Synthesis and antiprotozoal activities of dicationic bis(phenoxymethyl)benzenes, bis(phenoxymethyl)naphthalenes, and bis(benzyloxy)naphthalenes

Article abstract of DOI:10.1016/j.ejmech.2009.03.014

A series of 37 dicationically substituted bis(phenoxymethyl)benzene bis(phenoxymethyl)naphthalene, and bis(benzyloxy)naphthalene analogues of pentamidine was prepared and evaluated for antiprotozoal activities and cytotoxicity in in vitro. 1,3-Bis(4-amidinophenoxymethyl)benzene (1) was the most active against Trypanosoma brucei rhodesiense (IC₅₀ = 2.1 nM). 1,3-Bis[4-(N-isopropylamidino)phenoxymethyl]benzene (2) was most active against Plasmodium falciparum (IC₅₀ = 3.6 nM) and displayed a selectivity index more than 50 times greater than that of pentamidine. Several other compounds displayed lower antiplasmodial IC₅₀ values and higher selectivity indices relative to pentamidine. 1,4-Bis(4-amidinophenoxymethyl)benzene (14) was the most active against Leishmania donovani (IC₅₀ = 1.3 μM). Compound 2 displayed the greatest activity against T. b. rhodesiense in vivo, curing three of four infected mice dosed intraperitoneally at 5 mg/kg × 4 days.

Full text of DOI:10.1016/j.ejmech.2009.03.014

European Journal of Medicinal Chemistry 44 (2009) 3543–3551
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiprotozoal activities of dicationic bis(phenoxymethyl)benzenes,
bis(phenoxymethyl)naphthalenes, and bis(benzyloxy)naphthalenes
Donald A. Patrick a, Stanislav A. Bakunov a, Svetlana M. Bakunova a, E.V.K. Suresh Kumar a,
Heidi Chen a, Susan Kilgore Jones a, Tanja Wenzler b, Todd Barzcz c, Karl A. Werbovetz c,
,
Reto Brun b, Richard R. Tidwell a
*
a Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7525, USA
b Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland
c Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 37 dicationically substituted bis(phenoxymethyl)benzene bis(phenoxymethyl)naphthalene,
and bis(benzyloxy)naphthalene analogues of pentamidine was prepared and evaluated for antiprotozoal
activities and cytotoxicity in in vitro. 1,3-Bis(4-amidinophenoxymethyl)benzene (1) was the most active
Received 18 December 2008
Received in revised form
6 March 2009
Accepted 12 March 2009
Available online 24 March 2009
against
Trypanosoma
brucei
rhodesiense
(IC50 ¼ 2.1 nM).
1,3-Bis[4-(N-isopropylamidino)
phenoxymethyl]benzene (2) was most active against Plasmodium falciparum (IC50 ¼ 3.6 nM) and dis-
played a selectivity index more than 50 times greater than that of pentamidine. Several other compounds
displayed lower antiplasmodial IC50 values and higher selectivity indices relative to pentamidine. 1,4-
Bis(4-amidinophenoxymethyl)benzene (14) was the most active against Leishmania donovani
Keywords:
Diamidines
Xylene derivatives
Naphthalene derivatives
Antiprotozoal
(IC50 ¼ 1.3
of four infected mice dosed intraperitoneally at 5 mg/kg ꢀ 4 days.
Ó 2009 Elsevier Masson SAS. All rights reserved.
mM). Compound 2 displayed the greatest activity against T. b. rhodesiense in vivo, curing three
Antitrypanosomal
Antiplasmodial
Antileishmanial
1. Introduction
mostly those living in the poorest countries, are at risk of malaria. In
some African nations, malaria accounts for as much as 40% of the
public health expenditure [2]. An estimated 300–500 million severe
cases occur annually, with 1.5–2.0 million fatalities [2,5]. As many as
20 species of the Leishmania protozoa give rise to human leish-
maniasis, manifested mainly as the cutaneous, mucocutaneous, and
life-threatening visceral forms [6,7]. The WHO estimates that two
million new cases develop annually and 350 million people are at
risk [8]. Hundreds of cases individuals dually infected with HIV and
visceral leishmaniasis in the South American, European, and African
continents have been reported in recent years [9–14].
The need for safe, orally effective, and inexpensive drugs to
combat HAT (especially the late stage disease) is great. Suramin and
pentamidine, limited to treatment of early stage HAT, must be
administered parenterally [1], and adverse reactions to pentamidine
are well known [15]. Melarsoprol (an organoarsenical) is effective for
late stage HAT, but its disadvantages include a fatal encephalopathy
in up to 10% of cases and rising rates of treatment failure [4,15].
Eflornithine, a safer alternative available since 1990, is effective only
against late stage T. b. gambiense infections, must be administered in
high doses over long periods, and relapses have occurred [15,16].
The insect-vectored protozoal infections human African
trypanosomiasis (HAT), malaria, and leishmaniasis continue to
cause significant rates of morbidity and mortality. Both HAT and
malaria are most prevalent in sub-Saharan Africa [1,2]. The epide-
miology of HAT has been recently reviewed [3]. More than 90% of
reported cases of HAT, found in west and central Africa, are a chronic
infection due to Trypanosoma brucei gambinse, in which patients
may be asymptomatic for months or even years, and the disease is
often in an advanced state when symptoms first occur. An acute
infection due to Trypanosoma brucei rhodesiense, found in eastern
and southern Africa, represents less than 10% of reported cases [1].
Approximately 17 500 cases of HAT were reported in 2006 [4], while
the World Health Organization (WHO) estimates are between
50 000 and 70 000 cases [1]. Malaria is caused by several Plasmo-
dium species, with the most deadly infection due to Plasmodium
falciparum. The WHO estimates that 40% of the world’s population,
* Corresponding author. Tel.: þ1 919 966 4294; fax: þ1 919 966 0704.
E-mail address: Tidwell@med.unc.edu (R.R. Tidwell).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.014

3544
D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
A growing problem with treatment of malaria has been drug
Bis(phenoxymethyl)naphthalenes 20–22 (dimethylnaphthalene
derivatives) differ from the xylene derivatives by having a naph-
thalene system in place of the central benzene ring. Bis(benzyloxy)
naphthalenes 23–37 (dihydroxynaphthalene derivatives) differ
from compounds 20–22 not only by having opposite ring substi-
tution patterns on the naphthalene system but also by reversal of
the carbon and oxygen atoms bridging the naphthalene system and
the outer rings.
Compounds 1–37 were prepared as shown in Scheme 1. Wil-
liamson ether syntheses involving cyanophenols and a,a
0-dibro-
moxylenes or bis(bromomethyl)naphthalenes [33,34] gave the
previously known [27–29] dinitrile precursors to xylene deriva-
tives 1–19 and dimethylnaphthalene derivatives 20–22. Similar
resistance, especially to inexpensive and widely used drugs such as
chloroquine and sulfadoxine–pyrimethamine [2,17]. Thus more
expensive drugs or drug combinations (especially with artemisi-
nins) must be used [14,18,19]. Various antimalarial drugs which are
currently in clinical use or under development are chemically
related or have similar mechanisms of action (or resistance). Thus
the risk of cross-resistance and failure of new treatments are
increased [14,20].
Antileishmanial therapy has been summarized recently [21].
Parenteral pentavalent antimonial compounds have been the
primary therapy against visceral leishmaniasis. Resistance to ani-
timonial drugs in Bihar State, India (which accounts for 90% of
India’s and 45% of the world’s visceral cases), has led to alternate
therapies including amphotericin B (conventional and liposomal
formulations), paromomycin, and miltefosine (the only orally
administered drug).
