Synthesis and antiprotozoal activity of cationic 2-phenylbenzofurans

Article abstract of DOI:10.1021/jm800918v

A series of cationically substituted 2-phenylbenzofurans 1-49 have been synthesized, and their in vitro antiprotozoal properties against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, as well as cytotoxicity against mammalian cells, have been evaluated. Eight dications exhibited antitrypanosomal activities comparable to that of pentamidine and melarsoprol. Twenty-six compounds were more active than pentamidine, and seven dications demonstrated increased activities against P. falciparum than artemisinin. Five congeners were more active against L. donovani than pentamidine. Introduction of methoxy or hydroxy groups in the 7- and/or 2′-position afforded derivatives that were highly selective against T. b. rhodesiense, P. falciparum, and L. donovani. Fourteen 2-phenylbenzofurans displayed excellent in vivo efficacies in the acute mouse model of trypanosomiasis, curing 3/4 or 4/4 animals at 4 x 5 mg/kg. Diamidine 1 and di(N-isopropyl)amidine 45, administered at 4 x 1 mg/kg, exhibited potency comparable to that of melarsoprol, providing 3/4 and 2/4 cures, respectively.

Full text of DOI:10.1021/jm800918v

J. Med. Chem. 2008, 51, 6927–6944
6927
Synthesis and Antiprotozoal Activity of Cationic 2-Phenylbenzofurans
Stanislav A. Bakunov,† Svetlana M. Bakunova,† Tanja Wenzler,‡ Todd Barszcz,§ Karl A. Werbovetz,§ Reto Brun,‡ and
Richard R. Tidwell*,†
Department of Pathology and Laboratory Medicine, School of Medicine, The UniVersity of North Carolina, Chapel Hill, North Carolina
27599-7525, DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, 500 West 12th AVenue,
Columbus, Ohio 43210, and Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland
ReceiVed July 23, 2008
A series of cationically substituted 2-phenylbenzofurans 1-49 have been synthesized, and their in vitro
antiprotozoal properties against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania
donoVani, as well as cytotoxicity against mammalian cells, have been evaluated. Eight dications exhibited
antitrypanosomal activities comparable to that of pentamidine and melarsoprol. Twenty-six compounds were
more active than pentamidine, and seven dications demonstrated increased activities against P. falciparum
than artemisinin. Five congeners were more active against L. donoVani than pentamidine. Introduction of
methoxy or hydroxy groups in the 7- and/or 2′-position afforded derivatives that were highly selective against
T. b. rhodesiense, P. falciparum, and L. donoVani. Fourteen 2-phenylbenzofurans displayed excellent in
vivo efficacies in the acute mouse model of trypanosomiasis, curing 3/4 or 4/4 animals at 4 × 5 mg/kg.
Diamidine 1 and di(N-isopropyl)amidine 45, administered at 4 × 1 mg/kg, exhibited potency comparable
to that of melarsoprol, providing 3/4 and 2/4 cures, respectively.
Introduction
of resistance. This enhances the risk of development of cross-
resistanceandclinicalfailureofnewlyintroducedmedications.34-36
Treatments for HAT and leishmaniasis require parenteral
administration and suffer from toxicity, poor efficacy, and
increasing number of treatment failures.18,29,37,38 This is cur-
rently the case for visceral leishmaniasis, although new options
for its treatment have recently become available.39,40 In addition,
the overlap of parasitic infections and HIV in different parts of
the world results in growing number of cases of dually infected
patients.41-46 The need for safe and affordable antiprotozoal
therapies capable of overcoming parasite resistance makes the
identification of new drug candidates an urgent priority.
In our continuing search for novel dicationic compounds with
enhanced antiprotozoal potency and lowered toxicity, we
investigated a series of dicationic 2-phenylbenzofurans 1-49,
many of which are structurally similar to the well-known DNA
fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI). In the
early 1970s, syntheses of DAPI as well as three 2-phenylben-
zofurans 1, 3, and 11 were reported47,48 and the antitrypanosomal
properties of 2-(4-amidinophenyl)-5-amidinobenzofuran (1),
2-(4-amidinophenyl)-6-amidinobenzofuran (11), and DAPI were
published.47 Here, we report the lead optimization of these
dications. All 2-phenylbenzofurans 1-49 were tested in vitro
against T. b. rhodesiense (STIB900), chloroquine resistant
Plasmodium falciparum (K1), and axenic amastigotes of Leish-
mania donoVani (MHOM/SD/62/1S-CL2D) and evaluated for
cytotoxicity against rat myoblast cells (L6). Compounds exhibit-
ing high antitrypanosomal activities in vitro underwent in vivo
screening in the STIB900 acute mouse model of trypanosomiasis.
Although many aromatic diamidines have been reported to
possessawiderangeofantifungalandantiprotozoalproperties,1-14
only 1,5-bis(4-amidinophenoxy)pentane (pentamidine) has been
widely employed as a drug in humans. Pentamidine has been
used for more than 50 years against early stage Trypanosoma
brucei gambiense related human African trypanosomiasis (HATa
or sleeping sickness)15-18 as well as against antimony-resistant
leishmaniasis19,20 and pneumonia caused by the opportunistic
pathogen Pneumocystis jiroVeci (formerly P. carinii).21-23 The
drug demonstrated some activity against selected strains of
malaria,24-26 even though it was never approved to treat the
disease. Lack of oral bioavailability17 of pentamidine due to
the ionization of its two amidine groups at physiological pH
necessitates the parenteral administration, which is not practical
in remote areas where most cases of sleeping sickness and
leishmaniasis take place. Although several adverse effects, such
as hypotension, abdominal pain, vertigo, hypersalivation, hy-
poglycemia, nausea, and mild nephrotoxocity, have been
reported upon pentamidine administration,27-29 the drug is fairly
well tolerated by patients.
In recent years, the number of treatment failures of malaria,
HAT, and leishmaniasis has increased because of development
of resistant strains of parasites. Reduced efficiency of the most
commonly used antimalarial remedies, such as chloroquine and
sulfadoxine-pyrimethamine, requires the use of drug combina-
tions or more expensive artemisinin-containing therapies to slow
the development of resistance.30-33 Also, many current anti-
malarial drugs are structurally related or have similar modes of
action, which explains the appearance of the shared mechanisms
Chemistry
* To whom correspondence should be addressed. Phone: (919) 966-4294.
Fax: (919) 966-0704. E-mail: Tidwell@med.unc.edu.
Syntheses of compounds bearing cyano groups in the 5-posi-
tion of the benzofuran moiety and 4′-position of the phenyl ring
are based on the previously reported procedure by Dann,47 as
depicted in Scheme 1. Commercially available 5-bromosalicy-
laldehyde was reacted with R-bromo-p-tolunitrile followed by
base-catalyzed intramolecular ring closure to form 5-bromo-2-
(4-cyanophenyl)benzofuran (50). Compound 50 was treated with
†
The University of North Carolina.
Swiss Tropical Institute.
The Ohio State University.
‡
§
a Abbreviations: HAT, human African trypanosomiasis; HIV, human
immunodeficiency virus; DAPI, 4′,6-diamidino-2-phenylindole; IBX,
o-iodoxylbenzoic acid; PMD, pentamidine; MLSP, melarsoprol; CQ,
chloroquine; ATMS, artemisinin; PPT, podophyllotoxin.
10.1021/jm800918v CCC: $40.75
2008 American Chemical Society
Published on Web 10/09/2008

6928 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
Scheme 1a
a Reagents and conditions: (i) CuCN, DMF, reflux, 15 h; (ii) 1,4-dioxane, EtOH, HCl, 1-3 days; (iii) ethylenediamine, EtOH, 1-2 days or
1,2-diaminobenzene, MeOH, reflux, 15 h.
Scheme 2a
a Reagents and conditions: (i) Cu2O, pyridine, 100 °C, 15 h; (ii) Me2AlNH2, o-xylene, 80-100 °C, 3 h.
Scheme 3a
a Reagents and conditions: (i) Cu2O, pyridine, 100 °C, 15 h; (ii) hydroxylamine hydrochloride, pyridine; (iii) Ac2O, reflux, 15 h; (iv) pyridine hydrochloride,
120-140 °C, 3 h.
copper(I) cyanide in DMF at elevated temperature to yield
dinitrile 51.47 Conversion of nitrile 50 to an imidate ester under
modified Pinner conditions49 followed by reactions with eth-
ylenediamine or 1,2-diaminobenzene afforded 2-phenylbenzo-
furans 52 and 53, bearing imidazoline and benzimidazole
cationic groups in the 4′-positions. Reaction of bromobenzo-
furans 52 and 53 with copper(I) cyanide in DMF at elevated
temperature yielded the corresponding nitriles 54 and 55.
Unfortunately, extensive application of this synthetic strategy
is constrained by limited availability of 6-bromosalicylaldehyde
as a starting material. Also, the aforementioned approach is only
suitable for preparation of compounds bearing an electron-
withdrawing group in the p-position of the phenyl ring.
Consequently, our attempts to use it for synthesis of m-sub-
stituted 2-phenylbenzofurans were unsuccessful.
of the aromatic rings. The use of copper-catalyzed heteroan-
nulation (modified57,58 Castro reaction) to synthesize substituted
2-phenylbenzofurans is depicted in Schemes 2-4.
4-Hydroxy-3-iodobenzonitrile (56)59 was prepared by iodi-
nation of 4-cyanophenol following the procedure of Edgar and
Falling.60 Syntheses of 4-ethynylbenzonitrile (57) and 3-ethy-
nylbenzonitrile (58) were previously reported.61 Copper-
catalyzed heteroannulation of iodophenol 56 with phenylacet-
ylenes 57 and 58 under the conditions of the modified Castro
reaction afforded 5,4′- and 5,5′-substituted dinitriles 51 and 59
in moderate yields (Scheme 2).
A similar approach was employed to synthesize 6-substituted
derivatives. The commercially available 3-hydroxybenzoic acid
underwent iodination60 to 3-hydroxy-4-iodobenzoic acid, fol-
lowed by esterification to methyl benzoate 60 under acidic
conditions.62 Methyl 4-ethynylbenzoate (61) and methyl 3-ethy-
nylbenzoate (62) were synthesized following the established
protocol.59 Iodophenol 60 was reacted with phenylacetylenes
61 and 62 under modified Castro reaction conditions to provide
benzofuran diesters 63 and 64, which were directly converted
to dinitriles 65 and 66 by treatment with dimethylaluminum
An alternative method of synthesis of benzofurans involves
the palladium-catalyzed (Sonogashira reaction)50-54 or copper-
catalyzed (Castro reaction)55,56 interaction of o-halogenated
phenols with acetylenes. This approach allows greater structural
diversity and was therefore utilized to prepare 2-phenylbenzo-
furan congeners bearing cyano groups in the various positions

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6929
Scheme 4a
a Reagents and conditions: (i) Cu2O, pyridine, 100 °C, 15 h; (ii) pyridine hydrochloride, 120-140 °C, 3 h.
amide.63 A more expedient, one-step preparation of 65 and 66
involved modified Castro reactions between 3-hydroxy-4-
iodobenzonitrile (67)64 and phenylacetylenes 57 and 58, thus
circumventing the low-yield transformations of 63 and 64 to
65 and 66.
The synthesis of congeners bearing 7-methoxy and hydroxy
groups is depicted in Scheme 3. Commercially available
5-iodovanillin was reacted with the phenylacetylenes 57 and
6861 under modified Castro reaction conditions to form formyl-
benzofurans 69 and 70 in high yields. Reaction of compounds
69 and 70 with hydroxylamine hydrochloride in pyridine
afforded oximes 71 and 72, which were refluxed in Ac2O to
form dinitriles 73 and 74. Compounds 73 and 74 underwent
O-demethylation by treatment with molten pyridine hydrochlo-
ride to yield 7-hydroxydinitriles 75 and 76.
rhodesiense), chloroquine and artemisinin (P. falciparum), and
podophyllotoxin (L6). In addition to activities, three selectivity
indexes SIT, SIP, and SIL, reflecting the inhibition of the
particular parasite with respect to the L6 cells, were analyzed:
antitrypanosomal selectivity index SIT, expressed as the ratio
[IC50(L6-cells)/IC50(T. b. rhodesiense)]; antiplasmodial selectiv-
ity index SIP, expressed as the ratio [IC50(L6-cells)/IC50(P.
falciparum)]; and antileishmanial selectivity index SIL, expressed
as the ratio [IC50(L6-cells)/IC50(L. donoVani)].
Cytotoxicity Study. 2-Phenylbenzofurans 1-49 displayed a
range of in vitro cytotoxicities with IC50 values varying from
0.8 µM to >221 µM. Di(N-isopropyl)amidines were less
cytotoxic than diamidines and diimidazolines.59,61 Replacement
of amidine groups in the 2-phenylbenzofuran 1 with benzimi-
dazole or imidazole moieties in dications 4 and 5 afforded
derivatives exhibiting cytotoxicities comparable to that of
diamidine 1. Compound 7, bearing a benzimidazole substituent
in the 4′-position of the phenyl ring, was 30-fold less cytotoxic
than dication 1.
o-Iodophenols 56 and 67 were reacted with the methoxyphe-
nylacetylenes 68 and 77 to give the 2′-methoxy substituted
dinitriles 78-81 (Scheme 4). O-Demethylation using molten
pyridine hydrochloride afforded the corresponding 2′-hydroxy
derivatives 82-85.
The position of the cationic substituents mainly affected the
cytotoxicity of diamidines and diimidazolines but had lesser
effect on di(N-isopropyl)amidines. 5-Substituted diamidines and
diimidazolines exhibited lower cytotoxicity than the 6-substi-
tuted derivatives in all cases except diamidines 8 and 32 and
diimidazoline 34, which were more toxic than corresponding
6-substituted isomers 14, 44, and 46, respectively. Likewise,
diamidines and diimidazolines bearing one cationic group in
the 5′-position of the phenyl ring were less toxic than analogous
4′-substituted isomers except diamidine 44 and diimidazolines
3 and 46, which displayed higher cytotoxic IC50 values than
similar 5′-substituted derivatives 47, 10, and 49. Thus, 5,5′-
substituted diamidines 8 and 35 and 6,5′-substituted isomer 14
were 10-, 6-, and 160-fold less toxic than the corresponding
4′-substituted 2-phenylbenzofurans 1, 32, and 11. The position
of the cationic groups influenced the cytotoxicity of the
compounds 6 and 7. Attachment of the imidazoline moiety to
the 4′-position of the phenyl ring in the dication 6 afforded a
derivative nearly 10-fold more cytotoxic than compound 7,
bearing the imidazoline moiety in the 5-position of the benzo-
furan scaffold. Di(N-isopropyl)amidines exhibited comparable
cytotoxic IC50 values regardless of the position of cationic
fragments.
The effect of the methoxy and hydroxy groups on the
cytotoxicity of the 2-phenylbenzofurans 1-49 depended on the
type and the position of the cationic substituents. For instance,
introduction of the methoxy and hydroxy substituents in either
the 7-position of the benzofuran or 2′-position of the phenyl
ring of the 5,4′-substituted diamidine 1 resulted in a 15- to 100-
fold reduction in cytotoxicity (derivatives 17, 20, 26, and 32).
However, diamidine 23, bearing hydroxy groups in both the 7-
and 2′-positions, was equally cytotoxic as compound 1. Among
5,5′- and 6,4′-substituted diamidines, dications bearing methoxy
Dicationic 2-phenylbenzofurans 1-4 and 6-49 (Table 1)
were synthesized by the modified Pinner method49 (Scheme 5).
The dinitriles 51, 54, 55, 59, 65, 66, 73, 75, 76, 78-85 were
converted to imidate esters, which reacted with ethanolic
solutions of ammonia, isopropylamine, or ethylenediamine at
ambient temperature or with 1,2-diaminobenzene in refluxing
methanol. 5-(1H-Imidazol-2-yl)-2-[4-(1H-imidazol-2-yl)phenyl]-
benzofuran (5) was prepared from the corresponding 5-(4,5-
dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imidazol-2-
yl)phenyl]benzofuran (3) by oxidation with o-iodoxylbenzoic
acid (IBX),65 following a published protocol.66
Results and Discussion
In the case of pentamidine analogues and cationic diphenyl-
isoxazoles, the introduction of the methoxy groups resulted in
enhanced antiprotozoal activities24 and decreased cytotoxicities
of select compounds, therefore affording greater selectivity
toward specific parasites.61 We designed several cationic
substituted 2-phenylbenzofurans with the methoxy and hydroxy
groups attached to the aromatic rings to evaluate how this
structural variation would affect antiprotozoal properties of
compounds 1-49. Dications 4-7, bearing aromatic cationic
groups, were synthesized to investigate the effect of the reduced
basicity and increased lipophilicity of these congeners on their
activities against different parasites. The results of the in vitro
testing against bloodstream form trypomastigotes of T. b.
rhodesiense (STIB900), chloroquine resistant P. falciparum
(K1), and axenic amastigotes of L. donoVani (MHOM/SD/62/
1S-CL2D) as well as the assessment of cytotoxicity of the
compounds 1-49 against rat myoblast cells (L6) are sum-
marized in Table 1 and compared to the results of pentamidine
and DAPI. Other controls employed were melarsoprol (T. b.

6930 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
Table 1. Structures, Cytotoxicity, and in Vitro Antiprotozoal Activity of 2-Phenylbenzofurans 1-49

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6931
Table 1. Continued

6932 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 1. Continued
BakunoV et al.
a PMD, pentamidine. b DAPI, 4′,6-diamidino-2-phenylindole. c MLSP, melarsoprol. d CQ, chloroquine. e ATMS, artemisinin. f PPT, podophyllotoxin.
g Cytotoxicity (L6 rat myoblast cells). Average of duplicate determinations.76 h Trypanosoma brucei rhodesiense (STIB900). Average of duplicate
determinations.7,71,72 i Selectivity index70 for T. b. rhodesiense (SIT), expressed as the ratio [IC50(L6)/IC50(T. b. rhodesiense)]. j Chloroquine resistant Plasmodium
falciparum (K1). Average of duplicate determinations.7,73 k Selectivity index70 for P. falciparum (SIP), expressed as the ratio [IC50(L6)/IC50(P. falciparum)].
l Leishmania donoVani (MHOM/SD/62/1S-CL2D) axenic amastigotes. Average of duplicate determinations.74,75 m Selectivity index70 for L. donoVani (SIL),
expressed as the ratio [IC50(L6)/IC50(L. donoVani)].

