Structure-activity study of pentamidine analogues as antiprotozoal agents

2016

J. Med. Chem. 2009, 52, 2016–2035

Structure-Activity Study of Pentamidine Analogues as Antiprotozoal Agents

Svetlana M. Bakunova,^†Stanislav A. Bakunov,^†Donald A. Patrick,^†E. V. K. Suresh Kumar,^†Kwasi A. Ohemeng,^†

Arlene S. Bridges,^†Tanja Wenzler,^‡Todd Barszcz,^§Susan Kilgore Jones,^†Karl A. Werbovetz,^§Reto Brun,^‡and

Richard R. Tidwell*^,†

Department of Pathology and Laboratory Medicine, School of Medicine, The UniVersity of North Carolina, Chapel Hill, North Carolina 27599,

DiVision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State UniVersity, 500 West 12th AVenue, Columbus,

Ohio 43210, Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland

ReceiVed December 8, 2008

Diamidine 1 (pentamidine) and 65 analogues (2-66) have been tested for in vitro antiprotozoal activities

against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donoVani, and for

cytotoxicity against mammalian cells. Dications 32, 64, and 66 exhibited antitrypanosomal potencies equal

or greater than melarsoprol (IC₅₀) 4 nM). Nine congeners (2-4, 12, 27, 30, and 64-66) were more active

against P. falciparum than artemisinin (IC₅₀) 6 nM). Eight compounds (12, 32, 33, 44, 59, 62, 64, and 66)

exhibited equal or better antileishmanial activities than 1 (IC₅₀) 1.8 µM). Several congeners were more

active than 1 in vivo, curing at least 2/4 infected animals in the acute mouse model of trypanosomiasis. The

diimidazoline 66 was the most promising compound in the series, showing excellent in vitro activities and

high selectivities against T. b. rhodesiense, P. falciparum, and L. donoVani combined with high

antitrypanosomal efﬁcacy in vivo.

Introduction

Recently a growing problem of treatment failures due to

developing of resistance to common antiprotozoal drugs has

emerged around the world.^29-39Development of resistance to

the most commonly used antimalarial drugs, such as chloroquine

and sulfadoxine-pyrimethamine, represents a major obstacle in

controlling malaria. To slow the emergence of resistance, many

affected countries now rely on therapies that are more expensive

or the use of drug combinations (particularly with artemisin-

ins),^29,31,32,34which increases the economic burden of the

disease. Current treatments for HAT and leishmaniasis require

a long course of parenteral administration and suffer from un-

acceptable toxicity and poor efﬁcacy.^{8,11,16,40,41}Unresponsive-

ness to these medications and growing number of treatment

failures is becoming more prevalent because of development

of parasite resistance, although new options for treatment of

visceral leishmaniasis have recently become available.^42,43

Unfortunately, many drug formulations showing promise in

treating parasitic infections are too costly for routine chemo-

therapy in the developing world. Therefore, there is a need for

safe and affordable antiprotozoal treatments capable of over-

coming parasite resistance.

Throughout history, protozoan infections transmitted by

insects accompanied mankind, causing human suffering and

death. Malaria, human African trypanosomiasis (HAT^aor sleep-

ing sickness), and leishmaniasis are major protozoan diseases

that are prevalent in subtropical countries. Today, malaria

remains one of the leading causes of morbidity and mortality

in the world.^1-5The majority of those deaths occurs in sub-

Saharan Africa and is the result of the most severe and life-

threatening form of disease caused by Plasmodium falciparum.^4-6

Poverty and nutrient deﬁciency, wars and civil disturbances,

environmental and climatic changes, population growth in

malaria-endemic areas and traveling to these regions are the

factors contributing to the increased incidence of malaria.

Devastating epidemics of HAT depopulated many areas in

Africa at the beginning of the twentieth century.⁷Nowadays,

sleeping sickness, which is typically fatal without treatment, is

endemic in approximately 20 sub-Saharan countries.^8-11The

clinical manifestations of leishmaniasis depend on the species

of infecting Leishmania parasites and can range from self-

healingcutaneouslesionstolife-threateningvisceralinfections.^12-16

While the vast majority of leishmanial infections occur in the

developing world, leishmaniasis now exerts an increasing effect

on developed nations as well.¹⁷In addition, the overlap of

humanimmunodeﬁciencyvirus(HIV)withparasiticinfections^18-22

results in growing number of cases of dually infected individuals

in different parts of the globe.^23-28

1,5-Bis(4-amidinophenoxy)pentane (pentamidine)⁴⁴has been

used for more than half a century against early stage Trypano-

soma brucei gambiense related HAT,^11,36,45antimony resistant

leishmaniasis caused by Leishmania donoVani,^14,15,38and HIV

related pneumonia caused by the opportunistic pathogen Pneu-

mocystis jiroVeci (formerly Pneumocystis carinii).^46-48In early

studies, compound 1 (pentamidine) also displayed a curative

effect on Plasmodium infected monkeys⁴⁹and exhibited in vitro

activities against selected strains of the pathogen,^49-51but

availability of other effective drugs hindered its antimalarial

development. At physiological pH, both amidine groups in the

dication 1 are positively charged, thus decreasing membrane

permeability of the drug and diminishing its oral activity.^36,52

Consequently, compound 1 requires parenteral administration,

which makes the treatment less practical, especially in remote

areas where most cases of HAT and leishmaniasis take place.

It is fairly well tolerated by patients although some adverse

Advances in antiprotozoal chemotherapy in the middle of the

twentieth century helped to limit and control the spread of

parasitic infections, keeping the situation stable for decades.

* 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; WHO, World

Health Organization; HIV, human immunodeﬁciency virus; MAP, melar-

soprol; CQ, chloroquine; ATM, artemisinin; PPT, podophyllotoxin.

10.1021/jm801547t CCC: $40.75

2009 American Chemical Society

Published on Web 03/06/2009

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2017

Scheme 1^a

^aReagents and conditions: (i) dibromoalkane, K₂CO₃, DMF, reﬂux; (ii) HCl gas, EtOH, dioxane, 0 °C-ambient temp; (iii) appropriate amine, EtOH,

ambient temp; (iv) fuming HNO₃; (v) 70% HNO₃, TFA, Ac₂O.

effects, including hypotension, abdominal pain, vertigo, hyper-

salivation, hypoglycemia, nausea, and mild nephrotoxicity, have

been reported upon the drug administration.^52-54

34, 38, 65, and 66 were obtained using a modiﬁed Pinner

reaction.⁹⁵In this method, the dinitrile precursors 67-80 were

converted to imidate esters, which reacted with the appropriate

amines at ambient temperature to give corresponding dications

(Scheme 1).

Since the discovery of the antiprotozoal properties of the

diamidine 1, a large number of structurally related congeners

showing potency against a wide range of microorganisms,^55-64

including P. jiroVeci^65-77as well as fungi and bacteria,^78,79have

been synthesized. Aromatic diamidines also have been tested

as enzyme inhibitors^80-85and demonstrated antitumor poten-

tial.^86-90Over the years, a wealth of data on biological activity

of compounds related to 1 have been accumulated. Nevertheless,

sometimes an analysis of structure-activity relationships of

antiprotozoal properties of aromatic dications is difﬁcult because

compounds in a series may have been screened against either

different pathogens or different strains of the same pathogens

or have not been tested at all.

Dinitriles 67,^44,6668,⁴⁴69,^44,6670,^44,6676,⁹⁴and 77^94,96have

been reported before. Novel dinitriles 71-75 were obtained from

appropriate cyano phenols and dibromoalkanes by Williamson

ether syntheses. Compounds 78,⁶⁶79,⁶⁶and 80⁶⁶have been

prepared by the nitration of the dinitriles 67,^44,6668,⁴⁴and 69^44,66

following the published protocol. 1,5-Bis[4-(1H-tetrazol-5-

yl)phenoxy]pentane (6) was synthesized from 69 and sodium

azide in the solution of acetic acid in DMF under reﬂux in 82%

yield. The dications 7 and 8 were obtained by reaction of 1,5-

bis(4-aminophenoxy)pentane⁹⁷with two equivalents of the

hydrobromide salts of 2-naphthalenylmethyl esters of benzen-

ecarboximidothioic⁹⁸and ethanimidothioic⁹⁹acids in 77 and

17% yields, respectively, according to the published procedure.⁹⁸

1-(4-Amidinophenoxy)-5-(4-aminophenoxy)pentane (10) was

synthesized as previously described.⁸³A Williamson ether

synthesis involving 4-(4,5-dihydro-1H-imidazol-2-yl)-phenol¹⁰⁰

and 1-bromo-3-(4-cyanophenoxy)propane^44,96,101afforded 4-cy-

anophenoxy-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]pro-

pane (81), which was subjected to Pinner conditions⁹⁵to give

4-amidinophenoxy-[4-(4,5-dihydro-1H-imidazol-2-yl)phenox-

y]propane (15) in 64% yield. Similarly, 4-cyanophenoxy-(4-

ethoxycarbonylphenoxy)propane (82), prepared by reaction of

commercially available ethyl 4-hydroxybenzoate and 1-bromo-

3-(4-cyanophenoxy)propane^44,96,101in 54% yield, was converted

to 4-amidinophenoxy-(4-carboxyphenoxy)propane (16) by a

two-step process involving a Pinner reaction⁹⁵followed by acid

catalyzed hydrolysis of an ester group. The synthesis of 1,3-

bis[2-amino-4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]pro-

pane (36) also involved a two-step procedure. First, 1,3-bis(4-

cyano-2-nitrophenoxy)propane (78) was converted to the

corresponding 2,2′-dinitro substituted diimidazoline by a Pinner

method, which then underwent catalytic hydrogenation of its

The purpose of the current study was to evaluate diamidine

1 and a series of its congeners, both newly synthesized and

available from our in-house library, in the standard screens that

the Consortium for Parasitic Drug Development (CPDD) uses

to identify its antiprotozoal drug candidates. To analyze

structure-activity relationships with respect to antiprotozoal

activity, cytotoxicity, and parasite selectivity, dications 1-66

were screened in vitro against Trypanosoma brucei rhodesiense

(STIB900), chloroquine resistant Plasmodium falciparum (K1),

and axenic amastigotes of Leishmania donoVani (MHOM/SD/

62/1S-CL2_D) and evaluated for cytotoxicity against rat myoblast

cells (L6). Compounds exhibiting high antitrypanosomal activi-

ties in vitro underwent in vivo testing in the STIB900 acute

mouse model of infection.

Chemistry. Compounds 4,^66,915, ^58,6711,^92,9312,⁶⁶14,^50,66

17,^44,6618,⁶⁷19,⁶⁶33,⁸³35,⁶⁶37,⁶⁶39,^67,9440,⁶⁶42,⁶⁶43,^67,94

and 44⁶⁶were prepared earlier in this laboratory for other

purposes.

The previously described dications 1,⁴⁴20,⁶⁶22,⁶⁷23,⁶⁶25,⁶⁷

26,⁶⁶29,⁶⁶32, 41,⁶⁶45,⁶⁶46,⁶⁶47,^67,94and 64 as well as the

newly synthesized analogues 2, 3, 9, 13, 21, 24, 27, 28, 30, 31,

2018 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

Scheme 2^a

^aReagents and conditions: (i) dibromopentane, K₂CO₃, DMF, 100 °C; (ii) CuCN, DMF, reﬂux; (iii) HCl gas, EtOH, 1,4-dioxane, 0 °C-ambient temp;

(iv) ammonia or amine, EtOH, ambient temp; (v) 10% aq HCl, 70 °C.

Scheme 3^a

^aReagents and conditions: (i) dibromopentane, K₂CO₃, DMF, 100 °C; (ii) H₂, 10% Pd/C, EtOH; (iii) HCl gas, EtOH, 1,4-dioxane, 0 °C-ambient temp;

(iv) appropriate amine, EtOH, ambient temp.

nitro groups to form the 2,2′-diamino substituted congener 36

in 23% overall yield.

with 1,5-dibromopetane in the presence of potassium carbonate

in DMF to give 1,5-bis[(4-cyano)phenylthio]pentane (90), which

was oxidized with periodic acid and chromium trioxide in

acetonitrile¹⁰⁵to give the dinitrile sulfone 91 in 70% overall

yield. The dinitriles 90 and 91 were converted to the diamidines

62 and 63 using modiﬁed Pinner methodology.⁹⁵

The synthesis of compounds 48-55 is depicted in Scheme

2. Ethyl 5-bromo-2-hydroxybenzoate, prepared by acid-catalyzed

esteriﬁcation of commercially available 5-bromo-2-hydroxy-

benzoic acid, reacted with 1,5-dibromopentane to afford 1,5-

bis(4-bromo-2-ethoxycarbonylphenoxy)pentane (83) in 48%

yield. Compound 83 was converted to 1,5-bis(4-cyano-2-

ethoxycarbonylphenoxy)pentane (84) by reaction with CuCN

in DMF under reﬂux in 62% yield. An imidate ester prepared

from the dinitrile 84 by modiﬁed Pinner method reacted with

ammonia or appropriate amines to give dications 48-50. The

congeners 51-53 were synthesized by hydrolysis of the ester

groups in compounds 48-50 with aqueous HCl. Compounds

54 and 55 bearing one or two amide substituents were isolated

as byproducts formed during the reaction of the aforementioned

imidate ester with ammonia.

Results and Discussion

The effect of structural variations of aromatic dications on

activities against Trypanosoma, Plasmodium, and Leishmania

specieshavebeenpreviouslyinvestigatedbyothers^{44,91,96,97,106-111}

and us.^50,112However, a majority of these studies have been

conducted with different strains of parasites and many deriva-

tives have simply not been tested against all pathogens. Here,

we report a continued investigation of structure-activity

relationship of analogues related to compound 1. Structural

modiﬁcations of dications used in this study included substitution

on the cationic groups, increasing the length of the aliphatic

chain between the two aromatic rings from three to six carbon

atoms, varying the position of attachment of the cationic

substituents, introduction of substituents on the aromatic rings,

and replacement of oxygen atoms in the alkyl linker with

isosteric sulfur or nitrogen atoms. Diamidine 1 as a hydrochlo-

ride salt prepared in our laboratory following the published

procedure,⁴⁴and its congeners 2-66 were evaluated in vitro

for their activity against bloodstream form trypomastigotes of

T. b. rhodesiense (STIB900), chloroquine resistant P. falciparum

(K1), axenic amastigotes of L. donoVani (MHOM/SD/62/1S-

CL2_D), and for cytotoxicity against rat myoblast cells (L6). The

results of the screening are summarized in Tables 1-7 and

compared to the data of melarsoprol (T. b. rhodesiense),

chloroquine and artemisinin (P. falciparum), and podophyllo-

toxin (L6). In addition to activities, three selectivity indexes,¹¹³

The synthesis of the 3,3′-dihydroxy and 3,3′-diﬂuoro substi-

tuted dications 56-61 is shown in Scheme 3. Both 2-benzyloxy-

4-hydroxybenzonitrile (85), prepared by diazotization of 4-amino-

2-(benzyloxy)-benzonitrile¹⁰²in 52% yield, and commercially

available 2-ﬂuoro-4-hydroxybenzonitrile (86) reacted with 1,5-

dibromopentane to give 1,5-bis(4-cyano-3-benzyloxyphenoxy)

pentane (87) and 1,5-bis(4-cyano-3-ﬂuorophenoxy)pentane (89)

in 67 and 51% yields, respectively. The protection of phenol

groups in compound 87 was removed by catalytic hydrogenation

over 10% palladium on charcoal to afford 1,5-bis(4-cyano-3-

hydroxyphenoxy)pentane (88) in 97% yield. Dinitriles 88 and

89 underwent a Pinner reaction⁹⁵followed by interaction with

ammonia, i-propylamine, and ethylenediamine to yield the

dications 56-58 and 59-61, respectively.

The syntheses of the thio diamidine 62^91,103and the sulfone

63 are outlined in Scheme 4. 4-Mercaptobenzonitrile¹⁰⁴reacted

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2019

Scheme 4^a

^aReagents and conditions: (i) HCl gas, EtOH, dioxane, 0 °C-ambient temp; (ii) EtOH saturated with ammonia, ambient temp; (iii) H₅IO₆, CrO₃, MeCN.

