Antiprotozoal activity and DNA binding of dicationic acridones

Article abstract of DOI:10.1021/jm5018303

Dicationic acridone derivatives were synthesized and their antiparasitic activity was evaluated. Acridones displayed in vitro nanomolar IC₅₀ values against Trypanosoma brucei rhodesiense STIB900 with selectivity indices >1000. Compounds 1b, 3a,

Full text of DOI:10.1021/jm5018303

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Article
Antiprotozoal Activity and DNA Binding of Dicationic Acridones
Sandra Montalvo-Quirós, Andrea Taladriz-Sender, Marcel Kaiser, and Christophe Dardonville
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Feb 2015
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Antiprotozoal Activity and DNA Binding of
Dicationic Acridones
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Sandra Montalvo–Quirósa, Andrea Taladriz–Sendera§, Marcel Kaiserb,c and Christophe
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Dardonvillea*
a
Instituto de Química Médica, IQM–CSIC, Juan de la Cierva 3, E–28006 Madrid,
Spain.
b
Swiss Tropical and Public Health Institute, Socinstrasse, 57, CHꢀ4002 Basel,
Switzerland.
c University of Basel, Petersplatz 1, CHꢀ4003 Basel, Switzerland.
*
Corresponding author. Tel.: +34 912587490; Fax.: +34 915644853; eꢀmail:
dardonville@iqm.csic.es
§ Current address: Instituto de Química Orgánica General, IQOG–CSIC.
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Abstract
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Dicationic acridone derivatives were synthesized and their antiparasitic activity was
evaluated. Acridones displayed in vitro nanomolar IC50s against Trypanosoma brucei
rhodesiense STIB900 with selectivity indices >1000. Compounds 1b, 3a, and 3b were
as potent as the reference drug melarsoprol in this assay. Submicromolar range activities
were observed against wild type (NF54) and resistant (K1) strains of Plasmodium
falciparum whereas no significant activity was detected against Trypanosoma cruzi or
Leishmania donovani. Compounds 1a and 1b were curative in the STIB900 mouse
model for human African trypanosomiasis. UV spectrophotometric titrations and
circular dichroism (CD) experiments with fish sperm (FS)ꢀDNA showed that these
compounds form complexes with DNA with binding affinities in the 104 Mꢀ1 range. The
biological and biophysical data show that antiparasitic activity, toxicity, and DNA
binding of this series of acridones are dependent on the relative position of both
imidazolinium cations on the heterocyclic scaffold.
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Keywords: Trypanosoma brucei, Trypanosoma cruzi, Plasmodium falciparum,
Leishmania, sleeping sickness, malaria, antiprotozoal chemotherapy, imidazoline,
acridone, anthraquinone, UV titration, circular dichroism, DNA binder, intercalating
agent.
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Introduction
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Available chemotherapy for parasitic tropical diseases such as African and American
trypanosomiasis, leishmaniasis, and malaria is not satisfactory in many aspects; toxicity,
inefficiency against resistant strains, a parenteral mode of administration and
unaffordable price for lowꢀincome affected countries are all critical factors that
seriously limit the use of these drugs.1
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In the last years, our group has discovered very potent antitrypanosomal compounds
that cured murine models of acute sleeping sickness.2, 3 In addition, some of these
compounds were also very active in vitro against Plasmodium falciparum even though
this antimalarial activity was not always confirmed in vivo in the P. berghei mouse
model.3 These dicationic compounds (I–VIII, Figure 1), which contain a diphenyl
scaffold and two 2ꢀaminoimidazolinium moieties, bear a relatively short linker between
both phenyl rings and share structural characteristics (e.g. diphenyl dicationic
compound, short semiꢀrigid linker, crescent shape) with other related classes of
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antiprotozoal dicationic compounds such as the diamidines (Figure 1).4, In fact,
similarly to diamidines the bisꢀ2ꢀaminoimidazolines are very effective and selective
DNA minor groove binders.2, 3, 6, 7 Binding to kinetoplast DNA (kꢀDNA) has been
suggested to be in part responsible for the observed antitrypanosomal activity of the
diamidine class of compounds either by inhibition of topoisomerase II or by direct
inhibition of transcription.8, 9
Previous studies have shown that the Nꢀphenylbenzamide scaffold (I, Figure 1) gives
the most potent antitrypanosomal compounds in vivo3, 10 as well as the best ATꢀ
selective DNA minor groove binders.11, 12 In this work, we wanted to test whether
conformational restriction of the scaffold of the bisimidazoline compounds would have
an influence on their antiparasitic activity and/or DNA binding affinity. The acridone
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heterocycle was chosen as surrogate of the Nꢀphenylbenzamide linker between both
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cationic moieties as similar fused heterocyclic structures (e.g. acridone,13,
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anthraquinone,15 dihydroanthracene,3 9Hꢀfluorene,3 9Hꢀfluorenꢀ9ꢀone16) have
demonstrated antiprotozoal activities against trichomonads, trypanosomes and
Plasmodium species. The effect on antiparasitic activity and DNA binding of Nꢀ10
methylation and NH ↔ CO replacement was also studied (1a–c, 2b and 3a–c, Figure 1).
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Figure 1. Dicationic Diphenyl Compounds of the Diamidine and Bisꢀ2ꢀ
aminoimidazoline Classes. Example of Conformational Restriction Leading to New
Active Antiparasitic Compounds (compounds 1, 2, and 3).
In this study, the compounds were screened in vitro against a panel of parasites
(Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, and Plasmodium
falciparum). Their DNA binding affinity and binding mode were studied by UV
spectroscopy and circular dichroism (CD) experiments with fish sperm (FS) DNA.
Preliminary in vivo studies in mouse models of human African trypanosomiasis and
malaria.were also carried out with four molecules. The biological and biophysical data
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gathered in this work confirmed the high antiprotozoal potential of this series of
dicationic acridones and the importance of the substitution pattern on the observed
antiprotozoal activity and DNA binding properties of the compounds.
