Synthesis and antiparasitic activity of new bis-arylimidamides: DB766 analogs modified in the terminal groups

Article abstract of DOI:10.1016/j.ejmech.2014.06.022

Fifteen novel bis-arylimidamide derivatives with various 6-membered (7a-c) and 5-membered (7d-o) heterocyclic rings replacing the terminal pyridyl rings of the lead compound DB766{(2,5-bis[2-i-propoxy-4-(2-pyridylimino) aminophenylfuran]}, were prepared a

Full text of DOI:10.1016/j.ejmech.2014.06.022

Accepted Manuscript
Synthesis and antiparasitic activity of new bis-arylimidamides: DB766 analogs
modified in the terminal groups
Zong-ying Liu, Tanja Wenzler, Reto Brun, Xiaohua Zhu, David W. Boykin
PII:
S0223-5234(14)00541-8
10.1016/j.ejmech.2014.06.022
EJMECH 7065
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 January 2014
Revised Date: 6 June 2014
Accepted Date: 11 June 2014
Please cite this article as: Z.-y. Liu, T. Wenzler, R. Brun, X. Zhu, D.W. Boykin, Synthesis and
antiparasitic activity of new bis-arylimidamides: DB766 analogs modified in the terminal groups,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.022.
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ACCEPTED MANUSCRIPT
O
O
NH
NH
O
R
R
N
H
NH
R= various 5 and 6 membered ring heterocyclces
10 of 15 compounds are very active vs. Plasmodium falciparum (IC50 = 9-87 nM)

ACCEPTED MANUSCRIPT
Synthesis and antiparasitic activity of new bis-arylimidamides: DB766 analogs modified in the terminal
groups
Zong-ying Liua, b, Tanja Wenzlerc, d, Reto Brunc, d, Xiaohua Zhue, David W. Boykina, *
aDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
bInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, People’s Republic of China, cSwiss Tropical and Public Health Institute, Department
d
of Medical Parasitology and Infection Biology 4002 Basel, Switzerland, University of Basel, 4003 Basel,
e
Switzerland, Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, OH 43210, USA.
*Corresponding author. Tel.:+1 404-413-5498. Fax: +1 404-413-5505. E-mail address: dboykin@gsu.edu
Abstract: Fifteen novel bis-arylimidamide derivatives with various 6-membered (7a-c) and
5-membered (7d-o) heterocyclic rings replacing the terminal pyridyl rings of the lead
compound
DB766{(2,5-bis[2-i-propoxy-4-(2-pyridylimino)aminophenylfuran]},
were
prepared and evaluated versus Trypanosoma cruzi, Leishmania amazonensis, Trypanosoma
brucei rhodesiense and Plasmodium falciparum. Compound 7a with pyrimidine replacing the
pyridine rings showed good activity versus T. cruzi, T. brucei rhodesiense and P. falciparum
(IC50 = 200 nM, 32 nM and 8.5 nM, respectively). Three compounds (7g, 7i, 7j) with
thiazole replacing the pyridine rings gave low micromolar (0.17- 0.3 ꢀM) IC50 values versus
L. amazonensis, however only 7g exhibited an acceptable selectivity index (SI = 27).
Compounds 7a, 7j, and 7m exhibited potent activity against T. brucei rhodesiense (IC50 = 12-
60 nM). Ten of the 15 compounds with pyrimidine, pyrrole, thiazole and imidazole terminal
units were highly active against P. falciparum (IC50 = 9-87 nM). Both pyrimidine and
pyridine terminal groups are advantageous for anti-T. cruzi activity and several different
heterocyclic terminal units are effective versus P. falciparum, both findings merit further
investigation.
Keywords: bis-arylimidamides, DB766, antiparasitic agents
1. Introduction
Parasitic diseases including African sleeping sickness, Chagas’ disease, leishmaniasis, malaria and others have
been a major threat for mankind for centuries and continue to cause significant public health problems [1, 2].
Protozoan parasitic diseases are major causes of mortality and morbidity in tropical and subtropical countries,
and are also responsible for important economic losses [1-5]. Chagas’ disease is a neglected tropical illness
caused by Trypanosoma cruzi that affects approximately 9 million people in Latin America, with an expected
mortality of between 23 and 45 thousand individuals [5, 6]. Currently there are an estimated 300,000 cases in
the United States [7]. African sleeping sickness is caused by the protozoan parasite Trypanosoma brucei
exclusively in sub-Saharan Africa. The number of reported cases is decreasing and currently at less than 8000
per year but the estimated number is likely to be around 25,000 [8]. Leishmaniasis, a neglected tropical disease
caused by parasitic protozoa of the genus Leishmania, is responsible for 1- 2 million new clinical cases each
year in 88 countries where up to 350 million people are at risk of infection [9]. Malaria is a major public health
problem today in approximately 100 countries where it is estimated that 219 million clinical cases occur
annually, with mortality estimated at 660 thousand persons per year [10]. Approximately half the world
population is at risk of contract malaria [10]. However, up to now, for all of these parasitic diseases, effective
vaccines are lacking and the approved chemotherapeutic compounds are costly, toxic, require long periods of
treatment and generally are losing their effectiveness due to the development of resistant parasites. Therefore,
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new effective, more selective and safer antiparasitic agents are urgently needed.
Diamidines such as pentamidine (Figure 1) are DNA minor groove binders and represent a potent class of
antiparasitic agents with a broad spectrum of activities against human and veterinary pathogens [11]. However,
these compounds often have poor bioavailability and cause considerable side effects. Many analogues and
derivatives, including prodrugs, have been synthesized and screened in vitro and in vivo in order to overcome
these limitations [2, 11]. In our efforts to identify promising antiprotozoal agents from a library of aromatic
diamidines and their analogues, we have identified arylimidamides (AIAs), which have demonstrated high
potency against several parasites including T. cruzi [2, 12-14], Leishmania spp. [2,13-16], Neospora caninum
[17,18], Besnoitia besnoiti [19] and Echinococcus multilocularis [20]. The initial AIAs were modified versions
of furamidine (Figure 1) which were designed as DNA minor groove binders and which have pKa values of
approximately 7 in comparison to furamidine and other classical amidines which exhibit pKa values of 10-11
[21]. The lower pKa values were expected to improve the oral bioavailability of these modified analogues.
Furamidine and analogues showed only moderate anti-T.cruzi and anti-leishmanial effects [22, 23] and the
AIAs with altered physiochemical properties displayed improved activity. DB766 (Figure 1), one of the most
potent antileishmanial AIAs, exhibited in vitro activity with IC50 values of 0.036 and 0.087 ꢀM against L.dono
vani and L. amazonensis, respectively, and notably reduced liver parasitemia in both mice and hamsters [21].
DB766 also exhibited strong anti-T. cruzi activity and excellent selectivity for bloodstream trypomastigotes
and intracellular amastigotes (Y strain) [12]. Studies in vivo with DB766 demonstrated a reduction in the
parasite load levels in the blood and cardiac tissue with similar trypanocidal activity as that of benznidazole in
a mouse model of acute T. cruzi infection [12,13]. The excellent activity of DB766 motivated the design and
synthesis of novel structurally related compounds [16,24]. The mode of antiparasitic action of classical
diamidines has been suggested to involve, at least in part, kDNA binding leading to mitochondria
disruption [25], however the mode of action of AIAs, which has not been extensively studied, seems
likely to be different and more complex than that of the parent diamidines. For example, the anti-T. cruzi
activity of AIAs is not correlated with kDNA binding affinities [26]; despite the fact that AIAs do bind to
the minor groove of DNA in a highly structure dependent manner which has been studied in detail [27].
However, intracellular T. cruzi amastigotes exposed to AIAs showed kDNA disruption, mitochondrion
swelling and other manifestations including loss of subpellicular microtubules and induced an atypical
organization of multiple flagella in the trypomastigote stage [28]. AIA mechanistic studies appear to be
lacking against L. sp., T. brucei rhodesience and P. falciparum.
Figure 1 here
We have described the antileishmanial SAR for arylimidamides with multiple variations on the diphenylfuran
core of furamidine with the terminal groups held constant with 2-pyridyl groups [14-16]. We have recently
shown that relatively low molecular weight AIAs derived from DB766 can retain high activity against
Leishmania spp., however these smaller compounds are toxic [29]. We have also shown that DB766 analogues
that possess unsymmetrical substitutions on the diphenylfuran linker displayed nanomolar in vitro potency
against intracellular Leishmania with selectivity indexes >100 compared to J774 macrophages [16]. However,
relatively little variation in the terminal groups have been explored [15]. In order to accomplish our drug
development and molecular recognition goals, a variety of analogs of DB766 were prepared and are reported
herein, in which the terminal pyridyl groups have been replaced with various heterocyclic rings. The synthetic
compounds were evaluated for their activity in vitro against T. cruzi, L. amazonensis, T. brucei rhodesiense
and P. falciparum.
2. Results and discussion
2.1. Chemistry
The syntheses for the target bis-arylimidamides 7a-7o require the corresponding diamino compound 4 as the
key intermediate (Scheme 1). The preparation of the diamino compound 4 was conveniently achieved in two
steps starting with a Stille reaction between 1-bromo-2-isopropoxy-4-nitrobenzene (1) and 2,5-bis(tri-n-
butylstannyl)furan (2) to form the corresponding 2,5-bis [4-nitro-2-(2-propoxy)phenyl] furan 3 in good yield
(70%). Reduction of the 2,5-bis[4-nitro-2-(2-propoxy)phenyl]furan 3 by catalytic hydrogenation produced the
desired diamino compound 4 in excellent yield (96%). 1-Bromo-2-isopropoxy-4-nitrobenzene (1) was obtained
2