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6933
Scheme 5a
a Reagents and conditions: (i) 1,4-dioxane, EtOH, HCl, 1-3 days; (ii) appropriate amine, EtOH, 1-2 days.
(29 and 38) or hydroxy groups (35 and 44) in the 2′-position of
the phenyl ring exhibited lower cytotoxicity than compounds 8
and 11, respectively. Thus, 2′-hydroxy substituted dication 44
was 270-fold less toxic than 2-phenylbenzofuran 11. In the case
of 6,5′-substituted diamidines, addition of the methoxy (41) or
hydroxy (47) groups in the 2′-position of the molecule had little
effect on toxicity. Attachment of the methoxy or hydroxy
substituents to the 7- or/and 2′-positions of the aromatic rings
resulted in slightly increased cytotoxicity of di(N-isopropyl)a-
midines relative to 2-phenylbenzofurans 2, 9, 12, and 15.
Diimidazolines 19, 28, 31, 40, and 43 bearing methoxy
substituents were more cytotoxic than the parent compounds 3,
10, 13, and 16 regardless of the position of the cationic
fragments, while the introduction of hydroxy groups afforded
dications with lower cytotoxicity. For example, 2′-hydroxy
substituted diimidazoline 46 was nearly 40-fold less toxic than
the 2-phenylbenzofuran 13.
Antitrypanosomal Activity. All compounds 1-49 exhibited
in vitro antitrypanosomal potencies ranging from 2 nM to 5.1
µM with only two dications in the group showing IC50 values
exceeding 1 µM. Ten diamidines (1, 11, 17, 20, 26, 29, 32, 35,
38, and 44), one di(N-isopropyl)amidine (45), and three diimi-
dazolines (22, 25, and 34) displayed antitrypanosomal IC50
values equal to or less than 10 nM. Eight of these dications (1,
20, 22, 26, 29, 32, 38, and 44), all diamidines except diimida-
zoline 22, demonstrated antitrypanosomal potencies comparable
to or lower than that of melarsoprol (IC50 ) 4 nM). Six
2-phenylbenzofurans (1, 20, 26, 29, 32, and 38) exhibited
antitrypanosomal activities equal or superior to that of penta-
midine (IC50 ) 3 nM). 5-Substituted diamidines 29 and 32,
bearing methoxy and hydroxy groups in the 2′-position of the
phenyl ring, respectively, were the most potent compounds in
the series, with antitrypanosomal IC50 values of 2 nM.
Generally, diamidines were more active in vitro against T.
b. rhodesiense than di(N-isopropyl)amidines and diimidazo-
lines.59,61,67-69 Antitrypanosomal potency of 5′-substituted
dications decreased in the order Am > Im > i-PrAm, whereas
for compounds bearing one cationic group in the 4′-position of
the phenyl ring there was no apparent correlation between the
type of cationic substituents and activities against T. b. rhod-
esiense. Replacement of the amidine groups in 2-phenylben-
zofuran 1 with aromatic cationic moieties resulted in a 40- to
200-fold decrease of antitrypanosomal activities (compounds
4-7).
revealed higher antitrypanosomal activities relative to the
5-substituted derivatives 28, 31, and 37, respectively. Com-
pounds bearing one cationic group in the 4′-position of the
phenyl ring exhibited higher activities against T. b. rhodesiense
relative to 5′-substituted 2-phenylbenzofurans in all cases except
for dications 3, 26, 28, and 40, which were less active against
trypanosomes than the corresponding isomers 10, 29, 31, and
43, respectively.
In many cases, the attachment of the methoxy and hydroxy
groups resulted in enhanced antitrypanosomal activity of 2-phe-
nylbenzofurans. 5,4′-Substituted dications 17, 20, 26, and 32
with the methoxy or hydroxy groups in the 7-position of the
benzofuran moiety or 2′-position of the phenyl ring exhibited
antitrypanosomal activity comparable to that of the diamidine
1. Among the 5,5′-, 6,4′-, and 6,5′-substituted diamidines,
derivatives bearing methoxy groups in the 2′-position of the
phenyl ring were the most potent against T. b. rhodesiense. For
example, 2′-methoxy substituted diamidine 29 exhibited 12-
fold increase in the antitrypanosomal activity compared to the
diamidine 8. However, diamidine 23 bearing two hydroxy
groups in the 7- and 2′-positions was 6-fold less as potent against
T. b. rhodesiense as 2-phenylbenzofuran 1. 4′-Substituted di(N-
isopropyl)amidines and diimidazolines, bearing hydroxy groups
in the 7- or/and 2′-positions, displayed higher antitrypanosomal
activities than those with methoxy groups and the parent
dications 2 and 3. Thus, 2′-hydroxy substituted di(N-isopropyl)-
amidines 33 and 45 revealed antitrypanosomal potencies 6- and
16-fold greater than 2-phenylbenzofurans 2 and 12. Likewise,
attachment of methoxy groups to the 7-position of the benzo-
furan ring in the diimidazoline 22 or to the 2′-position of the
phenyl ring in the diimidazoline 34 resulted in increases activity
against T. b. rhodesiense by 47- and 24-fold, respectively,
relative to the parent dication 3.
All compounds 1-49 were selective against the Trypanosoma
parasite (selectivity index70 SIT, defined as a ratio of cytotoxic
IC50 to antitrypanosomal IC50 values, varied from 5 to 89 500).
Twelve dications (20, 22, 25, 26, 29, 32, 34, 35, 38, 44-46)
displayed antitrypanosomal selectivity indexes SIT greater than
that of pentamidine (SIP ) 15533).
Diamidines usually exhibited higher antitrypanosomal selec-
tivity indexes SIT compared to di(N-isopropyl)amidines and
diimidazolines. However, in the series of 5,4′- and 6,4′-
substituted dications, di(N-isopropyl)amidines 2 and 12 were
the most selective against T. b. rhodesiense. Also, among 5,4′-
substituted derivatives bearing hydroxy groups in the 2′- or/
and the 7-positions of the aromatic moieties, diimidazolines 22
and 34 displayed the highest antitrypanosomal selectivity.
2-Phenylbenzofurans 4-7 bearing aromatic cationic moieties
were 2- to 126-fold less selective against T. b. rhodesiense than
dication 1.
The position of the cationic groups affected the antitrypano-
somal properties of 2-phenylbenzofurans 1-49. Thus, 5-sub-
stituted diamidines and di(N-isopropyl)amidines were more
active against T. b. rhodesiense than 6-substituted derivatives
except diamidine 8 and di(N-isopropyl)amidines 27 and 33,
which displayed lower antitrypanosomal potencies than the
corresponding 6-substituted isomers 14, 39, and 45. In the case
of diimidazolines, 6-substituted derivatives 40, 43, and 49
The relative antitrypanosomal selectivity of 5- and 6-substi-
tuted dications as well as 4′- and 5′-substituted isomers depended

6934 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
on the type of the cationic substituents and the presence or
absence of the methoxy and hydroxy groups in the molecules.
5-Substituted 2-phenylbenzofurans were more selective against
T. b. rhodesiense compared to the 6-substituted congeners in
all cases except diamidines 8 and 32, di(N-isopropyl)amidines
27 and 33, and diimidazolines 28 and 37, which displayed lower
antitrypanosomal selectivity indexes SIT than the corresponding
6-substituted isomers 14, 44, 39, 45, 40, and 39, respectively.
In the case of diamidines and diimidazolines, relative antitry-
panosomal selectivities of 4′- and 5′-substituted isomers were
affected by the presence or absence of methoxy or hydroxy
substituents in the molecules. For instance, 5,5′-substituted
diamidines 8, 29, and 35 as well as the 6,5′-substituted diamidine
14 were more selective against T. b. rhodesiense than the
corresponding 4′-substituted isomers 1, 26, 32, and 11. Similarly,
5,5′- and 6,5′-substituted diimidazolines 10, 16, 31, and 43
exhibited higher selectivity indexes SIT than 4′-substituted
derivatives 3, 13, 28, and 40, respectively. All other 4′-
substituted 2-phenylbenzofurans exhibited higher antitrypano-
somal selectivity indexes SIT than corresponding 5′-substituted
dications.
two di(N-isopropyl)amidines (12 and 27), and two diimidazo-
lines (3 and 34). Seven of these dications (3, 11, 14, 17, 26,
33, and 34), all diamidines except diimidazolines 3 and 34,
exhibited antiplasmodial IC50 values superior to that of arte-
misinin (IC50 ) 6 nM). Diimidazoline 3, bearing cationic
substituents in the 5-position of the benzofuran moiety and the
4′-position of the phenyl ring, was the most potent compound
in the series, with an antiplasmodial IC50 value of 1 nM.
Diamidines and diimidazolines usually exhibited higher
antimalarial potency in vitro than di(N-isopropyl)amidines.
Replacement of the amidine groups in the 2-phenylbenzofuran
1 with aromatic cationic substituents reduced antiplasmodial
activities of the compounds 4-7.
The position of cationic groups influenced the antiplasmodial
properties of compounds 1-49. 6-Substituted diamidines and
di(N-isopropyl)amidines without the methoxy or hydroxy groups
in the 2′-position of the phenyl ring were more active against
P. falciparum than corresponding 5-substituted derivatives.
However, among the 2′-methoxy and 2′-hydroxy substituted
diamidines and di(N-isopropyl)amidines, compounds bearing
cationic substituents in the 5-position of the benzofuran ring
displayed higher antiplasmodial activities relative to the 6-sub-
stituted congeners in all cases except diamidines 29 and 35,
which were less active against P. falciparum than the corre-
sponding 6-substituted dications 41 and 47. All 5-substituted
diimidazolines exhibited antiplasmodial activities greater than
that of the 6-substituted derivatives. 2-Phenylbenzofurans bear-
ing one cationic group in the 4′-position of the phenyl ring
generally demonstrated higher antiplasmodial activities com-
pared to their 5′-substituted counterparts apart from diamidines
1 and 38 as well as diimidazoline 40, which were less effective
against P. falciparum than the corresponding dications 8, 41,
and 43.
Addition of the methoxy or hydroxy groups to the 2′- or/and
7-position of the aromatic rings in many cases improved
selectivity of 2-phenylbenzofurans 1-49 against T. b. rhod-
esiense. 5,4′-Substituted dications bearing the methoxy or
hydroxy groups in the 7-position of the benzofuran ring
displayed increased antitrypanosomal selectivity compared to
dications 1-3 in all cases except diimidazoline 19 and diamidine
23. Thus, 7-hydroxy substituted diamidine 20 and diimidazoline
22 exhibited antitrypanosomal selectivity indexes SIT that were
97- and 242-fold greater than those of the compounds 1 and 3,
respectively. In the cases of 5,4′- and 5,5′-substituted diamidines
bearing methoxy and hydroxy groups in the 2′-position of the
phenyl ring, the antitrypanosomal selectivity increased in the
order H < OH < OMe. Thus, 2′-methoxy substituted dications
26 and 29 were approximately 80- and 100-fold more selective
against T. b. rhodesiense than corresponding diamidines 1 and
8. The diamidine 29 not only exhibited the highest selectivity
index SIT of 89 500, which was nearly 6-fold greater than that
of pentamidine (SIT ) 15 533), but also was one of two the
most potent compounds in the series, displaying an antitrypa-
nosomal IC50 value of 2 nM. In the case of all 6,4′-substituted
dications, antitrypanosomal selectivity indexes SIT increased in
the order H < OMe < OH. For example, the antitrypanosomal
selectivities of 2-hydroxy substituted diamidine 44 and diimi-
dazoline 46 were nearly 500 times and more than 1200 times
greater than that of the corresponding dehydroxy compounds
11 and 13, respectively. The introduction of the hydroxy group
in the 7- and/or 2′-position of the aromatic moieties of the 4′-
substituted di(N-isopropyl)amidines improved their antitrypa-
nosomal selectivity indexes SIT. In the case of 5′-substituted
isomers, however, such transformation reduced the selectivity
of dications against the parasite compared to 2-phenylbenzo-
furans lacking substituents in the 2′-position.
Addition of methoxy and hydroxy substituents improved
antiplasmodial activities of some dications. For 5,4′-substituted
diamidines and di(N-isopropyl)amidines antiplasmodial poten-
cies increased in the order H < OH < OMe regardless of the
position of the methoxy and hydroxy groups. For example,
7-methoxy substituted diamidine 17 displayed antiplasmodial
activity nearly 8-fold greater than the dication 1. Similarly, the
diamidines 26 and 32 bearing methoxy and hydroxy groups in
the 2′-position were 16- and 12-fold more active against P.
falciparum than compound 1. However, addition of two hydroxy
groups to the 7- and 2′-position of the aromatic moieties in
2-phenylbenzofurans resulted in decreased antiplasmodial activi-
ties regardless of the nature of the cationic groups.
While the introduction of the methoxy and hydroxy groups
increased antiplasmodial activities of selected 2-phenylbenzo-
furans, such as 5,4′-substituted diamidines and di(N-isopropyl)-
amidines as well as 5,5′-, 6,4′-, and 6,5′-substituted diimida-
zolines, in some cases this structural modification afforded
congeners with lower potencies against P. falciparum compared
to the dications without methoxy or hydroxy substituents. For
example, antiplasmodial activities of 5,5′-, 6,4′-, and 6,5′-
substituted diamidines, 6,4′-, and 6,5′-substituted di(N-isopro-
pyl)amidines, as well as 5,4′-substituted diimidazolines de-
creased in the order H > OMe > OH relative to the corre-
sponding demethoxy and dehydroxy analogues.
Antiplasmodial Activity. Dications 1-49 exhibited potencies
against P. falciparum with in vitro IC50 values ranging from 1
to 793 nM (Table 1). Only 13 compounds in the series displayed
antimalarial IC50 values exceeding 100 nM. Twenty-six com-
pounds (1, 3-6, 8, 11, 12, 14, 17-22, 25-29, 32-34, 37, 38,
and 41) exhibited antiplasmodial activities higher than penta-
midine (IC50 ) 58 nM). Twelve compounds (3, 8, 11, 12, 14,
17, 26, 27, 32, 34, 38, and 41) demonstrated in vitro antiplas-
modial IC50 values equal to or less than 20 nM. This group
included eight diamidines (8, 11, 14, 17, 26, 32, 38, and 41),
All dications 1-49 were selective against P. falciparum
(selectivity index70 SIP, defined as a ratio of cytotoxic IC50 to
antiplasmodial IC50 values, varied from 34 to 52 333). Twenty-
six dications (2, 3, 7, 8, 12, 14, 15, 17, 18, 20-22, 25-27, 29,

