Bisguanidine, Bis(2-aminoimidazoline), and Polyamine Derivatives as Potent and Selective Chemotherapeutic Agents against Trypanosoma brucei rhodesiense. Synthesis and in Vitro Evaluation

Article abstract of DOI:10.1021/jm031024u

The in vitro screening for trypanocidal activity against Trypanosoma brucei rhodesiense of an in-house library of 62 compounds [i.e. alkane, diphenyl, and azaalkane bisguanidines and bis-(2-aminoimidazolines)], which were chosen for their structural similarity to the trypanocidal agents synthalin (1,10-decanediguanidine) and 4,4′-diguanidinodiphenylmethane and the polyamine N¹-(3-amino-propyl)propane-1,3-diamine, respectively, is reported. The original synthetic procedure for the preparation of 21 of these compounds is also reported. Most compounds displayed low micromolar antitrypanosomal activity, with five of them presenting a nanomolar inhibitory action on the parasite: 1,9-nonanediguanidine (1c), 1,12-dodecanediguanidine (1d), 4,4′ -bis[1,3-bis(tert-butoxycarbonyl)-2-imidazolidinylimino]diphenylamine (28a), 4,4′-bis(4,5-dihydro-1H-2-imidazolylamino)diphenylamine (28b), and 4,4′-diguanidino-diphenylamine (32b). Those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite [e.g. 1c (IC₅₀ = 49 nM; SI > 5294), 28b (IC₅₀ = 69 nM; SI = 3072), 32b (IC₅₀ = 22 nM; SI = 29.5), 41b (IC₅₀ = 118 nM; SI = 881)] represent new antitrypanosomal lead compounds.

Full text of DOI:10.1021/jm031024u

2296
J . Med. Chem. 2004, 47, 2296-2307
Bisgu a n id in e, Bis(2-a m in oim id a zolin e), a n d P olya m in e Der iva tives a s P oten t
a n d Selective Ch em oth er a p eu tic Agen ts a ga in st Tr ypa n osom a br u cei
r h od esien se. Syn th esis a n d in Vitr o Eva lu a tion
Christophe Dardonville*,† and Reto Brun‡
Instituto de Qu´ımica Me´dica, CSIC, J uan de la Cierva, 3, 28006-Madrid, Spain, and Swiss Tropical Institute,
Socinstrasse 57, CH-4002 Basel, Switzerland
Received September 9, 2003
The in vitro screening for trypanocidal activity against Trypanosoma brucei rhodesiense of an
in-house library of 62 compounds [i.e. alkane, diphenyl, and azaalkane bisguanidines and bis-
(2-aminoimidazolines)], which were chosen for their structural similarity to the trypanocidal
agents synthalin (1,10-decanediguanidine) and 4,4′-diguanidinodiphenylmethane and the
polyamine N1-(3-amino-propyl)propane-1,3-diamine, respectively, is reported. The original
synthetic procedure for the preparation of 21 of these compounds is also reported. Most
compounds displayed low micromolar antitrypanosomal activity, with five of them presenting
a nanomolar inhibitory action on the parasite: 1,9-nonanediguanidine (1c), 1,12-dode-
canediguanidine (1d ), 4,4′-bis[1,3-bis(tert-butoxycarbonyl)-2-imidazolidinylimino]diphenylamine
(28a ), 4,4′-bis(4,5-dihydro-1H-2-imidazolylamino)diphenylamine (28b), and 4,4′-diguanidino-
diphenylamine (32b). Those molecules that showed an excellent in vitro activity as well as
high selectivity for the parasite [e.g. 1c (IC50 ) 49 nM; SI > 5294), 28b (IC50 ) 69 nM; SI )
3072), 32b (IC50 ) 22 nM; SI ) 29.5), 41b (IC50 ) 118 nM; SI ) 881)] represent new
antitrypanosomal lead compounds.
In tr od u ction
The polyamine metabolic pathway of trypanosomatid
parasites has attracted much attention as drug target
for the last 15 years.16 This research led to the synthesis
and evaluation of many polyamine analogues as che-
motherapeutic agents against parasitic infections,17-20
with trypanothione reductase being a particularly tar-
geted enzyme (Figure 1).21-23
Sleeping sickness (human African trypanosomiasis,
HAT) is caused by two subspecies of African trypano-
somes, Trypanosoma brucei gambiense and Trypano-
soma brucei rhodesiense, responsible for the chronic and
acute form of the disease, respectively.1 Only four drugs
are licensed for the treatment of HAT2 (DFMO, suramin,
pentamidine, and melarsoprol), although other drugs
such as berenil (usually used against animal trypano-
somiasis) or the nitrofuran nifurtimox (registered for
use against Chagas’ disease) have also proved useful in
some limited cases. The actual situation of reemergence
of sleeping sickness in sub-Saharan Africa, with an
estimated 500 000 infected individuals,3 and the draw-
backs of the current HAT chemotherapy (e.g. toxicity,
increasing resistance, parenteral mode of administra-
tion, price) make the search for new trypanocidal drugs
urgently needed.4
Many diamidine, diguanidine, and polyamine com-
pounds have been investigated for their antitrypanoso-
mal activity as far as 65 years ago,5-11 giving rise, for
instance, to the aromatic diamidine drug pentamidine12
which is still used nowadays for the treatment of early
stage T.b. gambiense infections. However, this drug is
unable to cross the blood-brain barrier in sufficient
quantity to treat late-stage cases of HAT.13 Other
aromatic diamidines such as propamidine or berenil are
used for antiprotozoal chemotherapy in cattle, and the
trypanocidal activity of dicationic derivatives related to
pentamidine has been reported as well (Figure 1).9,14,15
There is a clear lack of research investment in the
field of tropical diseases in comparison to the number
of affected population.24,25 Thus, a reasonable approach
for the discovery of new antitrypanosomal lead com-
pounds at a lesser cost is the screening of already
available molecules for antiparasitic activity. Hence, a
rapid look at the structure of the polyamine, diguani-
dine, and diamidine compounds reported as antiproto-
zoal agents in the literature put into evidence the
potential as possible trypanocides of a series of bis-
guanidine and bis(2-aminoimidazoline) compounds pre-
viously synthesized in our laboratory for other pur-
poses.26-28 Moreover, some of the alkanediguanidine
[1,8-octanediguanidine (1b),29 1,12-dodecanediguanidine
(1d ),6,30,31 bis(guanidinopropyl)amine (6a )32] and di-
phenyl compounds [4,4′-diguanidinodiphenylmethane
(31b), 4,4′-diguanidinodiphenyl sulfone (33b)]8 available
in our in-house library had been previously reported for
their use as either trypanocide, microbicides, or fungi-
cides. Of particular interest was the recent report on
N,N′-bis(4-amidinophenyl)piperazine (Figure 1), which
proved to be a very effective antitrypanosomal agent in
vivo.15
Hence, in the search for new HAT chemotherapy, we
decided to carry out an in vitro screening of a total of
62 compounds against the parasite T. brucei rhodesiense
(Tables 1-5) taken from our in-house library and
* Corrresponding author. Tel.: +34 91-562-2900. Fax.: +34 91-564-
4853. E-mail: dardonville@iqm.csic.es.
† CSIC.
‡ Swiss Tropical Institute.
10.1021/jm031024u CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/26/2004

Chemotherapeutics against T. brucei rhodesiense
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2297
F igu r e 1. Examples of diamidine, guanidine, and polyamine compounds showing antiparasitic activity.
Ta ble 1. Structure, in Vitro Trypanocidal Activity, and Cytotoxicity of Guanidine and 2-Aminoimidazoline Alkane Derivatives 1a -3d
a
Controls: melarsoprol, IC50 ) 5.5 nM (SI ) 3456); diminazene diaceturate, IC50 ) 8.9 nM; CGP 40215, IC50 ) 4.5 nM (ref 44).
b
Selectivity index (SI) expressed as the ratio [IC50 L6-cells/IC50 T.b. rhodesiense].
structurally related to the trypanocidal agents synthalin
(1,10-decanediguanidine) and 4,4′-diguanidinodiphenyl-
methane (31b) and the polyamine N1-(3-aminopropyl)-
propane-1,3-diamine, respectively. We also describe here
the original synthesis of 21 molecules that had not been
previously reported.
11-13, 21-24 (Tables 1-4) as well as the diphenyl
derivatives 27a -33a , 27b-33b, and 34 (Table 5, entries
1-15) have been previously reported by us.26,27,28
Syn th esis of th e 3-Aza -1,6-h exa n ed ia m in e De-
r iva tives (Scheme 1). Guanylation of 3-aza-1,6-hex-
anediamine with an excess of DCC in CH3CN afforded
a mixture of the disubstituted dicyclohexylguanidine
derivative 5c and the trisubstituted compound 17.