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starting with the bromination of commercially available 2-amino-5-nitrophenol using sulfamic acid and
sodium nitrite in aqueous HBr to form 2-bromo-5-nitrophenol in 78% yield [16, 30, 31]. The phenolic group
was then alkylated with 2-iodopropane in the presence of potassium t-butoxide to give 1 in 68% yield [13, 15].
2,5-Bis-(tri-n-butylstannyl)furan 2 was prepared from furan via lithiation with sec-butyllithium and subsequent
treatment with tri-n-butyltin chloride as previously described [16, 32].
Scheme 1 here
The bis-arylimidamides 7a-7o (30-82% yields) were prepared in a one-step process in which 4 was reacted
with two equivalents of the various S-(2-naphthylmethyl)-2-thioimidatehydrobromides 6 in ethanol/acetonitrile
(Scheme 2) [33]. The S-(2-naphthylmethyl)-2-thioimidatehydrobromides 6 were obtained by reacting the
heterocyclicthioamides 5 with 2-(bromomethyl)naphthalene in refluxing CHCl3 according to the literature
procedure [33].
Scheme 2 here
2.2. Biological EvaluationsꢀIn vitro antiparasitic activities
The results for the evaluation of the 15 bis-arylimidamides against T. cruzi, L. amazonensis, T. brucei
rhodesiense and P. falciparum along with their cytotoxicity to L6 rat myoblast cells are shown in Table 1. For
comparison purposes, Table 1 includes data for DB766 and several standard antiparasitic drugs. Four (7a, 7c,
7j, 7m) of the 15 new compounds showed anti-T. cruzi IC50 values between 0.2 and 1.6 ꢀM, which are lower
than or comparable to that of benznidazole (IC50 = 1.87 ꢀM); however only 7a gave a reasonable selectivity
index (SI = 29). Compound 7a gave a quite low IC50 value of 0.2 ꢀM against T. cruzi, which approached that
of DB766 and is about 9-fold more potent than that of benznidazole. Eight (7a, 7g, 7i, 7j, 7k, 7l, 7m, 7o) of the
15 compounds gave IC50 values of 0.9 ꢀM or less against L. amazonensis, comparable to that of pentamidine
(IC50 = 0.83 ꢀM). The most active compound (which has an acceptable SI value of 27) of this set 7g (IC50
=
0.17 ꢀM) has an IC50 value comparable to amphotericin B but is two-fold less active than DB766. Three
compounds (7a, 7j, 7m) gave potent activity against T. brucei rhodesiense with IC50 values between 12 and 60
nM and acceptable selectivity indices (26-181) however; they were not as active as pentamidine (IC50 = 2.3
nM). Ten (7a, 7g-7o) of the 15 compounds showed good activity against P. falciparum with IC50 values
ranging from 9 to 87 nM all of which are more active than chloroquine (IC50 = 125 nM); all ten compounds
have acceptable selectivity indices (from 20 to 682). Compounds 7a and 7j are the most potent ones against P.
falciparum with IC50 values of 9 nM, 5-fold more potent than pentamidine.
Three new compounds (7a-7c), with nitrogen containing 6-membered rings similar to the lead compound
DB766, were prepared. When the pyridine rings of DB766 are replaced by pyrimidine rings (7a), with both
nitrogen atoms ortho to the amidine, a 2-fold loss of activity versus T. cruzi and an 8-fold decrease against L.
amazonensis is observed. The selectivity index for the T. cruzi activity is acceptable however that is not the
case for L. amazonensis. Interestingly, the activity of 7a against T. brucei rhodesiense and P. falciparum
shows an approximately 4-fold and 18-fold increase, respectively, compared to that of DB766. Both
compounds 7b (terminal pyrazines) and 7c (terminal pyridizines), which are isomeric to 7a, exhibited a
significant loss in potency against all four parasites tested compared to that of compound 7a.
Twelve analogues of DB766 were prepared in which the terminal pyridine rings were replaced with
various 5-membered ring heterocycles. When the pyridine rings were replaced with bromothiophenes (7d and
7e), essentially no antiparasitic activity was retained. On replacement of the pyridine rings with thiazoles (7f-
7k) significant variation in activity with structure and parasite was observed. None of the six thiazoles gave
anti-T. cruzi activity below 1 ꢀM and their selectivity indices were generally one or less. Three of the thiazoles
(7g, 7i, 7j) gave low micromolar (0.17- 0.3 ꢀM) IC50 values versus L. amazonensis, however only 7g exhibited
an acceptable selectivity index (SI = 27). Against T. brucei rhodesiense , 7j was the only thiazole to show
promising activity (IC50 = 12 nM; SI = 50). In contrast to the previous results, five of six thiazoles (7g-7k)
show promising activity (IC50 values between 9 and 86 nM) against P. falciparum and reasonable selectivity
3