Pentamidine (Fig. 1) has shown efficacy against all three
diseases. N-Hydroxy and -methoxy derivatives of amidines have
shown the potential to be orally active prodrugs of amidines
[22,23]. More recently, DB289, an N-methoxy (methamidoxime)
prodrug of furamidine (DB75) was in Phase III clinical trials against
early stage HAT and Phase II trials against malaria [24,25]. However,
the compound exhibited nephro- and hepatoxicities in a recent
expanded Phase I trial [26].
The present investigation involves bis(phenoxymethyl)benzene,
bis(phenoxymethyl)- and bis(benzyloxy)naphthalene derivatives
1–37 (Table 1). 1,3-Bis[(4-amidino)phenoxymethyl]benzene (1),
a pentamidine analogue bearing an aromatic ring between the
two oxygen atoms, is the lead compound for the series. The
syntheses of 24 novel compounds are described. Activities against
T. b. rhodesiense, P. falciparum, and Leishmania donovani, as well as
toxicity to rat myoblast cells in vitro are reported for the 37
compounds. Antitrypanosomal activities in vivo are reported for
select compounds.
reactions involving naphthalenediols and
a-bromotolunitriles
gave the dicyano precursors to dihydroxynaphthalene derivatives
23–37. Five of these seven intermediates had been prepared
previously [27]. The nitriles underwent Pinner reactions, and the
imidate intermediates were treated with the appropriate amines
to give compounds 1–3, 6, 8–26, and 28–37. Pinner syntheses
involving 1,4-bis[(3-cyano)phenoxymethyl]naphthalene failed to
give the desired products, the m-amidino and m-isopropylamidino
analogues of compounds 21 and 22, but gave 3-hydrox-
ybenzamidine and 3-hydroxy-N-isopropylbenzamidine. Thus, the
benzylic ether linkages of the starting material were unstable to
the strongly acidic reaction conditions. Williamson ether
syntheses involving protected 3-hydroxybenamidines and
1,4-bis(bromomethyl)naphthalene were also unsuccessful. Four
prodrugs were also prepared. Treatment of the corresponding
nitrile with hydroxylamine [35] gave the known amidoxime 4 [29].
Similar methodology was used in the preparation of 7. Amidoxime
27 was prepared from the corresponding nitrile by a Pinner
synthesis, followed by reaction of the imidate ester with hydrox-
ylamine. O-Methylation of amidoxime 4 using dimethyl sulfate
[35] gave compound methamidoxime 5. All cationic compounds
were isolated as their dihydrochloride salts.
2. Chemistry
3. Results and discussions
Dicationic compounds 1–37 (Table 1) are an expansion of
a smaller group of bis(phenoxymethyl)benzenes (xylene deriva-
tives) and naphthalene derivatives (1, 6, 14, 17, 20, 21, 23, 25, 29, 30,
33) prepared previously in this laboratory for other purposes [27].
Preparations of analogues 4, 8, 14, and 17 have also been reported
previously [28–31]. The in vitro activities of compounds 18, 25, 28,
31, and 34 against Leishmania infantum have been recently
published [32] but their syntheses (except for 25) have not been
described.
1,3-Bis(4-amidinophenoxymethyl)benzene (1), formally a deriv-
ative of m-xylene, is structurally similar to pentamidine but
contains an aromatic ring in the linkage between the oxygen atoms.
Such a linkage retains similar spacing between the oxygen atoms
but is more rigid. Variation of the nature and position of the two
cationic groups gives rise to congeners 2–7. o-Xylene derivatives
8–13 have a rigid four carbon spacing between the oxygen atoms,
while a six carbon spacing is present in p-xylene derivatives 14–19.
3.1. In vitro activities
The compounds were assayed in vitro against T. b. rhodesiense
STIB900 [36–39], chloroquine resistant P. falciparum K1 [36,40], and
L. donovani (MHOM/SD/62/1S-CL2D) axenic amastigotes [41,42] for
antiprotozoal activities, and against rat myoblast (L6) cells for
cytotoxicity [43] (Table 1). Selectivity indices [44] (ratios of cyto-
toxic IC50 values to antiprotozoal IC50 values) are shown for each
parasite. Antiprotozoal IC50 values and cytotoxic data are compared
to those of pentamidine and furamidine. Other positive controls
employed were melarsoprol (against T. b. rhodesiense), chloroquine
and artemisinin (against P. falciparum), and podophyllotoxin
(against L6 cells).
3.1.1. Antitrypanosomal activities
Xylene derivative 1 was the most active in vitro against
T. b. rhodesiense, with an IC50 value of 2.1 nM (Table 2), followed by
O
O
H2N
RN
NH2
NR
O
H2N
NH2
NH
NH
Pentamidine
R=H, Furamidine (DB 75)
R=OCH3, Pafuramidine (DB 289)
Fig. 1. Structures of 1,5-bis(4-amidinophenoxy)pentane (pentamidine), 2,5-bis(4-amidinophenyl)furan (furamidine, DB75), and 2,5-bis[4-(N-methoxyamidino)phenyl]furan
(pafuramidine, DB289).

D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
3545
regioisomer 14 (21 nM), imidazoline 3 (46 nM), and regioisomer 6
(47 nM). N-Isopropylamidines 2 and 15 and diamidine 17 exhibited
antitrypanosomal IC50 values between 68 and 78 nM. Of these
seven compounds, all but 6, 14 and 17 exhibited selectivity indices
higher than that of furamidine, but all were less selective for the
parasite than pentamidine. Compounds 8 and 11 displayed IC50
among the 17 least cytotoxic compounds. The most cytotoxic
compound (excluding prodrugs) was naphthalene derivative 33
(IC50 ¼ 6.9
mM). The xylene derivatives, as a whole, were more
selective for the respective parasites over L6 cells relative to the
naphthalene derivatives. Four xylene derivatives (1, 2, 3, and 15)
displayed antitrypanosomal selectivity indices greater than that of
furamidine. The antiplasmodial selectivity indices of five
analogues (2, 3, 9, 10, and 15) were greater than that of pentam-
idine. These and 13 other compounds (including eight naphtha-
lene derivatives) displayed antiplasmodial selectivity indices
greater than that of furamidine. Only two compounds, xylene
derivatives 3 and 10 exhibited antileishmanial selectivity indices
greater than that of pentamidine.
values around 0.14
mM, and all the other xylene derivatives were
less active, with IC50 values ranging from 0.16 to 24
mM. The
naphthalene derivatives, as a whole, were less active against T. b.
rhodesiense than the xylene derivatives. Analogue 35 was the most
active naphthalene derivative, with an IC50 value of 0.15
followed by congeners 30 and 25 with IC50 values between 0.16 and
0.20 M. A group of ten naphthalene derivatives displayed IC50
values between 0.2 and 0.4 M, and all the other naphthalene
derivatives were less active, with IC50 values between 0.8 and
2.4 M. Selectivity indices for all of the naphthalene derivatives
mM,
m
m
3.2. In vivo antitrypanosomal activities
m
were less than those of furamidine or pentamidine.
Select compounds were administered to mice infected with T. b.
rhodesiense STIB900 [26,36,37] (Table 2). This strain proved to be
more difficult to treat, relative to T. b. rhodesiense KETRI2537 and T.
b. brucei STIB795 [26]. All compounds were administered once daily
intraperitoneally except for the prodrugs 4, 5, 7, and 27, which were
given orally. A strong correlation between in vivo activities and the
in vitro data was not observed. For example, lead compound 1, the
most active against the trypanosome in the in vitro assay
(IC50 ¼ 2.1 nM) cured only one of four animals at the 4 ꢀ10 mg/kg
dosing, with a greater than 35 day mean survival. By contrast, the
corresponding N-isopropyl analogue 2, which displayed an IC50
value of 68 nM, cured three of four animals at both 4 ꢀ10 mg/kg
and 4 ꢀ 5 mg/kg doses, but the lower dose resulted in a lower mean
survival (greater than 60 days versus greater than 53 days). The
corresponding imidazoline 3 (IC50 value ¼ 46 nM) cured none of
the animals at 4 ꢀ 20 mg/kg. Compound 4, the amidoxime prodrug
of 1, displayed moderate activity, with one of four animals cured.
