Synthesis and in vitro antiprotozoal activity of bisbenzofuran cations

J. Med. Chem. 2007, 50, 5807-5823

5807

Synthesis and in Vitro Antiprotozoal Activity of Bisbenzofuran Cations

Svetlana M. Bakunova,^†Stanislav A. Bakunov,^†Tanja Wenzler,^‡Todd Barszcz,^§Karl A. Werbovetz,^§Reto Brun,^‡

James Edwin Hall,^†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 17, 2007

Forty three cationic bisbenzofurans were synthesized either by interaction of o-hydroxyaldehydes with

R-halogenated ketones followed by intramolecular ring closure or by a copper- or palladium-mediated

heteroannulation of substituted o-iodophenols with terminal acetylenes. In vitro antiprotozoal activities of

compounds 1-43 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania

donoVani and cytotoxicity against mammalian cells were influenced by the position and the type of cationic

substituents as well as the length of the carbon linker between aromatic moieties. One bisamidine displayed

an antitrypanosomal efficacy comparable to that of pentamidine and melarsoprol. Twenty two compounds

were more potent than pentamidine and seven dications were more effective than artemisinin against P.

falciparum. Eight bisbenzofurans displayed activity against L. donoVani superior to that of pentamidine.

Overall, bisamidines connected by two-carbon linkers exhibited the highest efficacies against T. b. rhodesiense,

P. falciparum, and L. donoVani.

Introduction

maniasis have been reported in United States and coalition forces

participating in Operation Iraqi Freedom through mid-April

2004.¹⁵

Protozoan parasitic infections malaria, human African try-

panosomiasis (HAT^aor sleeping sickness), and leishmaniasis

are re-emerging.^1-4Malaria occurs in over 90 countries

worldwide and is responsible for approximately 300 million

acute cases each year, with 1.5 to 2.0 million fatalities. About

90% of those deaths occur in sub-Saharan African regions and

are caused by Plasmodium falciparum, the species responsible

for the most severe and life-threatening form of disease.^3,5

African sleeping sickness remains one of the most neglected

diseases and is fatal if untreated.⁶There are two forms of HAT,

depending on the parasite involved. Trypanosoma brucei

gambiense causes a human chronic infection, endemic in western

and central Africa; T. b. rhodesiense has a vast animal reservoir

and causes acute illness in people in eastern and southern

African countries.^2,7,8Most episodes of HAT occur in the remote

rural areas, where the surveillance is weak or nonexistent, with

50 to 70 thousand estimated cases.⁹Leishmania species cause

a spectrum of disease ranging from self-healing cutaneous

lesions to life-threatening visceral infections, with the clinical

manifestations depending on the species of infecting parasite.

Approximately 1.5-2.0 million new cases of leishmaniasis

occur annually, with about 500 000 of them being of the

potentially fatal visceral form. The coexistence of HIV and

Leishmania species causing visceral disease¹⁰has in recent years

resulted in hundreds of cases of dually infected individuals in

Africa,¹¹Latin America,¹²and Southern Europe.^13,14In addition,

approximately 700 parasitologically confirmed cases of leish-

The emergence of resistance to the cheapest and most

commonly used drugs, such as chloroquine and sulfadoxine-

pyrimethamine, represents a major obstacle in controlling

malaria.^5,16Many countries must now rely on therapies that are

more expensive or use drug combinations (particularly with

artemisinins) to try to slow the development of resistance.^17,18

Various antimalarial drugs in current usage are chemically

related or have a similar mode of action, possibly sharing

resistance mechanisms, thus increasing the risk of cross-

resistance and clinical failure of newly introduced therapies.^3,19-21

Of the four drugs clinically approved for treating HAT, three

(suramin, pentamidine, and melarsoprol) were introduced more

than half a century ago. Because suramin and pentamidine are

ionized at physiological pH, they are unable to cross the blood-

brain barrier in therapeutic concentrations and are thus used

for the treatment of hemolymphatic early stage HAT, caused

by T. b. rhodesiense and T. b. gambiense infections, respectively.

The treatment of the second or neurological stage, when the

parasites invade the central nervous system (CNS), relies on

the organo-arsenical drug melarsoprol and the more recently

registered eflornithine. The latter is ineffective against T. b.

rhodesiense sleeping sickness and is used primarily to control

CNS-involved HAT caused by T. b. gambiense. These antitry-

panosomal therapies suffer from unacceptable toxicity, poor

efficacy, difficulties of administration, and increasing treatment

failures due to the development of parasite resistance.^{1,2,7,8,22,23}

Pentavalent antimonial compounds have been the mainstay

of antileishmanial chemotherapy since the 1940s (see refs 24,

25 for recent reviews of the chemotherapy of leishmaniasis),

but these agents require a long course of parenteral administra-

tion, and antimonial unresponsiveness is becoming more

prevalent, particularly in the Indian state of Bihar.²⁶Amphot-

ericin B has also been used as a second-line treatment, but it

must also be administered parenterally, and the nephrotoxicity

* To whom correspondence should be addressed. Phone: (919) 966-

4294. Fax: (919) 966-0704. E-mail: Tidwell@med.unc.edu

^†The University of North Carolina

^‡Swiss Tropical Institute

^§The Ohio State University.

^aAbbreviations: HAT, human African trypanosomiasis; CQ, chloroquine;

CNS, central nervous system; PMD, pentamidine; MLSP, melarsoprol;

ATMS, artemisinin; PPT, podophyllotoxin.

Published on Web 10/19/2007

5808 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

Scheme 1^a

^aReagents and conditions: (i) 1,3-Dichloroacetone, K₂CO₃, 2-butanone, reflux, 4 h; (ii) AlCl₃, LiAlH₄, ether, 25 °C, 1 h, then 2 N HCl; (iii) CuCN,

quinoline, reflux, 3 h.

caused by this agent is a serious limitation. Liposome-

encapsulated amphotericin B, which is much less toxic than

standard formulations, is too costly to be used for routine

antileishmanial chemotherapy in the developing world. Milte-

fosine was recently registered in India as the first oral treatment

for visceral leishmaniasis. Unfortunately, it cannot be used to

treat pregnant women, has severe gastrointestinal side effects,

and must also be given over a long course of treatment.²⁷

Since safe, effective, and affordable orally active therapies

capable of overcoming resistance are clearly needed, the

identification of new antiprotozoal drug candidates should be

an urgent priority. Aromatic diamidines demonstrate a wide

range of antifungal and antiprotozoal activities,^28-33although

they have never been used for treatment of malaria because other

effective therapies were available. The aromatic diamidine 1,5-

bis(4-amidinophenoxy)pentane (pentamidine) is employed widely

for treatment of early stage T. b. gambiense sleeping sickness,

as well as antimony-resistant leishmaniasis and Pneumocystis

carinii pneumonia. However, its poor oral activity⁷and the

necessity of parenteral administration makes the treatment less

practical, especially in remote areas, where most cases of HAT

and leishmaniasis occur. Despite some adverse effects, such as

hypotension, abdominal pain, vertigo, hypersalivation, hypogly-

cemia, nausea, and mild nephrotoxocity,^2,8,22,34pentamidine is

fairly well tolerated by patients.

The mechanism of antiprotozoal action of pentamidine is not

completely understood.^7,23,34,35It rapidly accumulates in milli-

molar concentrations inside African trypanosomes^36,37and

Plasmodium,^37,38inhibiting multiple targets in the para-

sites.^7,22,23,37Considering the low passive permeability of

pentamidine due to its positive charges, such accumulation

indicates the involvement of parasite specific transport mech-

anisms, which are important for selective activity of the

drug.^35,36,38,39

Phenyl oxygens in pentamidine are known to be a part of

the structural recognition motif for the aminopurine transporter

in Trypanosoma species and therefore are crucial for effective

uptake by the P2 carrier.^35,39-41Thus, replacement of these

oxygen atoms in pentamidine with methylene groups resulted

in a 20-fold loss in antitrypanosomal activity.⁴²On the other

hand, pentamidine analogues containing substituents in the ortho

position to the phenyl oxygens demonstrate similar or even

superior antiprotozoal properties as well as lower cytotoxicity

compared to the parent compound.^30,31

properties of bis(5-amidinobenzofuran-2-yl)methane 4 and 1,2-

bis(5-amidinobenzofuran-2-yl)ethane 8 were published,⁴⁴but

these compounds received very little attention since. Here we

report the synthesis of 4 as well as the new approach to 8 and

41 analogues 1-3, 5-7, 9-43. All dications were tested in

vitro against T. b. rhodesiense (STIB900), P. falciparum (K1,

resistant to chloroquine), and axenic amastigotes of L. donoVani

(MHOM/SD/62/1S-CL2_D) and evaluated for cytotoxicity against

rat myoblast cells (L6).

Chemistry

The benzofuran system was formed using two different

approaches. The first one included interaction of o-hydroxyal-

dehydes with R-halogenated ketones^43,46followed by intramo-

lecular ring closure and was employed for synthesis of

bisbenzofurans connected by a single carbon atom (Scheme 1).

In the second method, used to prepare compounds with multiple

methylene groups in the linker, the benzofuran ring was

constructed by the reaction of o-iodophenols with terminal

acetylenes. This approach was carried out under the conditions

of both copper-catalyzed (modified^47,48Castro reaction^49,50) and

palladium-catalyzed (Sonogashira-type reaction^51-57) heteroan-

nulation (Schemes 2, 3).

Bis(5-cyanobenzofuran-2-yl)methane 44 was prepared from

the commercially available 5-bromosalicylaldehyde following

the procedure described by Dann et al.^43,44

Hydroxyaldehydes 45 and 46 were converted to dinitriles 51

and 52 in three steps in 39% and 42% yields, respectively

(Scheme 1). Compounds 45 and 46, prepared from 3-bromophe-

nol by Reimer-Tiemann reaction,⁵⁸reacted with 1,3-dichloro-

acetone in refluxing 2-butanone⁴⁶providing ketones 47 and 48.

Reduction of compounds 47 and 48 with lithium aluminum

hydride and aluminum chloride in dry ether afforded bis-

(benzofuran-2-yl)methanes 49 and 50, which were converted

to dinitriles 51 and 52 by means of CuCN in boiling quinoline,

following the procedure outlined for the synthesis of 44.⁴⁴

The synthesis of bisbenzofurans connected by the ethylene

bridge is shown in Scheme 2. 4-Hydroxy-3-iodobenzoic acid^52,59

and 3-hydroxy-4-iodobenzoic acid^48,60,61were prepared from 4-

and 3-hydroxybenzoic acids, respectively,⁶⁰following the io-

dination protocol of Edgar and Falling,⁵⁹and were converted

then into methyl benzoates 53 and 54 by acid-catalyzed

esterification.⁶⁰The high yields (80%) on the iodination step

were obtained when 2 equiv of NaOH were used instead of

one, as suggested in the original procedure.⁵⁹Reaction of

o-iodophenols 53 and 54 with 4-pentyn-1-ol in refluxing

pyridine in the presence of cuprous oxide^47,48afforded benzo-

furan alcohols 55⁵²and 56, which were oxidized using the Swern

method⁶²to give aldehydes 57 and 58 in high yields. A one-

pot procedure⁶³for the conversion of aldehydes to alkynes was

employed in the synthesis of benzofurans 59 and 60 containing

In our continuing search for novel dicationic compounds with

enhanced efficacy and lowered toxicity as new antiprotozoal

drug candidates, we synthesized a series of dicationic bisben-

zofurans 1-43 as heterocyclic pentamidine analogues in which

phenoxy fragments were included into a benzofuran structural

motif. In the early 1970s, syntheses of six bisbenzofuran

dications were reported^43-45and the moderate antitrypanosomal

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5809

Scheme 2^a

^aReagents and conditions: (i) 4-Pentyn-1-ol, Cu₂O, pyridine, 100 °C, 15 h; (ii) 4-pentyn-1-ol, PdCl₂(PPh₃)₂, CuI, 1,1,3,3-tetramethylguanidine or

triethylamine, DMF, 25 °C or 80 °C, 3-24 h; (iii) 2 M oxalyl chloride in CH₂Cl₂, DMSO, CH₂Cl₂, -70 °C, 1 h, then NEt₃; (iv) dimethy1 1-diazo-2-

oxopropylphosphonate, K₂CO₃, MeOH; (v) 53 or 54, Cu₂O, pyridine, 100 °C, 15 h; (vi) Me₂AlNH₂, o-xylene, 80-100 °C, 3 h; (vii) 83 or 87, Cu₂O,

pyridine, 100 °C, 15 h.

Scheme 3^a

^aReagents and conditions: (i) Appropriate alkyldiyne, PdCl₂(PPh₃)₂, CuI, 1,1,3,3-tetramethylguanidine or triethylamine, DMF, 25 °C or 80 °C, 3-24 h;

(ii) appropriate alkyldiyne, Cu₂O, pyridine, 100 °C, 15 h; (iii) Me₂AlNH₂, o-xylene, 80-100 °C, 3 h; (iv) NH₂OH‚HCl, EtOH, pyridine; (v) Ac₂O, reflux,

2 h.

acetylenic side chains. Thus, interaction of aldehydes 57 and

58 with dimethyl-1-diazo-2-oxopropylphosphonate^64,65in the

presence of K₂CO₃in dry methanol afforded acetylenes 59 and

60. Compounds 59 and 60 were reacted with methyl benzoates

53 and 54 under the conditions of modified Castro reaction⁴⁷

to provide bisbenzofurans 61, 62, and 63, which were directly

converted to dinitriles 64, 65, and 66 in moderate yields (40-

50%) by treatment with dimethylaluminum amide.⁶⁶Overall

yield for the transformation of 53 to 64 was 9% over five steps

and 54 was converted to 65 and to 66 in 19% and 21% yields,

respectively. Although the Sonogashira reaction provided milder

conditions for the construction of the heterocyclic ring and

therefore represented a more attractive synthetic approach, the

modified Castro method worked better for the syntheses of

bisbenzofurans with a two-carbon linker, affording fewer

impurities and higher yields of the desired compounds.

The same synthetic strategy, utilizing heteroannulation of

o-iodophenol esters and terminal alkyldiynes, was employed for

the synthesis of dinitriles containing three and more methylene

groups in the linker (Scheme 3). Thus, interaction of 2 equiv

of methyl benzoates 53 and 54 with commercially available 1,6-

heptadiyne, 1,7-octadiyne, and 1,8-nonadiyne under the condi-

tions of Castro or Sonogashira reactions led to bisbenzofurans

67-72 in one step, affording comparable yields with both

methods. Treatment of esters 67-72 with dimethylaluminum

amide provided dinitriles 73, 74, 75,⁴⁵76-78 in moderate to

high yields. Likewise, 7,7′-dimethoxy-substituted nitrile 82 was

prepared from 5-iodovanillin 79 in three steps in 48% overall

5810 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

Scheme 4^a

^aReagents and conditions: (i) 1,4-Dioxane, EtOH, HCl, 1-3 days; (ii) appropriate amine, EtOH, 1-4 days.

yield. Heteroannulation of 79 and 1,8-nonadiyne, following the

modified Castro protocol,⁴⁷afforded dialdehyde 80. Dioxime

81, obtained by reaction of 80 with hydroxylamine, was

dehydrated by treatment with acetic anhydride to give 82

(Scheme 3).

amastigotes of L. donoVani (MHOM/SD/62/1S-CL2_D) as well

as the assessment of cytotoxicity of the compounds 1-43 against

rat myoblast cells (L6) are summarized in Table 2 and compared

to that of pentamidine. Other controls employed were melar-

soprol (T. b. rhodesiense), chloroquine and artemisinin (P.

falciparum), and podophyllotoxin (L6). In addition to activities,

four parameters included in Table 2 were calculated and

analyzed: antitrypanosomal selectivity index SI_T, expressed as

the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)]; antiplasmodial

selectivity index SI_P, expressed as the ratio [IC₅₀(L6)/IC₅₀(P.

falciparum)]; selectivity (S_P/T), expressed as ratio [IC₅₀(T. b.

rhodesiense)/IC₅₀(P. falciparum)]; and antileishmanial selectiv-

ity index SI_L, expressed as the ratio [IC₅₀(L6)/IC₅₀(L.

donoVani)]. Selectivity indexes SI_T, SI_Pand SI_Lreflect inhibition

In general, introduction of cyano groups required additional

synthetic steps and decreased the yields of the target compounds.

Employment of cyano-substituted halogenophenols as starting

materials allowed the synthetic pathway to be shortened and

improved the reaction outcome by eliminating the low yielding

transformations. 4-Hydroxy-3-iodobenzonitrile 83⁶⁷was pre-

pared by iodination of 4-cyanophenol following the procedure

of Edgar and Falling.⁵⁹3-Hydroxy-4-iodobenzonitrile 87 was

synthesized as previously described.⁶¹The Sonogashira reaction

of 83 with 4-pentyn-1-ol followed by the aforementioned

sequence (Scheme 2) afforded acetylene 86, which reacted both

with 83 and 87 to give dinitriles 64 and 65 in four steps in

30% overall yield for both compounds. A one-step reaction of

83 and 87 with 1,5-hexadiyne and the reaction of 87 with 1,6-

heptadiyne (Scheme 3) under the Castro conditions provided

dinitriles 64, 66, and 68 in 24%, 22%, and 62% yields,

respectively. The Sonogashira reaction of 83 with 1,8-nonadiyne

afforded dinitrile 77 in 77% yield. Because yields of benzofurans

were comparable in both the Castro and Sonogashira protocols

as the length of the carbon linker increased but the latter

occurred under milder conditions, the Sonogashira reaction was

chosen to prepare dinitriles 88-93. 1,9-Decadyine was com-

mercially available. 1,10-Undecadyine, 1,11-dodecadyine, 1,-

12-tridecadyine, 1,13-tetradecadyine, and 1,14-pentadecadyine

were prepared according to the published procedure.⁶⁸The

aforementioned alkyldiynes reacted with 83 to give 88-93 in

19-71% yields.

of the specific parasite relative to the L6 cells. Selectivity (S_P/T

)

reflects suppression of P. falciparum comparative to T. b.

rhodesiense.