Table 1. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 1-11

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

IC₅₀(µM) SI_P

271

L. donoVani^k

IC₅₀(µM)

h

j

l

compd

1^a

R¹

Am

R²

Am

IC₅₀(µM)

0.007

SI_T

SI_L

3.80

152

150

>178

>176

>229

3.1

>202

139

543

0.014

0.004

0.003

1.65

1.83

4.69

17.8

2.77

>100

126

2

3

4

5

MeAm

i-PrAm

Im

MeIm

TZ

MeAm

i-PrAm

Im

MeIm

TZ

0.024

0.027

0.053

>19.6

>229

0.147

3.04

6333

5556

>3358

9

1

21

38000

50000

>59333

107

32

8

>64

2

6

8.31

>28

2

7

8

9

10

BAm

AAm

AmNH₂

Am

BAm

AAm

AmNH₂

NH₂

0.201

0.048

1.41

15

>4208

99

<1

2

1

<1

>66

14

9.67

120

1.31

0.289

0.004

91

40

1945

2.24

0.234

54

50

>100

55.6

11

Am

AmOH

11.6

7.78

117

450

0.01

MAP^b

CQ^c

ATM^d

PPT^e

0.124

0.006

944

75000

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].¹¹³

reﬂecting the inhibition of the particular parasite with respect

to the L6 cells, were analyzed: antitrypanosomal selectivity

index SI_T, expressed as the ratio [IC₅₀(L6-cells)/IC₅₀(T. b.

rhodesiense)]; antiplasmodial selectivity index SI_P, expressed

as the ratio [IC₅₀(L6-cells)/IC₅₀(P. falciparum)]; and antile-

ishmanial selectivity index SI_L, expressed as the ratio [IC₅₀(L6-

cells)/IC₅₀(L. donoVani)]. Dications exhibiting high antitrypa-

nosomal activities in vitro underwent in vivo screening in the

STIB900 acute mouse model of trypanosomiasis (Table 8).

Structure-Activity Study. Modiﬁcations of Cationic

Moieties. The antiprotozoal activities of analogues bearing

various cationic substituents in the 4,4′-positions of aromatic

rings are presented in Table 1. Amidine groups in compound

1⁴⁴were replaced with (N-methyl)amidine (compound 2), (N-

isopropyl)amidine (compound 3), imidazoline (compound 4),^66,91

(N-methyl)imidazoline (compound 5),^58,67tetrazole (compound

6), and amidrazone (compound 9) motifs. Cationic substituents

of congeners 7 and 8 were attached to the 1,5-diphenoxypentane

motif through the nitrogen atoms on the amidine groups rather

than through the carbons. In the dications 10⁸³and 11,^92,93one

of the amidine moieties was replaced with amino or (N-

hydroxy)imidoyl groups, respectively.

The N-substitution on the cationic fragments signiﬁcantly

reduced cytotoxicity of the dications 2-6 and 9 compared to

the compound 1, corroborating previously published results.^66,68

For example, the bis(N-methyl)amidine 3 was 40-fold less

cytotoxic than diamidine 1. Similarly, cytotoxicity of congeners

4-6 bearing cyclic cationic scaffolds decreased 46- to 60-fold

compared to that of the compound 1. Cytotoxicity of compounds

7 and 8 depended on the type of the substituents on the cationic

fragments. While the phenyl substituted dication 7 exhibited

cytotoxicity comparable to that of 1, the corresponding methyl

analogue 8 was 50-fold less cytotoxic than the compound 1.

Cytotoxicity of congener 11 bearing amidine and (N-hydroxy)-

imidoyl groups was only slightly lower than that of diamidine

1, while compound 10 with amidine and amino substituents was

at least 30-fold less cytotoxic than dication 1.

2020 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

Table 2. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 12-19

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

L. donoVani^k

h

j

l

compd

R¹

Am

i-PrAm

Im

Am

Im

Am

R²

Am

i-PrAm

Im

CO₂H

Am

n

IC₅₀(µM)

SI_T

IC₅₀(µM)

SI_P

IC₅₀(µM)

SI_L

12

13

14

15

16

17

3

4

5

6

8.80

>192

75.3

71.6

167

4.70

62.7

3.80

10.5

7.78

117

0.007

0.093

0.110

0.018

>257

0.010

0.244

0.007

0.036

0.004

1257

2065

685

3978

0.6

470

257

543

292

0.006

0.025

0.010

0.018

>2.80

0.021

0.031

0.014

0.015

1467

7680

7530

3978

60

1.50

7.11

288

2.98

>100

NT^m

3.06

1.83

1.97

6

>27

26

24

<2

NT^m

20

2

224

18

Im

Am

2023

271

1^a

19

700

5

MAP^b

CQ^c

ATM^d

PPT^e

1945

0.124

0.006

944

450

0.01

75000

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].^{113 m}NT, not tested.

Table 3. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 20-31

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

IC₅₀(µM)

L. donoVani^k

IC₅₀(µM)

h

j

l

compd

R

n

IC₅₀(µM)

SI_T

SI_P

SI_L

20

21

22

23

Am

i-PrAm

Im

Am

i-PrAm

Im

Am

i-PrAm

Im

Am

3

4

5

6

16.2

>183

43.5

16.0

>171

>185

3.80

18.4

>175

100

17.1

107

99.0

7.78

117

0.052

0.543

4.30

0.056

0.619

1.94

0.007

0.065

0.416

1.49

0.075

0.258

0.875

0.004

312

>337

10

0.017

0.018

5.39

953

>10167

8

6.07

16.2

30.8

4.46

19.3

39.5

1.83

3.19

15.4

22.4

2.38

11.6

21.9

3

>11

1

4

>9

5

2

6

>11

4

7

286

>276

>95

543

283

421

67

228

415

113

1945

0.021

0.013

0.107

0.014

0.019

0.004

0.024

0.013

0.006

0.020

762

24

>13154

>1729

271

25

1^a

26

27

28

29

30

31

968

43750

4167

1315

17833

4950

i-PrAm

Im

9

5

MAP^b

CQ^c

ATM^d

PPT^e

0.124

0.006

944

75000

450

0.01

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].¹¹³

In this study, the N-alkylation of cationic fragments reduced

the activity of congeners 2-11 against T. b. rhodesiense and

L. donoVani compared to dication 1. Among compounds 2-11,

only bis(N-methyl)amidine 2, bis(N-isopropyl)amidine 3, and

diimidazoline 4 exhibited antitrypanosomal IC₅₀values less than

100 nM and antileishmanial potencies less than 20 µM. Previous

reports also demonstrated that N-substitution on the amidine

groups reduced antitrypanosomal and antileishmanial activities

of aromatic diamidines.^{50,91,107,108,110,112,114}While diimidazoline

4 was less potent against T. b. rhodesiense compared to bis(N-

methyl)amidine 2 and bis(N-isopropyl)amidine 3, it exhibited

greater antileishmanial activity than both congeners 2 and 3.

Derivatives 2-4 displayed selectivity against T. b. rhodesiense

6- to 12-fold greater than that of compound 1. They were also

4 to 32 times more selective against L. donoVani relative to 1.

Contrary to the antitrypanosomal and antileishmanial results,

the N-alkyl substitution on the amidine groups improved

activities of N-alkyl amidines 2 and 3 and diimidazoline 4

against P. falciparum. For example, dications 2-4 displayed

antiplasmodial IC₅₀values less than that of diamidine 1 (IC₅₀

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2021

Table 4. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners32-47

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

L. donoVani^k

IC₅₀(µM)

h

j

l

compd

R¹

Am

Im

Am

MeAm

Im

Am

Im

R²

Cl

n

IC₅₀(µM)

SI_T

IC₅₀(µM)

SI_P

SI_L

32

33

34

35

36

37

38

39

3

4

5

24.8

6.40

22.8

68.7

>167

114

0.004

0.019

0.188

0.156

0.813

0.023

0.057

0.090

0.042

0.181

0.270

0.068

0.007

0.044

0.098

0.010

0.040

0.004

6200

337

121

0.019

0.016

0.035

0.027

0.070

0.062

0.641

0.055

0.019

0.018

0.044

0.020

0.014

0.026

0.024

0.021

0.019

1305

400

651

2544

2386

1839

276

1909

574

2378

>4250

8200

271

1.56

1.17

30.9

NT^m

6.89

25.3

34.6

2.13

15.1

38.1

38.9

1.83

1.45

11.4

24.0

36.3

16

5

0.7

Br

NO₂

NH₂

OCH₃

Cl

NO₂

OCH₃

H

440

NT^m

17

7

>205

4957

3105

1167

260

177

105

3

5

3

>5

>4

2

8

3

40

41

42

10.9

42.8

>187

>164

3.80

11.2

37.3

162

>158

7.78

117

450

0.01

236

>693

>2412

543

255

381

16200

>3950

1945

43

1^a

Am

Im

44

45

46

47

Cl

431

NO₂

OCH₃

1554

7714

>8316

7

>4

MAP^b

CQ^c

ATM^d

PPT^e

0.124

0.006

944

75000

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparu)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].^{113 m}NT, not tested.

Table 5. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 48-55

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

L. donoVani^k

IC₅₀(µM) SI_L

h

j

l

compd

1^a

R¹

Am

R²

R³

IC₅₀(µM)

SI_T

543

>13

IC₅₀(µM)

SI_P

H

3.80

>152

>140

>138

>165

>144

>151

>165

>168

7.78

117

0.007

12.1

0.014

0.021

0.013

0.027

71.8

271

1.83

>50

2

3

48

49

50

51

52

53

54

55

Am

i-PrAm

Im

Am

i-PrAm

Im

CO₂Et

CO₂H

CO₂Et

CONH₂

CO₂Et

CO₂H

CONH₂

>7238

>10769

>5111

2

0.051

>15.3

>18.3

>16.0

>16.8

0.011

0.045

0.004

>2745

9

55.9

13.6

NT^m

>200

52.1

>50

2

10

NT^m

0.7

0.8

3

>1.60

>1.68

0.014

0.038

90

>11786

>4421

9

Am

>15000

>3733

1945

3

MAP^b

CQ^c

ATM^d

PPT^e

0.124

0.006

944

75000

450

0.01

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].^{113 m}NT, not tested.

) 14 nM) and artemisinin (ATM) (IC₅₀) 6 nM). Compounds

2-4 exhibited antiplasmodial selectivity indexes SI_Pthat were

140- to nearly 220-fold greater than that of dication 1. These

ﬁndings correlate with previously published results.^112,114

In the case of “reversed” amidines, phenyl derivative 7 was

20-fold more active against T. b. rhodesiense than the corre-

sponding methyl analogue 8. However, because of its higher

cytotoxicity, antitrypanosomal selectivity of the dication 7 was

2022 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

Table 6. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 56-61

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

IC₅₀(µM)

L. donoVani^k

h

j

l

compd

1^a

56

57

R¹

Am

i-PrAm

Im

Am

i-PrAm

Im

R²

IC₅₀(µM)

SI_T

SI_P

IC₅₀(µM)

SI_L

H

3.80

>195

>156

>169

6.90

131

23.2

7.78

117

0.007

7.96

14.7

543

>24

>11

>46

531

506

5

0.014

1.29

271

1.83

>100

1.88

15.0

52.6

2

4

9

OH

F

>151

>269

>1724

460

18714

43

0.581

0.098

0.015

0.007

0.544

58

59

60

61

3.70

0.013

0.259

4.46

0.4

MAP^b

CQ^c

ATM^d

PPT^e

0.004

1945

0.124

0.006

944

75000

450

0.01

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].¹¹³

Table 7. In Vitro Antiprotozoal Activity and Cytotoxicity of Congeners 62-66

cytotoxicity^f

T. b. rhodesiense^g

P. falciparumⁱ

IC₅₀(µM)

L. donoVani^k

IC₅₀(µM)

h

j

l

compd

R

X

IC₅₀(µM)

SI_T

SI_P

SI_L

1^a

62

63

64

65

66

Am

i-PrAm

Im

O

S

SO₂

NH

3.80

7.80

>174

0.95

125

34.5

7.78

117

0.007

0.005

28.1

<0.001

0.050

0.001

0.004

543

1560

>6

>950

2500

34500

1945

0.014

0.019

0.098

0.006

0.002

0.001

271

411

>1776

158

62500

34500

1.83

1.27

>100

0.502

5.75

2

6

2

22

136

0.254

MAP^b

CQ^c

ATM^d

PPT^e

0.124

0.006

944

75000

450

0.01

^aPentamidine hydrochloride. ^bMAP, melarsoprol. ^cCQ, chloroquine. ^dATM, artemisinin. ^ePPT, podophyllotoxin. ^fCytotoxicity (L6 rat myoblast cells).

Average of duplicate determinations.^{128 g}Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.^{129 h}Selectivity index for T. b.

rhodesiense (SI_T), expressed as the ratio [IC₅₀(L6)/IC₅₀(T. b. rhodesiense)].^{113 i}Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate

determinations.^{130,131 j}Selectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀(L6)/IC₅₀(P. falciparum)].^{113 k}Leishmania donoVani (MHOM/

SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.^{132,133 l}Selectivity index for L. donoVani (SI_L), expressed as the ratio [IC₅₀(L6)/

IC₅₀(L. donoVani)].¹¹³

3 times lower than that of the compound 8. Furthermore, methyl

analogue 8 displayed antimalarial potency that was 4 times

superior to that of phenyl congener 7. Antiplasmodial selectivity

index SI_Pof the compound 8 was 15-fold greater than that of

dication 1 and more than 280 times superior to that of the

dication 7. Both congeners 7 and 8 were inactive against L.

donoVani, despite the previous report in which “reversed”

diamidines possessing a diphenylfuran linker exhibited excellent

antileishmanial potencies.¹¹⁵

Bis(N-methyl)imidazoline 5, ditetrazole 6, diamidrazone 9,

and congener 10 were the least active compounds in the group

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

Dication 5 demonstrated lower DNA binding afﬁnity compared

to compound 1,¹¹⁶which could explain its reduced potency

toward the pathogens. Although a direct correlation between

DNA binding and antiprotozoal potency for analogues of

diamidine 1 has not been observed, it was concluded that a

possession of ∆T _mvalues above a certain threshold is required

for an activity.^108,117The reduced antiprotozoal potency of the

congener 6 compared to dication 1 is probably a result of

decreased basicity of tetrazole motifs, which are isosteres of

carboxyl groups. Among derivatives 10 and 11 bearing a single

amidine groups, dication 11 was 4 times more active against T.

b. rhodesiense, 10 times more effective against P. falciparum,

and 2 times more potent against L. donoVani than compound

10. These results can be explained by the fact that at physi-

ological pH only the amidine group in congener 10 is protonated,

although presence of at least two cationic fragments is required

for pronounced antiprotozoal activity.^110,114While being sig-

niﬁcantly less active in vitro compared to corresponding

amidines,^118-121aromatic amidoximes have been shown to be

rapidly converted to amidines in vivo.¹²²The use of amidoximes

as prodrugs for amidines creates an opportunity for developing

orally bioavailable drug candidates.^118,123-125

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2023

Table 8. Activity of Select Dications in the STIB900 Mouse Model of

Trypanosomiasis

Among compounds with linkers of the same length between

aromatic rings, substitution on the cationic fragments decreased

cytotoxicity of the dications 12-19.¹¹²For example, bis(N-

isopropyl)amidine 13 and diimidazoline 14 bearing a propylene

bridge demonstrated cytotoxicity 22 and 9 times lower than that

of diamidine 12. Similarly, diimidazoline 18 possessing a four-

carbon chain was 13 times less cytotoxic compared to dication

17. Replacement of a single amidine group in dication 12 with

an imidazoline fragment (compound 15) caused a signiﬁcant

reduction in cytotoxicity. Likewise, congener 16, bearing

amidine and carboxyl substituents, was 19 times less cytotoxic

than diamidine 12.