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(SI = 22-94). The results for the thiazoles against P. falciparum is an interesting finding and merits follow-up.
The antiparasitic results for the pyrrole analogue 7l are poor, similar to the thiophenes (7d-e), except against P.
falciparum, which yielded an IC50 value of 25 nM and acceptable selectivity (SI = 40). The two imidazole
analogues studied (7m and 7n) both show only modest activity against T. cruzi and L. amazonensis with poor
selectivity. The imidazole 7m shows reasonable activity and selectivity versus T .brucei rhodesience (IC50 =
60 nM; SI = 26) and both 7m (IC50 = 15 nM; SI = 106) and 7n (IC50 = 54 nM; SI = 148) provide good
activity and selectivity versus P. falciparum. The pyrazole 7o shows only modest activity against T. cruzi, L.
amazonensis and T. brucei rhodesiense, however it is reasonably active and selective for P. falciparum (IC50 =
70 nM; SI = 20).
Table 1 here
3. Conclusion
After evaluation of 15 new compounds in which the terminal pyridine groups of DB766 have been
replaced with various 5- and 6-membered heterocyclic rings, the pyridine terminal groups remain the best
choice for activity and selectivity against T. cruzi and L. amazonensis. Terminal pyrimidine units (7a) are
tolerated for anti-T. cruzi activity, albeit not as effect as pyridine, and the 2-methylthiazole system (7g)
performs similarly against L. amazonensis. Against T. brucei rhodesiense the pyridine unit (7a), the imidazole
system (7m) and the thiazole analogue (7j) show encouraging activity and selectivity. The major new finding
in this report is that this class of compounds seems generally active and selective against malaria with 10 of the
15 compounds giving IC50 values of 87 nM or less. These results suggest that lead optimization of this series
against malaria would likely be a fruitful endeavor. Our previous studies have shown that the number and
location of nitrogen atoms have a significant impact on the antitrypanosomal and antileshmanial activity of
related diamidines [37, 38]. The results from the current study of these bis-arylimidamides against T. cruzi, L.
amazonensis, T. brucei rhodesiense and P. falciparum are generally consistent with previous observations. For
compounds DB766, 7a, 7g, 7m, 7j, which show potent antiparasitic activity, one or two nitrogen atoms are
ortho to the amidine groups in the terminal rings, which suggests that nitrogen atoms ortho to the amidine
group is important for the antiparasitic activity of bis-arylimidamides.
4. Experimental section
4.1. Chemistry
All commercial solvents and reagents were used without purification. Melting points were recorded using a
1
Mel-Temp 3.0 capillary melting point apparatus and are uncorrected. H and 13C NMR spectra were recorded
TM
employing a Bruker 400 Ultrashield
instrument, chemical shifts (δ) are in ppm relative to internal TMS.
Mass spectra were recorded on a VG analytical 70-SE spectrometer. The compounds reported as salts
frequently analyzed correctly for fractional moles of water and/or other solvents; in each case 1H NMR spectra
were consistent with the presence of water or organic solvent(s). Elemental analyses were obtained from
Atlantic Microlab, Inc. (Norcross, GA) and are within ± 0.4 of the theoretical values.
4.1.1 2,5-Bis[4-nitro-2-(2-propoxy)phenyl]furan (3)
A mixture of 1-bromo-2-isopropoxy-4-nitrobenzene [16] (4.16 g, 16 mmol), 2,5-bis(tri- methylstannyl)furan
[30, 31] (3.16 g, 8 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (0.37 g) in dry 1,4-dioxane (50 ml)
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was heated under nitrogen at 80–90 for overnight, cooled to room temperature and filtered to give, after
rinsing with hexanes, an orange solid 3 (2.4 g, 70%). Mp 253-254 ; 1H NMR (CDCl3): δ 1.55 (d, J = 6.0 Hz,
12H), 4.89 (m, 2H), 7.39 (s, 2H), 7.85 (d, J = 2.0 Hz, 2H), 7.95 (dd, J = 2.0 Hz, 8.8 Hz, 2H), 8.13 (d, J = 8.8
Hz, 2H); 13C NMR (CDCl3): δ 21.9, 72.1, 108.5, 115.9, 126.0, 126.1, 147.6, 149.2, 154.1; HRMS (ESI) calcd.
for C22H23N2O7 (M++H): 427.1505, found: 427.1511.
4.1.2 2,5-Bis[4-amino-2-(2-propoxy)phenyl]furan (4)
To a suspension of 3 (3g, 7.0 mmol) in EtOAc (50 ml) and absolute EtOH (10 ml) was added Pd/C (10%) (0.3
