Trypanocidal activity of conformationally restricted pentamidine congeners

Article abstract of DOI:10.1021/jm020375q

A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bridge of pentamidine was replaced by trans-1,2-bismethylenecyclopropyl, phenyl, pyridinyl, piperazinyl, homopiperazinyl, and piperidinyl groups were synthesized

Full text of DOI:10.1021/jm020375q

J . Med. Chem. 2003, 46, 1041-1048
1041
Tr yp a n ocid a l Activity of Con for m a tion a lly Restr icted P en ta m id in e Con gen er s
Isaac O. Donkor,*,† Tien L. Huang,‡ Bin Tao,‡ Donna Rattendi,§ Schennella Lane,§ Marc Vargas,§
Burt Goldberg,§ and Cyrus Bacchi§
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 847 Monroe Avenue,
Room 327E, J ohnson Building, Memphis, Tennessee 38163, College of Pharmacy, Xavier University of Louisiana,
7325 Palmetto Street, New Orleans, Louisiana 70125, and Haskins Laboratories and Department of Biology, Pace University,
New York, New York 10038
Received August 30, 2002
A series of conformationally restricted congeners of pentamidine in which the flexible pentyl
bridge of pentamidine was replaced by trans-1,2-bismethylenecyclopropyl, phenyl, pyridinyl,
piperazinyl, homopiperazinyl, and piperidinyl groups were synthesized. The compounds were
evaluated for trypanocidal activity in vitro and in vivo against one drug-sensitive and three
drug-resistant trypanosome isolates. The DNA binding affinity of the compounds was also
studied using calf thymus DNA and poly(dA-dT). The nature of the linker influenced the DNA
binding affinity as well as the trypanocidal activity of the compounds. trans-1,2-Bis(4-
amidinophenoxymethylene)cyclopropane (1) was over 25-fold more potent than pentamidine
against the drug-resistant isolate KETRI 243As-10-3, albeit with comparable DNA binding
affinity. N,N′-Bis(4-amidinophenyl)homopiperazine (8) was the most potent trypanocide in vitro
against all four trypanosome isolates studied, but N,N′-bis(4-amidinophenyl)piperazine (6) was
the most effective agent in vivo against both drug-sensitive and drug-resistant trypanosomes.
In tr od u ction
used once the parasites have penetrated the CNS. The
fourth drug, eflornithine, is effective against the late-
stage disease caused by T. b. gambiense but is ineffective
against T. b. rhodesiense. Another drug, nifurtimox is
licensed for South American trypanosomiasis but has
also been used in trials against melarsoprol-refractory
late-stage disease. These therapies for African trypano-
somiasis are plagued by drawbacks such as toxicity and
the emergence of resistance;4-7 hence, the need for new
less-toxic agents is widely accepted.1
Human African trypanosomiasis or sleeping sickness
and the related cattle disease Nagana are caused by
subspecies of the parasitic hemoflagellate Trypanosoma
brucei. Each year between 300000-500000 cases of the
human disease are reported in sub-Saharan Africa1 and
the disease is resurgent.2 Trypanosoma brucei exhibits
a complex, digenetic life cycle that alternates between
the tsetse fly (Glossina spp.) vector and the mammalian
host. The life cycle is characterized by a complex series
of cell-type differentiations and variations in metabo-
lism. Infection with African trypanosomiasis starts with
the bite of an infected tsetse fly.3 The parasites move
from the site of infection to the draining lymphatic
vessels and the bloodstream, where they proliferate and
later invade other tissues including the central nervous
system (CNS). Once the parasites have established
residence in the CNS, there is progressive breakdown
of neurological function ending with death, which may
be preceded by coma. There are two forms of African
trypanosomiasis. One form is caused by Trypanosoma
brucei rhodesiense and is endemic in Eastern and
Southern Africa, in which parasites rapidly invade the
CNS, causing death within weeks if untreated. The
other form is caused by T. b. gambiense, which was
originally designated West African trypanosomiasis but
is also widespread in Central Africa. This form of the
disease proliferates more slowly and can take several
years before spreading to the CNS. Four drugs are
generally used to treat African trypanosomiasis, two
(pentamidine and suramin) of which are used prior to
CNS infection. The arsenic-based drug, melarsoprol is
Pentamidine, an aromatic diamidine, is used to treat
early-stage African trypanosomiasis, but the mechanism
of action of the drug is not well understood. It is,
however, known that pentamidine binds to the minor
groove of double helix DNA, and this has been suggested
to play a role in the mechanism of action of the drug.
Indeed, it has been postulated that aromatic diamidines
such as pentamidine may exert their biological activity
by first binding to DNA, which eventually leads to
inhibition of one or more of several DNA-dependent
enzymes (e.g., topoisomerases and nucleases) or by
direct inhibition of transcription.8-14 In support of this
hypothesis, Agbe and Yielding15 recently demonstrated
that an intact kinetoplast is required for the trypano-
cidal action of aromatic diamidines. We16 and others17
have recently reported the trypanocidal action of a series
of pentamidine-related aromatic diamidines. We18,19
have also reported the anti-Pneumocystis carinii activity
and DNA binding affinity of the pentamidine-related
aromatic dicationic compounds shown in Table 1. Be-
cause the interaction of aromatic diamidines with the
minor groove of double helix DNA8-14 and selective
inhibition of kinetoplast DNA synthesis20,21 is believed
to play a significant role in the antimicrobial action of
aromatic diamidines, we were interested in investigat-
ing the trypanocidal activity of the aromatic dicationic
* To whom correspondence should be addressed. Phone: (901) 448-
7736. Fax: (901) 448-6828. E-mail: idonkor@utmem.edu.
† University of Tennessee Health Science Center.
‡ Xavier University of Louisiana.
§ Pace University.
10.1021/jm020375q CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/13/2003

1042 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Donkor et al.