The corresponding methamidoxime prodrug 5 cured none of the
animals. Amidine 6 (a meta-amidino regioisomer of 1) and the
corresponding amidoxime prodrug 7 cured no animals at the tested
dosages. Of the xylene derivatives with different central ring
substitution patterns, all were inactive except for 11, which cured
one of four animals. This compound, as well as 15, proved to be
lethal at higher doses. Of the naphthalene derivatives tested, all
were inactive or toxic except for 25, which cured one of four
animals at 20 mg/kg.
3.1.2. Antiplasmodial activities
Xylene derivative 2 (the N-isopropyl derivative of 1) was the
most active against P. falciparum, displaying an IC50 value of 3.6 nM,
followed by imidazoline 10 (10 nM) and N-isopropylamidine 9
(11 nM). Four other xylene derivatives (3, 6, 15, and 16) exhibited
IC50 values between 10 and 20 nM, while congeners 1 and 18
displayed IC50 values between 25 and 50 nM. Thus, nine xylene
derivatives displayed IC50 values comparable to or lower than that
of pentamidine, and the IC50 values of three compounds were lower
than that of furamidine. The selectivity index of 2 was 100 times
greater than that of furamidine and nearly 50 times greater than
that of pentamidine, and those of 9, 10, and 15 were more than 20
times greater than that of furamidine. Compounds 9 and 10 dis-
played a 150- and 60-fold selectivity for P. falciparum over T. b.
rhodesiense, a property of potential usefulness in treating patients
with mixed infections. The IC50 values of analogues 14 and 17 were
between 60 and 70 nM, and all the other xylene derivatives were
less active, with IC50 values between 0.11 and 0.36 mM.
Among the naphthalene derivatives, the most active against P.
falciparum was N-isopropylamidine 24, with an IC50 value of 28 nM,
a selectivity index three times greater than that of furamidine and
a 45-fold selectivity for P. falciparum over T. b. rhodesiense. Eight
other analogues (20, 22, 23, 28, 30, 32, 36, and 37) were less active,
with IC50 values between 50 and 90 nM. The remaining naphtha-
lene derivatives displayed IC50 values between 0.11 and 0.57 mM.
3.1.3. Antileishmanial activities
3.3. Structure–activity relationship
Two xylene derivatives, amidine 14 and the corresponding
imidazoline 16, displayed antileishmanial IC50 values 1.3 and
As a whole, the xylene derivatives exhibited greater activity
against the three parasites and were less toxic than either group of
naphthalene derivatives. Lead compound 1 was the most active
against T. b. rhodesiense in vitro. All structural modifications
resulted in decreased antitrypanosomal activity. The corresponding
p-xylene and o-xylene derivatives 14 and 8 were 10 and 70 times
less active, respectively. Compound 6, a regioisomer of 1 with meta-
amidino groups was 20 times less active than 1. m-Xylene derivative
17 was only four times less active than its para-amidino counterpart
14, and the o-xylene derived isomers 8 and 11 were equal in activity.
All of the N-isopropylamidines and imidazolines were less active
than the corresponding simple amidines, consistent with previous
results from this laboratory [14,20] and elsewhere [45,46].
The introduction of a central fused ring system resulted in a 150-
fold loss of activity (20 versus 1). A smaller (10-fold) loss of activity
was observed between naphthalene derivative 21 and its xylene
derivative counterpart 14, and only a four-fold difference between
22 and 15. No significant activity was observed with any of the
dihydroxynaphthalene derivatives 21–37.
1.9
lower selectivity indices. Congeners 1, 3, 8, and 10 exhibited IC50
values between 3.3 and 4.0 M, and the selectivity indices of 3 and
10 were slightly higher than that of pentamidine. The other xylene
derivatives were less active, with IC50 values ranging from 5.5 M to
greater than 200 M. Among the naphthalene derivatives N-
sec-butyl amidine 37 was most active (IC50 ¼ 3.7 M), followed by
amidines 25 and 35 (4.5 and 4.8 M). The IC50 values of the other
naphthalene derivatives ranged from 5.7 M to greater than
200 M.
mM, comparable to that of pentamidine but with somewhat
m
m
m
m
m
m
m
3.1.4. Cytotoxicity and selectivity
All compounds 1–37 were less toxic to rat myoblast cells than
furamidine (IC50 ¼ 6.4 M) and 17 compounds (ten xylene deriv-
atives and seven naphthalene derivatives) were less cytotoxic
than pentamidine (IC50 ¼ 47 M). The least toxic compound was
N-isopropyl xylene derivative 2 (IC50 > 169 M) and nine other
N-isopropyl analogues (9, 12, 15, 18, 26, 28, 31, 34, and 36) were
m
m
m

3546
D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
Table 1
Structures and in vitro antiprotozoal activities of compounds 1–37.
4
m
4
3
R
m
O
m
3
O
p
R
O
m
R
2
O
p
p
R
p
Compounds 1-19
Compounds 20-22
p
R
m
m
R
p
O
2
1
7
6
O
5
Compounds 23-37
OH
N
OCH3
NH2
NH
NH
N
NH
NH2
Am
N
N
N
H
iPrAm
N
H
R =
NH2
AmOH
H
sBuAm
Im
P. falciparumb
IC50 M)
AmOMe
L. donovanic
Compd
Position
R
T. b. rhodesiensea
L6 cellsd
IC50 M)
9.14
>169
123
e
f
g
IC50 M)
(m
SIT
(
m
SIP
IC50
(m
M)
SIL
(m
1
2
3
4
5
6
7
8
1,3
1,3
1,3
1,3
1,3
1,3
1,3
1,2
1,2
1,2
1,2
1,2
1,2
1,4
1,4
1,4
1,4
1,4
1,4
1,3
1,4
1,4
1,5
1,5
1,5
1,5
1,5
1,6
2,6
2,6
2,6
2,6
2,7
2,7
2,7
2,7
2,7
p-Am
p-iPrAm
p-Im
p-AmOH
p-AmOMe
m-Am
m-AmOH
p-Am
p-iPrAm
p-Im
m-Am
m-iPrAm
m-Im
p-Am
p-iPrAm
p-Im
m-Am
m-iPrAm
m-Im
p-Am
p-Am
p-iPrAm
p-Am
p-iPrAm
m-Am
m-iPrAm
m-AmOH
p-iPrAm
p-Am
0.0021
0.0680
0.0462
>62.6
30.4
0.0473
7.71
0.144
1.68
4350
>2490
2670
<0.104
>1.11
1360
1.12
209
97.0
168
602
>126
2.75
600
0.0256
0.0036
0.0180
>10.4
>9.72
0.0191
2.09
0.153
0.0109
0.0104
0.109
357
>46 900
3.26