These products were separated by preparative reverse
phase HPLC.
Resu lts
Ch em istr y. The syntheses of the aliphatic com-
pounds 1a -d , 2a -d , 3b-d , 4a ,b, 5a ,b,d , 6a ,b, 7-9,

2298 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Dardonville and Brun
Ta ble 2. Structure, in Vitro Trypanocidal Activity, and Cytotoxicity of Guanidine and 2-Aminoimidazoline Azaalkane Derivatives
4a -7
a
Controls: melarsoprol, IC50 ) 5.5 nM (SI ) 3456); diminazene diaceturate, IC50 ) 8.9 nM; CGP 40215, IC50 ) 4.5 nM (ref 44).
b
Selectivity index (SI) expressed as the ratio [IC50 L6-cells/IC50 T.b. rhodesiense].
Sch em e 1a
a
Reagents and conditions: (a) PhCH2CO2CN (2 equiv), CH2Cl2; (b) HCHO, NaBH3CN, AcOH (pH <7), MeCN; (c) HCHO, NaBH3CN,
MeCN (pH >7); (d) H2, Pd-C 10%, MeOH, 1 M HCl; (e) 2-methylmercapto-4,5-dihydroimidazole iodide, MeOH, reflux; (f) DCC, MeCN,
rt; (g) 2-bromoethanol, MeCN, reflux; (h) chloroacetaldehyde (4 equiv), NaBH(OAc)3, AcOH, MeCN, rt; (i) bromoethylbenzene, K2CO3,
CH3CN, reflux; (j) S-methylisothiouronium sulfate, CH3CN, reflux
3-Aza-1,6-hexanediamine selectively protected on the
primary amino group with benzylcyanoformate28,33 (5d )
was used as starting material for the preparation of the
3-substituted derivatives 8-10, 14-16, 18-20, 25, and
26 (Scheme 1). Alkylation of 5d with bromoethylbenzene
or 2-bromoethanol yielded 8 (83%) and 20 (69%),
respectively. Hydrogenolysis of 8 with 10% Pd-C/1 M
HCl/MeOH afforded the amine 9, which was subse-
quently refluxed with S-methylisothiouronium sulfate
in CH3CN, yielding the bis-guanidine 10 (46%). When
5d was treated with chloroacetaldehyde in excess under
reductive conditions (NaBH(OAc)3/AcOH/CH3CN) at
room temperature, the chloroethyl derivative 19 (35%)
was obtained together with the acetate side product 18
(16%). In this reaction, the nucleophilic substitution of
chloroacetaldehyde (or 2-chloroethanol derived from the
reduction in situ of chloroacetaldehyde) by the second-
ary amine 5d , leading to a hydroxyethyl intermediate

Chemotherapeutics against T. brucei rhodesiense
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2299
Sch em e 2a
that can react with AcOH present in the reaction
medium, is a potentially competitive reaction. Working
at low temperature (0 °C) was unfavorable to the
competitive nucleophilic substitution, and 19 could be
obtained in 69% under these conditions.
An interesting feature was the methylation of 5d with
formaldehyde under reductive conditions (NaBH3CN/
CH3CN).34 Three main products were isolated depend-
ing on the pH of the reaction. Working in basic medium
(i.e. pH >7, no AcOH added) yielded the methylated
product 14 (51%), whereas acidic medium afforded both
five- and six-member ring aminals 25 and 26, respec-
tively. Structural characterization of both derivatives
was carried out by 1D (1H, 13C) and 2D NMR experi-
ments (i.e. HSQC, HMBC). A characteristic difference
between the 1H NMR spectra of 25 and 26 were the
aminal methylene protons, which appeared as a singlet
of two protons for the six-membered ring derivative 26,
whereas two singlets (separated by 14.8 Hz) were
observed for the five-membered ring counterpart 25.
This might account for the observation of two conform-
a
Reagents and conditions: (a) N-(2,2′-diethoxyethyl)carbodi-
imide (2.2 equiv); CH3SO3H (2 equiv); EtOH, reflux, 23 h; (b) (1)
6 M HCl, rt, 3 h; (2) 10% NaOH, rt, 1 h; (c) NBS, t-BuOOH, CCl4,
reflux; (d) tri-n-pentylphosphine (4 equiv); PhMe, reflux, 24 h.
Sch em e 3a
3
ers of the acyl derivative 25.35 Study of the J H-C
coupling constants between the aminal methylene pro-
tons and their neighbors allowed us to characterize both
compounds.
Syn th esis of th e Dip h en yl Der iva tives (Scheme
2). Guanidines 31c and 32c were obtained by reaction
of an ether solution of N-(2,2′-diethoxyethyl)carbodi-
imide (prepared from BrCN and aminoacetaldehyde
diethyl acetal)36 with 4,4′-diaminodiphenylamine and
4,4′-diaminodiphenymethane in the presence of CH3-
SO3H, respectively. Preparation of the 2-aminoimidazole
35 was carried out by base-catalyzed cyclization of the
guanidine precursor 31c following the methodology of
Munk et al.36 In this reaction, two cyclization products
could potentially form: (1H-imidazol-2-yl)arylamine
(“endocyclic” amino group) and 1-aryl-1H-imidazol-2-
a
Reagents and conditions: (a) DMSO, 100 °C, 60 h (73%); (b)
1
H2 (40 psi), 10% Pd-C, HCl, MeOH, rt (59%); (c) N,N′-bis(tert-
butoxycarbonyl)thiourea (2.2 equiv), HgCl2, Et3N, DMF, 0 °C then
rt (73%); (d) TFA, CH2Cl2 (88%); (e) N,N′-bis(tert-butoxycarbon-
yl)imidazoline-2-thione (2.2 equiv), HgCl2, Et3N, DMF, 0 °C then
rt (66%).
ylamine (“exocyclic” amino group). The H NMR spec-
trum of 35 showed a unique broad singlet for the 4-H
and 5-H imidazole protons at 6.76 ppm, whereas the
decoupled 13C NMR spectrum showed a unique signal
for both 4-C and 5-C imidazole carbons, indicating the
magnetic equivalence of these atoms. These data dem-
onstrated that the expected product 35 with the en-
docyclic amino group was obtained (e.g. if the isomer
with the exocyclic amino group were obtained, a typical
AB system would be observed for the 4-H and 5-H
imidazole protons).
Compound 37 has been previously described37 as a
B2 bradykinin receptor antagonist. The reported proce-
dure was quite lengthy, so we designed a three-step
synthesis starting from 4,4′-dimethylbenzophenone.
Radicalar bromination of 4,4′-dimethylbenzophenone
with NBS/t-BuOOH/CCl4 allowed the formation of the
dibromo compound 36, which was isolated by crystal-
lization from the reaction mixture (22% yield). The low
yield obtained could be explained by the formation of a
mixture of mono- and polyhalogenated derivatives (e.g.
three spots were observed by TLC of the crude reaction
mixture).38 The bis-phosphonium compound 37 was
obtained by nucleophilic substitution of the bromine
atoms of 36 with an excess of tri-n-pentylphosphine in
refluxing toluene. Tri-n-pentylphosphine was prepared
by a modification of the procedure described by Davies
et al.39 The reaction of an excess of bromopentane
Grignard’s reagent with phosphorus trichloride working
at -78 °C40 afforded the tri-n-pentylphosphine, which
was purified by fractional distillation.
The synthetic approach for the preparation of the
piperazine-based bisguanidine and bis(2-aminoimida-
zoline) compounds is depicted in Scheme 3. Aromatic
nucleophilic substitution of 1-fluoro-4-nitrobenzene with
commercially available 1-(4-nitrophenyl)piperazine in
DMSO at 100 °C afforded 38.41 Nitro groups were
reduced by catalytic hydrogenation (10% Pd-C/HCl/
MeOH), affording the amine 39.42 Introduction of the
Boc-protected guanidine and imidazoline moieties (com-
pounds 40a and 41a , respectively) was carried out in
good yield with N,N′-bis(tert-butoxycarbonyl)thiourea43
and N,N′-bis(tert-butoxycarbonyl)imidazoline-2-thione,26
respectively. Removal of the Boc-protecting groups was
accomplished by treatment with TFA, affording 40b and
41b as their trifluoroacetate salts.