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Results
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Chemistry. The dicationic target compounds 1a–c, and 2b were synthesized in two
steps from the diamines 4a–c and 5b, respectively (Scheme 1). Diamines 4a and 4b
were synthesized in three steps from 1ꢀchloroꢀ4ꢀnitrobenzoic acid whereas 4c was
prepared in four steps from diphenylmethane (Scheme 2).17 2,6ꢀDiaminoanthraquinone
(5b) was commercially available. An excess of the classical N,N′ꢀdi(tertꢀ
butoxycarbonyl)imidazolidineꢀ2ꢀthione reagent was used to introduce the Bocꢀprotected
imidazoline ring as previously reported.18 The isolated Bocꢀprotected 2ꢀ
aminoimidazoline compounds 6a–c and 7b were treated with TFA to yield the
trifluoroacetate salts of the target compounds. For in vivo assays, the pharmacologically
acceptable dihydrochloride salts of 1a–c and 2b were prepared in good yield with
strongly basic anion exchange resin (Clꢀ form). Nꢀmethylation of acridones 6a–c with
dimethylsulfate followed by acidic hydrolysis of the Bocꢀprotecting groups yielded the
Nꢀmethyl acridones 3a–c as trifluoroacetate salts.
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Scheme 1.a Synthesis of the Target Compounds
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S
O
9
O
X
BocN
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1
4
Boc
N
Boc
N
NBoc
7
2
3
NH2
H2N
N
N
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6
N
i
N
X
5
Boc
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Boc
4a: X = NH; 2,7-diamine
4b: X = NH; 2,6-diamine
4c: X = NH; 3,6-diamine
5b: X = CO, 2,6-diamine
6a: X = NH (52%)
6b: X = NH (26%)
6c: X = NH (32%)
7b: X = CO (10%)
iv
O
O
R
N
R
N
H
N
H
N
N
N
N
N
H
. 2Cl-
N
R
H
N
R
N
N
H
N
X
H
CH3
1a: X = NH, 2,7-disubstitution (77%)
1b: X = NH, 2,6-disubstitution (70%)
1c: X = NH, 3,6-disubstitution (70%)
2b: X = CO, 2,6-disubstitution (85%)
R = Boc
8a (64%)
8b (37%)
8c (48%)
R = H (TFA salt)
3a (87%)
ii
3b (93%)
3c (71%)
a
Reagents and conditions: (i) HgCl2, Et3N, DMF, 0 ºC→ rt; (ii) TFA, CH2Cl2; (iii)
strongly basic anion exchange resin (Clꢀ form), H2O, THF; (iv) Me2SO4, K2CO3, THF,
∆
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Scheme 2.a Synthesis of the Starting Material Diamines
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a
Reagents and conditions. (a) 1,4ꢀphenylenediamine or 1,3ꢀphenylenediamine,
K2CO3, Cu, CuSO4 cat., H2O, reflux; (b) Polyphosphoric acid, 100 ºC; (c) Na2S•9
H2O, NaOH, EtOH, H2O, reflux; (d) H2SO4 conc., 100 ºC; (e) KNO3, H2SO4, 15
ºC→70 ºC; (f) CrO3, AcOH, reflux; (g) HCl conc., SnCl2, 90–100 ºC; (h) EtOH,
HCl conc., 100 ºC; (i) 20 % aqueous NaOH, reflux.
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In Vitro Antiprotozoal Activity. The target compounds as well as seven synthetic
intermediates were screened in vitro against T. brucei, T. cruzi, L. donovani, and P.
falciparum. Cytotoxicity against rat L6ꢀcells was also evaluated in order to calculate the
selectivity index of the compounds (Table 1). None of the compound tested had
significant activity against T. cruzi or L. donovani amastigotes. The target acridones 1a–
1c displayed low nanomolar activities against bloodstream trypanomastigotes of T. b.
rhodesiense and excellent selectivities for the parasite (SI > 2000). Among these, the
most active was the 2,6ꢀdisubstituted compound 1b (IC50 = 7 nM) which was equipotent
to the reference drug melarsoprol and approximately 3ꢀtimes more potent than the Nꢀ
phenylbenzamide linked compound I (Figure 1, IC50 = 25 nM).3 In contrast, the 2,6ꢀ
disubstituted anthraquinone analogue 2b was 92ꢀ and 328ꢀfold less active than I and 1b,
respectively, indicating that NH ↔ CO replacement is not tolerated. Interestingly,
methylation at Nꢀ10 did not affect the activity for the 2,6ꢀ (3b) and 3,6ꢀ (3c)
disubstituted acridones whereas it did increase by 10ꢀfold the antiꢀT. brucei effect of the
2,7ꢀdisubstituted compound (3a). Hence, in this series the in vitro antitrypanosomal
activity profile was dependent on the substitution pattern of the acridone scaffold, with
2,6ꢀdisubstitution >> 3,6 > 2,7 for Nꢀ10(H) acridone derivatives 1a–c, and 2,7 ≈ 2,6 >
3,6 for Nꢀmethylated analogues 3a–c.
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In general, all of these compounds were less active against P. falciparum with
submicromolar IC50s except the anthraquinone derivative 2b that displayed a reversed
activity profile with nanomolar IC50 against P. falciparum (61 nM) and micromolar IC50
against T. brucei (2.29 µM). The aromatic amines 4a, 4b, 10 and 12 lacking the
imidazolinium moieties were mostly inactive against the parasites tested which
confirms that the presence of two cations is necessary to get nanomolar antiparasitic
activities.