7.27
3.99
2.80
>23.2
30.9
<0.0653
<0.336
6860
<0.628
>3.46
3361
4.12
197
15 000
10 500
775
>1470
184
194
>9820
1680
119
1830
175
1250
224
625
1060
1360
25.1
138
0.845
1290
44.7
153
654
174
>100
>100
6.53
>33.6
64.2
8.6
30.1
163
>50.0
<0.172
7.87
14.0
29.8
15.4
<5.41
<0.665
9.4
20.9
15.9
3.82
11.7
3.66
5.50
>30.0
>100
1.32
7.99
1.90
>200
14.8
>200
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
0.647
0.140
1.280
109
84.6
>162
66.5
12.4
>167
30.2
0.111
24.2
0.362
0.064
0.017
0.0180
0.0677
0.0474
0.113
0.0207
0.0748
0.380
0.0778
1.20
>2230
79.5
103
72.4
13.9
212
121
134
312
30.7
79.2
141
0.541
324
8.17
82.2
236
<0.0401
5.89
<0.099
8.02
87
1.42
19.8
68.8
28.3
41.8
78
38.2
14.4
50.7
0.325
0.234
0.313
0.250
1.24
0.182
0.359
12.4
0.0551
0.126
0.0669
0.0734
0.0280
0.575
0.367
7.930
0.0748
0.430
0.0870
0.112
0.0681
0.163
0.133
0.149
0.0738
0.0719
9.73
4.29
5.73
4.51
6.67
3.2
>100
<0.0670
11.4
2.70
<0.0665
6.17
2.02
6.70
0.298
2.36
8.49
7.12
>200
11.9
5.89
96.4
19.3
13.3
73.4
11.9
6.95
98.0
30.4
m-Am
0.162
0.311
0.960
0.219
0.223
0.146
0.348
0.812
m-iPrAm
m-sBuAm
p-Am
p-iPrAm
m-Am
12.4
31.7
440
208
390
16.9
42.6
739
205
1840
191
10.8
4.77
11.0
9.07
6.38
12.3
3.71
m-iPrAm
m-sBuAm
136
13.7
3.69
PMDh
FMDi
MLSPk
CQl
0.0028
0.0032
0.0064
16 600
1880
1220
0.0464
0.0138
1004
464
1.84
25.4
46.6
6.4
7.8
116.9
450.5
0.017
2.70j
0.1248
0.0043
937
105 000
ATMSm
PPTn

D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
3547
Br
m
1-3, 6
8-19
4
o
CN
p
a
b
p
O
HO
12-22
2
3
m
O
NC
m
m
p
Br
CN
p
m
CN
1
p
O
12
8
OH
m
Br
23-36
a
4
b
CN
7
HO
28-37
O
p
5
5
6
Diols
1,5
p
1,6
NC
m
2,6
2,7
m
m
O
O
O
O
O
m
m
c
NC
CN
R
R
p
(for 4 and 7)
p
p
p
4: R = p-AmOH
5: R = p-AmOMe
7: R = m-AmOH
d
b
NC
HOAm
O
O
O
CN
AmOH
27
Scheme 1. Reagents and conditions: (a) K2CO3 or Cs2CO3, DMF; (b) HCl, EtOH, 1,4-dioxane, then appropriate amine; (c) NH2OH$HCl, t-BuOK, DMSO; (d) Me2SO4, NaOH, DMF.
The alteration of central ring substitutions and the positions of
the amidine functions had a less pronounced impact upon the
antiplasmodial activities of the xylene derivatives relative to their
antitrypanosomal activities. In contrast, the introduction of alkyl
groups onto the amidine nitrogen atoms generally resulted in
increased antiplasmodial activity, consistent with previous results
from this laboratory [14,20]. Four of the five N-isopropylamidines
(2, 9, 15, and 18) and three of the imidazolines (3, 10, and 16) were
more active than the corresponding amidines, exhibiting anti-
plasmodial IC50 values less than 50 nM. Thus these seven
compounds displayed various degrees of selectivity for P. falcipa-
rum over T. b. rhodesiense, up to 150-fold in the case of N-iso-
propylamidine 9. The introduction of a central fused ring system
resulted in decreased antiplasmodial activities, although a much
smaller decrease relative to that observed with antitrypanosomal
activity. For example, only a two-fold decrease in antiplasmodial
activity was observed between compounds 20 and 1, in contrast to
a 150-fold decrease in antitrypanosomal activity. Among both
groups of naphthalene derivatives, only N-isopropylamidine 24
exhibited an antiplasmodial IC50 value less than 50 nM. Despite
their low activities, N-isopropylamidines 33 and 35 and N-sec-
butylamidines 32 and 37 were more active than the corresponding
amidines.
The N-isopropyl group had the most significant impact upon
decreased cytotoxicity. Ten of the 12 N-isopropylamidines in the
series were among the 17 compounds that were less cytotoxic than
pentamidine. The N-isopropyl derivatives were less cytotoxic than
the corresponding lamidines in 10 of 11 instances. Three imidazo-
lines (all xylene derivatives) were also among the 17 least cytotoxic
compounds. The N-sec-butyl group had a smaller impact upon
decreased cytotoxicity. Thirty-one of the 36 analogues were less
cytotoxic than lead compound 1.
4. Conclusions
The xylene derivatives, as a whole, were more active against the
three parasites in vitro and less cytotoxic than either group of the
naphthalene derivatives. While lead compound 1 was more active
a
Trypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations (Refs. [36–39]).
Plasmodium falciparum (K1, resistant to chloroquine). Average of duplicate determinations (Refs. [36,40]).
Leishmania donovani (MHOM/SD/62/1S-CL2D) axenic amastigotes. Average of duplicate determinations (Refs. [41,42]).
Rat myoblast cells, used as a measure of cytotoxicity. Average of duplicate determinations (Ref. [43]).
Selectivity index for T. b. rhodesiense (SIT), expressed as the ratio [IC50 (L6)/IC50 (T. b. rhodesiense)].
Selectivity index for P. falciparum (SIP), expressed as the ratio [IC50 (L6)/IC50 (P. falciparum)].
Selectivity index for L. donovani (SIL), expressed as the ratio [IC50 (L6)/IC50 (L. donovani)].
PMD, pentamidine.
FMD, furamidine.
Value reported in Ref. [7].
MLSP, melarsoprol.
CQ, chloroquine.
ATSM, artemisinin.
PPT, podophyllotoxin.
b
c
d
e
f
g
h
i
j
k
l
m
n

3548
D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
Table 2
HPLC chromatograms were recorded on a Hewlett–Packard 1090
Activities of select compounds against mice infected with T. b. rhodesiense
Series II chromatograph using a Zorbax Rx C8 column (4.6 ꢀ 75 mm,
(STIB900).a
3.5 mm) and UV photodiode array detection at 230, 254, 265, 290,
Compd
Doseb (mg/kg)
Routec
Cured/infectedd
MSDe (days)
and 320. Wavelengths reported are those at which the strongest
signals of the major products were observed. Mobile phases con-
sisted of mixtures of CH3CN (0–75%) in water containing formic acid
(80 mM), ammonium formate (20 mM) and triethylamine (15 mM).