Biologica l R esu lt s. In vit r o An t it r yp a n osom a l
Activity. The results of the determination of antitry-
panosomal activity against bloodstream-form trypomas-

2300 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Dardonville and Brun
Ta ble 3. Structure, in Vitro Trypanocidal Activity, and Cytotoxicity of 3-Aza-1,6-hexanediamine Derivatives 8-20
a
Controls: melarsoprol, IC50 ) 5.5 nM (SI ) 3456); diminazene diaceturate, IC50 ) 8.9 nM; CGP 40215, IC50 ) 4.5 nM (ref 44).
b
Selectivity index (SI) expressed as the ratio [IC50 L6-cells/IC50 T.b. rhodesiense].
Ta ble 4. Structure, in Vitro Trypanocidal Activity, and Cytotoxicity of 3-Aza-1,6-hexanediamine Cyclic Derivatives 21-26
a
Controls: melarsoprol, IC50 ) 5.5 nM (SI ) 3456); diminazene diaceturate, IC50 ) 8.9 nM; CGP 40215, IC50 ) 4.5 nM (ref 44).
b
Selectivity index (SI) expressed as the ratio [IC50 L6-cells/IC50 T.b. rhodesiense].
tigotes of T.b. rhodesisense (strain STIB 900) are
reported in Tables 1-5. All compounds displayed dose-
dependent activities against T.b. rhodesiense, with IC50
ranging from 0.022 to 113 µM, and were selective for
the parasite. Eight aliphatic (1b-d , 2b-d , 13, and 17)
and 12 diphenyl derivatives (27b, 28a ,b, 31b,c, 32a -
c, 34, 37, 40b, and 41b) showed an IC50 < 1 µM. Among
the latter, five compounds had an IC50 in the nanomolar
range (1c, 1d , 28a , 28b, and 32b) with a selectivity
index (SI) ranging from 13 (1d ) to more than 5000 (1c).
Alk a n e a n d Aza a lk a n e Der iva tives (Ta bles 1 a n d
2). The most potent compound within the alkane (Table
1) and azaalkane (Table 2) derivative series was 1,9-
nonanediguanidine (1c), with IC50 ) 49 nM and a
remarkable selectivity for the parasite (SI > 5294). In
these series, the guanidinium cation gave in general

Chemotherapeutics against T. brucei rhodesiense
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2301
Ta ble 5. Structure, in Vitro Trypanocidal Activity, and Cytotoxicity of Diphenyl Derivatives 27a -41b
a
Controls: melarsoprol, IC50 ) 5.5 nM (SI ) 3456); diminazene diaceturate, IC50 ) 8.9 nM; CGP40215, IC50 ) 4.5 nM (ref 44). b Selectivity
index (SI) expressed as the ratio [IC50 L6-cells/IC50 T.b. rhodesiense]
more active compounds (about 2-4-fold) than its 2-ami-
noimidazolinium counterpart (compare 1a -d vs 2a -
d ). This was also true for the azaalkane series (Table
2, 5a , 6a vs 5b, 6b). Increasing the chain length of the
methylene spacer (n ) 6, 8, 9, 12) between either
guanidinium or 2-aminoimidazolinium cations tended
to increase the activity in the following order: 6 < 8
(ca. 30-fold) < 9 ∼ 12 (Table 1). Regarding the selectiv-
ity, the nine-methylene spacer (1c and 2c) gave the best
SI (5294 and 71) in both series. Noteworthy was the
greater activity displayed by the dicationic derivatives
1b-d and 2b-d with respect to their monocationic
counterparts (Table 1). This behavior was also observed
for the azaalkane compound 6b,which was 3.5-fold more
active than 7 (Table 2).
Introduction of an unsubstituted nitrogen atom in the
methylene chain (e.g. in the azaalkane series, Table 2)
tended to reduce the activity compared to alkyl spacer.
This is exemplified by the activity of compounds 1a and
2a (8 and 19.3 µM, respectively) and their aza-analogues
5a and 5b (21.4 and 69.1 µM, respectively). Another
interesting result was that of the dicyclohexylguanidine
5c (IC50 ) 2.4 µM), which was 9-fold more active than
the guanidine analogue 5a (21.4 µM). This result might
reflect better pharmacokinetic properties of the more
lipophilic derivative 5c (i.e. to cross biological mem-
branes).
The dicyclohexylguanidine compound 17 (IC50 ) 0.98
µM) displayed the best activity and selectivity (SI > 82)
of all the 3-aza-1,6-hexanediamine derivatives (Table 3).
Again, it appeared that lipophilicity was an important
factor for good activity. In this series, substitution of
the secondary amino group with a phenethyl, 3-(2-
ethyl)indole, or methyl group afforded molecules slightly
more active than the parent compound (compare the
activities of 10/5a , 11/5b, and 16/5b).45 In addition, the
amines protected with a carbobenzyloxy group (Cbz)
were more active than their free amino counterparts
(compare 8/9, 12/13, and 14/15). Regarding the effect
of the substituent on the secondary nitrogen in the Cbz-
protected series, the following results, in order of
decreasing activity, were obtained: indole (13, 1 µM) >

2302 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Dardonville and Brun
methyl (14, 3.1 µM) ∼ phenethyl (8, 3.88 µM) > CH2-
CH2OAc (18, 7.1 µM) > CH2CH2OH (20, 14 µM).
Worth mentioning is the result obtained for the cyclic
analogues 25 and 26 (Table 4), which showed the same
range of activity as the parent compound 14 (IC50 ) 3.1
µM, Table 3) but a lower selectivity (SI ) 27 and 18.6
respectively, compared to 41 for 14). This behavior was
also observed with the cyclic analogue 23 (71.2 µM),
which displayed the same activity as the aliphatic
parent 5b (69.1 µM).
with 3c (SI > 5294) being more selective than the
control melarsoprol (SI ) 3456). The potency of 3c is to
be compared with that of synthalin (1,10-decanediguani-
dine)7 or 1,11-undecanediamidine, a trypanocidal drug
that proved able to cure mice and rabbits infected with
a strain of T.b. rhodesiense.6 The 1,9-nonanediguanidine
3c could be considered as the bio-isostere of 1,11-
undecanediamidine with the supplementary amino
groups of the guanidine moieties playing the role of the
two supplementary methylene units, thus keeping ap-
proximately the same chain length in both molecules.
Dip h en yl Der iva tives (Table 5). In this series of
bisguanidine and bis(2-aminoimidazoline) diphenyl ana-
logues (entries 1-17), the best activities were observed
for the compounds bearing a guanidinium group (from
3- to 10-fold with respect to the imidazoline analogues).
However, the 2-aminoimidazoline derivatives displayed,
in general, better selectivity than the guanidine coun-
terparts (compare the activity and selectivity of 31b/
27b, 32b/28b, 34/29b, and 32a /28a ). In addition, re-
placement of the guanidine or 2-aminoimidazoline with
a 2-aminoimidazole nucleus (compound 35, entry 18)
produced a loss of activity of 20- and 63-fold compared
to 28b and 32b, respectively. Interestingly, the very
lipophilic bis-phosphonium benzophenone derivative 37
showed a trypanocidal activity (IC50 ) 0.414 µM) similar
to that of the bisguanidinium diphenyl ketone 34 (0.206
mM) and a better selectivity (SI ) 28.5 versus 13.1).
Notable is the effect of the N-substitution of the
imidazoline and guanidine moieties (i.e. Boc, CH2CH-
(OEt)2). Boc protection afforded less active compounds
compared to unprotected counterparts (compare 27a /
27b, 29a /29b, 31a /31b, 32a /32b, 33a /33b, and 40a /40b)
with the exception of 28 and 30, in which the Boc
substituents produced a 1.4- and 12-fold increase in
activity respectively (IC50 ) 0.048 and 2.6 µM, respec-
tively) compared to the free imidazolinium cation (IC50
) 0.069 and 32.4 µM, respectively). Moreover, the Boc
substituents seemed to give somewhat less selective
compounds (SI ) 202 and 3072 for 28a and 28b,
respectively; SI ) 7.9 and 29.5 for 32a and 32b). On
the contrary, the 1,1-diethoxyethane substituent pro-
duced a great increase in selectivity, superior to 26- and
32-fold for 31c and 32c, respectively, with only a slight
loss in activity (2- and 10-fold, respectively) compared
to the unsubstituted parent compounds 31b and 32b.
Regarding the bridge linking both phenyl rings, the
same behavior was observed for the guanidinium and
2-aminoimidazolinium series, i.e., NH . CH2 > CO >
SO2 in order of decreasing activity (compare 27b-30b
and 31b-33b, 34). When a piperazine moiety was used
as bridge between both phenyl rings (Table 5, entries
21-25), the 2-aminoimidazolinium compound 41b
showed the best activity (0.118 µM) and also a 5 times
higher selectivity (SI ) 881) with respect to the guani-
dinium analogue (SI ) 172).