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Table 1. In Vitro Antiprotozoal Activity of the Target Compounds and Synthetic
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5
Intermediatesº
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7
8
9
Cytotox.f
T. b. rhod.a
T. cruzib L. don.c
P. falc.
Compound
K1d
NF54e
IC50 (Selectivity index)g µM
CC50 (µM)
75
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2.9
19.2
2.2
149
269
50
>230
>230
>230
104
nd
321
288
>127
177
73
113
145
nd
2.5
4.8
33
ndh
nd
nd
nd
4a
4b
6a
1a
23
73
0.069 (>3333)
0.007 (15257)
0.062 (2210)
2.3
0.007 (14843)
0.008 (12287)
0.060 (1647)
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0.34 (>666)
>230
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137
75
104
98
nd
nd
0.83 (128)
0.25 (546)
nd
1b
1c
2bi
0.06 (1229)
3ai
nd
nd
nd
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49
70
34
0.22 (468)
0.56 (175)
0.09 (1149)
nd
3bi
nd
nd
3ci
nd
nd
99
292
287
271
279
23
77
61
291
>365
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266
147
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11
19.5
124
67
0.005
nd
nd
nd
12
melarsoprol
benznidazole
miltefosine
chloroquine
1.7
0.45
0.13
0.012
podophyllotoxin
0.012
a
b
T. b. rhodesiense STIB900 trypomastigotes. T. cruzi Tulahuen C4 intracellular
c
d
amastigotes. L. donovani MHOMꢀETꢀ67/L82 axenic amastigotes. P. falciparum
e
intraerythrocytic stage: the K1 strain is resistant to chloroquine and pyrimethamine.
The NF54 strain is sensitive to all known antimalarials. f Rat skeletal myoblast L6ꢀcells.
g SI = [CC50 /IC50 (parasite)]. h Not determined. i Trifluoroacetate salt.
In Vivo Antitrypanosomal and Antimalarial Activity. A preliminary evaluation of
the in vivo potential of these dicationic acridones in the T. b. rhodesiense STIB900
mouse model was carried out with the three nonꢀmethylated regioisomers 1a–1c. This
model which reproduces the acute rhodesiense infection is quite difficult to cure and
represents a stringent model of first stage sleeping sickness. In this assay, mice were
treated intraperitoneally with the compounds for 4 consecutive days and parasitemia
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was monitored over 60 days. Mice that survived 60 days free of parasites were
considered as cured. The intraperitoneal administration of 20 mg/kg of the compounds
was curative for 1a (2/4 mice) and 1b (3/4 mice) whereas 1c showed acute toxicity at
this dose. At lower nonꢀtoxic dosage (5 mg/kg ip) 1c was not curative and increased
only moderately the mean time of relapse of parasitemia. Compound 1a was also tested
by oral dosage (50 mg/kg) showing a moderate effect although no cures were obtained
(Table 2). This indicates that the oral bioavailability of this class of dicationic acridones
is modest even though there is room for improvement.
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Compound 2b which was the best antimalarial compound from the in vitro screening
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was tested in the P. berghei mouse model by intraperitoneal administration.19,
Unfortunately, this compound was inactive on a four days treatment at 20 mg/kg/day
while higher dosage (50 mg/kg) was not tolerated.
Table 2. In Vivo Activity in the STIB900 Mouse Model of Stage 1 Sleeping Sickness.
Dosage
Dosage
(mg/kg)
ꢀ
4 × 20
4 × 50
4 × 20
4 × 20
4 × 5
b
Mean day
of relapse
7c
> 45.5
22
Compound
Curedb/Infected
routea
Control
1a
ꢀ
0/4
2/4
0/4
3/4
Td
ip
po
ip
1b
> 50
Td
ip
1c
0/4
14
a
ip = intraperitoneal, po = per os (oral); Number of mice that are alive and parasite
free after 60 days; c All mice were positive and euthanized at day 7. d Toxic after second
administration.
DNA Binding Studies. Since diphenyl bisꢀ2ꢀaminoimidazolines are DNA minor groove
3, 6, 11
binders2,
and acridone and anthraquinone derivatives generally bind by
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intercalation, we were interested in evaluating the binding affinity of the new
compounds with DNA quantitatively.
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Spectrophotometric Titrations. All of these acridone compounds have strong
absorption bands in the 250–300 nm range and weaker absorption bands above 300 nm.
The bands over 300 nm, which do not overlap with the DNA absorbance at 260 nm,
were useful for the spectrophotometric titrations. UVꢀvis spectra of compounds 1a-c,
2b, and 3a-c were recorded in the 250–500 nm wavelength range in the presence of
increasing amounts of FSꢀDNA. Addition of FSꢀDNA induced strong hypochromicity
(approximately 46–49% decrease in extinction coefficient at the compound peaks
wavelengths) and large bathochromic shifts for 1a, 2b, and 3a indicating the formation
of a complex with DNA (Figure 2 and Table 3). It is known that these effects are
generally more pronounced for intercalators than for groove binders.21ꢀ24 Hence, the
results may indicate a prevailing intercalative binding mode in FSꢀDNA for 1a, 2b, and
3a. In contrast, addition of FSꢀDNA to 1b, 1c, 3b, and 3c led to high hypochromicities
and weak bathochromic and hypsochromic shifts, respectively, indicating that binding
to DNA occurred, albeit in a different way. These results may indicate a mixed
intercalative and groove binding mode in FSꢀDNA. Apparently, these dicationic
compounds have different binding behaviours which appear to be dependent on the
respective position of the imidazoline rings on the acridone scaffold and the presence of
a methyl substituent at Nꢀ10 of the acridone ring. The binding constants calculated from
Scatchard plot analysis of the titration experiments using the (non–co–operative)
neighbour exclusion model of McGhee and von Hippel25 were in the range 9.6 – 31.4 ×
104 Mꢀ1 (Table 3) which is consistent with binding affinities found for other
intercalators.
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1a
3a
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0.05
0.00
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0.05
0.00
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Wavelength (nm)
Wavelength (nm)
1b
3b
0.20
0.15
0.10
0.05
0.00
0.20
406
408
0.15
0.10
0.05
0.00
332
332
388
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
1c
3c
0.3
0.3
0.2
0.1
0.0
333
335
334
333
380
388
404
387
0.2
0.1
0.0
381
397
399
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
2b
0.5
0.4
0.3
0.2
0.1
0.0
343
352
300
350
400
450
500
Wavelength (nm)
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Figure 2. Spectrophotometric titration of compounds 1a-c, 2b, and 3a-c (20 µM) with
FSꢀDNA in 10 mM phosphate buffer (pH 7) at 25 ºC. FSꢀDNA concentrations ranged
from 0 to 2.08 × 10ꢀ4 M (1a), 0 to 1.08 × 10ꢀ4 M (1b), 0 to 1.2 × 10ꢀ4 M (1c), 0 to 1.85 ×
10ꢀ4 M (2b), 0 to 3.47 × 10ꢀ4 M (3a), 0 to 2.28 × 10ꢀ4 M (3b), and 0 to 1.58 × 10ꢀ4 M
(3c). The arrows indicate increasing concentration of DNA.