g) and the mixture was hydrogenated on a Parr apparatus at an initial pressure of about 50 psi for 3 h, the
resulting solution was filtered over celite and the filtrate was concentrated in vacuo near dryness to give, after
dilution with hexanes, the pure diamino as a yellow/tan solid (2.47 g, 96%). Mp 77-79 ; 1H NMR (CDCl3): δ
1.44 (d, J = 6.0 Hz, 12H), 3.71 (s, 4H), 4.64 (heptet, J = 6.0 Hz, 2H), 6.32 (s, 2H), 6.37 (d, J = 8.2 Hz, 2H),
6.86 (s, 2H), 7.77 (d, J = 8.2 Hz, 2H); 13C NMR (CDCl3): δ 22.3, 70.2, 100.6, 107.5, 109.5, 112.7, 127.0,
146.2, 148.5, 154.8; Anal. Calcd. for C22H26N2O3. 0.25 H2O: C, 71.23; H, 7.20; N, 7.55. Found: C, 71.31; H,
7.13; N, 7.46.
4.1.3 General procedure for synthesis of S-2-naphthylmethyl thioimidatehydrobromides 6
To a stirred solution of thioacetamide (5 mmol) in CHCl3 (10 ml) was added 2-(bromomethyl)naphthalene (6
mmol). The mixture was heated to reflux for 1.5~10 h, cooled to room temperature and placed in an ice bath.
The resulting solid was collected and dried in vacuo to afford title compounds (68 ~ 96% in yield) as a solid
and used directly in the next step.
4.1.4 General procedure for compounds 7a-7o
To a solution of 2,5-bis(4-amino-2-(2-propoxy)phenyl)furan (1.5 mmol) in dry MeCN (8 mL) was added dry
EtOH (24 mL), and the solution was chilled briefly in an icewater bath. The appropriate S-(2-naphthylmethyl)
thioacetimidates (3.2 mmol) was then added, and the mixture was stirred overnight at room temperature. The
resulting solution was concentrated to near dryness, which was triturated with ether to give a yellow solid. The
solid was collected, dissolved in EtOH, and basified with NaOH (1 N), and the free base was extracted into
CHCl2. After drying (K2CO3) and partially concentrating, the resulting suspension was diluted with hexane to
give a fluffy yellow solid that was collected by filtration. The free base (1 mmol) was used directly by
suspending in EtOH (3 ml) and treated with HCl saturated ethanol (5 ml) for 1 h at room temperature. The
resulting solution was then concentrated under reduced pressure to near dryness to give a suspension that was
diluted with ether and filtered to yield an orange/ red powder.
4.1.4.1. 2,5-Bis[2-(2-propoxy)-4-(2-pyrimidylimino)aminophenyl]furan hydrochloride salt (7a)
Yield: 70%; mp 162-194 (dec.); 1H NMR (DMSO-d6): δ 1.44 (d, J = 6.0 Hz, 12H), 4.83 (m, 2H), 7.16 (dd, J
= 1.6 Hz, 8.4 Hz, 2H), 7.20 (s, 2H), 7.32 (d, J = 1.6 Hz, 2H), 8.00 (t, J = 5.0 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H),
9.23 (d, J = 5.0 Hz, 4H), 9.70 (s, 2H), 10.18 (s, 2H), 11.99 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.1, 111.7,
113.6, 118.2, 119.7, 125.5, 126.9, 134.6, 148.6, 153.6, 154.4, 157.3, 158.9; Anal. Calcd. for C32H32N8O3. 2HCl.
2.7H2O: C, 55.05; H, 5.69; N, 16.05. Found: C, 55.37; H, 5.54; N, 15.68.
4.1.4.2 2,5-Bis[2-(2-propoxy)-4-(2-pyrazinylimino)aminophenyl]furan hydrochloride salt (7b)
Yield: 30%; mp 191-193 (dec.); 1H NMR (DMSO-d6):δ 1.45 (d, J = 6.0 Hz, 12H), 4.84 (m, 2H), 7.17 (d, J =
8.4 Hz, 2H), 7.21 (s, 2H), 7.33 (s, 2H), 8.17 (d, J = 8.4 Hz, 2H), 9.01 (t, J = 2.2 Hz, 2H), 9.11 (d, J = 2.2 Hz,
2H), 9.52 (s, 2H), 9.59 (s, 2H), 10.28 (s, 2H), 12.05 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.1, 111.4, 113.6,
118.0, 119.7, 127.0, 134.7, 141.5, 144.9, 145.7, 148.6, 149.6, 154.5, 158.8; Anal. Calcd. for C32H32N8O3.
2HCl. 2.65H2O: C, 55.17; H, 5.69; N, 16.09. Found: C, 55.57; H, 5.63; N, 15.49.
4.1.4.3. 2,5-Bis[2-(2-propoxy)-4-(2-pyridazinylimino)aminophenyl]furan hydrochloride salt (7c)
o
Yield: 32%; mp 213-215 C (dec.); 1H NMR (DMSO-d6): δ 1.45 (d, J = 5.8 Hz, 12H), 4.87 (m, 2H), 7.19 (s,
2H), 7.21 (s, 2H), 7.36 (s, 2H), 8.15 (s, 2H), 8.17 (d, J = 8.4 Hz, 2H), 8.70 (d, J = 8.4 Hz, 2H), 9.63 (s, 2H),
9.64 (s, 2H), 10.48 (s, 2H), 12.32 (s, 2H); 13C NMR (DMSO-d6): δ 22.0, 70.7, 111.0, 113.2, 117.6, 119.3,
126.6, 128.1, 134.2, 148.2, 149.9, 154.1, 154.5, 158.7; Anal. Calcd. for C32H32N8O3. 2HCl. 4.05H2O: C, 53.19;
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H, 5.87; N, 15.50. Found: C, 53.58; H, 5.49; N, 14.96.
4.1.4.4. 2,5-Bis[2-(2-propoxy)-4-(5-bromo-2-thiophenylimino)aminophenyl]furan hydrochloride salt (7d)