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6935
32-34, 37-39, 41, 44, 46, 49) displayed antiplasmodial
selectivity indexes SIP greater than that of pentamidine (SIP )
803).
reduced antiplasmodial selectivity indexes SIP of selected
2-phenylbenzofurans. Thus, 5,5′-substituted diamidines and
di(N-isopropyl)amidines with the 2′-hydroxy group in the
molecules were less selective against the parasite compared to
the corresponding analogues lacking substitution in the 2′-
position. In the case of 6,5′-substituted diamidines as well as
6,4′- and 6,5′-substituted di(N-isopropyl)amidines, antiplasmo-
dial selectivity of the dications decreased in the order H > OMe
> OH.
Diamidines and diimidazolines usually exhibited higher
antimalarial selectivity indexes SIP than di(N-isopropyl)amidines.
Compounds 4-7 bearing aromatic cationic fragments displayed
reduced antiplasmodial selectivity, except compound 7, which
was slightly more selective against P. falciparum than
pentamidine.
Antileishmanial Activity. All 2-phenylbenzofurans except
dications 4, 5, 7, 16, 40, and 48 were active in the L. donoVani
axenic amastigote assay, demonstrating activities ranging from
0.99 to 60 µM. Diamidines 1, 26, and 32 as well as di(N-
isopropyl)amidines 2, 27, and 33 displayed antileishmanial IC50
values less than 2 µM. All these six compounds were 5,4′-
substituted derivatives with or without methoxy or hydroxy
groups in the 2′-position of the phenyl ring. Five 2-phenylben-
zofurans (1, 2, 27, 32, and 33) exhibited antileishmanial IC50
values equal or greater than that of pentamidine (IC50 ) 1.8
µM). Diamidine 1 revealed the highest antileishmanial potency
in the series, exhibiting an IC50 value of 0.99 µM.
In general, substitution on the amidine groups resulted in
decreased activity of the compounds 1-49 against L. donoVani
axenic amastigotes, although exceptions did occur. 2-Phenyl-
benzofurans 4-7 bearing aromatic cationic substituents pos-
sessed no antileishmanial activity except compound 6, with an
IC50 value of 5.7 µM.
The position of the cationic moieties had significant effect
on antileishmanial properties of 2-phenylbenzofurans 1-49.
Compounds bearing one of the cationic groups in the 5-position
of the benzofuran ring were more potent against L. donoVani
than 6-substituted derivatives in all cases except diamidine 29
and diimidazolines 31 and 37, which displayed lower antile-
ishmanial activity than the corresponding 6-substituted isomers
41, 43, and 49, respectively. Similarly, 4′-substituted 2-phenyl-
benzofurans exhibited higher activities against L. donoVani
compared to compounds having one cationic group attached to
the 5′-position of the phenyl ring on all occasions except for
diamidine 38 and diimidazolines 28 and 40, which displayed
lower antileishmanial activities than corresponding dications 41,
31, and 43.
Introduction of methoxy and hydroxy substituents in the 2′-
position of the phenyl ring and in the 7-position of the
benzofuran moiety resulted in decreased antileishmanial activi-
ties of the 5,4′-substituted diamidines.24 For example, diamidine
23 containing two hydroxy groups attached to the 7- and the
2′-position of the molecule was 14 times less active against L.
donoVani than dication 1. In the series of 5,5′- and 6,5′-
substituted diamidines and 5,4′-substituted di(N-isopropyl)a-
midines, compounds bearing hydroxy groups in the 2′-positions
of the phenyl ring displayed lower antileishmanial activities than
parent dications 8, 14, and 2. However, the introduction of the
2′-methoxy substituent in the molecules of the aforementioned
dications afforded congeners with improved potency against L.
donoVani. Thus, 2′-methoxy substituted diamidine 41 revealed
an antileishmanial activity nearly 4 times greater than dication
14. Antileishmanial activities of 6,4′-substituted diamidines,
5,5′-, 6,4′-, and 6,5′- substituted di(N-isopropyl)amidines as well
as 5,5′-substituted diimidazolines decreased in the order H >
OMe > OH.
The relative antiplasmodial selectivity of 5- and 6-substituted
dications as well as 4′- and 5′-substituted congeners depended
on the type of the cationic scaffolds and the presence of methoxy
and hydroxy groups in the molecules. For instance, 6-substituted
diamidines and di(N-isopropyl)amidines lacking methoxy and
hydroxy groups in the 2′-position exhibited higher antiplasmo-
dial selectivity indexes SIP than their 5-substituted counterparts,
while 5-substituted diimidazolines were more selective against
P. falciparum compared to the 6-substituted analogues. Among
the 2-phenylbenzofurans containing 2′-methoxy and hydroxy
groups, compounds bearing cationic substituents in the 5-posi-
tion of the benzofuran ring displayed higher antimalarial
selectivity in all cases except diamidines 29 and 35, which were
less selective against P. falciparum than corresponding 6-sub-
stituted isomers 41 and 47. In the case of diamidines and
diimidazolines, antiplasmodial selectivities of 4′- and 5′-
substituted isomers depended on the presence or absence of
methoxy or hydroxy groups in the molecules. For instance, 6,5′-
substituted diamidines 14 and 41 and diimidazolines 16 and 43
were more selective against P. falciparum than the correspond-
ing 6,4′-substituted isomers 11, 38, 13, and 40. Similarly, 5,5′-
substituted diamidine 8 and diimidazole 31 exhibited higher
selectivity indexes SIP than 5,4′-substituted derivatives 1 and
28, respectively. In all other cases diamidines and diimidazolines
bearing one cationic group in the 4′-position of the phenyl ring
were more selective against the parasite than the 5′-substituted
isomers. All 4′-substituted di(N-isopropyl)amidines exhibited
higher antiplasmodial selectivity indexes SIP than corresponding
5′-substituted dications.
In general, the position of the methoxy or hydroxy group in
the 7- or 2′-position of the aromatic rings increased selectivity
of some 2-phenylbenzofurans 1-49 against P. falciparum.
Among 5,4′-substituted diamidines and di(N-isopropyl)amidines
the selectivity against P. falciparum increased in the order H
< OH < OMe. Thus, 7-methoxy and hydroxy substituted
diamidines 17 and 20 exhibited antiplasmodial selectivity
indexes SIP that were almost 130- and 100-fold greater than
that of dication 1. Similarly, 2′- methoxy and hydroxy substi-
tuted dications 26 and 32 were 1300- and 200-fold more
selective against P. falciparum than the diamidine 1. Diamidine
26 not only was among the most potent compounds in the series,
with an antiplasmodial IC50 value of 3 nM, but also was the
most selective dication in the group, exhibiting an antiplasmodial
selectivity index 65 times greater than that of pentamidine.
However, introduction of two hydroxy groups in the 7- and 2′-
positions of the aromatic moieties reduced the selectivity of
diamidine 23 and di(N-isopropyl)amidine 24 compared to
dications 1 and 2. In the case of diimidazolines, antiplasmodial
selectivity indexes SIP of 5,5′-, 6,4′-, 6,5′-substituted derivatives
increased in the order H < OMe < OH. For example, the
introduction of hydroxy groups in the 2′-position of the phenyl
ring afforded diimidazolines 46 and 49, which were 48- and
30-fold as selective against P. falciparum as dications 13 and
16, respectively. However, in several cases the introduction of
methoxy or hydroxy groups in the 2′-position of the phenyl ring
All 2-phenylbenzofurans, except dications 4-7, 10, 11, 13,
16, 23, 28, 31, 40, and 43, were selective against L. donoVani
and displayed selectivity indexes70 (SIL, defined as a ratio of
cytotoxic IC50 to antileishmanial IC50 values) ranging from 2

6936 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
Table 2. Activity of Select 2-Phenylbenzofurans in the STIB900 Mouse
Model of Trypanosomiasis
to 125. Ten dications (2, 20, 22, 26, 27, 29, 33, 34, 38, and 41)
revealed antileishmanial selectivity indexes SIL greater than that
of pentamidine (SIL ) 25).
in vivob
in vitro,
dose
survival
(days)e
Diamidines and di(N-isopropyl)amidines generally exhibited
higher antileishmanial selectivity indexes SIL than diimidazo-
lines. Dications 4-7 bearing aromatic cationic motifs did not
show any selectivity against L. donoVani.
compd
IC50 (nM)a
(mg/kg)c
curesd
melarsoprol
4
4 × 8
4 × 2
4 × 1
4 × 20
4 × 5
4 × 20
4 × 5
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 20
4 × 5
4 × 5
4 × 5
4 × 1
1 × 10
4 × 5
4 × 5
4 × 5
4 × 1
1 × 10
4 × 20
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 1
1 × 10
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 1
1 × 10
4 × 5
4 × 5
4 × 5
4/4
4/4
2/4
2/4
2/4
4/4
0/4
3/4
3/4
2/4
3/4
0/4
0/4
0/4
0/4
0/4
4/4
0/4
1/4
1/4
0/4
4/4
0/4
2/4
0/4
4/4
1/4
1/4
4/4
0/4
2/4
3/4
1/4
3/4
3/4
1/4
2/4
4/4
0/4
2/4
0/4
2/4
0/4
2/4
0/4
3/4
0/4
2/4
3/4
0/4
0/4
3/4
0/4
0/4
4/4
0/4
2/4
1/4
1/4
0/4
1/4
0/4
2/4
3/4
2/4
3/4
0/4
1/4
0/4
>60
>60
>51.5
>57.5
>45
>60
>40.8
>53
>56
>51.8
>53
15.3
18.3
11
5.5
11
>60
26.5
>39.5
>38.5
33
>60
27.3
>44
11.5
>60
>30.8
>43
>60
19.8
>36.3
>49.8
>39
>51.3
>56.8
>33.5
>49.5
>60
26.3
>50.7
>21
>47
>39
>57
8.5
>54.5
34.8
>55.3
>50.5
18.5
26.8
>60
20
23.5
>60
26
>51.5
>34.5
>38
7
Antileishmanial selectivity of 5- and 6-substituted dications
as well as 4′- and 5′-substituted isomers depended on the type
of the cationic substituents and the presence or absence of
methoxy and hydroxy groups in the molecules. Compounds
bearing cationic groups at the 5-position were more selective
against L. donoVani in all cases except diamidines 8 and 29
and diimidazoline 37, which displayed lower antileishmanial
selectivity indexes SIL than corresponding 6-substituted isomers
14, 41, and 49. Likewise, 4′-substituted 2-phenylbenzofurans
usually exhibited higher antileishmanial selectivity indexes SIL
than corresponding 5′-substituted isomers apart from 5,4′-
substituted diamidine 1 and 6,4′-substituted diamidines 11 and
38, which were less selective against L. donoVani than corre-
sponding 5′-substituted derivatives 8, 14, and 41.
pentamidine
3
17
3
DAPI
1
2
125
3
189
249
122
7
4
6
11
12
14
17
148
21
6
Introduction of the methoxy and hydroxy substituents affected
the antileishmanial selectivity of the 2-phenylbenzofurans 1-49.
5,4′-Substituted diamidines and diimidazolines bearing methoxy
or hydroxy groups in the 7-position of the benzofuran ring were
more selective against L. donoVani than dications 1 and 3 in all
cases except diamidine 23, which contained hydroxy groups
both in 7- and in 2′-positions of the aromatic moieties. For
example, 7-hydroxy substituted diamidine 20 and diimidazoline
22 revealed antileishmanial selectivity indexes SIL that were
18- and 14-fold greater than those of compounds 1 and 3,
respectively. Conversely, di(N-isopropyl)amidines with substit-
uents in the 7-position of the benzofuran ring exhibited reduced
antileishmanial selectivity indexes compared to the compound
2. Thus, di(N-isopropyl)amidine 24, bearing the hydroxy groups
in the 7- and 2′-position of the molecule, was 31-fold less
selective against L. donoVani than dication 2. Introduction of
the methoxy and hydroxy groups at the 2′-position of the phenyl
ring also improved selectivity indexes SIL of some cationic
2-phenylbenzofurans. Antileishmanial selectivity of 5,4′-, 5,5′-,
and 6,4′-substituted diamidines bearing methoxy and hydroxy
groups in the 2′-position of the phenyl ring increased in the
order H < OH < OMe. For example, 2′-methoxy substituted
dications 26 and 29 were 42- and 9-fold more selective against
L. donoVani than corresponding diamidines 1 and 8. The
attachment of the hydroxy group to the 2′-position of the phenyl
ring of diimidazolines increased their antileishmanial selectivity
compared to unsubstituted dications, while the addition of the
2′-methoxy substituent resulted in less selective compounds.
Thus, 2′-hydroxy substituted diimidazoline 34 exhibited the
antileishmanial selectivity index SIL that was nearly 14-fold
higher than that of dication 3. In the case of di(N-isopropyl)a-
midines, the introduction of the 2′-methoxy or hydroxy groups
in the molecules reduced selectivity of these dications against
L. donoVani. Thus, di(N-isopropyl)amidine 2 not only was more
active than pentamidine against L. donoVani but also displayed
the highest selectivity index in the series (SIL ) 125), which
was 5-fold greater than that of pentamidine.
19
20
124
3
21
22
23
24
28
4
18
23
25
26
27
29
31
32
10
3
74
2
27
2
33
34
35
21
8
5
38
39
40
41
43
44
45
3
54
49
11
17
4
>48
25
>39
>55.5
>46.5
>51.8
23
9
46
47
49
11
29
83
>36
14
a Average of duplicate determinations. b STIB900 acute mouse model.7
c Intraperitoneal administration. d Number of mice that survive and are
parasite-free for 60 days. e Average days of survival; untreated controls
expired between days 7 and 8 after infection.
In Vivo Antitrypanosomal Activity. Select 2-phenylben-
zofurans exhibiting promising in vitro activities against T. b.
rhodesiense were evaluated in the STIB900 animal model of
African trypanosomiasis (Table 2). Initial screening was con-
ducted using intraperitoneal dosing at 20 mg/kg daily for 4 days.
However, because of the high curative rate and a significant
increase in the survival time of the infected animals, the
administered amount was reduced to 5 mg/kg daily for 4 days.

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6937
Even at this very low dosage 17 2-phenylbenzofurans (1, 2,
11, 17, 20-24, 26, 29, 32-35, 44, and 45) displayed excellent
in vivo efficacies, curing 2/4 animals or better. In addition,
dications 12 and 14 provided 3/3 and 3/4 cures, respectively,
when administered intraperitoneally at 20 mg/kg for 4 days (data
not shown).
L. donoVani. Promising antiprotozoal activity of cationic
substituted 2-phenylbenzofurans in vitro, excellent potency in
the acute mouse model of trypanosomiasis and the reduced
cytotoxicity compared to pentamidine warrant further investiga-
tion of this class of compounds.
Diamidines and di(N-isopropyl)amidines usually exhibited
higher in vivo efficacies than diimidazolines. For example,
diamidine 1 and di(N-isopropyl)amidine 2 provided 3/4 cures
when administered intraperitoneally at 5 mg/kg for 4 days, while
diimidazole 3 was inactive even at the dose of 20 mg/kg.
The position of attachment of cationic substituents influenced
the efficacies of 2-phenylbenzofurans in the acute mouse model
of trypanosomiasis. Thus, 5-substituted dications were more
effective in vivo than 6-substituted analogues and 4′-substituted
derivatives exhibited higher potencies than 5′-substituted iso-
mers. Overall, 12 out of 17 compounds active in the acute mouse
model had their cationic moieties attached to the 5- and 4′-
position of the aromatic rings.
Introduction of methoxy and hydroxy groups improved in
vivo potencies of 2-phenylbenzofurans. For example, diamidine
20 and di(N-isopropyl)amidine 21 bearing the hydroxy group
in the 7-position of the benzofuran moiety displayed improved
antitrypanosomal efficacy relative to that of dications 1 and 2,
curing 4/4 animals. Likewise, 7-hydroxy substituted and 2′-
hydroxy substituted diimidazolines 22 and 34 cured 3/4 animals,
while the aforementioned 2-phenylbenzofuran 3 was inactive
in the STIB900 model.
Fourteen dications providing 3/4 or 4/4 cures when admin-
istered at 5 mg/kg daily for 4 days were tested at an even lower
intraperitoneal dosage of 1 mg/kg daily for 4 days as well as at
a single-dose administration of 10 mg/kg. In the single-dose
regimen, seven dications (1, 17, 21, 23, 24, 32, and 35) provided
2/4 cures, while 7-hydroxy substituted diimidazoline 22 and 2′-
hydroxy substituted di(N-isopropyl)amidine 45 cured 3/4 mice.
Among the five 2-phenylbenzofurans (1, 20, 22, 23, and 45)
retaining curative ability in vivo at the regiment of 1 mg/kg
daily for 4 days, diamidine 1 and di(N-isopropyl)amidine 45
exhibited in vivo activity comparable to that of melarsoprol,
providing 3/4 and 2/4 cures, respectively.
Experimental Section
General Experimental Information. In vitro antitrypanosomal,
antiplasmodial, and antileishmanial activities and cytotoxicities were
determined following established protocols.7,71-76 In vivo antit-
rypanosomal testing was conducted as previously described.7 All
chemicals and solvents were purchased from Aldrich Chemical Co.,
Fisher Scientific, or Acros Organics and were used without further
purification. Uncorrected melting points were measured on a
Thomas-Hoover capillary melting point apparatus. 1H NMR spectra
were recorded on a Varian Gemini 2000 spectrometer operating at
300 MHz. Chemical shifts are reported in ppm relative to
tetramethylsilane. Anhydrous ethanol was distilled over Mg/I2
immediately prior to use. Reaction mixtures were monitored by
TLC using Whatman silica gel 250 µm UV254 plates or by reverse
phase HPLC. Organic layers of extraction mixtures were washed
with saturated NaCl solution and dried over Na2SO4 or MgSO4
before being evaporated under reduced pressure. Flash column
chromatography was performed using Davisil grade 633, type 60A
silica gel (200-425 mesh). Analytical HPLC chromatograms were
recorded on an Agilent 1200 chromatograph using an Agilent
Zorbax Rx C8 column (4.6 mm × 75 mm, 3.5 µm) and UV
photodiode array detection at 230, 254, 265, 290, and 320 nm. The
column temperature was maintained at 40 °C. Mobile phases
consisted of mixtures of acetonitrile (0-75%) in water containing
formic acid (80 mM), ammonium formate (20 mM), and triethy-
lamine (15 mM). Flow rates were maintained at 1.5 mL/min. In
method A, the concentration of acetonitrile was increased linearly
from 0 to 22.5% over 6 min and then from 22.5 to 56.25% over 4
min and finally maintained for 1 min. In method B, the concentra-
tion of acetonitrile was increased linearly from 22.5 to 75% over
10 min and then maintained for 2 min.
Preparative Reverse Phase HPLC. Preparative Reverse Phase
HPLC was performed on a Varian ProStar Chromatography
Workstation configured with two PS-215 pumps fitted with 50 mL
pump heads, a Dynamax Microsorb C18 (60 Å) column (41.4 mm
× 250 mm, 8 µm), PS-320 variable wavelength UV-vis detector,
and a PS-701 fraction collector. Mobile phases consisted of mixtures
of acetonitrile (0-75%) in water containing formic acid (40 mM)
and ammonium formate (10 mM). Flow rates were maintained at
40 mL/min. Detector wavelengths and mobile phase gradients were
optimized for the individual compounds. Select fractions were
analyzed for purity as described above for analytical HPLC.
Residues of evaporated pooled purified fractions were reconstituted
in water and lyophilized on a VirTis BenchTop 6K lyophilizer.
The lyophilized compounds were dissolved in ethanol and converted
into HCl salts with aqueous HCl.
Flash Chromatography of Amidines on C18 Reversed Phase
Silica Gel. The chromatographic column was half-filled with
acetonitrile and packed with a slurry of C18 silica gel (70 g) in
acetonitrile (70-100 mL). The excess acetonitrile was drained out,
and the top of the column was covered with a 2 cm pad of sand.
The column was equilibrated with 150 mL of initial mobile phase
that consisted of water containing formic acid (40 mM) and
ammonium formate (10 mM). A concentrated reaction mixture was
dissolved in the initial mobile phase. In the case of low solubility,
heating of the mixture and/or addition of a small amount of
methanol as a cosolvent was performed. After the reaction mixture
was applied to the column, the elution began with initial mobile
phase (150 mL) to remove the excess amine and then with a mobile
phase consisting of a mixture of acetonitrile (0-75%) in water
containing formic acid (40 mM) and ammonium formate (10 mM).
Acetonitrile concentrations varied for each individual compound
and contained 50-70% of calculated amount of acetonitrile at the
point of the retention time of the compound in analytical method
Conclusions
We have reported the synthesis and the in vitro evaluation
of the antiprotozoal properties of cationic substituted 2-phenyl-
benzofurans 1-49 against Trypanosoma brucei rhodesiense,
Plasmodium falciparum, and Leishmania donoVani as well as
the cytotoxicity study of these compounds against mammalian
cells. In our screens, diamidines usually exhibited higher
antitrypanosomal, antimalarial, and antileishmanial activities
compared to di(N-isopropyl)amidines and diimidazolines. Re-
placement of the amidine groups with aromatic cationic moieties
afforded more cytotoxic congeners with decreased antiparasitic
activity. The position of attachment of cationic groups influenced
the antiprotozoal properties of 2-phenylbenzofurans. Generally,
5-substituted dications were more active against T. b. rhod-
esiense, P. falciparum, and L. donoVani than the 6-substituted
congeners, and 4′-substituted derivatives exhibited higher activi-
ties than 5′-substituted isomers. In our cytotoxicity study,
however, 5,5′-substituted dications displayed higher antiparasitic
selectivity than 5,4′-substituted congeners. Introduction of the
methoxy or hydroxy substituents in the 7- or 2′-position of select
2-phenylbenzofurans reduced their cytotoxicity, enhanced an-
tiprotozoal activity, or achieved both, providing derivatives with
higher selectivity against T. b. rhodesiense, P. falciparum, and