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Table 3. Binding Constants and UVꢀvis Absorption Data Determined by UVꢀ
Spectrophotometric Titrations with FSꢀDNA
Cmpd λmax free λmax bound
ꢁλ Hypochromicitya
K ×104
(Mꢀ1) (n)b
(nm)
399
332
388
333
380
397
343
411
406
335
388
404
(nm)
407
332
ndc
(nm)
+8
0
(%)
49
47
nd
41
47
40
46
48
50
35
46
41
13.1 ± 1.0 (0.6–0.7)
1a
1b
31.4 ± 0.1 (1.2–1.7)
nd
+1
+1
+2
+9
+5
+2
ꢀ2
334
381
399
352
416
408
333
387
404
26.5 ± 3.2 (0.5–1.1)
1c
9.6 ± 0.5 (1.9–2.3)
25.0 ± 4.9 (0.5–1.0)
11.9 ± 0.4 (1.3–2.1)
2b
3a
3b
ꢀ1
17.5 ± 0.9 (2.3–2.4)
3c
0
a H = [(Absorbance cmpdfree ꢀ Absorbance complex) / Abs. cmpdfree]×100. b Fitting with
the McGhee & von Hippel neighbour exclusion model.25 The neighbour exclusion
c
parameter “n” (expressed in bp) is indicated between brackets. Not determined due to
overlapping bands.
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Circular Dichroism (CD) Spectroscopy. CD spectroscopy can provide useful
information on the mode of binding of achiral ligands to DNA. An induced CD (ICD)
signal observed in the presence of DNA indicates ligandꢀDNA interaction, the intensity
and sign of which depend on the binding mode. In general, minor groove BꢀDNA
binders with a transition moment oriented along the groove show strong positive ICD.
In contrast, intercalators display small ICD signals, the sign of which depends on the
orientation of the ligand transition moment, either parallel (positive ICD) or
perpendicular (negative ICD) to the dyad axis of DNA.26
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The binding mode of four representative compounds (1b, 1c, 2b, and 3c) with FSꢀDNA
was studied by CD spectroscopy in the wavelength range 230–500 nm. In these
experiments, the concentration of DNA was kept constant (30 µM) and different
ligand/DNA mixing ratios, r, were studied (Figure 3). These achiral compounds had no
CD signal when free in solution but all of them induced an increase in intensity of the
band at 275 nm when mixed with FSꢀDNA. However, it is difficult to say if this signal,
which ovelaps with the DNA band (λmax = 278 nm), was due to an ICD band of the
ligand or to a conformational change of the DNA.
The CD spectrum of 1b in presence of DNA showed a weak negative ICD band at 300
nm, the intensity of which augmented with increasing mixing ratio. A clear isoelliptic
point was also observed at 292 nm in the whole range of drug/DNA ratios indicating a
single dominant binding mode. This may be indicative of an intercalativeꢀbinding mode.
CD spectra of 1c showed variation depending on the binding ratio. At low ratio (0.5–
0.6) a positive band of unvarying intensity overlapping with the DNA band was
observed at 275 nm. At high drug/DNA ratio (1–2), the intensity of the positive band
decreased whereas a negative ICD band at 297 nm appeared gradually upon increasing
r. Red shift for r ≥ 1 and deviation of the isoelliptic point for all different ratio were also
observed possibly indicating heterogenous mode of binding (e.g. groove binding and
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intercalation). Even though the CD spectras of 1c and 3c showed similar profiles some
difference were apparent indicating similar but not identical modes of binding to FSꢀ
DNA. At drug/DNA ratio of 0.5–1, 3c showed a weak negative band at 297 nm with an
isoelliptic point at 286 nm, indicative of one dominant binding mode. At higher ratio (r
= 1.5) the negative band at 297 nm was red shifted and more pronounced, albeit less
intense than for 1c at the same binding ratio. These results are consistent with the
presence of mixed binding modes in DNA at higher r.
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CD spectra of the DNA–anthraquinone 2b complex were quite different from that
observed with acridones 1b, 1c, and 3c. No ICD signal was observed at r = 0.5–0.6
whereas a positive ICD signal at approximately 280 nm (overlapping with DNA band)
and a weak negative ICD at 380 nm appeared upon increment of r to 1 and 1.5. This
may indicate intercalation or external stacking at high compound to DNA concentration.
Altogether, these results are consistent with a prevailing intercalation mode of binding
even though other types of binding interaction (e.g. groove binding, electrostatic)
cannot be ruled out at this point.
In summary, UV titrations and CD results showed that these compounds bind to DNA
and suggest the existence of different binding modes (intercalation and/or minor
groove) depending on the structure and the compound to DNA ratio. Similar results
were described with the diamidine drug DAPI, an intercalator that also has very
favorable interaction in the minor groove at AT sequences.21, 23, 27, 28 Recent findings by
Nagle et al.29 with a series of rigid core (fluorene and dihydroanthracene) bisꢀ2ꢀ
aminoimidazolinium derivatives also support our observations that the relative position
of both cations influences the binding mode of these dicationic acridones. Further work
with specific oligonucleotides will be needed to elucidate the precise mode of binding to
DNA and sequence specificity of these dicationic acridones.