Ta ble 1. Structure, in Vitro Trypanocidal Activity, and DNA Binding Affinity of Conformationally Restricted Pentamidine
Congeners 1-13
a
b
Duplicate determinations with values within (10% of each other. CT is sonicated calf thymus DNA. c AT is sonicated poly(dA-dT).
d
Pent is pentamidine.
Ta ble 2. Structure, in Vitro Trypanocidal Activity, and DNA
Binding affinity of Amidinoalkyl derivatives of 8
mus DNA and poly(dA-dT)), and the trypanocidal activ-
ity of the aromatic dicationic agents 1-18 against one
drug-sensitive (Trypanosoma brucei brucei Lab 110
EATRO) and three drug-resistant (Trypanosoma brucei
rhodesiense) trypanosome isolates in vitro and in mouse
model infections.
Ch em istr y
The compounds were synthesized by following our
previously described procedures as summarized be-
low.18,19,22 Compounds 1-5 were synthesized as shown
in Scheme 1. Reaction of either 4-cyanophenol 19a or
4-cyanoaniline 19b with the appropriate dichlorides
20a -d gave the corresponding dinitriles 21a -d . Trans-
formation of the dinitriles to the corresponding imidate
ester followed by treatment with either ethanolic am-
monia or ethylenediamine afforded the appropriate
diamidines and diimidazolines 1-5 in 58-66% yield.
Compounds 6-9 and 14-18 were synthesized as out-
lined in Scheme 2. This involved aromatic nucleophilic
displacement reaction between 4-fluorobenzonitrile 22a
and piperazine 23a (or homopiperazine 23b) to give
dinitriles 24a and 24b, respectively. The dinitriles were
readily transformed into the corresponding imidate
a
Duplicate determinations with values within (10% of each
other. CT is sonicated calf thymus DNA. c AT is sonicated
poly(dA-dT).
b
compounds shown in Tables 1 and 2. In this report we
describe the synthesis, DNA binding affinity (calf thy-

Pentamidine Congeners
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1043
Sch em e 1a
a
Reagents: (a) Na/EtOH when X ) OH or DIEA/THF when X ) NH2; (b) HClg/EtOH/THF/CHCl3; (c) EtOH/NH3, reflux to give the
diamidine or (d) ethylenediamine/EtOH, reflux to give the diimidazoline. See Table 1 for the structure of Y.
Sch em e 2a
a
Reagents: (a) K2CO3/DMSO, 120 °C; (b) HCl(g)/EtOH/CHCl3/THF; (c) NH3(g)/EtOH, reflux; (d) ethylenediamine/EtOH, reflux; (e)
EtOH/appropriate alkylamine.
Sch em e 3a
a
Reagents: (a) K2CO3/DMSO, 120 °C, 6.5 h for 22a or DIEA, THF for 22b; (b) TFA, pyridine, DCC, DMSO/CH2Cl2 (1:2); (c) 31, 60%
NaH, THF; (d) 5% Pd/C, MeOH, H2, 2 h; (e) THF/EtOH, HCl gas, ethyleendiamine/EtOH reflux, 4.5 h.
ester hydrochlorides 25a and 25b, which upon reaction
with the appropriate amine gave target compounds 6-9
(56-81% yield) and 14-18 (69-81% yield). The syn-
thesis of 10 and 11 is shown in Scheme 3. Briefly, nu-
cleophilic aromatic displacement reaction between 4-flu-
orobenzonitrile 22a or 4-cyanobenzyl bromide 22b and
4-hydroxypiperidine 26 afforded 27a (94% yield) and
27b (97% yield), respectively. The hydroxyl groups of
these compounds were oxidized with DDC to give the
corresponding ketones 28a and 28b. Wittig reaction
between the ketones and phosphonate ester 31 gave
dinitriles 29a (70% yield) and 29b (68% yield), respec-
tively. Phosphonate ester 31 was obtained as a colorless
liquid in 97% yield by reacting 4-cyanobenzyl bromide
22b with triethyl phosphite 30 as shown in Scheme 4.23
Sch em e 4
The double bond of 29a was reduced with H2 over 5%
Pd/C, and the reduction product and 29b were trans-
formed into diimidazoles 10 and 11, respectively, as
described for the synthesis of 7. Compounds 12 and 13
were synthesized using 3-hydroxypiperidine and 4-fluo-
robenzonitrile as the starting materials and following
similar procedures described for the synthesis of 10 and

1044 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Donkor et al.
11. The E configuration of 12 was established by nuclear
243As10-3) or more effective than 11, which is a
4-piperidone derivative. Reduction of the double bond
of 12 gave 13, which was not resolved into its stereo-
isomers but was tested as a mixture of isomers. Com-
pound 13 (a 3-piperidone derivative) was also as effec-
tive or more effective than 10 (a 4-piperidone derivative)
depending on the trypanosome isolate. Thus, the aro-
matic diimiadzolines (12 and 13) derived from 3-piper-
idone were better trypanocidal agents compared to
aromatic diimidazolines (10 and 11) derived from 4-
piperidone. Compound 13 was also as effective as 12
versus all the trypanosome isolates except KETRI 269,
where it was about 6-fold more effective than 12.
Boykin et al.25 used a series of furamidine analogues
to demonstrate that increasing the DNA binding affinity
of aromatic diamidines by substituting alkyl residues
on the amidines enhances the biological action of the
compounds. The increased DNA binding affinity of the
alkylfuramidines was attributed to enhanced van der
Waals interactions between the N-alkyl groups and the
floor of the minor groove of double helix DNA. On the
basis of this precedence, 14-18 (Table 2) were synthe-
sized with the objective of enhancing the trypanocidal
potency of 8 via increased DNA binding affinity. The in
vitro trypanocidal action of these derivatives of 8 (i.e.,
14-16) was disappointing. None of the compounds was
more potent than 8. Compound 17 was the best member
of this set with IC50 values between 78.0 and 180 nM.