Yield: 82%; mp 203-205 (dec.); 1H NMR (DMSO-d6): δ 1.42 (d, J = 6.0 Hz, 12H), 4.81 (m, 2H), 7.09 (d, J
= 8.6 Hz, 2H), 7.17 (s, 2H), 7.27 (s, 2H), 7.57 (d, J = 4.0 Hz, 2H), 7.98 (d, J = 4.0 Hz, 2H), 8.10 (d, J = 8.6 Hz,
2H), 9.06 (s, 2H), 9.87 (s, 2H), 11.7 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.1, 111.3, 113.5, 117.9, 119.4,
121.2, 127.0, 131.1, 132.4, 135.1, 136.0, 148.6, 154.5, 155.8; Anal. Calcd. for C32H30Br2N4O3S2. 2HCl. 2H2O:
C, 45.14; H, 4.26; N, 6.58. Found: C, 45.00; H, 4.06; N, 6.44.
4.1.4.5. 2,5-Bis[2-(2-propoxy)-4-(4-bromo-2-thiophenylimino)aminophenyl]furan hydrochloride salt (7e)
Yield: 80%; mp 187-189 (dec.); 1H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 4.82 (m, 2H), 7.12 (d, J
= 8.4 Hz, 2H), 7.18 (s, 2H), 7.31 (s, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.24 (s, 2H), 8.30 (s, 2H), 9.18 (s, 2H),
10.05 (s, 2H), 11.90 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.0, 110.3, 111.3, 113.5, 117.8, 119.4, 126.9,
131.3, 132.3, 135.2, 136.7, 148.6, 154.4, 155.8; Anal. Calcd. for C32H30Br2N4O3S2. 2HCl. 1H2O: C, 46.11; H,
4.11; N, 6.72. Found: C, 46.20; H, 3.91; N, 6.65.
4.1.4.6. 2,5-Bis[2-(2-propoxy)-4-(4-methyl-2-thiazolylimino)aminophenyl]furan hydrochloride salt (7f)
1
Yield: 67%; mp 192-194 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 2.56 (s, 6H), 4.83 (m,
2H), 7.12 (d, J = 8.2 Hz, 2H), 7.18 (s, 2H), 7.29 (s, 2H), 7.99 (s, 2H), 8.11 (d, J = 8.2 Hz, 2H), 9.33 (s, 2H),
10.08 (s, 2H), 11.97 (s, 2H); 13C NMR (DMSO-d6): δ 17.0, 22.4, 71.0, 111.4, 113.5, 117.9, 119.5, 123.8, 126.9,
135.1, 148.6, 154.1, 154.5, 154.8; Anal. Calcd. for C32H34N6O3S2. 2HCl. 2.8H2O: C, 52.13; H, 5.69; N, 11.41.
Found: C, 52.24; H, 5.52; N, 11.36.
4.1.4.7. 2,5-Bis[2-(2-propoxy)-4-(2-methyl-4-thiazolylimino)aminophenyl]furan hydrochloride salt (7g)
1
Yield: 72%; mp 231-233 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 5.8 Hz, 12H), 2.82 (s, 6H), 4.84 (m,
2H), 7.12 (d, J = 8.6 Hz, 2H), 7.19 (s, 2H), 7.28 (s, 2H), 8.12 (d, J = 8.6 Hz, 2H), 9.01 (s, 2H), 9.12 (s, 2H),
9.93 (s, 2H), 11.69 (s, 2H); 13C NMR (DMSO-d6): δ 19.3, 22.4, 71.0, 111.7, 113.5, 118.2, 119.5, 126.9, 129.7,
134.6, 142.2, 148.6, 154.4, 154.9, 168.2; Anal. Calcd. for C32H34N6O3S2. 2HCl. 1.3 H2O: C, 54.12; H, 5.48; N,
11.84. Found: C, 54.34; H, 5.49; N, 11.64.
4.1.4.8. 2,5-Bis[2-(2-propoxy)-4-(2-amino-4-thiazolylimino)aminophenyl]furan hydrochloride salt(7h)
Yield: 60 %; mp 264-266 (dec.); 1H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 4.83 (m, 2H), 7.08 (d, J
= 8.2 Hz, 2H), 7.17 (s, 2H), 7.24 (s, 2H), 7.48 (s, 4H), 8.09 (d, J = 8.2 Hz, 2H), 8.92 (s, 2H), 9.51 (s, 2H),
11.25 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.0, 111.5, 113.4, 117.7, 118.0, 119.4, 126.8, 134.7, 138.6,
148.6, 154.4, 155.2, 169.1; Anal. Calcd. for C30H32N8O5S2. 2.6HCl. 2.2H2O: C, 47.97; H, 5.23; N, 14.92.
Found: C, 47.98; H, 5.10; N, 14.56.
4.1.4.9. 2,5-Bis[2-(2-propoxy)-4-(2-methanamine-4-thiazolylimino)aminophenyl]furan hydrochloride salt (7i)
1
Yield: 73%; mp 236-238 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 2.97 (s, 6H), 4.85 (m,
2H), 7.11 (d, J = 8.4 Hz, 2H), 7.18 (s, 2H), 7.27 (s, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.19 (s, 2H), 8.94 (s, 2H),
9.54 (s, 2H), 11.30 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 31.3, 71.0, 111.7, 113.5, 117.1, 118.2, 119.5, 126.9,
134.6, 138.5, 148.6, 154.4, 155.3, 169.4; Anal. Calcd. for C32H36N8O3S2. 2.5HCl. 3.6H2O: C, 47.99; H, 5.75;
N, 13.99. Found: C, 47.99; H, 5.39; N, 13.80.
4.1.4.10. 2,5-Bis[2-(2-propoxy)-4-(2-acetamide-4-thiazolylimino)aminophenyl]furan hydrochloride salt (7j)
1
Yield: 71%; mp 271-273 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 5.2 Hz, 12H), 2.26 (s, 6H), 4.84 (m,
2H), 7.13 (d, J = 8.2 Hz, 2H), 7.19 (s, 2H), 7.30 (s, 2H), 8.12 (d, J = 8.2 Hz, 2H), 8.65 (s, 2H), 9.17 (s, 2H),
9.72 (s, 2H), 11.54 (s, 2H), 12.53 (s, 2H); 13C NMR (DMSO-d6): δ 22.0, 22.6, 70.6, 111.1, 113.1, 117.6, 119.1,
122.7, 126.5, 134.2, 137.2, 148.2, 154.1, 155.2, 158.8, 169.8; Anal. Calcd. for C34H36N8O5S2. 2HCl. 3.9H2O:
C, 48.38; H, 5.47; N, 13.28. Found: C, 48.37; H, 5.48; N, 13.05.
4.1.4.11. 2,5-Bis[2-(2-propoxy)-4-(2-methylacetamide-4-thiazolylimino)aminophenyl]furan hydrochloride salt
(7k)
6