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1b
4
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8
9
DNA 30 ꢀM
r = 0.5
r = 0.6
r = 1
5
0
r = 1.5
r = 2
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-5
250
300
350
400
Wavelength (nm)
1c
10
DNA 30 ꢀM
r = 0.5
r = 0.6
r = 1
5
0
r = 1.5
r = 2
-5
-10
250
300
350
400
Wavelength (nm)
3c
10
5
DNA 30 ꢀM
r = 0.5
r = 0.6
r = 1
0
r = 1.5
-5
-10
250
300
350
400
Wavelength (nm)
2b
20
10
0
DNA 30 ꢀM
r = 0.5
r = 0.6
r = 1
r = 1.5
-10
300
400
500
Wavelength (nm)
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Figure 3. CD spectra of 1b, 1c, 3c, and 2b with FSꢀDNA (30 µM) at different mixing
ratio (r = 0.5, 0.6, 1.0, 1.5, 2.0). The spectra were recorded at 20 ºC in PBS containing
6% DMSO (v/v).
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Discussion
Dicationic bisimidazoline derivatives of the Nꢀphenylbenzamide scaffold have shown
great promise as antitrypanosomal agents and DNA minor groove binders.3 In this
study, we have tested the effect on antiprotozoal activity and DNA binding affinity of
conformational restriction of the Nꢀphenylbenzamide scaffold. The influence of the
relative position of both imidazolinium cations on the heterocyclic scaffold was also
studied. On the one hand, the conformational restriction produced by the introduction of
the acridone scaffold led to an increase in trypanocidal activity (about 3ꢀfold compared
to the Nꢀphenylbenzamide derivative I) for three compounds (1b, 3a, and 3b). In fact,
these acridones derivatives which have low nanomolar in vitro activity (< 10 nM)
against T. b. rhodesiense STIB900 and outstanding selectivity (> 12000) represent, with
the fluorene derivative reported before,3 the most potent class of bis(2ꢀ
aminoimidazolines) synthesized up to now. These IC50 values are comparable to that of
the reference drugs melarsoprol and pentamidine in this assay.30 On the other hand, the
substitution of the acridone by an anthraquinone heterocycle led to a drastic loss of
activity against T. brucei (100ꢀfold) but hardly affected the antiplasmodial activity (IC50
= 61 nM vs 28 nM for I). This CO ↔ NH replacement also had a dramatic effect on the
acute toxicity in vivo (e.g. the 2,6ꢀanthraquinone derivative 2b was toxic after the third
application at 50 mg/kg ip). Hence, the presence of nitrogen (Nꢀ10) in the scaffold
structure was favorable for high level antiꢀT. brucei action. Noteworthy, methylation at
Nꢀ10 did not modify significantly the antiꢀT. brucei activity and cytotoxicity except for
3a the activity and cytotoxicity of which was increased 10ꢀ and 2ꢀtimes, respectively.
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Remarkably, the relative disposition of both imidazoline cations with respect to the
heterocyclic scaffold was critical to the observed in vitro and in vivo activities. Clearly,
the acridone scaffold with a 2,6ꢀdisubstitution pattern gave the best results against T.
brucei in vitro (1b and 3b) and in vivo (1b). In contrast, the 2,6ꢀdisubstituted
anthraquinone derivative 2b moderately affected T. brucei growth whereas it showed a
pronounced in vitro activity against the P. falciparum chloroquineꢀresistant strain K1.
This result is consistent with a previous report by Winkelmann and Raether15 which
showed that 2,6ꢀdiguanidine anthraquinones have no effect on T. brucei. In this series,
the 3,6ꢀdisubstitution pattern was clearly the worst in vivo as it provoked acute toxicity
at low dosage (20 mg/kg). As preliminary screen, one compound, 1a, was tested by the
oral route in the STIB900 mouse model. Even though no cures were obtained, a
significant increase in the mean day of relapse of parasitemia was observed (22 vs 7
days for control mice) indicating weak oral bioavailability. Further work will be
necessary to determine whether other compounds of this series have better oral
bioavailability or if this property can be improved by structure modification.
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The bis(2ꢀaminoimidazoline) class of molecules binds strongly to the minor groove of
ATꢀrich DNA.2, 3, 6, 7, 11, 31, 32 Since dicationic compounds can accumulate to high levels
within the mitochondrion of T. brucei driven electrostatically by the negatively charged
environment of the inner mitochondrial matrix,33 this interaction with DNA could well
be the basis for their antitrypanosomal activity as ATꢀcontent of T. brucei kDNA is
approximately 72%.34 Based on the planar tricyclic structure of these derivatives, an
intercalation mode of binding was expected for the dicationic acridones and
anthraquinone analogues reported here. This mode of binding has been observed
previously with related 3,6ꢀbis(3ꢀalkylguanidino)acridines.35 In general, the effect of
nitrogen methylation at N–10 on DNA binding affinity was small indicating that
N(10)H does not contribute much to DNA binding. This is consistent with a prevalent
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intercalative mode of binding as the Nꢀmethyl group is coꢀplanar to the flat acridone
ring and, as such, should not have major influence on the intercalation between adjacent
base pairs. Our data support the view that the 3,6ꢀdisubstituted acridones 1c and 3c have
a different (e.g. minor groove) mode of binding to DNA compared to 2,6ꢀ and 2,7ꢀ
disubstituted acridones. In fact, 3,6ꢀdisubstituted acridones reported here have a
crescent shape similar to other dicationic minor groove binders such as 3,6ꢀ and 2,7ꢀ
bisimidazoline carbazoles,36 furamidine37, or VI7 (Figure 1) that match the groove
curvature and can fit into the minor groove at ATꢀrich sequences.
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Conclusions
We have shown here that the conformational restriction of the Nꢀphenylbenzamide
scaffold by transformation into an acridone heterocycle led to dicationic bisimidazoline
compounds with potent in vitro activity against T. brucei and P. falciparum. Compound
1b in particular was as active as melarsoprol in vitro and also curative in the STIB900
mouse model of stage 1 HAT. Clearly, the antiprotozoal activity depends on the relative
position of both imidazolinium cations on the fusedꢀheterocyclic scaffold, the 2,6ꢀ
disubtitution pattern giving the best results against T. brucei. Finally, we have shown
that these compounds bind to DNA with binding constants in the 104 Mꢀ1 range. The
substituents’ (cations) relative position also influenced the DNA binding mode even
though intercalation seemed to be the prevailing one. Since no apparent correlation was
observed between DNA binding affinity and in vitro activity against T. brucei, we
conclude that additional mechanisms of action may be involved in the antitrypanosomal
action of these dicationic acridones.
