New bis(2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity

Article abstract of DOI:10.1021/jm7013088

A series of 75 guanidine and 2-aminoimidazoline analogue molecules were assayed in vitro against Trypanosoma brucei rhodesiense STIB900 and Plasmodium falciparum K1. The dicationic diphenyl compounds exhibited the best activities with IC₅₀ values against T. b. rhodesiense and P. falciparum in the nanomolar range. Five compounds (7b, 9a, 9b, 10b, and 14b) cured 100% of treated mice upon ip administration at 20 mg/kg in the difficult to cure T. b. rhodesiense STIB900 mouse model. Overall, the compounds that bear the 2-aminoimidazoline cations benefit from better safety profiles than the guanidine counterparts. The observation of a correlation between DNA binding affinity at AT sites and trypanocidal activity for three series of compounds supported the view of a mechanism of antitrypanosomal action due in part to the formation of a DNA complex. No correlation between antiplasmodial activity and in vitro inhibition of ferriprotoporphyrin IX biomineralisation was observed, suggesting that additional mechanism of action is likely to be involved.

Full text of DOI:10.1021/jm7013088

J. Med. Chem. 2008, 51, 909–923
909
New Bis(2-aminoimidazoline) and Bisguanidine DNA Minor Groove Binders with Potent in
Vivo Antitrypanosomal and Antiplasmodial Activity
Fernando Rodríguez,† Isabel Rozas,† Marcel Kaiser,‡ Reto Brun,‡ Binh Nguyen,§ W. David Wilson,§ Rory Nelson García,| and
Christophe Dardonville*,
Centre for Synthesis and Chemical Biology, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland, Swiss Tropical Institute,
Socinstrasse, 57, CH-4002 Basel, Switzerland, Department of Chemistry, Georgia State UniVersity, Atlanta, Georgia 30303-3083,
Departamento de Parasitología, Facultad de Farmacia, UniVersidad Complutense de Madrid, AV. Complutense s/n, E-28040 Madrid, Spain,
and Instituto de Química Médica, CSIC, Juan de la CierVa 3, E-28006 Madrid, Spain
ReceiVed October 17, 2007
A series of 75 guanidine and 2-aminoimidazoline analogue molecules were assayed in vitro against
Trypanosoma brucei rhodesiense STIB900 and Plasmodium falciparum K1. The dicationic diphenyl
compounds exhibited the best activities with IC50 values against T. b. rhodesiense and P. falciparum in the
nanomolar range. Five compounds (7b, 9a, 9b, 10b, and 14b) cured 100% of treated mice upon ip
administration at 20 mg/kg in the difficult to cure T. b. rhodesiense STIB900 mouse model. Overall, the
compounds that bear the 2-aminoimidazoline cations benefit from better safety profiles than the guanidine
counterparts. The observation of a correlation between DNA binding affinity at AT sites and trypanocidal
activity for three series of compounds supported the view of a mechanism of antitrypanosomal action due
in part to the formation of a DNA complex. No correlation between antiplasmodial activity and in vitro
inhibition of ferriprotoporphyrin IX biomineralisation was observed, suggesting that additional mechanism
of action is likely to be involved.
Chart 1. General Structures of the Compounds Studied
Highlighting the Skeleton Common to the Different Scaffolds
Introduction
Infectious diseases caused by protozoan parasites are respon-
sible for great morbidity and mortality mainly in the least
developed countries. Despite the lack of significant research
investment on tropical diseases, rich countries recently started
to pay attention to malaria because this disease also represents
a potential threat for the developed world. Another tropical
disease, human African trypanosomiasis (HATa or sleeping
sickness) is only present in sub-Saharan Africa and affects
between 50 000 and 70 000 people.1 HAT belongs to the most
neglected diseases as defined by a World Health Organization/
Industry working group.2
Drugs available for HAT are obsolete and present unaccept-
able adverse effects, as well as increasing treatment failures due
to emergence of drug resistance or other reasons.3–5 On the other
hand, the chemotherapy of malaria is principally impaired by
the appearance of drug resistant strains of Plasmodium spp.
Hence, chloroquine, which was the most common antimalarial
drug for decades, is now practically ineffective and emergence
of resistance to other drugs such as mefloquine, halofantrine,
or artemisinin is beginning to appear.6 For those reasons, WHO
now recommends the use of antimalarial drug combinations
(e.g., artesunate/mefloquine, artesunate/amodiaquine) in order
to delay the development of resistant strains.5 Thus, the
discovery of new safe and efficient antiprotozoal agents to treat
HAT and malaria is a priority in international health.
Recent findings by our group have shown that bisguanidine
and especially bis(2-aminoimidazoline)diphenyl compounds
displayed potent antitrypanosomal activity in vitro and vivo
against T. b. rhodesiense, the causative agent of acute HAT.7,8
These studies revealed that compounds bearing 2-aminoimida-
zoline cations (scaffold A, Chart 1) had higher selectivity for
the parasite and similar activities with respect to their guanidine
counterparts. In addition, a correlation between antitrypanosomal
activity and DNA binding affinity was observed, suggesting a
possible mechanism of action for these compounds.7 Finally,
we showed that this class of compounds (i.e., 1a and 1b) entered
into trypanosomes via different transporters in addition to P2,
indicating that parasites that have lost the P2 transporter in
* To whom correspondence should be addressed. Phone: +34 912587490.
Fax: +34 915644853. E-mail: dardonville@iqm.csic.es.
†
Trinity College Dublin.
Swiss Tropical Institute.
Georgia State University.
Universidad Complutense de Madrid.
‡
§
|
Instituto de Química Médica.
a Abbreviations: CQ, chloroquine; FACS, fluorescence-activated cell
sorting; FPIX, ferriprotoporphyrin IX; HAT, human African trypanoso-
miasis; HEPES, 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid; MEM,
minimum essential medium; MOA, mechanism of action; µCi, microcurie;
Pip, piperidine; SI, selectivity index.
10.1021/jm7013088 CCC: $40.75
2008 American Chemical Society
Published on Web 02/05/2008

910 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
Table 1. In Vitro Antitrypanosomal and Antiplasmodial Activity of Diphenyl Dicationic Compounds (Scaffold A)
a Gua )
. Imi )
. (Boc)Gua )
. (Boc)Imi )
.
b T. brucei rhodesiense STIB900 strain.
Control: melarsoprol, IC50 ) 5.5 nM. c P. falciparum K1 strain. Control: chloroquine, IC50 ) 0.278 µM. d Data previously reported in ref 8 and included
here for comparison purposes e Data taken from ref 13.
selection of resistance to other drugs will not show cross-
resistance to this class of compounds. Encouraged by these
promising results, another series of 16 dicationic analogues (3–4,
7–11, 13b, and 14, Table 1) were evaluated against T. b.
rhodesiense, and their DNA binding affinity at AT-rich sites
was estimated by ∆Tm measurements with a nonalternating AT
sequence DNA polymer.9
Others have described excellent antiplasmodial activity of
related aromatic dicationic structures such as pentamidine or
DB75.10–13 For example, DB289, the neutral prodrug of DB75,

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 911
Scheme 1a
a Reagents and conditions: (a) N,N′-di(tert-butoxycarbonyl)thiourea or N,N′-di(tert-butoxycarbonyl)imidazoline-2-thione, HgCl2, Et3N, solvent; (b) CH2Cl2/
TFA (1:1); (c) IRA400 (Cl-) Amberlyte anion exchange resin; (d) CS2, CH2Cl2, 50 °C; (e) phenyl isothiocyanate (21d) or phenyl isocyanate (20d) or benzoyl
chloride (23d), CH2Cl2, 0 °C and then room temp, 18 h.
has been used to treat uncomplicated P. ViVax and P. falciparum
malaria.14 Stead et al. demonstrated that uptake of pentamidine
into infected erythrocytes proceeds via a route similar to the
new permeability pathway (NPP). In addition, it has been
proposed that these diamidines share a common MOA with
chloroquine by binding to ferriprotoporphyrin IX and inhibiting
the formation of hemozoin.15 These findings prompted us to
test the antiplasmodial potential of our dicationic diphenyl
compounds (Chart 1, scaffold A).9 The capacity of the com-
pounds to inhibit the formation of hemozoin as a possible
mechanism of antiplasmodial activity was also evaluated in vitro
with the ferriprotoporphyrin IX binding inhibition test (FBIT).16
Moreover, in order to extend our understanding of the SAR
of this class of antiprotozoal agents two new series of cationic
analogues, namely, 2-aminoimidazolinium compounds and their
guanidinium counterparts (Chart 1, scaffolds B and C) were
selected for in vitro screening on T. b. rhodesiense and P.
falciparum. As can be shown in Chart 1, the three analogous
series are strictly structurally related to one another; scaffold A
represents the “full” dicationic diphenyl model compound.
Scaffold B is analogous to A but devoid of one cationic group,
whereas scaffold C has “lost” one cationic moiety and one
phenyl ring. With these series in hand, we intend to demonstrate
the significance of each part of the molecule for the overall
activity of the dicationic diphenyl compounds and gain insight
into the SAR of this series of antiprotozoal compounds.
Finally, to check whether the activity was maintained in vivo,
the most active and selective compounds in vitro were assayed
in an acute HAT mouse model (T. b. rhodesiense STIB900) or
rodent malaria model (P. berghei GFP ANKA). Several lead
compounds with excellent in vivo activity emerged from this
screening.
The synthesis of compounds 3a,b, 4a,b, 15a,b, 17a,b, 18b,
24–26a,b, 29a,b, 31a,b, 33–35a,b, and their Boc-protected
precursors was described in a recent paper of Rodriguez et al.18
The synthesis of 10a,b, 13b, 14a,b, 16a,b, 27a,b, 28a, 32a,b,
36a,b, 37b, and their Boc-protected precurors will be reported
elsewhere.19 The synthesis of 7a,b, 8a, 9a,b, 11a,b, 19–23a,b
and 30a,b is described in Schemes 1 and 2. Among these
derivatives, 7a, 11a,b, 19a,b, 20a,b, 21a,b, 22b, and 23b (in
addition to all the Boc-protected precursors) are new, whereas
7b,20 8a,21 9a,22 9b,23 22a,24,25 and 23a22 have been previously
described in the literature using different synthetic strategies.
Briefly, our synthetic approach to introduce the guanidine and
2-aminoimidazoline groups relied on the reaction between
primary amines or diamines and N,N′-bis(tert-butoxycarbon-
yl)thiourea/HgCl2/Et3N26 or N,N′-bis(tert-butoxycarbonyl)imi-
dazoline-2-thione/HgCl2/Et3N,17 affording the Boc-protected
guanidines (7d, 9d, 11d, 19d, 20d, 22d, 23d, and 30d) and
2-aminoimidazolines (7e, 9e, 11e, 19e, 20e, 22e, 23e, and 30e),
respectively. Removal of the Boc protecting groups with TFA
followed by anion exchange chromatography afforded the
hydrochloride salts of the compounds 7a,b, 8a, 9a,b, 11a,b,
19a,b, 22a,b, 23a,b, and 30a,b. Compounds 20a, 20b, 21a, and
21b were studied as their trifluoroacetate salts (Scheme 1). In
the case of the thiourea derivatives 8d, 21d, and 21e, an
alternative strategy was employed to avoid the competitive
reaction occurring between the thiourea linker of the starting
material (i.e., 8 and 21) and the thiourea guanidine precursors
for the HgCl2 catalyst (Scheme 1). Hence, 8d was obtained by
condensation of the monomer 818 in an excess of carbon
disulfide. Alternatively, the reaction of 8 with 1 equiv of phenyl
isothiocyanate in CH2Cl2 afforded the guanidine derivative 21d
(68%). Compounds 20d and 23d could also be synthesized in
a similar way by reaction of 8 with phenyl isocyanate and
benzoyl chloride, respectively. A similar approach was used for
the synthesis of the 2-aminoimidazoline analogue 21e starting
from 8b. Thus, 1,4-phenylenediamine was reacted with 1 equiv
of N,N′-bis(tert-butoxycarbonyl)imidazoline-2-thione/HgCl2/
Results
Chemistry. Many of the compounds presented here were
previously synthesized by us for other purposes. The synthesis
of 1a-e, 2a-c, 5a-f, 6a-e, and 12a,b was described earlier.8,17