Alterations of the length of carbon chains connecting the

aromatic rings in compound 1 and its derivatives 3 and 4 reduced

antiprotozoal activities and selectivities of congeners 13-19

against T. b. rhodesiense, P. falciparum, and, except for the

bis(N-isopropyl)amidine 13, against L. donoVani. For example,

dication 13 exhibited antileishmanial activity 2 times greater

and was 3 times more selective against the pathogen than bis(N-

isopropyl)amidine 3. On the other hand, congener 12 was as

active as dication 1 against T. b. rhodesiense and demonstrated

superior potencies against P. falciparum and L. donoVani, at

the same time exhibiting greater selectivity to all three pathogens

than compound 1. These results correlate with the previously

published data on antiprotozoal properties of select aromatic

diamidines.⁵⁰

Among compounds with carbon chains of the same length,

N-substitution or replacement of the amidine groups reduced

activities of the dications 12-19 against T. b. rhodesiense,^108,112

P. falciparum, and L. donoVani. For example, the antitrypano-

somal potencies of bis(N-isopropyl)amidine 13 and diimidazo-

line 14, possessing a trimethylene linker, decreased 13- and 15-

fold, respectively, compared to diamidine 12. In addition,

diimidazoline 18 bearing a tetramethylene bridge was 24 times

less active against T. b. rhodesiense than dication 17. Smaller

declines in the antiplasmodial and the antileishmanial activities

of the congeners 13 and 14 with respect to compound 12 were

observed. Thus, bis(N-isopropyl)amidine 13 displayed 4 times

lower activities against both P. falciparum and L. donoVani,

and diimidazoline 14 was half as active against the parasites as

diamidine 12. At the same time, the dications 13 and 14 were

5 times more selective against P. falciparum and 4 times more

selective against L. donoVani than the compound 12. Interest-

ingly, dication 15, bearing amidine and imidazoline substituents,

was less active against P. falciparum than both diamidine 12

and diimidazoline 14. Congener 16, bearing amidine and

carboxyl substituents, exhibited almost no activity against all

three pathogens used in this study.

in vivo^b

in vitro

dose

survival

(days)^e

compd

cures^d

IC₅₀(nM)^a

(mg/kg)^c

melarsoprol

4

4 × 8

4/4

2/4

4/4

2/4

1/4

0/4

2/4

3/4

0/4

1/4

0/4

2/4

0/4

3/4

1/4

2/4

0/4

1/4

4/4

0/4

1/4

3/3

2/4

3/4

1/4

2/4

0/4

1/4

0/4

1/4

0/4

4/4

>60

4 × 2

4 × 1

>51.5

>44.3

>43.5

>60.0

>41.0

>41.3

>39.0

7.0

1

2

7

4 × 20

4 × 10

4 × 20

4 × 5

24

3

4

7

12

13

14

27

53

147

7

93

110

4 × 5

4 × 20

4 × 10

4 × 20

4 × 10

4 × 20

4 × 5

34.0

>38.5

>51.0

22.8

>26.8

24.8

15

17

18

10

244

>41.5

7.8

19

20

23

36

52

56

4 × 20

4 × 5

>51.8

>44.3

>32.8

>40.0

>43.0

20.0

18.5

>36.0

>60

13.0

>40.0

>60

>52.8

>60.0

>32.8

>41.5

22.0

17.5

18.3

29.5

26.0

>31.8

5.5 (toxic)

26.5

>33.0

32.3

>29.3

18.0

>60

4 × 20

4 × 10

4 × 20

4 × 10

4 × 20

4 × 5

26

29

32

33

35

37

65

75

4

19

156

23

4 × 5

4 × 20

4 × 5

39

40

90

42

4 × 20

4 × 10

4 × 20

4 × 10

4 × 20

4 × 5

42

43

44

46

47

49

55

59

62

64

65

66

270

68

44

10

40

51

45

13

5

4 × 10

4 × 20

4 × 10

4 × 5

<1

50

1

4 × 5

^aAverage of duplicate determinations.^{129 b}STIB900 acute mouse

model.^{134 c}Intraperitoneal administration. ^dNumber of mice that survive

and are parasite free for 60 days. ^eAverage days of survival; untreated

controls expired between day 7 and 10 post infection.

Change of the Position of Cationic Substituents. The

cytotoxicity and antiprotozoal activities of congeners bearing

cationic substituents in the 3,3′-positions of aromatic rings and

alkyl linkers ranging from three to six carbon atoms are

presented in Table 3.

Regardless of the length of the carbon bridge, placement of

the amidine groups in the 3,3′-positions reduced the cytotox-

icities of diamidines 20, 23, 26, and 29 relative to compound

1. Bis(N-isopropyl)amidines 21, 24, and 27, bearing three- to

ﬁve-carbon chains were less cytotoxic than dication 3. Further

elongation of the linker between the aromatic rings in compound

30 increased its cytotoxicity relative to congener 3. All the other

derivatives, 22, 28, and 31, except for the diimidazoline 25,

possessing a tetramethylene linker, were more cytotoxic than

the dication 4. Within the groups of 3,3′-substituted dications

with the same number of carbon atoms in the linker, the bis(N-

Alteration of the Length of Carbon Linkers. Antiprotozoal

activities of congeners bearing cationic substituents in the 4,4′-

positions of aromatic rings and an alkyl chain of three to six

carbon atoms are presented in Table 2. Previously, the nature

of the linker between the cationic groups had been shown to

affect the antiprotozoal properties of dications.^{44,58,111,112}In the

present study, there was no apparent correlation between the

number of methylene groups in the linker and the cytotoxicity

of compounds 12-19 with respect to analogues 1, 3, and 4.

Thus, diamidines 12, 14, and 19 bearing three-, four-, and six-

carbon chains were less cytotoxic than compound 1. Similarly,

bis(N-isopropyl)amidine 13 containing a propylene bridge

exhibited lower cytotoxicity compared to dication 3. On the

other hand, diimidazolines 14 and 18 with three- and four-carbon

chains, respectively, were more cytotoxic than congener 4.

2024 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

isopropyl)amidines and diimidazolines exhibited lower cyto-

toxicities than diamidines.

against L. donoVani usually increased with the number of carbon

atoms in the linker.

The 3,3′-substituted diamidines 20, 23, 26, and 29 exhibited

lower activities against T. b. rhodesiense than dication 1, as

did bis(N-isopropyl)amidines 21, 24, 27, and 30, as well as

diimidazolines 22, 25, 28, and 31 compared to 4,4′-substituted

bis(N-isopropyl)amidine 3 and diimidazoline 4, respectively.

Also, the 3,3′-substituted dications 20-22, 23, 25, and 29

displayed lower antitrypanosomal potencies and were less

selective against T. b. rhodesiense than the corresponding 4,4′-

substituted isomers 12-14, 17, 18, and 19. Within groups of

congeners with aliphatic linkers of the same length, the

antitrypanosomal properties of 3,3′-substituted dications 20-31

decreased in the order Am > i-PrAm > Im. Elongation of the

carbon chain between the aromatic rings reduced activities of

the 3,3′-substituted diamidines 20, 23, 26, and 29 against T. b.

rhodesiense. On the other hand, antitrypanosomal potencies of

bis(N-isopropyl)amidines 21, 24, 27, and 30 as well as diimi-

dazolines 22, 25, 28, and 31 improved as the number of carbon

atoms in the bridge increased. The 3,3′-substituted diamidines

and bis(N-isopropyl)amidines demonstrated comparable levels

of antitrypanosomal selectivity, while diimidazolines were

signiﬁcantly less selective against the pathogen.

Aromatic Ring Substituents. The cytotoxicity and antipro-

tozoal activities of congeners bearing substituents in the 2,2′-

or 3,3′-positions of aromatic rings and with alkyl linker chains

of three to ﬁve carbon atoms are presented in Tables 4-6.

2,2′-Substituted diamidines 32-35, 37, 40-42, and 44-46,

as well as bis(N-methyl)amidine 38 (Table 4) exhibited reduced

cytotoxicities relative to the corresponding dications 12 and 17

as well as to diamidine 1 in all cases except for the 2,2′-dibromo

substituted diamidine 33, which was slightly more cytotoxic

than compound 12. All 2,2′-substituted diimidazolines 36, 39,

43, and 47 demonstrated increased cytotoxicity relative to

analogue 4, whereas congeners 36, 39, and 43 were less

cytotoxic than the corresponding diimidazolines 14 and 18

containing the three- and four-carbon chains, respectively.

Within groups of dications with aliphatic linkers of the same

length, all 2,2′-dimethoxy substituted diimidazolines 39, 43, and

47 were more cytotoxic than the corresponding diamidines 37,

42, and 46, respectively. Conversely, the 2,2′-diamino diimi-

dazoline 36 exhibited lower cytotoxicity compared to the

diamidine 35.

The cytotoxicities of 2,2′-diethoxycarbonyl, -dicarboxy, and

-diaminocarbonyl diamidines 48, 51, 54, and 55 (Table 5) were

signiﬁcantly lower than that of compound 1, while bis(N-

isopropyl)amidine and diimidazoline congeners 49, 50, 52, and

53 were slightly more cytotoxic relative to the corresponding

analogues 3 and 4, lacking any 2,2′-substituents. Regardless of

the type of substituents in the 2,2′-positions, the diamidines

48-55 demonstrated comparable cytotoxicities. At the same

time, the introduction of the hydroxy or ﬂuoro groups in the

3,3′-positions produced different effects on cytotoxic properties

of dications 56-61 with regard to congeners 1, 3, and 4 (Table

6). Thus, cytotoxicity of the 3,3′-dihydroxy substituted diamidine

56 decreased more than 50-fold compared to diamidine 1, while

the bis(N-isopropyl)amidine 57 and the diimidazoline 58

displayed cytotoxicities similar to those of compounds 3 and

4. On the other hand, the addition of 3,3′-diﬂuoro groups in

diamidine 59 resulted in only a minor reduction of its cytotox-

icity relative to dication 1, whereas the congeners 60 and 61

were more cytotoxic than bis(N-isopropyl)amidine 3 and di-

imidazoline 4, respectively. Among the 3,3′-dihydroxy substi-

tuted dications 56-58, diamidine 56 exhibited the lowest

cytotoxicity, while in the group of 3,3′-diﬂuoro congeners

59-61, diamidine 59 was the most cytotoxic compound.

The antitrypanosomal properties of congeners 33-46 and

48-61 bearing substituents on the aromatic rings were dimin-

ished compared to compound 1, its N-substituted derivatives 3

and 4, diamidines 12 and 17, and their imidazoline analogues

14 and 18 (Tables 4-6). The only compounds that were more

active against T. b. rhodesiense than the corresponding deriva-

tives without 2,2′-substituents were 2,2′-dichloro diamidine 32

and 2,2′-dimethoxy diimidazolines 39, 43, and 47. The 2,2′-

diethoxycarbonyl dications 48 and 50, 2,2′-dicarboxy congeners

51-53, and the 3,3′-dihydroxy compounds 56-58 displayed

signiﬁcantly lower antitrypanosomal activities in the micromolar

range. While the 3,3′-diﬂuoro diamidine 59 was nearly as active

as dication 1 against T. b. rhodesiense, bis(N-isopropyl)amidine

60 and diimidazoline 61 were almost 10- and 80-fold less potent

against the pathogen than the corresponding congeners 3 and

4, respectively. The antitrypanosomal activities of the dications

59-61 decreased in the order Am > i-PrAm > Im. The

diamidine 32 displayed improved antitrypanosomal properties

and was 11- and 5-fold more selective for the T. b. rhodesiense

Antiplasmodial properties of 3,3′-substituted diamidines 20,

23, and 26 connected by the three-, four-, and ﬁve-carbon chains,

respectively, decreased with respect to diamidine 1, while the

analogue 29 bearing the hexamethylene linker was more active

against P. falciparum than 1. Contrary to the previously

published results,⁵⁰3,3′-substituted dications 20, 22, 25, and

29 were less active against P. falciparum than the corresponding

4,4′-substituted derivatives 12, 14, 18, and 19. At the same time,

bis(N-isopropyl)amidine 21 exhibited greater antimalarial po-

tency than the corresponding 4,4′-substituted analogue 13, and

diamidine 23 was as effective against the pathogen as congener

17. The 3,3′-substituted diamidines 23, 26, and 29 as well as

bis(N-isopropyl)amidine 21 were more selective for P. falci-

parum relative to the 4,4′-substituted congeners 17, 1, 19, and

13, respectively. Among the 3,3′-substituted dications bearing

four-, ﬁve-, and six-carbon linkers, the bis(N-isopropyl)amidines

exhibited the highest activity against P. falciparum, followed

by the diamidines and diimidazolines. Antiplasmodial selectivity

of these congeners decreased in the order i-PrAm > Im > Am.

At the same time, diamidine 20 and bis(N-isopropyl)amidine

21, bearing propylene bridge, displayed similar antiplasmodial

potencies, while the corresponding diimidazoline 22 was inactive

against the pathogen. Although activities of 3,3′-substituted

diamidines 20, 23, 26, and 29 against P. falciparum underwent

only minor changes as number of carbon atoms in the linker

increased, the antiplasmodial potencies of bis(N-isopropyl)a-

midines 21, 24, 27, and 30 increased with elongation of the

carbon chain. A similar improvement on an even greater scale

was observed for the diimidazolines 22, 25, 28, and 31.

Except for bis(N-isopropyl)amidines 21, 27, and 30, the 3,3′-

substituted congeners exhibited reduced activity against L.

donoVani compared to diamidine 1 and its N-substituted

derivatives 3 and 4. Antileishmanial properties of the dications

20-22, 25, and 29 also diminished with respect to their 4,4′-

substituted counterparts 12-14, 18, and 19,⁵⁰as did the

selectivity of the compounds 20-22 and 25 against the

pathogen. Among the 3,3′-substituted pentamidine congeners

with alkyl linkers of identical length, the antileishmanial

properties decreased in the order Am > i-PrAm > Im. On the

other hand, activities of the 3,3′-substituted congeners 20-31

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2025

than the dications 1 and 12, respectively. Similarly, the 2,2′-

dimethoxy substituted congeners as well as aminocarbonyl-

substituted dications 54 and 55 demonstrated greater antitry-

panosomal selectivity compared to the corresponding compounds

without 2,2′-substituents. Thus, the 2,2′-dimethoxy diamidine

46 and 2-aminocarbonyl-2′-ethoxycarbonyl diamidine 54 were

nearly 30- and 28-fold more selective against the pathogen than

compound 1.

46-fold less cytotoxic than compound 1. In contrast, the aza-

analogues 64-66 were up to 4 times more cytotoxic than the

corresponding dications 1, 3, and 4. N-Substitution on the

amidine groups reduced cytotoxicities of the congeners 65 and

66 relative to the diamidine 64. Thus, bis(N-isopropyl)amidine

65 and diimidazoline 66 were nearly 130 and about 35 times

less cytotoxic than dication 64.

The thio-analogue 62 demonstrated improved antitrypano-

somal and antileishmanial properties and a slight decline in

antiplasmodial potency compared to diamidine 1. Because of

its lower cytotoxicity, dication 62 displayed greater selectivity

toward T. b. rhodesiense, P. falciparum, and L. donoVani than

compound 1. In contrast, the sulfone derivative 63 was inactive

against both T. b. rhodesiense and L. donoVani. Although the

compound 63 exhibited only moderate antiplasmodial activity

(IC₅₀) 98 nM), it was 6 times more selective against P.

falciparum than diamidine 1.

Replacement of the linker oxygens in compound 1 with

nitrogen atoms afforded the aza-analogue 64, which was active

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

most signiﬁcant improvements in antiprotozoal properties were

observed in the antitrypanosomal and antileishmanial assays,

which is consistent with the previously reported data.⁵⁰Com-

pound 64 exhibited subnanomolar activity against T. b. rhod-

esiense, being nearly twice as selective against the pathogen as

diamidine 1. It was also almost 4 times more potent against L.

donoVani relative to dication 1, at the same time showing similar

parasite selectivity. While diamidine 64 displayed improved

antiplasmodial properties compared to compound 1, it demon-

strated lower selectivity toward P. falciparum than the dication

1.

Antiplasmodial activities of the 2,2′- and 3,3′-disubstituted

congeners 32-61 decreased relative to those of dications 1, 3,

and 4, although the potency against P. falciparum as well as

the parasite selectivity of the compounds 40, 41, and 43 bearing

four-carbon linkers improved compared to that of the diamidine

17 and diimidazoline 18.⁵⁰Substitution on the amidine frag-

ments often afforded derivatives with increased activities against

the pathogen. Thus, among dications 48-53 and 59-61, bis(N-

isopropyl)amidines 49, 52, and 60 displayed higher antiplas-

modial activities compared to the corresponding diamidines and

diimidazolines. By contrast, in the case of the 2,2′-dimethoxy

and 3,3′-dihydroxy congeners, the diimidazolines 39, 43, 47,

and 58 were the most active against P. falciparum in the

respective groups. While the antiplasmodial properties of the

2,2′-dichloro substituted congeners 32, 40, and 44 decreased

with the elongation of the alkyl linker, the activity of the 2,2′-

dimethoxy dications 37, 39, 42, 43, 46, and 47 versus P.

falciparum was enhanced as the length of the linker increased.

Also, the introduction of 2,2′-dimethoxy substituents resulted

in improved selectivity of diamidines 37, 42, and 46 for the

pathogen relative to the corresponding unsubstituted analogues.

Thus, congeners 42 and 46 were 19- and 28-fold more selective

for P. falciparum than diamidines 17 and 1, respectively.

The antileishmanial activities of congeners 34-43 and 45-47

bearing nitro, amino, and methoxy substituents on the aromatic

rings were lower relative to those of the unsubstituted dications

1, 4, 12, 14, and 18 (Tables 4-6). Compounds 48-58

possessing 2,2′-diethoxycarbonyl, 2,2′-dicarboxy, 2,2′-diami-

nocarbonyl, and 3,3′-dihydroxy substituents were completely

inactive against the pathogen. Conversely, the 2,2′-dichloro or

2,2′-dibromo substituted congeners 32, 33, and 44 bearing three-

and ﬁve-carbon chains were as active against L. donoVani as

dication 1.⁵⁰The 3,3′-diﬂuoro substituted diamidine 59 and

bis(N-isopropyl)amidine 60 exhibited antileishmanial IC₅₀values

comparable to those of the congeners 1 and 3, respectively, while

the corresponding diimidazoline 61 was less active against L.

donoVani than its unsubstituted counterpart 4. The antileish-

manial selectivity of dications was not signiﬁcantly affected by

the introduction of nitro, ethoxycarbonyl, carboxyl, and ami-

nocarbonyl groups to the 2,2′-positions of the aromatic rings.