With the exception of 18, which displayed DNA binding
affinity similar to that of 8, all of the other derivatives
were weaker DNA binders compared to 8. Unlike the
furamidine series, introduction of alkyl residues on the
amidine groups of 8 did not enhance DNA binding, and
as a result, no improvement in the trypanocidal potency
of the compounds was observed. This discrepancy could
be due in part to the conformations that the N-alkyl
derivatives of 8 adopt when bound to the minor groove
of DNA compared to the N-alkylfuramidines. The radius
of curvature of compounds that bind to the minor groove
of DNA has been shown to play a significant role in the
effectiveness of ligand binding to DNA.26 The central
homopiperazine ring, unlike the furan ring of the
furamidines, may not provide the required curvature
for locating the N-alkyl substituents in proximity to the
floor of the minor groove of double helix DNA.
Overhauser effect (NOE) experiments as described in
1
the Experimental Section. H NMR spectral data and
elemental analyses of all the compounds were satisfac-
tory.
Resu lts a n d Discu ssion
Aromatic diamidines incorporating conformationally
restricted linkers between the benzamidine groups were
synthesized and screened in vitro for trypanocidal
activity against one Trypanosoma brucei brucei pen-
tamidine-sensitive strain (EATRO Lab 110) and three
drug-resistant clinical isolates of Trypanosoma brucei
rhodesiense (KETRI 243, KETRI 269, and KETRI
243As-10-3). The results of the study are shown in
Tables 1 and 2. Generally, the nature of the linker
between the dicationic groups influenced the activity of
the compounds. Compound 1 and pentamidine had
similar DNA binding affinities, but they exhibited
different trypanocidal activity. Compound 1 was be-
tween 11-fold to 15-fold less potent than pentamidine
versus Lab 110 EATRO, KETRI 243, and KETRI 269,
but it was over 25-fold more potent than pentamidine
against the drug-resistant isolate KETRI 243As-10-3.
Compounds incorporating amide linkers (excluding 4)
were generally less effective against all of the trypano-
some isolates studied. For example, 2 and 3 with trans-
1,2-cyclopropane dicarboxamide linkers were virtually
inactive compared to 1 with a trans-1,2-bismethylenecy-
clopropyl ether linker. The planar and/or polar nature
of the linker between 2 and 3 compared to that of 1
might account for the observed difference in the try-
panocidal action of the compounds. This is consistent
with our previous report,16 which demonstrated that
introduction of polar groups in the linker between
aromatic diamidines is detrimental to trypanocidal
action. Alternatively, the difference in trypanocidal
action between 1 and 2 or 1 and 3 could be due to the
difference in the electron-rich phenoxy groups of 1
compared to the electron-poor acylated anilinyl groups
of 2 and 3. This interpretation can also explain the poor
trypanocidal action of 5 compared to 4 because the
pridine ring of 5 is π-deficient compared to the phenyl
ring of 4.24
Compounds 6-9 displayed good in vitro trypanocidal
action versus Lab 110 EATRO and the drug-resistant
clinical isolates of Trypanosoma brucei rhodesiense
(KETRI 243, KETRI 269, and KETRI 243As-10-3).
Compound 8 with a homopiperazine linker between the
benzamidine groups was the most potent agent of the
series versus most of the trypanosome isolates studied.
The compound was as effective as pentamidine against
KETRI 243 and about 2-fold more effective than pen-
tamidine against the drug-resistant isolate KETRI
243As-10-3. Also, 8 was a better trypanocidal agent
compared to 9, corroborating our previous observation16
that amidines are more effective trypanocidal agents
than imidazolines. Derivatives of 7 (i.e., 10-13) in which
the piperazine linker was replaced by substituted pi-
peridines were also studied. The compounds showed
moderate trypanocidal activity (IC50 ) 15-540 nM)
compared to 7 (IC50 ) 13-100 nM) depending on the
trypanosome isolate. Compound 12, which is a 3-piper-
idone derivative, was as effective (versus KETRI
It has been postulated that aromatic diamidines may
exert their biological action by first binding to double
helix DNA and causing inhibition of one or more of
several DNA-dependent enzymes (e.g., topoisomerases
and nucleases) or by direct inhibition of transcription.8-14
We therefore studied the affinity of the compounds in
Tables 1 and 2 for binding to sonicated calf thymus DNA
and poly(dA-dT) with the objective of investigating if
there is a correlation between the DNA affinity and the
trypanocidal activity of this series of compounds. All 18
compounds were found to bind to both calf thymus DNA
and poly(dA-dT). The compounds consistently displayed
stronger affinity for poly(dA-dT) compared to calf thy-
mus DNA, which is in agreement with the observation
that pentamidine and its analogues prefer AT-rich DNA
sequences.25,27,28 The most potent trypanocidal agents,
1 and 6-8, were also the strongest DNA binders in this
series, and the weakly active compounds, 2-5, were also
the weakest DNA binders. However, a direct correlation

Pentamidine Congeners
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1045
Ta ble 3. Activity of Conformationally Restricted Pentamidine
Congeners 6-9 versus T. b. brucei Lab 110 EATRO Mouse
Model Infectiona
Ta ble 5. Activity of Multiple Daily Dosing of 6 versus T. b.
rhodesiense KETRI 243 Mouse Model Infectiona
dosed
dosed
dosed
number cured/total (MSD)b
three times daily four times daily
five times daily
dose
(mg/kg) pent.