Flow rates were maintained at 1.5 mL/min at a column temperature
of 40 ꢁC. The concentration of CH3CN was increased linearly from
0 to 22.5% over 6 min, then from 22.5 to 56.25% over 4 min, then
maintained for 1 min. Preparative reverse phase HPLC was per-
formed on Varian ProStar Chromatography Workstation config-
ured with two PS-215 pumps fitted with 50 mL pumpheads,
1
2
4 ꢀ 10
4 ꢀ 10
4 ꢀ 5
i.p.
i.p.
i.p.
i.p.
p.o.
p.o.
i.p.
p.o.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
p.o.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p
1/4
3/4
3/4
0/4
1/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
3/4
0/4
>35.5
>60
>51.5
22.8
>38.3
6
3
4
5
6
7
8
4 ꢀ 20
4 ꢀ 100
4 ꢀ 100
4 ꢀ 5
17
4 ꢀ 100
4 ꢀ 20
4 ꢀ 10
4 ꢀ 20
4 ꢀ 5
14.5
13.5
>27.3
32
17
15
19.5
12
>60
7.8
6
13.5
18
11
14
15
16
17
20
25
27
28
30
34
35
MLSPg
PMDh
a Dynamax Microsorb C18 (60 Å) column (41.4 ꢀ 25 cm, 8
mm), PS-
4 ꢀ 20
4 ꢀ 20
4 ꢀ 20
4 ꢀ 20
4 ꢀ 100
4 ꢀ 5
320 variable wavelength UV–Vis detector, and a PS-701 fraction
collector. Mobile phases consisted of mixtures of CH3CN (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
4 ꢀ 5
4 ꢀ 5
using a Zorbax Rx C8 column (4.6 ꢀ 75 mm, 3.5
mm) and the latter
4 ꢀ 20
4 ꢀ 4
17.3
>60
42.8
mobile phases on an Agilent Technologies 1100 chromatograph.
Pooled purified fractions were evaporated under reduced pres-
sure, reconstituted in water, and lyophilized on a VirTis BenchTop
2K lyophilizer. Low resolution ESIþ mass spectra were recorded on
an Agilent Technologies 1100 Series LC/MSD Trap spectrometer or
an Applied Biosystems Series 100 spectrometer. Elemental anal-
yses were performed by Atlantic Microlab, Norcross, GA, and were
within ꢂ0.4% of calculated values.
4 ꢀ 20
fRef. [26].
a
Female NMRI mice.
Single daily doses.
intraperitoneal (i.p.) or oral (p.o.).
b
c
d
Mice that survived for 60 days after infection without showing a parasitemia
relapse.
e
Mean survival days post infection of the mice that showed a parasitemia relapse.
MLSP, melarsoprol.
PMD, pentamidine.
g
h
5.2. General procedure for compounds 1–3, 6, 8–26, and 27–37
than pentamidine against T. b. rhodesiense in vitro, all structural
modifications resulted in decreased activity. Analogue 2, the N-
isopropyl derivative of 1, was quite active in the acute mouse model
of the parasite. Disappointingly, none of the prodrugs exhibited
substantial oral activity. The greatest potential for these
compounds may lie in their antiplasmodial activities. Compound 2
displayed not only an IC50 value lower than that of all the reference
compounds, but also a selectivity index 47 times greater than that
of pentamidine. Nine other analogues exhibited IC50 values less
than 50 nM (activities comparable to or greater than that of pent-
amidine), four of which displayed selectivity indices between five
and 15 times greater than that of pentamidine. Compound 14 was
The nitrile was added to a mixture of anhydrous EtOH and 1,4-
dioxane that had been saturated with hydrogen chloride at 0 ꢁC in
a dry 3-neck flask equipped with a gas inlet tube, a thermometer,
and a drying tube, and cooled in an ice–salt bath. The reaction
mixture was then sealed, slowly warmed to ambient temperature,
and stirred until the nitrile was no longer detectable. The reaction
mixture was diluted with ether. The crude imidate was filtered off
under inert gas and dried under high vacuum over KOH. The imi-
date (or an aliquot thereof) was then reacted immediately with the
appropriate ammonia or the appropriate amine in EtOH. The reac-
tion mixture was diluted with ether, and the crude product was
filtered off. The product was purified by direct recyrstallization from
appropriate solvents or by preparative HPLC followed by conversion
to the dihydrochloride salt using aqueous or ethanolic HCl.
the most active against L. donovani (IC50 ¼ 1.3
mM), and eight other
analogues displayed IC50 values between 1.9 and 4.8 mM.
5. Experimental
5.2.1. 1,3-Bis[4-(N-isopropylamidino)phenoxymethyl]benzene
dihydrochloride (2)
5.1. Chemistry
Yield, 0.76 g (55%): mp 258–259 ꢁC; 1H NMR
d
9.42 (d, J ¼ 7.9 Hz,
Uncorrected melting points were measured on a Thomas–Hoo-
ver Capillary melting point apparatus. 1H NMR spectra were
recorded on a Varian Gemini 2000 (300 MHz) spectrometer. Spectra
were in DMSO-d6 (with 0.05% TMS) unless stated otherwise.
Anhydrous EtOH was distilled over Mg/I2 immediately prior to use.
Other anhydrous solvents were purchased from Aldrich Chemical
Co., Milwaukee, WI, in Sure-sealÒ containers and were used without
further purification. Reaction mixtures were monitored by TLC on
silica gel or by reverse phase HPLC. Organic layers of extraction
mixtures were washed with saturated NaCl solution and dried over
Na2SO4 or MgSO4 before being evaporated under reduced pressure.
Gravity and flash column chromatography were performed using
Davisil grade 633, type 60A silica gel (200–425 mesh). Analytical
2H), 9.32 (br s, 2H), 9.97 (br s, 2H), 7.73 (d, J ¼ 8.5 Hz, 4H), 7.59 (s,
1H), 7.40 (m, 2H), 7.45 (s, 3H), 7.22 (d, J ¼ 8.5 Hz, 4H), 5.26 (s, 4H),
4.05 (m, 2H), 1.26 (d, J ¼ 6.3 Hz, 12H); HPLC tR 8.25 min (98.7 area %
at 254 nM). Anal. Calcd for C28H34N4O2$2HCl: C, 63.27; H, 6.83; N,
10.54; Cl, 13.34. Found: C, 63.14; H, 6.64; N, 10.35; Cl, 13.34.
5.2.2. 1,3-Bis[4-(2-imidazolinyl)phenoxymethyl]benzene
dihydrochloride (3)
Yield, 1.52 g (82%): mp 241–243 ꢁC; 1H NMR
d 10.67 (s, 4H), 8.08
(d, J ¼ 8.2 Hz, 4H), 7.58 (s, 1H), 7.46 (s, 3H), 7.27 (d, J ¼ 8.2, 4H), 5.28
(s, 4H), 3.95 (s, 8H); HPLC tR 7.48 min (100 area % at 254 nM). Anal.
Calcd for C26H26N4O2$2HCl$H2O: C, 60.35; H, 5.84; N, 10.83; Cl,
13.70. Found: C, 60.28; H, 5.96; N, 10.87; Cl, 13.97.

D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
3549
5.2.3. 1,2-Bis[4-(N-isopropylamidino)phenoxymethyl]benzene
dihydrochloride (9)
5.24 (s, 4H), 4.07 (m, 2H), 1.27 (d, J ¼ 6.3 Hz, 12H); HPLC tR 8.09 min
(100 area % at 254 nM). Anal. Calcd for C28H34N4O2$2HCl$0.5H2O:
C, 62.22; H, 6.90; N, 10.36; Cl, 13.12. Found: C, 62.20; H, 6.88; N,
10.23; Cl, 12.97.
Yield, 0.06 g (3.7%): mp 196–198 ꢁC; 1H NMR
d 9.46
(d, J ¼ 7.7 Hz, 2H), 9.37 (br s, 2H), 9.03 (br s, 2H), 7.74 (d, J ¼ 8.5 Hz,
4H), 7.55 (m, 2H), 7.40 (m, 2H), 7.21 (d, J ¼ 8.5 Hz, 4H), 5.38 (s, 4H),
4.07 (m, 2H), 1.26 (d, J ¼ 6.3 Hz, 12H); MS m/z 459 (MHþ of free
base); HPLC tR 8.21 min (100 area % at 254 nM). Anal. Calcd for
C28H34N4O2$2HCl$1.4H2O: C, 60.41; H, 7.02; N, 10.06; Cl, 12.74.