912 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
Scheme 2a
Table 2. In Vitro Antitrypanosomal and Antiplasmodial Activity of
Diphenyl Monocationic Compounds (Scaffold B)
IC50 (µM)
cytotoxicity
L6-cells
compd
Ra
X
T.b.r.b
P.f.c
15a
15b
16a
16b
17a
17b
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
Gua
Imi
Gua
Imi
Gua
Imi
Imi
Gua
Imi
Gua
Imi
Gua
Imi
Gua
Imi
Gua
Imi
NH
NH
CH2
CH2
O
O
S
CO
CO
NH-CO-NH
NH-CO-NH
NH-CS-NH
NH-CS-NH
NH-CO
NH-CO
CO-NH
CO-NH
4.8
1.2
15.9
4.9
18.8
5.0
1.9
14.4
9.4
32.9
45.4
1.3
1.6
0.549
3.8
1.7
7.9
5.7
1.2
>18
5.2
4.1
3.4
8.7
3.1
13.3
>15
10.4
>15
49.5
90.7
43.9
73.0
59.9
166.7
28.5
118.1
225.8
>234
>219
>225
91.1
>309
>284
>309
>284
2.6
87.4
42.9
121.7
94.7
a Gua )
. Imi )
.
b T. brucei rhodesiense STIB900
a Reagents and conditions: (a) H2, 5% Pd-C, MeOH, room temp; (b)
HNO3, H2SO4; (c) 1-fluoro-4-nitrobenzene, DMF, 100 °C, 72 h; (d) phenyl
isocyanate, CH2Cl2, 0 °C; (e) aniline, Et3N, TBTU, CH2Cl2; (f) TFA,
CH2Cl2.
strain. Control: melarsoprol, IC50 ) 5.5 nM. c P. falciparum K1 strain.
Control: chloroquine, IC50 ) 0.278 µM.
observed in all the cases compared with the guanidinium
analogues [e.g., 3a (SI ) 8) vs 3b (SI ) 88), 9a (SI ) 319) vs
9b (SI ) 7731), 14a (SI ) 15) vs 14b (SI ) 624)]. This
observation was also true for the antiplasmodial activity of the
dicationic compounds.
Et3N17 to afford the monomer 8b. The crude product 8b was
reacted without further purification with 1 equiv of phenyl-
isothiocyanate, yielding 21e after chromatography on neutral
alumina (22% for two steps). Four of the starting material
amines (7,27 11,28,29 20, and 22) were obtained in a straight-
forward manner as depicted in Scheme 2.
Biological Results. In Vitro Activity. Structure–Activity
Relationships. The results of the in vitro antitrypanosomal and
antiplasmodial activity are presented in Tables 1-3. In order
to gain insights into the mechanism of antiplasmodial and
antitrypanosomal activity, the most active compounds (i.e.,
diphenyldicationic derivatives, scaffold A) were also tested as
inhibitors of ꢀ-hematin formation16 and as DNA minor groove
binders.30
Antitrypanosomal Activity. In general, the presence of two
cations (scaffold A) was essential to get nanomolar anti-T. brucei
activity. Accordingly, compounds with only one cation and no
phenyl ring in the para position (scaffold C) showed micromolar
IC50 values. The 1-(2,3-dihydro-1H-inden-5-yl)guanidine deriva-
tive 32a (IC50 ) 0.99 and 0.95 µM against T. b. rhodesiense
and P. falciparum, respectively) was the most active molecule
of this series. In contrast, scaffold A compounds displayed
nanomolar activities with the exception of the compounds with
an electron-attracting group such as SO2 (6a, 6b, and 6e) or
CO (5b) linking both phenyl rings. Removal of one cationic
moiety (scaffold B) or one phenyl cationic moiety (scaffold C)
led to a great loss of activity (e.g., compare 1a/15a/24a, 2a/
16a/27a, 2b/16b/27b). Interestingly, the decrease in activity
observed in a homologous series with the removal of one
cationic moiety (i.e., scaffold A f scaffold B) was less
pronounced for the bis(2-aminoimidazolinium) vs bisguani-
dinium compounds [e.g., compare 1a/15a (×218) vs 1b/15b
(×17), 2a/16a (×98) vs 2b/16b (×5.5), 3a/17a (×96) vs 3b/
17b (×10)]. Another remarkable effect of the 2-aminoimida-
zolinium cation was the higher selectivity for the parasite
Regarding the bridge linking both phenyl rings, the same
range of activity (IC50 in the low micromolar range) was
observed for electron donating groups: NH (1) ∼ CH2CH2 (10)
. piperidine (11) ∼ piperazine (12). Isosteric replacement of
the CH2 (2) by a sulfur (3) or oxygen atom (4) hardly changed
the activity. The best activity was observed with an amide (9a,
9b) or ethane bridge (10a, 10b). Fused ring dicationic com-
pounds 13 (fluorene) and 14 (dihydroanthracene) also gave
excellent antitrypanosomal activity. The bis(2-aminoimidazo-
line)fluorene derivative 13b was the best anti-T. brucei agent
in vitro (IC50 ) 4.9 nM, SI ) 17 000). This outstanding value
is to be compared with the guanidine analogue 13a (IC50 ) 24
nM, SI ) 196) previously described by Boykin and co-
workers.13 Hence, replacement of the guanidine cations by
2-aminoimidazoline cations led to a 5-fold increase in activity
and 86-fold increase in selectivity for this scaffold.
Dicationic guanidine compounds (scaffold A) with NH, CH2,
O, S, CO, or SO2 bridge linking both phenyl rings were 2- to
10-fold more potent than their 2-aminoimidazoline counterparts
(compare 1a/1b, 2a/2b, 3a/3b, 4a/4b, 5a/5b, and 6a/6b). The
opposite effect was observed for monocationic compounds
(scaffold B). However, no significant difference in antitrypa-
nosomal activities was observed between the bisguanidine and
bis(2-aminoimidazoline) counterparts for the compounds with
the urea (7a, 7b), amide (9a, 9b), or ethane bridge (10a, 10b).
Antiplasmodial Activity. In most cases, the best activities
for scaffolds A-C were observed for compounds bearing the
2-aminoimidazolinium cations except for the fluorene analogue
13b (IC50 ) 11.5 nM), the activity of which was 5-fold lower
than that reported by Boykin and co-workers for the guanidine
counterpart 13a (2.3 nM).13 Among 20 dicationic compounds

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 913
Table 3. In Vitro Antitrypanosomal and Antiplasmodial Activity of
Phenyl Monocationic Compounds (Scaffold C)
Table 4. In Vivo Antitrypanosomal Activity in the T. b. rhodesiense
(STIB900) Mouse Modela
dosage
compd routeb dosage (mg/kg) curedc/infected survival (days)d
control
7b
9a
0/4
4/4
4/4
4/4
4/4
2/4
4/4
7.5
ip
ip
ip
ip
ip
ip
4×20
4×20
4×20
4×20
4×20
4×20
>60
>60
>60
>60
>47.25
>60
IC50 (µM)
cytotoxicity
L6-cells
9b
compd Ra
X
Y
T.b.r.b P.f.c
10b
11b
14b
24a
25b
25a
25b
26a
26b
Gua NH2
Imi NH2
Gua (CH3)2NH
Imi (CH3)2NH
Gua Et2NH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
311.2 >26
131.8
>423
>358
>324
78.1
nd
15.0
>19
>18
5.7
a See Experimental Section for details of STIB 900 (T. b. rhodesiense)
model. b ip ) intraperitoneal. c Number of mice that survive and are parasite
free for 60 days. d Average days of survival.
20.1
46.6
2.4
Imi Et2NH
40.1
15.0
163.7 >27
262.4 >23
>444
>484
>425
309.6
239.7
173.9
>421
>375
231.8
>283
73.7
>378
>391
>351
>417
>372
>388
>349
189.8
>398
>455
27ad Gua CH3
Table 5. In Vivo Antiplasmodial Activity in the P. Berghei (ANKA
GFP) Mouse Modela
27b
28a
29a
29b
Imi CH3
Gua Et
45.7
5.9
7.3
9.0
1.3
1.7
dosage
routeb
dosage
(mg/kg)
curedc/
infected
% of
survival
(days)e
Gua CH3S
Imi CH3S
compd
control
chloroquine
activityd
30ad Gua CH3CO
0/4
0/4
0/4
0/4
0/4
0/4
0/4
Tf
6.2
9
20
7
6.7
7
11.3
155.4 >23
ip
ip
ip
ip
ip
ip
ip
ip
ip
ip
ip
ip
4 × 5
99.6
99.6
65.51
8.9
44.1
97.5
T
T
T
42.48
0
T
30b
31a
31b
32a
32b
33a
33b
Imi CH3CO
54.6
48.6
50.4
0.99
9.2
5.7
7.9
12.0
0.95
5.3
4 × 10
4 × 20
4 × 20
4 × 30
4 × 50
4 × 50
4 × 50
4 × 50
4 × 20
4 × 20
4 × 50
Gua 4-piperidin-1-yl
Imi 4-piperidin-1-yl
Gua fused cyclopentane
Imi fused cyclopentane
Gua Fused 1,4-dioxane
Imi Fused 1,4-dioxane
1b
2b
3b
4b
149.8 9.0
7a
68.4
>19
34ad Gua fused 1,3-dioxolane
10a
11a
12a
12b
14a
T
T
146.0 >23
34b
Imi fused 1,3-dioxolane
93.5
>20
35ad Gua CH3O
0/4
0/4
T
7
7
CH3O 237.0 14.8
35b
36a
36b
37b
Imi CH3O
Gua CH3
Imi CH3
CH3O 84.2
14.8
12.2
17.1
CH3
CH3
H
39.9
49.8
a See Experimental Section for details of ANKA GFP (P. berghei)
models. b ip ) intraperitoneal. c Number of mice that survive and are parasite
free for 60 days. d % of reduction of parasitemia. e Average days of survival.
f Toxic at the dose tested.
Imi
H
181.8 >25
a Gua )
. Imi )
.
b T. brucei rhodesiense STIB900
In Vivo Activity (Tables 4 and 5). The compounds display-
ing the best in vitro antitrypanosomal activities and selectivity
were administered intraperitoneally to mice infected with T. b.
rhodesiense STIB900. Five compounds (7b, 9a, 9b, 10b, and
14b) cured all mice at 20 mg/kg, whereas 11b only cured 2/4
mice at this dose (Table 4). These data were somehow surprising
because the in vitro activity and selectivity of 11b and the other
compounds tested were similar. Another interesting finding was
the enhanced antitrypanosomal activity and reduced toxicity of
the piperidine compound 11b compared to the piperazine
molecule 12a (i.e., 12a was toxic at 20 mg/kg in this model7).
Apparently, the presence of tertiary amino groups in 12a and
11b is not favorable for good in vivo activity, which may
possibly relate to unfavorable pharmacokinetic properties of the
compounds.
strain. Control: melarsoprol, IC50 ) 5.5 nM; c P. falciparum K1 strain.
Control: chloroquine, IC50 ) 0.278 µM; d the antiplasmodial action against
P. gallinaceum in chicks had been studied before by King, H. and Tonkin,
I. M. J. Chem. Soc. 1946, 1063–1069.
that displayed IC50 values less than 50 nM, diphenylamine
derivative 1b is the best antiplasmodial agent of the series (IC50
) 8.8 nM, SI ) 24 000). Most of the dicationic compounds
displayed excellent antimalarial activities regardless of the linker
X. However, compounds with an electron withdrawing linker
[SO2 (6), CO (5), NHCSNH (8)] exhibited somewhat lower
activity. The remarkable in vitro antiplasmodial activities of the
piperazine derivatives 12a and 12b (IC50 ) 15.2 and 12.3 nM,
respectively) were comparable to that of their diamidine
homologue reported by Mayence et al. (IC50 ) 4 nM).10 As
observed for the antitrypanosomal activity, the removal of one
cationic moiety from scaffold A compounds produced a dramatic
loss of activity [Tables 1 and 2; compare 1b/15b (×62), 2b/
16b (×108), 3a/17a (×171), 3b/17b (×150), 4b/18b (×48),
5b/19b (×40), 7a/20a (×42), 7b/20b (×121), 8a/21a (×14),
9a/22a (×241), 9b/22b (>535)].
Hence, the in vivo activity of the new compounds in the
difficult to cure STIB900 mouse model was comparable to that
of the lead compound 1b reported earlier.7 Additional experi-
ments to determine the minimum curative dose, oral bioavail-
ability, and potential activity in the chronic CNS mouse model
of sleeping sickness are ongoing.
The nanomolar activity observed for the uncharged Boc-
substituted guanidine (1d) and imidazoline (1e, 6e) derivatives
is worth noting. Interestingly, the bis(n-pentylphosphonium)
dicationic analogue 5f (IC50 ) 53 nM, SI ) 222) produced a
2-fold increase in activity together with a loss of selectivity with
respect to its imidazoline counterpart 5b (IC50 ) 129 nM, SI >
1658). Altogether, the data seem to indicate that lipophilic
groups, either cationic (phosphonium derivative 5f) or uncharged
(Boc-protected compounds 1d and 1e), are allowed for good in
vitro antiplasmodial activity of this class of symmetric diphenyl
compounds.
In addition, 10 compounds were tested in the P. berghei
mouse model to check whether the excellent in vitro antiplas-
modial activity of the dicationic compounds was retained in
vivo. Four compounds (1b, 3b, 4b, and 12a) reduced signifi-
cantly the parasitaemia upon ip treatment at 20–50 mg/kg, but
none of them was curative (Table 5). However, the bis(2-
aminoimidazoline) derivative 4b reduced parasitaemia drasti-
cally (97.5%) at 50 mg/kg and was able to increase mice survival
almost 2-fold compared to control animals. This activity
compared favorably with that of the control drug chloroquine
at 5 mg/kg. Besides, the ip administration of 3b (30 mg/kg)