Cytotoxicity Study. The results from cytotoxicity studies

clearly demonstrated that structural variations affected properties

of bisbenzofurans 1-43. In our survey 4-substituted dications

were more cytotoxic than corresponding 5- or 6-substituted

isomers. Compounds bearing cationic groups in the 5-position

of the aromatic ring were less cytotoxic than their 6-substituted

counterparts in all cases except derivatives 14 and 16, which

displayed lower cytotoxicity than that of 5,5′- or 5,6′-substituted

isomers 8, 11 and 10, 13. Compounds 11 and 12, containing

cationic groups in the 5- and 6′-positions were more cytotoxic

than comparable 5,5′- and 6,6′-substituted derivatives 8, 14 and

9, 15. Among different types of the tested cations, unsubstituted

bisamidines were more cytotoxic. Substitution on the amidine

moiety resulted in the reduction of cytotoxicity with N-isopropyl-

substituted amidines typically being the least cytotoxic in the

series. 5-Substituted bis(N-isopropyl)amidines 5, 9, 18, 24, and

30 displayed IC₅₀values against L6 cells exceeding 100 µM.

This group of compounds with lower cytotoxicity also included

bisamidine 17 and bisimidazoline 6. Nineteen 5-substituted

derivatives displayed IC₅₀values for cytotoxicity less than 100

µM. They included eleven bisamidines (4, 8, 23, 29, 32, 38-

43), one bis(N-isopropyl)amidine (33), and five bisimidazolines

(10, 19, 25, 31, and 34). In a series of 6-substituted cations

only two bis(N-isopropyl)amidines (15 and 21) and two bisimi-

dazolines (7 and 16) exhibited IC₅₀values against L6 cells

exceeding 100 µM. Those with the cytotoxicity values below

this level consisted of four bisamidines (14, 20, 26, and 32),

two bis(N-isopropyl)amidines (27 and 33), and three

bisimidazolines (22, 28, and 34). Among the 5,6′-substituted

bisbenzofurans 11-13 only bis(N-isopropyl)amidine 12

revealed a cytotoxic IC₅₀value exceeding 100 µM. Both

bisamidine 11 and bisimidazoline 13 scored below that

mark. Generally, elongation of the carbon bridge between

benzofuran rings resulted in increased cytotoxicity against L6

cells. Thus, bisamidines 38-43 with the linker containing from

All dicationic bisbenzofuran amidines (1-43) were synthe-

sized by the modified Pinner method⁶⁹(Scheme 4). Dinitriles

44, 51, 52, 64,^44,4565,⁴⁵66,⁴⁵73, 74, 75,⁴⁵76-78, 82, 88,⁴⁵

89-93 were converted to imidate esters, which reacted with

ethanolic solutions of ammonia, isopropylamine, or ethylene-

diamine to give the bisamidines 1, 4,⁴⁴8,⁴⁴11,⁴⁵14,⁴⁵17, 20,

23,⁴⁵26, 29, 32, 35, 38,⁴⁵39-43, bis(N-isopropyl)amidines 2,

5, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and bisimidazolines 3,

6, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, respectively (Table

1).

Results

A series of 43 dications bearing amidine (Am), N-isopropy-

lamidine (i-PrAm), or imidazoline (Im) substituents in positions

4, 5, or 6 of the benzofuran rings and with the length of the

alkyl linker between aromatic moieties ranging from one to

eleven methylene groups was evaluated for antiprotozoal activity

and cytotoxicity. The results of the in vitro testing against

bloodstream form trypomastigotes of T. b. rhodesiense (STIB900),

P. falciparum (K1, resistant to chloroquine), and axenic

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5811

Table 1. Structures of Bisbenzofurans 1-43

compd

n

R¹

Am

R²

R³

R⁴

R⁵

R⁶

1

2

3

4

5

6

7

8

9

1

H

1

2

3

4

5

6

7

8

9

10

11

i-PrAm

Im

H

Am

i-PrAm

Im

Am

i-PrAm

Im

H

Im

H

Im

H

Am

i-PrAm

Im

Am

i-PrAm

Im

Am

i-PrAm

Im

H

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

H

Am

i-PrAm

Im

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

Am

i-PrAm

Im

Am

i-PrAm

Im

Am

i-PrAm

Im

H

OMe

H

Am

i-PrAm

Im

H

Am

i-PrAm

Im

H

Am

H

six to eleven methylene groups were more cytotoxic than

pentamidine.

Four bisamidines (8, 11, 20 and 26) demonstrated antitrypa-

nosomal IC₅₀values less than 20 nM. The most potent

compound, bisamidine 8 (IC₅₀) 8 nM), exhibited efficacy

comparable to that of pentamidine and melarsoprol and was

the only derivative in the series with the antitrypanosomal IC₅₀

value less than 10 nM.

The position of substituents affected the trypanocidal proper-

ties of compounds 1-43. Bisbenzofurans containing cationic

substituents in the 5- or 6-positions were substantially more

effective against T. b. rhodesiense than their 4-substituted

counterparts. The relative antitrypanosomal activities of 5- and

6-substituted compounds were affected by the type of cationic

group and the length of the carbon linker in the molecules. Thus,

6-substituted bisamidines and bis(N-isopropyl)amidines were

generally more potent against T. b. rhodesiense than 5-substi-

tuted isomers, except bisamidine 8 and bis(N-isopropyl)amidines

12 and 30, which displayed lower antitrypanosomal IC₅₀values.

It is noteworthy that stepwise transition from the 6-substituted

Antitrypanosomal Activity. All compounds 1-43 displayed

antitrypanosomal activities in vitro, with IC₅₀values ranging

from 8 nM to 18.7 µM. Sixteen bisbenzofurans (4, 8, 11-15,

19, 20, 23-27, 35, and 38) displayed antitrypanosomal IC₅₀

values less than 100 nM. Among those compounds were nine

bisamidines (4, 8, 11, 14, 20, 23, 26, 35, and 38), four bis(N-

isopropyl) amidines (12, 15, 24, and 27) and three bisimida-

zolines (13, 19, and 25). Seven compounds (4, 8, 19, 23-25,

and 38) possessed substituents in 5-positions, six isomers (14,

15, 20, 26, 27, and 35) were 6-substituted, and three (11-13)

were mixed derivatives containing their cationic groups both

in 5- and 6′- positions. Eleven dications had a linker with an

even number of carbon atoms, of which six compounds (11-

16) were connected by an ethylene bridge and five compounds

possessed a tetramethylene chain. Four compounds had one (4),

three (19 and 20), or five (35) methylene groups in their linker.

5812 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

Table 2. Cytotoxicity and in Vitro Antiprotozoal Activity of Bisbenzofurans 1-43

cytotoxicity^a

T. b. rhodesiense^b

P. falciparum^d

L. donoVani^g

c

e

f

h

compd

IC₅₀(µM)

0.201

14.0

18.7

0.072

0.455

0.474

4.64

SI_T

IC₅₀(µM)

SI_P

S_P/T

3.0

15

IC₅₀(µM)

SI_L

1

2

3

4

5

6

7

8

22.5

158

74.6

94.2

>174

165

112

11

0.068

0.918

2.12

331

172

35

20.8

>100

3.80

11.3

13.8

11.1

1.06

4.14

5.31

1.54

1.97

3.59

0.887

2.46

3.88

4.40

31.1

13.2

2.38

5.68

4.65

1.34

12.7

1.97

1.52

3.87

9.54

2.49

6.85

1.83

1.79

5.66

1.86

5.10

8.10

3.76

4.30

2.79

12.2

3.16

2.31

2.97

1.83

1.1

>1.6

>0.75

25

4

8.8

2.6

4.5

14

211

2.7

1308

>382

348

0.028

0.102

0.034

0.022

0.003

0.011

0.002

0.006

0.046

0.004

0.005

0.034

0.041

0.009

0.004

0.037

0.036

0.353

0.032

0.010

0.026

0.058

0.076

0.164

0.067

0.020

0.067

0.066

0.133

0.110

0.057

0.018

0.133

0.364

0.279

0.694

0.296

0.287

0.219

0.058

3364

>1706

4853

5636

7167

>59667

3536

9500

20500

1263

19725

>34200

3353

2537

>18667

19725

1041

4306

231

>15

12

11

20

>43

124

27

21.5

>179

38.9

19.0

123

58.1

78.9

>171

114

0.008

0.148

0.127

0.015

0.040

0.090

0.031

0.060

0.415

0.145

0.133

0.031

0.019

0.122

0.355

0.051

0.099

0.073

0.011

0.082

0.321

0.127

0.101

0.120

0.164

0.104

0.231

0.078

0.408

0.531

0.083

0.158

0.220

0.119

0.178

0.210

0.003

0.004

2688

>1209

306

1267

3075

646

2545

>2850

275

9

49

12

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

PMDⁱ

MLSP^j

CQ^k

ATMS^l

PPT^m

7.3

7.5

12

62

16

89

>70

29

6.7

2.0

7.8

12

3.5

15

7.2

0.51

3.4

1.0

1.6

9.9

2.8

0.19

1.1

2.0

1.9

5.1

1.8

2.5

0.78

2.1

1.4

104

717

24

>168

78.9

38.5

155

>1263

2545

2026

1270

230

>5.4

>6.0

16

27

18

26

81.5

34.6

>163

35.4

12.0

27.9

30.9

33.0

>161

35.1

35.3

72.8

54.1

19.2

70.0

13.2

2.78

2.74

2.18

2.16

2.32

1.85

46.6

7.78

117

678

1081

>16300

1362

207

>1646

>13

18

7.9

485

1091

340

96

367

188

493

7.2

3.2

260

13

>24

19

20

13

29

>1594

>8050

293

524

215

700

234

246

172

25

33

17

535

547

492

337

3889

99

8

10

3

7.3

8

803

3.8

8.6

3.5

0.65

0.98

0.18

0.68

1

23

4.0

0.23

0.57

0.32

0.40

0.62

0.96

0.05

10

18

13

9

0.62

15533

1945

25

0.124

0.006

944

75000

450

0.01

^aCytotoxicity (L6 rat myoblast cells). Average of duplicate determinations.^{85 b}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate

determinations.^33,86,87^cSelectivity index for T. b. rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{70 d}Plasmodium falciparum

(K1, resistant to chloroquine). Average of duplicate determinations.⁸⁸^eSelectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P.

falciparum)].⁷⁰^fSelectivity (S_P/T), expressed as ratio [IC₅₀(T. b. rhodesiense)/IC₅₀(P. falciparum)]. ^gLeishmania donoVani (MHOM/SD/62/1S-CL2_D)

axenic amastigotes. Average of duplicate determinations.^89,90^hSelectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/IC₅₀(L. donoVani)].⁷⁰

ⁱPMD, pentamidine. ^jMLSP, melarsoprol. ^kCQ, chloroquine. ^lATMS, artemisinin. ^mPPT, podophyllotoxin.

bisamidine 14 to the 5,6′-substituted isomer 11 and the

5-substituted compound 8, involving moving each of two

amidine groups in the molecule into the 5 position, resulted in

a 2-fold increase in antitrypanosomal activity. On the other hand,

all 5,5′- and 5,6′-substituted bisimidazolines 6, 10, 13, 19, 25,

31, and 34, connected by the linker containing from one to five

methylene groups, demonstrated higher antitrypanosomal activ-

ity than the corresponding 6-substituted isomers 7, 16, 22, 28,

and 37.

panosomal efficacies, in the case of 6-substituted bisbenzofurans,

antitrypanosomal activity decreased in the order Am > i-PrAm

> Im.

The length of a carbon linker between aromatic rings affected

the trypanocidal properties of bisbenzofurans 1-43. Antitry-

panosomal activities of 5-substituted bis(N-isopropyl)amidines

and bisimidazolines increased with carbon chain elongation. The

lowest antitrypanosomal IC₅₀values in this group revealed

compounds 24 and 25, containing a tetramethylene linker. The

same tendency was observed in the series of 6-substituted

bisamidines, where compound 26 with a four-carbon bridge

displayed the lowest IC₅₀value of 11 nM. Overall, among

bisbenzofurans with identical cationic substituents in either 5-

or 6- positions, derivatives with an even number of carbon atoms

in the alkyl linker displayed higher antitrypanosomal activity.

Among bisbenzofurans with different types of cationic groups,

unsubstituted amidines exhibited higher antitrypanosomal activ-

ity in vitro. Substitution on the amidine fragment usually reduced

trypanocidal properties of the compounds 1-43. Although for

5-substituted derivatives there was no apparent correlation

between the type of the cationic substituents and their antitry-

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5813

All compounds 1-43 were selective against the Trypanosoma

parasite (selectivity index⁷⁰SI_T, defined as a ratio of cytotoxic

IC₅₀to antitrypanosomal IC₅₀values, varied from 4 to more

than 3000). Bisimidazolines were the least selective against T.

b. rhodesiense, except compounds 19, 31, and 34, in which cases

corresponding bisamidines 17, 29, and 32 exhibited the lowest

antitrypanosomal selectivity indexes. The most selective com-

pound in the group against T. b. rhodesiense was bis(N-

isopropyl)amidine 12 (SI_T) 3075), showing antitrypanosomal

IC₅₀value of 40 nM. This molecule possessed an ethylene linker

and contained substituents in 5- and 6′-positions. Bisamidine

8, the most potent compound in the series, demonstrated highest

antitrypanosomal selectivity index among bisamidines (SI_T)

2688). Overall, selectivity against T. b. rhodesiense decreased

with elongation of the carbon chain, particularly in case of

6-substituted dications.

Antiplasmodial Activity. Dications 1-43 displayed antima-

larial activities with IC₅₀values ranging from 2 nM to 2.1 µM

(Table 2). Twenty four compounds (4, 6-21, 23-26, 30, 35,

and 36) displayed antiplasmodial activity comparable or exceed-

ing that of pentamidine (IC₅₀) 58 nM). Among those

compounds there were nine bisamidines (4, 8, 11, 14, 17, 20,

23, 26, and 35), eight bis(N-isopropyl)amidines (9, 12, 15, 18,

21, 24, 30, and 36) and seven bisimidazolines (6, 7, 10, 13, 16,

19 and 25). Twelve compounds (8-12, 14, 15, 18, 19, 24, 30,

and 36) demonstrated in vitro antiplasmodial IC₅₀values less

than 20 nM. This group included three bisamidines (8, 11, and

14), seven bis(N-isopropyl)amidines (9, 12, 15, 18, 24, 30, and

36) and two bisimidazolines (10 and 19). Among them seven

compounds (8-10, 18, 19, 24, and 30) had their cationic

substituents in the 5 positions, three were 6-substituted isomers

(14, 15, and 36), and two were 5,6′-substituted derivatives (11

and 12). Of the eight compounds with an even number of

methylene units in the linker, seven (8-12, 14, 15) contained

an ethylene bridge. Two compounds (18 and 19) had a propylene

linker and two (30 and 36) were connected by five-carbon

chains.

Seven of these dications (8, 9, 11, 12, 14, 15, and 19), all

except bisimidazoline 19 connected by two-carbon linkers,

exhibited antiplasmodial IC₅₀values lower than that of arte-

misinin (ATMS) (IC₅₀) 6 nM). Bisamidine 11, bearing cationic

substituents in the 5- and 6′-positions, was the most potent

compound in the series, with an antiplasmodial IC₅₀value of 2

nM.

The position of the cationic substituents affected efficacies

of bisbenzofurans 1-43 against P. falciparum. While 4-sub-

stituted isomers were significantly less potent, 5- and 6-substi-

tuted bisamidines displayed comparable antimalarial properties.

5-Substituted bis(N-isopropyl)amidines and bisimidazolines were

more effective against Plasmodium than 6-substituted derivatives

in all cases except for compounds 7 and 36.

Among bisbenzofurans with different types of cationic

substituents, bisamidines or bis(N-isopropyl)amidines were

usually more potent against P. falciparum than bisimidazolines,

except compound 19, which revealed superior antiplasmodial

efficacy compared to corresponding derivatives 17 and 18.

Bisamidines exhibited the best antiplasmodial activity when

benzofuran moieties contained methylene or ethylene bridges.

However, this tendency changed with elongation of the alkyl

linkers. Thus, for both 5- and 6-substituted bisbenzofurans

connected by three or more carbon atoms, bis(N-isopropyl)-

amidines demonstrated better antiplasmodial efficacy.

a ratio of cytotoxic IC₅₀to antiplasmodial IC₅₀values), ranging

from 3 to nearly 60000. Regardless of the position of the cationic

substituents and the length of the alkyl chain between the

aromatic moieties, bis(N-isopropyl)amidines displayed the high-

est antimalarial selectivity among the dications in all cases,

except 4-substituted bisamidine 1 and 5-substituted bisimida-

zolines 6 and 19, connected by the methylene (1 and 6) or the

propylene linker (19). The most potent compound in the series,

bisamidine 11, possessed antiplasmodial selectivity index SI_P

nearly twelve fold greater than that of pentamidine. Despite

comparable potency of the most effective bisamidines and bis-

(N-isopropyl)amidines, the latter are attractive as potential

antimalarial drug candidates due to their low cytotoxicity as

well as high selectivity for the Plasmodium parasite. Thus, the

most potent of the bis(N-isopropyl)amidines compound 9 not

only demonstrated an antiplasmodial IC₅₀value of 3 nM but

also was the most selective in the group against the P.

falciparum (SI_P) 59667), showing antiplasmodial selectivity

index 74-fold greater than that of pentamidine.

Dications 2, 6, 7, 9, 10, 15, 16, 18, 24, and 36 displayed

selectivity (S_P/T) (ratio of antitrypanosomal IC₅₀to antiplasmo-

dial IC₅₀values) equal or greater than 10-fold. The most

discriminatory of these compounds, bisimidazoline 7, demon-

strating 215-fold S_P/Tratio, exhibited an antiplasmodial IC₅₀of

22 nM. Bisbenzofurans 9, 15, 18, 24, and 36, with selectivity

(S_P/T) between 10 and 46-fold, were the most effective of the

ten dications as well as the least cytotoxic, with compound 9

providing the best combination of high antiplasmodial activity,

high selectivity (S_P/T), and low cytotoxicity.