In these series, the guanidine moiety afforded in
general better trypanocidal drugs than the 2-aminoimi-
dazoline one. Moreover, the presence of two cations was
required for potent activity, which is in agreement with
the results previously obtained by King et al.6 This
assumption could probably be extended to the diphenyl
series, according to the previous findings reported for
aromatic diamidines and diguanidines,6,9,10 although
this hypothesis was not tested here because mono-
cationic aromatic compounds were not available in our
library.
For short methylene chains (n ) 5-7), introduction
of a secondary nitrogen atom into the alkyl spacer
afforded less active molecules, although further substi-
tution of this nitrogen could increase slightly the activity
(compounds 8-20). Conformational restriction of the
azaalkane molecules did not affect nor increase the
trypanocidal action compared to their linear analogues
(compounds 23, 25, and 26). The importance of the
lipophilicy of these molecules, facilitating drug uptake
by the parasite by passive diffusion, was exemplified
by the higher antitrypanosomal activity of 5c with
respect to 5a .
The most interesting results probably came from the
diphenyl series with a NH bridge. The bis(2-aminoimi-
dazoline) derivative 28b was extremely potent (IC50
)
69 nM) and also highly selective for the parasite (SI )
3072). The Boc-protected counterpart 28a had the same
range of activity but a lower selectivity index (SI ) 202).
This result in particular might be relevant because of
the higher lipophilicity of the Boc-protected compound
28a . Late-stage cases of HAT involve central nervous
system (CNS) infection and hence require drugs able
to cross the blood-brain barrier. However, the Boc-
protecting group is probably stable under the conditions
of the in vitro assay but potentially could be metabolized
in vivo to afford the unprotected derivative 28b.
Changing the 2-aminoimidazolinium cations for guani-
dinium ones led to the most active compound of this
screening, 32b (22 nM, SI ) 29.5). The nature of the
bridge linking both phenyl rings had a clear influence
on the trypanocidal action of these compounds. Electron-
donating groups such as NH, piperazine, or CH2 af-
forded better trypanocides than electron-withdrawing
groups such as CdO or SO2. Such behavior was consis-
tent with the findings of Donkor et al. in the pentami-
dine congener series, where electron-rich phenyl groups
(e.g. phenoxy) afforded better trypanocides than electron-
poor phenyl rings (e.g. acetylated aniline or pyridine).15
Discu ssion
Some of the compounds described in this paper were
available in our in-house library. Since few of these
molecules had been previously reported in the literature
for their anti-trypanosomal activity (e.g. 1d ,5 31b,8
33b10), we anticipated that our compounds would dis-
play trypanocidal action. Indeed, simple aliphatic
diguanidines were potent and selective trypanocides,
If we compare the different cationic species studied
(i.e. guanidinium, 2-aminoimidazolinium, phosphoni-
um), the good activity and selectivity displayed by the
bis-phosphonium derivative 37 (IC50 ) 0.414 µM, SI )

Chemotherapeutics against T. brucei rhodesiense
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2303
28.5) is of particular interest. These results suggest that
lipophilic bis-phosphonium diphenyl derivatives might
be a good alternative (with potentially better pharma-
cokinetic properties) to the guanidine or 2-aminoimi-
dazoline derivatives. With respect to the guanidine
cation, N-substitution with a diethoxyethane moiety
afforded highly selective antitrypanosomal agents (SI
> 554 and 767 for 31c and 32c, respectively).
human brain membranes.27 Finally, several azaalkane
derivatives (Tables 2 and 3, 4a ,b, 5a ,b, 6a ,b, 7, 10, 11,
23) were tested for analgesic activity in mice.28 These
data are relevant and should be taken into consideration
when choosing possible lead compounds for in vivo
assays.
Con clu sion
We have reported here the screening for trypanocidal
activity against T.b. rhodesiense of an in-house library
of 62 compounds [i.e. alkane, diphenyl, and azaalkane
bisguanidine and bis(2-aminoimidazoline)] that were
chosen for their structural similarity to the trypanocidal
agents synthalin (1,10-decanediguanidine), and 4,4′-
diguanidinodiphenylmethane and the polyamine N1-(3-
aminopropyl)propane-1,3-diamine, respectively. The origi-
nal synthetic procedure for the preparation of 21 of these
compounds was also reported.
The results of the determination of in vitro antitry-
panosomal activity allowed drawing some conclusions
about the SAR of these series of molecules. Most
compounds displayed low micromolar antitrypanosomal
activity, with five of them presenting a nanomolar
inhibitory action on the parasite (1c, 1d , 28a , 28b, and
32b). A few of these compounds, which showed an
excellent in vitro activity as well as high selectivity, e.g.
1c (IC50 ) 49 nM; SI > 5294), 28b (IC50 ) 69 nM; SI )
3072), 32b (IC50 ) 22 nM; SI ) 29.5), 41b (IC50 ) 118
nM; SI ) 881), are promising lead compounds for
antitrypanosomal chemotherapy. The results of in vivo
activity of these molecules will be reported in due
course.
The antitrypanocidal efficacy of a drug depends on
its effective uptake by the parasite. It is known that
diamidines such as pentamidine, which have a very slow
rate of diffusion across biological membranes, can be
transported into the cell by a P2-amino-purine trans-
porter that specifically recognizes the main H2N-
C(R1)dNR2 motif.46,47 The guanidine molecules reported
here also present this recognition motif. In the case of
the diphenyl derivatives, most of the Boc-protected
molecules (i.e. the most lipophilic) showed a weaker
activity than the charged, unprotected, guanidinium
analogues. This might account for a more efficient
transport of the unprotected derivatives through the P2
transporter, although affinity assays for this transporter
remain to be done.
It is still too early to propose a mode of action of the
compounds presented here, and further studies are
needed. However, a number of dicationic molecules
belonging to the diamidine family (e.g. pentamidine) are
known to bind to the minor-groove of DNA, and their
antiprotozoal activity is thought to be the result of that
interaction (e.g. inhibition of DNA-dependent enzymes
or inhibition of transcription).48-50 In a recent article,
Donkor et al. studied the trypanocidal activity of a series
of conformationally restricted congeners of pentami-
dine.15 Although a direct correlation between the DNA
binding affinity and the trypanocidal activity was not
observed, the authors concluded that compounds with
strong DNA affinity generally showed good trypanocidal
activity in that series. In particular, N,N′-bis(4-amidi-
nophenyl)piperazine (Figure 1) and N,N′-bis(4-imida-
zolinophenyl)piperazine were the most potent trypano-
cides and also the strongest DNA binders in this series.
According to the results of Donkor et al., we might
expect good DNA binding affinity for compounds 40b
and 41b, which are the guanidine and 2-aminoimida-
zoline analogues of these congeners, respectively (vide
infra). However, this hypothesis will need experimental
confirmation.
Tropical diseases mainly affect Third-World countries,
which usually lack research capacities and financial
resources for investigation. The lack of available funds
and research in this field put into light the importance
of screening in-house libraries of molecules already
available in order to save time and money in the
discovery of new lead compounds for neglected diseases
such as HAT.
Exp er im en ta l Section
Ch em istr y. All reaction solvents were purchased anhy-
drous and used as received. Other solvents used were reagent
grade. Reactions were monitored by TLC using precoated silica
gel 60 F254 plates. Chromatography was performed either
with silica gel 60 PF254 (particle size 40-63 µm) or with a
medium-pressure chromatography system using KP-Sil 40S
or 40M cartridges (particle size 32-63 µm, 60 Å). All reactions
requiring anhydrous conditions or an inert atmosphere were
Several compounds presented in this paper were first
and foremost studied for different activities on the CNS
(i.e. R1-adrenergic antagonism, I2-imidazoline binding
site affinity or analgesic properties). The knowledge of
these interactions (i.e. possible side effects) is of impor-
tance, because useful antitrypanocidal agents are ex-
pected to penetrate the CNS to cure late-stage cases of
HAT. The diphenyl compounds (Table 5, entries 5-8
and 12-15) present R1-adrenergic antagonist activity
in various tissues.26,51 In particular, the blood pressure
and heart rate responses of two compounds (29b and
31b) had been tested on rats in vivo, suggesting a
smaller magnitude of cardiovascular effects than the R1-
adrenergic antagonist doxazosin at the same dose.52 On
the other hand, the alkane derivatives (Table 1, 1a -d
and 2a -d ) showed a moderate to good affinity for the
I2-imidazoline binding sites and R2-adrenoceptors in
1
performed under a positive pressure of N2. H NMR and 13C
NMR spectra were recorded at 200 and 50 MHz, respectively,
unless otherwise noted. Chemical shifts of the 1H NMR spectra
were internally referenced to the residual proton resonance
of the deuterated solvents: CDCl3 (7.26 ppm), D2O (δ 4.6 ppm),
CD3OD (3.49 ppm), and DMSO (δ 2.49 ppm). Chemical shifts
of 13C NMR and 31P spectra were referenced with a capilar of
DMSO-d6 (δ 39.5 ppm) and H3PO4 (δ 0 ppm), respectively. IR
spectra were recorded as KBr pellets or neat. Melting points
were determined with a Reichert-J ung Thermovar apparatus
and are uncorrected. Mass spectra were recorded on a Hewlett-
Packard Series 1100 MSD spectrometer (ES, APCI) and on a
VG Autospec spectrometer (FAB). Elemental analysis was
performed on a Heraeus CHN-O rapid analyzer. Analytical
results were within (0.4% of the theoretical values unless
otherwise noted. Analytical HPLC was run on a Beckman LC-
168 HPLC with either a Waters Delta Pak 5µ-C18, 100 Å (3.9
× 150 mm) (column I) or a Varian Microsorb-MV-C18, 100Å

2304 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Dardonville and Brun
column (column II) using the following conditions: gradient
time ) 40 and 15 min for columns I and II, respectively, H2O/
CH3CN (100:0 f 0:100) (TFA 0.1%), flow rate ) 1 mL/min, λ
) 214 and 254 nm. Preparative HPLC (compounds 5c and 17)
was carried out using a Waters Deltaprep apparatus with a
Waters prepak-RCM Base column and detection at 214 nm.