914 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
Table 6. DNA Binding Affinity and Selectivity Index of the Diphenyl Dicationic Compounds
a Pentamidine: ∆Tm ) 32.3 °C (see ref 7). b Data taken from ref 7. c Selectivity index ) (IC50 L6-cells)/(IC50 T. b. rhodesiense).
produced an activity of 44.1% similar to its diamidine coun-
terpart administered subcutaneously at 40 mg/kg.31 Altogether,
the data confirm the antimalarial potential of the diphenyl amine,
diphenyl ether, and diphenyl sulfide scaffolds.
The rest of the compounds were either toxic at the tested
dose of 50 mg/kg (7a, 10a, 11a, and 14a) or inactive (12b).
Taken together, the in vivo results suggest that in this series
guanidine derivatives are more toxic than their cyclic congeners,
in agreement with the higher SI observed in vitro for the
2-aminoimidazoline analogues.
Insights into the Mechanism of Action. (1) DNA Binding
Affinity. Many aromatic diamidines and diguanidines are strong
DNA minor groove binders.11,32,33 Some evidence suggests that
this interaction is responsible to some extent for the antiprotozoal
activity frequently displayed by this class of compounds. We
previously reported the existence of a correlation between DNA
binding affinity to AT-rich sites and in vitro antitrypanosomal
activity of some of the dicationic compounds presented here
(1a, 1c, 2a, 2c, 5f and 1b, 2b, 5a, 12a, 12b).7 In order to extend
our knowledge of the structural requirements responsible for
high-quality DNA minor groove binding, and possibly good
antiprotozoal activity, the thermal melting curves (∆Tm) of the
new set of compounds (3a,b, 4a,b, 7–11a,b, 13b, and 14a,b)
were determined using a nonalternating AT sequence DNA
polymer. We used the same experimental conditions (i.e., low
salt buffer) as reported before,7 thus allowing the comparison
of the ∆Tm values of the entire series. The results of DNA
binding of the previous set of compounds (1, 2, 5, and 12) and
the new series (3, 4, 7–11, 13, 14) are presented in Table 6.
Overall, strong DNA binding was observed for this new series
of dicationic compounds with ∆Tm values ranging from 20.1
to 47.1 °C. The 2-aminoimidazoline derivatives consistently

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 915
Figure 2. Plot of SI vs Tm for the bis(2-aminoimidazoline) compounds
showing a good correlation between in vitro selectivity (SI ) (IC50
L6-cells)/(IC50 T. brucei)) and Tm increase: (9) 1b, 2b, 3b, 4b, 7b, 9b,
10b, 11b, 12b, 13b, and 14b.
Table 7. Ferriprotoporphyrin IX Biomineralization Inhibition and
Selectivity Index of the Diphenyl Dicationic Compounds
inhibition FPIXa
IC50 (µM)b
selectivityc
SI
compound
Figure 1. Plot of log(1/IC50) vs ∆Tm showing the correlation between
in vitro antitrypanosomal activity and Tm increase (a) for the set of
bis(2-aminoimidazoline) compounds, Y ) 0.0857x - 4.8776, R )
0.9056, and (b) for the set of bisguanidine derivatives, Y ) 0.03767x
– 3.0968, R ) 0.5994.
1a
1b
1c
1d
1e
0%
429.2
161.8
45%
170.7
0%
36
24091
>1548
48
166
88
4051
>4861
36
1079
76
932
40
>1658
223
>500
>35
1025
>2547
3714
26
209
6893
57
2a
2b
2c
3a
3b
4a
4b
5a
5b
5f
6a
6b
6e
7a
7b
8a
NTd
15%
0%
displayed higher ∆Tm values (2-7 °C) than the guanidine
counterparts, indicating that the ethylene bridge of the 2-ami-
noimidazoline group confers some special features, possibly
extra hydrophobic contacts with the walls of the groove that
contribute positively to the binding of these compounds.34
Curiously, the opposite effect is usually observed when compar-
ing series of dicationic benzamidines and their imidazolines
analogues, the amidine group conferring habitually better minor
groove binders than the imidazoline counterparts.35–38
Most interesting was the correlation observed between in vitro
antiprotozoal activity and DNA binding affinity for the set of
2-aminoimidazoline compounds. Hence, plotting ∆Tm versus
IC50 against trypanosomes revealed the two to be correlated as
shown in Figure 1a. In addition, a clear trend showing an
increase in activity with an increase in ∆Tm was observed for
the guanidine set of compounds (Figure 1b). The most exciting
finding from this DNA binding affinity study was observed with
the 2-aminoimidazoline set of compounds, the SI of which
correlated in a fairly good manner with the Tm increase (Figure
2). In contrast, no correlation was observed for the guanidine
set of analogues.
(2) Inhibition of ꢀ-Hematin Formation. Earlier studies have
shown that several diphenyldiamidine compounds displaying
antiplasmodial activity bind effectively to FPIX in vitro,
inhibiting the crystallization of toxic FPIX into nontoxic
hemozoin. The result of this interaction would be responsible
for the antimalarial activity of that class of compounds.10,15 We
decided to test whether our bisguanidine and bis(2-aminoimi-
dazoline) derivatives might work in the same way to evaluate
this hypothesis. A simple in vitro assay,16 the detection by
optical density measurement of solubilized ꢀ-hematin remaining
after contact with drugs, was used to assess the capacity of our
compounds to inhibit hemozoin biomineralization (Table 7).
0%
0%
952.1
NT
117.2
219.2
42%
NT
27%
0%
393.3
686.3
0%
704.7
0%
0%
0%
0%
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
2181
585
12000
3053
8455
2043
7252
83
0%
127.5
NT
491
0%
161
2000
a Chloroquine: IC50 ) 17.9 µM. b The percentage of inhibition at 1 mg/
mL (highest concentration tested) is given when the IC50 could not be
determined. c Selectivity index ) IC50(L6-cells)/IC50 (P. falciparum). d Not
tested.
Some of the compounds inhibited hemozoin formation at
micromolar concentrations comparable to that of quinine (IC50
) 324 µM)16 though higher than chloroquine (IC50 ) 17.9 µM).
However, no correlation between in vitro antiplasmodial activity
and hemozoin inhibition was observed.

916 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
workers have also reported micromolar in vitro anti-P. falci-
parum activity of a monocationic diphenylurea compound. That
compound, WR268961, the structure of which is related to 20
(i.e., scaffold B), was an inhibitor of the recombinant P.
falciparum aspartic protease plasmepsin II.42 This is relevant
because plasmepsins are validated targets for antimalarial
therapy43–45 and the activity of 7, 20, and 21 might be related
to plasmepsin inhibition, even though this would need experi-
mental confirmation.
Another exciting finding was the nanomolar activity exhibited
by the Boc-protected compounds (1d, 1e, and 6e) against P.
falciparum K1. The fair FPIX biomineralization inhibition
displayed by 1e in the FBIT assay (IC50 ) 170.7 µM) suggests
that this compound could interfere in the heme detoxification
process of the parasite. However, since these highly lipophilic
precursors are uncharged molecules, one might expect their
pharmacodynamics to be different from the dicationic derivatives.
Despite the lack of correlation between FPIX biomineraliza-
tion inhibition and in vitro activity against P. falciparum, the
binding to FPIX might be responsible to some extent for the
observed antiplasmodial activity and/or for the accumulation
of the compounds into the parasites as shown previously for
some diamidine drugs.15 However, care should be taken in the
interpretation of these results because we have no evidence at
this time that these inhibitors are able to reach the food vacuole
of the parasite to target the heme detoxification pathway. Indeed,
additional MOA is likely to be involved because our dicationic
compounds were less potent FPIX inhibitors than CQ, whereas
they showed nanomolar range activity about 1 order of
magnitude superior to CQ against the P. falciparum CQ-resistant
strain K1.
The diphenyl dicationic compounds retained only moderate
activity in the P. berghei murine model, in contrast to CQ, which
may possibly indicate unfavorable pharmacokinetic properties
or a different MOA. On the other hand, the modest in vivo
activity observed after ip administration in the P. berghei model
does not necessarily mean that these diphenyldiguanidine
compounds are poor antimalarials. In fact, some recent evidence
suggests that the P. berghei mouse model might not be optimal
to predict the activity of aromatic diamidines against human
malaria.11 For instance, DB289, which showed only modest
activity in the P. berghei mouse model, has shown remarkable
activity in a human trial against P. falciparum.14 For that reason,
we believe that these families of compounds deserve more
thorough investigations of their in vivo antimalarial action.
Figure 3. SAR against T. b. rhodesiense and P. falciparum.
Discussion
Encouraged by the excellent in vitro and in vivo trypanocidal
activity of a series of dicationic diphenyl compounds previously
discovered by our group,7 we report a continued investigation
of 2-aminoimidazoline and guanidine analogues as potential
antiplasmodial and antitrypanosomal agents. In vitro and in vivo
data from three new series of derivatives revealed some
important trends for the SAR of these compounds. In all the
cases, the diphenyl dicationic compounds (scaffold A) were the
most active antitrypanosomal and antiplasmodial compounds
with IC50 values in the nanomolar range. These compounds were
more efficient than their monocationic analogues which showed
IC50 in the micromolar range (scaffold B), highlighting once
more that the presence of two cationic groups is essential for
consistent activity in this series. The same trend was reported
earlier with amidine containing diphenyl ureas active against
P. falciparum,39 guanidine diphenyl derivatives active against
T. equiperdum,21,40 or furamidine analogues.33,41 In addition,
some basic SAR emerged relating to the best linker between
both phenyl rings (i.e., NH, NHCO, and CH2CH2) and the best
cation (i.e., 2-aminoimidazoline) to get selective compounds
with good in vivo antitrypanosomal activity (Figure 3). Fused
ring dicationic compounds (13 and 14) also exhibited excellent
activity in agreement with the results reported earlier by Arafa
et al.13 Interestingly, with regard to the linker between the two
phenyl rings, our results agree to some extent with the findings
of Turner27 for a series of bis[1,6-dihydro-6,6-dimethyl-1,3,5-
triazine-2,4-diamines]. Thus, it appears that the 1,2-diphenyle-
thane and fluorene moieties should be considered as good
scaffolds for the design of symmetric dicationic antitrypano-
somal drugs regardless of the cationic moiety present in the
molecule.
We do not have a clear explanation concerning the extra
selectivity and lower in vivo toxicity observed in all the cases
with the 2-aminoimidazoline cations in comparison with the
guanidine one. One may speculate whether the higher lipophi-
licity and/or a reduced H-bond donating capacity plays a role
in the reduced toxicity of the imidazoline derivatives. On the
other hand, the observation that the 2-aminoimidazoline deriva-
tives consistently displayed higher DNA binding affinity than
the guanidine counterparts and the correlation observed between
selectivity and Tm increase for this series suggest that the
formation of a DNA complex may be an important issue to
explain the MOA and selectivity profile of these dicationic
derivatives. This new and quite exciting observation indicates
that more investigations on 2-aminoimidazolines as DNA minor
groove binders and antiprotozoal agents are warranted.
Conclusion
In order to extend our understanding of the SAR of the bis(2-
aminoimidazoline) lead compounds reported earlier,7,8 we have
screened a new series of symmetric dicationic guanidine and
2-aminoimidazoline aromatic analogues against T. b. rhod-
esiense and P. falciparum. We have shown that in this series
the 2-aminoimidazoline derivatives were safer (higher SI) and
more potent in vivo against T. b. rhodesiense than their
guanidine counterparts. Moreover, a correlation between DNA
binding affinity and selectivity toward the parasite was observed
indicating that high affinity binding to the minor groove of DNA
may be part of their mechanism of antitrypanosomal action. This
view7 was also supported by the observation of a correlation
between DNA binding affinity and trypanocidal activity of two
series of compounds.
Of note was the excellent in vitro antiplasmodial activity of
the urea dicationic derivatives 7a and 7b (IC50 ) 96 and 28
nM, respectively). Similar activities had been reported for
amidine-containing diphenylureas.39 Interestingly, Jiang and co-
Five new dicationic lead compounds (7b, 9a, 9b, 10b, and
14b), upon ip administration of 20 (mg/kg)/day, cured 100%
of treated mice in the T. b. rhodesiense STIB900 model. In