6938 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
A. After the purification was completed, the column was washed
with acetonitrile (3 × 100 mL), ethanol (100 mL), deionized water
(2 × 100 mL) and kept in acetonitrile or acetonitrile-water mixture.
Select fractions were analyzed for purity as described above for
analytical HPLC. Residues of evaporated pooled purified fractions
were reconstituted in water and lyophilized on a VirTis BenchTop
6K lyophilizer. The lyophilized compounds were dissolved in
ethanol and converted into HCl salts with aqueous HCl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[3-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]benzofuran Dihydrochloride (10). White solid
(0.28 g, 56%): mp 356-358 °C. 1H NMR (DMSO-d6) δ 11.04 (br
s, 4H), 8.90 (s, 1H), 8.54 (d, J ) 1.6 Hz, 1H), 8.31 (d, J ) 7.7 Hz,
1H), 8.15 (d, J ) 8.2 Hz, 1H), 8.08 (dd, J ) 8.2 and 1.6 Hz, 1H),
7.95 (d, J ) 8.2 Hz, 1H), 7.94 (s, 1H), 7.82 (dd, J ) 7.7 and 7.7
Hz, 1H), 4.06 (s, 4H), 4.04 (s, 4H). HPLC (method A) tR ) 5.27
min (100 area %). Anal. (C20H18N4O·2HCl·1H2O) C, H, N, Cl.
2-(4-Amidinophenyl)-6-amidinobenzofuran Dihydrochloride
(11).47 White solid (1.07 g, 62%): mp >355 °C (lit.47 365-370
°C). 1H NMR (DMSO-d6) δ 9.56 (br s, 4H), 9.34 (br s, 4H), 8.28
(s, 1H), 8.22 (d, J ) 8.2 Hz, 2H), 8.04 (d, J ) 8.2 Hz, 2H), 7.96
(d, J ) 8.2 Hz, 1H), 7.90 (s, 1H), 7.82 (dd, J ) 8.2 and 1.6 Hz,
1H). HPLC (method A) tR ) 4.02 min (100 area %). Anal.
(C16H14N4O·2HCl·1.2H2O) C, H, N, Cl.
Elemental analyses were performed by Atlantic Microlab,
Norcross, GA, and were within (0.4% of calculated values.
General Procedure for Syntheses of Diamidines (1-3, 8-49).
2-(4-Amidinophenyl)-5-amidinobenzofuran Dihydrochloride
(1).47 A mixture of dry 1,4-dioxane (100 mL) and dry EtOH (40
mL) in a three-neck 500 mL flask equipped with a gas inlet tube,
a thermometer, and a drying tube was saturated with gaseous HCl
at 0 °C. 2-(4-Cyanophenyl)benzofuran-5-carbonitrile (51) (5.00 g,
20.5 mmol) was added in one portion, the flask was sealed, and
the mixture was stirred at ambient temperature until the starting
material was no longer detectable by HPLC. The reaction mixture
was diluted with dry ether and a solid product was filtered off under
argon, washed with diethyl ether, and dried under high vacuum to
afford a diimidate ester (8.37 g, 100%), which was reacted
immediately with appropriate amines.
To a suspension of the diimidate ester (2.00 g, 4.89 mmol) in
dry EtOH (30 mL) was added dry EtOH saturated with gaseous
ammonia (20 mL), and the reaction mixture was stirred at ambient
temperature. The progress of the reaction was monitored by HPLC.
Upon completion, the reaction mixture was concentrated to give
crude product, which was purified by preparative HPLC to afford
1 as a white solid (1.14 g, 66%): mp >350 °C (lit.47 355-358
°C). 1H NMR (DMSO-d6) δ 9.55 (br s, 4H), 9.37 (br s, 4H), 8.30
(d, J ) 1.6 Hz, 1H), 8.21 (d, J ) 8.8 Hz, 2H), 8.05 (d, J ) 8.8 Hz,
2H), 7.94 (s, 1H), 7.93 (d, J ) 8.8 Hz, 1H), 7.87 (dd, J ) 8.8 and
1.6 Hz, 1H). HPLC (method A) tR ) 4.04 min (100 area %). Anal.
(C16H14N4O·2HCl·0.4H2O) C, H, N, Cl.
2-(4-N-Isopropylamidinophenyl)-5-N-isopropylamidinoben-
zofuran Dihydrochloride (2). White solid (0.67 g, 42%): mp
355-357 °C. 1H NMR (DMSO-d6) δ 9.77 (br s, 1H), 9.70 (d, J )
7.5 Hz, 1H), 9.63 (br s, 1H), 9.57 (br s, 1H), 9.30 (br s, 1H), 9.24
(br s, 1H), 8.19 (d, J ) 8.2 Hz, 1H), 8.18 (s, 1H), 8.17 (d, J ) 1.6
Hz, 1H), 7.92 (d, J ) 8.8 Hz, 4H), 7.74 (dd, J ) 8.2 and 1.6 Hz,
1H), 4.16 (m, 2H), 1.31 (d, J ) 6.3 Hz, 6H), 1.30 (d, J ) 6.3 Hz,
6H). HPLC (method A) tR ) 6.20 min (100 area %). Anal.
(C22H26N4O·2HCl·2.3H2O) C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]benzofuran Dihydrochloride (3).48 White solid
(1.12 g, 76%): mp >350 °C (lit.48 no melting point). 1H NMR
(DMSO-d6) δ 10.95 (s, 4H), 8.55 (d, J ) 1.6 Hz, 1H), 8.25 (s,
4H), 8.10 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.99 (s, 1H), 7.97 (d, J )
8.2 Hz, 1H), 4.03 (s, 8H). HPLC (method A) tR ) 5.19 min (100
area %). Anal. (C20H18N4O·2HCl·1H2O) C, H, N, Cl.
2-(4-N-Isopropylamidinophenyl)-6-N-isopropylamidinoben-
zofuran Dihydrochloride (12). White solid (0.27 g, 24%): mp
338-340 °C. 1H NMR (DMSO-d6) δ 9.71 (br s, 6H), 8.19 (d, J )
8.2 Hz, 2H), 8.17 (s, 1H), 7.93 (d, J ) 8.2 Hz, 3H), 7.88 (s, 1H),
7.69 (dd, J ) 8.2 and 1.6 Hz, 1H), 4.15 (m, 2H), 1.32 (d, J ) 6.3
Hz, 6H), 1.31 (d, J ) 6.3 Hz, 6H). HPLC (method A) tR ) 6.06
min (100 area %). Anal. (C22H26N4O·2HCl·0.9H2O) C, H, N, Cl.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]benzofuran Dihydrochloride (13). White solid
1
(0.51 g, 47%): mp >330 °C (dec). H NMR (DMSO-d6) δ 10.95
(s, 4H), 8.52 (s, 1H), 8.28 (d, J ) 8.2 Hz, 2H), 8.23 (d, J ) 8.2
Hz, 2H), 8.01 (d, J ) 8.2 Hz, 1H), 8.00 (d, J ) 8.2 Hz, 1H), 7.93
(s, 1H), 4.04 (s, 8H). HPLC (method A) tR ) 5.15 min (100 area
%). Anal. (C20H18N4O·2HCl·1.8H2O) C, H, N, Cl.
2-(3-Amidinophenyl)-6-amidinobenzofuran Dihydrochloride
(14). White solid (1.07 g, 62%): mp >355 °C. 1H NMR (DMSO-
d6) δ 9.51 (br s, 8H), 8.51 (br s, 1H), 8.32 (d, J ) 8.2 Hz, 1H),
8.27 (s, 1H), 7.97 (d, J ) 8.2 Hz, 1H), 7.94 (d, J ) 8.2 Hz, 1H),
7.84 (s, 1H), 7.81 (dd, J ) 8.2 and 8.2 Hz, 1H), 7.80 (dd, J ) 8.2
and 1.6 Hz, 1H). HPLC (method A) tR ) 4.08 min (100 area %).
Anal. (C16H14N4O·2HCl·0.3H2O) C, H, N, Cl.
2-(3-N-Isopropylamidinophenyl)-6-N-isopropylamidinoben-
zofuran Dihydrochloride (15). White solid (0.35 g, 41%): mp
1
325-327 °C. H NMR (DMSO-d6) δ 9.91 (d, J ) 7.5 Hz, 1H),
9.75 (d, 1H), 9.74 (s, 1H), 9.64 (s, 1H), 9.40 (s, 1H), 9.29 (s, 1H),
8.37 (dd, J ) 1.6 and 1.6 Hz, 1H), 8.28 (dd, J ) 7.7 and 1.6 Hz,
1H), 8.17 (s, 1H), 7.93 (d, J ) 8.2 Hz, 1H), 7.84 (s, 1H), 7.83 (dd,
J ) 7.7 and 1.6 Hz, 1H), 7.78 (dd, J ) 7.7 and 7.7 Hz, 1H), 7.69
(dd, J ) 8.2 and 1.6 Hz, 1H), 4.13 (m, 2H), 1.31 (d, J ) 6.3 Hz,
6H), 1.30 (d, J ) 6.3 Hz, 6H). HPLC (method A) tR ) 6.01 min
(100 area %). Anal. (C22H26N4O·2HCl·0.8H2O) C, H, N, Cl.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[3-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]benzofuran Dihydrochloride (16). White solid
1
(0.81 g, 74%): mp >330 °C (dec). H NMR (DMSO-d6) δ 11.11
(br s, 4H), 8.86 (s, 1H), 8.53 (s, 1H), 8.32 (d, J ) 7.7 Hz, 1H),
8.16 (d, J ) 7.7 Hz, 1H), 8.00 (d, J ) 8.2 Hz, 1H), 7.97 (d, J )
8.2 Hz, 1H), 7.86 (s, 1H), 7.82 (dd, J ) 7.7 and 7.7 Hz, 1H), 4.05
(s, 4H), 4.03 (s, 4H). HPLC (method A) tR ) 5.16 min (100 area
%). Anal. (C20H18N4O·2HCl·1.4H2O) C, H, N, Cl.
2-(3-Amidinophenyl)-5-amidinobenzofuran Dihydrochloride
(8). White solid (0.17 g, 33%): mp 294-296 °C. 1H NMR (DMSO-
d6) δ 9.70 (s, 2H), 9.53 (s, 2H), 9.46 (s, 2H), 9.33 (s, 2H), 8.52
(dd, J ) 1.6 and 1.6 Hz, 1H), 8.32 (d, J ) 7.7 Hz, 1H), 8.31 (s,
1H), 7.94 (d, J ) 8.8 Hz, 1H), 7.93 (d, J ) 7.7 Hz, 1H), 7.88 (s,
1H), 7.85 (dd, J ) 8.8 and 1.6 Hz, 1H), 7.79 (dd, J ) 7.7 and 7.7
Hz, 1H). HPLC (method A) tR ) 4.23 min (100 area %). Anal.
(C16H14N4O·2HCl·1.2H2O·0.1EtOH) C, H, N, Cl.
2-(4-Amidinophenyl)-5-amidino-7-methoxybenzofuran Dihy-
drochloride (17). White solid (0.40 g, 54%): mp 303-305 °C. 1H
NMR (DMSO-d6) δ 9.57 (br s, 4H), 9.34 (br s, 2H), 9.29 (br s,
2H), 8.19 (d, J ) 8.2 Hz, 2H), 8.02 (d, J ) 8.2 Hz, 2H), 7.91 (s,
1H), 7.89 (s, 1H), 7.52 (s, 1H), 4.11 (s, 3H). HPLC (method A) tR
) 4.59 min (100 area %). Anal. (C17H16N4O2 ·1.9HCl·1.5H2O) C,
H, N, Cl.
2-(3-N-Isopropylamidinophenyl)-5-N-isopropylamidinoben-
zofuran Dihydrochloride (9). White solid (0.34 g, 53%): mp
265-268 °C. H NMR (DMSO-d6) δ 9.87 (d, J ) 7.5 Hz, 1H),
2-(4-N-Isopropylamidinophenyl)-5-N-isopropylamidino-7-
methoxybenzofuran Dihydrochloride (18). White solid (0.48 g,
1
1
54%): mp 330-331 °C (dec). H NMR (DMSO-d6) δ 9.73 (br s,
9.71 (s, 1H), 9.70 (d, J ) 7.5 Hz, 1H), 9.57 (s, 1H), 9.39 (s, 1H),
9.25 (s, 1H), 8.37 (s, 1H), 8.29 (dd, J ) 7.7 and 1.6 Hz, 1H), 8.18
(d, J ) 1.6 Hz, 1H), 7.92 (d, J ) 8.8 Hz, 1H), 7.87 (s, 1H), 7.82
(dd, J ) 7.7 and 1.6 Hz, 1H), 7.77 (dd, J ) 7.7 and 7.7 Hz, 1H),
7.73 (dd, J ) 8.8 and 1.6 Hz, 1H), 4.16 (m, 2H), 1.32 (d, J ) 6.3
Hz, 6H), 1.31 (d, J ) 6.3 Hz, 6H). HPLC (method A) tR ) 6.14
min (98.0 area %). Anal. (C22H26N4O·2HCl·0.9H2O) C, H, N, Cl.
2H), 9.59 (br s, 2H), 9.21 (br s, 2H), 8.17 (d, J ) 8.2 Hz, 2H),
7.91 (d, J ) 8.2 Hz, 2H), 7.88 (s, 1H), 7.74 (s, 1H), 7.39 (s, 1H),
4.11 (m, 2H), 4.09 (s, 3H), 1.31 (d, J ) 6.0 Hz, 6H), 1.30 (d, J )
6.0 Hz, 6H). HPLC (method A) tR ) 6.55 min (100 area %). Anal.
(C23H28N4O2 ·2HCl·0.4H2O) C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]-7-methoxybenzofuran Dihydrochloride (19).