Compounds 1a -d , 2a -d , 3b-d , 4a -6a ,53 4b, 5b,d , 6b,
7-9, 11-13, 21-24, 27a -33a , 27b-33b, and 34 were pre-
pared as previously reported.26-28,53
45.2 (t), 42.6 (t), 40.2 (q), 37.2 (t); LRMS (FAB+) m/z 268 [(M
+ H)]. Anal. (C24H31N13O14) C, H, N.
3-Aza h exa n e-1,7-(N,N′-d icycloh exyl)d igu a n id in e (5c).
A solution of 3-(2-aminoethylamino)propylamine (1 mL, 8.5
mmol) and DCC (3.7 g, 17.9 mmol) in dry CH3CN (25 mL) was
stirred for 4 days at room temperature under argon atmo-
sphere. The solvent was removed by reduce pressure and the
crude oil was dissolved in Et2O. A current of HClg was bubbled
into the solution for 2 min. The white precipitate was collected,
rinsed with Et2O, and dried in vacuo, affording a mixture of
the di- and trisubstituted compounds 5c and 17, which were
separated by preparative HPLC using the following eluent
system: H2O/CH3CN (100:0 f 0:100) (TFA 0.1%). Trifluoro-
acetate of 5c: white solid; mp 88-93 °C; 1H NMR (400 MHz,
CDCl3) δ 3.45 (t, 2H, J ) 6.3 Hz), 3.6-3.25 (m, 10H), 3.18 (t,
2H, J ) 7 Hz), 2.95 (br t, 2H), 1.82 (q, 2H, J ) 7.8 Hz), 1.69
(br s, 8H), 1.60 (br d, 8H), 1.46 (br d, 4H), 1.16 (t, 16H, J ) 10
Hz), 1.0 (br m, 5H); 13C NMR (100 MHz, CDCl3) δ 163.7 (TFA),
154.0 (s), 154.0 (s), 117.6 (TFA), 52.6 (d), 52.5 (d), 47.3 (t), 46.5
(t), 39.3 (t), 38.9 (t), 33.4 (t), 33.4 (t), 26.5 (t), 25.8 (t), 25.7 (t);
LRMS (ES+) m/z 265.9 [(M + 2H), 100], 644.6 [(M + TFA)],
758.6 [(M + 2TFA)]; HPLC (column II) tR ) 9.37 min (100%).
3-Azah exan e-1,3,7-(N,N′-dicycloh exyl)tr igu an idin e (17).
Trifluoroacetate of 17: white flocculent solid; 1H NMR (500
MHz, CD3OD) δ 3.73-3.60 (m, 8H), 3.56-3.44 (m, 6H), 2.14-
1.8 (m, 30H), 1.7-1.3 (m, 32H); 13C NMR (125 MHz, CD3OD)
δ 160.1 (s), 154.2 (s), 56.0 (d), 52.6 (d), 52.6 (d), 49.8 (t), 47.7
(t), 39.9 (t), 39.7 (t), 34.5 (t), 33.9 (t), 33.9 (t), 28.2 (t), 26.2 (t),
26.2 (t), 26.1 (t), 26.1 (t), 26.0 (t); LRMS (ES+) m/z 369 [(M +
2H), 100], 246.5 [(M + 3H)]; HPLC (column I) tR ) 30.55 min
(99.58%).
{3-[(2-Ben zyloxyca r bon yla m in oeth yl)(2-ch lor oeth yl)-
a m in o]p r op yl}ca r ba m ic Acid Ben zyl Ester (19). Chloro-
acetaldehyde (50% in water, 1.5 mL, 11.6 mmol) was added
to a solution of 5d (1.11 g, 2.9 mmol) in CH3CN (20 mL). After
a few minutes, AcOH (0.5 mL, 8.5 mmol)) was added, followed
5 min later by NaBH(OAc)3 (1.24 g, 5.8 mmol). The reaction
mixture was stirred for 4 h at room temperature, and the pH
was adjusted to 5-6 with AcOH during the course of the
reaction. The reaction was quenched by careful addition of 5%
NaHCO3 and diluted with CH2Cl2. The organic phase was
separated and the aqueous phase was extracted three times
with CH2Cl2. Combined organic extracts were washed with
brine, dried (Na2SO4), and concentrated by reduce pressure.
Chromatography (40M cartridge) with petroleum ether/acetone
(80:20) yielded the acetyl side product 18 (16%) and the
expected chloro derivative 19 as an oil that solidified as a
yellowish pasty residue (450 mg, 35%); IR (KBr) ν 1680, 1415,
1240, 755, 713, 675 cm-1; 1H NMR (CDCl3) δ 7.29 (br s, 10H),
5.67 (br, NH), 5.60 (br, NH), 5.09 (s, 2H), 5.08 (s, 2H), 3.43 (t,
2H, J ) 6.1 Hz), 3.3-3.1 (m, 4H), 2.68 (t, 2H, J ) 6.3 Hz),
2.6-2.4 (m, 4H), 1.56 (quint, 2H, J ) 6.4 Hz); 13C NMR (CDCl3)
δ 157.0 (2C), 137.1, 128.8, 128.3, 66.8, 56.0, 54.0, 52.4, 42.5,
39.7, 39.3, 27.5; LRMS (ES+) m/z 448.5 [(M + HCl), 100], 412
[(M + H)]. Anal. (C23H30N3O4Cl/H2O) Calcd: C, 59.29; H, 6.92;
N, 9.02. Found: C, 59.27; H, 7.26; N, 9.02.
N -{3-[(2-G u a n i d i n o e t h y l)p h e n e t h y la m i n o ]p r o p -
yl}gu a n id in e (10). A solution of 9 (0.5 mmol) and S-
methylisothiouronium sulfate (148 mg, 0.53 mmol) in dry
MeOH (7 mL) was heated for 12 h at reflux. The solvent was
removed by reduce pressure, and the crude product dissolved
in a mixture of H2O/EtOH was treated with a few drops of 5%
H2SO4. The solution was allowed to stand for 3 days in the
refrigerator and the supernatant was discarded. Acetone was
added and the oily residue was triturated with a spatula until
the product crystallized. The solid was dried in vacuo, affording
10 as a highly hygroscopic colorless solid (104 mg, 46%): 1H
NMR (D2O) δ 7.3-7.0 (m, 5H), 3.6-2.8 (m, 12H), 1.87 (m, 2H);
13C NMR (D2O) δ 155.9 (br), 135.0, 128.1, 127.7, 126.4, 53.0,
50.6, 49.9, 37.1, 35.4, 28.2, 22.7. LRMS (ES+) m/z 307 [(M +
H)], 100].