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 917
8.17 (d, 2H, J ) 7.8 Hz, Ar); 13C NMR (DMSO-d6) δ 33.0 (C3Pip),
42.7 (C4Pip), 48.4 (C2Pip), 113.9, 124.9, 127.2, 129.5, 137.6, 147.3,
154.8, 155.7 (Ar).
contrast, despite their excellent in vitro antiplasmodial activity
and capacity for reducing the parasitaemia of mice infected with
P. berghei, the dicationic compounds 1b, 2b, 3b, 4b, and 12a
did not cure the animals in this model. However, the P. berghei
mouse model may not be the best predictive model for this kind
of dicationic compounds as we have pointed out before.
In this study we also described the excellent in vitro
antiplasmodial activity of noncationic Boc-protected guanidines
and imidazolines as well as phosphonium derivatives, the in
vivo antimalarial efficacy of which is currently being evaluated.
Hence, more thorough investigation on the antiplasmodial
activity of this class of compounds is warranted.
In light of these promising results, we believe that bis(2-
aminoimidazoline) derivatives deserve more investigation as
antiprotozoal agents and DNA minor groove binders. The
synthesis and study of new derivatives and prodrugs of our lead
compounds is ongoing and will be reported in due course.
1,4-Bis(4-aminophenyl)piperidine (11). A suspension of 1.5 g
(4.6 mmol) of 1,4-bis(4-nitrophenyl)piperidine 11f in methanol in
the presence of 5% Pd-C (185 mg) was hydrogenated at 3 bar
and room temperature for 22 h. Afterward, the catalyst was filtered
off and the solvent removed in vacuo to afford 1.198 g (98%) of
11 as a navy-blue solid: mp 156–158 °C; MS (ESI+) m/z 268.1802
[M + H]+; IR (Nujol) ν 3365, 3297, 3201 (NH2) cm-1; 1H NMR
(DMSO-d6) δ 1.63–1.80 (m, 4H, H3Pip), 2.32–2.43 (m, 1H, H4Pip),
2.53–2.61 (m, 2H, H2ePip), 3.38–3.49 (m, 2H, H2aPip), 4.58 (broad
s, 2H, NH2), 4.85 (broad s, 2H, NH2), 6.46–6.54 (m, 4H, Ar), 6.73
(d, 2H, J ) 8.8 Hz, Ar), 6.92 (d, 2H, J ) 8.3 Hz, Ar); 13C NMR
(DMSO-d6) δ 35.1 (C3Pip), 42.1 (C4Pip), 53.3 (C2Pip), 115.4, 116.2,
120.0, 128.4, 134.9, 143.4, 144.5, 148.0 (Ar).
1-(4-Aminophenyl)-3-phenylurea (20). An amount of 1.10 mL
(10.0 mmol) of phenyl isocyanate was added under an inert
atmosphere and at 0 °C over a solution of 1.082 g (10.0 mmol) of
1,4-phenylenediamine in dry CH2Cl2 (10 mL). The mixture was
allowed to reach room temperature and was stirred for 10 min.
The white solid precipitated was filtered and washed with cold
CH2Cl2 to afford 2.15 g (95%) of the pure 1-(4-aminophenyl)-3-
phenylurea: mp, decomposes at 212 °C (lit.,47 260 °C); IR (Nujol)
ν 3353, 3292, 3181 (NH, NH2), 1622 (CO, lit.,48 1630) cm-1; 1H
NMR (DMSO-d6) δ 4.80 (broad s, 2H, NH2), 6.56 (d, 2H, J ) 8.5
Hz, Ar), 6.88–6.99 (m, 1H, Ar), 7.12 (d, 2H, J ) 8.5 Hz, Ar),
7.21–7.30 (m, 2H, Ar), 7.46 (d, 2H, J ) 8.0 Hz, Ar), 8.17 (broad
s, 1H, NH), 8.51 (broad s, 1H, NH); 13C NMR (DMSO-d6) δ 115.5,
119.3, 122.2, 122.7, 129.9, 130.1, 141.5, 145.4 (Ar), 154.3 (CO).
4-(tert-Butoxycarbonylamino)benzamide (22c). Aniline (930
mg, 10 mmol) and TBTU (3.211 g, 10 mmol) were added at 0 °C
and under argon over a solution containing 4-[(tert-butoxycarbo-
nyl)amino]benzoic acid (2.373 g, 10.0 mmol) and Et3N (5.6 mL,
40 mmol) in 15 mL of dry CH2Cl2. The reaction mixture was stirred
at room temperature for 7 h. After that time a white precipitate
was formed. This precipitate turned out to be the desired compound
and was filtered and washed with cold hexane to yield 2.3 g (74%)
of 4-(tert-butoxycarbonylamino)benzamide as a white solid: mp
Experimental Section
Chemistry. All the commercial chemicals were obtained from
Sigma-Aldrich, Fluka, or Lancaster and were used without further
purification. Deuterated solvents for NMR use were purchased from
Apollo. Dry solvents were prepared using standard procedures
according to Vogel,46 with distillation prior to use. Chromatographic
columns were run using silica gel 60 (230–400 mesh ASTM) or
aluminum oxide (activated, Neutral Brockman I STD grade 150
mesh). Solvents for synthesis purposes were used at GPR grade.
Analytical TLC was performed using Merck Kieselgel 60 F254 silica
gel plates or Polygram Alox N/UV254 aluminum oxide plates.
Visualization was by UV light (254 nm). NMR spectra were
recorded in a Bruker DPX-400 Avance spectrometer operating at
400.13 and 600.1 MHz for 1H NMR and 100.6 and 150.9 MHz for
13C NMR. Shifts are referenced to the internal solvent signals. NMR
data were processed using Bruker Win-NMR 5.0 software. Elec-
trospray mass spectra were recorded on a Mass Lynx NT V 3.4 on
a Waters 600 controller connected to a 996 photodiode array
detector with methanol, water, or ethanol as carrier solvents. Melting
points were determined using an Electrothermal IA9000 digital
melting point apparatus and are uncorrected. Infrared spectra were
recorded on a Mattson Genesis II FTIR spectrometer equipped with
a Gateway 2000 4DX2-66 workstation and on a PerkinElmer
Spectrum One FT-IR spectrometer equipped with Universal ATR
sampling accessory. Sample analysis was carried out in Nujol using
NaCl plates. Elemental analysis was carried out at the Microanalysis
Laboratory, School of Chemistry and Chemical Biology, University
College Dublin. Analytical results were within (0.4% of the
theoretical (calcd) values unless otherwise noted.
205–207 °C; IR (Nujol) ν 3352, 3330 (NH), 1704, 1646 (CO) cm-1
.
1H NMR (DMSO-d6) δ 1.51 (s, 9H, (CH3)3), 7.06–7.15 (m, 1H,
Ar), 7.32–7.42 (m, 2H, Ar), 7.54–7.67 (m, 2H, Ar), 7.74–7.85 (m,
2H, Ar), 7.89–7.99 (m, 2H, Ar), 9.72 (broad s, 1H, NH), 10.09
(broad s, 1H, NH); 13C NMR (DMSO-d6) δ 29.4 ((CH3)3), 80.9
(C(CH3)3), 118.5, 121.7, 124.8, 129.4, 129.9, 130.0, 140.7, 144.1
(Ar), 154.0 ((CH3)3COCO), 166.3 (PhNHCOPh).
4-Aminobenzanilide (22). A solution of 3.113 g (10.0 mmol)
of 22c in 35 mL of CH2Cl2/TFA (1:1) was stirred at room
temperature for 6 h. After that time, the solvent was eliminated
under vacuum to generate the trifluoroacetate salt. This salt was
redissolved in 20 mL of CH2Cl2 and was washed with a 2 M NaOH
solution (2 × 15 mL). The organic layer was dried over anhydrous
Na2SO4 and filtered and the solvent was evaporated to afford 1.961
g (93%) of 4-aminobenzanilide as a white solid: mp 136–138 °C
(lit.,49 138–140 °C); IR (Nujol) ν 3393, 3350, 3184 (NH, NH2),
(1) Synthesis of the Starting Material Amines. 1,3-Bis(4-
aminophenyl)urea (7).27 A suspension of 5 g (16.5 mmol) of 1,3-
bis(4-nitrophenyl)urea in methanol in the presence of 5% Pd-C
(480 mg) was hydrogenated at atmospheric pressure and room
temperature for 22 h. Afterward, the catalyst was filtered through
a pad of Celite. The filter cake was rinsed with MeOH/DMF (1:1)
and the solvent removed in vacuo to afford 3.9 g (97%) of 1,3-
1644 (CO) cm-1 1H NMR (DMSO-d6)50 δ 5.79 (broad s, 2H,
;
1
NH2), 6.64 (d, 2H, J ) 8.8 Hz, Ar), 7.02–7.11 (m, 1H, Ar),
7.31–7.40 (m, 2H, Ar), 7.72–7.86 (m, 4H, Ar), 9.80 (broad s, 1H,
NH); 13C NMR (DMSO-d6) δ 113.9, 121.5, 122.4, 124.2, 129.8,
130.7, 141.1, 153.5 (Ar), 166.7 (CO).
bis(4-aminophenyl)urea 7 as a white solid. H NMR (DMSO-d6)
δ 4.73 (broad s, 4H, NH2), 6.51 (d, 4H, J ) 8.0 Hz, Ar), 7.07 (d,
4H, J ) 8.0 Hz, Ar), 8.08 (broad s, 2H, NH); 13C NMR (DMSO-
d6) δ 115.6, 121.8, 130.6, 145.0 (Ar), 154.8 (CO).
(2) Synthesis of Boc-Protected Precursors and New Tested
Compounds. General Method A for the Synthesis of the
Boc-Protected Guanidine and Boc-Protected 2-Aminoimidazo-
line Precursors. An amount of 6.6 mmol of HgCl2 was added over
a solution of 3.0 mmol of the corresponding diamine, 6.0 mmol of
N,N′-di(tert-butoxycarbonyl)thiourea (for 7d, 9d, and 11d), or N,N′-
di(tert-butoxycarbonyl)imidazolidine-2-thione (for 7e, 9e, and 11e)
and 2.1 mL (15.0 mmol) of Et3N in DMF (10 mL) at 0 °C. The
resulting mixture was stirred at 0 °C for 1 h and for the appropriate
duration at room temperature. Then the reaction mixture was diluted
with EtOAc and filtered through a pad of Celite. The filter cake
1,4-Bis(4-nitrophenyl)piperidine (11f). A solution of 2.5 g (12.1
mmol) of 4-(4-nitrophenyl)piperidine28,29 and 1.712 g (12.1 mmol)
of 1-fluoro-4-nitrobenzene in DMF (8 mL) was heated at 100 °C
for 72 h. Afterward, the solvent was removed in vacuo and the
residue obtained was recrystallized from CH3CN to yield 2.95 g
(74%) of 1,4-bis(4-nitrophenyl)piperidine 11f as a yellowish solid:
mp 170–172 °C; MS (ESI+) m/z 350.1105 [M + Na]+; IR (Nujol)
ν 1597, 1516 (NO2) cm-1; 1H NMR (DMSO-d6) δ 1.62–1.80 (m,
2H, H3Pip), 1.83–1.97 (m, 2H, H3Pip), 2.99–3.19 (m, 3H, H2ePip
+ H4Pip), 4.16–4.31 (m, 2H, H2aPip), 7.08 (d, 2H, J ) 8.8 Hz,
Ar), 7.56 (d, 2H, J ) 7.8 Hz, Ar), 8.06 (d, 2H, J ) 8.8 Hz, Ar),

