun, R. Antitrypanosomal activity of fexinidazole, a new oral
nitroimidazole drug candidate for treatment of sleeping sickness.
Antimicrob. Agents Chemother. 2011, 55 (12), 5602−5608.
2548
dx.doi.org/10.1021/jm101439s | J. Med. Chem. 2012, 55, 2538−2548