However, 2,2′-dichloro or 2,2′-dimethoxy substituted diamidines

32, 37, 44, and 46 displayed higher antileishmanial selectivities

compared to both diamidines 12 and 1.

Compounds 65 and 66, the N-substituted congeners of 64,

displayed slightly different antiprotozoal properties. Thus, bis(N-

isopropyl)amidine 65 demonstrated lower antitrypanosomal and

antileishmanial activities while at the same time showing

improved selectivity for T. b. rhodesiense and L. donoVani with

respect to diamidines 1 and 64. On the other hand, the

antiplasmodial activity of 65 increased 7- and 3-fold relative to

that of 1 and 64, respectively. Dication 65, which was

signiﬁcantly less cytotoxic than diamidine 64, exhibited the

highest antiplasmodial selectivity among all congeners (SI_P)

62500). It was 230 times more selective for P. falciparum than

compound 1. Conversely, diimidazoline 66 displayed high

activities against all three parasites used in this study. Compound

66 not only exhibited the second highest antitrypanosomal

activity in the series with the IC₅₀value of 1 nM but also

demonstrated the highest parasite selectivity (SI_T) 34500),

being 63 times more selective against T. b. rhodesiense than

diamidine 1. Diimidazoline 66 was also the most active

compound in the group against P. falciparum (IC₅₀) 1 nM)

and L. donoVani, where the dications 64 and 66 were the only

two congeners with antileishmanial IC₅₀values less than 1 µM.

The compound 66 possessed antileishmanial selectivity index

(SI_L) 136), which was 68-fold greater than that of dication 1.

The aza-analogues 64-66 exhibited higher activity against P.

falciparum and L. donoVani relative to compound 1 and its bis(N-

isopropyl)amidine and diimidazoline congeners 3 and 4. While

diimidazoline 4 displayed greater antiplasmodial selectivity com-

pared to its counterpart 66, the (N-isopropyl)amidine 65 was more

selective for the parasite than the corresponding derivative 3. At

the same time, the bis(N-isopropyl)amidine 65 and the diimida-

zoline 66 were 2-3 times more selective against L. donoVani than

the congeners 3 and 4. Compound 65 exhibited 2-fold decrease in

antileishmanial activity and selectivity for the pathogen relative to

Replacement of Oxygen Atoms in the Linker Region. The

cytotoxicity and antiprotozoal activities of dications, in which

oxygen atoms in the linker region were replaced with sulfur

atoms (compound 62), sulfonyl groups (compound 63), and

amino groups (compounds 64, 65, and 66) are presented in Table

7. The oxygen atoms in the aliphatic linker are part of the

recognitionmotifforP2transporterinTrypanosomaspecies^11,49,64,126

and are important for the activity of diamidine 1 against the

pathogen.¹²⁷

The replacement of oxygens produced opposite effects on

cytotoxicities of the sulfur- and nitrogen-containing congeners

62-66. While cytotoxicity of the thio-analogue 62 declined only

slightly compared to diamidine 1, the sulfone derivative 63 was

2026 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

dication 3. Conversely, diamidine 64 and diimidazoline 66 were

more potent against T. b. rhodesiense than dications 1 and 4,

respectively. Aza-analogue 66 displayed antitrypanosomal selectiv-

ity index SI_Lmore than 10-fold greater than that of the corre-

sponding congener 4. Overall, diimidazoline 66 was the most

promising compound in the series, showing excellent activities and

high selectivities against T. b. rhodesiense, P. falciparum, and L.

donoVani.

somal activity both in vitro and in vivo, providing cures to 4/4

animals when administered at 5 mg/kg daily for four days.

Conclusions

In this study, a series of dications has been tested in vitro

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

well as for cytotoxicity against mammalian cells. Generally,

N-substituted congeners were less cytotoxic compared to the

unsubstituted diamidines. Conversely, the substitution on aro-

matic rings increased cytotoxicity of select analogues. Thus,

the cytotoxicity of 2,2′-substituted dications increased in the

order OMe, CO₂Et, CO₂H, CONH₂< NO₂< Cl. Replacement

of oxygens in the aliphatic linker of compound 1 with sulfur

atoms or sulfone groups reduced cytotoxicity of dications as

well.

Unsubstituted amidines were more active in vitro against T.

b. rhodesiense and L. donoVani compared to the N-substituted

derivatives. In several cases, bis(N-isopropyl)amidines exhibited

superior antiplasmodial potency relative to the corresponding

diamidines and diimidazolines. Diamidine 12 (possessing a

trimethylene linker) demonstrated improved activities against

all three pathogens compared to compound 1. Placement of the

cationic fragments in the 3,3′-position of the aromatic rings

reduced antitrypanosomal and antileishmanial properties of

dications, while their activity against P. falciparum was similar

to that of the 4,4′-substituted isomers. Introduction of 2,2′-

dichloro substituents further improved antitrypanosomal proper-

ties of diamidine 12. Similarly, placement of the halogen groups

in the 2,2′-positions also increased activities of dications against

L. donoVani. The replacement of oxygen atoms in the alkyl chain

with secondary amino groups improved activities of the resulting

congeners against T. b. rhodesiense, P. falciparum, and L.

donoVani. 2,2′-Dichloro substituted diamidine 32 and diimida-

zoline 66 exhibited excellent in vivo efﬁcacies in the acute

mouse model of trypanosomiasis, providing cures of all infected

animals.

In Vivo Antitrypanosomal Activity. Select dications ex-

hibiting promising in vitro activities against T. b. rhodesiense

were evaluated in vivo in the STIB900 mouse model of African

trypanosomiasis (Table 8). The screening was conducted using

intraperitoneal dosing at 20 mg/kg daily for four days. Diamidine

1 and 12 congeners (2, 13, 14, 18, 19, 20, 23, 32, 37, 39, 40,

and 66) displayed promising in vivo efﬁcacies, curing at least

2 out of 4 mice. Because of the signiﬁcant curative rate and

substantial increase in the survival time of the infected animals,

the administered daily dosage was reduced to 10 mg/kg or even

further to 5 mg/kg. At the 10 mg/kg daily for 4 days regimen,

six dications (2, 13, 20, 32, 37, and 66) provided cures to at

least 2 out of 4 mice, while at the 5 mg/kg daily for four days

treatment, four congeners (2, 32, 37, and 66) cured 2 or more

out of 4 animals.

In several cases, N-substitution on the cationic fragments

increased antitrypanosomal efﬁcacies of tested compounds. For

example, the bis(N-methyl)amidine 2 provided 4/4 cures when

administered intraperitoneally at 20 mg/kg for four days, while

diamidine 1 cured only 2/4 animals at this dosage. Similarly,

the diimidazolines 14, 18, and 66 displayed better in vivo

activities against T. b. rhodesiense compared to the correspond-

ing diamidines 12, 17, and 64, respectively. The position of

attachment of cationic substituents and the length of the alkyl

bridge inﬂuenced the efﬁcacies of dications in the acute mouse

model of trypanosomiasis. Thus, the 3,3′-substituted diamidines

(compounds 20, 23, 26, and 29) were more effective in vivo

than the 4,4′-substituted analogues (dications 12, 17, 1, and 19)

when connected by the tri- or tetramethylene linkers and

displayed lower activities when the aromatic rings were con-

nected by ﬁve- or six-carbon chains. Similarly, the introduction

of 2,2′-dichloro substituents improved the efﬁcacies of dia-

midines 32 and 40 against T. b. rhodesiense compared to that

of dications 12 and 17, while the same structural variation

reduced the in vivo activity of diamidine 44 compared to

compound 1. The 2,2′-dimethoxy analogues 37 and 39 with a

three-carbon linker were more active than their unsubstituted

counterparts 12 and 14. However, the presence of methoxy

groups with longer four- and ﬁve-carbon linkers had no effect

on activity (42, 43, and 47 versus 17, 18, and 4, respectively).

The antitrypanosomal efﬁcacies of the unsubstituted diimida-

zolines 14, 18, and 4, the 2,2′-dichloro substituted diamidines

32, 40, and 44, and the 2,2′-dimethoxy substituted diamidines

and diimidazolines 37, 39, 42, 43, 46, and 47 decreased as the

length of the carbon linker between the aromatic moieties

increased. Replacement of the oxygen atoms in the aliphatic

linker with sulfur atoms afforded the diamidine 62, which,

despite promising antitrypanosomal activities in vitro, was

inactive in the in vivo model. Among the congeners 64-66

possessing secondary amino groups in place of oxygen atoms,

the diamidine 64 cured only 1 out of 4 mice at 4 × 5 mg/kg

and bis(N-isopropyl)amidine 65 was inactive at this dosage.

Conversely, diimidazoline 66 displayed excellent antitrypano-

Experimental Section

Biology. Preparation of Compounds. Compounds were dis-

solved in 100% dimethylsulfoxide (DMSO) and ﬁnally diluted in

culture medium prior to the assay. The DMSO concentration never

exceeded 1% in the in vitro assays. For in vivo experiments, the

compounds were dissolved in DMSO and further diluted with

distilled H₂O to a ﬁnal DMSO concentration of 10% prior to

injection into the animals.

In Vitro Cytotoxicity Assay (L6 Rat Myoblast Cells). IC₅₀

values were determined using the Alamar blue assay¹²⁸and were

carried out twice independently and in duplicate. Brieﬂy, 4000 L6

cells were seeded in RPMI 1640 medium supplemented with

L-glutamine 2 mM, HEPES 5.95 g/L, NaHCO₃2 g/L, and 10%

fetal bovine serum in 96-well microtiter plates. The serial drug

dilutions were incubated for 70 h at 37 °C under a humidiﬁed 5%

CO₂atmosphere. The viability marker Alamar blue (12.5 mg

resazurin [Sigma] dissolved in 100 mL phosphate buffered saline)

(10 µL) was then added to each well and the plate was incubated

for additional 2-3 h. The plates were read in a Spectramax Gemini

XS microplate ﬂuorescence scanner (Molecular Devices) using an

excitation wavelength 536 nm and an emission wavelength 588

nm. The IC₅₀values were calculated from the sigmoidal inhibition

curves using the SoftmaxPro software.

In Vitro Growth Inhibition Assay of T. b. rhodesiense

(STIB900). IC₅₀values were determined using the Alamar blue

assay and were carried out twice independently and in duplicate.

Brieﬂy, the compounds were tested in minimum essential medium

with Earle’s salts, supplemented as previously described¹²⁹with

the following modiﬁcations: 2-mercaptoethanol 0.2 mM, sodium

pyruvate 1 mM, hypoxanthine 0.5 mM, and 15% heat-inactivated

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2027

horse serum. Serial drug dilutions were prepared in 96-well

microtiter plates and each well inoculated with 2000 bloodstream

forms and incubated for 70 h at 37 °C under a humidiﬁed 5% CO₂

atmosphere. The viability marker Alamar blue (12.5 mg resazurin

[Sigma] dissolved in 100 mL phosphate buffered saline) (10 µL)

was then added to each well and the plate was incubated for

additional 2-6 h. The plates were read in a Spectramax Gemini

XS microplate ﬂuorescence scanner (Molecular Devices) using an

excitation wavelength of 536 nm and an emission wavelength of

588 nm. The IC₅₀values were calculated from the sigmoidal

inhibition curves using the SoftmaxPro software.

before being evaporated under reduced pressure. Flash column

chromatography was performed using Davisil grade 633, type 60A

silica gel (200-425 mesh). Analytical HPLC chromatograms were

recorded on an Agilent 1090 or 1200 chromatograph using an

Agilent Zorbax Rx C8 column (4.6 mm × 75 mm, 3.5 µm) and

UV photodiode array detection at 230, 254, 265, 290, and 320 nm.

The column temperature was maintained at 40 °C. Mobile phases

consisted of mixtures of acetonitrile (0-75%) in water containing

formic acid (80 mM), ammonium formate (20 mM), and triethy-

lamine (15 mM). Flow rates were maintained at 1.5 mL/min. In

method A, the concentration of acetonitrile was increased linearly

from 0 to 22.5% over 6 min, then from 22.5 to 56.25% over 4

min, and ﬁnally maintained for 1 min. In method B, the concentra-

tion of acetonitrile was increased linearly from 22.5 to 75% over

10 min and then maintained for 2 min.

Preparative Reverse Phase HPLC. Preparative reverse phase

HPLC was performed on a Varian ProStar Chromatography

Workstation conﬁgured with two PS-215 pumps ﬁtted with 50 mL

pump heads, a Dynamax Microsorb C18 (60 Å) column (41.4 mm

× 250 mm, 8 µm), PS-320 variable wavelength UV-vis detector,

and a PS-701 fraction collector. Mobile phases consisted of mixtures

of acetonitrile (0-75%) in water containing formic acid (40 mM)

and ammonium formate (10 mM). Flow rates were maintained at

40 mL/min. Detector wavelengths and mobile phase gradients were

optimized for the individual compounds. Select fractions were

analyzed for purity as described above for analytical HPLC.

Residues of evaporated pooled puriﬁed fractions were reconstituted

in water and lyophilized on a VirTis BenchTop 2K or 6K

lyophilizer. The lyophilized compounds were dissolved in ethanol

and converted into HCl salts with aqueous HCl.

Flash Chromatography of Amidines on C₁₈Reversed Phase

Silica Gel. The chromatographic column was half-ﬁlled with

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

acetonitrile (70-100 mL). The excess acetonitrile was drained out,

and the top of the column was covered with a 2 cm pad of sand.

The column was equilibrated with 150 mL of initial mobile phase

consisting 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 the excess amine and then with a mobile phase

consisting of a mixture of acetonitrile (0-75%) in water containing

formic acid (40 mM) and ammonium formate (10 mM). Acetonitrile

concentrations varied for each individual compound and contained

50-70% of the 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), deionized water (2

× 100 mL), and kept in acetonitrile or acetonitrile-water mixture.

Select fractions were analyzed for purity as described above for

analytical HPLC. Residues of evaporated pooled puriﬁed fractions

were reconstituted in water and lyophilized on a VirTis BenchTop

6K lyophilizer. The lyophilized compounds were dissolved in

ethanol and converted into HCl salts with aqueous HCl.

In Vitro Growth Inhibition Assay of P. falciparum (K1). The

determination of IC₅₀values against erythrocytic stages of P.

falciparum was carried out twice independently and in duplicate

using the [³H]-hypoxanthine incorporation assay.^130,131Brieﬂy, the

compounds were tested in RPMI 1640 medium 10.44 g/L,

supplemented with Hepes 5.94 g/L, Albumax II 5 g/L, sodium

bicarbonate 2.1 g/L, and neomycin 100 mg/L in 96-well microtiter

plates. Infected human red blood cells in medium (hematocrit

1.25%, parasitemia 0.3%) were incubated with the drug dilutions

in an atmosphere of 93% N₂, 4% CO₂, and 3% O₂at 37 °C. After

48 h, [³H]-hypoxanthine (0.5 µCi/well) was added and the plates

were incubated for additional 24 h under the same conditions. The

wells were harvested with a Betapalte cell harvester and transferred

on a glass ﬁber ﬁlter. Viability was assessed by measuring the

incorporation of [³H]-hypoxanthine by a Betaplate liquid scintil-

lation counter (Wallac, Zurich, Switzerland). The IC₅₀values were

calculated from the sigmoidal inhibition curves using MS Excel.

Antileishmanial Assay. Axenic amastigotes of L. donoVani

(WHO designation MHOM/SD/62/1S-CL2_D) were adapted from

promastigotes and grown in the amastigote medium described

previously¹³²at 37 °C. In a ﬁnal volume of 60 µL, 6 × 10⁴parasites

were added to each well of a 96-well plate except for negative

control wells. Standard and test compounds were added as

appropriate using 2-fold dilutions to allow a range of concentrations

to be tested. Plates were then incubated at 37 °C for 72 h in a

humidiﬁed environment containing 5% CO₂. The tetrazolium dye-

based CellTiter reagent (Promega, Madison, WI) was used to assess

parasite growth.¹³³Several hours after adding 12 µL of the CellTiter

reagent to each well of the plate, absorbance readings were taken

at 490 nm using a SpectraMax Plus 384 microplate reader

(Molecular Devices, Sunnyvale, CA). SoftMax Pro software

(Amersham Biosciences, Piscataway, NJ) was used to calculate IC₅₀

values by employing the dose-response equation y ) [(a - d)/(1

+ (x/c)^b)] + d, where x ) compound concentration, y ) absorbance

at 490 nm, a ) upper asymptote, b ) slope, c ) IC₅₀value, and

d ) lower asymptote.