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6939
1
White solid (0.68 g, 80%): mp >350 °C. H NMR (DMSO-d6) δ
10.84 (br s, 4H), 8.24 (d, J ) 8.2 Hz, 2H), 8.18 (d, J ) 8.2 Hz,
2H), 8.10 (s, 1H), 7.97 (s, 1H), 7.78 (s, 1H), 4.10 (s, 3H), 4.04 (s,
4H), 4.03 (s, 4H). HPLC (method A) tR ) 5.66 min (100 area %).
Anal. (C21H20N4O2 ·2HCl·1.7H2O) C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)-2-methoxyphenyl]benzofuran Dihydrochloride (28).
White solid (0.60 g, 70%): mp >350 °C (dec). 1H NMR (DMSO-
d6) δ 10.86 (br s, 4H), 8.48 (s, 1H), 8.18 (d, J ) 8.2 Hz, 1H), 8.03
(s, 1H), 8.02 (d, J ) 8.2 Hz, 1H), 7.93 (d, J ) 8.2 Hz, 1H), 7.81
(d, J ) 8.2 Hz, 1H), 7.73 (s, 1H), 4.15 (s, 3H), 4.04 (s, 8H). HPLC
(method A) tR ) 5.74 min (100 area %). Anal. (C21H20-
N4O2 ·2HCl·2H2O) C, H, N, Cl.
2-(5-Amidino-2-methoxyphenyl)-5-amidinobenzofuran Dihy-
drochloride (29). White solid (0.57 g, 78%): mp 252-254 °C (dec).
1H NMR (DMSO-d6) δ 9.49 (br s, 2H), 9.48 (br s, 2H), 9.27 (br s,
2H), 9.24 (br s, 2H), 8.47 (d, J ) 2.2 Hz, 1H), 8.27 (d, J ) 1.6 Hz,
1H), 7.98 (dd, J ) 8.8 and 2.2 Hz, 1H), 7.92 (d, J ) 8.2 Hz, 1H),
7.83 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.66 (s, 1H), 7.49 (d, J ) 8.8
Hz, 1H), 4.14 (s, 3H). HPLC (method A) tR ) 4.52 min (100 area
%). Anal. (C17H16N4O2 ·2HCl·1.3H2O) C, H, N, Cl.
2-(5-N-Isopropylamidino-2-methoxyphenyl)-5-N-isopropyla-
midinobenzofuran Dihydrochloride (30). White solid (0.48 g,
54%): mp 240-242 °C (dec). 1H NMR (DMSO-d6) δ 9.68 (d, J )
8.0 Hz, 2H), 9.54 (d, J ) 8.2 Hz, 2H), 9.20 (d, J ) 8.5 Hz, 2H),
8.34 (d, J ) 2.2 Hz, 1H), 8.16 (d, J ) 1.6 Hz, 1H), 7.91 (d, J )
8.2 Hz, 1H), 7.86 (dd, J ) 8.8 and 2.2 Hz, 1H), 7.72 (dd, J ) 8.2
and 1.6 Hz, 1H), 7.67 (s, 1H), 7.47 (d, J ) 8.8 Hz, 1H), 4.13 (s,
3H), 4.12 (m, 2H), 1.31 (d, J ) 6.0 Hz, 6H), 1.30 (d, J ) 6.0 Hz,
6H). HPLC (method A) tR ) 6.65 min (100 area %). Anal.
(C23H28N4O2 ·2HCl·2.1H2O) C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[5-(4,5-dihydro-1H-imid-
azol-2-yl)-2-methoxyphenyl]benzofuran Dihydrochloride (31).
White solid (0.55 g, 67%): mp 329-330 °C (dec). 1H NMR
(DMSO-d6) δ 10.68 (br s, 4H), 8.63 (d, J ) 2.2 Hz, 1H), 8.43 (d,
J ) 1.6 Hz, 1H), 8.12 (dd, J ) 8.8 and 2.2 Hz, 1H), 7.99 (dd, J )
8.8 and 1.6 Hz, 1H), 7.93 (d, J ) 8.8 Hz, 1H), 7.68 (s, 1H), 7.56
(d, J ) 8.8 Hz, 1H), 4.16 (s, 3H), 4.04 (s, 4H), 4.02 (s, 4H). HPLC
(method A) tR ) 5.66 min (100 area %). Anal. (C21H20N4-
O2 ·2HCl·0.9H2O) C, H, N, Cl.
2-(4-Amidino-2-hydroxyphenyl)-5-amidinobenzofuran Dihy-
drochloride (32). White solid (0.41 g, 56%): mp 346-347 °C (dec).
1H NMR (DMSO-d6) δ 11.67 (br s, 1H), 9.47 (br s, 4H), 9.26 (br
s, 4H), 8.28 (d, J ) 1.6 Hz, 1H), 8.08 (d, J ) 8.2 Hz, 1H), 7.90 (d,
J ) 8.8 Hz, 1H), 7.81 (dd, J ) 8.8 and 1.6 Hz, 1H), 7.74 (s, 1H),
7.53 (d, J ) 1.1 Hz, 1H), 7.39 (dd, J ) 8.2 and 1.1 Hz, 1H). HPLC
(method A) tR ) 4.22 min (100 area %). Anal. (C16H14N4-
O2 ·2HCl·1.9H2O) C, H, N, Cl.
2-(2-Hydroxy-4-N-Isopropylamidinophenyl)-5-N-isopropyla-
midinobenzofuran Dihydrochloride (33). White solid (0.65 g,
74%): mp 317-318 °C (dec). 1H NMR (DMSO-d6) δ 11.67 (br s,
1H), 9.70 (d, J ) 8.2 Hz, 1H), 9.67 (d, J ) 7.7 Hz, 1H), 9.51 (br
s, 2H), 9.16 (br s, 2H), 8.15 (d, J ) 1.6 Hz, 1H), 8.06 (d, J ) 8.2
Hz, 1H), 7.88 (d, J ) 8.8 Hz, 1H), 7.72 (s, 1H), 7.70 (dd, J ) 8.8
and 1.6 Hz, 1H), 7.48 (d, J ) 1.1 Hz, 1H), 7.30 (dd, J ) 8.2 and
1.1 Hz, 1H), 4.10 (m, 2H), 1.30 (d, J ) 6.0 Hz, 6H), 1.29 (d, J )
6.0 Hz, 6H). HPLC (method A) tR ) 6.40 min (100 area %). Anal.
(C22H26N4O2 ·2HCl·2.2H2O) C, H, N, Cl.
2-(4-Amidinophenyl)-5-amidinobenzofuran-7-ol Dihydrochlo-
ride (20). White solid (0.23 g, 40%): mp >350 °C (dec). 1H NMR
(DMSO-d6) δ 11.12 (br s, 1H), 9.55 (br s, 2H), 9.38 (br s, 4H),
9.20 (br s, 2H), 8.20 (d, J ) 8.2 Hz, 2H), 8.04 (d, J ) 8.2 Hz, 2H),
7.85 (s, 1H), 7.66 (s, 1H), 7.26 (s, 1H). HPLC (method A) tR
3.90 min (100 area %). Anal. (C16H14N4O2 ·2.1HCl ·0.6H2O) C, H,
)
N, Cl.
2-(4-N-Isopropylamidinophenyl)-5-N-isopropylamidinoben-
zofuran-7-ol Dihydrochloride (21). White solid (0.20 g, 28%):
mp 321-323 °C (dec). H NMR (DMSO-d6) δ 11.14 (br s, 1H),
9.74 (d, J ) 7.7 Hz, 1H), 9.59 (d, J ) 7.0 Hz, 2H), 9.43 (br s, 1H),
9.24 (br s, 1H), 9.09 (br s, 1H), 8.17 (d, J ) 8.2 Hz, 2H), 7.92 (d,
J ) 8.2 Hz, 2H), 7.83 (s, 1H), 7.54 (d, J ) 1.6 Hz, 1H), 7.17
(d, J ) 1.6 Hz, 1H), 4.10 (m, 2H), 1.30 (d, J ) 6.0 Hz, 6H), 1.29
(d, J ) 6.0 Hz, 6H). HPLC (method A) tR ) 5.77 min (100 area
%). Anal. (C22H26N4O2 ·2HCl·2H2O) C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)phenyl]benzofuran-7-ol Dihydrochloride (22). White
solid (0.30 g, 47%): mp 329-330 °C (dec). 1H NMR (DMSO-d6)
δ 11.13 (br s, 1H), 10.77 (br s, 4H), 8.23 (br s, 4H), 7.88 (s, 2H),
7.39 (s, 1H), 4.02 (s, 4H), 3.99 (s, 4H). HPLC (method A) tR
5.03 min (100 area %). Anal. (C20H18N4O2 ·2HCl·1.4H2O) C, H,
1
)
N, Cl.
2-(4-Amidino-2-hydroxyphenyl)-5-amidinobenzofuran-7-ol Di-
hydrochloride (23). White solid (0.46 g, 44%): mp 333-335 °C
(dec). H NMR (DMSO-d6) δ 9.60-8.60 (br s, 8H), 8.11 (d, J )
8.2 Hz, 1H), 7.68 (s, 1H), 7.67 (d, J ) 1.6 Hz, 1H), 7.50 (d, J )
1.6 Hz, 1H), 7.40 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.21 (d, J ) 1.6
Hz, 1H). HPLC (method A) tR ) 4.08 min (100 area %). Anal.
(C16H14N4O3 ·2HCl·1.6H2O) C, H, N, Cl.
2-(2-Hydroxy-4-N-Isopropylamidinophenyl)-5-N-isopropyla-
midinobenzofuran-7-ol Dihydrochloride (24). Yellow solid (0.52
g, 41%): mp 319-320 °C. H NMR (DMSO-d6) δ 11.61 (s, 1H),
11.04 (s, 1H), 9.70 (d, J ) 7.7 Hz, 1H), 9.57 (d, J ) 8.0 Hz, 1H),
9.52 (s, 1H), 9.41 (s, 1H), 9.19 (s, 1H), 9.08 (s, 1H), 8.08 (d, J )
8.2 Hz, 1H), 7.65 (s, 1H), 7.55 (d, J ) 1.6 Hz, 1H), 7.46 (d, J )
1.6 Hz, 1H), 7.32 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.14 (d, J ) 1.6
Hz, 1H), 4.08 (m, 2H), 1.28 (d, J ) 6.0 Hz, 12H). HPLC (method
A) tR ) 5.92 min (100 area %). Anal. (C22H26N4O3 ·2HCl·2H2O)
C, H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)-2-hydroxyphenyl]benzofuran-7-ol Dihydrochloride
(25). Yellow solid (0.65 g, 55%): mp >360 °C (dec). H NMR
(DMSO-d6) δ 8.17 (d, J ) 8.2 Hz, 1H), 7.91 (d, J ) 1.6 Hz, 1H),
7.68 (s, 1H), 7.65 (d, J ) 1.6 Hz, 1H), 7.61 (dd, J ) 8.2 and 1.6
Hz, 1H), 7.40 (d, J ) 1.6 Hz, 1H), 4.00 (s, 8H). HPLC (method
A) tR ) 5.19 min (100 area %). Anal. (C20H18N4O3 ·2HCl ·1.7H2O)
C, H, N, Cl.
2-(4-Amidino-2-methoxyphenyl)-5-amidinobenzofuran Dihy-
drochloride (26). White solid (0.50 g, 65%): mp 337-338 °C (dec).
1H NMR (DMSO-d6) δ 9.70-9.15 (br s, 8H), 8.26 (d, J ) 1.6 Hz,
1H), 8.16 (d, J ) 8.2 Hz, 1H), 7.91 (d, J ) 8.2 Hz, 1H), 7.83 (dd,
J ) 8.2 and 1.6 Hz, 1H), 7.74 (s, 1H), 7.73 (d, J ) 1.1 Hz, 1H),
7.64 (dd, J ) 8.2 and 1.1 Hz, 1H), 4.16 (s, 3H). HPLC (method
A) tR ) 4.57 min (100 area %). Anal. (C17H16N4O2 ·2HCl ·0.2H2O)
C, H, N, Cl.
2-(4-N-Isopropylamidino-2-methoxyphenyl)-5-N-isopropyla-
midinobenzofuran Dihydrochloride (27). White solid (0.80 g,
87%): mp 325-326 °C (dec). H NMR (DMSO-d6) δ 9.90-9.12
(br s, 6H), 8.16 (d, J ) 1.6 Hz, 1H), 8.14 (d, J ) 8.2 Hz, 1H), 7.89
(d, J ) 8.2 Hz, 1H), 7.73 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.72 (s,
1H), 7.65 (d, J ) 1.1 Hz, 1H), 7.53 (dd, J ) 8.2 and 1.1 Hz, 1H),
4.17 (s, 3H), 4.16 (m, 2H), 1.32 (d, J ) 6.0 Hz, 6H), 1.31 (d, J )
6.0 Hz, 6H). HPLC (method A) tR ) 6.79 min (100 area %). Anal.
(C23H28N4O2 ·2HCl·1.1H2O) C, H, N, Cl.
1
1
1
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)-2-hydroxyphenyl]benzofuran Dihydrochloride (34).
1
White solid (0.57 g, 68%): mp >360 °C. H NMR (DMSO-d6) δ
11.78 (br s, 1H), 10.83 (br s, 4H), 8.52 (d, J ) 1.6 Hz, 1H), 8.10
(d, J ) 8.2 Hz, 1H), 8.05 (dd, J ) 8.8 and 1.6 Hz, 1H), 7.94 (d, J
) 8.8 Hz, 1H), 7.74 (s, 1H), 7.64 (s, 1H), 7.57 (d, J ) 8.2 Hz,
1H), 4.03 (s, 4H), 4.01 (s, 4H). HPLC (method A) tR ) 5.36 min
(100 area %). Anal. (C20H18N4O2 ·2HCl·1H2O) C, H, N, Cl.
2-(5-Amidino-2-hydroxyphenyl)-5-amidinobenzofuran Dihy-
drochloride (35). White solid (0.30 g, 44%): mp 323-325 °C (dec).
1H NMR (DMSO-d6) δ 12.19 (br s, 1H), 9.46 (br s, 2H), 9.35 (br
s, 2H), 9.22 (br s, 2H), 9.08 (br s, 2H), 8.42 (d, J ) 2.2 Hz, 1H),
8.26 (d, J ) 1.6 Hz, 1H), 7.91 (d, J ) 8.2 Hz, 1H), 7.81 (d, J )
8.8 Hz, 1H), 7.80 (d, J ) 8.8 Hz, 1H), 7.68 (s, 1H), 7.38 (d, J )
8.2 Hz, 1H). HPLC (method A) tR ) 3.85 min (100 area %). Anal.
(C16H14N4O2 ·2.1HCl·2.4H2O) C, H, N, Cl.
1