918 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
was rinsed with EtOAc. The organic phase was extracted with water
(2 × 30 mL), washed with brine (1 × 30 mL), dried over anhydrous
Na2SO4, and concentrated under vacuum to give a residue that was
purified by column chromatography as specified.
4,4′-Bis[2,3-di(tert-butoxycarbonyl)guanidino]benzanilide (9d).
Following the general synthetic method A, the crude residue was
purified by silica gel column chromatography, eluting with hexane/
EtOAc (2:1) to yield 1.3 g (61%) of 9d as a white solid: mp >300
°C; MS (ESI+) m/z 712.3635 [M + H]+; IR (Nujol) ν 3368, 3291,
3261 (NH), 1738, 1720, 1645, 1627, 1608 (CO, CN) cm-1; 1H NMR
(CDCl3) δ 1.48 (s, 9H, (CH3)3), 1.51 (s, 9H, (CH3)3), 1.56 (s, 18H,
(CH3)3), 7.54 (d, 2H, J ) 8.6 Hz, Ar), 7.64 (d, 2H, J ) 8.0 Hz, Ar),
7.68 (d, 2H, J ) 8.0 Hz, Ar), 7.82 (d, 2H, J ) 8.6 Hz, Ar), 8.19
(broad s, 1H, PhNHCOPh), 10.30 (broad s, 1H, NHGu), 10.51 (broad
s, 1H, NHGu), 11.63 (broad s, 2H, NHGu); 13C NMR (CDCl3) δ 27.9,
28.0 ((CH3)3), 79.6, 80.0, 83.6, 84.0 (C(CH3)3), 120.6, 122.1, 123.4,
128.1, 131.2, 132.1, 135.9, 139.3 (Ar), 153.2, 153.7, 154.0
((CH3)3COCO), 163.1, 163.4 (CN), 165.2 (PhNHCOPh).
General Method B for the Removal of the Boc-Protecting
Groups and Regeneration of the Hydrochloride Salts. A solution
of the corresponding Boc-protected derivative (0.5 mmol) in 20
mL of CH2Cl2/TFA (1:1) was stirred at room temperature for the
appropriate duration. After that time, the solvent was eliminated
under vacuum to generate the trifluoroacetate salt. This salt was
dissolved in 20 mL of water and treated for 24 h with IRA400
Amberlyte resin in its Cl- form. Then the resin was removed by
filtration and the aqueous solution washed with CH2Cl2 (2 × 10
mL). Evaporation of the water afforded the pure dihydrochloride
salt as a hygroscopic solid. Absence of the trifluoroacetate salt was
checked by 19F NMR.
Alternative Method C for the Synthesis of the Boc-Protected
Guanidines 20d, 21d, and 23d. A solution of 4-[2,3-di(tert-
butoxycarbonyl)guanidino]aniline 8 in dry CH2Cl2 was treated under
an inert atmosphere and at 0 °C with the corresponding electrophile
(phenyl isocyanate for 20d, phenyl isothiocyanate for 21d, and
benzoyl chloride for 23d) and with Et3N (only in the case of 23d).
After the reaction mixture was allowed to reach room temperature,
it was stirred for the appropriate duration. Further workup followed
by column cromatography as specified afforded the corresponding
Boc-protected guanidine.
4,4′-Bis[1,3-di(tert-butoxycarbonyl)-2-imidazolidinylimino]ben-
zanilide (9e). Following the general synthetic method A, the crude
residue was purified by neutral alumina column flash chromatog-
raphy, eluting with hexane/EtOAc (1:4). The residue obtained after
the column was recrystallized from Et2O to yield 1.040 g (45%) of
9e as a white solid: mp 198–200 °C; IR (Nujol) ν 3346 (NH), 1733,
1
1709, 1689, 1663, 1598 (CO, CN) cm-1; H NMR (DMSO-d6) δ
1.30 (s, 36H, (CH3)3), 3.77 (s, 4H, CH2), 3.80 (s, 4H, CH2), 6.83
(d, 2H, J ) 8.5 Hz, Ar), 6.92 (d, 2H, J ) 8.5 Hz, Ar), 7.66 (d, 2H,
J ) 8.5 Hz, Ar), 7.89 (d, 2H, J ) 8.5 Hz, Ar), 9.92 (broad s, 1H,
NH); 13C NMR (DMSO-d6) δ 28.4, 28.5 ((CH3)3), 43.9, 44.0 (CH2),
82.4, 82.6 (C(CH3)3), 121.3, 121.6, 121.7, 128.6, 129.3, 134.8,
139.8, 141.0 (Ar), 145.1, 150.4, 150.6, 152.9 ((CH3)3COCO), CN),
165.4 (PhNHCOPh).
1,3-Bis(4-[2,3-di(tert-butoxycarbonyl)guanidino]phenyl)urea
(7d). Following the general synthetic method A, the crude residue
was purified by silica gel column chromatography, eluting with
hexane/EtOAc (3:1) to yield 1.24 g (57%) of 7d as a white solid:
mp >300 °C; MS (ESI+) m/z 727.3777 [M + H]+; IR (Nujol) ν
1,4-Bis[4-(N2,N3-bis(tert-butyloxycarbonyl)guanidino)phe-
nyl]piperidine (11d). General method A was used, with 598 mg
(2.2 mmol) of HgCl2, 268 mg (1.0 mmol) of 11, 553 mg (2.0 mmol)
of N,N′-di(tert-butoxycarbonyl)thiourea, and 0.9 mL (6.4 mmol)
of Et3N in CH2Cl2/DMF (1:2) (8 mL) at 0 °C. The resulting mixture
was stirred at 0 °C for 1 and 36 h more at room temperature. After
general workup, the residue was purified by silica gel column
chromatography, eluting with hexane/EtOAc (3:1) to yield 483 mg
(64%) of 11d as a white solid: mp, decomposes at 220 °C; MS
(ESI+) m/z 752.4326 [M + H]+; IR (Nujol) ν 3291, 3266 (NH),
3368, 3307, 3264 (NH), 1720, 1644, 1624, 1605 (CO, CN) cm-1
;
1H NMR (CDCl3) δ 1.46 (s, 18H, (CH3)3), 1.58 (s, 18H, (CH3)3),
7.25 (d, 4H, J ) 8.5 Hz, Ar), 7.29 (d, 4H, J ) 8.5 Hz, Ar), 7.40
(broad s, 2H, NHCONH), 10.14 (broad s, 2H, NHGu), 11.64 (broad
s, 2H, NHGu); 13C NMR (CDCl3) δ 28.0, 28.1 ((CH3)3), 80.0, 83.8
(C(CH3)3), 118.8, 124.8, 129.9, 137.6 (Ar), 152.1, 153.1
((CH3)3COCO), 155.3, 163.2 (NHCONH, CN).
1
1728, 1638, 1608 (CO, CN) cm-1; H NMR (CDCl3) δ 1.52 (s,
1,3-Bis(4-[1,3-di(tert-butoxycarbonyl)-2-imidazolidinylimi-
no]phenyl)urea (7e). Following the general synthetic method A,
the crude residue was purified by neutral alumina column flash
chromatography, eluting with CH2Cl2/EtOAc (1:5). The residue
obtained after the column was precipitated with cold hexane to yield
1.25 g (63%) of 7e as a white solid: mp 180–182 °C; IR (Nujol) ν
3357 (NH), 1757, 1704, 1648 (CO, CN) cm-1; 1H NMR (CDCl3)
δ 1.33 (s, 36H, (CH3)3), 3.83 (s, 8H, CH2), 6.90 (d, 4H, J ) 7.0
Hz, Ar), 7.14–7.26 (m, 6H, 4Ar + 2NH); 13C NMR (CDCl3) δ
27.8 ((CH3)3), 43.1 (CH2), 82.9 (C(CH3)3), 120.6, 121.8, 133.9,
139.3 (Ar), 143.4 ((CH3)3COCO), 150.2, 153.5 (NHCONH, CN).
Dihydrochloride Salt of 1,3-Bis(4-guanidinophenyl)urea (7a).
Following general method B, an amount of 210 mg (95%) of the
pure dihydrochloride salt of 7a was obtained as a white solid: mp,
18H, (CH3)3), 1.55 (s, 18H, (CH3)3), 1.82–2.00 (m, 4H, H3Pip),
2.56–2.69 (m, 1H, H4Pip), 2.71–2.89 (m, 2H, H2ePip), 3.71–3.82
(m, 2H, H2aPip), 6.95 (d, 2H, J ) 8.0 Hz, Ar), 7.22 (d, 2H, J )
7.0 Hz, Ar), 7.48 (d, 2H, J ) 8.0 Hz, Ar), 7.54 (d, 2H, J ) 7.0 Hz,
Ar), 10.19 (broad s, 1H, NH), 10.29 (broad s, 1H, NH), 11.68 (broad
s, 2H, NH); 13C NMR (CDCl3) δ 28.0, 28.1, 28.2 ((CH3)3), 33.1
(C3Pip), 41.8 (C4Pip), 50.8 (C2Pip), 79.3, 79.4, 83.3, 83.5 (C(CH3)3),
117.0, 122.4, 123.3, 127.2, 128.7, 134.7, 142.5, 149.0 (Ar), 153.2,
153.3, 153.4, 153.5 (CO), 163.5, 163.6 (CN).
Di-tert-butyl-2-(4-[4-(4-[1,3-bis(tert-butyloxycarbonyl)tetrahy-
dro-1H-2-imidazolyliden]aminophenyl)piperidino]phenylimino)-
1,3-imidazolidinedicarboxylate (11e). Method A was used, with
598 mg (2.2 mmol) of HgCl2, 268 mg (1.0 mmol) of 11, 605 mg
(2.0 mmol) of N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione,
and 0.9 mL (6.4 mmol) of Et3N in CH2Cl2/DMF (1:2) (8 mL) at 0
°C. The resulting mixture was stirred at 0 °C for 1 and 41 h more
at room temperature. The reaction was worked up and purified by
silica gel column chromatography, eluting with hexane/EtOAc (1:1).
The residue obtained after the column was recrystallized from
hexane/Et2O (1:1) to yield 523 mg (65%) of 11e as a white solid:
mp 191–193 °C; MS (ESI+) m/z 804.4681 [M + H]+; IR (Nujol)
1
decomposes at 248 °C; MS (ESI+) m/z 327.1928 [M + H]+; H
NMR (D2O) δ 7.27 (d, 4H, J ) 8.5 Hz, Ar), 7.43 (d, 4H, J ) 8.5
Hz, Ar); 13C NMR (D2O) δ 121.6, 126.4, 129.1, 137.0 (Ar), 155.0,
156.0 (CO, CN). Anal. (C15H20Cl2N8O·2.3H2O) C, H, N.
1,3-Bis(4-[2,3-di(tert-butoxycarbonyl)guanidino]phenyl)thiourea-
(8d). A solution of 701 mg (2.0 mmol) of 4-[2,3-di(tert-butoxy-
carbonyl)guanidino]aniline 8 and 3.0 mL (50.0 mmol) of carbon
disulfide in 20 mL of CH2Cl2 was stirred at reflux for 54 h.
Afterward, the solvent was evaporated to give a residue that was
purified by silica gel column chromatography, eluting with hexane/
EtOAc (3:2) to yield 240 mg (32%) of 8d as a white solid: mp
>300 °C; MS (ESI+) m/z 743.3412 [M + H]+; IR (Nujol) ν 3396,
1
ν 1753, 1704, 1662, 1606 (CO, CN) cm-1; H NMR (CDCl3) δ
1.32 (s, 18H, (CH3)3), 1.34 (s, 18H, (CH3)3), 1.83–1.98 (m, 4H,
H3Pip), 2.49–2.61 (m, 1H, H4Pip), 2.66–2.79 (m, 2H, H2ePip),
3.60–3.71 (m, 2H, H2aPip), 3.83 (s, 8H, CH2), 6.85–7.0 (m, 6H,
Ar), 7.11 (d, 2H, J ) 8.0 Hz, Ar); 13C NMR (CDCl3) δ 27.8, 27.9
((CH3)3), 33.4 (C3Pip), 41.8 (C4Pip), 43.0 (CH2), 51.8 (C2Pip), 82.5,
82.7 (C(CH3)3), 117.8, 121.4, 122.2, 126.9, 138.4, 139.0, 140.4,
141.1 (Ar), 146.4, 148.0 (CO); 150.3, 150.4 (CN).
3291, 3169 (NH), 1720, 1646, 1630, 1152 (CO, CN, CS) cm-1
;
1H NMR (CDCl3) δ 1.50 (s, 18H, (CH3)3), 1.55 (s, 18H, (CH3)3),
7.34 (d, 4H, J ) 8.0 Hz, Ar), 7.61 (d, 4H, J ) 8.0 Hz, Ar), 7.98
(broad s, 2H, NHCSNH), 10.36 (broad s, 2H, NHGu), 11.63 (broad
s, 2H, NHGu); 13C NMR (CDCl3) δ 27.8, 27.9 ((CH3)3), 79.6, 83.6
(C(CH3)3), 123.4, 124.9, 134.0, 134.8 (Ar), 152.9, 153.8 (CO), 163.1
(CN), 179.4 (CS).
Trihydrochloride Salt of 1,4-Bis(4-guanidinophenyl)piperi-
dine (11a). Following method B, an amount of 224 mg (90%) of
the pure hydrochloride salt of 11a was obtained as a brown solid:

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 919
1
mp, decomposes at 220 °C; H NMR (D2O) δ 2.19–2.33 (m, 4H,
H3Pip), 3.10–3.24 (m, 1H, H4Pip), 3.80–3.93 (m, 4H, H2Pip), 7.32
(d, 2H, J ) 8.0 Hz, Ar), 7.48 (d, 2H, J ) 8.0 Hz, Ar), 7.54 (d, 2H,
J ) 8.5 Hz, Ar), 7.78 (d, 2H, J ) 8.5 Hz, Ar); 13C NMR (D2O) δ
29.9 (C3Pip) 37.4 (C4Pip), 56.3 (C2Pip) 122.3, 125.8, 126.6, 127.8,
132.3, 135.9, 139.4, 142.9 (Ar), 155.6, 155.9 (CN). Anal.
(C19H28Cl3N7 ·2H2O) C, H, N.
mL (9.3 mmol) of Et3N in DMF (6 mL) at 0 °C. The resulting
mixture was stirred at 0 °C for 1 and 26 h more at room temperature.
Then the reaction mixture was diluted with EtOAc and filtered
through a pad of Celite. The filter cake was rinsed with EtOAc.
The organic phase was extracted with water (2 × 30 mL), washed
with brine (1 × 30 mL), dried over anhydrous Na2SO4, and
concentrated under vacuum to give a residue that was purified by
silica gel column chromatography, eluting with hexane/EtOAc (1:1)
to yield 940 mg (67%) of 20d as a white solid.
Method C is as follows. An amount of 0.33 mL (3.0 mmol) of
phenyl isocyanate was added under an inert atmosphere and at 0
°C over a solution of 1.052 g (3.0 mmol) of 4-[2,3-di(tert-
butoxycarbonyl)guanidino]aniline 8 in dry CH2Cl2 (10 mL). The
mixture was allowed to reach room temperature and was stirred
for 10 h. Then the solvent was removed in vacuo and the residue
obtained was purified by silica gel column chromatography, eluting
with hexane/EtOAc (1:1). The pure compound 20d was obtained
as a white solid (1.034 g, 73%) by recrystallization from hexane/
CH2Cl2: mp >300 °C; MS (ESI+) m/z 470.2425 [M + H]+; IR
(Nujol) ν 3300 (NH), 1726, 1633, 1600, 1568 (CO, CN) cm-1; 1H
NMR (DMSO-d6) δ 1.42 (s, 9H, (CH3)3), 1.53 (s, 9H, (CH3)3),
6.94–7.02 (m, 1H, Ar), 7.24–7.33 (m, 2H, Ar), 7.38–7.53 (m, 6H,
Ar), 8.69 (broad s, 1H, NHCONH), 8.71 (broad s, 1H, NHCONH),
9.96 (broad s, 1H, NHGu), 11.54 (broad s, 1H, NHGu); 13C NMR
(DMSO-d6) δ 28.9, 29.2 ((CH3)3), 80.0, 84.6 (C(CH3)3), 119.5,
119.6, 123.1, 125.1, 130.1, 131.8, 138.2, 141.0 (Ar), 153.6, 153.8,
154.5, 164.2 ((CH3)3COCO, CN, NHCONH).
Trihydrochloride Salt of 1,4-Bis[4-(4,5-dihydro-1H-2-imida-
zolylamino)phenyl]piperidine (11b). Following method B, an
amount of 279 mg (94%) of the pure hydrochloride salt of 11b
was obtained as a brown solid: mp 101–103 °C; MS (ESI+) m/z
404.2578 [M + H]+; 1H NMR (D2O) δ 2.14–2.29 (m, 4H, H3Pip),
3.07–3.19 (m, 1H, H4Pip), 3.72 (s, 4H, CH2), 3.76 (s, 4H, CH2),
3.74–3.86 (m, 4H, H2Pip), 7.26 (d, 2H, J ) 8.6 Hz, Ar), 7.43 (d,
2H, J ) 8.0 Hz, Ar), 7.49 (d, 2H, J ) 8.6 Hz, Ar), 7.75 (d, 2H, J
) 8.0 Hz, Ar); 13C NMR (D2O) δ 29.8 (C3Pip) 37.3 (C4Pip), 42.2,
42.3 (CH2), 56.3 (C2Pip) 122.2, 124.0, 125.0, 127.7, 133.2, 136.5,
138.9, 142.3 (Ar), 157.8, 158.2 (CN). Anal. (C23H32Cl3N7 ·2.3H2O)
C, H, N.
4-[2,3-Di(tert-butoxycarbonyl)guanidino]benzophenone (19d).
Method A was used, with 896 mg (3.3 mmol) of HgCl2, 592 mg
(3.0 mmol) of 4-aminobenzophenone, 830 mg (3.0 mmol) of N,N′-
di(tert-butoxycarbonyl)thiourea, and 1.3 mL (9.3 mmol) of Et3N
in DMF (5 mL) at 0 °C. The resulting mixture was stirred at 0 °C
for 1 and 48 h more at room temperature. The reaction was worked
up and purified by silica gel column chromatography, eluting with
hexane/EtOAc (2:1) to yield 976 mg (74%) of 19d as a white solid:
mp 151–153 °C; IR (Nujol) ν 3197, 3154 (NH), 1718, 1656, 1643,
1-(4-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]phe-
nyl)-3-phenylurea (20e). Method A was used, with 896 mg (3.3
mmol) of HgCl2, 682 mg (3.0 mmol) of 20, 907 mg (3.0 mmol) of
N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione, and 1.3 mL (9.3
mmol) of Et3N in DMF (6 mL) at 0 °C. The resulting mixture was
stirred at 0 °C for 1 and 38 h more at room temperature. Then the
reaction mixture was diluted with EtOAc and filtered through a
pad of Celite. The filter cake was rinsed with EtOAc. The organic
phase was extracted with water (2 × 30 mL), washed with brine
(1 × 30 mL), dried over anhydrous Na2SO4, and concentrated under
vacuum to give a residue that was purified by neutral alumina
column flash chromatography, eluting with hexane/EtOAc/CH2Cl2
(6:4:1) to yield 924 mg (62%) of 20e as a white solid: mp 162–164
°C; IR (Nujol) ν 3365 (NH), 1739, 1728, 1708, 1694 (CO, CN)
1595 (CO, CN) cm-1; H NMR (CDCl3) δ 1.53 (s, 9H, (CH3)3),
1
1.56 (s, 9H, (CH3)3), 7.44–7.51 (m, 2H, Ar), 7.54–7.60 (m, 1H,
Ar), 7.75–7.86 (m, 6H, Ar), 10.63 (broad s, 1H, NH), 11.66 (broad
s, 1H, NH); 13C NMR (CDCl3) δ 28.0, 28.1 ((CH3)3), 80.0, 84.1
(C(CH3)3), 120.9, 128.1, 129.8, 131.3, 132.1, 133.2, 137.8, 140.8
(Ar), 153.1, 153.2 ((CH3)3COCO), 163.2 (CN), 195.6 (PhCOPh).
4-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]ben-
zophenone (19e). Method A was used, with 896 mg (3.3 mmol)
of HgCl2, 592 mg (3.0 mmol) of 4-aminobenzophenone, 907 mg
(3.0 mmol) of N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione,
and 1.3 mL (9.3 mmol) of Et3N in DMF (5 mL). The resulting
mixture was stirred at 0 °C for 1 and 48 h more at room temperature.
The reaction was worked up and purified by neutral alumina column
flash chromatography, eluting with hexane/EtOAc (2:3). The residue
obtained after the column was precipitated and washed with cold
hexane/Et2O (1:1) to yield 600 mg (43%) of 19e as a white solid:
mp 49–51 °C; IR (Nujol) ν 1750, 1719, 1644 (CO, CN) cm-1; 1H
NMR (CDCl3) δ 1.28 (s, 18H, (CH3)3), 3.78 (s, 4H, CH2), 6.96 (d,
2H, J ) 8.5 Hz, Ar), 7.33–7.39 (m, 2H, Ar), 7.41–7.48 (m, 1H,
Ar), 7.61–7.70 (m, 4H, Ar); 13C NMR (CDCl3) δ 27.5 ((CH3)3),
42.9 (CH2), 82.6 (C(CH3)3), 120.6, 127.7, 129.2, 130.7, 131.1,
131.4, 138.1, 140.1 (Ar), 149.5 ((CH3)3COCO), 152.9 (CN), 195.4
(PhCOPh).
1
cm-1; H NMR (DMSO-d6) δ 1.29 (s, 18H, (CH3)3), 3.75 (s, 4H,
CH2), 6.79 (d, 2H, J ) 7.0 Hz, Ar), 6.92–7.01 (m, 1H, Ar),
7.23–7.36 (m, 4H, Ar), 7.45 (d, 2H, J ) 7.5 Hz, Ar), 8.51 (broad
s, 1H, NH), 8.57 (broad s, 1H, NH); 13C NMR (DMSO-d6) δ 28.8
((CH3)3), 44.2 (CH2), 82.7 (C(CH3)3), 119.4, 120.0, 122.5, 122.9,
130.1, 135.5, 140.0, 141.2 (Ar), 144.3, 151.0, 153.8 ((CH3)3COCO,
CN, NHCONH).
Trifluoroacetate Salt of 1-(4-Guanidinophenyl)-3-phenylurea
(20a). A solution of 235 mg (0.5 mmol) of 20d in 20 mL of CH2Cl2/
TFA (1:1) was stirred at room temperature for 3 h. After that time,
the solvent was eliminated under vacuum and cold water was added
to generate a precipitate that was filtered, washed with an additional
100 mL of cold water, and dried to yield 176 mg (91%) of the
pure trifluoroacetate salt as a purple solid: mp 116–118 °C; MS
Hydrochloride Salt of 4-Guanidinobenzophenone (19a). Fol-
lowing method B, 145 mg (97%) of the pure hydrochloride salt of
19a was obtained as a white solid: mp 187–189 °C; MS (ESI+)
m/z 240.1208 [M + H]+; H NMR (D2O) δ 7.15 (d, 2H, J ) 8.0
1
1
(ESI+) m/z 270.1328 [M + H]+; H NMR (CD3OD) δ 6.96–7.03
Hz, Ar), 7.23–7.31 (m, 2H, Ar), 7.40 (d, 2H, J ) 8.0 Hz, Ar),
7.44–7.52 (m, 3H, Ar); 13C NMR (D2O) δ 122.7, 127.9, 129.5,
131.4, 132.9, 133.7, 135.6, 138.7 (Ar), 154.9 (CN), 197.6 (CO).
Hydrochloride Salt of 4-(2-Imidazolidinylimino)benzophe-
none (19b). Following method B, 156 mg (92%) of the pure
hydrochloride salt of 19b were obtained as a yellow solid: mp 64–66
(m, 1H, Ar), 7.18 (d, 2H, J ) 8.5 Hz, Ar), 7.23–7.31 (m, 2H, Ar),
7.36–7.45 (m, 2H, Ar), 7.43 (d, 2H, J ) 8.0 Hz, Ar); 13C NMR
(CD3OD) δ 120.5, 121.4, 123.8, 124.0, 127.7, 129.8, 140.3, 140.4
(Ar), 155.3, 158.3 (CN, CO); 19F NMR (CD3OD) δ -79.35. Anal.
(C16H16F3N5O3 ·0.2H2O) C, H, N.
°C; MS (ESI+) m/z 266.1324 [M + H]+; H NMR (D2O) δ 3.64
1
Trifluoroacetate Salt of 1-[4-(2-imidazolidinylimino)phenyl]-
3-phenylurea (20b). A solution of 248 mg (0.5 mmol) of 20e in
20 mL of CH2Cl2/TFA (1:1) was stirred at room temperature for
3 h. After that time, the solvent was eliminated under vacuum and
cold water was added to generate a precipitate that was filtered,
washed with an additional 100 mL of cold water, and dried to yield
194 mg (89%) of the pure trifluoroacetate salt as a gray solid: mp
102–104 °C; MS (ESI+) m/z 296.1522 [M + H]+; 1H NMR
(CD3OD) δ 3.73 (s, 4H, CH2), 6.96–7.04 (m, 1H, Ar), 7.18 (d, 2H,
J ) 9.0 Hz, Ar), 7.22–7.30 (m, 2H, Ar), 7.44 (d, 2H, J ) 7.6 Hz,
(s, 4H, CH2), 7.01 (d, 2H, J ) 8.8 Hz, Ar), 7.18–7.25 (m, 2H, Ar),
7.31 (d, 2H, J ) 7.2 Hz, Ar), 7.34–7.43 (m, 3H, Ar); 13C NMR
(D2O) δ 42.2 (CH2), 120.7, 127.9, 129.4, 131.4, 132.8, 133.0, 135.5,
139.2 (Ar), 156.5 (CN), 197.1 (CO). Anal. (C16H16ClN3O·2.0H2O)
C, H, N.
1-(4-[2,3-Di(tert-butoxycarbonyl)guanidino]phenyl)-3-phenyl-
urea (20d). Method A was used, with 896 mg (3.3 mmol) of HgCl2,
682 mg (3.0 mmol) of 1-(4-aminophenyl)-3-phenylurea 20, 830
mg (3.0 mmol) of N,N′-di(tert-butoxycarbonyl)thiourea, and 1.3