STIB900 Acute Mouse Model of Trypanosomiasis. Experi-

ments were performed as previously reported¹³⁴with minor

modiﬁcations. Brieﬂy, female NMRI mice were infected intrap-

eritoneally (ip) with 2 × 10⁴STIB900 bloodstream forms.

Experimental groups of four mice were treated ip with tested

dications on 4 consecutive days from day 3 to day 6 postinfection.

A control group was infected but remained untreated. The tail blood

of all mice was checked for parasitemia until 60 days postinfection.

Surviving and aparasitemic mice at day 60 were considered cured

and then euthanized. Death of animals (including the aparasitemic

mice, >60) was recorded to calculate the mean survival time in

days.

Chemistry. General Experimental Information. All chemicals

and solvents were purchased from Aldrich Chemical Co., Fisher

Scientiﬁc, or Acros Organics and were used without further

puriﬁcation. Uncorrected melting points were measured on a

Thomas-Hoover capillary melting point apparatus. ¹H NMR

spectra were recorded on a Varian Gemini 2000 spectrometer

operating at 300 MHz. Chemical shifts are reported in ppm relative

to tetramethylsilane. Anhydrous ethanol was distilled over Mg/I₂

immediately prior to use. Reaction mixtures were monitored by

TLC using Whatman silica gel 250 µm UV₂₅₄plates or by reverse

phase HPLC. Organic layers of extraction mixtures were washed

with saturated NaCl solution and dried over Na₂SO₄or MgSO₄

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.

General Procedure for Syntheses of Diamidines (1-5, 9-53,

55-60, 64-66). 1,5-Bis(benzamidine-4-oxy)pentane (Pentami-

dine) Dihydrochloride (1).⁴⁴A mixture of dry 1,4-dioxane (230

mL) and dry EtOH (20 mL) in a 3-neck 500 mL ﬂask equipped

with a gas inlet tube, a thermometer, and a drying tube was saturated

with gaseous HCl at 0 °C. 1,5-Bis(4-cyanophenoxy)pentane (69)

(6.00 g, 20.0 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. The reaction mixture

was diluted with dry diethyl ether and cooled in a freezer overnight.

The resulting precipitate was ﬁltered off under Ar, washed with

2028 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

dry diethyl ether, and dried under high vacuum for 3 h to yield a

diimidate ester (9.10 g, 98%), which was reacted immediately with

ammonia, appropriate amines, or hydrazine.

above for the compound 7. A crude product was puriﬁed by

preparative HPLC and recrystallized from 1 N HCl to yield

1

compound 8 (0.10 g, 17%): mp 208-211 °C. H NMR (DMSO-

d₆), δ 11.56 (s, 2H), 9.53 (s, 2H), 8.33 (s, 2H), 7.23 (d, J ) 8.8

Hz, 4H), 7.05 (d, J ) 8.8 Hz, 4H), 4.03 (t, J ) 6.0 Hz, 4H), 2.35

(s, 6H), 1.80 (m, 4H), 1.58 (m, 2H). HPLC (method A) t_R) 6.46

min (100 area %); m/z 368.7 (MH⁺of free base). Anal.

(C₂₁H₂₈N₄O₂·2HCl·0.25H₂O) C, H, N, Cl.

The diimidate ester (9.10 g, 19.0 mmol) was suspended in dry

EtOH (80 mL) and saturated ethanolic ammonia (70 mL) was

added. The reaction mixture was sealed and stirred at ambient

temperature. The progress of the reaction was monitored by HPLC.

After 2 days, the mixture was diluted with dry diethyl ether and

cooled in a freezer. A formed precipitate was ﬁltered off, dried

under high vacuum, and recrystallized from 1 N HCl/EtOH and

then from water/i-PrOH to yield the compound 1 (5.10 g, 63%):

mp 245.5 °C (lit.⁴⁴233-234 °C dec). ¹H NMR (DMSO-d₆), δ 9.25

(s, 4H), 9.03 (s, 4H), 7.85 (d, J ) 8.2 Hz, 4H), 7.15 (d, J ) 8.2

Hz, 4H), 4.13 (t, J ) 6.2 Hz, 4H), 1.82 (m, 4H), 1.61 (m, 2H).

HPLC (method A) t_R) 6.28 min (100 area %). Anal.

(C₁₉H₂₄N₄O₂·2HCl·1H₂O) C, H, N, Cl.

1,5-Bis[(N-methyl)benzamidine-4-oxy]pentane Dihydrochlo-

ride (2). Compound 2 was prepared from 1,5-bis(4-cyanophenoxy)-

pentane (69)⁴⁴as a white solid (3.00 g, 91%): mp 205-207 °C.

¹H NMR (DMSO-d₆), δ 9.99 (s, 2H), 9.48 (s, 2H), 8.95 (s, 2H),

7.85 (d, J ) 8.8 Hz, 4H), 7.10 (d, J ) 8.8 Hz, 4H), 4.10 (t, J ) 6.2

Hz, 4H), 2.99 (s, 6H), 1.81 (m, 4H), 1.60 (m, 2H). HPLC (method

A) t_R) 6.80 min (98.5 area %); m/z 369.2 (MH⁺of free base).

Anal. (C₂₁H₂₈N₄O₂·2HCl) C, H, N.

1,5-Bis(benzamidrazone-4-oxy)pentane Dihydrochloride (9).

Compound 9 was prepared from 1,5-bis(4-cyanophenoxy)pentane

(69)⁴⁴as a white solid (7.00 g, 81%): mp 195 °C. ¹H NMR (DMSO-

d₆), δ 7.78 (d, J ) 8.8 Hz, 4H), 7.14 (d, J ) 8.8 Hz, 4H), 6.7 (br

s, 10H), 4.11 (t, J ) 6.2 Hz, 4H), 1.80 (m, 4H), 1.05 (m, 2H).

HPLC (method A) t_R) 6.19 min (100 area %); m/z 371.5 (MH⁺

of free base). Anal. (C₁₉H₂₆N₆O₂·2HCl·1.4H₂O) C, H, N, Cl.

1-(4-Amidinophenoxy)-5-(4-aminophenoxy)pentane Dihydro-

chloride (10).⁸³Compound 10 was prepared according to the

published procedure as a white solid (2.63 g, 83%): mp 218-220

°C (lit.⁸³125-126 °C). ¹H NMR (DMSO-d₆), δ 10.22 (br s, 3H),

9.24 (s, 2H), 9.02 (s, 2H), 7.85 (d, J ) 9.0 Hz, 2H), 7.31 (d, J )

8.9 Hz, 2H), 7.15 (d, J ) 9.0 Hz, 2H), 7.03 (d, J ) 9.0 Hz, 2H),

4.12 (t, J ) 6.4 Hz, 2H), 4.00 (t, J ) 6.3 Hz, 2H), 1.79 (m, 4H),

1.57 (m, 2H). HPLC (method A) t_R) 6.64 min (100 area %). Anal.

(C₁₈H₂₃N₃O₂·2HCl) C, H, N, Cl.

1,3-Bis[(N-isopropyl)benzamidine-4-oxy]propane Dihydro-

chloride (13). Compound 13 was prepared from 1,3-bis(4-cy-

anophenoxy)propane (67).⁴⁴A crude product was recrystallized

from 1 N HCl to yield compound 13 (1.08 g, 96%): mp 263-266

°C. ¹H NMR (DMSO-d₆), δ 9.50 (m, 4H), 9.20 (s, 2H), 7.85 (d, J

) 8.8 Hz, 4H), 7.15 (d, J ) 8.8 Hz, 4H), 4.30 (t, J ) 6.2 Hz, 4H),

4.20 (m, 2H), 2.25 (m, 2H), 1.27 (d, J ) 6.3 Hz, 12H); m/z 397.2

(MH⁺of free base). Anal. (C₂₃H₃₂N₄O₂·2HCl) C, H, N.

1,5-Bis[(N-isopropyl)benzamidine-4-oxy]pentane Dihydro-

chloride (3). Compound 3 was prepared from 1,5-bis(4-cyanophe-

noxy)pentane (69).⁴⁴The oily crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

1

3 (0.30 g, 71%): mp 195 °C (dec). H NMR (DMSO-d₆), δ 9.41

(d, J ) 7.9 Hz, 2H), 9.31 (s, 2H), 8.97 (s, 2H), 7.73 (d, J ) 8.8

Hz, 4H), 7.13 (d, J ) 8.8 Hz, 4H), 4.11 (t, J ) 6.2 Hz, 4H), 4.06

(m, 2H), 1.82 (m, 4H), 1.59 (m, 2H), 1.27 (d, J ) 6.3 Hz, 12H).

HPLC (method A) t_R) 7.98 min (100 area %). Anal.

(C₂₅H₃₆N₄O₂·2.4HCl·2H₂O) C, H, N, Cl.

1-(4-Amidinophenoxy)-3-[(2-imidazolinyl)benzene-4-oxy]pro-

pane Dihydrochloride (15). Compound 15 was prepared from 1-(4-

cyanophenoxy)-3-[(2-imidazolinyl)benzene-4-oxy]propane (81). A

crude product was recrystallized from 1 N HCl to yield compound

15 (0.47 g, 64%): mp 166-170 °C. ¹H NMR (DMSO-d₆), δ 10.79

(s, 2H), 9.36 (s, 2H), 9.17 (s, 2H), 8.13 (d, J ) 8.8 Hz, 2H), 7.89

(d, J ) 8.8 Hz, 2H), 7.20 (d, J ) 8.8 Hz, 2H), 7.17 (d, J ) 8.8 Hz,

2H), 4.29 (t, J ) 5.5 Hz, 2H), 4.25 (t, J ) 5.5 Hz, 2H), 3.95 (s,

4H), 2.24 (m, 2H). HPLC (method A) t_R) 5.39 min (98.3 area

1,5-Bis-[(1H-tetrazolyl-5)benzene-4-oxy]pentane (6). A sus-

pension of 1,5-bis(4-cyanophenoxy)pentane (69)⁴⁴(0.20 g, 0.65

mmol), sodium azide (0.13 g, 2.00 mmol), and glacial acetic acid

(0.12 g, 2.00 mmol) in DMF (2 mL) was reﬂuxed overnight. The

mixture was cooled to ambient temperature, diluted with water (5

mL), and treated with 1 N NaOH (2 mL) to dissolve a formed

precipitate. The solution was extracted with benzene (2 × 10 mL)

and then acidiﬁed with 1N HCl to yield a white precipitate, which

was ﬁltered off, washed with water (3 × 15 mL), and dried under

high vacuum. The crude product was recrystallized from i-PrOH

%);

m/z

339.4

(MH⁺

of

free

base).

Anal.

(C₁₉H₂₂N₄O₂·2HCl·1.5H₂O) C, H, N.

1

1-(4-Amidinophenoxy)-3-(4-carboxyphenoxy)propane Hy-

drochloride (16). 1-(4-Cyanophenoxy)-3-(4-ethoxycarbonylphe-

noxy)propane (87) was subjected to Pinner conditions followed by

reaction with ammonia as described above for 1 to yield 1-(4-

amidinophenoxy)-3-(4-ethoxycarbonylphenoxy)propane (0.45 g,

69%); mp 153-154 °C. ¹H NMR (DMSO-d₆), δ 9.31 (s, 2H), 9.14

(s, 2H), 7.98 (d, J ) 8.8 Hz, 2H), 7.90 (d, J ) 8.8 Hz, 2H), 7.25

(d, J ) 8.8 Hz, 2H), 7.10 (d, J ) 8.8 Hz, 2H), 4.29 (m, 6H), 2.25

(m, 2H), 1.30 (t, J ) 7.2 Hz, 3H).

to yield compound 6 (0.21 g, 82%): mp 246-247 °C (dec). H

NMR (DMSO-d₆), δ 7.96 (d, J ) 8.7 Hz, 4H), 7.16 (d, J ) 8.7

Hz, 4H), 4.10 (t, J ) 6.2 Hz, 4H), 1.84 (m, 4H), 1.61 (m, 2H).

HPLC (method B) t_R) 4.69 min (98.6 area %). Anal. (C₁₉H₂₀N₈O₂)

C, H, N.

1,5-Bis[4-(benzimidoylamino)-benzene-1-oxy]pentane Dihy-

drochloride (7). To an ice-cold solution of 1,5-bis(4-aminophe-

noxy)pentane⁹⁷(0.20 g, 0.70 mmol) in dry MeCN (5 mL) and dry

EtOH (15 mL) was added hydrobromide salt of benzenecarbox-

imidothioic acid 2-naphthalenylmethyl ester⁹⁸(0.80 g, 2.20 mmol).

The mixture was stirred at ambient temperature overnight. The

resulting solution was concentrated to give an oily residue, which

was triturated with diethyl ether to yield a yellow precipitate. The

solid was taken in a hot water (200 mL) and washed with diethyl

ether (2 × 100 mL). The water layer was concentrated to give a

white solid, which was recrystallized from 1 N HCl to yield

A suspension of the 1-(4-amidinophenoxy)-3-(4-ethoxycarbon-

ylphenoxy)propane in 2 N aqueous HCl was kept at 70 °C

overnight. The mixture was concentrated and a residue was

recrystallized from 1 N HCl to yield compound 16 (0.30 g, 45%);

1

mp 278-280 °C. H NMR (DMSO-d₆), δ 12.65 (br s. 1H), 9.29

(s, 2H), 9.12 (s, 2H), 7.87 (m, 4H), 7.16 (d, J ) 8.8 Hz, 2H), 7.14

(d, J ) 8.8 Hz, 2H), 4.25 (m, 4H), 2.23 (m, 2H). HPLC (method

A) t_R) 8.06 min (100 area %); m/z 315.1 (MH⁺of free base).

Anal. (C₁₇H₁₈N₂O₄·1HCl) C, H, N, Cl.

1

compound 7 (0.31 g, 77%): mp 143-145 °C. H NMR (DMSO-

d₆), δ 11.23 (s, 2H), 9.71 (s, 2H), 8.24 (s, 2H), 7.90 (dd, J₁) 7.1

Hz, J₂) 2.1 Hz, 4H), 7.79 (tt, J₁) 7.4 Hz, J₂) 2.1 Hz, 2H), 7.67

(dd, J₁) 7.6 Hz, J₂) 7.1 Hz, 4H), 7.40 (d, J ) 8.8 Hz, 4H), 7.13

(d, J ) 8.8 Hz, 4H), 4.72 (t, J ) 6.2 Hz, 4H), 1.82 (m, 4H), 1.59

(m, 2H). HPLC (method B) t_R) 2.84 min (98.0 area %); m/z 493.0

(MH⁺of free base). Anal. (C₃₁H₃₂N₄O₂·2HCl·2H₂O) C, H, N, Cl.

1,5-Bis[4-(acetimidoylamino)-benzene-1-oxy]pentane Dihy-

drochloride (8). Compound 8 was prepared from 1,5-bis(4-

aminophenoxy)pentane⁹⁷and hydrobromide salt of ethanimidothioic

acid 2-naphthalenylmethyl ester⁹⁹following the procedure described

1,3-Bis(benzamidine-3-oxy)propane Dihydrochloride (20).⁶⁶

Compound 20 was prepared from 1,3-bis(3-cyanophenoxy)propane

(71).⁶⁶A crude product was puriﬁed by ﬂash chromatography on

C₁₈reversed phase silica gel to yield compound 20 (0.38 g, 66%);

mp 189-191 °C (lit.⁶⁶300 °C). H NMR (DMSO-d₆), δ 9.47 (s,

1

4H), 9.30 (s, 4H), 7.53 (t, J ) 7.7 Hz, 2H), 7.48 (s, 2H), 7.43 (d,

J ) 7.7 Hz, 2H), 7.32 (d, J ) 7.7 Hz, 2H), 4.27 (t, J ) 6.0 Hz,

4H), 2.24 (m, 2H). HPLC (method A) t_R) 4.36 min (100 area %).

Anal. (C₁₇H₂₀N₄O₂·2HCl·2H₂O) C, H, N, Cl.