6940 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
2-(2-Hydroxy-5-N-Isopropylamidinophenyl)-5-N-isopropyla-
midinobenzofuran Dihydrochloride (36). White solid (0.39 g,
45%): mp 273-275 °C (dec). 1H NMR (DMSO-d6) δ 12.11 (br s,
1H), 9.65 (d, J ) 8.2 Hz, 1H), 9.55 (d, J ) 8.2 Hz, 1H), 9.53 (br
s, 1H), 9.42 (br s, 1H), 9.21 (br s, 1H), 9.08 (br s, 1H), 8.27 (d, J
) 2.2 Hz, 1H), 8.15 (d, J ) 1.6 Hz, 1H), 7.89 (d, J ) 8.8 Hz, 1H),
7.72-7.65 (m, 2H), 7.67 (s, 1H), 7.40 (d, J ) 8.8 Hz, 1H), 4.13
(m, 2H), 1.30 (d, J ) 6.0 Hz, 12H). HPLC (method A) tR ) 6.00
min (100 area %). Anal. (C22H26N4O2 ·2HCl ·1.2H2O·0.6EtOH) C,
H, N, Cl.
5-(4,5-Dihydro-1H-imidazol-2-yl)-2-[5-(4,5-dihydro-1H-imid-
azol-2-yl)-2-hydroxyphenyl]benzofuran Dihydrochloride (37).
White solid (0.52 g, 67%): mp 278-280 °C (dec). 1H NMR
(DMSO-d6) δ 12.40 (br s, 1H), 11.00-10.50 (br s, 4H), 8.58 (d, J
) 2.2 Hz, 1H), 8.47 (d, J ) 1.6 Hz, 1H), 8.03 (dd, J ) 8.8 and 1.6
Hz, 1H), 8.00 (dd, J ) 8.2 and 2.2 Hz, 1H), 7.90 (d, J ) 8.2 Hz,
1H), 7.66 (s, 1H), 7.40 (d, J ) 8.8 Hz, 1H), 4.03 (s, 4H), 4.00 (s,
4H). HPLC (method A) tR ) 5.13 min (100 area %). Anal.
(C20H18N4O2 ·2HCl·1.8H2O) C, H, N, Cl.
2-(4-Amidino-2-hydroxyphenyl)-6-amidinobenzofuran Dihy-
drochloride (44). Light-yellow solid (0.31 g, 45%): mp >300 °C
(dec). 1H NMR (DMSO-d6) δ 11.70 (s, 1H), 9.49 (s, 4H), 9.27 (s,
4H), 8.23 (s, 1H), 8.09 (d, J ) 7.7 Hz, 1H), 7.95 (d, J ) 8.2 Hz,
1H), 7.78 (d, J ) 7.7 Hz, 1H), 7.56 (s, 1H), 7.52 (d, J ) 1.6 Hz,
1H), 7.39 (dd, J ) 8.2 and 1.6 Hz, 1H). HPLC (method A) tR )
4.08 min (100 area %). Anal. (C16H14N4O2 ·2HCl·2.2H2O) C, H,
N, Cl.
2-(2-Hydroxy-4-N-Isopropylamidinophenyl)-6-N-isopropyla-
midinobenzofuran Dihydrochloride (45). White solid (0.41 g,
48%): mp 258-261 °C. 1H NMR (DMSO-d6) δ 11.75 (s, 1H), 9.73
(d, J ) 8.2 Hz, 1H), 9.68 (d, J ) 8.2 Hz, 1H), 9.57 (s, 1H), 9.55
(s, 1H), 9.25 (s, 2H), 8.13 (s, 1H), 8.06 (d, J ) 8.2 Hz, 1H), 7.92
(d, J ) 8.2 Hz, 1H), 7.73 (s, 1H), 7.66 (d, J ) 8.2 Hz, 1H), 7.50
(s, 1H), 7.31 (d, J ) 8.2 Hz, 1H), 4.17 (m, 2H), 1.31 (d, J ) 6.0
Hz, 6H), 1.29 (d, J ) 6.0 Hz, 6H). HPLC (method A) tR ) 6.08
min (100 area %). Anal. (C22H26N4O2 ·2.1HCl·2.1H2O) C, H, N,
Cl.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)-2-hydroxyphenyl]benzofuran Dihydrochloride (46).
2-(4-Amidino-2-methoxyphenyl)-6-amidinobenzofuran Dihy-
drochloride (38). Light-yellow solid (0.39 g, 56%): mp >300 °C
1
Yellow solid (0.45 g, 57%): mp 255-257 °C. H NMR (DMSO-
1
(dec). H NMR (DMSO-d6) δ 9.66 (s, 2H), 9.52 (s, 2H), 9.38 (s,
d6) δ 11.76 (s, 1H), 10.76 (s, 2H), 10.70 (s, 2H), 8.41 (s, 1H), 8.15
(d, J ) 8.2 Hz, 1H), 8.00 (d, J ) 8.2 Hz, 1H), 7.92 (dd, J ) 8.2
and 1.1 Hz, 1H), 7.80 (s, 1H), 7.56 (d, J ) 1.1 Hz, 1H), 7.54 (dd,
J ) 8.2 and 1.1 Hz, 1H), 4.04 (s, 4H), 4.01 (s, 4H). HPLC (method
A) tR ) 5.21 min (98.0 area %). Anal. (C20H18N4O2 ·2.3HCl ·2H2O)
C, H, N, Cl.
2H), 9.30 (s, 2H), 8.24 (s, 1H), 8.16 (d, J ) 8.2 Hz, 1H), 7.95 (d,
J ) 8.2 Hz, 1H), 7.79 (d, J ) 8.2 Hz, 1H), 7.78 (s, 1H), 7.74 (s,
1H), 7.66 (d, J ) 8.2 Hz, 1H), 4.16 (s, 3H). HPLC (method A) tR
) 4.76 min (100 area %). Anal. (C17H16N4O2 ·2HCl·1.7H2O) C,
H, N, Cl.
2-(4-N-Isopropylamidino-2-methoxyphenyl)-6-N-isopropyla-
midinobenzofuran Dihydrochloride (39). White solid (0.46 g,
57%): mp >250 °C (dec). 1H NMR (DMSO-d6) δ 9.84 (d, J ) 7.7
Hz, 1H), 9.71 (s, 1H), 9.67 (d, J ) 8.2 Hz, 1H), 9.56 (s, 1H), 9.31
(s, 1H), 9.21 (s, 1H), 8.14 (d, J ) 8.2 Hz, 1H), 8.13 (s, 1H), 7.93
(d, J ) 8.2 Hz, 1H), 7.73 (s, 1H), 7.67 (d, J ) 8.2 Hz, 1H), 7.65
(s, 1H), 7.54 (d, J ) 8.2 Hz, 1H), 4.16 (s, 3H), 4.13 (m, 2H), 1.32
(d, J ) 6.0 Hz, 6H), 1.30 (d, J ) 6.0 Hz, 6H). HPLC (method A)
tR ) 6.82 min (98.7 area %). Anal. (C23H28N4O2 ·2HCl·1.7H2O)
C, H, N, Cl.
2-(5-Amidino-2-hydroxyphenyl)-6-amidinobenzofuran Dihy-
drochloride (47). White solid (0.31 g, 45%): mp >300 °C (dec).
1H NMR (DMSO-d6) δ 12.16 (s, 1H), 9.48 (s, 2H), 9.38 (s, 2H),
9.22 (s, 2H), 9.07 (s, 2H), 8.39 (s, 1H), 8.20 (s, 1H), 7.95 (d, J )
7.7 Hz, 1H), 7.80 (d, J ) 7.7 Hz, 1H), 7.77 (d, J ) 8.2 Hz, 1H),
7.69 (s, 1H), 7.35 (d, J ) 8.2 Hz, 1H). HPLC (method A) tR
)
4.09 min (100 area %). Anal. (C16H14N4O2 ·2HCl·2H2O) C, H, N,
Cl.
2-(2-Hydroxy-5-N-Isopropylamidinophenyl)-6-N-isopropyla-
midinobenzofuran Dihydrochloride (48). White solid (0.48 g,
1
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[4-(4,5-dihydro-1H-imid-
azol-2-yl)-2-methoxyphenyl]benzofuran Dihydrochloride (40).
57%): mp >275 °C (dec). H NMR (DMSO-d6) δ 12.12 (s, 1H),
9.69 (d, J ) 8.2 Hz, 1H), 9.62 (d, J ) 8.2 Hz, 1H), 9.59 (s, 1H),
9.46 (s, 1H), 9.23 (s, 1H), 9.10 (s, 1H), 8.25 (d, J ) 2.2 Hz, 1H),
8.13 (s, 1H), 7.92 (d, J ) 8.2 Hz, 1H), 7.69 (dd, J ) 7.7 and 2.2
Hz, 1H), 7.68 (s, 1H), 7.66 (d, J ) 7.7 Hz, 1H), 7.40 (d, J )
8.2 Hz, 1H), 4.13 (m, 2H), 1.32 (d, J ) 6.0 Hz, 6H), 1.30 (d, J )
6.0 Hz, 6H). HPLC (method A) tR ) 5.84 min (98.7 area %). Anal.
(C22H26N4O2 ·2.1HCl·1.5H2O) C, H, N, Cl.
1
Light-yellow solid (0.51 g, 65%): mp >255 °C (dec). H NMR
(DMSO-d6) δ 11.08 (s, 2H), 10.90 (s, 2H), 8.46 (s, 1H), 8.20 (d,
J ) 8.2 Hz, 1H), 8.03 (d, J ) 1.1 Hz, 1H), 7.99 (d, J ) 8.8 Hz,
1H), 7.97 (d, J ) 8.8 Hz, 1H), 7.83 (dd, J ) 8.2 and 1.1 Hz, 1H),
7.77 (s, 1H), 4.14 (s, 3H), 4.05 (s, 8H). HPLC (method A) tR
5.79 min (99.1 area %). Anal. (C21H20N4O2 ·2.4HCl·3.1H2O) C,
H, N, Cl.
)
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[5-(4,5-dihydro-1H-imid-
azol-2-yl)-2-hydroxyphenyl]benzofuran Dihydrochloride (49).
Tan solid (0.45 g, 57%): mp 270-273 °C. 1H NMR (DMSO-d6) δ
12.41 (s, 1H), 11.06 (s, 2H), 10.73 (s, 2H), 8.58 (d, J ) 1.6 Hz,
1H), 8.48 (s, 1H), 8.01 (dd, J ) 8.8 and 1.6 Hz, 1H), 7.98 (d, J )
8.8 Hz, 1H), 7.96 (d, J ) 8.8 Hz, 1H), 7.69 (s, 1H), 7.42 (d,
J ) 8.8 Hz, 1H), 4.03 (s, 4H), 4.00 (s, 4H). HPLC (method A) tR
) 5.24 min (98.5 area %). Anal. (C20H18N4O2 ·2.3HCl·2.6H2O)
C, H, N, Cl.
2-(5-Amidino-2-methoxyphenyl)-6-amidinobenzofuran Dihy-
drochloride (41). Purple solid (0.41 g, 59%): mp >230 °C (dec).
1H NMR (DMSO-d6) δ 9.55 (s, 2H), 9.53 (s, 2H), 9.32 (s, 2H),
9.26 (s, 2H), 8.46 (s, 1H), 8.24 (s, 1H), 8.00 (d, J ) 8.8 Hz, 1H),
7.95 (d, J ) 8.2 Hz, 1H), 7.79 (d, J ) 8.2 Hz, 1H), 7.68 (s, 1H),
7.50 (d, J ) 8.8 Hz, 1H), 4.14 (s, 3H). HPLC (method A) tR
)
5.03 min (100 area %). Anal. (C17H16N4O2 ·2.1HCl ·1.7H2O) C, H,
N, Cl.
2-(5-N-Isopropylamidino-2-methoxyphenyl)-6-N-isopropyla-
midinobenzofuran Dihydrochloride (42). White solid (0.35 g,
41%): mp >240 °C (dec). 1H NMR (DMSO-d6) δ 9.71 (br s, 2H),
9.57 (br s, 2H), 9.21 (s, 1H), 9.16 (s, 1H), 8.31 (d, J ) 1.6 Hz,
1H), 8.14 (s, 1H), 7.93 (d, J ) 8.2 Hz, 1H), 7.87 (dd, J ) 8.8 and
1.6 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J ) 8.2 Hz, 1H), 7.48 (d, J )
8.8 Hz, 1H), 4.13 (m, 2H), 4.12 (s, 3H), 1.31 (d, J ) 6.0 Hz, 12H).
HPLC (method A) tR ) 6.66 min (100 area %). Anal.
(C23H28N4O2 ·2.1HCl·2.6H2O) C, H, N, Cl.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-[5-(4,5-dihydro-1H-imid-
azol-2-yl)-2-methoxyphenyl]benzofuran Dihydrochloride (43).
Light-yellow solid (0.61 g, 77%): mp 252-255 °C. 1H NMR
(DMSO-d6) δ 11.08 (s, 2H), 10.88 (s, 2H), 8.64 (s, 1H), 8.50 (s,
1H), 8.21 (d, J ) 8.2 Hz, 1H), 8.00 (d, J ) 8.8 Hz, 1H), 7.95 (d,
J ) 8.2 Hz, 1H), 7.66 (s, 1H), 7.53 (d, J ) 8.8 Hz, 1H), 4.14 (s,
3H), 4.03 (s, 8H). HPLC (method A) tR ) 5.95 min (100 area %).
Anal. (C21H20N4O2 ·2HCl·2.5H2O) C, H, N, Cl.
5-(1H-Benzimidazolyl)-2-[4-(1H-benzimidazolyl)phenyl]ben-
zofuran Dihydrochloride (4). A mixture of an imidate ester (1.91
g, 4.66 mmol), prepared from 51 (1.20 g, 4.91 mmol) following
the procedure described above for 1, and 1,2-diaminobenzene (1.11
g, 10.3 mmol) in dry MeOH (100 mL) was refluxed overnight. A
precipitate formed, was separated, washed with ether, and dried to
give 4 as a yellow solid (1.75 g, 75%): mp >340 °C (dec). 1H
NMR (DMSO-d6) δ 8.57 (d, J ) 1.6 Hz, 1H), 8.36 (d, J ) 8.8 Hz,
2H), 8.24 (dd, J ) 8.8 and 1.6 Hz, 1H), 8.20 (d, J ) 8.8 Hz, 2H),
7.91 (d, J ) 8.8 Hz, 1H), 7.81 (s, 1H), 7.80-7.65 (m, 4H),
7.36-7.25 (m, 4H). HPLC (method A) tR ) 5.24 min (98.5 area
%). Anal. (C28H18N4O·2HCl·1.8H2O) C, H, N, Cl.
5-(1H-Imidazol-2-yl)-2-[4-(1H-imidazol-2-yl)phenyl]benzofu-
ran Dihydrochloride (5). To a suspension of 3 (free base) (2.38
g, 7.20 mmol) in dry DMSO (60 mL) was added o-iodoxylbenzoic
acid65 (8.05 g, 28.8 mmol), and the mixture was stirred at 50-60
°C for 48 h. Saturated aqueous Na2S2O3 (30 mL) was added upon

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6941
cooling, and the mixture was basified with 1 M KOH (50 mL),
diluted with iced water (300 mL), and stirred for 1 h. A precipitate
formed, was separated, washed with water, and dried. The crude
product was recrystallized from DMF and converted to a hydro-
chloride salt by recrystallization from 1 M HCl/EtOH to give 5 as
recrystallized from EtOH/DMF to afford 53 as a white solid (3.04
g, 85%): mp 300-301 °C. H NMR (DMSO-d6) δ 8.32 (d, J )
8.5 Hz, 2H), 8.13 (d, J ) 8.5 Hz, 2H), 7.92 (d, J ) 2.0 Hz, 1H),
7.65 (d, J ) 8.8 Hz, 1H), 7.60 (m, 2H), 7.57 (s, 1H), 7.50 (dd, J
) 8.8 and 2.0 Hz, 1H), 7.23 (m, 2H). HPLC (method B) tR ) 8.47
min (100 area %). Anal. (C21H13BrN2O·0.4H2O) C, H, N, Br.
1
1
a tan solid (1.33 g, 46%): mp >340 °C (dec). H NMR (DMSO-
d6) δ 8.61 (d, J ) 1.6 Hz, 1H), 8.37 (d, J ) 8.8 Hz, 2H), 8.25 (d,
J ) 8.8 Hz, 2H), 8.22 (dd, J ) 8.8 and 1.6 Hz, 1H), 7.97 (d, J )
8.8 Hz, 1H), 7.91 (s, 1H), 7.85 (s, 2H), 7.82 (s, 2H). HPLC (method
A) tR ) 5.82 min (98.2 area %). Anal. (C20H14N4O·2HCl·1.8H2O)
C, H, N, Cl.
General Procedure for the Copper-Mediated Heteroannula-
tion. 2-(4-Cyanophenyl)benzofuran-5-carbonitrile (51).47,48
A
mixture of methyl-4-hydroxy-3-iodobenzonitrile (56) (9.80 g, 40.0
mmol), 4-ethynylbenzonitrile (57) (6.00 g, 47.2 mmol), and
copper(I) oxide (3.43 g, 24.0 mmol) in dry pyridine (180 mL) was
refluxed overnight. The mixture was allowed to cool and concen-
trated. The residue was diluted with 1 M HCl (50 mL), and a brown
precipitate was filtered off and washed with water, 10% aqueous
ammonia, water, and EtOH. The product was purified by flash
chromatography, eluting with CHCl3 followed by recrystallization
from MeCN to afford 51 as a white solid (4.24 g, 43%): mp
230-232 °C. (lit.47 243-245 °C, lit.48 235-240 °C). 1H NMR
(DMSO-d6) δ 8.32 (d, J ) 1.6 Hz, 1H), 8.14 (d, J ) 8.2 Hz, 2H),
8.01 (d, J ) 8.2 Hz, 2H), 7.90 (d, J ) 8.8 Hz, 1H), 7.84 (dd, J )
8.8 and 1.6 Hz, 1H), 7.83 (s, 1H). HPLC (method B) tR ) 8.31
min (100 area %). Anal. (C16H8N2O) C, H, N.
2-(3-Cyanophenyl)benzofuran-5-carbonitrile (59). Purification
was by flash chromatography, eluting with hexanes/CHCl3, to give
59 as a white solid (1.50 g, 45%): mp 173-175 °C. 1H NMR
(DMSO-d6) δ 8.46 (dd, J ) 1.6 and 1.6 Hz, 1H), 8.31 (d, J ) 1.1
Hz, 1H), 8.28 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.93 (dd, J ) 8.2 and
1.1 Hz, 1H), 7.88 (d, J ) 8.2 Hz, 1H), 7.83 (dd, J ) 8.2 and 1.6
Hz, 1H), 7.78 (s, 1H), 7.76 (dd, J ) 8.2 and 8.2 Hz, 1H). HPLC
(method B) tR ) 8.38 min (100 area %). Anal. (C16H8N2-
O·0.1EtOH) C, H, N.
5-(1H-Benzimidazolyl)-2-[4-(4,5-dihydro-1H-imidazol-2-yl)phe-
nyl]benzofuran Dihydrochloride (6). A mixture of 52 (2.60 g,
7.62 mmol) and copper(I) cyanide (2.05 g, 22.9 mmol) in dry DMF
(100 mL) was refluxed for 48 h under an Ar atmosphere. The
reaction mixture, after successive treatments with ethylenediamine
and sodium cyanide solutions, was filtered, and a solid residue was
washed with 15% aqueous NH3 (3 × 150 mL) and water. The dry
solid was recrystallized from DMF to give 5-cyano-2-[4-(4,5-
dihydro-1H-imidazol-2-yl)phenyl]benzofuran (54), which was con-
verted to an imidate ester following the procedure described above
for 1. A mixture of the imidate ester (1.15 g, 2.83 mmol) and 1,2-
diaminobenzene (0.34 g, 3.11 mmol) in dry MeOH (60 mL) was
refluxed overnight. A precipitate formed, was separated, washed
with ether, and dried to give 6 as a tan solid (0.39 g, 23%): mp
1
>350 °C (dec). H NMR (DMSO-d6) δ 10.84 (br s, 2H), 8.78 (d,
J ) 1.6 Hz, 1H), 8.40 (dd, J ) 8.8 and 1.6 Hz, 1H), 8.25 (d, J )
8.8 Hz, 2H), 8.20 (d, J ) 8.8 Hz, 2H), 8.03 (d, J ) 8.8 Hz, 1H),
8.01 (s, 1H), 7.90-7.80 (m, 2H), 7.60-7.45 (m, 2H), 4.03 (s, 4H).
HPLC (method A) tR ) 7.78 min (98.5 area %). Anal.
(C24H18N4O·2HCl·1.7H2O) C, H, N, Cl.
2-[4-(1H-Benzimidazolyl)phenyl]-5-(4,5-dihydro-1H-imidazol-
2-yl)benzofuran Dihydrochloride (7). A mixture of 53 (2.51 g,
6.45 mmol) and copper(I) cyanide (1.73 g, 19.3 mmol) in dry DMF
(120 mL) was refluxed for 48 h under an Ar atmosphere. The
reaction mixture, after successive treatments with ethylenediamine
and sodium cyanide solutions, was filtered, and a solid residue was
washed with 15% aqueous NH3 (3 × 150 mL) and water. The dry
solid was recrystallized from DMF to give 5-cyano-2-[4-(1H-
benzimidazolyl)phenyl]benzofuran (55), which was converted to
an imidate ester following the procedure described above for 1. A
mixture of the imidate ester (1.41 g, 3.10 mmol) and ethylenedi-
amine (2 mL) in dry EtOH (50 mL) was stirred at ambient
temperature for 48 h. The reaction mixture was concentrated and
the residue was purified by preparative HPLC to give 7 as a tan
Methyl-2-(4-(methoxycarbonyl)phenyl)benzofuran-6-carbox-
ylate (63). Purification was by flash chromatography, eluting with
CH2Cl2, to give 63 as a white solid (11.0 g, 71%): mp 200-202
°C. 1H NMR (DMSO-d6) δ 8.21 (s, 1H), 8.13 (s, 2H), 8.12 (s,
2H), 7.92 (dd, J ) 8.2 and 1.1 Hz, 1H), 7.85 (d, J ) 8.2 Hz, 1H),
7.78 (s, 1H), 3.91 (s, 3H), 3.90 (s, 3H). HPLC (method B) tR
)
9.58 min (100 area %). Anal. (C18H14O5) C, H.
Methyl-2-(3-(methoxycarbonyl)phenyl)benzofuran-6-carbox-
ylate (64). Purification was by flash chromatography, eluting with
hexanes/CH2Cl2, to give 64 as a white solid (5.30 g, 69%): mp
1
159-161 °C. H NMR (DMSO-d6) δ 8.49 (dd, J ) 1.6 and 1.6
Hz, 1H), 8.26 (dd, J ) 8.2 and 1.6 Hz, 1H), 8.22 (s, 1H), 8.03 (dd,
J ) 8.2 and 1.6 Hz, 1H), 7.90 (dd, J ) 8.2 and 1.1 Hz, 1H), 7.81
(d, J ) 8.2 Hz, 1H), 7.73 (d, J ) 1.1 Hz, 1H), 7.71 (dd, J ) 8.2
and 8.2 Hz, 1H), 3.93 (s, 3H), 3.90 (s, 3H). HPLC (method B) tR
) 9.59 min (100 area %). Anal. (C18H14O5) C, H.
1
solid (0.12 g, 4%): mp >340 °C (dec). H NMR (DMSO-d6) δ
10.72 (s, 4H), 8.47 (d, J ) 8.8 Hz, 2H), 8.46 (s, 1H), 8.29 (d, J )
8.8 Hz, 2H), 8.00 (s, 2H), 7.93 (s, 1H), 7.80 (m, 2H), 7.48 (m,
2H), 4.04 (s, 4H). HPLC (method A) tR ) 8.29 min (100 area %).
Anal. (C24H18N4O·2HCl·3H2O) C, H, N, Cl.
2-(4-Cyanophenyl)-5-formyl-7-methoxybenzofuran (69). Pu-
rification was by flash chromatography, eluting with hexanes/
CH2Cl2, to give 69 as a white solid (5.00 g, 69%): mp 198-200
°C (CHCl3/EtOH). 1H NMR (DMSO-d6) δ 10.04 (s, 1H), 8.11 (d,
J ) 8.8 Hz, 2H), 7.99 (d, J ) 8.8 Hz, 2H), 7.96 (d, J ) 1.1 Hz,
1H), 7.88 (s, 1H), 7.46 (d, J ) 1.1 Hz, 1H), 4.06 (s, 3H). HPLC
(method B) tR ) 8.09 min (100 area %). Anal. (C17H11NO3 ·0.8H2O)
C, H, N.
5-Bromo-2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]benzofu-
ran (52). A mixture of an imidate ester (3.48 g, 9.14 mmol),
prepared from 5-bromo-2-(4-cyanophenyl)benzofuran 50 following
the procedure described above for 1, and ethylenediamine (5 mL)
in dry EtOH (100 mL) was stirred at ambient temperature overnight
and then refluxed for 2 h. A precipitate formed, was separated,
rinsed with ether, and dried. The residue was washed with 1 M
NaOH (50 mL), water, EtOH, and dried to give crude product,
which was recrystallized from EtOH/DMF to afford 52 as a white
solid (2.93 g, 94%): mp 298-300 °C. 1H NMR (DMSO-d6) δ 7.98
(d, J ) 8.8 Hz, 2H), 7.95 (d, J ) 8.8 Hz, 2H), 7.90 (d, J ) 1.9 Hz,
1H), 7.63 (d, J ) 8.8 Hz, 1H), 7.52 (s, 1H), 7.49 (dd, J ) 8.8 and
1.9 Hz, 1H), 7.05 (br s, 1H), 3.63 (s, 4H). HPLC (method B) tR )
4.87 min (100 area %). Anal. (C17H13BrN2O) C, H, N, Br.
5-Bromo-2-[4-(1H-benzimidazolyl)phenyl]benzofuran (53). A
mixture of an imidate ester (3.48 g, 9.14 mmol), prepared from 50
following the procedure described above for 1, and 1,2-diami-
nobenzene (1.10 g, 10.2 mmol) in dry MeOH (100 mL) was
refluxed overnight. A precipitate formed, was separated, rinsed with
ether, and dried. The residue was washed with 1 M NaOH (50
mL), water, EtOH and dried to give crude product, which was
2-(4-Cyano-2-methoxyphenyl)-5-formyl-7-methoxybenzofu-
ran (70). Purification was by flash chromatography, eluting with
hexanes/CH2Cl2, to give 70 as a white solid (6.63 g, 80%): mp
1
310-312 °C (DMF). H NMR (DMSO-d6) δ 10.03 (s, 1H), 8.07
(d, J ) 8.2 Hz, 1H), 7.93 (s, 1H), 7.69 (s, 1H), 7.67 (s, 1H), 7.55
(d, J ) 8.2 Hz, 1H), 7.46 (s, 1H), 4.08 (s, 6H). HPLC (method B)
tR ) 8.78 min (100 area %). Anal. (C18H13NO4) C, H, N.
2-(4-Cyano-2-methoxyphenyl)benzofuran-5-carbonitrile (78).
1
Yellow solid (8.72 g, 92%): mp 282-283 °C (DMF). H NMR
(DMSO-d6) δ 8.29 (d, J ) 1.6 Hz, 1H), 8.12 (d, J ) 8.2 Hz, 1H),
7.88 (d, J ) 8.2 Hz, 1H), 7.83 (dd, J ) 8.2 and 1.6 Hz, 1H), 7.75
(d, J ) 1.1 Hz, 1H), 7.68 (s, 1H), 7.61 (dd, J ) 8.2 and 1.1 Hz,
1H), 4.08 (s, 3H). HPLC (method B) tR ) 8.86 min (100 area %).
Anal. (C17H10N2O2) C, H, N.