Experimental section
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Chemistry. All dry solvents and reagents were purchased from Aldrich or Fluka in
Sure/Seal bottles. All reactions requiring anhydrous conditions or an inert atmosphere
were performed under a positive pressure of N2. All reactions were monitored by
HPLC–MS or Thin Layer Chromatography (TLC) using silica gel 60 F254 plates
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1
(Merck). Chromatography was performed with Isolute SI prepacked columns. H and
13C NMR spectra were recorded on a Bruker Advance 300 or Varian Inova 500
1
spectrometer. Chemical shifts of the H NMR spectra were internally referenced to
TMS (δ 0 ppm) for CDCl3 or the residual proton resonance of the deuterated solvents:
D2O (δ 4.79 ppm), CD3OD (δ 3.31 ppm) and DMSOꢀd6 (δ 2.50 ppm). Signal splitting
patterns are described as: singlet (s), broad singlet (br s), doublet (d), triplet (t),
quadruplet (q), multiplet (m), or combination thereof. J values are given in Hz. Melting
points were determined in open capillary tubes with a SMP3–Stuart Scientific apparatus
or Mettler Toledo MP70 melting point system, and are uncorrected. All compounds are
>95% pure by HPLC or combustion analysis otherwise noted. Elemental analysis was
performed on a Heraeus CHN–O Rapid analyser. Analytical results were within ± 0.4 %
of the theoretical values unless otherwise noted. Analytical HPLC–MS was run with an
Xbridge C18–3.5 ꢀm (2.1×100 mm) column on a Waters 2695 separation module
coupled with a Waters Micromass ZQ spectrometer using electrospray ionization
(ESI+). The following HPLC conditions were used: column temperature = 30 ºC,
gradient time = 5 min, H2O/CH3CN (10:90 → 90:10) (HCO2H 0.1 %), flow rate = 1
mL/min, UV detection: photodiode array (λ = 190−400 nm). Accurate mass were
measured with an Agilent Technologies Q–TOF 6520 spectrometer using electrospray
ionization.
The starting material diamines (4a–c) were synthesized according to the procedures
described in a patent by Neidle et al.17 The synthesis and characterization of 4a–c and
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the Bocꢀprotected imidazoline intermediates (6a–c, 7b, and 8a–c) is given as
4
5
Supporting Information.
6
7
8
General Procedure for the Removal of the Boc-protecting Groups and
Regeneration of the Hydrochloride Salts. The Bocꢀprotected compound (6a, 6b, 6c,
or 7b) dissolved in CH2Cl2 (2 mL) was treated with TFA (2 mL). The clear solution was
stirred at room temperature for 5 h. The volatiles were removed under vacuum and Et2O
was added to the flask. The crude residue was triturated with a spatula to yield the TFA
salt of the product. The etheral phase was discarded and the solid dissolved in 30 mL of
water (for 1a, 1c, 2b) was stirred overnight with Amberlyte IRA400 strongly basic
anion exchange resin (Clꢀ form) [Note that for 1b, 1 mL of THF was added as coꢀ
solvent to dissolve the TFA salt before adding the resin]. The resin was filtered off and
washed with water. The aqueous filtrate was extracted successively with CH2Cl2 (10
mL) and EtOAc (10 mL), and filtered through a flutted paper. Rotatory evaporation of
the water yielded the dihydrochloride salt of the compound as a solid.
9
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Dihydrochloride salt of 2,7-bis((4,5-dihydro-1H-imidazol-2-yl)amino)acridin-
9(10H)-one (1a). Starting from 6a (111.5 mg) and following the general procedure, the
dihydrochloride salt of 1a was obtained as a yellow solid (49 mg, 77%). HPLC > 95%;
1
mp > 250 ºC (decomp.). H NMR (300 MHz, D2O) δ 8.19 (br s, 2H, ArH), 7.73 (br s,
4H, ArH), 3.88 (s, 8H, CH2CH2). 13C NMR (75 MHz, D2O + CD3OD) δ 178.6, 159.5,
139.6, 131.2, 130.5, 130.4, 120.3, 120.2, 43.8. LRMS (ES+) m/z 362 (M+H)+, 182
[(M+2H)2+, 100%]. ESI–HRMS m/z 362.1745 [M+H]+ (C19H20N7O requires 362.1724).
Dihydrochloride salt of 2,6-bis((4,5-dihydro-1H-imidazol-2-yl)amino)acridin-
9(10H)-one (1b). Starting from 6b (244 mg) and following the general procedure, the
dihydrochloride salt of 1b was obtained as a greenish solid (97 mg, 70%). HPLC >
1
95%; mp > 350 ºC. H NMR (300 MHz, DMSO) δ 12.37 (s, 1H, NH), 11.31 (s, 1H,
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NH), 10.64 (s, 1H, NH), 8.70 (s, 2H, NH), 8.37 (s, 2H, NH), 8.25 (d, J = 8.7, 1H, ArH),
8.03 (d, J = 2.4, 1H, ArH), 7.66 (m, 2H, ArH), 7.45 (s, 1H, ArH), 7.16 (dd, J = 1.9, 8.8,
6
7
1
1H, ArH), 3.73 (s, 4H, CH2CH2), 3.68 (s, 4H, CH2CH2). H NMR (300 MHz, D2O) δ
8
9
7.49 (d, J = 8.4, 1H, ArH), 7.27 (s, 1H, ArH), 7.19 (d, J = 8.4, 1H, ArH), 6.92 (d, J =
8.9, 1H, ArH), 6.70 (d, J = 8.9, 1H, ArH), 6.55 (s, 1H, ArH), 3.86 (s, 4H, CH2CH2),
3.82 (s, 4H, CH2CH2). 13C NMR (75 MHz, D2O) δ 176.9, 158.2, 157.3, 140.8, 140.7,
138.4, 129.6, 129.5, 127.9, 119.5, 119.2, 118.3, 116.4, 115.6, 107.2, 43.2. LRMS (ES+)
m/z 362.4 (M+H)+, 181.5 [(M+2H)2+, 100%]. ESI–HRMS m/z 362.1737 [M+H]+
(C19H20N7O requires 362.1724).