{3-[(2-Ben zyloxyca r b on yla m in oet h yl)m et h yla m in o]-
p r op yl}ca r ba m ic Acid Ben zyl Ester (14). NaBH3CN (100
mg, 1.49 mmol) was added to a solution of amine 5d (443 mg,
1.15 mmol) and 37% aqueous formaldehyde (0.4 mL, 4.6
mmol). The reaction was stirred for 4 h at room temperature,
and the solvents were removed by reduce pressure. The crude
residue was partitioned between CHCl3 and water. The organic
phase was collected and the aqueous phase was extracted three
times with CHCl3. Organic extracts were washed with brine,
dried (Na2SO4), and concentrated by reduce pressure. Flash
chromatography (40S cartridge) with CH2Cl2/MeOH (95:5)
afforded the methylated amine 14 as a colorless solid (236 mg,
51%): mp 60-62 °C; IR (KBr) ν 3300, 2900, 2725, 1665, 1515,
1250, 1120, 960, 720, 685, 670 cm-1; 1H NMR (CDCl3) δ 7.4-
7.2 (m, 10H), 5.6 (br, NH), 5.4 (br, NH), 5.07 (s, 2H), 5.05 (s,
2H), 3.3-3.1 (m, 4H), 2.5-2.3 (m, 4H), 2.17 (s, 3H), 1.62 (quint,
2H, J ) 6 Hz); 13C NMR (CDCl3) δ 157.1, 137.3, 137.2, 129.0,
128.6, 128.5, 67.1, 67.0, 57.3, 55.9, 42.2, 40.1, 38.9, 27.4; LRMS
(ES+) m/z 400 [(M + H), 100]. Anal. (C22H29N3O4) C, H, N.
N1-(2-Am in oeth yl)-N1-m eth ylp r op a n e-1,3-d ia m in e (15).
Catalytic hydrogenation of a suspension of 14 (230 mg, 0.57
mmol), 10% Pd-C (23 mg), and 1 M HCl (1 mL) in MeOH (30
mL) under 36 psi hydrogen pressure for 24 h at room
temperature afforded the HCl salt of 15 as a colorless oil (118
mg, quantitative): 1H NMR (D2O) δ 3.0-2.8 (m, 4H), 2.58 (t,
2H, J ) 6.8 Hz), 2.46 (t, 2H, J ) 7.6 Hz), 2.16 (s, 3H), 1.74
(quint, 2H, J ) 7.6 Hz); 13C NMR (D2O) δ 54.8 (t), 54.5 (t),
41.0 (q), 38.5 (t), 37.0 (t), 24.5 (t); LRMS (APCI+) m/z 132 [(M
+ H), 100]. Anal. (C6H20N3Cl3‚0.3H2O) Calcd: C, 29.47; H, 8.49;
N, 17.19. Found: C, 29.35; H, 8.40; N, 17.00.
N,N′-Bis[2-(4,5-d ih yd r o-1H-im id a zol-2-yla m in o)eth yl]-
N′-m eth ylp r op a n e-1,3-d ia m in e (16). A solution of 15 (110
mg, 0.84 mmol) and 2-methylmercapto-4,5-dihydro-1H-imida-
zole iodide (410 mg, 1.76 mmol) in EtOH (10 mL) was heated
for 24 h at reflux (CAUTION: the noxious gas CH3SH is
evolved during the reaction and it should be trapped with a
concentrated aqueous NaOH solution). The solvent was re-
moved by reduce pressure and the crude compound was
purified by formation of its picrate salt: a hot solution of picric
acid (400 mg in 5 mL of H2O) was added to the hot reaction
mixture and the flask was allowed to stand in the refrigerator
for 1 week. The crystals were collected by filtration and rinsed
successively with water, hexane, and Et2O. Picrate of 16:
yellow solid (302 mg, 53%); mp 81-83 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.94 (br s, 1H), 9.25 (br s, 1H), 8.54 (s, 4H), 8.18
(t, 1H, J ) 5.7 Hz), 8.13 (t, 1H, J ) 4.8 Hz), 7.65 (br, 1H), 3.80
(s, 3H), 3.58 (s, 4H), 3.55 (s, 4H), 3.1 (m, 4H), 2.8-2.7 (m, 4H),
1.8 (br, 2H); 13C NMR (50 MHz, DMSO-d6) δ 170.5, 160.9 (s),
159.4 (s), 141.5 (s), 125.3 (d), 124.8 (s), 53.6 (br, t), 53.0 (t),
Acet ic a cid 2-[(2-Ben zyloxyca r b on yla m in oet h yl)(3-
b en zyloxyca r b on yla m in op r op yl)a m in o]et h yl
est er
1
(18): 223 mg, 16%; H NMR (CDCl3) δ 7.28 (br s, 10H), 5.68
(br, NH), 5.52 (br, NH), 5.04 (s, 2H), 5.02 (s, 2H), 4.03 (t, 2H,
J ) 5.6 Hz), 3.25-3.05 (m, 4H), 2.60 (t, 2H, J ) 5.6 Hz), 2.55-
2.35 (m, 4H), 1.90 (s, 3H), 1.55 (quint, 2H); 13C NMR (CDCl3)
δ 171.5, 157.0, 156.9, 137.1, 128.9, 128.5, 128.4, 66.95, 66.87,
62.4, 54.0, 52.7, 43.5, 39.9, 39.2, 27.3, 21.2; LRMS (ES+) m/z
472 [(M + H), 100]. Anal. (C25H33N3O6) C, H, N.
{3-[(2-Ben zyloxycar bon ylam in oeth yl)(2-h ydr oxyeth yl)-
a m in o]p r op yl}ca r ba m ic Acid Ben zyl Ester (20). A solu-
tion of bromoethanol (0.03 mL, 0.42 mmol) in CH3CN (1 mL)
was added to a solution of 5d (208 mg, 0.54 mmol) in CH3CN
(4 mL). The reaction mixture was refluxed for 12 h and the
solvent was removed by reduce pressure. The crude product
was purified by chromatography with EtOAc/MeOH (80:20),
and the resulting compound dissolved in a little CH2Cl2 was
filtered on a path of Celite, affording the pure product 20 (124

Chemotherapeutics against T. brucei rhodesiense
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2305
mg, 69%): 1H NMR (300 MHz, CDCl3) δ 7.22 (br s, 10H), 5.63
(br, 1H), 5.46 (br, 1H), 4.97 (s, 4H), 3.44 (t, 2H, J ) 5 Hz), 3.1
(m, 5H), 2.5-2.3 (m, 6H), 1.49 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ 156.76, 156.57, 136.5, 128.3, 127.9, 66.4, 59.1, 55.8,
53.7, 51.3, 38.9, 38.6, 27.0; IR (neat) ν 3500-3300 (br), 2905,
1680, 1515, 1235, 715, 675 cm-1; LRMS (ES+) m/z 430 [(M +
H)], 542 [(M + Na), 100]. Anal. (C23H31N3O5‚H2O) Calcd: C,
61.73; H, 7.43; N, 9.39. Found: C, 61.83; H, 6.98; N, 8.82.
127.8, 126.9, 118.9, 101.3, 65.0, 44.9, 15.2; LRMS (ES+) m/z
516 [(M + H), 100]. Anal. (C28H49N7O10S2‚2H2O) Calcd: C,
45.21; H, 7.18; N, 13.18; S, 8.62. Found: C, 44.80; H, 7.02; N,
12.70; S, 9.08.
4,4′-Bis(2-im id a zolyla m in o)d ip h en yla m in e (35). In a
flask cooled to 0 °C, was dissolved the guanidine 32c (350 mg,
0.68 mmol) in 6 M HCl (5 mL). After stirring for 3 h at room
temperature, 10% NaOH was added until a precipitate formed
(pH > 11). The reaction mixture was stirred for 75 min and
was poured into a 1 M NaOH solution. The aqueous phase
was extracted with CH2Cl2. The crude product was collected
by filtration of the aqueous phase. The crude solid was
dissolved in boiling water (10 mL) and the flask was allowed
to stand overnight at room temperature. The product was
collected by filtration, washed several times with H2O, and
dried in vacuo at 50 °C, affording the free base of 35 as a purple
solid (82 mg). The hydrochloride salt was prepared in the
following manner: to 35 dissolved in H2O was added 3 N HCl
until pH 2 was reached. The compound was lyophilized,
dissolved in MeOH, and purified by Et2O-mediated precipita-
tion: purple solid (51 mg, 19%); mp >200 °C (dec); IR (KBr) ν
3-(3-Ben zyloxyca r bon yla m in op r op yl)im id a zolid in e-1-
ca r boxylic Acid Ben zyl Ester (25). To a solution of amine
5d (1.06 g, 2.7 mmol) and formaldehyde (37% in H2O, 1.5 mL,
13.5 mmol) in CH3CN was added NaBH3CN (226 mg, 3.6
mmol). After 10 min, a few drops of AcOH were added to the
cloudy solution to adjust the pH to 6-7. The reaction was
stirred for 17 h at room temperature and the solvent was
removed by reduce pressure. The crude residue was treated
with water and 1 M NaOH was added to adjust the pH to 10-
11. The aqueous phase was extracted three times with CH2-
Cl2, and the organic extracts were dried (Na2SO4) and con-
centrated by reduce pressure. The crude oil was chromato-
graphed (40M cartridge) with CH2Cl2/MeOH (98:2). The six-
membered heterocycle 26 was eluted first, followed by 25.