6
7
8
9
dose
(mg/kg) MSD
no. cured/
total
no. cured/
total
no. cured/
total
MSD
MSD
1.0
2.5
5.0
10.0
25.0
5/5 (-) 2/3 (16.0) 0/3 (10.3)
0/3 (8.3)
1/3 (16.5) 0/3 (4.3)
2/3 (12.0) 0/3 (7.7)
0/3 (4.7)
5/5 (-) 3/3 (-)
5/5 (-) 3/3 (-)
3/3 (-)
3/3 (-)
3/3 (-)
5
10
25
14.0
18.0
26.0
0/3
0/3
2/3
14.7
22.0
>39
0/3
0/3
3/3
14.7
21.3
>39
0/3
0/3
3/3
c
c
3/3 (-)
3/3 (-)
3/3 (-)
1/3 (14.0)
3/3 (-)
0/3 (19.7*) 3/3 (-)
a
Conditions were the same as in Table 3 except for the dosing
a
Mice were infected (2.5 × 105 parasites), and the infections
were allowed to progress 24 h before treatment began. Compounds
were dissolved in distilled water (vehicle) and injected ip once a
day for 3 days. Mice treated with vehicle as control all died with
frequency.
animals when administered 48 h after infection, but the
mean survival times in days varied from 5 days to over
35 days depending on the dose of the drug. Compound
6 was, however, not effective when it was administered
72 h after infection. In a second study, 6 and 8 were
protective but not curative versus mice infected with
the drug-resistant isolate T. b. rhodesiense KETRI 243
(data not shown). For example, when mice were infected
with KETRI 243 and treated with 10 mg/kg of either 6
or 8, they survived for an average period of 20 and 15
days, respectively, compared to vehicle-treated mice,
which had an average life span of 9 days after infection.
Compound 6 was further studied versus infection due
to the drug-resistant isolate T. b. rhodesiense KETRI
243. In this study, the dose and the frequency of
administration of 6 were increased. As shown in Table
5, when mice infected with the drug-resistant isolate
T. b. rhodesiense KETRI 243 were treated with 25 mg/
kg of 6, three times daily for 3 days, 67% of the mice
were cured. When the frequency of drug administration
was increased from three times daily to four or five
times daily for 3 days, all of the mice were cured. The
required multiple daily dosing of 6 to achieve effective
trypanocidal activity may be due to rapid elimination
of the compound. Derivatives of 6 that do not require
multiple daily dosing may be clinically useful trypano-
cides. Our laboratory is currently investigating such
agents.
In summary, a series of aromatic amidines and
imidazolines incorporating conformationally restricted
linkers between the cationic groups were synthesized
as constrained congeners of pentamidine. The DNA
binding affinity and the in vitro and in vivo trypanocidal
activity of the compounds were studied. The nature of
the linker between the amidine or imidazoline groups
influenced the DNA binding affinity as well as the
trypanocidal activity of the compounds. Generally,
compounds with strong affinity for DNA also showed
good trypanocidal activity. Compounds with dicarboxa-
mide linkers were not good DNA binders and were also
generally ineffective trypanocides compared to the
compounds without such linkers (e.g., 1 versus 2).
However, a clear correlation between in vitro trypano-
cidal activity and in vitro DNA binding affinity was not
observed.
b
MSD values ranging between 5 and 13 days. MSD is the mean
survival time (in days) of animals dying of trypanosomiasis
exclusive of cured animals. Animals surviving >30 days beyond
the death of the last control with no parasites in tail blood smears
were considered cured. Three mice were studied at each drug dose
with MSD values for each study group shown in parentheses. For
pentamidine (pent.), five mice were studied at each drug dose. The
dash (-) means all the animals were cured as defined above. The
asterisk (/) indicates that the compound was toxic. c Not deter-
mined.
Ta ble 4. Effect of Delayed Dosing of 6 on T. b. brucei Lab 110
EATRO Toxicitya
24 h after
infection
48 h after
infection
72 h after
infection
dose
(mg/kg) MSD
no. cured/
total
no. cured/
no. cured/
total
MSD
total
MSD
2.5
5.0
10.0
>35
>35
>35
3/3
3/3
3/3
>35
3/3
2/3
2/3
4-5
4-5
4-5
0/3
1/3
0/3
5
7
a
Dosing was once daily for 3 days, starting at 24, 48, or 72 h
after infection. Other methods were as in Table 3.
between the trypanocidal activity and the DNA binding
affinity of the compounds in this series was not ob-
served.
Compounds 6-9 displayed good in vitro trypanocidal
activity, so they were selected for in vivo evaluation
versus T. b. brucei Lab 110 EATRO and T. b. rho-
desiense KETRI 243 isolates in mouse model infections.
The compounds were administered intraperitoneally as
single daily injections over 3 days except where indi-
cated otherwise. The results of this study are shown in
Table 3. Compounds 6 and 7 were as effective as
pentamidine in curing infections due to T. b. brucei Lab
110 EATRO at doses ranging from 2.5 to 25 mg/kg per
day, but 7 was toxic at 25 mg/kg per day. Compound 8
was protective at doses between 1.0 and 5.0 mg/kg per
day and was curative at doses of 10.0 or 25 mg/kg per
day. Compound 9 was the least effective member. It was
curative only at a dose of 25 mg/kg per day. In contrast
to the results of the in vitro studies, 6 was a more
effective trypanocide in vivo compared to 8, which was
the best trypanocidal agent of the series in vitro. The
lack of correlation between the in vitro and in vivo
activities of the compounds may be attributed to differ-
ences in their pharmacokinetic properties; however, this
awaits experimental confirmation.