ACCEPTED MANUSCRIPT
1
Yield: 68 %; mp >300 ; H NMR (DMSO-d6): δ 1.44 (d, J = 5.4 Hz, 12H), 2.46 (s, 6H), 3.82 (s, 6H), 4.87
(m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.21 (s, 2H), 7.34 (s, 2H), 8.17 (d, J = 8.0 Hz, 2H), 8.57 (s, 2H), 9.05 (s,
2H), 9.77 (s, 2H), 11.37 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 23.3, 35.9, 71.1, 112.1, 113.7, 118.6, 119.8,
124.1, 127.1, 134.3, 136.6, 148.6, 154.6, 155.6, 160.5, 172.2; Anal. Calcd. for C36H40N8O5S2. 2HBr: C, 48.54;
H, 4.75; N, 12.58. Found: C, 48.63; H, 4.75; N, 12.22.
4.1.4.12. 2,5-Bis[2-(2-propoxy)-4-(1H-2-pyrrolylimino)aminophenyl]furan hydrochloride salt (7l)
1
Yield: 76%; mp 231-233 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 4.84 (m, 2H), 6.41 (m,
2H), 7.09 (dd, J = 1.6 Hz, 8.4 Hz, 2H), 7.17(s, 2H), 7.24 (d, J = 1.6 Hz, 2H), 7.42 (s, 2H), 7.44 (s, 2H), 8.11
(d, J = 8.4 Hz, 2H), 8.63 (s, 2H), 9.45 (s, 2H), 11.25 (s, 2H), 12.65 (s, 2H); 13C NMR (DMSO-d6): δ 22.4,
71.0, 111.4, 113.3, 117.8, 117.9, 119.2, 119.5, 126.9, 127.2, 134.8, 148.6, 153.5, 154.5; Anal. Calcd. for
C32H34N6O3. 2HCl. 1.5H2O: C, 59.15; H, 6.05; N, 12.94. Found: C, 59.13; H, 5.93; N, 12.81.
4.1.4.13. 2,5-Bis[2-(2-propoxy)-4-(1H-2-imidazolylimino)aminophenyl]furan hydrochloride salt (7m)
Yield: 69%; mp 254-256 (dec.); 1H NMR (DMSO-d6): δ 1.43 (d, J = 3.6 Hz, 12H), 4.84 (m, 2H), 7.12 (d, J
= 7.6 Hz, 2H), 7.18 (s, 2H), 7.28 (s, 2H), 7.61 (s, 4H), 8.11 (d, J = 7.6 Hz, 2H), 9.26 (s, 2H), 9.98 (s, 2H),
11.80 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 71.0, 111.4, 113.5, 118.0, 119.4, 121.9, 126.9, 134.3, 135.3,
148.6, 151.4, 154.4; Anal. Calcd. for C30H32N8O3. 2HCl. 2.2H2O: C, 54.17; H, 5.82; N, 16.85. Found: C,
54.20; H, 5.60; N, 16.64.
4.1.4.14. 2,5-Bis[2-(2-propoxy)-4-(1-methyl-1H-4-imidazylimino)aminophenyl]furan hydrochloride salt (7n)
1
Yield: 75%; mp 191-193 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 6.0 Hz, 12H), 3.84 (s, 6H), 4.84 (m,
2H), 7.09 (dd, J = 1.4 Hz, 8.2 Hz, 2H), 7.17 (s, 2H), 7.22 (d, J = 1.4 Hz, 2H), 8.05 (s, 2H), 8.09 (d, J = 8.2 Hz,
2H), 8.42 (s, 2H), 8.77 (s, 2H), 9.57 (s, 2H), 11.34 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 34.5, 71.0, 111.5,
113.3, 118.0, 119.2, 126.8, 127.9, 129.4, 134.8, 140.7, 148.6, 154.4, 155.7; Anal. Calcd. for C32H36N8O3. 2HCl.
2.3H2O: C, 55.30; H, 6.18; N, 16.12. Found: C, 55.62; H, 6.01; N, 15.77.
4.1.4.15. 2,5-Bis[2-(2-propoxy)-4-(1-methyl-1H-3-pyrazolylimino)aminophenyl]furan hydrochloride salt (7o)
1
Yield: 75%; mp 196-198 (dec.); H NMR (DMSO-d6): δ 1.43 (d, J = 5.8 Hz, 12H), 4.06 (s, 6H), 4.85 (m,
2H), 7.11 (d, J = 8.2 Hz, 2H), 7.18 (s, 2H), 7.26 (s, 2H), 7.42 (s, 2H), 8.10 (d, J = 8.2 Hz, 2H), 8.11 (s, 2H),
9.00 (s, 2H), 9.83 (s, 2H), 11.63 (s, 2H); 13C NMR (DMSO-d6): δ 22.4, 39.6, 71.0, 109.1, 111.7, 113.4, 118.2,
119.4, 126.9, 134.5, 134.6, 139.9, 148.6, 154.4, 155.4; Anal. Calcd. for C32H36N8O3. 2HCl. 3.6H2O: C, 53.55;
H, 6.35; N, 15.62. Found: C, 53.46; H, 6.13; N, 15.26.
4.2. Biology
4.2.1. In vitro antiparasitic activity:
In vitro antiprotozoal activities against intracellular amastigote T. cruzi strain Tulahuen LacZ/C4, the
trypomastigote bloodstream form of T. b. rhodesiense strain STIB900 and the erythrocytic stages of the
chloroquine resistant P. falciparum strain K1, were measured following established protocols [35]. Serial
drug dilutions were tested against the relevant parasite forms and plates incubated at 37°C. The drug
exposure time of the intracellular T. cruzi assay was 4 days. The endpoint of the reporter gene assay was
measured by addition of the substrate (CPRG) for β-galactosidase resulting in a color change. The drug
exposure time was 3 days in the axenic T. b. rhodesiense Alamar blue viability assay. Viability of P.
falciparum was assessed after 3 days drug exposure using the tritium labeled hypoxanthine
incorporation assay [35]. The in vitro activities against intracellular L. amazonensis were determined
following established methodology [36]. The IC50 values are the mean of two independent assays.
Coefficients of variation were less than 50%.
4.2.2. In vitro cytotoxicity assay:
7