Found: C, 60.40; H, 6.88; N, 10.03; Cl, 12.60.
5.2.11. 1,4-Bis[3-(2-imidazolinyl)phenoxymethyl]benzene
dihydrochloride (19)
Yield, 0.90 g (70%): mp 293 ꢁC, dec; 1H NMR
d
10.83 (s, 4H), 7.82
(s, 2H), 7.55 (m, 6H), 7.51 (s, 4H), 5.24 (s, 4H), 4.00 (s, 8H); HPLC tR
7.25 min (100 area at 254 nM). Anal. Calcd for
%
5.2.4. 1,2-Bis[4-(2-imidazolinyl)phenoxymethyl]benzene
C26H26N4O2$2HCl$1.5H2O: C, 59.32; H, 5.94; N, 10.64; Cl, 13.47.
dihydrochloride (10)
Found: C, 59.38; H, 6.09; N, 10.56; Cl, 13.23.
Yield, 0.99 g (81%): mp 185–190 ꢁC; 1H NMR
d 10.67 (s, 4H), 8.07
(dd, J ¼ 8.7 and 2.1 Hz, 4H), 7.56 (m, 2H), 7.41 (m, 2H), 7.26
(dd, J ¼ 8.7 and 2.1 Hz, 4H), 5.38 (s, 4H), 3.95 (s, 8H); MS m/z 427
(MHþ of free base); HPLC tR 7.39 min (96.02 area % at 254 nM). Anal.
Calcd for C26H26N4O2$2HCl$0.7H2O: C, 60.99; H, 5.79; N, 10.94;
Cl, 13.83. Found: C, 60.94; H, 5.75; N, 10.91; Cl, 13.98.
5.2.12. 1,4-Bis[4-(N-isopropylamidino)phenoxymethyl]naphthalene
dihydrochloride (22)
Yield 0.11 g (15%): mp 294–295 ꢁC; 1H NMR
d
9.42 (d, J ¼ 7.6 Hz,
2H), 9.32 (br s, 2H), 8.96 (br s, 2H), 8.17 (m, 2H), 7.74 (d, J ¼ 8.8 Hz,
4H), 7.73 (s, 2H), 7.66 (m, 2H), 7.32 (d, J ¼ 8.8 Hz, 4H), 5.71 (s, 4H),
4.04 (m, 2H), 1.27 (d, J ¼ 6.3 Hz, 12H); MS m/z 509 (MHþ of free
base); HPLC tR 8.83 min (100 area % at 254 nM). Anal. Calcd for
C32H36N4O2$2HCl$0.8H2O: C, 64.49; H, 6.70; N, 9.40; Cl, 11.90.
Found: C, 64.35; H, 6.66; N, 9.29; Cl, 12.07.
5.2.5. 1,2-Bis (3-amidinophenoxymethyl)benzene dihydrochloride
(11)
Yield, 0.19 g (14%): mp 238–240 ꢁC.; 1H NMR
d 9.48 (br s, 4H),
9.13 (br s, 4H), 7.61 (m, 2H), 7.57 (m, 2H), 7.51 (d, J ¼ 7.9 Hz, 2H), 7.41
(m, 6H); MS m/z 375 (MHþ of free base); HPLC tR 6.53 min (97 area
% at 230 nM). Anal. Calcd for C22H22N4O2$2HCl$0.7H2O: C, 57.45;
H, 5.57; N, 12.18. Found: C, 57.42; H, 5.63; N, 12.00.
5.2.13. 1,5-Bis[4-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (24)
Yield 0.39 g (30%): mp 304–305 ꢁC; 1H NMR
d 9.63
(d, J ¼ 8.0 Hz, 2H), 9.49 (br s, 2H), 9.19 (br s, 2H), 7.82 (d, J ¼ 8.0 Hz,
2H), 7.79 (d, J ¼ 8.3 Hz, 4H), 7.76 (d, J ¼ 8.3 Hz, 4H), 7.44
(t, J ¼ 8.0 Hz, 2H), 7.13 (d, J ¼ 8.0 Hz, 2H), 5.45 (s, 4H), 4.08 (m, 2H),
1.28 (d, J ¼ 6.4 Hz, 12H); MS m/z 509 (MHþ of free base); HPLC tR
5.2.6. 1,2-Bis[3-(N-isopropylamidino)phenoxymethyl]benzene
dihydrochloride (12)
Yield, 0.35 g (24%): mp indef.; 1H NMR
d
9.68 (d, J ¼ 7.7 Hz, 2H),
9.54 (br s, 2H), 9.18 (br s, 2H), 7.58 (m, 2H), 7.42 (m, 4H), 7.36
(m, 6H), 5.41 (s, 4H), 4.07 (m, 2H), 1.27 (d, J ¼ 6.3 Hz, 12H); MS m/z
459 (MHþ of free base); HPLC tR 8.00 min (100 area % at 230 nM).
Anal. Calcd for C28H34N4O2$2HCl$1.3H2O: C, 60.60; H, 7.01; N, 10.10;
Cl, 12.78. Found: C, 60.31; H, 6.97; N, 10.25; Cl, 12.13.
8.79 min (97.8 area
%
at 230 nM). Anal. Calcd for
C32H36N4O2$2HCl: C, 66.09; H, 6.59; N, 9.63; Cl, 12.19. Found:
C, 65.89; H, 6.50 N, 9.44; Cl, 12.11.
5.2.14. 1,5-Bis[3-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (26)
5.2.7. 1,2-Bis[3-(2-imidazolinyl)phenoxymethyl]benzene
Yield, 0.16 g (12%): mp 249–250 ꢁC; 1H NMR
d
9.71 (d, J ¼ 7.7 Hz,
dihydrochloride (13)
2H), 9.56 (br s, 2H), 9.25 (br s, 2H), 7.94 (s, 2H), 7.87 (m, 4H), 7.74
(d, J ¼ 8.0 Hz, 2H), 7.67 (t, J ¼ 7.6 Hz, 2H), 7.45 (t, J ¼ 8.0 Hz, 2H), 7.17
(d, J ¼ 8.0 Hz, 2H), 5.39 (s, 4H), 4.13 (m, 2H), 1.29 (d, J ¼ 6.6 Hz, 12H);
MS m/z 509 (MHþ of free base); HPLC tR 8.80 min (100 area % at
230 nM). Anal. Calcd for C32H36N4O2$2HCl$H2O: C, 64.10; H, 6.72;
N, 9.34; Cl, 11.83. Found: C, 64.06 H, 6.66 N, 9.25; Cl, 11.87.
Yield, 0.77 g (65%): mp 289 ꢁC; 1H NMR
d 10.67 (s, 4H), 7.94
(br s, 2H), 7.57 (m, 6H), 7.42 (m, 4H), 5.41 (s, 4H), 4.00 (s, 8H);MS m/
z 427 (MHþ of free base); HPLC tR 7.23 min (100 area % at 254 nM).
Anal. Calcd for C26H26N4O2$2HCl$1.4H2O: C, 59.52; H, 5.92;
N, 10.68; Cl, 13.51. Found: C, 59.46; H, 5.92; N, 10.63; Cl, 13.63.
5.2.8. 1,4-Bis[4-(N-isopropylamidino)phenoxymethyl]benzene
dihydrochloride (15)
5.2.15. 1,5-Bis[3-(N-hydroxyamidino)benzyloxy]naphthalene
dihydrochloride (27)
Yield, 0.95 g (36%): mp 318 ꢁC dec.; 1H NMR
d
9.42 (d, J ¼ 8.8 Hz,
The crude imidate ester was prepared from the corresponding
nitrile (1.50 g, 8.85 mmol) following the general procedure, then
suspended in ethanol (10 mL). To this mixture was added a solution
of hydroxylamine (50 mL of a 0.68 M ethanolic solution, prepared
from hydroxylamine hydrochloride and sodium ethoxide, followed
by filtration). The mixture was refluxed for 1 h and allowed to cool.