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2029

1,3-Bis[(N-isopropyl)benzamidine-3-oxy]propane Dihydro-

chloride (21). Compound 21 was prepared from 1,3-bis(3-cy-

anophenoxy)propane (71).⁶⁶An oily crude material was puriﬁed

by ﬂash chromatography on C₁₈reversed phase silica gel to yield

compound 21 (0.35 g, 50%); mp 146 °C (dec). ¹H NMR (DMSO-

d₆), δ 9.65 (s, 2H), 9.53 (s, 2H), 9.27 (s, 2H), 7.50 (t, J ) 7.7 Hz,

2H), 7.30 (m, 6H), 4.27 (br s, 4H), 4.13 (br s, 2H), 2.24 (br s, 2H),

1.27 (d, J ) 6.0 Hz, 12H). HPLC (method A) t_R) 6.20 min (100

area %). Anal. (C₂₃H₃₂N₄O₂·2.1HCl·1H₂O) C, H, N, Cl.

4H), 3.99 (s, 8H), 1.83 (m, 4H), 1.60 (m, 2H). HPLC (method A)

t_R) 6.72 min (100 area %). Anal. (C₂₃H₂₈N₄O₂·2HCl·2H₂O) C,

H, N, Cl.

1,6-Bis(benzamidine-3-oxy)hexane Dihydrochloride (29).⁶⁶

Compound 29 was prepared from 1,6-bis(3-cyanophenoxy)hexane

(74). A crude product was recrystallized from 1 N HCl to yield

compound 29 (0.75 g, 95%); mp 247-249 °C (lit.⁶⁶268 °C). H

1

NMR (DMSO-d₆), δ 9.41 (s, 4H), 9.24 (s, 4H), 7.52 (dd, J₁) 8.2

Hz, J₂) 7.6 Hz, 2H), 7.42 (s, 2H), 7.40 (d, J ) 7.6 Hz, 2H), 7.29

(d, J ) 8.2 Hz, 2H), 4.09 (t, J ) 6.0 Hz, 4H), 1.78 (m, 4H), 1.51

(br s, 4H). HPLC (method A) t_R) 6.79 min (100 area %). Anal.

(C₂₀H₂₆N₄O₂·2HCl) C, H, N, Cl.

1,3-Bis-[(2-imidazolinyl)benzene-3-oxy]propane Dihydrochlo-

ride (22). Compound 22 was prepared from 1,3-bis(3-cyanophe-

noxy)propane (71).⁶⁶A crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

36 (0.48 g, 73%); mp 306-307 °C. ¹H NMR (DMSO-d₆), δ 10.93

(s, 4H), 7.78 (s, 2H), 7.66 (d, J ) 7.1 Hz, 2H), 7.55 (dd, J₁) 8.2

Hz, J₂) 7.1 Hz, 2H), 7.35 (d, J ) 8.2 Hz, 2H), 4.28 (t, J ) 5.5

Hz, 4H), 3.99 (s, 8H), 2.23 (m, 2H). HPLC (method A) t_R) 5.17

min (100 area %). Anal. (C₂₁H₂₄N₄O₂·2HCl·2.2H₂O) C, H, N, Cl.

1,4-Bis(benzamidine-3-oxy)butane Dihydrochloride (23).⁶⁶

Compound 23 was prepared from 1,4-bis(3-cyanophenoxy)butane

(72).⁶⁶A crude product was recrystallized from 1 N HCl to yield

1,6-Bis[(N-isopropyl)benzamidine-3-oxy]hexane Dihydrochlo-

ride (30). Compound 30 was prepared from 1,6-bis(3-cyanophe-

noxy)hexane (74). A crude product was recrystallized from 1 N

1

HCl to yield compound 30 (0.65 g, 69%); mp 150-153 °C. H

NMR (DMSO-d₆), δ 9.60 (d, J ) 8.2 Hz, 2H), 9.49 (s, 2H), 9.21

(s, 2H), 7.50 (dd, J₁) 8.2 Hz, J₂) 7.6 Hz, 2H), 7.23 (m, 6H),

4.15 (m, 2H), 4.09 (t, J ) 6.0 Hz, 4H), 1.78 (br s, 4H), 1.51 (br s,

4H), 1.27 (d, J ) 6.0 Hz, 12H). HPLC (method A) t_R) 7.94 min

(100 area %). Anal. (C₂₆H₃₈N₄O₂·2HCl·0.5H₂O) C, H, N, Cl.

compound 23 (0.85 g, 98%); mp 245-246 °C (lit.⁶⁶257 °C). H

1

1,6-Bis-[(2-imidazolinyl)benzene-3-oxy]hexane Dihydrochlo-

ride (31). Compound 31 was prepared from 1,6-bis(3-cyanophe-

noxy)hexane (74). A crude product was recrystallized from 1 N

NMR (DMSO-d₆), δ 9.45 (s, 4H), 9.27 (s, 4H), 7.52 (dd, J₁) 8.2

Hz, J₂) 7.6 Hz, 2H), 7.46 (s, 2H), 7.42 (d, J ) 7.6 Hz, 2H), 7.30

(d, J ) 8.2 Hz, 2H), 4.17 (s, 4H), 1.92 (s, 4H). HPLC (method A)

t_R) 5.16 min (100 area %). Anal. (C₁₈H₂₂N₄O₂·2HCl·1.9H₂O)

C, H, N, Cl.

1

HCl to yield compound 31 (0.85 g, 96%); mp 243-244 °C. H

NMR (DMSO-d₆), δ 10.84 (s, 4H), 7.70 (s, 2H), 7.61 (d J ) 7.6

Hz, 2H), 7.54 (dd, J₁) 8.2 Hz, J₂) 7.6 Hz, 2H), 7.31 (d J ) 8.2

Hz, 2H), 4.09 (t, J ) 6.0 Hz, 4H), 3.99 (s, 8H), 1.78 (br s, 4H),

1.50 (br s, 4H). HPLC (method A) t_R) 7.33 min (100 area %).

Anal. (C₂₄H₃₀N₄O₂·2HCl·2H₂O) C, H, N, Cl.

1,4-Bis[(N-isopropyl)benzamidine-3-oxy]butane Dihydrochlo-

ride (24). Compound 24 was prepared from 1,4-bis(3-cyanophe-

noxy)butane (72).⁶⁶An oily crude material was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

1,3-Bis(3-chlorobenzamidine-4-oxy)propane Dihydrochloride

(32). Compound 32 was prepared from 1,3-bis(2-chloro-4-cy-

anophenoxy)propane (75).⁶⁶A crude product was recrystallized

from 1 N HCl to yield compound 32 (1.07 g, 84%); mp 299-301

1

24 (0.86 g, 81%); mp 163-165 °C. H NMR (DMSO-d₆), δ 9.59

(d, J ) 6.6 Hz, 2H), 9.47 (s, 2H), 9.17 (s, 2H), 7.51 (dd, J₁) 8.2

Hz, J₂) 7.6 Hz, 2H), 7.30 (m, 6H), 4.16 (br s, 4H), 4.08 (m, 2H),

1.92 (br s, 4H), 1.27 (d, J ) 6.6 Hz, 12H). HPLC (method A) t_R

) 6.98 min (100 area %). Anal. (C₂₄H₃₄N₄O₂·2.3HCl·1.7H₂O) C,

H, N, Cl.

1

°C. H NMR (DMSO-d₆), δ 9.42 (s, 4H), 9.24 (s, 4H), 8.05 (d, J

) 1.6 Hz, 2H), 7.91 (dd, J₁) 8.8 Hz, J₂) 1.6 Hz, 2H), 7.44 (d,

J ) 8.8 Hz, 2H), 4.40 (t, J ) 5.5 Hz, 4H), 2.32 (m, 2H). HPLC

(method A) t_R

(C₁₇H₁₈N₄O₂Cl₂·2HCl) C, H, N, Cl.

)

5.92 min (98.4 area %). Anal.

1,4-Bis-[(2-imidazolinyl)benzene-3-oxy]butane Dihydrochlo-

ride (25).⁶⁷Compound 25 was prepared from 1,4-bis(3-cyanophe-

noxy)butane (72).⁶⁶A crude product was recrystallized from 1 N

HCl/EtOH to yield compound 25 (0.97 g, 98%); mp 333 °C (dec)

1,3-Bis(3-nitrobenzamidine-4-oxy)propane Dihydrochloride

(34). Compound 34 was prepared from 1,3-bis(2-nitro-4-cyanophe-

noxy)propane (78).⁶⁶A crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

(lit.⁶⁷>300 °C). H NMR (DMSO-d₆), δ 10.86 (s, 4H), 7.75 (s,

1

2H), 7.62 (d J ) 8.2 Hz, 2H), 7.55 (t, J ) 8.2 Hz, 2H), 7.33 (d, J

) 8.2 Hz, 2H), 4.18 (br s, 4H), 3.99 (s, 8H), 1.92 (br s, 4H). HPLC

1

34 (0.12 g, 22%); mp 281-283 °C. H NMR (DMSO-d₆), δ 9.54

(method A) t_R

(C₂₂H₂₆N₄O₂·2HCl·2H₂O) C, H, N, Cl.

)

5.99 min (100 area %). Anal.

(s, 4H), 9.30 (s, 4H), 8.46 (d, J ) 1.6 Hz, 2H), 8.18 (dd, J₁) 9.3

Hz, J₂) 1.6 Hz, 2H), 7.61 (d, J ) 9.3 Hz, 2H), 4.47 (t, J ) 5.5

Hz, 4H), 2.29 (m, 2H). HPLC (method A) t_R) 4.42 min (100 area

1,5-Bis(benzamidine-3-oxy)pentane Dihydrochloride (26).⁶⁶

Compound 26 was prepared from 1,5-bis(3-cyanophenoxy)pentane

(73).⁴⁴A crude product was recrystallized from 1 N HCl to yield

compound 26 (1.12 g, 77%); mp 88-92 °C (lit.⁶⁶133-134 °C).

¹H NMR (DMSO-d₆), δ 9.46 (s, 4H), 9.26 (s, 4H), 7.52 (t, J ) 8.2

Hz, 2H), 7.44 (s, 2H), 7.42 (d, J ) 8.2 Hz, 2H), 7.29 (d, J ) 8.2

Hz, 2H), 4.11 (t, J ) 6.2 Hz, 4H), 1.83 (m, 4H), 1.58 (m, 2H).

HPLC (method A) t_R) 5.81 min (100 area %). Anal.

(C₁₉H₂₄N₄O₂·2HCl·1H₂O) C, H, N, Cl.

%);

m/z

403.2

(MH⁺

of

free

base).

Anal.

(C₁₇H₁₈N₆O₆·2HCl·0.3H₂O) C, H, N, Cl.

1,3-Bis-[3-amino-(2-imidazolinyl)benzene-4-oxy]propane Tet-

rahydrochloride (36). 1,3-Bis(2-nitro-4-cyanophenoxy)propane

(78)⁶⁶(1.80 g, 4.90 mmol) underwent Pinner reaction followed by

the interaction with ethylenediamine as described above for

compound 1. A crude material was recrystallized from 1 N aqueous

HCl to afford 1,3-bis-[3-nitro-(2-imidazolinyl)benzene-4-oxy]pro-

pane dihydrochloride (0.68 g, 34%). ¹H NMR (DMSO-d₆), δ 10.65

(s, 4H), 8.59 (d, J ) 1.8 Hz, 2H), 8.25 (dd, J₁) 8.2 Hz, J₂) 1.8

Hz, 2H), 7.67 (d, J ) 8.2 Hz, 2H), 4.47 (t, J ) 6.0 Hz, 4H), 3.98

(s, 8H), 2.30 (m, 2H); m/z 455.0 (MH⁺of free base).

1,5-Bis[(N-isopropyl)benzamidine-3-oxy]pentane Dihydro-

chloride (27). Compound 27 was prepared from 1,5-bis(3-cy-

anophenoxy)pentane (73).⁴⁴A crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

1

27 (0.43 g, 58%); mp 152-155 °C. H NMR (DMSO-d₆), δ 9.56

A suspension of 1,3-bis-[3-nitro-(2-imidazolinyl)benzene-4-ox-

y]propane dihydrochloride (0.68 g, 1.50 mmol) and 10% Pd/C (0.1

g) in MeOH (150 mL) was stirred at 50 psi of H₂for 1 h. The

mixture was ﬁltered through a pad of celite and a ﬁltrate was

concentrated to give a crude product, which was recrystallized from

1 N HCl/EtOH to yield compound 36 (0.40 g, 67%); mp 283-286

(d, J ) 8.2 Hz, 2H), 9.45 (s, 2H), 9.17 (s, 2H), 7.47 (t, J ) 7.7 Hz,

2H), 7.25 (m, 6H), 4.08 (m, 6H), 1.78 (m, 4H), 1.57 (m, 2H), 1.24

(d, J ) 6.0 Hz, 12H). HPLC (method A) t_R) 7.49 min (100 area

%). Anal. (C₂₅H₃₆N₄O₂·2HCl·1H₂O) C, H, N, Cl.

1,5-Bis-[(2-imidazolinyl)benzene-3-oxy]pentane Dihydrochlo-

ride (28). Compound 28 was prepared from 1,5-bis(3-cyanophe-

noxy)pentane (73).⁴⁴A crude product was recrystallized from 1 N

HCl to yield compound 28 (0.75 g, 85%); mp 91-93 °C. ¹H NMR

(DMSO-d₆), δ 10.93 (br s, 4H), 7.75 (s, 2H), 7.63 (br s, 2H), 7.54

(t, J ) 8.2 Hz, 2H), 7.31 (d J ) 8.2 Hz, 2H), 4.11 (t, J ) 6.0 Hz,

1

°C. H NMR (DMSO-d₆), δ 10.45 (s, 4H), 7.51 (dd, J₁) 8.2 Hz,

J₂) 1.8 Hz, 2H), 7.41 (d, J ) 1.8 Hz, 2H), 7.21 (d, J ) 8.2 Hz,

2H), 4.37 (t, J ) 6.0 Hz, 4H), 4.11 (br s, 6H), 3.99 (s, 8H), 2.29

(m, 2H). HPLC (method A) t_R) 4.88 min (98.2 area %); m/z 395.2

(MH⁺of free base). Anal. (C₂₁H₂₆N₆O₂·4HCl) C, H, N.

2030 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

(m, 4H), 1.65 (m, 2H), 1.28 (m, 18H). HPLC (method B) t_R

1,3-Bis[3-methoxy-(N-methyl)benzamidine-4-oxy]propane Di-

hydrochloride (38). Compound 38 was prepared from 1,5-bis(2-

methoxy-4-cyanophenoxy)pentane (76).⁹⁴A crude material was

recrystallized from EtOH/diethyl ether followed by treatment with

3 N HCl to yield compound 38 (1.20 g, 50%); mp 167-169 °C.

¹H NMR (DMSO-d₆) (free base), δ 9.50 (br s, 4H), 7.57 (s, 1H),

7.51 (d, J ) 8.2 Hz, 1H), 7.40 (d, J ) 6.7 Hz, 1H), 7.39 (s, 1H),

7.18 (d, J ) 8.0 Hz, 1H), 7.17 (d, J ) 8.2 Hz, 1H), 4.21 (t, J ) 5.0

Hz, 4H), 3.88 (s, 3H), 3.81 (s, 3H), 3.40 (s, 6H), 2.22 (m, 2H).

HPLC (method A) t_R) 5.17 min (98.2 area %); m/z 401.2 (MH⁺

of free base). Anal. (C₂₁H₂₈N₄O₄·2HCl·2H₂O) C, H, N.

1,4-Bis(3-nitrobenzamidine-4-oxy)butane Dihydrochloride

(41).⁶⁶Compound 41 was prepared from 1,5-bis(2-nitro-4-cy-

anophenoxy)butane (79).⁶⁶A crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

41 (0.35 g, 49%); mp 300-301 °C (lit.⁶⁶298 °C). ¹H NMR

(DMSO-d₆), δ 9.53 (s, 4H), 9.28 (s, 4H), 8.46 (d, J ) 2.2 Hz, 2H),

8.18 (dd, J₁) 8.8 Hz, J₂) 2.2 Hz, 2H), 7.67 (d, J ) 8.8 Hz, 2H),

4.38 (br s, 4H), 1.94 (br s, 4H). HPLC (method A) t_R) 4.96 min

(100 area %); m/z 417.3 (MH⁺of free base). Anal.

(C₁₈H₂₀N₆O₆·2HCl) C, H, N, Cl.

1,5-Bis(3-nitrobenzamidine-4-oxy)pentane Dihydrochloride

(45).⁶⁶Compound 45 was prepared from 1,5-bis(2-nitro-4-cy-

anophenoxy)pentane (80).⁶⁶A crude product was puriﬁed by ﬂash

chromatography on C₁₈reversed phase silica gel to yield compound

45 (0.30 g, 33%); mp 261-263 °C (lit.⁶⁶255 °C). ¹H NMR

(DMSO-d₆), δ 9.49 (s, 4H), 9.23 (s, 4H), 8.44 (d, J ) 2.2 Hz, 2H),

8.14 (dd, J₁) 9.3 Hz, J₂) 2.2 Hz, 2H), 7.63 (d, J ) 9.3 Hz, 2H),

4.32 (t, J ) 6.0 Hz, 4H), 1.84 (m, 4H), 1.58 (m, 2H). HPLC (method

A) t_R) 6.02 min (100 area %); m/z 431.7 (MH⁺of free base).