Antileishmanial Activity. All compounds except bis(N-

isopropyl)amidine 2 and bisimidazoline 3, both of which

contained a mono-methylene linker, were active in the L.

donoVani axenic amastigote assay, demonstrating activity rang-

ing from 0.89 to 31 µM. Ten bisbenzofurans 8, 11, 12, 14, 23,

25, 26, 31, 32, and 34 displayed antileishmanial IC₅₀values

less than 2 µM. Among those compounds there were six

bisamidines (8, 11, 14, 23, 26, and 32), one bis(N-isopropyl)-

amidine (12), and three bisimidazolines (25, 31, and 34). Six

compounds (8, 23, 25, 31, 32, and 34) contained their substit-

uents in the 5,5′ positions, two were 6,6′-substituted bisamidines

(14 and 26), and two were mixed 5,6′-substituted derivatives

(11 and 12). Seven bisbenzofurans possessed aliphatic linkers

containing two (8, 11, 12, and 14) and four (23, 25, 26)

methylene groups, and three compounds (31, 32, and 34)

contained a five-carbon chain. Five bisamidines (8, 11, 14, 23,

26, and 32) as well as two bisimidazolines (31 and 34) exhibited

antileishmanial IC₅₀values equal or superior to that of penta-

midine (IC₅₀) 1.83 µM). Bisbenzofurans 8 and 14, both

connected by the ethylene bridge, were the most potent

compounds among 5- and 6-substituted isomers, respectively.

It is noteworthy that 5,6′-substituted bisamidine 11 displayed

lower antileishmanial activity than both 5-substituted compound

8 and 6-substituted isomer 14. Bisamidine 14 revealed the

highest antileishmanial activity in the series, exhibiting an IC₅₀

value of 887 nM.

Both 5- and 6-substituted isomers displayed comparable

antileishmanial properties. However, placement of the

cationic substituents in the 4-position of the benzofuran rings

caused a significant loss of efficacy, especially for derivatives

2 and 3.

Similar to T. b. rhodesiense, introduction of substituents in

the cationic moiety usually afforded less potent compounds

against L. donoVani axenic amastigotes, although exceptions did

occur. For both 5- and 6-substituted bisamidines and bis(N-

All bisbenzofurans 1-43 were selective against the P.

falciparum and displayed selectivity indexes⁷⁰(SI_P, defined as

5814 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

isopropyl)amidines, compounds connected by a linker containing

two or four methylene groups displayed better activity. In case

of bisimidazolines, however, the most effective compounds (31

and 37) contained a five-carbon chain.

and antitrypanosomal selectivity indexes SI_Tshows that six of

the synthesized dications (8, 12, 14, 15, 19, and 20) were less

cytotoxic and more selective against T. b. rhodesiense than

melarsoprol. None of the bisbenzofurans 1-43 reached the level

of antitrypanosomal selectivity of pentamidine. The P. falci-

parum strain K1 (resistant to chloroquine) was in general very

susceptible to the bisbenzofurans screened in this study. Twenty

two dications (4, 6-21, 23-25, 30, and 36) displayed greater

potency and were more selective against P. falciparum than

pentamidine. Seven bisbenzofurans (8, 9, 11, 12, 14, 15, and

19) exhibited antiplasmodial IC₅₀values superior to that of

artemisinin. In areas where the risk of cross-infection is high,

it is important to possess therapies that are highly selective for

P. falciparum over T. b. rhodesiense to treat patients infected

with both organisms. Ten dications 2, 6, 7, 9, 10, 15, 16, 18,

24, and 36 displayed selectivity (S_P/T) equal or greater than 10-

fold. In this group of discriminatory compounds bisbenzofurans

9, 15, 18, and 24, revealing selectivity (S_P/T) between 10 and

46-fold, were the most effective against P. falciparum as well

as the least cytotoxic. Ten bisbenzofurans (8, 11, 12, 14, 23,

25, 26, 31, 32, and 34) were equally or more potent than

pentamidine against L. donoVani axenic amastigotes. Eleven

bisbenzofurans (4, 9, 12, 14-17, 21, 23, 30, and 34) displayed

antileishmanial selectivity comparable or superior to that of

pentamidine.

All bisbenzofurans, except 4-substituted derivatives 1-3 and

5-substituted bisamidines 38-43, containing six to eleven

methylene groups in the alkyl linker, were selective against L.

donoVani and displayed selectivity indexes⁷⁰(SI_L, defined as a

ratio of cytotoxic IC₅₀to antileishmanial IC₅₀values), ranging

from 3 to 89. Among bisbenzofurans with different types of

cationic substituents, bisamidines or bis(N-isopropyl)amidines

were generally more selective against L. donoVani than bisimi-

dazolines. The only exception was 7-methoxy-substituted bi-

simidazoline 34, which exhibited a higher antileishmanial

selectivity index SI_Lthan that of corresponding dications 32

and 33. Although there was no clear correlation between the

length of the carbon linker connecting benzofuran rings and

the selectivity of 5-substituted derivatives, in the case of

6-substituted dications the selectivity against L. donoVani

decreased with elongation of the carbon chain. Among eleven

compounds with antileishmanial selectivity indexes SI_Lcom-

parable or superior to that of pentamidine (SI_L) 25), five

dications (9, 12, 14, 15, and 16) had ethylene linkers, two (30

and 34) were connected by five-carbon bridges, and two (17

and 21) contained three methylene groups in their alkyl chains.

Two bisamidines (4 and 23) possessed a one- and four-carbon

linker, respectively. Bisamidine 14 was not only the most potent

compound in the series but also demonstrated the greatest

selectivity against the L. donoVani axenic amastigotes (SI_L)

89), which was more than 3-fold higher than that of pentamidine.

Three major structural variations were explored: the place-

ment of substituents, the type of cationic groups, and the length

of the linker between two benzofuran moieties. The evaluation

of the effect of the position of cationic groups on the antipro-

tozoal efficacies of bisbenzofurans 1-43 demonstrated that

4-substituted derivatives 1-3 were significantly less active

against all three parasites as well as more cytotoxic against L6

cells than their 5- and 6-substituted counterparts. These results

revealed that the placement of cationic substituents affects the

antiprotozoal properties of dications 1-43. Both 5- and

6-substituted bisbenzofurans exhibited comparable activities

against T. b. rhodesiense, P. falciparum, and L. donoVani. These

findings correspond to the relative antimalarial activities of para-

versus meta-substituted pentamidine congeners.³⁰

Discussion

The absence of widespread resistance to pentamidine in the

field, despite its massive use as an HAT prophylactic,^7,37makes

the drug an attractive lead compound in a search for new

antiprotozoal therapies.^6,17,37,71The mechanism of action of

pentamidine is not completely understood.^7,23,34,35Its rapid

accumulation inside Trypanosoma and Plasmodium species,

resultingintheinhibitionofmultipletargetsintheparasites,^22,23,36-38

suggests the involvement of parasite specific uptake mechanisms

that are critical for the activity of pentamidine and absence of

the prevalent resistance to it.^35,36,38,39

In order to improve efficacy and selectivity against individual

parasites, heterocyclic cationic pentamidine analogues containing

benzofuran rings have been synthesized. This structural modi-

fication also intended to protect phenoxy groups from metabolic

deactivation by including them in the aromatic rings. Such an

alteration would allow bisbenzofurans to retain affinity for the

aminopurine P2 transporter and therefore their antitrypanosomal

activity.

In T. brucei species the P2 transporter partially mediates

uptakeofpentamidineandmelaminophenylarsenicals,^{35-37,39,40,72-74}

that share structural recognition motifs for the carrier.^35,39-41

Cross-resistance between pentamidine and arsenicals was ob-

served in laboratory strains of Trypanosoma species^75-77and

was attributed to the loss of the P2 delivery system.^{35,36,39,73,78}

Pentamidine, however, accumulates even in strains without

significant P2-transporter activity,³⁵suggesting the presence of

other routes of uptake. The existence of additional modes of

accumulation^{37,39,73,74,78}coupled with multiple modes of action

might explain this lack of widespread resistance to pentamidine.

Data presented in Table 2 indicate that bisbenzofuran 8

displayed in vitro antitrypanosomal activity comparable to that

of pentamidine and melarsoprol. Analysis of L6 cytotoxicity

Introduction of the substituents on the amidine groups usually

decreased antitrypanosomal^28,79-81and antileishmanial³²activi-

ties of bisbenzofurans 1-43. It is tempting to explain the

reduced antitrypanosomal efficacy of bis(N-isopropyl)amidines

and bisimidazolines by the higher affinity of unsubstituted

bisamidines for the aminopurine transporter. However, as was

previously shown, there is no direct correlation between the

affinity for the P2 carrier and the antitrypanosomal efficacies

of the compounds.^82,83Antimalarial properties of bisbenzofurans

1-43 were affected by substitution on the cationic groups in a

different way. Bis(N-isopropyl)amidines displayed antiplasmo-

dial activities comparable to that of unsubstituted amidines. One

possible explanation could be the different mechanism of uptake

of diamidines in P. falciparum, where pentamidine penetrates

the Plasmodium-infected erythrocyte membrane through a

parasite-induced pore with properties of the new permeability

pathway (NPP).^37,38Because the uptake through the NPP is

favorable for compounds with increased hydrophobicity,⁸⁴one

can speculate that substituted bisbenzofurans in which polar

amidine groups are shielded by aliphatic substituents might

exhibit higher activity compared to unsubstituted ones if

antiplasmodial efficacy depended only on the parasite uptake.

However, this assumption does not explain the lower antiplas-

modial activities of bisimidazolines compared to that of both

bisamidines and bis(N-isopropyl)amidines. Substitution on the

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5815

cationic groups also decreased the cytotoxicity of bisbenzofurans

1-43 against L6 cells.

against P. falciparum. Seven dications (8, 9, 11, 12, 14, 15,

and 19) revealed higher antiplasmodial efficacies than that of

artemisinin. Bisbenzofurans 2, 6, 7, 9, 10, 15, 16, 18, 24, and

36 were discriminatory for P. falciparum over T. b. rhodesiense,

showing parasite selectivity ratio (S_P/T) equal or greater than

10-fold. Bis(N-isopropyl)amidine 9 provided the best combina-

tion of low cytotoxicity, high antiplasmodial activity (IC₅₀) 3

nM), and high selectivity (S_P/T) 46). Compounds 8, 11, 14,

23, 26, 31, 32, and 34 displayed activity against L. donoVani

axenic amastigotes superior to that of pentamidine (IC₅₀) 1.83

µM). Bisamidine 14, exhibiting an antileishmanial IC₅₀value

of 887 nM, was the most potent compound in the series against

L. donoVani. It was twice as active and more than three times

as selective against the parasite as pentamidine. Promising in

vitro efficacies of selected bisbenzofuran cations against T. b.

rhodesiense, P. falciparum, and L. donoVani as well as their

reduced cytotoxicity compared to pentamidine warrant further

investigation of the antiprotozoal properties of these compounds

in vivo and the evaluation of oral activities of their prodrugs.

The length of the linker between aromatic rings influenced

the antiprotozoal properties of bisbenzofurans 1-43. Bisa-

midines connected by two-carbon chains demonstrated the

highest activity against T. b. rhodesiense, P. falciparum, and

L. donoVani. The length of the alkyl bridge and therefore the

distance between the two cationic moieties might affect not only

the efficacies of bisbenzofurans but also the development of

parasite resistance to this class of compounds. It was shown

that T. brucei strains resistant to propamidine remain sensitive

to pentamidine.^37,39This flexibility of pentamidine plays an

important role in the absence of the widespread resistance to

the drug. Other diamidines with lesser flexibility are all prone

to multidrug resistance.^37,39Elongation of the carbon linker also

decreased the selectivity of bisbenzofurans 1-43 against T. b.

rhodesiense, P. falciparum, and L. donoVani. Clearly, the

distance between two cationic groups in bisbenzofurans influ-

ences the efficacy and cytotoxicity of the compounds and might

as well affect the development of parasite resistance.

Currently, we cannot offer any reasonable explanation of how

and why the substitution on the amidine groups affects the

antiprotozoal properties of bisbenzofurans 1-43. To better

understand the mode of action and thus improve the efficacy

of aromatic diamidines, further studies of the influence of the

type of cationic substituents and the distance between aromatic

moieties on uptake and intracellular distribution of dicationic

bisbenzofurans are needed.

Experimental Section

General Experimental. In vitro antitrypanosomal, antiplasmo-

dial, and antileishmanial activities and cytotoxicities were deter-

mined following established protocols.^33,85-90

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/I₂immediately prior to

use. Reaction mixtures were monitored by TLC using Whatman

silica gel 250 µm UV₂₅₄plates or by reverse phase HPLC. Organic

layers of extraction mixtures were washed with saturated NaCl

solution and dried over Na₂SO₄or MgSO₄before being evaporated

under reduced pressure. Gravity and flash column chromatography

were performed using Davisil grade 633 type 60A silica gel (200-

425 mesh). Analytical HPLC chromatograms were recorded on a

Hewlett-Packard 1090 Series II 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. In method C, the

concentration of acetonitrile was increased linearly from 45 to 75%

over 10 min and then maintained for 2 min. In method D, the

concentration of acetonitrile was increased linearly from 45 to 75%

over 7 min and then maintained for 6 min. Chromatographic data

were recorded and analyzed using HP ChemStation software.

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

using an Agilent Zorbax Rx C8 column (4.6 mm × 75 mm, 3.5

µm) and the latter mobile phases on an Agilent Technologies 1100

chromatograph. Residues of evaporated pooled purified fractions

were reconstituted in water and lyophilized on a VirTis BenchTop

Conclusions

We have reported the synthesis and the results of the in vitro

testing of bisbenzofurans 1-43 against T. b. rhodesiense, P.

falciparum, and L. donoVani as well as the cytotoxicity of these

compounds against L6 mammalian cells. For the synthesis of

bisbenzofurans connected by a single carbon atom, o-hydroxy-

aldehydes reacted with R-halogenated ketones followed by

intramolecular ring closure. Compounds with multiple methyl-

ene groups in the linker were synthesized by means of copper-

or palladium-mediated heteroannulation of substituted o-io-

dophenols with terminal acetylenes. We found that the position

and the type of the cationic substituents in the molecule and

the length of the linker between the aromatic moieties affected

cytotoxicity and in vitro antiprotozoal activity of the compounds

1-43. 5-Substituted bisbenzofurans were generally less cyto-

toxic than compounds bearing their substituents in the 4- or 6-

positions. Cytotoxicity also decreased with substitution on the

cationic groups, with bis(N-isopropyl)amidines usually being

the least cytotoxic in the series, and increased with the

elongation of the carbon linker. Consequently, the selectivity

of bisbenzofurans 1-43 against T. b. rhodesiense, P. falciparum,

and L. donoVani decreased as number of methylene groups in

the alkyl bridge increased. 4-Substituted bisbenzofurans were

significantly less active against all parasites than corresponding

5- and 6-substituted isomers. Substitution on the cationic

moieties of bisbenzofurans resulted in compounds with inferior

antitrypanosomal and antileishmanial activity. Bisamidines and

bis(N-isopropyl)amidines were more efficient against P. falci-

parum than bisimidazolines. Bisamidines connected by two-

carbon chains demonstrated the highest activity against T. b.

rhodesiense, P. falciparum, and L. donoVani. Bisamidine 8

displayed an antitrypanosomal IC₅₀value of 8 nM comparable

to that of pentamidine and melarsoprol. Bisbenzofurans 4, 6-21,

23-25, 30, and 36 exhibited antiplasmodial activity exceeding

that of pentamidine and were up to 74-fold more selective

5816 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

2K lyophilizer. Acquired bisbenzofurans, as free bases, were

dissolved in ethanol and converted into HCl salts with aqueous

HCl.

M/Z 417.2 (MH⁺of free base). HPLC (method A) t_R6.29 min

(100 area %). Anal. (C₂₅H₂₈N₄O₂‚2HCl‚1.5H₂O‚0.5C₂H₅OH) C, H,

N, Cl.

Bis(4-(2-imidazolinyl)benzofuran-2-yl)methane Dihydrochlo-

ride (3). Light yellow solid (0.48 g, 70%): mp 333-335 °C (1.5

Flash Chromatography of Amidines on C₁₈Reversed Phase

Silica Gel. Chromatographic column was half-filled with acetonitrile

and packed with a slurry of C₁₈silica gel (70 g) in acetonitrile

(70-100 mL). Excess of acetonitrile was drained out, and the top

of the column was covered with 2 cm pad of sand. The column

was balanced 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 case of low solubility, heating of the mixture

and/or addition of a small amount of methanol as a cosolvent were

performed. After the reaction mixture was applied to the column,

the elution began with initial mobile phase (150 mL) to remove

excess of amine and then with a mobile phase consisting of 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 A. After the purification was

completed, the column was washed with acetonitrile (3 × 100 mL),

ethanol (100 mL), and deionized water (2 × 100 mL) and kept in

acetonitrile or acetonitrile-water mixture. Select fractions were

analyzed for purity using an Agilent Zorbax Rx C8 column (4.6

mm × 75 mm, 3.5 µm) and described mobile phases on an Agilent

Technologies 1100 chromatograph. Residues of evaporated pooled

purified fractions were reconstituted in water and lyophilized on a

VirTis BenchTop 2K lyophilizer. Acquired bisbenzofurans, as free

bases, were dissolved in ethanol and converted into HCl salts with

aqueous HCl.

Low-resolution ESI mass spectra were recorded on an Agilent

Technologies 1100 Series LC/MSD Trap spectrometer. Elemental

analyses were performed by Atlantic Microlab, Norcross, GA, and

were within (0.4% of calculated values.

Materials. All chemicals and solvents were purchased from

Aldrich Chemical Co., Fisher Scientific, or Acros Organics and

were used without further purification.

General Procedure for Syntheses of Diamidines (1-43). Bis-

(4-amidinobenzofuran-2-yl)methane Dihydrochloride (1). A

mixture of dry 1,4-dioxane (40 mL) and dry EtOH (10 mL) in a

three-neck 250 mL flask equipped with a gas inlet tube, a

thermometer, and a drying tube was saturated with gaseous HCl at

0 °C. Bis(4-cyanobenzofuran-2-yl)methane (41) (1.42 g, 4.8 mmol)

was added in one portion, the flask was sealed, and the mixture

was stirred at room temperature until the starting material was no

longer detectable by HPLC (1 day). After dilution of the reaction

mixture with dry ether, the diimidate (2.11 g, 96%), was filtered

off under argon, washed with diethyl ether, dried under high

vacuum, and reacted immediately with appropriate amines.