The amidoxime base was precipitated by dilution with ether, then
converted to the HCl salt using ethanolic HCl. Yield, 0.31 g (15%):
2H), 9.32 (br s, 2H), 8.99 (br s, 2H), 7.73 (d, J ¼ 8.8 Hz, 4H), 7.50
(s, 4H), 7.21 (d, J ¼ 8.8 Hz, 4H), 5.25 (s, 4H), 4.07 (m, 2H), 1.26
(d, J ¼ 6.3 Hz, 12H); HPLC tR 8.20 min (97.8 area % at 254 nM). Anal.
Calcd for C28H34N4O2$2HCl$0.5H2O: C, 62.22; H, 6.90; N, 10.36;
Cl, 13.12. Found: C, 62.41; H, 6.79; N, 10.28; Cl, 12.99.
5.2.9. 1,4-Bis[4-(2-imidazolinyl)phenoxymethyl]benzene
dihydrochloride (16)
mp > 200 ꢁC (dec.); 1H NMR
d 11.27 (s, 1H), 9.05 (2, 2H), 7.93 (s, 2H),
Yield, 0.70 g (28%): mp 268–170 ꢁC; 1H NMR
d
10.60 (s, 4H), 8.05
7.87 (m, 4H), 7.74 (d, J ¼ 8.0 Hz, 2H), 7.67 (t, J ¼ 7.7 Hz, 2H), 7.45 (t,
J ¼ 8.0 Hz, 2H), 7.16 (d, J ¼ 8.0 Hz, 2H), 5.38 (s, 4H); MS m/z 457
(MHþ of free base); HPLC tR 3.68 min (97.3% at 254 nM). Anal. Calcd
for C26H24N4O4$2HCl$0.6H2O$0.3C2H5OH: C, 57.66; H, 5.28;
N, 10.11; Cl, 12.80. Found: C, 57.68; H, 5.04; N, 10.13; Cl, 12.57.
(d, J ¼ 8.8 Hz, 4H), 7.51 (s, 4H), 7.28 (d, J ¼ 8.8 Hz, 4H), 5.27 (s, 4H),
3.96 (s, 8H); HPLC tR 7.35 min (97.5 area % at 265 nM). Anal. Calcd
for C26H26N4O2$2HCl$1.5H2O: C, 59.32; H, 5.94; N, 10.64; Cl, 13.47.
Found: C, 59.07; H, 6.02; N, 10.45; Cl, 13.19.
5.2.10. 1,4-Bis[3-(N-isopropylamidino)phenoxymethyl]benzene
dihydrochloride (18)
5.2.16. 1,6-Bis[4-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (28)
Yield, 0.99 g (73%): mp 173 ꢁC; 1H NMR
d
9.60 (d, J ¼ 7.9 Hz, 2H),
Yield, 1.08 g (61%): mp 218–220 ꢁC; 1H NMR
d
9.61
9.48 (br s, 2H), 9.19 (br s, 2H), 7.52 (s, 4H), 7.42 (s, 2H), 7.38 (m, 6H),
(d, J ¼ 8.4 Hz, 2H), 9.48 (br s, 2H), 9.16 (br s, 2H), 8.15 (d, J ¼ 9.2 Hz,

3550
D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
1H), 7.75 (m, 8H), 7.41 (d, J ¼ 2.5 Hz,1H), 7.37 (d, J ¼ 4.4 Hz, 2H), 7.28
(dd, J ¼ 9.2 and 2.4 Hz, 1H), 6.92 (t, J ¼ 4.3 Hz, 1H), 5.43 (s, 2H), 5.37
(s, 2H), 4.07 (m, 2H), 1.27 (d, J ¼ 6.2 Hz, 12H); MS m/z 509 (MHþ of
free base); HPLC tR 8.84 min (100 area % at 230 nM). Anal. Calcd for
C32H36N4O2$2HCl$1.6H2O: C, 62.97; H, 6.80; N, 9.18; Cl, 11.62.
Found: C, 63.01; H, 6.70; N, 9.14; Cl, 11.74.
5.30 (s, 4H), 3.90 (m, 2H), 1.68 (m, 4H), 1.25 (d, J ¼ 6.3 Hz, 6H), 0.94
(t, J ¼ 6.3 Hz, 6H); MS m/z 537 (MHþ of free base); HPLC tR 9.44 min
(100 area % at 230 nM). Anal. Calcd. for C34H40N4O2$2HCl$H2O:
C, 65.25; H, 7.05; N, 8.95. Found: C, 65.16; H, 6.99; 8.86.
5.3. 1,3-Bis[4-(N-methoxyamidino)phenoxymethyl]benzene
dihydrochloride (5)
5.2.17. 2,6-Bis[3-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (31)
Dimethyl sulfate (1.18 g, 9.39 mmol) was added dropwise to
a solution of 1,3-bis[(4-{N-hydroxy}amidino)phenoxymethyl]ben-
zene (4) [29] in DMSO (30 mL) and 2 M NaOH (10 mL) at 0 ꢁC. After
removal of the ice bath, the mixture was stirred at ambient
temperature for 5 h and diluted with ice water (300 mL). The
resulting precipitate was filtered off, washed with water (100 mL)
and dried overnight. The crude product was purified by flash
chromatography (EtOAc/hexanes [2:3]), followed by recrystalliza-
tion from EtOAc/hexanes to give 0.80 g (59%) of free base. The
product was recrystallized from EtOH/1 M HCl to give 0.89 g of
Yield, 0.24 g (24%): mp 194–197 ꢁC; 1H NMR
d 9.67
(d, J ¼ 8.5 Hz, 2H), 9.50 (br s, 2H), 9.14 (br s, 2H), 7.83 (m, 6H), 7.68
(m, 4H), 7.45 (d, J ¼ 2.4 Hz, 2H), 7.25 (dd, J ¼ 8.9 and 2.4 Hz, 2H),
5.28 (s, 4H), 4.07 (m, 2H),1.28 (d, J ¼ 6.4 Hz,12H); MS m/z 509 (MHþ
of free base); HPLC tR 8.72 min (97.1 area % at 230 nM). Anal. Calcd
for C32H36N4O2$2HCl$H2O: C, 64.10; H, 6.72; N, 9.34; Cl, 11.83.
Found: C, 64.17; H, 6.69; N, 9.20; Cl, 11.61.
5.2.18. 2,6-Bis[3-(N-sec-butylamidino)benzyloxy]naphthalene
dihydrochloride (32)
dihydrochloride (5): mp 218–220 ꢁC; 1H NMR
d 9.05 (br s, 4H), 7.79
Yield, 0.20 g (19%): mp 258–259 ꢁC; 1H NMR
d
9.65 (br s, 2H),
(d, J ¼ 9.0 Hz, 4H), 7.58 (s, 1H), 7.45 (s, 3H), 7.21 (d, J ¼ 9.0, 4H), 5.25
(s, 4H), 3.85 (s, 6H); HPLC tR 10.31 min (100 area % at 254 nM). Anal.
Calcd for C24H26N4O4$2HCl$0.4H2O: C, 56.01; H, 5.64; N, 10.89;
Cl, 13.78. Found: C, 56.11; H, 5.51; N, 10.81; Cl, 13.61.