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The in vitro cytotoxicity of the compounds was determined in a 3 day drug exposure Alamar blue
viability assay as previously described [34]. The IC50 values are the mean of two independent assays.
Coefficients of variation were less than 50%.
Acknowledgements
The Bill and Melinda Gates Foundation supported this work through a subcontract with the Consortium of
Parasitic Drug Development (R.B., D.W.B.). The sponsors had no role in study design; in the collection,
analysis and interpretation of data; in the writing of this report; nor in the decision to publish.
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Table 1. Antiparasitic activities and cytotoxicity data for bis-arylimidamides DB766 analogs.
Cyto
Code
T. c.
L. a.
T. b. r
P. f.
toxicity
a
b
c
b
b
IC50
IC50
IC50
IC50
(µM)
0.12
IC50
SI d
SI e
SI f
25
SI g
(µM)
3.0
5.8
>129
1.8
>110
>108
>122
4.7
3.7
0.8
0.6
0.8
1.0
1.6
8.0
1.4
46.6
(µM)
(µM)
0.087
0.7
(µM)
0.149
0.0085
0.552
0.582
0.914
0.992
0.999
0.087
0.039
0.031
0.009
0.035
0.025
0.015
0.054
0.070
0.0464
ND
DB766
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
0.1
0.2
16
1.5
30
29
8.1
1.2
1
34.5
8.29
34.8
1.64
ND
20.13
682.3
233.7
3.09
120
108.9
122.1
54.0
94.9
25.8
66.7
22.6
40
0.032
8.077
1.70
181.3
15.97
1.06
6.47
2.46
1.55
6.36
10.4
8.69
50
10.3
8.69
26. 7
74.1
3.21
20173
3.7
1.1
>110
>108
>122
2.3
ND h
ND
17.0
43.9
78.6
0.74
1
1
ND
>10
0.17
1.7
0.30
0.22
0.81
0.90
0.37
5.0
>12.2
27.6
2.18
2.67
2.73
0.99
1.11
4.32
1.6
2.04
0.66
0.35
0.38
0.26
0.24
2.67
0.44
0.23
6.53
5.6
2.3
1.6
3.1
4.1
0.6
18
6.1
0.357
0.092
0.012
0.078
0.115
0.060
0.108
0.436
0.0023
ND
106.6
148.1
20
7o
0.74
0.83
0.14
1.89
55.9
pentamidine
amphotericin B
7.1
ND
1004
benznidazole
chloroquine
melarsoprol
>150
76.5
5.1
1.87
ND
>80
ND
ND
ND
ND
0.004
ND
0.125
612
1275
podophyllotoxin
0.017
a Cytotoxicity was evaluated using cultured L6 rat myoblast cells [34].
b The in vitro activities were obtained using intracellular amastigote T. cruzi strain Tulahen
LacZ/C4, the trypomastigote bloodstream form of T. brucei rhodesiense strain STIB900 and the
erythrocytic stages of the chloroquine resistant P. falciparum strain K1. The IC50 values are the
mean of two independent assays. Coefficients of variation were less than 50% [35].
c Intracellular L. amazonensis were used and some values are reported in ref [15]; all were
determined as described in ref [36].
d Selectivity index for T. cruzi expressed as the ratio: IC50 (L6)/IC50 (T. cruzi).
e Selectivity index for L. amazonensis expressed as the ratio: IC50 (L6)/IC50 (L. . amazonensis).
f Selectivity index for T. brucei rhodesiense expressed as the ratio: IC50 (L6)/IC50 (T. brucei
rhodesiense.).
g Selectivity index for P. falciparum expressed as the ratio: IC50 (L6)/IC50 (P. falciparum).
h ND, not determined.