Compound 1 was the most effective agent against the
drug-resistant isolate KETRI 243As-10-3. It was over
25-fold more potent than pentamidine versus this
isolate. Compound 8 with a homopiperazine linker
showed a broad spectrum of trypanocidal activity. It was
also the most potent agent in vitro. However, 6 with a
piperazine linker emerged as the most effective agent
in vivo. Compound 6 cured infections due to the drug-
The effect of delayed dosing of 6 on infection due to
T. b. brucei Lab 110 EATRO at doses ranging from 2.5
to 10 mg/kg per day was studied. The data (Table 4)
indicated that 6 effectively cured the mice at all of the
doses studied if the drug was administered 24 h after
infection. Compound 6 also cured 67-100% of the

1046 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Donkor et al.
piperidine. The product was dissolved in DMSO/benzene
mixture (1:2), and DCC and pyridine were added under a
nitrogen atmosphere. The mixture was cooled to 5 °C, and TFA
was added dropwise. Following this, the mixture was allowed
to warm to room temperature and stirring was continued for
24 h. EtOAc was added to precipitate N,N′-dicyclohexylurea,
which was filtered off. The filtrate was washed with brine
(3 × 100 mL), and the organic layer was left at room temper-
ature overnight. The piperidone that separated out was
collected, dried, and reacted with 4-cyanobenzyl phosphonate
(31), which was obtained by heating a mixture of 4-cyanoben-
zyl bromide (1 equiv) and triethyl phosphite (1 equiv) at 160-
170 °C for 8 h followed by distillation to give 31 (bp 165 °C/
0.1 mmHg). The piperidone was then reacted with 31 as
follows. NaH (1 equiv) was added to dry THF, the slurry was
cooled to 20 °C, and 31 (1 equiv) was added dropwise with
stirring. After the addition, the mixture was stirred at room
temperature for 30 min. The resulting yellow-brown solution
was maintained below 25 °C, and a solution of the piperidone
(1 equiv) in dry THF was added dropwise. The mixture was
stirred at room temperature for 30 min and refluxed overnight.
After the mixture was cooled, the THF layer was recovered
and concentrated to dryness and the residue was recrystallized
from a hexanes/acetone mixture (1:2) to give the corresponding
dinitrile.
resistant isolate T. b. rhodesiense KETRI 243, but its
effectiveness was dependent on the dosage and the
frequency of administration. Derivatives of 6 that do not
require multiple daily dosing may be clinically useful
trypanocides.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Haake-
Buchler melting point apparatus and are uncorrected. The 1H
NMR spectra were recorded on Varian Gemini-300 and GE
Omega-500 spectrometers, and DMSO-d6 was used as the
solvent unless otherwise noted. The chemical shifts (δ) are
reported in ppm relative to TMS, 0.00 ppm. Elemental
analyses (C, H, N) were performed by M-H-W Laboratories,
Phoenix, AZ, and are within (0.4% of the theoretical values.
All chemicals and solvents were purchased from Aldrich
Chemical Co. THF was distilled from Na and benzophenone
before use, and other chemicals were used as received.
Anhydrous ethanol and methanol were used throughout this
study unless stated otherwise.
t r a n s-1,2-Bis(4-cya n op h e n oxym e t h yle n e )cyclop r o-
p a n e (21a ). This dinitrile precursor to the synthesis of 1 was
prepared as previously reported.18 Yield, 76%; mp 150-152
°C; 1H NMR (CDCl3) δ 7.58-7.55 (m, 4H), 6.92-6.90 (m, 4H),
4.07-4.05 (m, 4H), 2.00-1.98 (m, 4H).
N -(4-Cya n op h e n yl)-4-(4-cya n ob e n zylid in yl)p ip e r i-
Gen er a l P r oced u r e for th e Syn th esis of Din itr iles
Requ ir ed for th e Syn th esis of 2-5. Following a known
procedure,29 a solution of the appropriate acid dichloride in
THF was added to a stirred solution of 4-aminobenzonitrile
(2 equiv) and DIEA (2 equiv) in THF at 0 °C under a N2
atmosphere. After 30 min, another portion of DIEA (2 equiv)
in THF was added and the mixture was stirred overnight. The
solvent was removed under reduced pressure followed by
recrystallization of the residue to give the corresponding
dinitrile.
N,N′-Bis(4-cya n op h en yl)-tr a n s-1,2-cyclop r op a n ed ia -
m id e (21b, d in itr ile p r ecu r sor for th e syn th esis of 2 a n d
3): yield 62%; mp >305 °C; 1H NMR δ 10.80 (s, 2H), 7.75-
7.71 (m, 8H), 2.31 (t, 2H), 1.34 (t, 2H).
N,N′-Bis(4-cya n op h en yl)-1,3-ben zod ia m id e (21c, d in i-
tr ile p r ecu r sor for th e syn th esis of 4): yield 77%; mp 298-
299 °C; 1H NMR δ 10.80 (s, 2H), 8.51(s, 1H), 8.16 (dd, 2H),
7.98 (d, 4H), 7.82 (d, 4H), 7.72 (t, 1H).
N,N′-Bis(4-cyan oph en yl)-2,6-pyr idin ediam ide (21d, din i-
tr ile p r ecu r sor for th e syn th esis of 5): yield 72%; mp 304-
306 °C; 1H NMR δ 11.30 (s, 2H), 8.41 (d, 2H), 8.32 (t, 1H),
8.16 (d, 4H), 7.91 (d, 4H).
Gen er a l P r oced u r e for th e Syn th esis of Din itr iles
Requ ir ed for th e Syn th esis of 6-9. Under a N2 atmosphere,
a mixture of either piperazine or homopiperazine (1 equiv),
4-fluorobenzonitrile (2 equiv), and K2CO3 (3 equiv) in dry
DMSO (100 mL) was heated at 120 °C for 6 h. After the
mixture was cooled to room temperature, an excess of water
was added and the precipitated solid was filtered, washed with
water, and dried to give the appropriate dinitrile.
d in e (29a , d in itr ile p r ecu r sor for th e syn th esis of 10):
1
yield 70%; mp 124-125 °C; H NMR (CDCl3) δ 7.62 (d, 2H),
7.50 (d, 2H), 7.30 (d, 2H), 6.85 (d, 2H), 6.40 (s, 1H), 3.51 (t,
2H), 3.42 (t, 2H), 2.59 (m, 2H), 2.52 (t, 2H).