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Dihydrochloride salt of 3,6-bis((4,5-dihydro-1H-imidazol-2-yl)amino)acridin-
9(10H)-one (1c). Starting from 6c (102 mg) and following the general procedure, the
dihydrochloride salt of 1c was obtained as a yellow solid (40 mg, 70%). HPLC > 95%;
mp > 250 ºC (decomp.). 1H NMR (300 MHz, D2O) δ 7.66 (d, J = 8.8, 2H, ArH), 6.80
(dd, J = 2.1, 8.8, 2H, ArH), 6.67 (d, J = 2.1, 2H, ArH), 3.90 (s, 8H, CH2CH2). 13C NMR
(75 MHz, D2O) δ 177.0, 157.5, 141.1, 140.8, 128.0, 117.1, 115.7, 107.5, 43.2. LRMS
(ES+) m/z 362.5 (M+H)+, 181.6 [(M+2H)2+, 100%]. ESI–HRMS m/z 362.1739 [M+H]+
(C19H20N7O requires 362.1724).
Dihydrochloride
salt
of
2,6-bis((4,5-dihydro-1H-imidazol-2-
yl)amino)anthraquinone (2b). Starting from 7b (94 mg) and following the general
procedure, the dihydrochloride salt of 2b was obtained as an orangish solid (46 mg,
1
85%). HPLC > 95%; mp > 250 ºC (decomp.). H NMR (300 MHz, D2O) δ 8.09 (d, J =
8.5, 2H, ArH), 7.80 (d, J = 2.3, 2H, ArH), 7.61 (dd, J = 8.5, 2.3, 2H, ArH), 3.90 (s, 8H,
CH2CH2). 13C NMR (75 MHz, D2O) δ 182.8, 157.9, 142.3, 134.7, 130.2, 129.9, 127.3,
119.2, 43.3. LRMS (ES+) m/z 375 [(M+H)+, 100%]. ESI–HRMS m/z 375.1555 [M+H]+
(C20H19N6O2 requires 375.1564).
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Ditrifluoroacetate salt of 2,7-bis((4,5-dihydro-1H-imidazol-2-yl)amino)-10-
methylacridin-9(10H)-one (3a). A solution of 8a (13 mg, 0.167 mmol) dissolved in
CH2Cl2 (0.5 mL) and trifluoroacetic acid (0.2 mL) was stirred at room temperature for
66 h. The solvents were removed under vacuum and the crude residue dissolved in a
little MeOH was filtered through a plug of cotton to remove insoluble matter. The clear
solution was concentrated to ca. 0.2 mL and diethyl ether was added to precipitate the
product. The flask was allowed to stand for a few hours in the refrigerator, the
supernatant was removed, and the solid precipitate was rinsed with Et2O. Compound 3a
was obtained as a yellow amorphous solid (8.8 mg, 87%). HPLC > 95%; mp 234 ºC. 1H
NMR (500 MHz, CD3OD) δ 8.31 (d, J = 2.7, 2H, ArH), 7.98 (d, J = 9.2, 2H, ArH), 7.78
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(dd, J = 2.7, 9.2, 2H, ArH), 4.06 (s, 3H, NCH3), 3.83 (s, 8H, CH2CH2). C NMR (126
MHz, CD3OD) δ 178.56, 160.53, 142.75, 132.12, 131.17, 123.44, 122.90, 119.39,
44.26, 34.91. LRMS (ES+) m/z 376.5 [(M+H)+], 188.7 [(M+2H)2+, 100%]. ESI–HRMS
m/z 376.1896 [M+H]+ (C20H22N7O requires 376.1880).
Ditrifluoroacetate salt of 2,6-bis((4,5-dihydro-1H-imidazol-2-yl)amino)-10-
methylacridin-9(10H)-one (3b). Using the same procedure as above starting from 8b
(8 mg, 0.103 mmol) yielded 3b as a yellowish amorphous solid (5.8 mg, 93%). HPLC =
1
93%; mp 166–170 ºC. H NMR (500 MHz, CD3OD) δ 8.51 (d, J = 8.6, 1H, ArH), 8.31
(d, J = 2.7, 1H, ArH), 7.96 (d, J = 9.2, 1H, ArH), 7.76 (dd, J = 2.7, 9.2, 1H, ArH), 7.70
(d, J = 1.9, 1H, ArH), 7.28 (dd, J = 1.9, 8.7, 1H, ArH), 4.03 (s, 3H, NCH3), 3.88 (s, 4H,
CH2CH2), 3.82 (s, 4H, CH2CH2). 13C NMR (126 MHz, CD3OD) δ 178.38, 160.51,
159.72, 145.12, 143.10, 143.04, 131.94, 131.15, 130.44, 123.86, 122.98, 120.94,
119.26, 117.62, 110.29, 44.27, 44.26, 34.83. LRMS (ES+) m/z 376.6 [(M+H)+], 188.8
[(M+2H)2+, 100%]. ESI–HRMS m/z 376.1892 [M+H]+ (C20H22N7O requires 376.1880).
Ditrifluoroacetate salt of 3,6-bis((4,5-dihydro-1H-imidazol-2-yl)amino)-10-
methylacridin-9(10H)-one (3c). Using the same procedure as above starting from 8c
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(10 mg, 0.129 mmol) yielded 3c as a brownish amorphous solid (5.5 mg, 71%). HPLC
= 93%; mp 180–181 ºC. 1H NMR (300 MHz, CD3OD) δ 8.49 (d, J = 8.6, 2H, ArH),
7.67 (d, J = 1.9, 2H, ArH), 7.27 (dd, J = 1.9, 8.6, 2H, ArH), 3.97 (s, 3H, NCH3), 3.87 (s,
8H, CH2CH2). 13C NMR (126 MHz, CD3OD) δ 178.20, 159.72, 145.34, 142.92, 130.40,
121.30, 117.57, 110.34, 44.27, 34.77. LRMS (ES+) m/z 376.5 [(M+H)+], 188.8
[(M+2H)2+, 100%]. ESI–HRMS m/z 376.1899 [M+H]+ (C20H22N7O requires 376.1880).