Compound 25: 1H NMR (300 MHz, CDCl3) δ 7.25 (m, 10H),
5.3 (br, 1H, NH), 5.04 (s, 2H), 4.99 (s, 2H), 3.94 (s, 1H), 3.89
(s, 1H), 3.37 (td, 2H, J ) 8.9 and 9.3 Hz), 3.17 (td, 2H, J ) 9.0
and 9.6 Hz), 2.70 (m, 2H), 2.43 (br t, 2H, J ) 9 Hz), 1.58 (quint,
2H, J ) 9.7 Hz); 13C NMR (300 MHz, CDCl3) δ 157.0, 154.4,
137.2, 129.0, 128.6, 68.8, 68.5, 67.4, 67.1, 53.4, 52.6, 52.2, 44.9,
44.7, 43.6, 40.2, 28.8; LRMS (ES+) m/z 398 [(M + H), 100].
3-(2-B e n z y lo x y c a r b o n y la m i n o e t h y l)t e t r a h y d r o -
p yr im id in e-1-ca r boxylic a cid ben zyl ester (26): 1H NMR
(300 MHz, CDCl3) δ 7.3 (br s, 10H, aro), 5.5 (br, 1H, NH), 5.1
[s, 2H, PhCH2OC(O)NH], 5.06 [s, 2H, PhCH2OC(O)N], 4.12
(s, 2H, NCH2N), 3.48 (br t, 2H, J ) 8.2 Hz, CbzNCH2), 3.22
(br m, 2H, CH2NHCbz), 2.67 (br t, 2H, NCH2CH2), 2.49 (m,
2H, NCH2CH2NHCbz), 1.53 (m, 2H, CH2CH2CH2); 13C NMR
(75 MHz, CDCl3) δ 156.7 [s, C(dO)NH], 155.28 [s, C(dO)N],
136.94 (s, aro), 136.86 (s, aro), 128.7 (d, aro), 128.3 (d, aro),
128.1 (d, aro), 67.4 [t, PhCH2OC(O)NH], 66.7 [t, PhCH2OC-
(O)N], 65.1 (t, NCH2N), 51.9 (t, NCH2CH2), 44.1 (t, CbzNCH2),
38.3 (t, CH2NHCbz), 22.5 (t, CH2CH2CH2); LRMS (ES+) m/z
398 [(M + H), 100].
1
1650, 1590, 1500, 1310, 815, 670 cm-1; H NMR (D2O) δ 7.15
(br s, 8H), 6.76 (br s, 4H); 13C NMR (D2O) δ 145.9 (s), 142.5
(s), 130.2 (s), 125.4 (d), 119.8 (d), 114.0 (d); LRMS (ES+) m/z
332 [(M + H), 100], 166.6 [(M + 2H)]. Anal. (C18H20Cl3N7)
Calcd: C, 49.05; H, 4.57; N, 22.25. Found: C, 49.71; H, 4.45;
N, 21.66.
Bis(4-br om om eth ylp h en yl)m eth a n on e (36). A solution
of 4,4′-dimethylbenzophenone (1 g, 4.8 mmol), NBS (1.71 g,
9.6 mmol), and four drops of t-BuOOH in CCl4 (15 mL) was
heated at reflux for 18 h under argon atmosphere. The
insoluble succinimide was filterered off and the solvent was
removed by reduce pressure. The pure product was obtained
by crystallization from CH2Cl2 as colorless needles (387 mg,
22%): mp 135-137 °C; IR (KBr) ν 1630, 1585, 1390, 1255,
1
1155, 905, 665 cm-1; H NMR (CDCl3) δ 7.78 (d, 4H, J ) 8.4
Hz), 7.51 (d, 4H, J ) 8.4 Hz), 4.57 (s, 4H); 13C NMR (CDCl3)
δ 195.8, 142.9, 137.8, 131.1, 129.6, 32.9. Anal. (C15H12Br2O‚
0.5H2O) Calcd: C, 47.78; H, 3.47. Found: C, 47.87; H, 3.08.
Tr i-n -p en tylp h osp h in e. To a suspension of magnesium
(1.96 g) in dry THF (50 mL) under argon was added a solution
of 1-bromopentane (10 mL, 80.7 mmol) in THF (20 mL). The
resulting reaction mixture was heated at reflux for 20 min.
Then, the reaction was cooled to -78 °C and a solution of
phosphorus trichloride (1.74 mL, 20 mmol) in THF (10 mL)
was added dropwise. The reaction was stirred 30 min at -78
°C and the cold bath was removed. The reaction was allowed
to warm to room temperature and was then heated at reflux
for 30 min. The reaction was quenched with saturated NH4Br
solution (20 mL). The precipitate was filtered off under argon
atmosphere and the crude product was distilled under vacuum,
affording the tri-n-pentylphosphine as a colorless oil (1.305 g,
27%). The product was conserved under argon in the refrigera-
4,4′-Bis[N 3-(2,2-d ie t h oxye t h yl)gu a n id in o]d ip h e n yl-
m eth a n e (31c). A 1 M ether solution of 2,2-diethoxyethyl-
carbodiimide (11.1 mL, 11.1 mmol) was added to a solution of
4,4′-diaminodiphenylmethane (1.05 g, 5.3 mmol) in dry EtOH
under N2. Methanesulfonic acid (0.69 mL, 10.6 mmol) was
added dropwise to the clear reaction mixture and a white
precipitate formed immediately. The reaction was refluxed for
44 h and then poured into 0.5 M aqueous NaOH solution. The
aqueous phase was extracted (3 × CH2Cl2). Organic extracts
were dried (Na2SO4) and concentrated by reduce pressure. The
guanidine 31c was crystallized with CH2Cl2 and washed with
Et2O. Some more compound was obtained by precipitation of
the mother liquor with Et2O: colorless solid (926 mg, 34%);
1
tor: bp (3 mmHg) 115-125 °C; H NMR (CDCl3) δ 1.61 (m,
6H), 1.36 (m, 18H), 0.88 (m, 9H); 13C NMR (D2O) δ 33.9 (d,
J 31P-13C ) 13.7 Hz), 29.1, 27.8, 22.8, 22.0, 14.5; 31P NMR
(CDCl3) δ 50.23.
1
mp 188-190 °C; H NMR (CDCl3/CD3OD) δ 7.0 (d, 4H, J )
7.5 Hz), 6.73 (d, 4H, J ) 7.5 Hz), 4.44 (t, 2H, J ) 4.8 Hz), 3.75
(s, 2H), 3.8-3.4 (m, 8H), 3.19 (d, 4H, J ) 4.8 Hz), 1.12 (t, 12
H, J ) 7 Hz); 13C (CDCl3/CD3OD) δ 154.5, 146.6, 136.1, 130.2,
124.2, 102.6, 63.8, 45.1, 41.2, 15.6; LRMS (EI) m/z 514 [(M+•),
70], 485 [(M - 29), 100]. Anal. (C27H42N6O4) C, H, N.
4,4′-Bis(tr i-n -p en tylp h osp h on iu m )ben zop h en on e Br o-
m id e (37). A solution of 36 (344 mg, 0.93 mmol) and tri-n-
pentylphosphine (1.02 g, 4.2 mmol) in dry toluene (10 mL) was
heated at reflux for 24 h. The precipitate that had formed while
the reaction mixture cooled was triturated with a spatula until
a solid formed. The solid was collected by filtration, rinsed with
dry toluene, and dried in vacuo at 70 °C: colorless hygroscopic
solid (748 mg, 94%); spectroscopic data are in agreement with
the literature;37 LRMS (FAB+) m/z 695.5 [(M+); 100]. Anal.
(C45H78OP2Br2) C, H.
1,4-Bis(4-n itr op h en yl)p ip er a zin e (38).54 A solution of
1-(4-nitrophenyl)piperazine (2.58 g, 12.5 mmol) and 1-fluoro-
4-nitrobenzene (599 mg, 4.16 mmol) in DMSO (15 mL) was
heated 60 h at 100 °C. The cool reaction was poured into water
(50 mL). The precipitate was collected by filtration and rinsed
with a small quantity of water. The product was first crystal-
lized with PhMe/EtOH and rinsed with cold toluene and cold
4,4′-Bis[N 3-(2,2-d ie t h oxye t h yl)gu a n id in o]d ip h e n yl-
a m in e (32c). The same procedure as for 31c starting from
4,4′-diaminodiphenylamine (421 mg, 2.1 mmol), 2,2-diethoxy-
ethylcarbodiimide (4.6 mL, 4.6 mmol), and methanesulfonic
acid (0.27 mL, 4.2 mmol) was used. The crude product
dissolved in EtOH was treated with Et2O. The solid precipitate
was filtered off and the mother liquor was concentrated by
reduce pressure. The residue was dissolved in MeOH and 32c
was isolated by Et2O-mediated precipitation (707 mg, 65%).