1

M HCl). H NMR (DMSO-d₆) δ 10.70 (s, 4H), 8.00 (d, J ) 8.2

Hz, 2H), 7.79 (d, J ) 7.7 Hz, 2H), 7.56 (dd, J ) 8.2 and 7.7 Hz,

2H), 7.26 (s, 2H), 4.68 (s, 2H), 4.04 (s, 8H). HPLC (method A) t_R

5.41 min (98.2 area %). Anal. (C₂₃H₂₀N₄O₂‚2HCl‚0.5H₂O) C, H,

N, Cl.

Bis(5-amidinobenzofuran-2-yl)methane Dihydrochloride (4).⁴⁴

Yellow solid (0.18 g, 19%): mp 212-214 °C (lit.⁴⁴201-206 °C).

¹H NMR (DMSO-d₆) δ 9.40 (s, 4H), 9.16 (s, 4H), 8.15 (d, J ) 1.6

Hz, 2H), 7.80 (d, J ) 8.8 Hz, 2H), 7.73 (dd, J ) 8.8 and 1.6 Hz,

2H), 7.02 (s, 2H), 4.59 (s, 2H). Anal. (C₁₉H₁₆N₄O₂‚2HCl‚0.6H₂O)

C, H, N, Cl.

Bis(5-(N-isopropyl)amidinobenzofuran-2-yl)methane Dihy-

drochloride (5). White solid (0.75 g, 65%): mp 238-240 °C. ¹H

NMR (DMSO-d₆) δ 9.58 (d, J ) 7.8 Hz, 2H), 9.44 (s, 2H), 9.09

(s, 2H), 8.03 (d, J ) 1.6 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H), 7.63

(dd, J ) 8.8 and 1.6 Hz, 2H), 7.00 (s, 2H), 4.59 (s, 2H), 4.08 (m,

2H), 1.29 (d, J ) 6.0 Hz, 12H). HPLC (method B) t_R1.49 min

(100 area %). Anal. (C₂₅H₂₈N₄O₂‚2HCl‚1.5H₂O) C, H, N, Cl.

Bis(5-(2-imidazolinyl)benzofuran-2-yl)methane Dihydrochlo-

1

ride (6). Light yellow solid (0.27 g, 25%): mp 233-235 °C. H

NMR (DMSO-d₆) δ 10.80 (s, 4H), 8.38 (s, 2H), 7.96 (d, J ) 8.8

Hz, 2H), 7.85 (d, J ) 8.8 Hz, 2H), 7.06 (s, 2H), 4.60 (s, 2H), 4.01

(s, 8H). HPLC (method A) t_R5.83 min (99.6 area %). Anal.

(C₂₃H₂₀N₄O₂‚2HCl‚1.1H₂O) C, H, N, Cl.

Bis(6-(2-imidazolinyl)benzofuran-2-yl)methane Dihydrochlo-

1

ride (7). Light brown solid (0.13 g, 50%): mp 341-343 °C. H

NMR (DMSO-d₆) δ 10.82 (s, 4H), 8.40 (s, 2H), 7.96 (d, J ) 8.2

Hz, 2H), 7.88 (d, J ) 8.2 Hz, 2H), 7.03 (s, 2H), 4.66 (s, 2H), 4.01

(s, 8H). HPLC (method A) t_R5.65 min (100 area %). Anal.

(C₂₃H₂₀N₄O₂‚2HCl‚2H₂O) C, H, N, Cl.

1,2-Bis(5-amidinobenzofuran-2-yl)ethane Dihydrochloride (8)⁴⁴

was prepared from dinitrile 59 (0.40 g, 1.28 mmol) to give, after

preparative HPLC and recrystallization from aqueous HCl, a yellow

solid (0.17 g, 32%): mp 356 °C (dec) (lit.⁴⁴293-295 °C (dec)).

¹H NMR (DMSO-d₆) δ 9.35 (s, 4H), 9.10 (s, 4H), 8.06 (s, 2H),

7.77 (d, J ) 8.8 Hz, 2H), 7.68 (d, J ) 8.8 Hz, 2H), 6.87 (s, 2H),

3.34 (s, 4H). HPLC (method A) t_R5.89 min (100 area %). Anal.

(C₂₀H₁₈N₄O₂‚2HCl‚1.3H₂O) C, H, N, Cl.

1,2-Bis(5-(N-isopropyl)amidinobenzofuran-2-yl)ethane Dihy-

drochloride (9) was prepared from dinitrile 59 (0.40 g, 1.28 mmol)

to give, after preparative HPLC and recrystallization from aqueous

HCl, a white solid (0.18 g, 29%): mp 350 °C (dec) (1.5 M HCl).

¹H NMR (DMSO-d₆) δ 9.54 (d, J ) 8.0 Hz, 2H), 9.41 (s, 2H),

9.06 (s, 2H), 7.95 (d, J ) 1.6 Hz, 2H), 7.75 (d, J ) 8.8 Hz, 2H),

7.59 (dd, J ) 8.8 and 1.6 Hz, 2H), 6.85 (s, 2H), 4.07 (m, 2H), 3.34

(s, 4H), 1.27 (d, J ) 6.0 Hz, 12H). HPLC (method A) t_R5.41 min

(100 area %). Anal. (C₂₆H₃₀N₄O₂‚2HCl) C, H, N, Cl.

1,2-Bis(5-(2-imidazolinyl)benzofuran-2-yl)ethane Dihydro-

chloride (10) was prepared from dinitrile 59 (0.40 g, 1.28 mmol)

to give, after preparative HPLC and recrystallization from aqueous

HCl, a beige solid (0.25 g, 42%): mp 366 °C (dec) (1.5 M HCl).

¹H NMR (DMSO-d₆) δ 10.68 (s, 4H), 8.29 (s, 2H), 7.90 (d, J )

8.8 Hz, 2H), 7.82 (d, J ) 8.8 Hz, 2H), 6.94 (s, 2H), 4.00 (s, 8H),

3.34 (s, 4H). HPLC (method A) t_R4.69 min (100 area %). Anal.

(C₂₄H₂₂N₄O₂‚2HCl‚1.7H₂O) C, H, N, Cl.

The diimidate (0.70 g, 1.5 mmol) was suspended in dry EtOH

saturated with gaseous ammonia (19 mL). The sealed reaction

mixture was stirred at room temperature. The progress of the

reaction was monitored by HPLC. After 4 days, the reaction mixture

was diluted with diethyl ether (50 mL) and placed in a freezer

overnight. The resulting precipitate was collected by filtration,

washed with diethyl ether, and dried under vacuum to give 0.45 g

(89%) of crude material, which was recrystallized from 1.5 M HCl

1

to afford 1, as a brown solid (0.14 g, 21%): mp 310-311 °C. H

1-(5-Amidinobenzofuranyl-2)-2-(6-amidinobenzofuranyl-2)-

ethane Dihydrochloride (11)⁴⁵was prepared from dinitrile 65. The

crude product was purified by flash chromatography on C₁₈reversed

phase silica gel to afford 11 (0.24 g, 46%): mp >300 °C (1.5 M

NMR (DMSO-d₆) δ 9.43 (s, 4H), 9.36 (s, 4H), 7.91 (d, J ) 8.2

Hz, 2H), 7.61 (d, J ) 7.7 Hz, 2H), 7.50 (dd, J ) 8.2 and 7.7 Hz,

2H), 7.00 (s, 2H), 4.64 (s, 2H). HPLC (method A) t_R4.67 min

(100 area %). Anal. (C₁₉H₁₆N₄O₂‚2HCl‚1H₂O) C, H, N, Cl.

1

HCl). H NMR (DMSO-d₆) δ 9.38 (s, 2H), 9.34 (s, 2H), 9.14 (s,

Bis(4-(N-isopropyl)amidinobenzofuran-2-yl)methane Dihy-

drochloride (2). Brown solid (0.06 g, 8%): mp 210 °C (dec)

4H), 8.09 (s, 1H), 8.08 (s, 1H), 7.76 (d, J ) 8.2 Hz, 2H), 7.70 (m,

2H), 6.87 (s, 2H), 3.33 (d, J ) 7.5 Hz, 4H). HPLC (method A) t_R

5.83 min (100 area %). Anal. (C₂₀H₁₈N₄O₂‚2HCl‚2H₂O) C, H, N,

Cl.

1

(diethyl ether/EtOH/1 M HCl). H NMR (DMSO-d₆) δ 9.77 (d, J

) 8.4 Hz, 2H), 9.55 (s, 2H), 9.41 (s, 2H), 7.87 (d, J ) 7.7 Hz,

2H), 7.55 (d, J ) 7.1 Hz, 2H), 7.48 (dd, J ) 7.7 and 7.1 Hz, 2H),

6.96 (s, 2H), 4.66 (s, 2H), 4.18 (m, 2H), 1.31 (d, J ) 6.0 Hz, 12H).

1-(5-N-Isopropylamidinobenzofuranyl-2)-2-(6-N-isopropyla-

midinobenzofuranyl-2)ethane Dihydrochloride (12) was prepared

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5817

from dinitrile 65. The crude product was purified by flash

chromatography on C₁₈reversed phase silica gel to afford 12 (0.20

g, 32%): mp 220 °C (1.5 M HCl). H NMR (DMSO-d₆) δ 9.57

1,3-Bis(6-(N-isopropyl)amidinobenzofuran-2-yl)propane Di-

hydrochloride (21). White solid (0.52 g, 41%): mp 289-291 °C

1

(dec). H NMR (DMSO-d₆) δ 9.54 (d, J ) 6.6 Hz, 2H), 9.42 (s,

(m, 2H), 9.48 (s, 1H), 9.43 (s, 1H), 9.12 (d, J ) 7.2 Hz, 2H), 7.97

(d, J ) 8.2 Hz, 2H), 7.74 (d, J ) 8.2 Hz, 2H), 7.59 (m, 2H), 6.85

(s, 2H), 4.10 (m, 2H), 3.35 (d, J ) 7.5 Hz, 4H), 1.29 (d, J ) 6.0

Hz, 12H). HPLC (method A) t_R7.68 min (100 area %). Anal.

(C₂₆H₃₀N₄O₂‚2HCl‚2.5H₂O) C, H, N, Cl.

2H), 9.06 (s, 2H), 7.97 (s, 2H), 7.75 (d, J ) 8.2 Hz, 2H), 7.57 (d,

J ) 8.2 Hz, 2H), 6.85 (s, 2H), 4.06 (m, 2H), 2.96 (t, J ) 7.1 Hz,

4H), 2.20 (p, J ) 7.1 Hz, 2H), 1.29 (d, J ) 6.0 Hz, 12H). HPLC

(method B) t_R2.33 min (100 area %). Anal. (C₂₇H₃₂N₄O₂‚2HCl‚

1H₂O) C, H, N, Cl.

1-(5-(2-Imidazolinyl)benzofuranyl-2)-2-(6-(2-imidazolinyl)-

benzofuranyl-2)ethane Dihydrochloride (13) was prepared from

dinitrile 65. The crude product was purified by flash chromatog-

raphy on C₁₈reversed phase silica gel to afford 13 (0.15 g, 25%):

1,3-Bis(6-(2-imidazolinyl)benzofuran-2-yl)propane Dihydro-

1

chloride (22). White solid (0.69 g, 59%): mp >300 °C (dec). H

NMR (DMSO-d₆) δ 10.75 (s, 4H), 8.31 (s, 2H), 7.86 (d, J ) 8.2

Hz, 2H), 7.77 (d, J ) 8.2 Hz, 2H), 6.86 (s, 2H), 4.02 (s, 8H), 2.98

(t, J ) 6.6 Hz, 4H), 2.20 (p, J ) 6.6 Hz, 2H). HPLC (method B)

t_R1.45 min (100 area %). Anal. (C₂₅H₂₄N₄O₂‚2HCl‚2H₂O) C, H,

N, Cl.

1

mp 225 °C (1.5 M HCl). H NMR (DMSO-d₆) δ 10.79 (s, 2H),

10.72 (s, 2H), 8.35 (s, 1H), 8.30 (s, 1H), 7.91 (d, J ) 8.2 Hz, 1H),

7.87 (d, J ) 8.2 Hz, 1H), 7.78 (d, J ) 8.8 Hz, 1H), 7.77 (d, J )

8.8 Hz, 1H), 6.90 (s, 1H), 6.88 (s, 1H), 3.98 (s, 8H), 3.34 (s, 4H).

HPLC (method A) t_R6.61 min (100 area %). Anal. (C₂₄H₂₂N₄O₂‚

2HCl‚2.5H₂O) C, H, N, Cl.

1,4-Bis(5-amidinobenzofuran-2-yl)butane Dihydrochloride

(23).⁴⁵White solid (0.13 g, 38%): mp 325-327 °C (dec) (1.5 M

1

HCl) (lit.⁴⁵335 °C (dec)). H NMR (DMSO-d₆) δ 9.35 (s, 4H),

1,2-Bis(6-amidinobenzofuran-2-yl)ethane Dihydrochloride (14)⁴⁵

was prepared from dinitrile 60 (0.13 g, 0.42 mmol) to give, after

preparative HPLC and recrystallization from aqueous HCl, a light

9.13 (s, 2H), 9.08 (s, 2H), 8.08 (d, J ) 1.6 Hz, 2H), 7.76 (d, J )

8.2 Hz, 2H), 7.69 (dd, J ) 8.2 and 1.6 Hz, 2H), 6.82 (s, 2H), 2.91

(br s, 4H), 1.81 (br s, 4H). HPLC (method B) t_R1.75 min (100

area %). Anal. (C₂₂H₂₂N₄O₂‚2HCl‚0.5H₂O) C, H, N, Cl.

1

yellow solid (0.12 g, 60%): mp 362 °C (dec) (1.5 M HCl). H

NMR (DMSO-d₆) δ 9.39 (s, 4H), 9.15 (s, 4H), 8.10 (d, J ) 1.1

Hz, 2H), 7.77 (d, J ) 8.2 Hz, 2H), 7.69 (dd, J ) 8.2 and 1.1 Hz,

2H), 6.88 (s, 2H), 3.34 (s, 4H). HPLC (method A) t_R5.53 min

(100 area %). Anal. (C₂₀H₁₈N₄O₂‚2HCl‚1H₂O) C, H, N, Cl.

1,2-Bis(6-(N-isopropyl)amidino benzofuran-2-yl)ethane Di-

hydrochloride (15) was prepared from dinitrile 60 (0.13 g, 0.42

mmol) to give, after preparative HPLC and recrystallization from

aqueous HCl, a light yellow solid (0.13 g, 54%): mp 346 °C (dec)

1,4-Bis(5-(N-isopropyl)amidinobenzofuran-2-yl)butane Dihy-

drochloride (24). White solid (0.17 g, 41%): mp 283-285 °C

1

(dec) (1.5 M HCl). H NMR (DMSO-d₆) δ 9.56 (d, J ) 8.2 Hz,

2H), 9.42 (s, 2H), 9.09 (s, 2H), 7.96 (d, J ) 1.6 Hz, 2H), 7.73 (d,

J ) 8.2 Hz, 2H), 7.59 (dd, J ) 8.2 and 1.6 Hz, 2H), 6.80 (s, 2H),

4.08 (m, 2H), 2.91 (br s, 4H), 1.81 (br s, 4H), 1.29 (d, J ) 6.0 Hz,

12H). Anal. (C₂₈H₃₄N₄O₂‚2HCl‚1.2H₂O) C, H, N, Cl.

1,4-Bis(5-(2-imidazolinyl)benzofuran-2-yl)butane Dihydro-

chloride (25). White solid (0.25 g, 63%): mp 323-324 °C (dec).

¹H NMR (DMSO-d₆) δ 10.78 (s, 4H), 8.33 (d, J ) 1.6 Hz, 2H),

7.94 (dd, J ) 8.8 and 1.6 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H), 6.84

(s, 2H), 4.01 (s, 8H), 2.91 (br s, 4H), 1.82 (br s, 4H). HPLC (method

A) t_R7.82 min (100 area %). Anal. (C₂₆H₂₆N₄O₂‚2HCl‚2.5H₂O)

C, H, N, Cl.

1

(1.5 M HCl). H NMR (DMSO-d₆) δ 9.58 (d, J ) 8.2 Hz, 2H),

9.47 (s, 2H), 9.11 (s, 2H), 7.98 (s, 2H), 7.75 (d, J ) 8.2 Hz, 2H),

7.58 (d, J ) 8.2 Hz, 2H), 6.86 (s, 2H), 4.10 (m, 2H), 3.34 (s, 4H),

1.29 (d, J ) 6.6 Hz, 12H). HPLC (method A) t_R7.51 min (100

area %). Anal. (C₂₆H₃₀N₄O₂‚2HCl‚1.2H₂O) C, H, N, Cl.

1,2-Bis(6-(2-imidazolinyl)benzofuran-2-yl)ethane Dihydro-

chloride (16). Yellow solid (0.21 g, 84%) (EtOH/DMSO/3 M

HCl): mp >360 °C. ¹H NMR (DMSO-d₆/D₂O) δ 10.60 (br s, 4H),

8.14 (d, J ) 1.1 Hz, 2H), 7.82 (d, J ) 8.2 Hz, 2H), 7.75 (dd, J )

8.2 and 1.1 Hz, 2H), 6.91 (s, 2H), 4.02 (s, 8H), 3.38 (s, 4H). HPLC

(method A) t_R6.27 min (100 area %). Anal. (C₂₄H₂₂N₄O₂‚1.9HCl‚

1H₂O) C, H, N, Cl.

1,4-Bis(6-amidinobenzofuran-2-yl)butane Dihydrochloride (26).

White solid (0.45 g, 73%): mp 328-330 °C (dec) (diethyl ether/

1

EtOH/1.5 M HCl). H NMR (DMSO-d₆) δ 9.38 (s, 4H), 9.14 (s,

4H), 8.09 (s, 2H), 7.76 (d, J ) 8.2 Hz, 2H), 7.68 (d, J ) 8.2 Hz,

2H), 6.81 (s, 2H), 2.93 (br s, 4H), 1.82 (br s, 4H). HPLC (method

A) t_R7.15 min (100 area %). Anal. (C₂₂H₂₂N₄O₂‚2HCl‚1.5H₂O)

C, H, N, Cl.