Anal. (C₁₉H₂₂N₆O₆·2HCl·1H₂O) C, H, N, Cl.

1,5-Bis(3-methoxybenzamidine-4-oxy)pentane Dihydrochlo-

ride (46).⁶⁶Compound 46 was prepared from 1,5-bis(2-methoxy-

4-cyanophenoxy)pentane (77).⁹⁴A crude product was puriﬁed by

ﬂash chromatography on C₁₈reversed phase silica gel to yield

compound 46 (0.45 g, 51%); mp 233-234 °C (lit.⁶⁶258-259 °C).

¹H NMR (DMSO-d₆), δ 9.29 (s, 4H), 9.04 (s, 4H), 7.51 (d, J )

8.2 Hz, 2H), 7.50 (s, 2H), 7.18 (d, J ) 8.2 Hz, 2H), 4.10 (m, 4H),

3.86 (s, 6H), 1.83 (m, 4H), 1.57 (m, 2H). HPLC (method A) t_R)

5.91 min (100 area %). Anal. (C₂₁H₂₈N₄O₄·2HCl·0.1H₂O) C, H,

N, Cl.

)

2.74 min (98.5 area %). Anal. (C₃₁H₄₄N₄O₆·2HCl) C, H, N, Cl.

1,5-Bis[3-ethoxycarbonyl-(2-imidazolinyl)benzene-4-oxy]pen-

tane Dihydrochloride (50). Compound 50 was prepared from 1,5-

bis(4-cyano-2-ethoxycarbonylphenoxy)pentane (84). A crude ma-

terial was recrystallized from EtOH saturated with HCl to yield

compound 50 (0.65 g, 33%); mp 232-235 °C. ¹H NMR (DMSO-

d₆), δ 10.58 (s, 4H), 8.31 (d, J ) 2.7 Hz, 2H), 8.19 (dd, J₁) 9.3

Hz, J₂) 2.7 Hz, 2H), 7.45 (d, J ) 9.3 Hz, 2H), 4.29 (q, J ) 7.1

Hz, 4H), 4.21 (t, J ) 6.0 Hz, 4H), 3.98 (s, 8H), 1.83 (m, 4H), 1.65

(m, 2H), 1.27 (t, J ) 7.1 Hz, 6H). HPLC (method B) t_R) 2.12

min (100 area %). Anal. (C₂₉H₃₆N₄O₆·2HCl·2.5H₂O) C, H, N, Cl.

Syntheses of amidinoacids (51-53). 1,5-Bis(4-amidino-2-

carboxyphenoxy)pentane (51). A suspension of 1,5-bis(4-amidino-

2-ethoxycarbonylphenoxy)pentane dihydrochloride (48) (0.07 g,

0.13 mmol) in 3 N HCl was kept at 70 °C overnight. The solvent

was evaporated and a light-brown residue was recrystallized from

1 N HCl to yield compound 51 (0.015 g, 24%); mp 233-235 °C.

¹H NMR (DMSO-d₆), δ 9.26 (s, 4H), 8.85 (s, 4H), 8.14 (d, J )

2.2 Hz, 2H), 7.97 (dd, J₁) 8.8 Hz, J₂) 2.2 Hz, 2H), 7.37 (d, J )

8.8 Hz, 2H), 4.18 (br s, 4H), 1.83 (m, 4H), 1.61 (m, 2H). HPLC

(method A) t_R

(C₂₁H₂₄N₄O₆·2HCl·2.5H₂O) C, H, N.

)

3.72 min (100 area %). Anal.

1,5-Bis[3-carboxy-(N-isopropyl)benzamidine-4-oxy]pentane Di-

hydrochloride (52). Following the procedure described above for

the compound 51, 52 was prepared from 1,5-bis[3-ethoxycarbon-

yl(N-isopropyl)benzamidine-4-oxy]pentane (49). A crude product

was recrystallized from 1 N HCl/EtOH to yield compound 52 (0.12

1

g, 66%); mp 198-200 °C. H NMR (DMSO-d₆), δ 9.49 (d, J )

6.6 Hz, 2H), 9.35 (s, 2H), 8.97 (s, 2H), 8.00 (s, 2H), 7.87 (d, J )

8.2 Hz, 2H), 7.34 (d, J ) 8.2 Hz, 2H), 4.16 (br s, 4H), 4.01 (m,

2H), 1.82 (m, 4H), 1.62 (m, 2H), 1.26 (d, J ) 4.9 Hz, 12H). HPLC

(method A) t_R

(C₂₇H₃₆N₄O₆·2HCl·0.9H₂O·0.5EtOH) C, H, N, Cl.

)

5.50 min (100 area %). Anal.

1,5-Bis[3-carboxy-(2-imidazolinyl)benzene-4-oxy]pentane Di-

hydrochloride (53). Following the procedure described above for

the compound 51, 53 was prepared from 1,5-bis[3-ethoxycarbonyl-

(2-imidazolinyl)benzene-4-oxy]pentane (50). A crude product was

recrystallized twice from 1 N HCl to yield compound 53 (0.12 g,

1

26%); mp 187-189 °C. H NMR (DMSO-d₆), δ 10.56 (s, 4H),

8.30 (s, 2H), 8.17 (d, J ) 8.2 Hz, 2H), 7.42 (d, J ) 8.2 Hz, 2H),

4.18 (br s, 4H), 3.97 (s, 8H), 1.82 (m, 4H), 1.63 (m, 2H). HPLC

1,5-Bis-[3-methoxy-(2-imidazolinyl)benzene-4-oxy]pentane Di-

hydrochloride (47).^67,94Compound 47 was prepared from 1,5-

bis(2-methoxy-4-cyanophenoxy)pentane (77).⁹⁴A crude material

was recrystallized from 1 N HCl to yield compound 47 (2.40 g,

62%); mp 175 °C (lit.⁹⁴188 °C). ¹H NMR (DMSO-d₆), δ 9.45 (s,

4H), 7.83 (s, 2H), 7.75 (d, J ) 8.2 Hz, 2H), 7.20 (d, J ) 8.2 Hz,

2H), 4.17 (m, 4H), 4.00 (s, 8H), 3.85 (s 6H), 1.80 (m 6H). HPLC

(method A) t_R) 6.97 min (98.4 area %); m/z 453.2 (MH⁺of free

base). Anal. (C₂₅H₃₂N₄O₄·2HCl·2.5H₂O) C, H, N.

(method A) t_R

(C₂₅H₂₈N₄O₆·2.2HCl·2H₂O) C, H, N, Cl.

)

4.44 min (100 area %). Anal.

1-(4-Amidino-2-ethoxycarbonylphenoxy)-5-(4-amidino-2-ami-

nocarbonylphenoxy)pentane Dihydrochloride (54). The reaction

mixture described above for the compound 48 was separated by

preparative HPLC and a product was recrystallized from 1 N HCl

1

to yield compound 54 (0.10 g, 4%); mp 168-170 °C. H NMR

(DMSO-d₆), δ 9.30 (s, 4H), 8.94 (s, 2H), 8.90 (s, 2H), 8.25 (s,

1H), 8.13 (s, 1H), 8.01 (m, 2H), 7.70 (m, 2H), 7.39 (m, 2H), 4.28

(q, J ) 7.1 Hz, 2H), 4.19 (m, 4H), 1.84 (m, 4H), 1.63 (m, 2H),

1.29 (t, J ) 7.1 Hz, 3H). HPLC (method B) t_R) 0.80 min (100

area %); m/z 456.4 (MH⁺of free base). Anal.

(C₂₃H₂₉N₅O₅·2HCl·1H₂O) C, H, N, Cl.

1,5-Bis(4-amidino-2-ethoxycarbonylphenoxy)pentane Dihy-

drochloride (48). Compound 48 was synthesized from 1,5-bis(4-

cyano-2-ethoxycarbonylphenoxy)pentane (84). A reaction product,

containing a mixture of amides 48, 54, and 55, was separated by

preparative HPLC. The puriﬁed component was recrystallized from

1 N HCl/EtOH to give compound 48 (0.08 g, 3%); mp 133-135

1,5-Bis(4-amidino-2-aminocarbonylphenoxy)pentane Dihy-

drochloride (55). The reaction mixture described above for the

compound 48 was separated by preparative HPLC and a product

was recrystallized from 1 N HCl to yield compound 55 (0.13 g,

6%); mp 208-210 °C. ¹H NMR (DMSO-d₆), δ 9.33 (s, 4H), 9.02

(s, 4H), 8.24 (d, J ) 2.7 Hz, 2H), 7.96 (dd, J₁) 8.8 Hz, J₂) 2.7

Hz, 2H), 7.76 (s, 2H), 7.64 (s, 2H), 7.38 (d, J ) 8.8 Hz, 2H), 4.25

(m, 4H), 1.88 (m, 4H), 1.60 (m, 2H). HPLC (method B) t_R) 0.57

min (100 area %); m/z 427.4 (MH⁺of free base). Anal.

(C₂₁H₂₆N₆O₄·2HCl·2H₂O) C, H, N, Cl.

1

°C. H NMR (DMSO-d₆), δ 9.34 (s, 4H), 9.06 (s, 4H), 8.14 (s,

2H), 8.03 (d, J ) 8.8 Hz, 2H), 7.40 (d, J ) 8.8 Hz, 2H), 4.29 (q,

J ) 7.1 Hz, 2H), 4.27 (q, J ) 7.1 Hz, 2H), 4.20 (t, J ) 5.5 Hz,

4H), 1.84 (m, 4H), 1.65 (m, 2H), 1.28 (t, J ) 7.1 Hz, 3H), 1.27 (t,

J ) 7.1 Hz, 3H). HPLC (method A) t_R) 7.31 min (100 area %).

Anal. (C₂₅H₃₂N₄O₆·2HCl·2H₂O) C, H, N, Cl.

1,5-Bis[3-ethoxycarbonyl(N-isopropyl)benzamidine-4-oxy-

]pentane Dihydrochloride (49). Compound 49 was prepared from

1,5-bis(4-cyano-2-ethoxycarbonylphenoxy)pentane (84). A very

hygroscopic crude material was obtained, which was recrystallized

from dry EtOH saturated with HCl to yield compound 49 (0.49 g,

1,5-Bis(2-hydroxybenzamidine-4-oxy)pentane Dihydrochlo-

ride (56). Compound 56 was prepared from 1,5-bis(4-cyano-3-

hydroxyphenoxy)pentane (88). A crude product was separated by

preparative HPLC and treated with ethanol saturated with HCl to

1

53%); mp 220-222 °C. H NMR (DMSO-d₆), δ 9.53 (d, J ) 8.2

Hz, 2H), 9.40 (s, 2H), 9.02 (s, 2H), 7.99 (d, J ) 2.2 Hz, 2H), 7.90

(dd, J₁) 8.8 Hz, J₂) 2.2 Hz, 2H), 7.37 (d, J ) 8.8 Hz, 2H), 4.29

(q, J ) 7.1 Hz, 4H), 4.18 (t, J ) 6.0 Hz, 4H), 4.03 (m, 2H), 1.84

1

yield compound 56 (0.13 g, 27%); mp 145-148 °C. H NMR

(DMSO-d₆), δ 9.00 (br s, 6H), 8.70 (s, 4H), 7.61 (d, J ) 8.8 Hz,

SAR Study of Pentamidine Congeners

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2031

2H), 6.62 (d, J ) 2.3 Hz, 2H), 6.55 (dd, J₁) 8.8 Hz, J₂) 2.3 Hz,

2H), 4.03 (t, J ) 5.5 Hz, 4H), 1.75 (m, 4H), 1.53 (m, 2H). HPLC

(method A) t_R) 6.24 min (100 area %); m/z 373.7 (MH⁺of free

base). Anal. (C₁₉H₂₄N₄O₄·2HCl·0.9H₂O) C, H, N, Cl.

(100 area %); m/z 437.0 (MH⁺of free base). Anal.

(C₁₉H₂₄N₄S₂O₄·2HCl·0.4H₂O) C, H, N, Cl.

1,5-Bis(benzamidine-4-amino)pentane Tetrahydrochloride

(64).^50,66Compound 64was prepared from 1,5-bis(1-cyanobenzene-

4-amino)pentane.⁶⁶A crude product was puriﬁed by ﬂash chro-

matography on C₁₈reversed phase silica gel to yield compound 64

(0.43 g, 49%); mp 215 °C (dec) (lit.⁶⁶295 °C). ¹H NMR (DMSO-

d₆), δ 8.89 (s, 4H), 8.70 (s, 4H), 8.19 (br s, 4H), 7.69 (d, J ) 8.8

Hz, 4H), 6.69 (d, J ) 8.8 Hz, 4H), 3.10 (br s, 4H), 1.58 (br s, 4H),

1.46 (br s, 2H). HPLC (method A) t_R) 5.69 min (100 area %).

Anal. (C₁₉H₂₆N₆·4HCl·0.2H₂O) C, H, N, Cl.

1,5-Bis[(N-isopropyl)benzamidine-4-amino]pentane Tetrahy-

drochloride (65). Compound 65 was prepared from 1,5-bis(1-

cyanobenzene-4-amino)pentane.⁶⁶A crude product was puriﬁed by

ﬂash chromatography on C₁₈reversed phase silica gel to yield

compound 65 (0.47 g, 49%); mp 199-202 °C. ¹H NMR (DMSO-

d₆), δ 9.03 (br s, 4H), 8.65 (s, 2H), 7.56 (d, J ) 8.8 Hz, 4H), 7.29

(br s, 4H), 6.70 (d, J ) 8.8 Hz, 4H), 4.03 (m, 2H), 3.11 (br s, 4H),

1.58 (br s, 4H), 1.46 (br s, 2H), 1.25 (d, J ) 6.0 Hz, 12H). HPLC

(method A) t_R) 6.94 min (100 area %). Anal. (C₂₅H₃₈N₆·

4HCl·2H₂O) C, H, N, Cl.

1,5-Bis[4-(2-imidazolinyl)benzene-1-amino]pentane Dihydro-

chloride (66). Compound 66 was prepared from 1,5-bis(1-cy-

anobenzene-4-amino)pentane.⁶⁶A crude product was puriﬁed by

ﬂash chromatography on C₁₈reversed phase silica gel to yield

compound 66 (0.57 g, 58%); mp 182-185 °C. ¹H NMR (DMSO-

d₆), δ 10.06 (s, 4H), 7.78 (d, J ) 8.8 Hz, 4H), 7.06 (br s, 2H), 6.69

(d, J ) 8.8 Hz, 4H), 3.88 (s, 8H), 3.11 (br s, 4H), 1.58 (m, 4H),

1.46 (m, 2H). HPLC (method A) t_R) 5.86 min (100 area %). Anal.

(C₂₃H₃₀N₆·2HCl·1.5H₂O) C, H, N, Cl.

1,5-Bis[2-hydroxy-(N-isopropyl)benzamidine-4-oxy]pentane

Dihydrochloride (57). Compound 57 was prepared from 1,5-bis(4-

cyano-3-hydroxyphenoxy)pentane (88). A crude product was

separated by preparative HPLC and treated with ethanol saturated

with HCl to yield compound 57 (0.11 g, 29%); mp 123-125 °C.

¹H NMR (DMSO-d₆), δ 11.02 (br s, 2H), 9.18 (d, J ) 7.6 Hz,

2H), 8.97 (s, 2H), 8.81 (s, 2H), 7.34 (d, J ) 8.6 Hz, 2H), 6.68 (d,

J ) 2.3 Hz, 2H), 6.54 (dd, J₁) 8.6 Hz, J₂) 2.3 Hz, 2H), 3.99 (t,

J ) 6.5 Hz, 4H), 3.97 (m, 2H), 1.75 (m, 4H), 1.53 (m, 2H), 1.22

(d, J ) 6.0 Hz, 12H). HPLC (method A) t_R) 7.87 min (100 area

%); m/z 457.3 (MH⁺of free base). Anal. (C₂₅H₃₆N₄O₄· 2HCl ·

2.6H₂O) C, H, N, Cl.

1,5-Bis-[2-hydroxy(2-imidazolinyl)benzene-4-oxy]pentane Di-

hydrochloride (58). Compound 58 was prepared from 1,5-bis(4-

cyano-3-hydroxyphenoxy)pentane (88). A crude product was

separated by preparative HPLC and treated with ethanol saturated

with HCl to yield compound 58 (0.11 g, 29%); mp 128-131 °C.