6942 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
2-(5-Cyano-2-methoxyphenyl)benzofuran-5-carbonitrile (79).
Yellow solid (8.72 g, 92%): mp 220-222 °C (DMF). H NMR
(DMSO-d6) δ 8.29 (d, J ) 1.6 Hz, 1H), 8.24 (s, 1H), 7.94 (dd, J
) 8.2 and 1.6 Hz, 1H), 7.84 (d, J ) 8.8 Hz, 1H), 7.80 (dd, J ) 8.2
and 1.6 Hz, 1H), 7.56 (s, 1H), 7.43 (d, J ) 8.8 Hz, 1H), 4.10 (s,
3H). HPLC (method B) tR ) 8.57 min (100 area %). Anal.
(C17H10N2O2) C, H, N.
2-(4-Cyano-2-methoxyphenyl)-5-formyl-7-methoxybenzofu-
ran Oxime (72). White solid (5.16 g, 80%): mp 255-257 °C. 1H
NMR (DMSO-d6) δ 11.17 (s, 1H), 8.21 (s, 1H), 8.05 (d, J ) 7.7
Hz, 1H), 7.69 (s, 1H), 7.59 (s, 1H), 7.56 (d, J ) 7.7 Hz, 1H), 7.46
(s, 1H), 7.27 (s, 1H), 4.06 (s, 3H), 4.00 (s, 3H). HPLC (method B)
tR ) 7.66 min (100 area %). Anal. (C18H14N2O4 ·0.5H2O) C, H, N.
General Procedure for Synthesis of Dinitriles (73, 74).
2-(4-Cyanophenyl)-7-methoxybenzofuran-5-carbonitrile (73). A
mixture of 71 (4.00 g, 13.7 mmol) in acetic anhydride (100 mL)
was refluxed overnight. The reaction mixture was cooled, diluted
with water (100 mL), and stirred for 1 h at ambient temperature. A
precipitate formed, was separated, washed with water, dried, and
recrystallized from CHCl3 to yield 73 as a tan solid (3.35 g, 90%):
mp 251-253 °C. 1H NMR (DMSO-d6) δ 8.10 (d, J ) 8.8 Hz,
2H), 7.98 (d, J ) 8.8 Hz, 2H), 7.87 (d, J ) 1.1 Hz, 1H), 7.78 (s,
1H), 7.47 (d, J ) 1.1 Hz, 1H), 4.04 (s, 3H). HPLC (method B) tR
) 8.48 min (100 area %). Anal. (C17H10N2O2 ·0.1H2O) C, H, N.
2-(4-Cyano-2-methoxyphenyl)-7-methoxybenzofuran-5-carbo-
nitrile (74). Tan solid (7.15 g, 84%): mp 325-326 °C (DMF). 1H
NMR (DMSO-d6) δ 8.07 (d, J ) 8.2 Hz, 1H), 7.84 (s, 1H), 7.70
(s, 1H), 7.63 (s, 1H), 7.57 (d, J ) 8.2 Hz, 1H), 7.43 (s, 1H), 4.08
(s, 3H), 4.05 (s, 3H). HPLC (method B) tR ) 9.09 min (100 area
%). Anal. (C18H12N2O3) C, H, N.
1
2-(4-Cyano-2-methoxyphenyl)benzofuran-6-carbonitrile (80).
Purification was by flash chromatography, eluting with CHCl3, to
give 80 as a yellow solid (3.79 g, 41%): mp 256-258 °C (CHCl3).
1H NMR (DMSO-d6) δ 8.26 (s, 1H), 8.09 (d, J ) 7.7 Hz, 1H),
7.90 (d, J ) 8.2 Hz, 1H), 7.74 (s, 1H), 7.70 (d, J ) 7.7 Hz, 1H),
7.69 (s, 1H), 7.60 (d, J ) 8.2 Hz, 1H), 4.08 (s, 3H). HPLC (method
B) tR ) 8.81 min (100 area %). Anal. (C17H10N2O2) C, H, N.
2-(5-Cyano-2-methoxyphenyl)benzofuran-6-carbonitrile (81).
Purification was by flash chromatography, eluting with CHCl3, to
give 81 as a yellow solid (5.10 g, 57%): mp 197-198 °C (EtOAc).
1H NMR (DMSO-d6) δ 8.30 (s, 1H), 8.24 (s, 1H), 7.96 (d, J ) 8.8
Hz, 1H), 7.89 (d, J ) 8.2 Hz, 1H), 7.70 (d, J ) 8.2 Hz, 1H), 7.62
(s, 1H), 7.44 (d, J ) 8.8 Hz, 1H), 4.10 (s, 3H). HPLC (method B)
tR ) 8.53 min (100 area %). Anal. (C17H10N2O2) C, H, N.
2-(4-Cyanophenyl)benzofuran-6-carbonitrile (65).47 Method
1. Conversion of Benzofuran Esters to Dinitriles with Dimethy-
laluminum Amide. Anhydrous NH3 was bubbled through dry
o-xylene (280 mL) for 20 min at 0 °C. A 2.0 M solution of AlMe3
in toluene (120 mL, 240 mmol) was added, and the NH3 was passed
through the mixture for 20 min. Then the solution was stirred at
room temperature for 1 h. Methyl-2-(4-(methoxycarbonyl)phenyl)-
benzofuran-6-carboxylate (63) (16.4 g 52.8 mmol) was added in
one portion. The mixture was stirred at 100-110 °C for 3 h, allowed
to cool to ambient temperature, and diluted with CHCl3 (400 mL).
Water (100 mL) was added dropwise with vigorous stirring, and a
formed precipitate was filtered off. The filtrate was concentrated
to give crude product, which was purified by flash chromatography,
eluting with hexanes/CH2Cl2, to give 65 as a white solid (3.70 g,
General Procedure for the Demethylation of 2-Phenyl Ben-
zofurans 75, 76, 82-85. 2-(4-Cyanophenyl)-7-hydroxybenzofu-
ran-5-carbonitrile (75). To molten pyridine hydrochloride (12 g)
was added 73 (2.00 g, 7.29 mmol), and the reaction mixture was
kept at 160-180 °C for 3 h, cooled to 80-90 °C, and diluted with
water (100 mL). A precipitate formed, was separated, washed with
water, and dried to give 75 as a gray solid (1.58 g, 83%): mp
1
330-332 °C (DMF). H NMR (DMSO-d6) δ 11.09 (s, 1H), 8.14
(d, J ) 8.8 Hz, 2H), 8.02 (d, J ) 8.8 Hz, 2H), 7.77 (s, 1H), 7.73
(d, J ) 1.6 Hz, 1H), 7.12 (d, J ) 1.6 Hz, 1H). HPLC (method B)
tR ) 6.85 min (100 area %). Anal. (C16H8N2O2 ·0.1H2O) C, H, N.
2-(4-Cyano-2-hydroxyphenyl)-7-hydroxybenzofuran-5-carbo-
nitrile (76). Gray solid (2.83 g, 78%): mp >360 °C (EtOH/DMF).
1H NMR (DMSO-d6) δ 11.49 (s, 1H), 11.00 (br s, 1H), 8.06 (d, J
) 8.2 Hz, 1H), 7.71 (d, J ) 1.1 Hz, 1H), 7.64 (s, 1H), 7.45 (dd, J
) 8.2 and 1.1 Hz, 1H), 7.35 (d, J ) 1.6 Hz, 1H), 7.08 (d, J ) 1.6
Hz, 1H). HPLC (method B) tR ) 5.99 min (100 area %). Anal.
(C16H8N2O3 ·0.4H2O) C, H, N.
1
29%): mp 243-245 °C (acetone) (lit.47 221-223 °C). H NMR
(DMSO-d6) δ 8.30 (br s, 1H), 8.14 (d, J ) 8.2 Hz, 2H), 8.02 (d, J
) 8.2 Hz, 2H), 7.90 (d, J ) 8.2 Hz, 1H), 7.85 (s, 1H), 7.72 (dd, J
) 8.2 and 1.6 Hz, 1H). HPLC (method B) tR ) 8.38 min (100 area
%). Anal. (C16H8N2O) C, H, N.
Method 2. Copper-Mediated Heteroannulation. Following the
procedure described above for 51, 65 was prepared from 3-hydroxy-
4-iodobenzonitrile (67) (6.00 g, 24.5 mmol), 57 (3.74 g, 29.4 mmol),
and Cu2O (2.10 g, 14.7 mmol) in pyridine (120 mL). Crude product
was purified by flash chromatography, eluting with hexanes/CH2Cl2,
to give 65 as a white solid (4.57 g, 76%).
2-(4-Cyano-2-hydroxyphenyl)benzofuran-5-carbonitrile (82).
Gray solid (3.19 g, 84%): mp 302-303 °C (EtOH/DMF). 1H NMR
(DMSO-d6) δ 11.51 (s, 1H), 8.28 (d, J ) 1.6 Hz, 1H), 8.02 (d, J
) 8.2 Hz, 1H), 7.85 (d, J ) 8.2 Hz, 1H), 7.80 (dd, J ) 8.2 and 1.6
Hz, 1H), 7.70 (s, 1H), 7.43 (dd, J ) 8.2 and 1.1 Hz, 1H), 7.36 (d,
J ) 1.1 Hz, 1H). HPLC (method B) tR ) 7.54 min (100 area %).
Anal. (C16H8N2O2) C, H, N.
2-(3-Cyanophenyl)benzofuran-6-carbonitrile (66). Following
the procedure described above for 65 in method 1, 66 was prepared
from 64 (15.5 g, 50.0 mmol) as a white solid (3.65 g, 30%): mp
2-(5-Cyano-2-hydroxyphenyl)benzofuran-5-carbonitrile (83).
1
1
204-205 °C (acetone). H NMR (DMSO-d6) δ 8.45 (d, J ) 1.6
White solid (3.21 g, 86%): mp 257-260 °C (EtOAc). H NMR
Hz, 1H), 8.28 (s, 1H), 8.27 (d, J ) 8.2 Hz, 1H), 7.93 (d, J ) 7.7
Hz, 1H), 7.91 (d, J ) 7.7 Hz, 1H), 7.79 (s, 1H), 7.76 (dd, J ) 8.2
and 8.2 Hz, 1H), 7.71 (dd, J ) 8.2 and 1.6 Hz, 1H). HPLC (method
B) tR ) 8.40 min (100 area %). Anal. (C16H8N2O) C, H, N.
Following the procedure described above for 51, 66 was prepared
from 67 (6.00 g, 24.5 mmol), 58 (3.74 g, 29.4 mmol), and Cu2O
(2.10 g, 14.7 mmol) in pyridine (120 mL). Crude product was
purified by flash chromatography, eluting with hexanes/CH2Cl2, to
give 66 as a white solid (3.53 g, 59%).
General Procedure for Synthesis of Oximes (71, 72). 2-(4-
Cyanophenyl)-5-formyl-7-methoxybenzofuran Oxime (71). A
mixture of 69 (4.28 g, 15.4 mmol) and NH2OH·HCl (1.18 g, 17.0
mmol) in dry Py (70 mL) was stirred at ambient temperature for
2 h. The mixture was diluted with iced water and stirred for 1 h. A
precipitate formed, was separated, washed with water, and dried.
Recrystallization was from CHCl3 to yield 71 as a white solid (3.56
g, 79%): mp 205-206 °C (CHCl3). 1H NMR (DMSO-d6) δ 11.19
(s, 1H), 8.23 (s, 1H), 8.08 (d, J ) 8.8 Hz, 2H), 7.97 (d, J ) 8.8
Hz, 2H), 7.73 (s, 1H), 7.46 (d, J ) 1.6 Hz, 1H), 7.29 (d, J ) 1.6
Hz, 1H), 4.01 (s, 3H). HPLC (method B) tR ) 7.11 min (100 area
%). Anal. (C17H12N2O3) C, H, N.
(DMSO-d6) δ 11.93 (s, 1H), 8.26 (s, 1H), 8.24 (d, J ) 1.6 Hz,
1H), 7.84 (d, J ) 8.2 Hz, 1H), 7.78 (dd, J ) 8.2 and 1.6 Hz, 1H),
7.75 (dd, J ) 8.2 and 1.1 Hz, 1H), 7.59 (s, 1H), 7.19 (d, J ) 8.2
Hz, 1H). HPLC (method B) tR ) 7.13 min (100 area %). Anal.
(C16H8N2O2 ·0.2 EtOAc) C, H, N.
2-(4-Cyano-2-hydroxyphenyl)benzofuran-6-carbonitrile (84).
White solid (1.52 g, 80%): mp >300 °C (EtOH). 1H NMR (DMSO-
d6) δ 11.57 (s, 1H), 8.26 (s, 1H), 8.05 (d, J ) 8.2 Hz, 1H), 7.92 (d,
J ) 8.2 Hz, 1H), 7.74 (s, 1H), 7.71 (d, J ) 8.2 Hz, 1H), 7.44 (d,
J ) 8.2 Hz, 1H), 7.36 (s, 1H). HPLC (method B) tR ) 7.44 min
(100 area %). Anal. (C16H8N2O2 ·0.3EtOH) C, H, N.
2-(5-Cyano-2-hydroxyphenyl)benzofuran-6-carbonitrile (85).
White solid (1.56 g, 82%): mp >300 °C (EtOH). 1H NMR (DMSO-
d6) δ 11.99 (s, 1H), 8.25 (d, J ) 1.6 Hz, 1H), 8.24 (s, 1H), 7.90 (d,
J ) 8.2 Hz, 1H), 7.76 (dd, J ) 8.6 and 1.6 Hz, 1H), 7.70 (dd, J )
8.2 and 1.1 Hz, 1H), 7.65 (d, J ) 1.1 Hz, 1H), 7.20 (d, J ) 8.6 Hz,
1H). HPLC (method B) tR ) 7.07 min (100 area %). Anal.
(C16H8N2O2 ·0.2 H2O) C, H, N.
Acknowledgment. This work was supported by the Bill and
Melinda Gates Foundation and the Medicines for Malaria Venture.