920 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
Ar), 7.53 (d, 2H, J ) 9.0 Hz, Ar); 13C NMR (CD3OD) δ 44.1
(CH2), 120.5, 121.2, 124.0, 126.3, 129.8, 130.9, 140.1, 140.3 (Ar),
155.3, 160.4 (CN, CO); 19F NMR (CD3OD) δ -78.90. Anal.
(C18H18F3N5O3 ·1.4H2O) C, H, N.
4-[2,3-Di(tert-butoxycarbonyl)guanidino]benzanilide (22d).
Method A was used, with 896 mg (3.3 mmol) of HgCl2, 637 mg
(3.0 mmol) of 4-aminobenzanilide, 830 mg (3.0 mmol) of N,N′-
di(tert-butoxycarbonyl)thiourea, and 1.3 mL (9.3 mmol) of Et3N
in CH2Cl2/DMF (5:1) (6 mL) at 0 °C. The resulting mixture was
stirred at 0 °C for 1 and 22 h more at room temperature. The
reaction was worked up and purified by silica gel column chro-
matography, eluting with hexane/EtOAc (2:1) to yield 1.015 g
(74%) of 22d as a white solid: mp >300 °C; MS (ESI+) m/z
477.2269 [M + Na]+; IR (Nujol, cm-1) ν 3375, 3187 (NH), 1718,
1658, 1643, 1596 (CO, CN); 1H NMR (CDCl3) δ 1.54 (s, 9H,
(CH3)3), 1.57 (s, 9H, (CH3)3), 7.11–7.20 (m, 1H, Ar), 7.33–7.42
(m, 2H, Ar), 7.63–7.78 (m, 4H, Ar), 7.80–7.93 (m, 2H, Ar), 8.08
(broad s, 1H, PhNHCOPh), 10.55 (broad s, 1H, NHGu), 11.65 (broad
s, 1H, NHGu); 13C NMR (CDCl3) δ 27.9, 28.0 ((CH3)3), 80.1, 84.1
(C(CH3)3), 120.1, 122.4, 124.1, 128.0, 128.8, 131.3, 138.4, 139.3
(Ar), 153.1, 153.8 ((CH3)3COCO), 163.1 (CN), 165.6 (PhNHCOPh).
1-(4-[2,3-Di(tert-butoxycarbonyl)guanidino]phenyl)-3-phe-
nylthiourea (21d). Method C was used, with 0.37 mL (3.0 mmol)
of phenyl isothiocyanate and 1.052 g (3.0 mmol) of 4-[2,3-di(tert-
butoxycarbonyl)guanidine]aniline 8 in dry CH2Cl2 (10 mL). The
mixture was allowed to reach room temperature and was stirred
for 18 h. Then the solvent was removed in vacuo and the residue
obtained was purified by silica gel column chromatography, eluting
with hexane/EtOAc (1:1). The pure compound 21d was obtained
as a white solid (988 mg, 68%) by recrystallization from hexane/
CH2Cl2: mp 120–122 °C; MS (ESI+) m/z 486.2248 [M + H]+; IR
(Nujol) ν 3291, 3247, 3165, 3099 (NH), 1717, 1644, 1599, 1150
(CO, CN, CS) cm-1; 1H NMR (DMSO-d6) δ 1.43 (s, 9H, (CH3)3),
1.54 (s, 9H, (CH3)3), 7.09–7.17 (m, 1H, Ar), 7.29–7.40 (m, 2H,
Ar), 7.43–7.62 (m, 6H, Ar), 9.82 (broad s, 1H, NHCSNH), 9.83
(broad s, 1H, NHCSNH), 10.02 (broad s, 1H, NHGu), 11.50 (broad
s, 1H, NHGu); 13C NMR (DMSO-d6) δ 29.0, 29.2 ((CH3)3), 80.1,
84.7 (C(CH3)3), 124.2, 124.9, 125.2, 125.7, 129.7, 134.2, 137.6,
140.8 (Ar), 153.5, 154.2 (CO), 164.1 (CN), 180.8 (CS).
1-(4-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]phe-
nyl)-3-phenylthiourea (21e). An amount of 896 mg (3.3 mmol)
of HgCl2 was added over a solution of 325 mg (3.0 mmol) of 1,4-
phenylenediamine, 907 mg (3.0 mmol) of N,N′-di(tert-butoxycar-
bonyl)imidazolidine-2-thione, and 1.3 mL (9.3 mmol) of Et3N in
DMF (5 mL) at 0 °C. The resulting mixture was stirred at 0 °C for
1 and 22 h more at room temperature. Then the reaction mixture
was diluted with EtOAc and filtered through a pad of Celite. The
filter cake was rinsed with EtOAc. The organic phase was extracted
with water (2 × 30 mL), washed with brine (1 × 30 mL), dried
over anhydrous Na2SO4, and concentrated under vacuum to give a
dark residue. This residue was dissolved in 10 mL of dry CH2Cl2,
and 0.37 mL (3.0 mmol) of phenyl isothiocyanate was added under
an inert atmosphere and at 0 °C. The mixture was allowed to reach
room temperature and was stirred for 19 h. Then the solvent was
removed in vacuo and the new residue obtained was purified by
neutral alumina column flash chromatography, eluting with hexane/
EtOAc (3:7) to yield 347 mg (22%) of 21e as a white solid: mp
118–120 °C; IR (Nujol) ν 3311 (NH), 1756, 1716, 1151 (CO, CN,
CS) cm-1; 1H NMR (CDCl3) δ 1.34 (s, 18H, (CH3)3), 3.82 (s, 4H,
CH2), 6.99 (d, 2H, J ) 8.0 Hz, Ar), 7.16–7.25 (m, 3H, Ar),
7.30–7.42 (m, 4H, Ar), 7.89 (broad s, 1H, NH), 8.16 (broad s, 1H,
NH); 13C NMR (CDCl3) δ 27.8 ((CH3)3), 43.1 (CH2), 82.9
(C(CH3)3), 122.2, 124.7, 125.9, 126.1, 128.8, 130.8, 137.8, 140.0
(Ar), 147.5, 149.8 (CO, CN), 179.8 (CS).
4-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]benza-
nilide (22e). Method A was used, with 896 mg (3.3 mmol) of
HgCl2, 637 mg (3.0 mmol) of 4-aminobenzanilide, 907 mg (3.0
mmol) of N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione, and
1.3 mL (9.3 mmol) of Et3N in CH2Cl2/DMF (5:1) (6 mL) at 0 °C.
The resulting mixture was stirred at 0 °C for 1 and 25 h more at
room temperature. The reaction was worked up and purified by
neutral alumina column flash chromatography, eluting with hexane/
EtOAc (6:4). The residue obtained after the column was recrystal-
lized from hexane/Et2O (1:1) to yield 843 mg (58%) of 22e as a
white solid: mp 163–165 °C; IR (Nujol) ν 3338 (NH), 1737, 1707,
1
1670, 1637 (CO, CN) cm-1; H NMR (CDCl3) δ 1.34 (s, 18H,
(CH3)3), 3.85 (s, 4H, CH2), 7.03 (d, 2H, J ) 8.5 Hz, Ar), 7.08–7.16
(m, 1H, Ar), 7.29–7.39 (m, 2H, Ar), 7.66 (d, 2H, J ) 8.0 Hz, Ar),
7.80 (d, 2H, J ) 8.5 Hz, Ar), 8.04 (broad s, 1H, NH); 13C NMR
(CDCl3) δ 27.8 ((CH3)3), 43.2 (CH2), 83.0 (C(CH3)3), 120.0, 121.2,
123.9, 127.8, 128.1, 128.8, 138.3, 140.2 (Ar), 149.8 ((CH3)3COCO),
151.9 (CN), 165.3 (PhNHCOPh).
Hydrochloride Salt of 4-(2-Imidazolidinylimino)benzanilide
(22b). Following method B, an amount of 158 mg (94%) of the
pure hydrochloride salt of 22b was obtained as a white solid: mp,
1
decomposes at 270 °C; MS (ESI+) m/z 281.1369 [M + H]+; H
NMR (D2O) δ 3.71 (s, 4H, CH2), 7.22–7.32 (m, 3H, Ar), 7.41–7.52
(m, 4H, Ar), 7.74–7.83 (m, 2H, Ar); 13C NMR (D2O) δ 42.1 (CH2),
121.7, 121.8, 125.3, 128.6, 128.7, 130.7, 136.3, 138.4 (Ar), 157.0
(CN), 167.3 (CO). Anal. (C16H17ClN4O·1.0H2O) C, H, N.
4′-[2,3-Di(tert-butoxycarbonyl)guanidino]benzanilide (23d).
Method A was used, with 896 mg (3.3 mmol) of HgCl2, 637 mg
(3.0 mmol) of 4′-aminobenzanilide, 830 mg (3.0 mmol) of N,N′-
di(tert-butoxycarbonyl)thiourea, and 1.3 mL (9.3 mmol) of Et3N
in CH2Cl2/DMF (5:1) (6 mL) at 0 °C. The resulting mixture was
stirred at 0 °C for 1 and 22 h more at room temperature. The
reaction was worked up and purified by silica gel column chro-
matography, eluting with hexane/EtOAc (3:1) to yield 850 mg
(62%) of 23d as a white solid.
Method C is as follows. Amounts of 0.28 mL (2.0 mmol) of
Et3N and 0.23 mL (2.0 mmol) of benzoyl chloride were added under
an inert atmosphere and at 0 °C to a solution of 701 mg (2.0 mmol)
of 4-[2,3-di(tert-butoxycarbonyl)guanidino]aniline 8 in dry CH2Cl2
(10 mL). The reaction mixture was allowed to reach room
temperature and stirred for 16 h. Then an additional 20 mL of
CH2Cl2 was added and the organic phase was washed with water
(2 × 15 mL) and dried over anhydrous Na2SO4. The solvent was
removed in vacuo and the residue obtained was purified by silica
gel column chromatography, eluting with hexane/EtOAc (3:1) to
yield 820 mg (90%) of 23d as a white solid: mp >300 °C; MS
(ESI+) m/z 477.2126 [M + Na]+; IR (Nujol) ν 3379, 3261, 3170
(NH), 1718, 1645, 1627, 1575 (CO, CN) cm-1; 1H NMR (CDCl3)
δ 1.36 (s, 9H, (CH3)3), 1.57 (s, 9H, (CH3)3), 7.25 (d, 2H, J ) 9.0
Hz, Ar), 7.38–7.52 (m, 3H, Ar), 7.56 (d, 2H, J ) 8.5 Hz, Ar), 7.81
(d, 2H, J ) 7.0 Hz, Ar), 8.74 (broad s, 1H, PhNHCOPh), 10.03
(broad s, 1H, NHGu), 11.50 (broad s, 1H, NHGu); 13C NMR (CDCl3)
δ 27.9, 28.0 ((CH3)3), 79.7, 83.7 (C(CH3)3), 120.7, 124.5, 127.1,
Trifluoroacetate Salt of 1-(4-Guanidinophenyl)-3-phenylth-
iourea (21a). A solution of 243 mg (0.5 mmol) of 21d in 20 mL
of CH2Cl2/TFA (1:1) was stirred at room temperature for 3 h. After
that time, the solvent was eliminated under vacuum and cold water
was added to generate a precipitate that was filtered, washed with
an additional 100 mL of cold water, and dried to yield 197 mg
(94%) of the pure trifluoroacetate salt as a white solid: mp 166–168
1
°C; MS (ESI+) m/z 286.1183 [M + H]+; H NMR (CD3OD) δ
7.16–7.28 (m, 3H, Ar), 7.31–7.47 (m, 4H, Ar), 7.53 (d, 2H, J )
7.5 Hz, Ar); 13C NMR (CD3OD) δ 122.4, 123.6, 124.2, 124.6,
126.6, 129.9, 136.5, 136.9 (Ar), 158.4 (CN), 182.4 (CS); 19F NMR
(CD3OD) δ -78.97. Anal. (C16H16F3N5O2S·1.0H2O) C, H, N.
Trifluoroacetate Salt of 1-[4-(2-Imidazolidinylimino)phenyl]-
3-phenylthiourea (21b). A solution of 256 mg (0.5 mmol) of 21e
in 20 mL of CH2Cl2/TFA (1:1) was stirred at room temperature
for 3 h. After that time, the solvent was eliminated under vacuum
and cold water was added to generate a precipitate that was filtered,
washed with an additional 100 mL of cold water, and dried to yield
221 mg (92%) of the pure trifluoroacetate salt as a yellow solid:
1
mp 138–140 °C; MS (ESI+) m/z 312.1379 [M + H]+; H NMR
(D2O) δ 3.72 (s, 4H, CH2), 7.26–7.40 (m, 7H, Ar), 7.41–7.48 (m,
2H, Ar); 13C NMR (D2O) δ 42.2 (CH2), 124.7, 125.8, 127.1, 127.4,
129.1, 133.3, 136.3, 136.6 (Ar), 158.2 (CN), 179.4 (CS); 19F NMR
(D2O) δ -76.15. Anal. (C18H18F3N5O2S·3.0H2O) C, H, N.