1,3-Bis(5-amidinobenzofuran-2-yl)propane Dihydrochloride

(17). White solid (0.47 g, 71%): mp 168-170 °C (1.5 M HCl).

¹H NMR (DMSO-d₆) δ 9.34 (s, 4H), 9.06 (s, 4H), 8.08 (d, J ) 1.6

Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H), 7.69 (dd, J ) 8.8 and 1.6 Hz,

2H), 6.88 (s, 2H), 2.95 (t, J ) 7.1 Hz, 4H), 2.17 (p, J ) 7.1 Hz,

2H). HPLC (method B) t_R1.31 min (100 area %). Anal. (C₂₁H₂₀N₄O₂‚

2HCl‚1H₂O) C, H, N, Cl.

1,4-Bis(6-(N-isopropyl)amidinobenzofuran-2-yl)butane Dihy-

drochloride (27). White solid (0.37 g, 51%): mp 282-283 °C

(dec) (diethyl ether/EtOH/1.5 M HCl). ¹H NMR (DMSO-d₆) δ 9.55

(d, J ) 8.2 Hz, 2H), 9.45 (s, 2H), 9.09 (s, 2H), 7.97 (s, 2H), 7.75

(d, J ) 8.2 Hz, 2H), 7.58 (d, J ) 8.2 Hz, 2H), 6.81 (s, 2H), 4.08

(m, 2H), 2.93 (br s, 4H), 1.81 (br s, 4H), 1.29 (d, J ) 6.0 Hz,

12H). HPLC (method B) t_R2.92 min (100 area %). Anal.

(C₂₈H₃₄N₄O₂‚2HCl‚0.5H₂O) C, H, N, Cl.

1,4-Bis(6-(2-imidazolinyl)benzofuran-2-yl)butane Dihydro-

chloride (28). White solid (0.37 g, 54%): mp 343-345 °C (dec).

¹H NMR (DMSO-d₆) δ 10.66 (s, 4H), 8.28 (s, 2H), 7.84 (d, J )

8.2 Hz, 2H), 7.80 (d, J ) 8.2 Hz, 2H), 6.84 (s, 2H), 4.02 (s, 8H),

2.94 (br s, 4H), 1.83 (br s, 4H). HPLC (method B) t_R1.94 min

(100 area %). Anal. (C₂₆H₂₆N₄O₂‚2HCl‚0.7H₂O) C, H, N, Cl.

1,5-Bis(5-amidinobenzofuran-2-yl)pentane Dihydrochloride

(29). White solid (0.40 g, 67%): mp 156-157 °C (1.5 M HCl).

¹H NMR (DMSO-d₆) δ 9.36 (s, 4H), 9.14 (br s, 4H), 8.07 (s, 2H),

7.75 (d, J ) 8.8 Hz, 2H), 7.68 (dd, J ) 8.8 and 1.6 Hz, 2H), 6.80

(s, 2H), 2.84 (t, J ) 7.7 Hz, 4H), 1.78 (m, 4H), 1.45 (m, 2H).

HPLC (method B) t_R2.29 min (100 area %). Anal. (C₂₃H₂₄N₄O₂‚

2HCl‚1.6H₂O) C, H, N, Cl.

1,3-Bis(5-(N-isopropyl)amidinobenzofuran-2-yl)propane Di-

hydrochloride (18). White solid (0.42 g, 53%): mp 195-197 °C

1

(1.5 M HCl). H NMR (DMSO-d₆) δ 9.58 (d, J ) 7.7 Hz, 2H),

9.45 (s, 2H), 9.13 (s, 2H), 7.98 (s, 2H), 7.74 (d, J ) 8.8 Hz, 2H),

7.60 (d, J ) 8.8 Hz, 2H), 6.86 (s, 2H), 4.10 (m, 2H), 2.95 (t, J )

7.1 Hz, 4H), 2.17 (p, J ) 7.1 Hz, 2H), 1.29 (d, J ) 6.6 Hz, 12H).

HPLC (method B) t_R2.52 min (100 area %). Anal. (C₂₇H₃₂N₄O₂‚

2HCl‚1H₂O) C, H, N, Cl.

1,3-Bis(5-(2-imidazolinyl)benzofuran-2-yl)propane Dihydro-

1

chloride (19). White solid (0.72 g, 97%): mp 203-205 °C. H

NMR (DMSO-d₆) δ 10.80 (s, 4H), 8.32 (d, J ) 1.6 Hz, 2H), 7.94

(dd, J ) 8.8 and 1.6 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H), 6.88 (s,

2H), 4.01 (s, 8H), 2.96 (t, J ) 7.1 Hz, 4H), 2.19 (p, J ) 7.1 Hz,

2H). HPLC (method A) t_R7.25 min (100 area %). Anal.

(C₂₅H₂₄N₄O₂‚2HCl‚1.2H₂O) C, H, N, Cl.

1,3-Bis(6-amidinobenzofuran-2-yl)propane Dihydrochloride

1,5-Bis(5-(N-isopropyl)amidinobenzofuran-2-yl)pentane Di-

hydrochloride (30) was prepared from dinitrile 72 (0.48 g, 1.36

mmol) to give, after preparative HPLC and recrystallization from

aqueous HCl, a white solid (0.20 g, 32%): mp 193-195 °C (1.5

M HCl). ¹H NMR (DMSO-d₆) δ 9.58 (d, J ) 7.0 Hz, 2H), 9.46 (s,

2H), 9.16 (s, 2H), 7.97 (s, 2H), 7.72 (d, J ) 8.2 Hz, 2H), 7.59 (d,

J ) 8.2 Hz, 2H), 6.79 (s, 2H), 4.11 (m, 2H), 2.85 (m, 4H), 1.77

1

(20). Light yellow solid (0.56 g, 53%): mp >300 °C (dec). H

NMR (DMSO-d₆) δ 9.42 (s, 4H), 9.21 (s, 4H), 8.11 (s, 2H), 7.76

(d, J ) 8.2 Hz, 2H), 7.70 (d, J ) 8.2 Hz, 2H), 6.85 (s, 2H), 2.97

(t, J ) 7.1 Hz, 4H), 2.18 (p, J ) 7.1 Hz, 2H). HPLC (method B)

t_R1.14 min (100 area %). Anal. (C₂₁H₂₀N₄O₂‚2HCl‚0.3H₂O) C, H,

N, Cl.

5818 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

(m, 4H), 1.45 (m, 2H) 1.29 (d, J ) 6.0 Hz, 12H). HPLC (method

A) t_R8.57 min (98.0 area %). Anal. (C₂₉H₃₆N₄O₂‚2HCl‚0.7H₂O)

C, H, N, Cl.

303 °C. ¹H NMR (DMSO-d₆) δ 9.41 (s, 4H), 9.23 (s, 4H), 8.09 (s,

2H), 7.75 (d, J ) 8.2 Hz, 2H), 7.70 (d, J ) 8.2 Hz, 2H), 6.79 (s,

2H), 2.82 (t, J ) 6.6 Hz, 4H), 1.71 (br s, 4H), 1.33 (s, 8H). HPLC

(method A) t_R8.82 min (100 area %). Anal. (C₂₆H₃₀N₄O₂‚2.3HCl)

C, H, N, Cl.

1,5-Bis(5-(2-imidazolinyl)benzofuran-2-yl)pentane Dihydro-

chloride (31). White solid (0.22 g, 37%): mp 155-157 °C (1.5 M

1

HCl). H NMR (DMSO-d₆) δ 10.69 (s, 4H), 8.28 (d, J ) 1.6 Hz,

1,9-Bis(5-amidinobenzofuranyl-2)nonane Dihydrochloride (41).

The crude product was purified by flash chromatography on

2H), 7.89 (dd, J ) 8.8 and 1.6 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H),

6.82 (s, 2H), 4.01 (s, 8H), 2.85 (t, J ) 7.1 Hz, 4H), 1.78 (m, 4H),

1.46 (m, 2H). HPLC (method A) t_R8.25 min (100 area %). Anal.

(C₂₇H₂₈N₄O₂‚2HCl‚2H₂O) C, H, N, Cl.

C

₁₈reversed phase silica gel to afford 41 (0.63 g, 55%): mp 167-

170 °C. ¹H NMR (DMSO-d₆) δ 9.36 (s, 4H), 9.12 (s, 4H), 8.07 (s,

2H), 7.75 (d, J ) 8.7 Hz, 2H), 7.69 (d, J ) 8.7 Hz, 2H), 6.79 (s,

2H), 2.81 (t, J ) 7.1 Hz, 4H), 1.68 (br s, 4H), 1.31 (br s, 10H).

HPLC (method B) t_R4.36 min (98.5 area %). Anal. (C₂₇H₃₂N₄O₂‚

2HCl‚1.5H₂O) C, H, N, Cl.

1,5-Bis(5-amidino-7-methoxybenzofuranyl-2)pentane Dihy-

drochloride (32). White solid (0.31 g, 60%): mp 187-189 °C

1

(dec) (1.5 M HCl). H NMR (DMSO-d₆) δ 9.43 (s, 4H), 9.20 (s,

4H), 7.70 (s, 2H), 7.36 (s, 2H), 6.77 (s, 2H), 4.02 (s, 6H), 2.83 (t,

J ) 7.1 Hz, 4H), 1.76 (m, 4H), 1.43 (m, 2H). HPLC (method A)

t_R8.25 min (100 area %). Anal. (C₂₅H₂₈N₄O₄‚2HCl) C, H, N, Cl.

1,5-Bis(5-N-isopropylamidino-7-methoxybenzofuranyl-2)pen-

tane Dihydrochloride (33). The crude product was purified by

preparative HPLC to yield a white solid (0.13 g, 22%): mp 165-

170 °C (1.5 M HCl). ¹H NMR (DMSO-d₆) δ 9.58 (d, J ) 7.7 Hz,

2H), 9.48 (s, 2H), 9.13 (s, 2H), 7.56 (s, 2H), 7.23 (s, 2H), 6.76 (s,

2H), 4.09 (m, 2H), 4.02 (s, 6H), 2.83 (t, J ) 7.1 Hz, 4H), 1.76 (m,

4H), 1.43 (m, 2H), 1.29 (d, J ) 6.6 Hz, 12H). HPLC (method A)

t_R9.29 min (100 area %). Anal. (C₃₁H₄₀N₄O₄‚2.1HCl‚2.4H₂O) C,

H, N, Cl.

1,10-Bis(5-amidinobenzofuranyl-2)decane Dihydrochloride

(42). The crude product was purified by flash chromatography on

C

₁₈reversed phase silica gel to afford 42 (0.75 g, 55%): mp 281-

283 °C. ¹H NMR (DMSO-d₆) δ 9.36 (s, 4H), 9.13 (s, 4H), 8.07 (s,

2H), 7.76 (d, J ) 8.2 Hz, 2H), 7.68 (d, J ) 8.2 Hz, 2H), 6.79 (s,

2H), 2.81 (t, J ) 7.1 Hz, 4H), 1.69 (br s, 4H), 1.29 (m, 12H).

HPLC (method B) t_R4.81 min (98.4 area %). Anal. (C₂₈H₃₄N₄O₂‚

2HCl‚0.2H₂O) C, H, N, Cl.

1,11-Bis(5-amidinobenzofuranyl-2)undecane Dihydrochloride

(43). The crude product was purified by flash chromatography on

reversed phase silica gel to afford 43 (0.43 g, 48%): mp 230-

233 °C. ¹H NMR (DMSO-d₆) δ 9.36 (s, 4H), 9.13 (s, 4H), 8.07 (s,

2H), 7.76 (d, J ) 8.8 Hz, 2H), 7.68 (d, J ) 8.8 Hz, 2H), 6.79 (s,

2H), 2.81 (t, J ) 7.1 Hz, 4H), 1.68 (m, 4H), 1.27 (m, 14H). HPLC

(method B) t_R5.27 min (100 area %). Anal. (C₂₉H₃₆N₄O₂‚2.1HCl‚

1.3H₂O) C, H, N, Cl.

1,5-Bis(5-(2-imidazolinyl)-7-methoxybenzofuranyl-2)pen-

tane Dihydrochloride (34). The crude product was purified by

preparative HPLC to yield a white solid (0.17 g, 30%): mp 197-

1

200 °C (1.5 M HCl). H NMR (DMSO-d₆) δ 10.80 (s, 4H), 7.90

(s, 2H), 7.65 (s, 2H), 6.79 (s, 2H), 4.01 (s, 14H), 2.83 (t, J ) 7.7

Hz, 4H), 1.76 (m, 4H), 1.43 (m, 2H). HPLC (method A) t_R8.36

min (100 area %). Anal. (C₂₉H₃₂N₄O₄‚2HCl‚2.5H₂O) C, H, N, Cl.

1,5-Bis(6-amidinobenzofuran-2-yl)pentane Dihydrochloride

(35). White solid (0.25 g, 26%): mp 243-245 °C (dec) (EtOH/1

General Procedure for Synthesis of Bisbenzofuranmetha-

nones (47, 48). Bis(4-bromobenzofuran-2-yl)methanone (47).

Dried K₂CO₃(7.00 g, 50.1 mmol) was added to a solution of

2-bromo-6-hydroxybenzaldehyde (45) (6.57 g, 32.3 mmol) in dry

2-butanone (35 mL). The suspension was refluxed for 30 min and

allowed to cool. A solution of 1,3-dichloroacetone (3.00 g, 24.2

mmol) in dry 2-butanone (5 mL) was added. The reaction mixture

was refluxed for 4 h, cooled, filtered through a pad of Celite

(2 cm), and concentrated. A dark residue was recrystallized

from CHCl₃to give 47 as a brown solid (5.05 g, 76%): mp 223-

225 °C. ¹H NMR (DMSO-d₆) δ 8.10 (s, 2H), 7.92 (d, J ) 8.2 Hz,

2H), 7.70 (d, J ) 7.7 Hz, 2H), 7.57 (dd, J ) 8.2 and 7.7 Hz, 2H).

HPLC (method B) t_R9.83 min (100 area %). Anal. (C₁₇H₈Br₂O₃)

C, H, Br.

1

M HCl). H NMR (DMSO-d₆) δ 9.42 (s, 4H), 9.22 (s, 4H), 8.10

(s, 2H), 7.76 (d, J ) 8.2 Hz, 2H), 7.70 (d, J ) 8.2 Hz, 2H), 6.79

(s, 2H), 2.86 (t, J ) 7.1 Hz, 4H), 1.78 (m, 4H), 1.44 (m, 2H).

Anal. (C₂₃H₂₄N₄O₂‚2HCl‚0.7H₂O) C, H, N, Cl.

1,5-Bis(6-(N-isopropyl)amidinobenzofuran-2-yl)pentane Di-

hydrochloride (36). White solid (0.48 g, 40%): mp 193-195 °C

1

(dec) (i-PrOH/1.5 M HCl). H NMR (DMSO-d₆) δ 9.55 (d, J )

7.0 Hz, 2H), 9.45 (s, 2H), 9.10 (s, 2H), 7.96 (s, 2H), 7.73 (d, J )

8.2 Hz, 2H), 7.57 (d, J ) 8.2 Hz, 2H), 6.79 (s, 2H), 4.10 (m, 2H),

2.86 (m, 4H), 1.78 (m, 4H), 1.46 (m, 2H), 1.29 (d, J ) 6.0 Hz,

12H). HPLC (method B) t_R3.47 min (100 area %). Anal.

(C₂₉H₃₆N₄O₂‚2HCl‚1.3H₂O) C, H, N, Cl.

Bis(6-bromobenzofuran-2-yl)methanone (48). Yellow solid

1

(2.16 g, 76%): mp 176-177 °C (CH₂Cl₂). H NMR (DMSO-d₆)

δ 8.26 (s, 2H), 8.17 (br s, 2H), 7.87 (d, J ) 8.8 Hz, 2H), 7.61 (dd,

J ) 8.7 and 1.6 Hz, 2H). HPLC (method B) t_R9.62 min (100 area

%). Anal. (C₁₇H₈Br₂O₃) C, H, Br.

1,5-Bis(6-(2-imidazolinyl)benzofuran-2-yl)pentane Dihydro-

chloride (37). White solid (0.62 g, 49%): mp 308-310 °C (dec)

1

(i-PrOH/1.5 M HCl). H NMR (DMSO-d₆) δ 10.74 (s, 4H), 8.31

General Procedure for Synthesis of Bisbenzofuranmethanes

(49, 50). Bis(4-bromobenzofuran-2-yl)methane (49). A solution

of aluminum chloride (4.80 g, 11.4 mmol) in dry diethyl ether (70

mL) was added dropwise under Ar to a stirred suspension of lithium

aluminum hydride (0.98 g, 25.8 mmol) in dry diethyl ether (100

mL). Vacuum-dried 47 (4.80 g, 11.4 mmol) was added in small

portions. The reaction mixture was stirred at room temperature for

1 h followed by careful addition of 2 M HCl (100 mL) and

extraction with diethyl ether (2 × 100 mL). The ether solution was

dried over CaCl₂and concentrated to provide 49 as a white solid

(3.84 g, 83%): mp 142-143 °C (diethyl ether) ¹H NMR (DMSO-

d₆) δ 7.60 (d, J ) 8.2 Hz, 2H), 7.47 (d, J ) 7.7 Hz, 2H), 7.24 (dd,

J ) 8.2 and 7.7 Hz, 2H), 6.80 (s, 2H), 4.52 (s, 2H). HPLC (method

B) t_R10.67 min (100 area %). Anal. (C₁₇H₁₀Br₂O₃) C, H, Br.

Bis(6-bromobenzofuran-2-yl)methane (50). White solid (1.43

g, 99%): mp 146-147 °C (diethyl ether). ¹H NMR (DMSO-d₆) δ

7.85 (br s, 2H), 7.55 (d, J ) 8.2 Hz, 2H), 7.39 (dd, J ) 8.2 and 1.6

Hz, 2H), 6.82 (s, 2H), 4.44 (s, 2H). HPLC (method B) t_R10.43

min (100 area %). Anal. (C₁₇H₁₀Br₂O₃) C, H, Br.