Methanesulfonate salt of 32c: purple solid; IR (KBr) ν 1625,
1
1600, 1480, 1175, 1160, 1025, 1015, 750, 737 cm-1; H NMR
(D2O) δ 7.11 (s, 8H), 4.65 (m, 2H), 3.8-3.4 (m, 8H), 3.33 (m,
4H), 1.11 (t, 12H, J ) 7.1 Hz); 13C NMR (D2O) δ 156.7, 143.1,

2306 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Dardonville and Brun
EtOH, respectively. The pure compound was obtained as a red
solid by crystallization with CH3CN (1 g, 73%): mp 265-266
°C (lit.54 mp 261 °C, PhNO2); 1H NMR (DMSO-d6) δ 8.1 (d,
4H, J ) 9 Hz), 7.0 (d, 4H, J ) 9 Hz), 3.71 (s, 8H); 13C NMR
(DMSO-d6) δ 153.9, 136.8, 125.6, 111.9, 45.1. Anal. (C16H16N4O4‚
0.8H2O) Calcd: C, 56.10; H, 5.18; N, 16.36. Found: C, 56.12;
H, 5.35; N, 16.24.
Biologica l Tests. In Vitr o An titr yp a n osom a l Activity
a ga in st Tr ypa n osom a br u cei r h od esien se. Minimum es-
sential 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 Na-pyruvate, 0.1
mM hypoxanthine, and 15% heat inactivated horse serum was
added to each well of a 96-well microtiter plate. Three-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 incubated at 37 °C under a 5% CO2
atmosphere for 72 h. Alamar Blue (10 µL) 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 are analyzed using the microplate
reader software Softmax Pro (Molecular Devices Cooperation,
Sunnyvale, CA).
1,4-Bis(4-a m in op h en yl)p ip er a zin e (39).42 The nitro com-
pound 38 (705 mg, 2.1 mmol) was dissolved in HCl-saturated
methanolic solution (70 mL). The solution was hydrogenated
(40 psi H2) in the presence of 10% Pd-C (165 mg) for 14 h at
room temperature. The catalyst was filtered off and the solvent
was removed by reduce pressure, affording the crude hydro-
chloride of 39. Recrystallization with EtOH afforded the pure
1
HCl salt of 39 (300 mg, 41%): mp >350 °C; H NMR (D2O) δ
7.24 (s, 8H), 3.49 (s, 8H); LRMS (ES+) m/z 269 [(M + H), 100],
135 [(M + 2H)]. Anal. (C16H24Cl4N4‚1.7H2O) Calcd: C, 43.40;
H, 6.24; N, 12.65. Found: C, 43.08; 5.49; N, 12.83.
1,4-Bis[4-(N2,N3-bis(ter t-bu tyloxyca r bon yl)gu a n id in o)-
p h en yl]p ip er a zin e (40a ). To a solution of 39 (54 mg, 0.2
mmol), N,N′-bis(tert-butoxycarbonyl)thiourea (122 mg, 0.44
mmol), and Et3N (0.14 mL, 1 mmol) in DMF (2 mL) at 0 °C
under N2 was added HgCl2 (119 mg, 0.44 mmol) at once. A
precipitate formed immediately. The resulting dark reaction
mixture was stirred for 30 min at 0 °C and 2.5 days at room
temperature. The reaction was diluted with CH2Cl2 and
filtered through a path of Celite. The filter cake was rinsed
with CH2Cl2. The organic phase was washed with brine, dried
(MgSO4), and concentrated. Nonmobile impurities were re-
moved by short flash chromatography on silica with hexane/
EtOAc (75:25). The pure product was obtained by crystalliza-
tion from hexane: yellowish solid (110 mg, 73%); mp >300 °C
dec; 1H NMR (CDCl3) δ 11.64 (br, 2H), 10.17 (br, 2H), 7.47 (d,
4H, J ) 8.9 Hz), 6.92 (d, 4H, J ) 8.9 Hz), 3.27 (s, 8H), 1.5 (br
s, 36H); 13C NMR (CDCl3) δ 164.2, 154.1, 153.9, 149.2, 130.0,
124.1, 117.4, 84.0, 79.9, 50.3, 28.7; LRMS (ES+) m/z 753 [(M
+ H)]. Anal. (C38H56N8O8‚0.7C6H14) Calcd: C, 62.37; H, 8.16;
N, 13.79. Found: C, 62.23; H, 8.80; N, 14.11.
1,4-Bis(4-gu a n id in op h en yl)p ip er a zin e (40b). TFA (2
mL) was added to a stirred solution of 40a (37 mg, 0.049 mmol)
in CH2Cl2 (3 mL). After 2 days, the volatiles were removed by
reduce pressure, and the product was precipitated by addition
of Et2O. The compound was dried in vacuo, affording 40b as
a greenish hygroscopic solid (25 mg, 88%). Trifluoroacetic salt
of 40b: 1H NMR (D2O) δ 7.19 (m, 8H), 3.36 (s, 8H); 13C NMR
(D2O) δ 156.2, 148.4, 128.2, 127.1, 118.7, 49.4; LRMS (ES+)
m/z 353 [(M + H)], 177 [(M + 2H), 100]. Anal. (C24H27F6N8O6)
Calcd: C, 41.51; H, 3.92; N, 16.13. Found: C, 41.52; H, 4.33;
N, 17.04.
Di-ter t-bu tyl 2-(4-[4-(4-[1,3-bis(ter t-bu tyloxyca r bon yl)-
tetr ah ydr o-1H-2-im idazolyliden ]am in oph en yl)piper azin o]-
p h en ylim in o)-1,3-im id a zolid in ed ica r boxyla te (41a ). The
same procedure as for 40a starting from the HCl salt of 39
(111 mg, 0.27 mmol), Et3N (0.37 mL, 2.7 mmol), and HgCl2
(160 mg, 0.59 mmol) and using N,N′-bis(tert-butoxycarbonyl)-
imidazoline-2-thione (178 mg, 0.59 mmol) as reagent for the
introduction of the imidazoline nucleus was used. Flash
chromatography with hexane/EtOAc (50:50) afforded the
product as a colorless solid (143 mg, 66%): 1H NMR (CDCl3)
δ 6.9 (m, 8H), 3.79 (s, 8H), 3.20 (s, 8H), 1.31 (s, 36H); 13C NMR
(50 MHz, CDCl3) δ 150.3 (s), 147.2 (s), 141.5 (s), 138.5 (s), 122.1
(d), 117.3 (d), 82.4 (s), 50.4 (t), 42.9 (t), 27.7 (q). Anal.
(C42H60N8O8) C, H, N.
1,4-Bis[4-(4,5-d ih yd r o-1H-2-im id a zolyla m in o)p h en yl]-
p ip er a zin e (41b). TFA (2 mL) was added to a stirred solution
of 41a (65 mg, 0.08 mmol) in CH2Cl2 (3 mL). After 12 h, the
volatiles were removed by reduce pressure, and the product
dissolved in water was extracted with CH2Cl2 to remove
organic soluble impurities. The water was evaporated and the
product was dried in vacuo to afford 41b as a greenish
hygroscopic solid: 1H NMR (D2Ã) δ 7.3-7.0 (br m, 8H), 3.63
(s, 8H), 3.59 (s, 8H); 13C NMR (CD3OD, 75 MHz) δ 161.1, 152.5,
128.7, 127.4, 118.6, 50.6, 44.5; LRMS (ES+) m/z 405 [(M + H)],
203.2 [(M + 2H), 100]. Anal. (C26H30F6N8O4) C, H, N.
In Vitr o Cytotoxicity w ith 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), 10% fetal bovine serum, and 4 × 104 L-6
cells (rat skeletal 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 incuba-
tion the plates were inspected under an inverted microscope
to ensure growth of the controls and sterile conditions. Then
10 µL of Alamar Blue was added to each well, and the plates
were incubated for another 2 h. 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).
Ack n ow led gm en t. We gratefully acknowledge the
European Commission (Marie Curie Research Training
Grant, Category 20, ERBFMBICT961676), the Spanish
MECD (SB2001-0174), and the UNDP/World Bank/
WHO Special Program for Research and Training in
Tropical Diseases (TDR). The excellent technical as-
sistance of Elke Gobright at the Swiss Tropical Institute
is highly acknowledged.
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