N -(4-Cya n ob e n zyl)-4-(4-cya n ob e n zylid in yl)p ip e r i-
d in e (29b, d in itr ile p r ecu r sor for th e syn th esis of 11):
1
yield 68%; H NMR (CDCl3) δ 7.58 (d, 2H), 7.42 (d, 2H), 7.31
(d, 2H), 6.83 (d, 2H), 6.38 (s, 1H), 3.50 (s, 2H), 2.66 (m, 4H),
2.20 (m, 4H).
N -(4-Cya n op h e n yl)-3-(4-cya n ob e n zylid in yl)p ip e r i-
d in e (d in itr ile p r ecu r sor for th e syn th esis of 12 a n d 13):
1H NMR (CDCl3) δ 7.56 (d, 2H), 7.44 (d, 2H), 7.34 (d, 2H), 6.81
(d, 2H), 6.40 (s, 1H), 3.98 (s, 2H), 3.43 (t, 2H), 2.82 (t, 2H),
1.97 (m, 2H).
Gen er a l P r oced u r e for th e Syn th esis of th e Dia m i-
d in es, N-Alk yla m id in es, a n d Diim id a zolin es. Anhydrous
HCl gas was bubbled through an ice-cooled solution of the
dinitrile in EtOH, dry THF, and/or CHCl3 for 1 h. The solution
was allowed to warm to room temperature and stirred for 3-5
days. The solvent was concentrated to near-dryness below 40
°C under reduced pressure, and ether was added to precipitate
the imidate ester as the hydrochloride salt. The solid was
immediately dissolved in MeOH or EtOH after filtration. For
the synthesis of amidines, anhydrous ammonia gas was
bubbled through the solution for 1 h, during which time the
solution was gently refluxed for 4 h. After the mixture was
cooled, the solvent was removed under reduced pressure and
ether was added to precipitate the crude product, which was
purified by recrystallization to give the amidine as the dihy-
drochloride salt. For the synthesis of imidazolines, excess
ethylenediamine was added to a solution of the imidate ester
hydrochloride in MeOH or EtOH. The mixture was refluxed
for 2-4 h before the solvent was evaporated. Ether was added
to the residue to afford the imidazoline free base, which was
refluxed in saturated HCl/EtOH solution for 2-4 h. The
solvent was removed under reduced pressure, and the residue
was purified by recrystallization to give the desired imidazoline
as the dihydrochloride salt, with the exception of 2, which was
obtained as a free base without refluxing in HCl/EtOH. For
the synthesis of N-alkyl-substituted amidines 14-18, the
imidate ester of N,N′-bis(4-cyanophenyl)homopiperazine was
reacted with the appropriate alkylamine (2 equiv) to give the
desired products.
N,N′-Bis(4-cya n op h en yl)p ip er a zin e (24a , d in itr ile p r e-
cu r sor for th e syn th esis of 6 a n d 7): yield 83%; mp 271-
1
273 °C; H NMR (CDCl3) δ 7.53(d, 4H), 6.87(d, 4H), 3.52 (s,
8H).
N,N′-Bis(4-cya n op h en yl)h om op ip er a zin e (24b, d in i-
tr ile p r ecu r sor for th e syn th esis of 8 a n d 9): yield 80%;
mp 208-209 °C; 1H NMR (CDCl3) δ 7.48 (d, 4H), 6.72 (d, 4H),
3.71 (s, 4H), 3.49 (t, 4H), 2.13-2.10 (m, 2H).
Gen er a l P r oced u r e for th e Syn th esis of Din itr iles
Requ ir ed for th e Syn th esis of 10-13. Under a N2 atmo-
sphere, a mixture of either 3-hydroxypiperidine or 4-hydroxy-
piperidine (1 equiv), 4-fluorobenzonitrile (1 equiv) or 4-cy-
anobenzyl bromide (1 equiv), and K2CO3 (1.5 equiv) in dry
DMSO (100 mL) was heated at 120 °C for 6 h. After the
reaction mixture was cooled to room temperature, it was
poured into excess water and the precipitate that separated
out was filtered, washed with water, dried, and recrystallized
from acetone to give the corresponding N-substituted hydroxy-
tr a n s-1,2-Bis(4-a m id in op h en oxym eth ylen e)cyclop r o-
p a n e (1): yield 66%; mp 257-259 °C; 1H NMR δ 9.23 (s, 4H),
9.02 (s, 4H), 7.83 (d, 4H), 7.12 (d, 4H), 4.14 (m, 4H), 1.88 (m,
4H). Anal. (C19H22N4O2‚2HCl‚2H2O) C, H, N.

Pentamidine Congeners
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1047
N,N′-Bis(4-a m id in op h en yl)-tr a n s-1,2-cyclop r op a n e d i-
ca r boxa m id e (2): yield 64%; mp >300 °C; H NMR δ 11.10
(s, 2H), 9.15 (s, 8H), 7.79 (m, 8H), 2.46 (m, 2H), 1.38 (m, 2H).
5.16 (bs, 2H), 3.74 (s, 4H), 3.61 (t, 4H), 3.51 (m, 4H), 3.44 (t,
4H), 1.93 (m, 2H). Anal. (C23H32N6O2‚2HCl‚0.5H2O) C, H, N.
1
N ,N ′-Bis[4-(N -n -b u t yla m id in o)p h e n yl]h om op ip e r a -
1
Anal. (C19H20N6O2‚2HCl) C, H, N.
zin e (17): yield, 81%; mp >300 °C; H NMR δ 9.14 (s, 2H),
N ,N ′-Bis(4-im id a zolin op h e n yl)-t r a n s-1,2-cyclop r o-
8.84 (s, 2H), 8.46 (s, 2H), 7.50 (d, 4H), 6.82 (d, 4H), 3.68 (s,
4H), 3.45 (s, 4H), 3.26-3.28 (m, 4H), 1.88 (m, 2H), 1.51-1.55
(m, 4H), 1.27-1.32 (m, 4H), 0.82-0.87 (m, 6H). Anal. (C27H40N6‚
2HCl‚0.6H2O) C, H, N.