(s, 2H), 7.86 (d, J ) 8.2 Hz, 2H), 7.78 (d, J ) 8.2 Hz, 2H), 6.81

(s, 2H), 4.02 (s, 8H), 2.87 (t, J ) 7.1 Hz, 4H), 1.79 (m, 4H), 1.45

(m, 2H). Anal. (C₂₇H₂₈N₄O₂‚2HCl‚1.5H₂O‚0.3i-PrOH) C, H, N, Cl.

1,6-Bis(5-amidinobenzofuranyl-2)hexane Dihydrochloride

(38).⁴⁵The crude product was purified by flash chromatography

on C₁₈reversed phase silica gel to afford 38 (0.54 g, 48%): mp

307-309 °C (lit.⁴⁵305 °C (dec)). ¹H NMR (DMSO-d₆) δ 9.39 (s,

4H), 9.19 (s, 4H), 8.08 (s, 2H), 7.75 (d, J ) 8.2 Hz, 2H), 7.69 (d,

J ) 8.2 Hz, 2H), 6.79 (s, 2H), 2.83 (t, J ) 6.6 Hz, 4H), 1.73 (br

s, 4H), 1.42 (br s, 4H). HPLC (method A) t_R8.19 min (100 area

%). Anal. (C₂₄H₂₆N₄O₂‚2HCl‚0.2H₂O) C, H, N, Cl.

1,7-Bis(5-amidinobenzofuranyl-2)heptane Dihydrochloride

(39). The crude product was purified by flash chromatography on

C

₁₈reversed phase silica gel to afford 39 (0.69 g, 74%): mp 130-

132 °C. ¹H NMR (DMSO-d₆) δ 9.36 (s, 4H), 9.12 (s, 4H), 8.07 (s,

2H), 7.75 (d, J ) 8.8 Hz, 2H), 7.68 (d, J ) 8.8 Hz, 2H), 6.79 (s,

2H), 2.83 (t, J ) 7.1 Hz, 4H), 1.71 (br s, 4H), 1.37 (s, 6H). HPLC

(method A) t_R8.47 min (99.0 area %). Anal. (C₂₅H₂₈N₄O₂‚2HCl‚

2H₂O) C, H, N, Cl.

General Procedure for Synthesis of Bisbenzofuranmethane

Carbodinitriles (51, 52). Bis(4-cyanobenzofuran-2-yl)methane

(51). A mixture of bis(4-bromobenzofuran-2-yl)methanone (49)

(3.50 g, 8.62 mmol) and CuCN (6.12 g, 69.0 mmol) was heated

1,8-Bis(5-amidinobenzofuranyl-2)octane Dihydrochloride (40).

The crude product was purified by flash chromatography on

C₁₈reversed phase silica gel to afford 40 (0.76 g, 82%): mp 302-

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5819

under reflux in dry quinoline (10 mL) for 2 h. The hot reaction

mixture was poured into 2 M HCl and stirred at 50-70 °C for 1 h.

A precipitate formed was separated, dried under vacuum, and

suspended in chlorobenzene (300 mL). The mixture was stirred

for 1 h at ambient temperature and filtered. The filtrate was

concentrated, and a dark solid residue (2.00 g, 78%) was purified

by column chromatography eluting with CHCl₃to produce 51 as a

57 (8.50 g, 36.6 mmol) and K₂CO₃(10.2 g, 74.0 mmol) in dry

methanol (500 mL). The mixture was stirred at ambient temperature

until complete conversion was attained according to HPLC (2 h)

and concentrated. The residue was diluted with diethyl ether (500

mL), washed with water (2 × 100 mL), and dried over CaCl₂. The

solvent was removed and the residue was recrystallized from (9:1)

hexanes/EtOAc to produce 59 as a white solid (7.66 g, 92%): mp

57-58 °C. ¹H NMR (DMSO-d₆) δ 8.23 (d, J ) 1.6 Hz, 1H), 7.88

(dd, J ) 8.8 and 1.6 Hz, 1H), 7.63 (d, J ) 8.8 Hz, 1H), 6.82 (s,

1H), 3.86 (s, 3H), 3.00 (t, J ) 7.1 Hz, 2H), 2.84 (t, J ) 2.8 Hz,

1H), 2.63 (td, J ) 7.1 and 2.8 Hz, 2H). HPLC (method B) t_R6.73

min (100 area %). Anal. (C₁₄H₁₂O₃‚0.2H₂O) C, H.

6-Methoxycarbonyl-2-(3-butynyl-1)benzofuran (60). Follow-

ing the procedure described above for 59, 60 was prepared from

58 as a white solid (0.40 g, 98%): mp 68-69 °C (hexanes/EtOAc).

¹H NMR (DMSO-d₆) δ 8.05 (d, J ) 1.1 Hz, 1H), 7.84 (dd, J )

8.2 and 1.1 Hz, 1H), 7.68 (d, J ) 8.2 Hz, 1H), 6.83 (s, 1H), 3.87

(s, 3H), 3.02 (t, J ) 7.1 Hz, 2H), 2.86 (t, J ) 2.8 Hz, 1H), 2.63

(td, J ) 7.1 and 2.8 Hz, 2H). HPLC (method B) t_R6.71 min (97.6

area %). Anal. (C₁₄H₁₂O₃‚0.1H₂O) C, H.

1

light yellow solid (1.62 g, 63%): mp 173-174 °C (CHCl₃). H

NMR (DMSO-d₆) δ 7.96 (d, J ) 8.2 Hz, 2H), 7.77 (d, J ) 7.7 Hz,

2H), 7.46 (dd, J ) 8.2 and 7.7 Hz, 2H), 7.09 (s, 2H), 4.64 (s, 2H).

HPLC (method B) t_R7.92 min (100 area %). Anal. (C₁₉H₁₀N₂O₂)

C, H, N.

Bis(6-cyanobenzofuran-2-yl)methane (52). Yellow solid (0.55

1

g, 56%): mp 207-209 °C (CHCl₃). H NMR (DMSO-d₆) δ 8.19

(s, 2H), 7.80 (d, J ) 8.2 Hz, 2H), 7.66 (d, J ) 8.2 Hz, 2H), 7.00

(s, 2H), 4.61 (s, 2H). HPLC (method B) t_R7.92 min (96.6 area %).

Anal. (C₁₉H₁₀N₂O₂‚0.3H₂O) C, H, N.

General Procedure for the Copper-Mediated Heteroannula-

tion. 5-Methoxycarbonyl-2-(3-hydroxypropyl-1)benzofuran (55).⁵²

A mixture of methyl 4-hydroxy-3-iodobenzoate (53) (35.0 g, 126

mmol), 4-pentyn-1-ol (11.0 g, 131 mmol), and copper(I) oxide

(12.6 g, 88.2 mmol) in dry pyridine (150 mL) was stirred at 100-

120 °C overnight. The mixture was allowed to cool to ambient

temperature, diluted with EtOAc (200 mL), filtered through a Celite

pad (5 cm), and concentrated. The residue was dissolved in EtOAc

(300 mL), washed with 2 M HCl (50 mL) and brine (100 mL),

dried over Na₂SO₄, and concentrated. The residue was purified by

column chromatography, eluting with (1:1) hexanes/EtOAc, fol-

lowed by recrystallization from (2:1) hexanes/diethyl ether to

produce 55 as a light yellow solid (14.4 g, 49%). ¹H NMR (DMSO-

d₆) δ 8.20 (s, 1H), 7.86 (d, J ) 8.2 Hz, 1H), 7.61 (d, J ) 8.2 Hz,

1H), 6.74 (s, 1H), 4.59 (br s, 1H), 3.86 (s, 3H), 3.49 (br s, 2H),

2.84 (t, J ) 7.1 Hz, 2H), 1.85 (m, 2H). HPLC (method B) t_R4.20

min (100 area %).

1,2-Bis(5-methoxycarbonylbenzofuran-2-yl)ethane (61). Fol-

lowing the procedure described above for 55, 61 was prepared from

53 and 59 as a white solid (5.24 g, 43%): mp 165-167 °C

1

(hexanes/EtOAc). H NMR (DMSO-d₆) δ 8.20 (s, 2H), 7.87 (d, J

) 8.8 Hz, 2H), 7.63 (d, J ) 8.8 Hz, 2H), 6.82 (s, 2H), 3.86 (s,

6H), 3.33 (s, 2H), 3.29 (s, 2H). Anal. (C₂₂H₁₈O₆‚0.8H₂O) C, H.

1-(5-Methoxycarbonylbenzofuran-2-yl)-2-(6-methoxycarbo-

nylbenzofuran-2-yl)ethane (62). Following the procedure de-

scribed above for 55, 62 was prepared from 53 and 54 as a white

solid (1.70 g, 69%): mp 108-109 °C (CH₂Cl₂). ¹H NMR (DMSO-

d₆) δ 8.20 (s, 1H), 8.05 (s, 1H), 7.86 (d, J ) 8.8 Hz, 1H), 7.84 (d,

J ) 8.8 Hz, 1H), 7.66 (d, J ) 8.8 Hz, 1H), 7.63 (d, J ) 8.8 Hz,

1H), 6.83 (s, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 3.30 (s, 4H). HPLC

(method B) t_R7.86 min (100 area %). Anal. (C₂₂H₁₈O₆) C, H.

1,2-Bis(6-methoxycarbonylbenzofuran-2-yl)ethane (63). Fol-

lowing the procedure described above for 55, 63 was prepared from

54 and 60 as a white solid (2.00 g, 48%): mp 175-177 °C (CH₂-

Cl₂). ¹H NMR (DMSO-d₆) δ 8.04 (br s, 2H), 7.83 (dd, J ) 8.2 and

1.1 Hz, 2H), 7.65 (d, J ) 8.2 Hz, 2H), 6.83 (s, 2H), 3.87 (s, 6H),

3.33 (s, 4H). HPLC (method B) t_R9.16 min (100 area %). Anal.

(C₂₂H₁₈O₆) C, H.

6-Methoxycarbonyl-2-(3-hydroxypropyl-1)benzofuran (56).

Following the procedure described above for 55, 56 was prepared

from 54 and 4-pentyn-1-ol as a light yellow solid (8.90 g, 78%):

1

mp 63-65 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ 8.04 (s,

1H), 7.81 (d, J ) 8.2 Hz, 1H), 7.65 (d, J ) 8.2 Hz, 1H), 6.74 (s,

1H), 4.59 (br s, 1H), 3.87 (s, 3H), 3.49 (t, J ) 6.0 Hz, 2H), 2.86

(t, J ) 7.7 Hz, 2H), 1.85 (m, 2H). HPLC (method B) t_R4.17 min

(96.5 area %). Anal. (C₁₃H₁₄O₄‚0.1H₂O) C, H.

1,2-Bis(5-cyanobenzofuran-2-yl)ethane (64).⁴⁴Method 1. Con-

version of Bisbenzofuran Esters to Dinitriles with Dimethyla-

luminum Amide. Anhydrous NH₃was bubbled through dry

o-xylene (100 mL) for 20 min at 0 °C. A 2.0 M solution of AlMe₃

in toluene (27 mL, 54.0 mmol) was added, the NH₃was passed

through the mixture for 20 min, and then the solution was stirred

at room temperature for 1 h. 1,2-Bis(5-methoxycarbonylbenzofuran-

2-yl)ethane (61) (5.00 g 13.2 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(250 mL). Water (100

mL) was added dropwise with vigorous stirring, and a precipitate

formed was filtered off. The filtrate was concentrated to give crude

64 (4.00 g, 97%), which was purified by column chromatography,

eluting with CH₂Cl₂, to give the desired product as a light yellow

solid (1.96 g, 48%): mp 243-244 °C (CH₂Cl₂) (lit.⁴⁴248-250

5-Methoxycarbonyl-2-(2-formylethyl-1)benzofuran (57). Ox-

alyl chloride (2 M solution in CH₂Cl₂, 40 mL, 80 mmol) was placed

under Ar into a reaction flask, equipped with addition funnel, inlet

thermometer, and drying tube charged with Drierite and cooled to

-70 °C. A mixture of DMSO (12.2 mL, 171 mmol) and dry CH₂-

Cl₂(30 mL) was added dropwise. After 30 min a solution of 55

(14.3 g, 61.0 mmol) in CH₂Cl₂was slowly added and the mixture

was stirred at -70 °C for 2 h. After that time, triethylamine (45.2

mL, 405 mmol) was added dropwise at -60 °C. The mixture was

allowed to warm to ambient temperature, water (100 mL) was added

carefully, and the stirring was continued for 10 min. The phases

were separated, and the aqueous layer was extracted with CH₂Cl₂

(2 × 75 mL). Combined organic layers were washed with 2 M

HCl (30 mL), dried over Na₂SO₄, and concentrated. The residue

was purified by column chromatography, eluting with (5:1) hexanes/

EtOAc, followed by recrystallization from (3:1) hexanes/EtOAc

to produce 52 as a light yellow solid (12.9 g, 91%): mp. 48-49

°C. ¹H NMR (DMSO-d₆) δ 9.70 (s, 1H), 8.20 (s, 1H), 7.87 (d, J )

8.2 Hz, 1H), 7.62 (d, J ) 8.2 Hz, 1H), 6.75 (s, 1H), 3.87 (s, 3H),

3.07 (m, 2H), 2.96 (m, 2H). Anal. (C₁₃H₁₂O₄‚0.1H₂O) C, H.

6-Methoxycarbonyl-2-(2-formylethyl-1)benzofuran (58). Fol-

lowing the procedure described above for 57, 58 was prepared from

56 as a light yellow solid (2.78 g, 85%): mp 57-58 °C (hexanes/

1

°C). H NMR (DMSO-d₆) δ 8.10 (br s, 2H), 7.73 (d, J ) 8.2 Hz,

2H), 7.68 (dd, J ) 8.2 and 1.6 Hz, 2H), 6.82 (s, 2H), 3.31 (s, 4H).

HPLC (method B) t_R8.15 min (100 area %). Anal. (C₂₀H₁₂N₂O₂‚

0.6H₂O) C, H, N.

Method 2. Copper-Mediated Heteroannulation. A mixture of

4-hydroxy-3-iodobenzonitrile (83) (10.0 g, 40.0 mmol), Cu₂O (3.15

g, 22.0 mmol), and 1,5-hexadiene (50% solution in pentane, 4.00

g, 25.0 mmol) in pyridine (100 mL) was stirred at room temperature

for 5 h and then refluxed overnight. The mixture was concentrated,

diluted with chloroform, filtered through a pad of Celite (2 cm),

and concentrated. An oily residue was treated with acetonitrile (50

mL) to form a precipitate, which was recrystallized from CH₂Cl₂

to give pure 64 (1.90 g, 24%).

1

EtOAc). H NMR (DMSO-d₆) δ 9.76 (s, 1H), 8.04 (s, 1H), 7.83

(d, J ) 8.2 Hz, 1H), 7.66 (d, J ) 8.2 Hz, 1H), 6.75 (s, 1H), 3.87

(s, 3H), 3.09 (m, 2H), 2.95 (m, 2H). Anal. (C₁₃H₁₂O₄) C, H.

5-Methoxycarbonyl-2-(3-butynyl-1)benzofuran (59). A solu-

tion of dimethyl 1-diazo-2-oxopropylphosphonate⁶⁴(8.90 g, 46.4

mmol) in dry methanol (50 mL) was added to a stirring mixture of

Method 3. Palladium-Catalyzed Heteroannulation. 4-Hy-

droxy-3-iodobenzonitrile (83) (2.52 g, 10.3 mmol) was added to a

5820 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

stirred suspension of DMF (8 mL), triethylamine (6 mL), PdCl₂-

(PPh₃)₂(0.28 g, 2 mol %), and CuI (0.28 g). The resulting mixture

was stirred at room temperature for 15 min, and 5-cyano-2-(3-

butynyl-1)benzofuran (86) (2.00 g, 10.2 mmol) was added at once.

The resulting mixture was heated at 80 °C for 15 min, slowly cooled

to ambient temperature, and stirred for 2 days. A precipitate formed

was filtered off, washed with ethyl acetate, and dried under vacuum

to yield 64 (2.00 g, 63%).

1-(5-Cyanobenzofuranyl-2)-2-(6-cyanobenzofuranyl-2)-

ethane (65) was prepared from 61 following the procedure

described above for 64 in Method 1. Light yellow solid (0.65 g,

46%): mp 235-237 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆) δ 8.14 (s,

1H), 8.10 (s, 1H), 7.74 (d, J ) 8.2 Hz, 2H), 7.67 (dd, J ) 8.2 and

1.6 Hz, 1H), 7.61 (dd, J ) 8.2 and 1.6 Hz, 1H), 6.86 (s, 1H), 6.82

(s, 1H), 3.31 (s, 4H). HPLC (method B) t_R6.43 min (100 area %).

Anal. (C₂₀H₁₂N₂O₂‚0.2H₂O) C, H, N.

Following the procedure described above for 64 in Method 3,

65 was prepared from 5-cyano-2-(3-butynyl-1)benzofuran (86) and

3-hydroxy-4-iodobenzonitrile (87) as a light yellow solid (2.22 g,

63%).

1,2-Bis(6-cyanobenzofuran-2-yl)ethane (66).⁴⁵Following the

procedure described above for 64 in Method 1, 66 was prepared

from 63 as a light yellow solid (0.56 g, 27%): mp 266-267 °C

(CH₂Cl₂) (lit.⁴⁵272-274 °C). ¹H NMR (DMSO-d₆) δ 8.16 (s, 2H),

7.75 (d, J ) 8.2 Hz, 2H), 7.63 (d, J ) 8.2 Hz, 2H), 6.88 (s, 2H),

3.35 (s, 4H). HPLC (method B) t_R8.26 min (100 area %). Anal.

(C₂₀H₁₂N₂O₂‚0.1H₂O) C, H, N.

Following the procedure described above for 64 in Method 3,

70 was prepared from 54 and 1,7-octadiyne as a white solid (5.62

g, 45%).