¹H NMR (DMSO-d₆), δ 12.12 (s, 2H), 9.74 (s, 4H), 7.85 (d, J )

9.0 Hz, 2H), 6.79 (d, J ) 2.4 Hz, 2H), 6.63 (dd, J₁) 9.0 Hz, J₂)

2.4 Hz, 2H), 4.04 (t, J ) 6.4 Hz, 4H), 3.89 (s, 8H), 1.78 (m, 4H),

1.53 (m, 2H). HPLC (method A) t_R) 7.10 min (100 area %); m/z

425.3 (MH⁺of free base). Anal. (C₂₃H₂₈N₄O₄·2HCl·2H₂O) C, H,

N, Cl.

1,5-Bis(2-ﬂuorobenzamidine-4-oxy)pentane Dihydrochloride

(59). Compound 59 was prepared from 1,5-bis(4-cyano-3-ﬂuo-

rophenoxy)pentane (89). A crude product was separated by prepara-

tive HPLC and treated with ethanol saturated with HCl to yield

6-Bis(3-cyanophenoxy)hexane (74). Potassium carbonate (5.00

g, 36.0 mmol) was added to a solution of 3-cyanophenol (3.00 g,

25.0 mmol) in dry DMF (50 mL) at 130 °C. The suspension was

stirred at 130 °C for 20 min, and a solution of 1,6-dibromohexane

(3.20 g, 13.0 mmol) in dry DMF (20 mL) was added dropwise.

The reaction mixture was kept at 130-140 °C for 3 h, cooled to

ambient temperature, and poured into ice-water mixture. A formed

precipitate was ﬁltered off, washed with water, dried, and recrystal-

lized from 70% aqueous EtOH to yield compound 74 (3.60 g, 89%);

1

compound 59 (0.10 g, 9%); mp 245-247 °C. H NMR (DMSO-

d₆), δ 9.30 (s, 4H), 9.24 (s, 4H), 7.63 (m, 2H), 7.70 (d, J ) 11.8

Hz, 2H), 6.98 (d, J ) 10.2 Hz, 2H), 4.11 (t, J ) 6.2 Hz, 4H), 1.81

(m, 4H), 1.55 (m, 2H). HPLC (method A) t_R) 6.27 min (100 area

%); m/z 377.2 (MH⁺of free base). Anal. (C₁₉H₂₂F₂N₄O₂·2HCl)

C, H, N, Cl, F.

1,5-Bis[2-ﬂuoro-(N-isopropyl)benzamidine-4-oxy]pentane Di-

hydrochloride (60). Compound 60 was prepared from the com-

pound (89). A crude product was separated by preparative HPLC

and treated with ethanol saturated with HCl to yield compound 60

1

mp 91-93 °C. H NMR (DMSO-d₆), δ 7.48 (dd, J₁) 8.2 Hz, J₂

) 7.6 Hz, 2H), 7.39 (d, J ) 1.6 Hz, 2H), 7.37 (d, J ) 7.6 Hz, 2H),

7.27 (dd, J₁) 8.2 Hz, J₂) 1.6 Hz, 2H), 4.04 (t, J ) 6.6 Hz, 4H),

1.74 (m, 4H), 1.47 (br s, 4H). HPLC (method B) t_R) 9.15 min

(100 area %). Anal. (C₂₀H₂₀N₂O₂) C, H, N.

1

(0.23 g, 20%); mp 254-256 °C. H NMR (DMSO-d₆), δ 9.69 (d,

J ) 7.6 Hz, 2H), 9.46 (s, 2H), 9.29 (s, 2H), 7.56 (t, J ) 8.5 Hz,

2H), 7.12 (dd, J₁) 12.8 Hz, J₂) 2.2 Hz, 2H), 6.96 (dd, J₁) 8.6

Hz, J₂) 2.0 Hz, 2H), 4.10 (t, J ) 6.5 Hz, 4H), 4.06 (m, 2H), 1.79

(m, 4H), 1.57 (m, 2H), 1.24 (d, J ) 6.0 Hz, 12H). HPLC (method

A) t_R) 8.02 min (100 area %); m/z 461.8 (MH⁺of free base).

Anal. (C₂₅H₃₄F₂N₄O₂·2HCl·0.85H₂O) C, H, N, Cl, F.

1-(4-Cyanophenoxy)-3-[(2-imidazolinyl)benzene-4-oxy]pro-

pane (81). A mixture of 4-(4,5-dihydro-1H-imidazol-2-yl)-phenol¹⁰⁰

(1.35 g, 8.30 mmol) and sodium bicarbonate (1.43 g, 17.0 mmol)

in absolute ethanol (50 mL) was reﬂuxed for 2 h. A solution of

1-bromo-3-(4-cyanophenoxy)propane^44,96,101(2.00 g, 8.30 mmol)

in absolute ethanol (50 mL) was added and the resulting mixture

was reﬂuxed for 7 days. A formed precipitate was collected,

recrystallized twice from 50% EtOH, and was dried under high

vacuum to yield compound 81 (0.70 g, 22%); mp 194-197 °C. ¹H

NMR (DMSO-d₆), δ 8.05 (d, J ) 8.8 Hz, 2H), 7.80 (d, J ) 8.8

Hz, 2H), 7.25 (d, J ) 8.8 Hz, 2H), 7.17 (d, J ) 8.8 Hz, 2H), 4.20

(m, 4H), 4.05 (s, 4H), 2.25 (m, 2H). HPLC (method B) t_R) 3.67

min (98.0 area %).

1,5-Bis-[2-ﬂuoro(2-imidazolinyl)benzene-4-oxy]pentane Di-

hydrochloride (61). Compound 61 was prepared from the dinitrile

89. A crude product was recrystallized from 1 N HCl to yield

compound 61 (0.27 g, 27%); mp 216-218 °C. ¹H NMR (DMSO-

d₆), δ 10.40 (s, 4H), 7.98 (t, J ) 8.8 Hz, 2H), 7.18 (dd, J₁) 9.0

Hz, J₂) 2.4 Hz, 2H), 7.05 (dd, J₁) 8.9 Hz, J₂) 2.2 Hz, 2H),

4.15 (t, J ) 6.4 Hz, 4H), 3.95 (s, 8H), 1.83 (m, 4H), 1.56 (m, 2H).

HPLC (method A) t_R) 7.19 min (98.0 area %). Anal.

(C₂₃H₂₆F₂N₄O₂·2HCl·0.9H₂O) C, H, N, Cl, F.

1,5-Bis(benzamidine-4-thio)pentane Dihydrochloride (62).^91,103

Compound 62 was prepared from 1,5-bis[(4-cyano)phenylthio]pen-

tane (90) as a white solid (1.88 g, 41%); mp 150 °C (dec). ¹H NMR

(DMSO-d₆), δ 9.34 (s, 4H), 9.09 (s, 4H), 7.77 (d, J ) 8.7 Hz, 4H),

7.49 (d, J ) 8.7 Hz, 4H), 3.10 (t, J ) 6.8 Hz, 4H), 1.64 (m, 6H).

HPLC (method A) t_R) 7.45 min (99.4 area %); m/z 373.3 (MH⁺

of free base). Anal. (C₁₉H₂₄N₄S₂·2HCl·0.75H₂O) C, H, N, Cl.

1,5-Bis(benzamidine-4-sulfonyl)pentane Dihydrochloride (63).

Compound 63 was prepared from 1,5-bis[(4-cyano)phenylsulfo-

nyl]pentane (91). The crude product was puriﬁed by preparative

HPLC to yield compound 63 as a white solid (0.48 g, 37%); mp

1-(4-Cyanophenoxy)-3-(4-ethoxycarbonylphenoxy)propane (82).

A mixture of ethyl 4-hydroxybenzoate (0.70 g, 4.20 mmol) and

KOH (0.24 g, 4.30 mmol) in dry ethanol (20 mL) was reﬂuxed for

1 h. A solution of 4-(3-bromopropoxy)-benzonitrile⁴⁴(1.00 g, 4.20

mmol) in dry ethanol (10 mL) was added dropwise. The resulting

mixture was reﬂuxed for 1 day and a solid residue was ﬁltered off.

A ﬁltrate was cooled to 0 °C and a formed precipitate was ﬁltered

off and recrystallized from EtOH to yield compound 82 (0.75 g,

54%); mp 109-110 °C. ¹H NMR (CDCl₃), δ 8.13 (d, J ) 8.8 Hz,

2H), 7.65 (d, J ) 8.8 Hz, 2H), 7.01 (d, J ) 8.8 Hz, 2H), 6.96 (d,

J ) 8.8 Hz, 2H), 4.40 (q, J ) 6.5 Hz, 2H), 4.27 (t, J ) 6.0 Hz,

4H), 2.32 (m, 2H), 1.39 (t, J ) 6.5 Hz, 3H). HPLC (method B) t_R

) 8.99 min (98.0 area %).

1

225 °C (dec). H NMR (DMSO-d₆), δ 9.64 (s, 4 H), 9.42 (s, 4H),

8.12 (d, J ) 8.5 Hz, 4H), (8.06, d, J ) 8.5 Hz, 4H), 3.42 (m, 4H),

1.54 (m, 4 H), 1.44 (m, 2H). HPLC (method A) t_R) 3.37 min

2032 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

BakunoVa et al.

1,5-Bis(4-bromo-2-ethoxycarbonylphenoxy)pentane (83). To

a stirred solution of ethyl 5-bromosalicylate (16.0 g, 65.0 mmol)

in dry DMF (100 mL) at 125-130 °C was added dried K₂CO₃

(16.0 g, 65.3 mmol). The mixture was stirred under argon at this

temperature for 25 min, and a solution of 1,5-dibromopentane (7.59

g, 33.0 mmol) in DMF (50 mL) was added dropwise over 1.5 h. A

progress of the reaction was monitored by HPLC. After about 1.5 h,

the reaction mixture was cooled to ambient temperature, diluted

with ethyl acetate (400 mL), and washed with water (3 × 100 mL).

The organic solution was dried over MgSO₄and concentrated to

give a crude oily product (19.3 g). The crude material was puriﬁed

by column chromatography eluting with EtOAc/hexanes (gradient

5 to 20%) to yield compound 83 (8.90 g, 48%); mp 107-108 °C

(hexanes/EtOAc). ¹H NMR (DMSO-d₆), δ 7.73 (m, 2H), 7.68 (m,

2H), 7.12 (m, 2H), 4.23 (m, 4H), 4.05 (m, 4H), 1.78 (br s, 4H),

1.61 (br s, 2H), 1.25 (m, 6H). HPLC (method B) t_R) 10.98 min

(100 area %). Anal. (C₂₃H₂₆Br₂O₆) C, H, Br.

1,5-Bis(4-cyano-3-ﬂuorophenoxy)pentane (89). Following the

procedure described above for the synthesis of compound 69,

dinitrile 89 was prepared from 2-ﬂuoro-4-hydroxybenzonitrile and

1,5-dibromopentane (0.64 g, 51%); mp 97-98 °C. ¹H NMR

(DMSO-d₆), δ 7.81 (t, J ) 8.2 Hz, 2H), 7.15 (dd, J₁) 12 Hz, J₂

) 2.4 Hz, 2H), 6.96 (dd, J₁) 8.8 Hz, J₂) 2.4 Hz, 2H), 4.11 (t,

J ) 6.3 Hz, 4H), 1.80 (m, 4H), 1.56 (m, 2H). HPLC (method B)

t_R) 8.76 min (100 area %). Anal. (C₁₉H₁₆F₂N₂O₂) C, H, N.

1,5-Bis[(4-cyano)phenylthio]pentane (90). Following the pro-

cedure described above for the synthesis of dinitrile 74, compound

90 was prepared from 4-mercaptobenzonitrile¹⁰⁴and 1,5-dibro-

mobenzene. A crude product was recrystallized from acetonitrile

1

to give 90 as white crystals (9.56 g, 94%); mp 121 °C. H NMR

(DMSO-d₆), δ 7.73 (d, J ) 8.4 Hz, 4H), 7.44 (d, J ) 8.4 Hz, 4H),

3.07 (t, J ) 7.0 Hz, 4H), 1.65 (m, 4H), 1.58 (m, 2H). HPLC (method

B) t_R) 9.38 min (100 area %). Anal. (C₁₉H₁₈N₂S₂) C, H, N.

1,5-Bis[(4-cyano)phenylsulfonyl]pentane (91). A solution of

dinitrile (90) (3.39 g, 10.0 mmol) in acetonitrile (150 mL) and

ethanol (150 mL) was added dropwise to a mixture of periodic

acid (10.0 g, 43.9 mmol) and chromium trioxide (0.25 mg, 2.50

mmol) in acetonitrile (200 mL). After the mixture was stirred

overnight, more periodic acid (5.00 g) and chromium oxide (0.27

g) were added. After 2 h, the mixture was ﬁltered through a sintered

funnel. The ﬁltrate was washed with saturated solution of Na₂S₂O₃

and then with brine. The organic solution was dried over MgSO₄

and concentrated. The product was recrystallized from ethanol with

a little acetonitrile to give 91 as white crystals (2.97 g, 74%); mp

217 °C. ¹H NMR (DMSO-d₆), δ 8.16 (d, J ) 8.3 Hz, 4H), 8.07 (d,

J ) 8.3 Hz, 4H), 3.38 (m, 4H), 1.51 (m, 4H), 1.39 (m, 2H). HPLC

(method B) t_R) 5.22 min (100 area %). Anal. (C₁₉H₁₈N₂O₄S₂) C,

H, N.

1,5-Bis(4-cyano-2-ethoxycarbonylphenoxy)pentane (84). To a

suspension of compound 83 (7.40 g, 13.0 mmol) in dry DMF was

added dried CuCN (3.70 g, 41.0 mmol). The mixture was reﬂuxed

for 22 h, cooled to ambient temperature, diluted with EtOAc (500

mL), and washed with concentrated aqueous ammonia (300 mL)

and water (2 × 300 mL). The combined organic phases were dried

over MgSO₄and concentrated to give a crude brown product (5.40

g, 90%). The crude material was puriﬁed by column chromatog-

raphy eluting with EtOAc/hexanes (1/1) to yield compound 84 (3.70

1

g, 62%); mp 114-115 °C (hexanes/EtOAc). H NMR (DMSO-

d₆), δ 8.04 (d, J ) 2.2 Hz, 2H), 7.99 (dd, J₁) 8.8 Hz, J₂) 2.2

Hz, 2H), 7.33 (d, J ) 8.8 Hz, 2H), 4.25 (q, J ) 7.1 Hz, 4H), 4.16

(t, J ) 6.0 Hz, 4H), 1.81 (m, 4H), 1.61 (m, 2H), 1.26 (t, J ) 7.1

Hz, 6H). HPLC (method B) t_R) 8.57 min (98.7 area %). Anal.

(C₂₅H₂₆N₂O₆) C, H, N.

Acknowledgment. This work was supported by the Bill and

Melinda Gates Foundation and the Medicines for Malaria

Venture Foundation.

2-(Benzyloxy)-4-hydroxybenzonitrile (85). To a suspension of

4-amino-2-(benzyloxy)benzonitrile¹⁰²(1.00 g, 4.50 mmol) in ice

water (20 mL) was slowly added cold 30% H₂SO₄(15 mL) to

maintain temperature below 5-7 °C. A solution of NaNO₂(1.00

g, 15.0 mmol) in water (15 mL) was added dropwise, and the

resulting mixture was stirred for 30 min at temperature below 5-7

°C. After that, the reaction mixture was warmed to ambient

temperature and then kept at 80 °C for 1 h. The mixture was cooled,

diluted with EtOAc (200 mL), washed with brine (2 × 100 mL),

dried over MgSO₄, and concentrated to give crude material (1.10

g). The crude product was puriﬁed by column chromatography

eluting with EtOAc/hexanes (3/7) and recrystallized from a mixture

of chloroform/hexane (3/1) to yield compound 85 (0.52 g, 52%);

Supporting Information Available: Elemental analysis data.

This material is available free of charge via the Internet at http://

pubs.acs.org.

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mp 108-110 °C. H NMR (CDCl₃) δ 7.41 (m, 6H), 6.48 (s, 1H),

6.45 (dd, J₁) 2.2 Hz, J₂) 1.0 Hz, 1H), 5.73 (s, 1H), 5.16 (s, 2H).

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122-124 °C. H NMR (DMSO-d₆), δ 7.65 (d, J ) 8.6 Hz, 2H),

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) 1.9 Hz, 2H), 5.27 (s, 4H), 4.08 (t, J ) 6.6 Hz, 4H), 1.78 (m,

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8.2 Hz, J₂) 1.8 Hz, 2H), 6.49 (d, J ) 1.8 Hz, 2H), 4.00 (t, J )

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5.79 min (100 area %). Anal. (C₁₉H₁₈N₂O₄·0.2H₂O) C, H, N.

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