1
p a n e d ica r boxa m id e (3): yield 63%; mp >360 °C; H NMR
δ 11.16 (s, 2H), 10.55 (bs, 4H), 8.00 (d, 4H), 7.87 (d, 4H), 3.97
(s, 8H), 2.45 (m, 2H), 1.40 (m, 2H). Anal. (C23H24N6O2‚2HCl)
C, H, N.
N,N′-Bis[4-(N-cyclop r op yla m id in o)p h en yl]h om op ip er -
a zin e (18): yield, 69%; mp >300 °C; 1H NMR δ 9.56 (bs, 2H),
9.31 (bs, 2H), 8.72 (bs, 2H), 7.67 (d, 4H), 6.87 (d, 4H), 3.73 (s,
4H), 3.50 (s, 4H), 2.71 (m, 2H), 1.91 (m, 2H), 0.89 (m, 4H),
0.76 (m, 4H). Anal. (C25H32N6‚2HCl‚0.8H2O) C, H, N.
Th er m a l Den a tu r a tion Stu d ies. Pentamidine, EDTA,
Tris-HCl, calf thymus DNA, and poly(dA-dT) used in this study
were purchased from Sigma Chemical Company. The method
used for the determination of the binding affinity (∆Tm) of
1-18 to calf thymus DNA and the nucleic acid homopolymer
poly(dA-dT) has been described previously.18,30,31 The binding
affinity was measured by determining the change in the
midpoint (Tm) of the thermal denaturation curves of calf
thymus DNA as well as poly(dA-dT) at a 1:5 compound to base
pair ratio. Each ∆Tm value reported in Table 1 represents the
mean of at least two experimental determinations.
N,N′-Bis(4-am idin oph en yl)-1,3-ben zodicar boxam ide (4):
yield 63%; mp >360 °C; 1H NMR δ 11.23 (s, 2H), 9.30 (s, 4H),
9.02 (s, 4H), 8.94 (s, 1H), 8.22 (d, 2H), 8.20 (d, 4H), 7.88 (d,
4H), 7.74 (t, 1H). Anal. (C22H20N6O2‚2HCl‚2H2O) C, H, N.
N,N′-Bis(4-a m id in op h en yl)-2,6-p yr id in ed ica r b oxa m -
1
id e (5): yield 58%; mp 318 °C; H NMR δ 11.72 (s, 2H), 9.33
(s, 4H), 9.04 (s, 4H), 8.44 (d, 4H), 8.32 (m, 3H), 7.93 (d, 4H).
Anal. (C21H19N7O2‚2HCl) C, H, N.
N,N′-Bis(4-a m id in op h en yl)p ip er a zin e (6): yield 81%; mp
356 °C; 1H NMR δ 9.02 (s, 4H), 8.69 (s, 4H), 7.79 (d, 4H), 7.15
(d, 4H), 3.58 (s, 8H). Anal. (C18H22N6‚2HCl) C, H, N.
N,N′-Bis(4-im idazolin oph en yl)piper azin e (7): yield 65%;
1
mp >360 °C; H NMR δ 10.17 (s, 4H), 7.87 (d, 4H), 7.09 (d,
4H), 3.91 (s, 8H), 3.61 (s, 8H). Anal. (C22H26N6‚2HCl‚2H2O) C,
H, N.
N,N′-Bis(4-a m id in op h en yl)h om op ip er a zin e (8): yield
56%; mp 340 °C; 1H NMR δ 8.91 (s, 4H), 8.61 (s, 4H), 7.73 (d,
4H), 6.92 (d, 4H), 3.76 (s, 4H), 3.53 (m, 4H), 1.96 (m, 2H). Anal.
(C19H24N6‚2HCl‚2H2O) C, H, N.
P h ar m acology. Trypanosoma brucei brucei Lab 110 EATRO
strain (pentamidine-sensitive) and clinical isolates of Trypa-
nosoma brucei rhodesiense were used in this study. T. b.
rhodesiense isolates were obtained from A. R. Njogu of the
Kenya Trypanosomiasis Research Institute (KETRI, Muguga,
Kenya). These included KETRI 243 (DFMO, melarsoprol,
pentamidine, and berenil resistant), KETRI 243As-10-3, which
is a cloned subpopulation of KETRI 243 and is refractory to
arsenicals and aromatic diamidines such as berenil and
pentamidine, and KETRI 269 (DFMO and pentamidine resis-
tant).32
N,N′-Bis(4-im idazolin oph en yl)h om opiper azin e (9): yield
1
71%; mp 326 °C; H NMR δ 10.14 (s, 4H), 7.85 (d, 4H), 6.94
(d, 4H), 3.89 (s, 8H), 3.78 (s, 4H), 3.55 (m, 4H), 1.93 (m, 2H).
Anal. (C23H28N6‚2HCl‚H2O) C, H, N.
N-(4-Im id a zolin op h en yl)-4-(4-im id a zolin ob en zyl)p i-
p er id in e (10): yield 32%; mp 240 °C; 1H NMR δ 10.80 (s,
2H), 10.26 (s, 2H), 8.04 (d, 2H), 7.90 (d, 2H), 7.50 (d, 2H), 7.07
(d, 2H), 4.22 (t, 2H), 4.01 (s, 4H), 3.93 (s, 4H), 2.89 (t, 2H),
2.69 (d, 2H), 1.91 (m, 1H), 1.65 q, 2H), 1.24 (q, 2H). Anal.
(C24H29N5‚2HCl) C, H, N.