1,5-Bis(5-methoxycarbonylbenzofuran-2-yl)pentane (71). Fol-

lowing the procedure described above for 64 in Method 2, 71 was

prepared from 53 and 1,8-nonadiyne as a yellow solid (6.15 g,

1

72%): mp 83-85 °C (EtOH). H NMR (DMSO-d₆) δ 8.16 (d, J

) 1.6 Hz, 2H), 7.84 (dd, J ) 8.8 and 1.6 Hz, 2H), 7.59 (d, J ) 8.8

Hz, 2H), 6.71 (s, 2H), 3.86 (s, 6H), 2.81 (t, J ) 7.1 Hz, 4H), 1.76

(m, 4H), 1.43 (m, 2H). HPLC (method B) t_R10.79 min (100 area

%). Anal. (C₂₅H₂₄O₆) C, H.

Following the procedure described above for 64 in Method 3,

71 was prepared from 53 and 1,8-nonadiyne as a yellow solid (0.71

g, 75%)

1,5-Bis(6-methoxycarbonylbenzofuran-2-yl)pentane (72). Fol-

lowing the procedure described above for 64 in Method 2, 72 was

prepared from 54 and 1,8-nonadiyne as a yellow solid (8.19 g,

84%): mp 83-85 °C (EtOH). ¹H NMR (DMSO-d₆) δ 8.01 (s, 2H),

7.82 (d, J ) 8.2 Hz, 2H), 7.62 (d, J ) 8.2 Hz, 2H), 6.73 (s, 2H),

3.87 (s, 6H), 2.84 (t, J ) 7.1 Hz, 4H), 1.77 (m, 4H), 1.45 (m, 2H).

HPLC (method B) t_R10.63 min (100 area %). Anal. (C₂₅H₂₄O₆) C,

H.

Following the procedure described above for 64 in Method 3,

71 was prepared from 54 and 1,8-nonadiyne as a yellow solid (8.00

g, 99%)

1,3-Bis(5-cyanobenzofuran-2-yl)propane (73). Following the

procedure described above for 64 in Method 1, 73 was prepared

from 67 as a white solid (2.47 g, 49%): mp 153-154 °C (hexanes/

EtOAc). ¹H NMR (DMSO-d₆) δ 8.07 (d, J ) 1.6 Hz, 2H), 7.72 (d,

J ) 8.8 Hz, 2H), 7.65 (dd, J ) 8.8 and 1.6 Hz, 2H), 6.79 (s, 2H),

2.93 (t, J ) 7.1 Hz, 4H), 2.15 (p, J ) 7.1 Hz, 2H). HPLC (method

B) t_R8.69 min (96.1 area %). Anal. (C₂₁H₁₄N₂O₂‚0.2EtOAc) C, H,

N.

1,3-Bis(6-cyanobenzofuran-2-yl)propane (74). Following the

procedure described above for 64 in Method 1, 74 was prepared

from 68 as a white solid (2.92 g, 58%): mp 199-201 °C (hexanes/

EtOAc). ¹H NMR (DMSO-d₆) δ 8.11 (s, 2H), 7.71 (d, J ) 7.7 Hz,

2H), 7.60 (d, J ) 7.7 Hz, 2H), 6.83 (s, 2H), 2.95 (t, J ) 7.7 Hz,

4H), 2.17 (p, J ) 7.7 Hz, 2H). HPLC (method B) t_R8.64 min (100

area %). Anal. (C₂₁H₁₄N₂O₂) C, H, N.

Following the procedure described above for 64 in Method 2,

74 was prepared from 3-hydroxy-4-iodobenzonitrile (87) and 1,6-

heptadiyne as a white solid (5.38 g, 62%).

Following the procedure described above for 64 in Method 3,

74 was prepared from 3-hydroxy-4-iodobenzonitrile (87) and 1,6-

heptadiyne as a white solid (0.78 g, 47%).

1,4-Bis(5-cyanobenzofuran-2-yl)butane (75).⁴⁵Following the

procedure described above for 64 in Method 1, 75 was prepared

from 69 as a white solid (2.02 g, 40%): mp 187-188 °C (hexanes/

EtOAc) (lit.⁴⁵190-192 °C). ¹H NMR (DMSO-d₆) δ 8.10 (s, 2H),

7.73 (d, J ) 8.2 Hz, 2H), 7.69 (d, J ) 8.2 Hz, 2H), 6.76 (s, 2H),

2.90 (m, 4H), 1.80 (m, 4H). HPLC (method B) t_R9.40 min (100

area %). Anal. (C₂₂H₁₆N₂O₂‚0.3H₂O) C, H, N.

1,4-Bis(6-cyanobenzofuran-2-yl)butane (76). Following the

procedure described above for 64 in Method 1, 76 was prepared

from 70 as a white solid (1.57 g, 38%): mp 215-217 °C (CHCl₃).

¹H NMR (DMSO-d₆) δ 8.11 (s, 2H), 7.72 (d, J ) 8.2 Hz, 2H),

7.61 (d, J ) 8.2 Hz, 2H), 6.80 (s, 2H), 2.91 (br s, 4H), 1.80 (br s,

4H). HPLC (method B) t_R9.32 min (100 area %). Anal. (C₂₂H₁₆N₂O₂‚

0.4H₂O) C, H, N.

1,5-Bis(5-cyanobenzofuran-2-yl)pentane (77). Following the

procedure described above for 64 in Method 1, 77 was prepared

from 71 as a white solid (1.71 g, 42%): mp 133-134 °C (hexanes/

EtOAc). ¹H NMR (DMSO-d₆) δ 8.06 (s, 2H), 7.68 (d, J ) 8.2 Hz,

2H), 7.64 (d, J ) 8.2 Hz, 2H), 6.71 (s, 2H), 2.83 (m, 4H), 1.77 (m,

4H), 1.44 (m, 2H). HPLC (method B) t_R9.90 min (100 area %).

Anal. (C₂₃H₁₈N₂O₂‚0.1H₂O) C, H, N.

Following the procedure described above for 64 in Method 3,

77 was prepared from 3-hydroxy-4-iodobenzonitrile (83) and 1,8-

nonadiyne as a light yellow solid (13.7 g, 77%).

Following the procedure described above for 64 in Method 2,

66 was prepared from 3-hydroxy-4-iodobenzonitrile (87) and 1,5-

hexadiyne as a light yellow solid (1.77 g, 22%)

1,3-Bis(5-methoxycarbonylbenzofuran-2-yl)propane (67). Fol-

lowing the procedure described above for 64 in Method 2, 67 was

prepared from 53 and 1,6-heptadiyne as a white solid (6.37 g,

1

75%): mp 87-88 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ

8.17 (d, J ) 1.6 Hz, 2H), 7.86 (dd, J ) 8.8 and 1.6 Hz, 2H), 7.61

(d, J ) 8.8 Hz, 2H), 6.79 (s, 2H), 3.86 (s, 6H), 2.92 (t, J ) 7.1 Hz,

4H), 2.15 (m, 2H). HPLC (method B) t_R9.67 min (100 area %).

Anal. (C₂₃H₂₀O₆‚0.5H₂O) C, H.

1,3-Bis(6-methoxycarbonylbenzofuran-2-yl)propane (68). Fol-

lowing the procedure described above for 64 in Method 2, 68 was

prepared from 54 and 1,6-heptadiyne as a white solid (6.10 g,

1

44%): mp 100-101 °C (hexanes/benzene). H NMR (DMSO-d₆)

δ 8.01 (s, 2H), 7.82 (d, J ) 8.2 Hz, 2H), 7.64 (d, J ) 8.2 Hz, 2H),

6.79 (s, 2H), 3.87 (s, 6H), 2.94 (t, J ) 7.1, 4H), 2.17 (m, 2H).

HPLC (method B) t_R9.63 min (93.0 area %). Anal. (C₂₃H₂₀O₆) C,

H.

Following the procedure described above for 64 in Method 3,

68 was prepared from 54 and 1,6-heptadiyne. The reaction mixture

was stirred at 80 °C for 3 days, cooled to ambient temperature,

diluted with water (100 mL), and extracted with ethyl acetate (3 ×

150 mL). The extracts were dried over MgSO₄and concentrated

to give a crude that was purified by column chromatography on

silica gel eluting with (1:1) hexanes/chloroform to yield 68 as a

white solid (8.50 g, 66%).

1,4-Bis(5-methoxycarbonylbenzofuran-2-yl)butane (69). Fol-

lowing the procedure described above for 64 in Method 2, 69 was

prepared from 53 and 1,7-octadiyne as a white solid (6.0 g, 79%):

1

mp 130-131 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ 8.19

(d, J ) 1.1 Hz, 2H), 7.86 (dd, J ) 8.8 and 1.1 Hz, 2H), 7.61 (d, J

) 8.8 Hz, 2H), 6.75 (s, 2H), 3.86 (s, 6H), 2.88 (br s, 4H), 1.80 (br

s, 4H). Anal. (C₂₄H₂₂O₆‚0.6H₂O) C, H.

1,4-Bis(6-methoxycarbonylbenzofuran-2-yl)butane (70). Fol-

lowing the procedure described above for 64 in Method 2, 70 was

prepared from 54 and 1,7-octadiyne as a white solid (4.96 g,

70%): mp 130-131 °C (hexanes/EtOAc). ¹H NMR (DMSO-d₆) δ

8.01 (br s, 2H), 7.83 (dd, J ) 8.2 and 1.1 Hz, 2H), 7.65 (d, J ) 8.2

Hz, 2H), 6.75 (s, 2H), 3.86 (s, 6H), 2.90 (m, 4H), 1.80 (m, 4H).

HPLC (method B) t_R10.19 min (100 area %). Anal. (C₂₄H₂₂O₆) C,

H.

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5821

1,5-Bis(6-cyanobenzofuran-2-yl)pentane (78). Following the

procedure described above for 64 in Method 1, 78 was prepared

from 72 as a white solid (3.30 g, 50%): mp 106-107 °C (hexanes/

from 83 and 1,10-undecadiyne⁶⁸as a light yellow solid (2.01 g,

43%): mp 133 °C (hexanes/EtOAc). ¹H NMR (DMSO-d₆) δ 8.08

(s, 2H), 7.71 (d, J ) 8.8 Hz, 2H), 7.65 (d, J ) 8.8 Hz, 2H), 6.72

(s, 2H), 2.80 (t, J ) 7.1 Hz, 4H), 1.68 (br s, 4H), 1.35 (m, 6H).

HPLC (method B) t_R10.98 min (100 area %). Anal. (C₂₅H₂₂N₂O₂‚

0.2hexane) C, H, N.

1

EtOAc). H NMR (DMSO-d₆) δ 8.11 (br s, 2H), 7.73 (d, J ) 8.2

Hz, 2H), 7.62 (dd, J ) 8.2 and 1.0 Hz, 2H), 6.79 (s, 2H), 2.87 (t,

J ) 7.1 Hz, 4H), 1.78 (m, 4H), 1.44 (m, 2H). HPLC (method B)

t_R9.89 min (95.2 area %). Anal. (C₂₃H₁₈N₂O₂) C, H, N.

1,8-Bis(5-cyanobenzofuranyl-2)octane (90). Following the pro-

cedure described above for 64 in Method 3, 90 was prepared from

83 and 1,11-dodecadiyne⁶⁸as a light yellow solid (1.50 g, 23%):

1,5-Bis(5-formyl-7-methoxybenzofuran-2-yl)pentane (80). Fol-

lowing the procedure described above for 64 in Method 2, 80 was

prepared from 5-iodovaniline 79 as a white solid (6.01 g, 78%):

1

mp 167-168 °C (EtOAc). H NMR (DMSO-d₆) δ 8.08 (s, 2H),

1

mp 110-112 °C (EtOAc). H NMR (DMSO-d₆) δ 9.98 (s, 2H),

7.72 (d, J ) 8.2 Hz, 2H), 7.66 (d, J ) 8.2 Hz, 2H), 6.72 (s, 2H),

2.80 (t, J ) 7.1 Hz, 4H), 1.68 (m, 4H), 1.32 (br s, 8H). HPLC

(method B) t_R11.91 min (100 area %). Anal. (C₂₆H₂₄N₂O₂‚

0.3EtOAc) C, H, N.

7.75 (s, 2H), 7.33 (s, 2H), 6.76 (s, 2H), 3.99 (s, 6H), 2.82 (t, J )

7.1 Hz, 4H), 1.76 (m, 4H), 1.42 (m, 2H). Anal. (C₂₅H₂₄O₆‚1H₂O)

C, H.

1,5-Bis(5-formyl-7-methoxybenzofuran-2-yl)pentane Bisoxime

(81). Bisaldehyde 80 (5.50 g, 13.0 mmol) and hydroxylamine

hydrochloride (2.10 g, 30.0 mmol) were dissolved in a mixture of

dry pyridine (20 mL) and dry ethanol (20 mL). The reaction mixture

was stirred under Ar at ambient temperature for 2 h. The mixture

was concentrated to half of its volume and diluted with water to

form an oily residue. The liquid was decanted, and the residue was

dissolved in ethanol (30 mL), filtered through a paper filter, and

diluted with water (100 mL). The ethanol was evaporated. During

the concentration, a precipitation occurred. The precipitate was

filtered off and recrystallized from aqueous EtOH to yield 81 as a

white solid (5.00 g, 85%): mp 125-127 °C. ¹H NMR (DMSO-d₆)

δ 11.06 (s, 2H), 8.16 (s, 2H), 7.27 (d, J ) 1.6 Hz, 2H), 7.13 (d, J

) 1.6 Hz, 2H), 6.59 (s, 2H), 3.92 (s, 6H), 2.77 (t, J ) 7.1 Hz, 4H),

1.73 (m, 4H), 1.42 (m, 2H). Anal. (C₂₅H₂₆N₂O₆‚0.3H₂O) C, H, N.

1,5-Bis(5-cyano-7-methoxybenzofuran-2-yl)pentane (82). A

mixture of 81 (3.00 g, 6.71 mmol) in acetic anhydride (10 mL)

was refluxed for 2 h. The reaction mixture was cooled down, diluted

with water (100 mL) and stirred for 2 h at ambient temperature. A

precipitate formed was separated and recrystallized from a mixture

of hexanes and EtOAc to yield 82 as a white solid (2.30 g, 83%):

1,9-Bis(5-cyanobenzofuranyl-2)nonane (91). Following the

procedure described above for 64 in Method 3, 91 was prepared

from 83 and 1,12-tridecadiyne⁶⁸as a light yellow solid (1.20 g,

1

24%): mp 95-97 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ

8.08 (s, 2H), 7.72 (d, J ) 8.2 Hz, 2H), 7.66 (d, J ) 8.2 Hz, 2H),

6.72 (s, 2H), 2.79 (t, J ) 7.1 Hz, 4H), 1.67 (m, 4H), 1.29 (br s,

10H). HPLC (method C) t_R11.40 min (100 area %). Anal.

(C₂₇H₂₆N₂O₂) C, H, N.

1,10-Bis(5-cyanobenzofuranyl-2)decane (92). Following the

procedure described in Method 3 for 64, 92 was prepared from 83

and 1,13-tetradecadiyne⁶⁸as a light yellow solid (1.91 g, 36%):

1

mp 141-143 °C (EtOAc). H NMR (DMSO-d₆) δ 8.08 (s, 2H),

7.71 (d, J ) 8.8 Hz, 2H), 7.66 (d, J ) 8.8 Hz, 2H), 6.72 (s, 2H),

2.80 (t, J ) 7.1 Hz, 4H), 1.67 (m, 4H), 1.25 (m, 12H). HPLC

(method D) t_R9.93 min (100 area %). Anal. (C₂₈H₂₈N₂O₂‚

0.1EtOAc) C, H, N.

1,11-bis(5-cyanobenzofuranyl-2)undecane (93). Following the

procedure described above for 64 in Method 3, 93 was prepared

from 83 and 1,14-pentadecadiyne⁶⁸as a white solid (1.01 g, 19%):

1

mp 87-89 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ 8.07 (s,

2H), 7.70 (d, J ) 8.2 Hz, 2H), 7.65 (d, J ) 8.2 Hz, 2H), 6.72 (s,

2H), 2.79 (t, J ) 7.1 Hz, 4H), 1.68 (m, 4H), 1.25 (m, 14H). HPLC

(method D) t_R10.12 min (97.9 area %). Anal. (C₂₉H₃₀N₂O₂‚

0.3hexane) C, H, N.

1

mp 136-137 °C. H NMR (DMSO-d₆) δ 7.66 (s, 2H), 7.31 (s,

2H), 6.69 (s, 2H), 3.97 (s, 6H), 2.80 (t, J ) 7.1 Hz, 4H), 1.71 (m,

4H), 1.43 (m, 2H). Anal. (C₂₅H₂₂N₂O₄‚0.2AcOH) C, H, N.

5-Cyano-2-(3-hydroxypropyl-1)benzofuran (84). Following the

procedure described above for 64 in Method 3, 84 was prepared

from 4-hydroxy-3-iodobenzonitrile 83 and 4-pentyn-1-ol as a white

Acknowledgment. This work was supported by the Bill and

Melinda Gates Foundation and the Medicines for Malaria

Venture.

1

solid (8.91 g, 74%): mp 65-66 °C (hexanes/EtOAc). H NMR

(DMSO-d₆) δ 8.08 (d, J ) 1.0 Hz, 1H), 7.72 (d, J ) 8.2 Hz, 1H),

7.66 (dd, J ) 8.2 and 1.0 Hz, 1H), 6.73 (s, 1H), 4.58 (t, J ) 4.9

Hz, 1H), 3.48 (q, J ) 6.0 Hz, 2H), 2.86 (t, J ) 7.1 Hz, 2H), 1.85

(m, 2H). HPLC (method B) t_R3.72 min (100 area %). Anal. (C₁₂H₁₁-

NO₂) C, H, N.

Supporting Information Available: Elemental analysis data.

This material is available free of charge via the Internet at http://

pubs.acs.org.

5-Cyano-2-(2-formylethyl-1)benzofuran (85). Following the

procedure described above for 57, 85 was prepared from 84 as a

light yellow solid (6.51 g, 84%): mp 52-53 °C (hexanes/EtOAc).

¹H NMR (DMSO-d₆) δ 9.75 (s, 1H), 8.10 (br s, 1H), 7.73 (d, J )

8.2 Hz, 1H), 7.67 (dd, J ) 8.2 and 1.6 Hz, 1H), 6.75 (s, 1H), 3.09

(t, J ) 7.1 Hz, 2H), 2.95 (t, J ) 7.1 Hz, 2H). Anal. (C₁₂H₉NO₂) C,

H, N.