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Biology
In vitro antiprotozoal activity. An Alamar blueꢀbased assay was used to determine
the in vitro activity against bloodstream forms T. b. rhodesiense38 and axenically grown
amastigotes of L. donovani,39 and the cytoxicity to rat myoblast L6ꢀcells as previously
reported.20 Activity against intracellular amastigotes of T. cruzi Tulahuen strain C2C4
containing the βꢀgalactosidase (LacZ) gene40 was determined with the colorimetric
assay using chlorophenyl red βꢀD
ꢀgalactopyranoside (CPRG)ꢀNonidet as reported.20
Antimalarial activity against erithrocytic stage of P. falciparum (NF54 chloroquine
sensitive strain and K1 chloroquine/pyrimethamine resistant strain) was determined
with a [3H]hypoxanthine incorporation assay.20, 41
In vivo activity against T. b. rhodesiense. Four female NMRI mice were used per
experimental group. Each mouse was inoculated i.p. with 104 bloodstream forms of
STIB900, respectively. Heparinized blood from a donor mouse with approximately 5 ×
106 /mL parasitaemia was suspended in PSG to obtain a trypanosome suspension of 1 ×
105 /mL. Each mouse was injected with 0.25 mL. Compounds were formulated in 100%
DMSO and diluted 10ꢀfold in distilled water. Compound treatment was initiated 3 days
postꢀinfection on four consecutive days for all administration routes (i.p., p.o.) in a
volume of 0.1 mL/10 g. Three mice served as infectedꢀuntreated controls. They were
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not injected with the vehicle alone since we have established in our laboratory (over
many years) that these vehicles do not affect parasitaemia nor the mice. Parasitaemia
was monitored using smears of tailꢀsnip blood twice a week after treatment for two
weeks followed by once a week until 60 days postꢀinfection. Mice were considered
cured when there was no parasitaemia relapse detected in the tail blood over the 60ꢀday
observation period. Mean relapse days (MRD) were determined as day of relapse postꢀ
infection of mice.
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All the in vivo efficacy studies in mice were conducted at the Swiss Tropical and Public
Health Institute (Basel) according to the rules and regulations for the protection of
animal rights ("Tierschutzverordnung") of the Swiss "Bundesamt für Veterinärwesen".
They were approved by the veterinary office of Canton BaselꢀStadt, Switzerland.
DNA binding
Spectrophotometric titrations. UVꢀvis spectra were measured on a Perkin–Elmer
Lambda 35 UV–vis spectrophotometer in a 1.5 mL quartz cuvette (1 cm pathlength) in
10 mM phosphate buffer (pH 7) previously degassed by sonication. A stock solution of
FSꢀDNA (23 mg in 5 mL of phosphate buffer) was prepared and shaken gently for 1 h.
The solution was diluted 100× and the UV spectrum at 260 nm was recorded (Abs260
=
1.1016). The concentration of FSꢀDNA stock solution (8.6 mM) was worked out from
the following equation: Abs260 = 100 × C × d × ε260 using the extinction coefficient
12800 M(bp)ꢀ1.cmꢀ1 at 260 nm for FSꢀDNA.42 Stock solutions of the compounds 1aꢀc,
2b, and 3aꢀc (1 mL, C = 20 µM in phosphate buffer) were prepared from 1 mM stock
solutions in DMSO by dilution with 10 mM phosphate buffer. The final amount of
DMSO in the stock solution was 2%.
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Spectrophotometric titrations were performed by sequential addition of aliquots of DNA
solution (C = 860 µM) to 800 µL of the compound (20 µM) until saturation was
observed. A 1 min incubation time was allowed between each addition. The
experiments were performed at 25 ºC. The data was processed with Excel and the
percentage of hypochromicity (peak to peak) and bathochromic shift was calculated. K
values were determined using Scatchard plots and non linear curve fitting with the
(non–co–operative) McGhee and von Hippel neighbour exclusion model.25
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CD experiments. Circular dichroism studies were recorded using a Jasco Jꢀ815
spectropolarimeter from 220 to 500 nm with 1 cm quartz cuvettes at 20 ºC. The spectra
were averaged over three scans with a scan speed of 200 nm/min and a buffer blank
correction. A 30 ꢂM FSꢀDNA solution in 10 mM phosphate buffer (pH 7) with 6%
DMSO was first scanned; the compounds at increasing concentration ratios were then
titrated into the same cuvette, and the complexes were scanned under the same
conditions. For each compound (1b, 1c, 2b, and 3c), six dilutions in phosphate buffer
with 6% DMSO (15, 20, 30, 45, and 60 µM) were prepared starting from 1 mM stock
solutions in DMSO. The desired ratios of compound to DNA (r = 0.5, 0.6, 1.0, 1.5, and
2.0) were obtained by adding compound to the cell containing a constant amount of
DNA. FSꢀDNA was purchased from ACROS (CAS 68938ꢀ01ꢀ2).
Acknowledgements. This work was supported by grants from the Spanish Ministerio
de Ciencia e Innovación (Grant SAF2009ꢀ10399). SMQ was recipient of a JAEꢀIntro
fellowship from the CSIC.
Supporting information available. Synthesis and characterization of starting material
diamines (4a, 4b, and 4c) and Bocꢀprotected intermediates 6a–c, 7b, and 8a–c.
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Corresponding Author
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*Phone: +34 912587490. Fax. +34 915644853. Eꢀmail: dardonville@iqm.csic.es
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Abbreviations Used
FS–DNA, fish sperm–DNA; ICD signal, induced circular dichroism signal; k–DNA,
kinetoplast–DNA; r, ligand/DNA ratio.
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