DNA Minor GrooVe Binders
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 921
128.2, 131.6, 131.2, 135.5, 136.5 (Ar), 153.0, 154.8 ((CH3)3COCO),
163.1 (CN), 166.0 (PhNH C OPh).
path length was filled with a 1 mL solution of DNA polymer or
DNA-compound complex. The DNA polymer (40 µM base) and
the compound solution (12 µM) were prepared in a low salt buffer
(0.01 M [2-(N-morpholino)ethanesulfonic acid], 0.001 M disodium
EDTA, adjusted to pH 6.25) so that a compound to DNA base
ratio of 0.3 was obtained. This effectively saturates all minor groove
binding sites for these compounds. The thermal melting tempera-
tures of the duplex or duplex-compound complex obtained from
the first derivative of the melting curves are reported.
4′-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]benza-
nilide (23e). Method A was used, with 896 mg (3.3 mmol) of
HgCl2, 637 mg (3.0 mmol) of 4′-aminobenzanilide 23, 907 mg (3.0
mmol) of N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione, and
1.3 mL (9.3 mmol) of Et3N in CH2Cl2/DMF (5:1) (6 mL) at 0 °C.
The resulting mixture was stirred at 0 °C for 1 and 25 h more at
room temperature. The reaction was worked up and purified by
neutral alumina column flash chromatography, eluting with hexane/
EtOAc (3:7). The residue obtained after the column was recrystal-
lized from EtOAc to yield 697 mg (48%) of 23e as a white solid:
mp 115–117 °C; IR (Nujol) ν 3309 (NH), 1752, 1718, 1696 (CO,
CN) cm-1; 1H NMR (DMSO-d6) δ 1.30 (s, 18H, (CH3)3), 3.77 (s,
4H, CH2), 6.85 (d, 2H, J ) 8.5 Hz, Ar), 7.49–7.62 (m, 3H, Ar),
7.67 (d, 2H, J ) 8.0 Hz, Ar), 7.98 (d, 2H, J ) 7.0 Hz, Ar), 10.14
(broad s, 1H, NH); 13C NMR (DMSO-d6) δ 28.8 ((CH3)3), 44.2
(CH2), 82.7 (C(CH3)3), 122.0, 122.1, 128.9, 129.6, 132.6, 134.9,
136.4, 140.3 (Ar), 145.8 ((CH3)3COCO), 151.0 (CN), 166.3
(PhNHCOPh).
Ferriprotoporphyrin IX Biomineralization Inhibition Test
(FBIT). The FBIT was performed according to Deharo et al.16 In
a 96-well plate was incubated at 37 °C for 18–24 h a mixture
containing 50 µL of 0.5 mg/mL of hemin chloride (Sigma H 5533)
freshly dissolved in DMSO, 100 µL of 0.5 M sodium acetate buffer
(pH 4.4) and 50 µL of different concentrations of drug solution or
50 µL of solvent (for control). The plate was centrifuged at 1600g
for 5 min, and the supernatant was discarded. The remaining pellet
was resuspended with 200 µL of DMSO to eliminate unreacted
hemin. Then the plate was centrifuged once again and the
supernatant similarly discarded. The precipitate (ꢀ-hematin) was
dissolved in 150 µL of 0.1 N NaOH, and the absorbance was read
at 405 nm with an ELISA reader (ELX 800 Biotech Instruments).
The percentage of inhibition of ferriprotoporphyrin IX biominer-
alization was calculated with the following equation:
Hydrochloride Salt of 4′-(2-Imidazolidinylimino)benzanil-
ide (23b). Following method B, 157 mg (96%) of the pure
hydrochloride salt of 23b were obtained as a white solid: mp,
1
decomposes at 180 °C; MS (ESI+) m/z 281.1369 [M + H]+; H
NMR (D2O) δ 3.70 (s, 4H, CH2), 7.23 (d, 2H, J ) 8.6 Hz, Ar),
7.48–7.58 (m, 4H, Ar), 7.60–7.65 (m, 1H, Ar), 7.80 (d, 2H, J )
7.0 Hz, Ar); 13C NMR (D2O) δ 42.1 (CH2), 122.8, 124.2, 126.9,
128.3, 131.5, 132.0, 133.0, 135.4 (Ar), 158.0 (CN), 168.9 (CO).
Anal. (C16H17ClN4O·0.5H2O) C, H, N.
(
)
{[(
)
inhibition % ) 100 ×
Abs of control -
(
)]
(
)}
Abs of drug ⁄ Abs of control
where Abs is absorbance. IC50 values were determined using the
TENDANCE function of the Excel program.
4-[2,3-Di(tert-butoxycarbonyl)guanidino]acetophenone (30d).
Method A was used, with 896 mg (3.3 mmol) of HgCl2, 406 mg
(3.0 mmol) of 4-aminoacetophenone 30, 830 mg (3.0 mmol) of
N,N′-di(tert-butoxycarbonyl)thiourea, and 1.3 mL (9.3 mmol) of
Et3N in DMF (5 mL) at 0 °C. The resulting mixture was stirred at
0 °C for 1 and 41 h more at room temperature. The reaction was
worked up and purified by silica gel column chromatography,
eluting with hexane/EtOAc (3:1) to yield 943 mg (83%) of 30d as
a white solid: mp, decomposes at 176 °C; IR (Nujol) ν 3278, 3146
(NH), 1718, 1686, 1637, 1598 (CO, CN) cm-1; 1H NMR (CDCl3)
δ 1.51 (s, 9H, (CH3)3), 1.53 (s, 9H, (CH3)3), 2.55 (s, 3H, CH3),
7.74 (d, 2H, J ) 8.5 Hz, Ar), 7.92 (d, 2H, J ) 8.0 Hz, Ar), 10.56
(broad s, 1H, NH), 11.61 (broad s, 1H, NH); 13C NMR (CDCl3) δ
26.3 (CH3), 27.9, 28.0 ((CH3)3), 79.9, 84.0 (C(CH3)3), 121.0, 129.3,
132.9, 141.2 (Ar), 153.1 ((CH3)3COCO), 163.1 (CN), 196.8
(CH3CO).
In Vitro Activity against T. brucei rhodesiense STIB900. This
trypanosoma strain was isolated in 1982 from a human patient in
Tanzania. Minimum essential medium (50 µL) supplemented with
25 mM HEPES, 1 g/L additional glucose, 1% MEM nonessential
amino acids (100×), 0.2 mM 2-mercaptoethanol, 2 mM sodium
pyruvate, 0.1 mM hypoxanthine, and 15% heat inactivated horse
serum was added to each well of a 96-well microtiter plate. The
3-fold serial drug dilutions were prepared in duplicate in the
columns covering a range from 90 to 0.123 µg/mL. Then 104
bloodstream forms of T. b. rhodesiense STIB 900 in 50 µL was
added to each well and the plate was incubated at 37 °C under a
5% CO2 atmosphere for 72 h. An amount of 10 µL of resazurin
solution (12.5 mg of resazurin dissolved in 100 mL of distilled
water) was then added to each well, and incubation continued for
a further 2–4 h. Then the plates were read with a Spectramax
Gemini XS microplate fluorometer (Molecular Devices Cooperation,
Sunnyvale, CA) using an excitation wavelength of 536 nm and an
emission wavelength of 588 nm. Data were analyzed using the
microplate reader software Softmax Pro (Molecular Devices
Cooperation, Sunnyvale, CA).
In Vitro Activity against P. falciparum K1. In vitro activity
against erythrocytic stages of P. falciparum was determined using
a 3H-hypoxanthine incorporation assay, using the chloroquine and
pyrimethamine resistant K1 strain and the standard drug chloroquine
(Sigma C6628). Compounds were dissolved in DMSO at 10 mg/
mL and added to parasite cultures incubated in RPMI 1640 medium
without hypoxanthine, supplemented with HEPES (5.94 g/L),
NaHCO3 (2.1 g/L), neomycin (100 U/mL), AlbuMax (5 g/L), and
washed human red cells A+ at 2.5% hematocrit (0.3% parasitemia).
Serial doubling dilutions of each drug were prepared in 96-well
microtiter plates and incubated in a humidified atmosphere at 37
°C, 4% CO2, 3% O2, 93% N2. After 48 h, 50 µL of 3H-hypoxanthine
()0.5 µCi) was added to each well of the plate. The plates were
incubated for a further 24 h under the same conditions. The plates
were then harvested with a Betaplate cell harvester (Wallac, Zurich,
Switzerland) and the red blood cells transferred onto a glass fiber
filter and then washed with distilled water. The dried filters were
inserted into a plastic foil with 10 mL of scintillation fluid and
counted in a Betaplate liquid scintillation counter (Wallac, Zurich,
Switzerland). IC50 values were calculated from sigmoidal inhibition
curves using Microsoft Excel.
4-[1,3-Di(tert-butoxycarbonyl)-2-imidazolidinylimino]acetophe-
none (30e). Method A was used, with 896 mg (3.3 mmol) of HgCl2,
406 mg (3.0 mmol) of 4-aminoacetophenone 30, 907 mg (3.0 mmol)
of N,N′-di(tert-butoxycarbonyl)imidazolidine-2-thione, and 1.3 mL
(9.3 mmol) of Et3N in DMF (5 mL) at 0 °C. The resulting mixture
was stirred at 0 °C for 1 and 25 h more at room temperature. The
reaction was worked up and purified by neutral alumina column
flash chromatography, eluting with hexane/EtOAc (3:2). The residue
obtained after the column was precipitated with Et2O and washed
with cold hexane to yield 613 mg (50%) of 30e as a white solid:
mp 113–115 °C; IR (Nujol) ν 1744, 1705, 1670 (CO, CN) cm-1
;
1H NMR (CDCl3) δ 1.31 (s, 18H, (CH3)3), 2.53 (s, 3H, CH3), 3.84
(s, 4H, CH2), 6.99 (d, 2H, J ) 8.0 Hz, Ar), 7.85 (d, 2H, J ) 8.0
Hz, Ar); 13C NMR (CDCl3) δ 26.3 (CH3), 27.8 ((CH3)3), 43.2
(CH2), 83.0 (C(CH3)3), 121.0, 129.4, 131.2, 140.2 (Ar), 149.8
((CH3)3COCO), 153.3 (CN), 197.1 (CH3CO).
DNA Binding Assays. Thermal melting experiments were
conducted with a Varian Cary 300 Bio spectrophotometer equipped
with a 6 × 6 multicell temperature-controlled block. Temperature
was monitored with a thermistor inserted into a 1 mL quartz cuvette
containing the same volume of water as in the sample cells.
Absorbance changes at 260 nm were monitored from 20 to 95 °C
with a heating/cooling rate of 0.5 °C/min and a data collection rate
of two points per °C. The poly(dA)-poly(dT) DNA polymer was
purchased from Amersham Pharmacia Biotech Inc., NJ (extinction
coefficient ꢀ260) 6000 cm-1 M-1 base). A quartz cell with a 1 cm

922 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Rodríguez et al.
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phenomenon of treatment failures in human African trypanosomiasis.
Trop. Med. Int. Health 2001, 6, 906–914.
In Vitro Cytotoxicity with L-6 Cells. Assays were performed
in 96-well microtiter plates, each well containing 100 µL of RPMI
1640 medium supplemented with 1% L-glutamine (200 mM) and
10% fetal bovine serum and 4 × 10 L-6 cells (rat skeletal
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myoblasts) with or without a serial drug dilution columns covering
a range from 90 to 0.123 µg/mL. Each compound was tested in
duplicate. After 72 h of incubation, the plates were inspected under
an inverted microscope to ensure growth of the controls and sterile
conditions. An amount of 10 µL of Alamar blue (12.5 mg of
resazurin in 100 mL of phosphate buffered saline) was then added
to each well, and the plates were incubated for another 2 h. Then
the plates were read with a Spectramax Gemini XS microplate
fluorometer (Molecular Devices Cooperation, Sunnyvale, CA) using
an excitation wavelength of 536 nm and an emission wavelength
of 588 nm. Data were analyzed using the microplate reader software
Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA).
In Vivo Activity against T. brucei. All efficacy studies were
approved by the institutional animal experimentation ethics com-
mittee. Female NMRI mice weighting 22–25 g were infected with
cryopreserved stabilates of T. brucei rhodesiense STIB 900. Each
mouse was infected intraperitoneally with 2 × 104 (STIB 900)
bloodstream forms. Groups of four mice were treated intraperito-
neally with the compounds on days 3-6. A control group remained
untreated. The parasitemia of all animals was checked every second
day up to day 14 postinfection and two times a week thereafter
until 60 days. Death of animals was recorded to calculate the mean
survival time. Surviving and aparasitemic mice were considered
cured at 60 days and then euthanized.
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In Vivo Antimalarial Efficacy Studies. All efficacy studies were
approved by the institutional animal experimentation ethics com-
mittee. In vivo antimalarial activity was assessed basically as
previously described.51 Groups of three female NMRI mice (20–22
g) were intravenously infected with 2 × 107 parasitized erythrocytes
on day 0 with GFP-transfected P. berghei strain ANKA.52
Compounds were formulated in 100% DMSO, diluted 10-fold in
distilled water, and administered intraperitoneally in a volume of
10 mL kg-1 on four consecutive days (4, 24, 48, and 72 h
postinfection). Parasitemia was determined on day 4 postinfection
(24 h after last treatment) by FACS analysis. Activity was calculated
as the difference between the mean percent parasitaemia for the
control (n ) 5 mice) and treated groups expressed as a percent
relative to the control group. The survival time in days was also
recorded up to 30 days after infection. A compound was considered
curative if the animal survived to day 30 after infection with no
detectable parasites.
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Acknowledgment. We are grateful to the UNICEF/UNDP/
World Bank/WHO Special Programme for Research and Train-
ing in Tropical Diseases (TDR) (RB), the CSIC-I3 program
(Grant PIE200680I121), and the Spanish Ministerio de Edu-
cación y Ciencia (Grant SAF2006-04698) for funding. F.R.
thanks the Consejería de Educación Cultura y Deporte de la
Comunidad Autónoma de La Rioja for his grant. Research on
DNA interactions is supported by National Institutes of Health
Grant AI064200 (W.D.W.). We thank J. C. Janse, Leiden
University, for providing the GFP transfected P. berghei strain
(RB).
Supporting Information Available: Synthesis and characteriza-
tion of the compounds 7b, 8a, 9a, 9b, 22a, 23a, 30a, and 30b and
combustion analysis data for compounds 7a, 11a, 11b, 19b, 20a,
21a, 21b, 22b, and 23b. This material is available free of charge
via the Internet at http://pubs.acs.org.
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