5-Cyano-2-(3-butynyl-1)benzofuran (86). Following the pro-

cedure described above for 59, 86 was prepared from 85 as a white

solid (4.70 g, 77%): mp 110-112 °C (hexanes/EtOAc). ¹H NMR

(DMSO-d₆) δ 8.13 (br s, 1H), 7.74 (d, J ) 8.2 Hz, 1H), 7.68 (dd,

J ) 8.2 and 1.6 Hz, 1H), 6.82 (s, 1H), 3.02 (t, J ) 7.1 Hz, 2H),

2.84 (t, J ) 2.2 Hz, 1H), 2.63 (m, 2H). HPLC (method B) t_R6.31

min (100 area %). Anal. (C₁₃H₉NO) C, H, N.

References

(1) Barrett, M. P. The fall and rise of sleeping sickness. Lancet 1999,

353, 1113-1114.

(2) Legros, D.; Ollivier, G.; Gastellu-Etchegorry, M.; Paquet, C.; Burri,

C.; Jannin, J.; Buscher, P. Treatment of human African trypanoso-

miasis-present situation and needs for research and development.

Lancet Infect. Dis. 2002, 2, 437-440.

(3) Bloland, P. B. Drug resistance in malaria. http://www.who.int/csr/

resources/publications/drugresist/malaria.pdf

2007).

(4) Choi, C. M.; Lerner, E. A. Leishmaniasis as an emerging infection.

J. InVestig. Dermatol. Symp. Proc. 2001, 6, 175-182.

(5) Greenwood, B.; Mutabingwa, T. Malaria in 2002. Nature 2002, 415,

670-672.

(accessed

May

(6) Brun, R.; Balmer, O. New developments in human African trypa-

nosomiasis. Curr. Opin. Infect. Dis. 2006, 19, 415-420.

(7) Fairlamb, A. H. Chemotherapy of human African trypanosomiasis:

current and future prospects. Trends Parasitol. 2003, 19, 488-494.

(8) 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.

1,6-Bis(5-cyanobenzofuran-2-yl)hexane (88). Following the

procedure described above for 64 in Method 3, 88 was prepared

from 83 and 1,9-decadiyne⁶⁸as a light yellow solid (4.31 g, 71%):

1

mp 184-185 °C (hexanes/EtOAc). H NMR (DMSO-d₆) δ 8.08

(s, 2H), 7.72 (d, J ) 8.2 Hz, 2H), 7.66 (d, J ) 8.2 Hz, 2H), 6.73

(s, 2H), 2.81 (t, J ) 7.1 Hz, 4H), 1.71 (br s, 4H), 1.40 (br s, 4H).

HPLC (method B) t_R10.54 min (100 area %). Anal. (C₂₄H₂₀N₂O₂‚

0.2EtOAc) C, H, N.

(9) WHO. African trypanosomiasis (sleeping sickness). http://

www.who.int/mediacentre/factsheets/fs259/en/(accessed

2007).

(10) 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.

May

1,7-Bis(5-cyanobenzofuran-2-yl)heptane (89). Following the

procedure described above for 64 in Method 3, 89 was prepared

5822 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

BakunoVa et al.

(11) Wolday, D.; Berhe, N.; Akuffo, H.; Desjeux, P.; Britton, S. Emerging

Leishmania/HIV co-infection in Africa. Med. Microbiol. Immunol.

2001, 190, 65-67.

(12) Rabello, A.; Orsini, M.; Disch, J. Leishmania/HIV co-infection in

Brazil: an appraisal. Ann. Trop. Med. Parasitol. 2003, 97 Suppl 1,

17-28.

(13) Pintado, V.; Lopez-Velez, R. HIV-associated visceral leishmaniasis.

Clin. Microbiol. Infection 2001, 7, 291-300.

(14) Desjeux, P.; Alvar, J. Leishmania/HIV co-infections: epidemiology

in Europe. Ann. Trop. Med. Parasitol. 2003, 97 Suppl 1, 3-15.

(15) Weina, P. J.; Neafie, R. C.; Wortmann, G.; Polhemus, M.; Aronson,

N. E. Old world leishmaniasis: an emerging infection among

deployed US military and civilian workers. Clin. Infect. Dis. 2004,

39, 1674-1680.

(16) May, J.; Meyer, C. G. Chemoresistance in falciparum malaria. Trends

Parasitol. 2003, 19, 432-435; discussion 435-436.

(17) Biagini, G. A.; O’Neill, P. M.; Bray, P. G.; Ward, S. A. Current

drug development portfolio for antimalarial therapies. Curr. Opin.

Pharmacol. 2005, 5, 473-478.

(38) Stead, A. M.; Bray, P. G.; Edwards, I. G.; de Koning, 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.

(39) de Koning, H. P. Transporters in African trypanosomes: role in drug

action and resistance. Int. J. Parasitol. 2001, 31, 512-522.

(40) de Koning, H. P.; Jarvis, S. M. Adenosine transporters in bloodstream

forms of Trypanosoma brucei brucei: substrate recognition motifs

and affinity for trypanocidal drugs. Mol. Pharmacol. 1999, 56, 1162-

1170.

(41) Carter, N. S.; Barrett, M. P.; de Koning, H. P. A drug resistance

determinant in Trypanosoma brucei. Trends Microbiol. 1999, 7, 469-

471.

(42) Zhang, X.; Berger, B. J.; Ulrich, P. Synthesis and trypanocidal activity

of the bis-carba analogue of pentamidine. Bioorg. Med. Chem. Lett.

1996, 6, 1035-1036.

(43) Dann, O.; Fernbach, R.; Pfeifer, W.; Demant, E.; Bergen, G.;

Lang, S.; Lurding, G. Trypanocide diamidine mit drei ringen in

zwei isolierten ringsystemen. Liebigs Ann. Chem. 1972, 760, 37-

87.

(44) Dann, O.; Volz, G.; Demant, E.; Pfeifer, W.; Bergen, G.; Fick, H.;

Walkenhorst, E. Trypanocide diamidine mit vier ringen in einem oder

zwei ringsystemen. Liebigs Ann. Chem. 1973, 1112-1140.

(45) Dann, O.; Char, H.; Griebmeier, H. Synthesen biskationisher,

trypanocider 1-benzofuran-verbindungen. Liebigs Ann. Chem. 1982,

1836-1869.

(46) Guillaumel, J.; Royer, R. Synthese d’ethers diethylamino ethyliques

de bibenzofurannes et de bis-benzofuryl cetones, comme analogues

pharmacochimiques de la Tilorone. J. Heterocycl. Chem. 1986, 23,

1277-1282.

(47) 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.

(48) 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.

(49) 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.

(50) 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.

(51) Sonogashira, K.; Tohda, Y.; Hagihara, N. A convenient synthesis of

acetylenes: catalytic substitutions of acetylenic hydrogen with

bromoalkenes, iodoarenes and bromopyridines. Tetrahedron Lett.

1975, 16, 4467-4470.

(52) Fancelli, D.; Fagnola, M. D.; Severino, D.; Bedeschi, A. Solid phase

synthesis of 2-substituted benzofurans via the palladium-catalysed

heteroannulation of acetylenes. Tetrahedron Lett. 1997, 38, 2311-

2314.

(18) 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.

(19) Le Bras, J.; Durand, R. The mechanisms of resistance to antimalarial

drugs in Plasmodium falciparum. Fundam. Clin. Pharmacol. 2003,

17, 147-153.

(20) Hyde, J. E. Drug-resistant malaria. Trends Parasitol. 2005, 21, 494-

498.

(21) Arav-Boger, R.; Shapiro, T. A. Molecular mechanisms of resistance

in antimalarial chemotherapy: the unmet challenge. Annu. ReV.

Pharmacol. Toxicol. 2005, 45, 565-585.

(22) Burchmore, R. J.; Ogbunude, P. O.; Enanga, B.; Barrett, M. P.

Chemotherapy of human African trypanosomiasis. Curr. Pharm. Des.

2002, 8, 257-267.

(23) Docampo, R.; Moreno, S. N. Current chemotherapy of human African

trypanosomiasis. Parasitol. Res. 2003, 90 Supp 1, S10-13.

(24) Ouellette, M.; Drummelsmith, J.; Papadopoulou, B. Leishmaniasis:

Drugs in the clinic, resistance and new developments. Drug Resist.

Update 2004, 7, 257-266.

(25) Werbovetz, K. Promising therapeutic targets for antileishmanial drugs.

Expert Opin. Ther. Targets 2002, 6, 407-422.

(26) Sundar, S.; More, D.; Singh, M.; Singh, V.; Sharma, S.; Makharia,

A.; Kumar, P.; Murray, H. Failure of pentavalent antimony in visceral

leishmaniasis in India: Report from the center of the Indian epidemic.

Clin. Infect. Dis. 2000, 31, 1104-1107.

(27) Singh, S.; Sivakumar, R. Challenges and new discoveries in the

treatment of leishmaniasis. J. Infect. Chemother. 2004, 10, 307-

315.

(28) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity of

2,5-bis(4-guanylphenyl)furans. J. Med. Chem. 1977, 20, 531-536.

(29) Anne, J.; De Clercq, E.; Eyssen, H.; Dann, O. Antifungal and

antibacterial activities of diarylamidine derivatives. Antimicrob.

Agents Chemother. 1980, 18, 231-239.

(30) 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.

(31) Tidwell, R. R.; Jones, S. K.; Geratz, J. D.; Ohemeng, K. A.; Cory,

M.; Hall, J. E. Analogues of 1,5-bis(4-amidinophenoxy)pentane

(pentamidine) in the treatment of experimental Pneumocystis carinii

pneumonia. J. Med. Chem. 1990, 33, 1252-1257.

(32) 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.

(33) 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.

(34) Bouteille, B.; Oukem, O.; Bisser, S.; Dumas, M. Treatment perspec-

tives for human African trypanosomiasis. Fundam. Clin. Pharmacol.

2003, 17, 171-181.

(35) Barrett, M. P.; Fairlamb, A. H. The biochemical basis of arsenical-

diamidine crossresistance in African trypanosomes. Parasitol. Today

1999, 15, 136-140.

(36) Carter, N. S.; Berger, B. J.; Fairlamb, A. H. Uptake of diamidine

drugs by the P2 nucleoside transporter in melarsen-sensitive and

-resistant Trypanosoma brucei brucei. J. Biol. Chem. 1995, 270,

28153-28157.

(37) 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.

(53) Katritzky, A. R.; Fali, C. N.; Li, J. General synthesis of polysubsti-

tuted benzo[â]furans. J. Org. Chem. 1997, 62, 8205-8209.

(54) Kundu, N. G.; Pal, M.; Mahanty, J. S.; De, M. Palladium-catalysed

heteroannulation with acetylenic compounds: synthesis of benzo-

furans. J. Chem. Soc., Perkin Trans. 1 1997, 2815-2820.

(55) Lutjens, H.; Scammells, P. J. Synthesis of natural products

possessing a benzo[â]furan skeleton. Tetrahedron Lett. 1998, 39,

6581-6584.

(56) Inoue, M.; Carson, M. W.; Frontier, A. J.; Danishefsky, S. J. Total

synthesis and determination of absolute configuration of frondosin

B. J. Am. Chem. Soc. 2001, 123, 1878-1889.

(57) Sonogashira, K. Development of Pd-Cu catalyzed cross-coupling

of terminal acetylenes with sp²-carbon halides. J. Organomet. Chem.

2002, 653, 46-49.

(58) Kobayashi, S.; Azekawa, M.; Morita, H. The Reimer-Tiemann

reaction of m-halophenols. II. With m-bromophenol. Chem. Pharm.

Bull. 1969, 17, 89-93.

(59) Edgar, K. J.; Falling, S. N. An efficient and selective method for

the preparation of iodophenols. J. Org. Chem. 1990, 55, 5287-5291.

(60) Diaz, P.; Gendre, F.; Stella, L.; Charpentier, B. New synthetic

retinoids obtained by palladium-catalyzed tandem cyclisation-hydride

capture process. Tetrahedron 1998, 54, 4579-4590.

(61) 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.;

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.

(62) Mancuso, A. J.; Swern, D. Activated dimethyl sulfoxide: useful

reagents for synthesis. Synthesis 1981, 165-185.

Antiprotozoal Bisbenzofuran Cations

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5823

(63) Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. An improved

one-pot procedure for the synthesis of alkynes from aldehydes. Synlett

1996, 521-522.

(64) Callant, P. D. H. L.; Vandewalle, M. An efficient preparation and

the intramolecular cyclopropanation of R-diazo-â-ketophosphonates

and R-diazophoshonoacetates. Synth. Commun. 1984, 14, 155-

161.

(65) Kitamura, M.; Tokunaga, M.; Noyori, R. Asymmetric hydrogenation

of â-keto phosphonates: a practical way to fosfomycin. J. Am. Chem.

Soc. 1995, 117, 2931-2932.

(78) de Koning, H. P.; Jarvis, S. M. Uptake of pentamidine in Trypano-

soma brucei brucei is mediated by the P2 adenosine transporter and

at least one novel, unrelated transporter. Acta Trop. 2001, 80, 245-

250.

(79) Steck, E. A.; Kinnamon, K. E.; Rane, D. S.; Hanson, W. L.

Leishmania donoVani, Plasmodium berghei, Trypanosoma rhod-

esiense: antiprotozoal effects of some amidine types. Exp. Parasitol.

1981, 52, 404-413.

(80) Donkor, I. O.; Assefa, H.; Rattendi, D.; Lane, S.; Vargas, M.;

Goldberg, B.; Bacchi, C. Trypanocidal activity of dicationic com-

pounds related to pentamidine. Eur. J. Med. Chem. 2001, 36, 531-

538.

(81) 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.

(82) Klenke, B.; Stewart, M.; Barrett, M. P.; Brun, R.; Gilbert, I. H.

Synthesis and biological evaluation of s-triazine substituted polyamines

as potential new anti-trypanosomal drugs. J. Med. Chem. 2001, 44,

3440-3452.

(83) Baliani, A.; Bueno, G. J.; Stewart, M. L.; Yardley, V.; Brun, R.;

Barrett, M. P.; Gilbert, I. H. Design and synthesis of a series of

melamine-based nitroheterocycles with activity against Trypanoso-

matid parasites. J. Med. Chem. 2005, 48, 5570-5579.

(84) Kirk, K. Membrane transport in the malaria-infected erythrocyte.

Physiol. ReV. 2001, 81, 495-537.

(66) Wood, J. L.; Khatri, N. A.; Weinreb, S. M. A direct conversion of

esters to nitriles. Tetrahedron Lett. 1979, 20, 4907-4910.

(67) 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.

(68) Hellbach, B.; Gleiter, R.; Rominger, F. Cyclic diynes by alkyne

metathesis. Synthesis 2003, 2535-2541.

(69) Dox, A. W.; Whitmore, F. C. Acetamidine Hydrochloride. In Organic

Syntheses, 2nd ed.; Blatt, A. H., Ed.; John Wiley & Sons: New York,

1941; Vol. 1, pp 5-7.

(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.

(85) Sperandeo, N. R.; Brun, R. Synthesis and biological evaluation of

pyrazolylnaphthoquinones as new potential antiprotozoal and cyto-

toxic agents. Chembiochem 2003, 4, 69-72.

(71) Soeiro, M. N.; De Souza, E. M.; Stephens, C. E.; Boykin, D. W.

Aromatic diamidines as antiparasitic agents. Expert Opin. InVestig.

Drugs 2005, 14, 957-972.

(72) Carter, N. S.; Fairlamb, A. H. Arsenical-resistant trypanosomes lack

an unusual adenosine transporter. Nature 1993, 361, 173-176.

(73) de Koning, H. P. Uptake of pentamidine in Trypanosoma brucei

brucei is mediated by three distinct transporters: implications for

cross-resistance with arsenicals. Mol. Pharmacol. 2001, 59, 586-

592.

(74) Matovu, E.; Stewart, M. L.; Geiser, F.; Brun, R.; Maser, P.; Wallace,

L. J.; Burchmore, R. J.; Enyaru, J. C.; Barrett, M. P.; Kaminsky, R.;

Seebeck, T.; de Koning, H. P. Mechanisms of arsenical and diamidine

uptake and resistance in Trypanosoma brucei. Eukaryot. Cell. 2003,

2, 1003-1008.

(75) Rollo, I. M.; Williamson, J. Acquired resistance to ‘Melarsen’,

tryparsamide and amidines in pathogenic trypanosomes after treat-

ment with ‘Melarsen’ alone. Nature 1951, 167, 147-148.

(76) Fairlamb, A. H.; Carter, N. S.; Cunningham, M.; Smith, K.

Characterization of melarsen-resistant Trypanosoma brucei brucei

with respect to cross-resistance to other drugs and trypanothione

metabolism. Mol. Biochem. Parasitol. 1992, 53, 213-222.

(77) Pospichal, H.; Brun, R.; Kaminsky, R.; Jenni, L. Induction of

resistance to melarsenoxide cysteamine (Mel Cy) in Trypanosoma

brucei brucei. Acta Trop. 1994, 58, 187-197.

(86) Raz, B.; Iten, M.; Grether-Buhler, Y.; Kaminsky, R.; Brun, R. The

Alamar Blue assay to determine drug sensitivity of African trypa-

nosomes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop.

1997, 68, 139-147.

(87) 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.

(88) Matile, H.; Pink, J. R. L. Plasmodium falciparum malaria parasite

culture and their use in immunology. In Immunological Methods;

Academic Press: San Diego, 1990; pp 221-234.

(89) Werbovetz, K.; Brendle, J.; Sackett, D. Purification, characterization,

and drug susceptibility of tubulin from Leishmania. Mol. Biochem.

Parasitol. 1999, 98, 53-65.

(90) 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. Pharm. 2003, 64, 1325-1333.

JM0708634

Article Doi

Synthesis and in vitro antiprotozoal activity of bisbenzofuran cations

DOI: 10.1021/jm0708634

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm0708634

Full text of DOI:10.1021/jm0708634

Products guided by the article

R&D Labs maybe for 66639-67-6

Relevant to this article

Hot Product