N-(4-Im idazolin oben zyl)-4-(4-im idazolin oben zylidin yl)-
p ip er id in e (11): yield 40%; mp 150-151 °C; 1H NMR δ 11.00
(s, 2H), 10.82 (s, 2H), 8.15 (d 2H), 8.04 (d, 2H), 7.94 (d, 2H),
7.50 (d, 2H), 6.53 (s, 1H), 4.43 (s, 2H), 3.99 (s, 4H), 3.96 (s,
4H), 2.48-3.01 (m, 8H). Anal. (C25H29N5‚2HCl) C, H, N.
N-(4-Im idazolin oph en yl)-3-(4-im idazolin oben zylidin yl)-
p ip er id in e (12): yield 21%; mp 180-181 °C; 1H NMR δ 10.71
(s, 2H), 10.17 (s, 2H), 8.00 (d, 2H), 7.87 (d, 2H), 7.50 (d, 2H),
7.10 (d, 2H), 6.67 (s, 1H), 4.20 (s, 2H), 3.97 (s, 4H), 3.89 (s,
4H), 3.58 (t, 2H), 2.60 (t, 2H), 1.73 (q, 2H). NOE experiments
showed that 12 assumed the E configuration. The free base of
12 in CD3OD was used in these experiments. Irradiation of
the alkene proton (δ 6.53) of 12 caused enhancement of the
methylene protons (δ 3.98) adjacent to the nitrogen atom of
the piperidine ring. Similarly, irradiation of the methylene
protons caused enhancement of the alkene proton. Anal.
(C24H27N5‚2HCl) C, H, N.
N-(4-Im id a zolin op h en yl)-3-(4-im id a zob en zyl)p ip er i-
d in e (13): yield 38%; mp 260-261 °C; 1H NMR δ 10.89 (s,
2H), 10.37 (s, 2H), 8.04 (d, 2H), 7.79 (d, 2H), 7.48 (d, 2H), 6.89
(d, 2H), 3.93 (s, 4H), 3.81 (s, 4H), 2.58-2.85 (m, 6H), 1.80 (m,
1H), 1.19-1.60 (m, 4H). Anal. (C24H29N5‚2HCl) C, H, N.
In Vitr o Stu d ies. The compounds were tested against
trypanosome isolates grown as blood forms in HMI-18 me-
dium33 containing 20% horse serum in 24-well microplates at
37 °C. Wells were inoculated with 1 × 105 trypanosomes. The
compounds were diluted in the medium at the appropriate
concentration and replaced daily. Cell counts were made daily
with a Coulter counter, model Z1 (Beckman Coulter, Miami,
FL). Cells were diluted with Isoton I buffer (Beckman Coulter),
and the aperture was standardized at 5.14 µm. Background
checks were performed daily on 1:10 dilutions of medium.
Counts, normalization, and coincident counts were accounted
for by the standardized Coulter analysis program. Occasion-
ally, hemocytometer counts were performed as a check on the
validity of the Coulter program. IC50 values were determined
after 48 h from semilog plots, and the values are the result of
duplicate determinations. Initially a broad concentration curve
was used and then a close concentration curve from which IC50
values were determined. Assays were done in duplicate, and
each point was the average of the two. Control cells grew to 5
× 106/mL.
In Vivo Stu d ies. Female Swiss-Webster mice (weight, 20
g) were infected (intraperitoneally) with 2.5 × 105 trypano-
somes from rat blood, and the infection was allowed to develop
for 24 h before drug treatment was begun. Groups of three
mice each were injected (intraperitoneally) with each drug
concentration. All of the experiments included a group of
untreated controls. Untreated mice died 5-13 days after
infection, depending on the isolate. Parasitemia of animals
dying of infection averaged (0.5-1.0) × 109/mL of blood. These
infections are very predictable, and daily counts were not done
because of the labor involved. The procedures used are
standard for our laboratory.32 Animals were monitored weekly
for parasites in tail vein blood smears. Mice were considered
cured if they survived more than 30 days after the death of
the last control with no parasites in tail vein blood smears.
MSD (mean survival in days) was also recorded for each group.
It is the average time of survival of the animals in the group,
exclusive of cured animals.
N,N′-Bis[4-(N-isop r op yla m id in o)p h en yl]h om op ip er a -
1
zin e (14): yield, 71%; mp >300 °C; H NMR δ 9.09 (m, 4H),
8.66 (s, 2H), 7.60 (d, 4H), 6.90 (d, 4H), 4.02 (m, 2H), 3.73 (s,
4H), 3.49 (m, 4H), 1.93 (m, 2H), 1.22 (d, 12H). Anal. (C25H36N6‚
2HCl‚H2O) C,H, N.
N,N′-Bis[4-(N-ter t-b u t yla m id in o)p h en yl]h om op ip er -
1
a zin e (15): yield, 78%; mp 271-273 °C; H NMR δ 8.40 (bs,
2H), 7.97 (s, 4H), 7.64 (d, 4H), 6.76 (q, 4H), 3.67 (s, 4H), 3.43
(m, 4H), 1.98 (m, 2H), 1.26 (m, 18H). Anal. (C27H40N6‚2HCl‚
H2O) C, H, N.
N,N′-Bis[4-(N-2-h yd r oxyet h yla m id in o)p h en yl]h om o-
p ip er a zin e (16): yield, 73%; mp 281-283 °C; 1H NMR δ 9.23
(s, 2H), 8.90 (s, 2H), 8.58 (s, 2H), 7.65 (d, 4H), 6.90 (d, 4H),

1048 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Donkor et al.
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Ack n ow led gm en t. The study was funded by NIH
Grant 1R15 AI/OD 39683 to I.O.D., NIH Grants DA07970
and 2S06GM08008 to T.L.H., and Grant 950504 from
the World Health Organization/World Bank/United
Nations Development Programme Special Programme
for Tropical Disease Research to C.J .B).
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