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Figure 1. Antiparasitic Leads
O
O
HN
NH
Pentamidine
NH2
NH2
O
O
NH
HN
O
N
N
H
H
N
N
DB766
NH
O
HN
H2N
NH2
Furamidine

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Highlights
. 15 new DB766 analogues with 5- or 6-heterocyclic ring terminal groups were made
. compounds evaluated vs. T. cruzi, T. b. rhodesiense, P. falciparum and L. amazonensis
. generally the pyridine terminal unit remains the most active
. major finding: 10 of 15 compounds are very active vs. P. falciparum ( IC50 = 9-87 nM)

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Synthesis and antiparasitic activity of new bis-arylimidamides: DB766 analogs modified in
the terminal groups
Zong-yingLiua, b, TanjaWenzlerc, d, RetoBrunc, d, XiaohuaZhue, David W. Boykina, *
aDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
bInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100050, People’s Republic of China,cSwiss Tropical and Public Health
Institute, Department of Medical Parasitology and Infection Biology 4002 Basel, Switzerland,
dUniversity of Basel, 4003 Basel, Switzerland,eDivision of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
*Corresponding author. Tel.:+1 404-413-5498. Fax: +1 404-413-5505. E-mail address:
dboykin@gsu.edu
1H NMR and 13C NMR scans for 7a-7o follow.

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Figure 1: 1H NMR (400 MHz, DMSO-d6) of 7a.
Figure 2: 13C NMR (400 MHz, DMSO-d6) of 7a.

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Figure 3: 1H NMR (400 MHz, DMSO-d6) of 7b.
Figure 4: 13C NMR (400 MHz, DMSO-d6) of 7b.

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Figure 5: 1H NMR (400 MHz, DMSO-d6) of 7c.
Figure 6: 13C NMR (400 MHz, DMSO-d6) of 7c.

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Figure 7: 1H NMR (400 MHz, DMSO-d6) of 7d.
Figure 8: 13C NMR (400 MHz, DMSO-d6) of 7d.

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Figure 9: 1H NMR (400 MHz, DMSO-d6) of 7e.
Figure 10: 13C NMR (400 MHz, DMSO-d6) of 7e.

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Figure 11: 1H NMR (400 MHz, DMSO-d6) of 7f.
Figure 12: 13C NMR (400 MHz, DMSO-d6) of 7f.
Figure 13: 1H NMR (400 MHz, DMSO-d6) of 7g.

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Figure 14: 13C NMR (400 MHz, DMSO-d6) of 7g.

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Figure 15: 1H NMR (400 MHz, DMSO-d6) of 7h.
Figure 16: 13C NMR (400 MHz, DMSO-d6) of 7h.

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Figure 17: 1H NMR (400 MHz, DMSO-d6) of 7i.
Figure 18: 13C NMR (400 MHz, DMSO-d6) of 7i.

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Figure 19: 1H NMR (400 MHz, DMSO-d6) of 7j.
Figure 20: 13C NMR (400 MHz, DMSO-d6) of 7j.

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Figure 21: 1H NMR (400 MHz, DMSO-d6) of 7k.
Figure 22: 13C NMR (400 MHz, DMSO-d6) of 7k.

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Figure 23: 1H NMR (400 MHz, DMSO-d6) of 7l.
Figure 24: 13C NMR (400 MHz, DMSO-d6) of 7l.

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Figure 25: 1H NMR (400 MHz, DMSO-d6) of 7m.
Figure 26: 13C NMR (400 MHz, DMSO-d6) of 7m.

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Figure 27: 1H NMR (400 MHz, DMSO-d6) of 7n.
Figure 28: 13C NMR (400 MHz, DMSO-d6) of 7n.