Antiprotozoal Cationic 2-Phenylbenzofurans
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6943
factors in human immunodeficiency virus-infected patients who were
receiving intravenous pentamidine therapy for Pneumocystis carinii
pneumonia. Clin. Infect. Dis. 1997, 24, 854–859.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(24) Bell, C. A.; Hall, J. E.; Kyle, D. E.; Grogl, M.; Ohemeng, K. A.;
Allen, M. A.; Tidwell, R. R. Structure-activity relationships of analogs
of pentamidine against Plasmodium falciparum and Leishmania
mexicana amazonensis. Antimicrob. Agents Chemother. 1990, 34,
1381–1386.
(25) Stead, A. M.; Bray, P. G.; Edwards, I. G.; DeKoning, H. P.; Elford,
B. C.; Stocks, P. A.; Ward, S. A. Diamidine compounds: selective
uptake and targeting in Plasmodium falciparum. Mol. Pharmacol.
2001, 59, 1298–1306.
References
(1) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity of 2,5-
bis(4-guanylphenyl)furans. J. Med. Chem. 1977, 20, 531–536.
(2) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity of 2,5-
bis(4-guanylphenyl)thiophenes and -pyrroles. J. Med. Chem. 1977, 20,
1219–1221.
(3) Anne, J.; De Clercq, E.; Eyssen, H.; Dann, O. Antifungal and
antibacterial activities of diarylamidine derivatives. Antimicrob. Agents
Chemother. 1980, 18, 231–239.
(26) Bray, P. G.; Barrett, M. P.; Ward, S. A.; de Koning, H. P. Pentamidine
uptake and resistance in pathogenic protozoa: past, present and future.
Trends Parasitol. 2003, 19, 232–239.
(4) Bell, C. A.; Cory, M.; Fairley, T. A.; Hall, J. E.; Tidwell, R. R.
Structure-activity relationships of pentamidine analogs against Giardia
lamblia and correlation of antigiardial activity with DNA-binding
affinity. Antimicrob. Agents Chemother. 1991, 35, 1099–1107.
(5) Bell, C. A.; Dykstra, C. C.; Naiman, N. A.; Cory, M.; Fairley, T. A.;
Tidwell, R. R. Structure-activity studies of dicationically substituted
bis-benzimidazoles against Giardia lamblia: correlation of antigiardial
activity with DNA binding affinity and giardial topoisomerase II
inhibition. Antimicrob. Agents Chemother. 1993, 37, 2668–2673.
(6) Brendle, J. J.; Outlaw, A.; Kumar, A.; Boykin, D. W.; Patrick, D. A.;
Tidwell, R. R.; Werbovetz, K. A. Antileishmanial activities of several
classes of aromatic dications. Antimicrob. Agents Chemother. 2002,
46, 797–807.
(7) Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F. A.; Wilson,
W. D.; Boykin, D. W. Synthesis and antiprotozoal activity of aza-
analogues of furamidine. J. Med. Chem. 2003, 46, 4761–4769.
(8) Ismail, M. A.; Brun, R.; Wenzler, T.; Tanious, F. A.; Wilson, W. D.;
Boykin, D. W. Dicationic biphenyl benzimidazole derivatives as
antiprotozoal agents. Bioorg. Med. Chem. 2004, 12, 5405–5413.
(9) Ansede, J. H.; Anbazhagan, M.; Brun, R.; Easterbrook, J. D.; Hall,
J. E.; Boykin, D. W. O-Alkoxyamidine prodrugs of furamidine: in
vitro transport and microsomal metabolism as indicators of in vivo
efficacy in a mouse model of Trypanosoma brucei rhodesiense
infection. J. Med. Chem. 2004, 47, 4335–4338.
(10) Arafa, R. K.; Brun, R.; Wenzler, T.; Tanious, F. A.; Wilson, W. D.;
Stephens, C. E.; Boykin, D. W. Synthesis, DNA affinity, and
antiprotozoal activity of fused ring dicationic compounds and their
prodrugs. J. Med. Chem. 2005, 48, 5480–5488.
(11) Salom-Roig, X. J.; Hamze, A.; Calas, M.; Vial, H. J. Dual molecules
as new antimalarials. Comb. Chem. High Throughput Screening 2005,
8, 49–62.
(12) Ismail, M. A.; Arafa, R. K.; Brun, R.; Wenzler, T.; Miao, Y.; Wilson,
W. D.; Generaux, C.; Bridges, A.; Hall, J. E.; Boykin, D. W. Synthesis,
DNA affinity, and antiprotozoal activity of linear dications: terphenyl
diamidines and analogues. J. Med. Chem. 2006, 49, 5324–5332.
(13) Werbovetz, K. Diamidines as antitrypanosomal, antileishmanial and
antimalarial agents. Curr. Opin. InVest. Drugs 2006, 7, 147–157.
(14) Rodriguez, F.; Rozas, I.; Kaiser, M.; Brun, R.; Nguyen, B.; Wilson,
W. D.; Garcia, R. N.; Dardonville, C. New bis(2-aminoimidazoline)
and bisguanidine DNA minor groove binders with potent in vivo
antitrypanosomal and antiplasmodial activity. J. Med. Chem. 2008,
51, 909–923.
(15) Doua, F.; Miezan, T. W.; Sanon Singaro, J. R.; Boa Yapo, F.; Baltz,
T. The efficacy of pentamidine in the treatment of early-late stage
Trypanosoma brucei gambiense trypanosomiasis. Am. J. Trop. Med.
Hyg. 1996, 55, 586–588.
(16) Bouteille, B.; Oukem, O.; Bisser, S.; Dumas, M. Treatment perspectives
for human African trypanosomiasis. Fundam. Clin. Pharmacol. 2003,
17, 171–181.
(17) Fairlamb, A. H. Chemotherapy of human African trypanosomiasis:
current and future prospects. Trends Parasitol. 2003, 19, 488–494.
(18) Barrett, M. P.; Boykin, D. W.; Brun, R.; Tidwell, R. R. Human African
trypanosomiasis: pharmacological re-engagement with a neglected
disease. Br. J. Pharmacol. 2007, 152, 1155–1171.
(19) Croft, S. L.; Yardley, V. Chemotherapy of leishmaniasis. Curr. Pharm.
Des. 2002, 8, 319–342.
(20) Singh, S.; Sivakumar, R. Challenges and new discoveries in the
treatment of leishmaniasis. J. Infect. Chemother. 2004, 10, 307–315.
(21) Goa, K. L.; Campoli-Richards, D. M. Pentamidine isethionate. A
review of its antiprotozoal activity, pharmacokinetic properties and
therapeutic use in Pneumocystis carinii pneumonia. Drugs 1987, 33,
242–258.
(22) Monk, J. P.; Benfield, P. Inhaled pentamidine. An overview of its
pharmacological properties and a review of its therapeutic use in
Pneumocystis carinii pneumonia. Drugs 1990, 39, 741–756.
(23) O’Brien, J. G.; Dong, B. J.; Coleman, R. L.; Gee, L.; Balano, K. B.
A 5-year retrospective review of adverse drug reactions and their risk
(27) Burchmore, R. J.; Ogbunude, P. O.; Enanga, B.; Barrett, M. P.
Chemotherapy of human African trypanosomiasis. Curr. Pharm. Des.
2002, 8, 257–267.
(28) Legros, D.; Ollivier, G.; Gastellu-Etchegorry, M.; Paquet, C.; Burri,
C.; Jannin, J.; Buscher, P. Treatment of human African trypanoso-
miasisspresent situation and needs for research and development.
Lancet Infect. Dis. 2002, 2, 437–440.
(29) Burri, C.; Brun, R. Human African Trypanosomiasis. In Manson’s
Tropical Diseases; Cook, G. C., Zumla, A., Eds.; W. B. Saunders:
London, 2003; pp 1303-1323.
(30) Greenwood, B.; Mutabingwa, T. Malaria in 2002. Nature 2002, 415,
670–672.
(31) May, J.; Meyer, C. G. Chemoresistance in falciparum malaria. Trends
Parasitol 2003, 19, 432–435 (discussion 435-436).
(32) Biagini, G. A.; O’Neill, P. M.; Bray, P. G.; Ward, S. A. Current drug
development portfolio for antimalarial therapies. Curr. Opin. Phar-
macol. 2005, 5, 473–478.
(33) Woodrow, C. J.; Krishna, S. Antimalarial drugs: recent advances in
molecular determinants of resistance and their clinical significance.
Cell. Mol. Life Sci. 2006, 63, 1586–1596.
(34) Le Bras, J.; Durand, R. The mechanisms of resistance to antimalarial
drugs in Plasmodium falciparum. Fundam. Clin. Pharmacol. 2003,
17, 147–153.
(35) Hyde, J. E. Drug-resistant malaria. Trends Parasitol. 2005, 21, 494–
498.
(36) Arav-Boger, R.; Shapiro, T. A. Molecular mechanisms of resistance
in antimalarial chemotherapy: the unmet challenge. Annu. ReV.
Pharmacol. Toxicol. 2005, 45, 565–585.
(37) Loiseau, P. M.; Bories, C. Mechanisms of drug action and drug
resistance in Leishmania as basis for therapeutic target identification
and design of antileishmanial modulators. Curr. Top. Med. Chem.
2006, 6, 539–550.
(38) Mishra, J.; Saxena, A.; Singh, S. Chemotherapy of leishmaniasis: past,
present and future. Curr. Med. Chem. 2007, 14, 1153–1169.
(39) Murray, H. W.; Berman, J. D.; Davies, C. R.; Saravia, N. G. Advances
in leishmaniasis. Lancet 2005, 366, 1561–1577.
(40) Sundar, S.; Jha, T. K.; Thakur, C. P.; Sinha, P. K.; Bhattacharya, S. K.
Injectable paromomycin for visceral leishmaniasis in India. N. Engl.
J. Med 2007, 356, 2571–2581.
(41) Sartori, A. M.; Ibrahim, K. Y.; Nunes Westphalen, E. V.; Braz, L. M.;
Oliveira, O. C., Jr.; Gakiya, E.; Lopes, M. H.; Shikanai-Yasuda, M. A.
Manifestations of Chagas disease (American trypanosomiasis) in
patients with HIV/AIDS. Ann. Trop. Med. Parasitol. 2007, 101, 31–
50.
(42) Bronzan, R. N.; Taylor, T. E.; Mwenechanya, J.; Tembo, M.; Kayira,
K.; Bwanaisa, L.; Njobvu, A.; Kondowe, W.; Chalira, C.; Walsh, A. L.;
Phiri, A.; Wilson, L. K.; Molyneux, M. E.; Graham, S. M. Bacteremia
in Malawian children with severe malaria: prevalence, etiology, HIV
coinfection, and outcome. J. Infect. Dis. 2007, 195, 895–904.
(43) Wolday, D.; Berhe, N.; Akuffo, H.; Desjeux, P.; Britton, S. Emerging
Leishmania/HIV co-infection in Africa. Med. Microbiol. Immunol.
2001, 190, 65–67.
(44) Desjeux, P.; Alvar, J. Leishmania/HIV co-infections: epidemiology
in Europe. Ann. Trop. Med. Parasitol. 2003, 97, 3–15.
(45) Rabello, A.; Orsini, M.; Disch, J. Leishmania/HIV co-infection in
Brazil: an appraisal. Ann. Trop. Med. Parasitol 2003, 97, 17–
28.
(46) Cruz, I.; Nieto, J.; Moreno, J.; Canavate, C.; Desjeux, P.; Alvar, J.
Leishmania/HIV co-infections in the second decade. Indian J. Med.
Res 2006, 123, 357–388.
(47) Dann, O.; Bergen, G.; Demant, E.; Volz, G. Trypanocide diamidine
des 2-phenyl-benzofurans, 2-phenyl-indens und 2-phenyl-indols. Lie-
bigs Ann. Chem. 1971, 749, 68–89.
(48) Dann, O.; Char, H.; Griessmeier, H. Synthesen biskationisher, try-
panocider 1-benzofuran-verbindungen. Liebigs Ann. Chem. 1982,
1836–1869.

6944 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
BakunoV et al.
(49) Dox, A. W.; Whitmore, F. C. Acetamidine Hydrochloride. In Organic
Synthesis, 2nd ed.; Blatt, A. H., Ed.; John Wiley & Sons: New York,
1941; Vol. 1, pp 5-7.
(50) Sonogashira, K.; Tohda, Y.; Hagihara, N. A convenient synthesis of
acetylenes: catalytic substitutions of acetylenic hydrogen with bro-
moalkenes, iodoarenes and bromopyridines. Tetrahedron Lett. 1975,
16, 4467–4470.
(51) Lutjens, H.; Scammells, P. J. Synthesis of natural products possessing
a benzo[ꢀ]furan skeleton. Tetrahedron Lett. 1998, 39, 6581–6584.
(52) Verner, E.; Katz, B. A.; Spencer, J. R.; Allen, D.; Hataye, J.;
Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli,
A.; Radika, K.; Rai, R.; She, M.; Shrader, W.; Sprengeler, P. A.; Trapp,
S.; Wang, J.; Young, W. B.; Mackman, R. L. Development of serine
protease inhibitors displaying a multicentered short (< 2.3 Å) hydrogen
bond binding mode: inhibitors of urokinase-type plasminogen activator
and factor Xa. J. Med. Chem. 2001, 44, 2753–2771.
(53) Sonogashira, K. Development of Pd-Cu catalyzed cross-coupling of
terminal acetylenes with sp2-carbon halides. J. Organomet. Chem.
2002, 653, 46–49.
(54) Bates, C. G.; Saejueng, P.; Murphy, J. M.; Venkataraman, D. Synthesis
of 2-arylbenzo[ꢀ]furans via copper(I)-catalyzed coupling of o-io-
dophenols and aryl acetylenes. Org. Lett. 2002, 4, 4727–4729.
(55) Castro, C. E.; Gaughan, E. J.; Owsley, D. C. Indoles, benzofurans,
phthalides, and tolanes via copper(I) acetylides. J. Org. Chem. 1966,
31, 4071–4078.
(56) Castro, C. E.; Havlin, R.; Honwad, V. K.; Malte, A. M.; Moje, S. W.
Copper(I) substitutions. Scope and mechanism of cuprous acetylide
substitutions. J. Am. Chem. Soc. 1969, 91, 6464–6470.
(57) Doad, G. J. S.; Barltrop, J. A.; Petty, C. M.; Owen, T. C. A versatile
and convenient synthesis of benzofurans. Tetrahedron Lett. 1989, 30,
1597–1598.
Shima, Y.; Tashiro, K.; Yamamoto, H.; Yoshimoto, R.; Iwata, S.; Tsuji,
T.; Sakurai, K.; Shoji, M. Rational design, synthesis, and structure-
activity relationships of novel factor Xa inhibitors: (2-substituted-4-
amidinophenyl)pyruvic and -propionic acids. J. Med. Chem. 2003, 46,
1845–1857.
(65) Frigerio, M.; Santagostino, M.; Sputore, S. A user-friendly entry to
2-iodoxybenzoic acid (IBX). J. Org. Chem. 1999, 64, 4537–4538.
(66) Nicolaou, K. C.; Mathison, C. J. N.; Montagnon, T. o-Iodoxybenzoic
acid (IBX) as a viable reagent in the manipulation of nitrogen- and
sulfur-containing substrates: scope, generality, and mechanism of IBX-
mediated amine oxidations and dithiane deprotections. J. Am. Chem.
Soc. 2004, 126, 5192–5201.
(67) Steck, E. A.; Kinnamon, K. E.; Rane, D. S.; Hanson, W. L. Leishmania
donoVani, Plasmodium berghei, Trypanosoma rhodesiense: antipro-
tozoal effects of some amidine types. Exp. Parasitol. 1981, 52, 404–
413.
(68) Donkor, I. O.; Assefa, H.; Rattendi, D.; Lane, S.; Vargas, M.; Goldberg,
B.; Bacchi, C. Trypanocidal activity of dicationic compounds related
to pentamidine. Eur. J. Med. Chem. 2001, 36, 531–538.
(69) Donkor, I. O.; Huang, T. L.; Tao, B.; Rattendi, D.; Lane, S.; Vargas,
M.; Goldberg, B.; Bacchi, C. Trypanocidal activity of conforma-
tionally restricted pentamidine congeners. J. Med. Chem. 2003, 46,
1041–1048.
(70) Kaminsky, R.; Schmid, C.; Brun, R. An “in vitro selectivity index”
for evaluation of cytotoxicity of antitrypanosomal compounds. In Vitro
Toxicol. 1996, 9, 315–324.
(71) Baltz, T.; Baltz, D.; Giroud, C.; Crockett, J. Cultivation in a semi-
defined medium of animal infective forms of Trypanosoma brucei, T.
equiperdum, T. eVansi, T. rhodesiense and T. gambiense. EMBO J.
1985, 4, 1273–1277.
(58) Pilling, R. J.; Whiting, D. A. FIQ and FIQ2, new Q-site inhibitors for
photosynthetic electron transport: synthesis and the relationship
between stereochemistry and biological activity. J. Chem. Soc., Perkin
Trans. 1 1999, 2077–2086.
(59) Bakunova, S. M.; Bakunov, S. A.; Wenzler, T.; Barszcz, T.; Wer-
bovetz, K. A.; Brun, R.; Hall, J. E.; Tidwell, R. R. Synthesis and in
vitro antiprotozoal activity of bisbenzofuran cations. J. Med. Chem.
2007, 50, 5807–5823.
(72) Raz, B.; Iten, M.; Grether-Buhler, Y.; Kaminsky, R.; Brun, R. The
Alamar Blue assay to determine drug sensitivity of African trypano-
somes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop.
1997, 68, 139–147.
(73) Matile, H.; Pink, J. R. L. Plasmodium falciparum Malaria Parasite
Culture and Their Use in Immunology. In Immunological Methods;
Lefkovits, I., Pernis, B., Eds.; Academic Press: San Diego, CA, 1990;
pp 221-234.
(60) Edgar, K. J.; Falling, S. N. An efficient and selective method for the
preparation of iodophenols. J. Org. Chem. 1990, 55, 5287–5291.
(61) Patrick, D. A.; Bakunov, S. A.; Bakunova, S. M.; Kumar, E. V.;
Lombardy, R. J.; Jones, S. K.; Bridges, A. S.; Zhirnov, O.; Hall, J. E.;
Wenzler, T.; Brun, R.; Tidwell, R. R. Synthesis and in vitro
antiprotozoal activities of dicationic 3,5-diphenylisoxazoles. J. Med.
Chem. 2007, 50, 2468–2485.
(62) Diaz, P.; Gendre, F.; Stella, L.; Charpentier, B. New synthetic retinoids
obtained by palladium-catalized tandem cyclisation-hydride capture
process. Tetrahedron 1998, 54, 4579–4590.
(63) Wood, J. L.; Khatri, N. A.; Weinreb, S. M. A direct conversion of
esters to nitriles. Tetrahedron Lett. 1979, 20, 4907–4910.
(64) Sagi, K.; Nakagawa, T.; Yamanashi, M.; Makino, S.; Takahashi, M.;
Takayanagi, M.; Takenaka, K.; Suzuki, N.; Oono, S.; Kataoka, N.;
Ishikawa, K.; Shima, S.; Fukuda, Y.; Kayahara, T.; Takehana, S.;
(74) Werbovetz, K.; Brendle, J.; Sackett, D. Purification, characterization,
and drug susceptibility of tubulin from Leishmania. Mol. Biochem.
Parasitol. 1999, 98, 53–65.
(75) Werbovetz, K.; Sackett, D.; Delfin, D.; Bhattacharya, G.; Salem, M.;
Obrzut, T.; Rattendi, D.; Bacchi, C. Selective antimicrotubule activity
of N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (GB-II-5)
against kinetoplastid parasites. Mol. Pharmacol. 2003, 64, 1325–
1333.
(76) Sperandeo, N. R.; Brun, R. Synthesis and biological evaluation of
pyrazolylnaphthoquinones as new potential antiprotozoal and cytotoxic
agents. ChemBioChem 2003, 4, 69–72.
JM800918V