9.53 (br s, 2H), 9.20 (br s, 2H), 7.88 (m, 2H), 7.85 (d, J ¼ 7.9 Hz, 2H),
7.79 (dd, J ¼ 9.0 and 1.9 Hz, 2H), 7.72 (dm, J ¼ 7.7 Hz, 2H), 7.66
(t, J ¼ 7.6 Hz, 2H), 7.45 (s, 2H), 7.25 (dd, J ¼ 9.0 and 1.9 Hz), 5.28
(s, 4H), 3.89 (m, 2H), 1.65 (m, 4H), 1.25 (d, J ¼ 6.3 Hz, 6H), 0.94
(t, J ¼ 7.3 Hz, 6H); MS m/z 537 (MHþ of free base); HPLC tR 9.31 min
(100 area % at 230 nM). Anal. Calcd for C34H40N4O2$2HCl$1.2H2O:
C, 64.69; H, 7.09; N, 8.88. Found: C, 64.47; H, 6.86; N, 8.92.
5.4. 1,3-Bis[3-(N-hydroxyamidino)phenoxymethyl]benzene
dihydrochloride (7)
Potassium tert-butoxide (7.80 g, 69.5 mmol) was added to
a solution of hydroxylamine hydrochloride (5.40 g, 77.7 mmol) in
DMSO (100 mL). The mixture was stirred at ambient temperature
for 1 h prior to the addition of 1,3-bis(3-cyanophenox-
ymethyl)benzene (3.75 g, 11.0 mmol). The mixture was stirred at
ambient temperature overnight and poured over ice water. The
resulting precipitate was filtered off and dried to give an off-white
5.2.19. 2,7-Bis[4-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (34)
Yield, 1.23 g (52%): mp 236–238 ꢁC; 1H NMR
d 9.60
(d, J ¼ 8.0 Hz, 2H), 9.47 (br s, 2H), 9.14 (br s, 2H), 7.78
(d, J ¼ 9.1 Hz, 2H), 7.77 (d, J ¼ 8.4 Hz, 4H), 7.71 (d, J ¼ 8.4 Hz, 4H),
7.29 (d, J ¼ 2.3 Hz, 2H), 7.11 (dd, J ¼ 8.8 and 2.3 Hz, 2H), 5.34 (s, 4H),
4.06 (m, 2H), 1.27 (d, J ¼ 6.4 Hz, 12H); MS m/z 509 (MHþ of free
base); HPLC tR 8.94 min (100 area % at 230 nM). Anal. Calcd for
C32H36N4O2$2HCl$0.5H2O: C, 65.08; H, 6.66; N, 9.29; Cl, 12.01.
Found: C, 65.25; H, 6.48; N, 9.29; Cl, 11.83.
solid as the free base (4.28 g, 96% crude). 1H NMR
d 9.64 (s, 2H), 7.56
(s, 1H), 7.43 (s, 3H), 7.33 (s, 2H), 7.29 (s, 2H), 7.28 (s, 2H), 7.03
(m, 2H), 5.82 (s, 4H), 5.15 (s, 4H). An aliquot of the free base (1.02 g,
2.50 mmol) was suspended in warm ethanol (20 mL) and treated
with concentrated ethanolic HCl (10 mL). The suspension was
heated and water (5 mL) was added until solids dissolved. Upon
cooling, white crystals precipitated as the dihydrochloride salt
5.2.20. 2,7-Bis[(3-amidino)benzyloxy]naphthalene dihydrochloride
(35)
Yield, 0.54 g (64%): mp 265–268 ꢁC; 1H NMR
d
9.48 (br s, 4H),
(0.88 g, 74%): mp 208–210 ꢁC (dec.): 1H NMR
d 11.15 (br s,1.1H), 8.94
9.22 (br s, 4H), 8.01 (s, 2H), 7.83 (m, 6H), 7.67 (t, J ¼ 7.7 Hz, 2H), 7.39
(d, J ¼ 2.2 Hz, 2H), 7.11 (dd, J ¼ 8.9 and 2.3 Hz, 2H), 5.30 (s, 4H); MS
m/z 425 (MHþ of free base); HPLC tR 7.86 min (100 area % at
230 nM). Anal. Calcd for C26H24N4O2$2HCl$0.7H2O: C, 61.23; H,
5.41; N, 10.98; Cl, 13.90. Found: C, 61.36; H, 5.47; N, 10.91; Cl, 13.80.
(br s, 2.5H), 7.53 (br s, 1H), 7.49 (dd, J ¼ 8.0 and 8.0 Hz, 2H), 7.43 (m,
3H), 7.39 (m, 2H), 7.30 (dd, J ¼ 8.0 and 8.0 Hz, 4H), 5.19 (s, 4H); MS
m/z 407 (MHþ of free base), HPLC tR 7.88 min (100 area % at
254 nM). Anal. Calcd for C22H22N4O4$2HCl: C, 55.12; H, 5.05;
N, 11.69; Cl, 14.79. Found: C, 55.40; H, 5.03; N, 11.66; Cl, 14.72.
5.2.21. 2,7-Bis[3-(N-isopropylamidino)benzyloxy]naphthalene
dihydrochloride (36)
5.5. General method for nitrile precursors to compounds 23–37
Yield, 0.53 g (54%): mp indef.; 1H NMR
d
9.71 (d, J ¼ 8.1 Hz, 2H),
A solution of the a-bromo-tolunitrile in DMF was added to
9.55 (br s, 2H), 9.20 (s, 2H), 7.90 (s, 2H), 7.84 (d, J ¼ 7.6 Hz, 2H), 7.79
(d, J ¼ 8.9 Hz, 2H), 7.72 (d, J ¼ 7.7 Hz, 2H), 7.65 (t, J ¼ 7.7 Hz, 2H), 7.41
(d, J ¼ 2.2 Hz, 2H), 7.11 (dd, J ¼ 8.8 and 1.9 Hz, 2H), 5.29 (s, 4H), 4.10
(m, 2H), 1.29 (d, J ¼ 6.3 Hz, 12H); MS m/z 509 (MHþ of free base);
a mixture of the naphthalenediol and K2CO3 or Cs2CO3 in DMF
under N2 at 90–120 ꢁC. The reaction mixture was maintained until
starting materials were no longer detectable. The cooled reaction
mixture was paritioned between water and CH2Cl2. The product
was isolated by column chromatography eluting with CH2Cl2 or
CHCl3 followed by recrystallization from an appropriate solvent.
HPLC tR 8.89 min (97.5 area
% at 230 nM). Anal. Calcd for
C32H36N4O2$2HCl$0.9H2O: C, 64.29; H, 6.71; N, 9.37; Cl, 11.86.
Found: C, 64.18; H, 6.76; N, 9.37; Cl, 12.04.
5.5.1. 1,6-Bis[(4-cyano)benzyloxy]naphthalene
5.2.22. 2,7-Bis[3-(N-sec-butylamidino)benzyloxy]naphthalene
dihydrochloride (37)
Yield, 6.22 g (64%): mp 188–189 ꢁC; 1H NMR (CDCl3)
d 8.26
(d, J ¼ 9.4 Hz, 1H), 7.72 (d, J ¼ 8.3 Hz, 2H), 7.71 (d, J ¼ 8.3 Hz, 2H),
7.62 (t, J ¼ 8.3 Hz, 4H), 7.35 (s, 1H), 7.33 (d, J ¼ 1.6 Hz, 1H), 7.24 (dd,
J ¼ 9.1 and 2.5 Hz, 1H), 7.16 (d, J ¼ 2.4 Hz, 1H), 6.72 (m, 1H), 5.30
(s, 2H), 5.26 (s, 2H). Anal. Calcd for C26H18N2O2: C, 79.98; H, 4.65;
N, 7.17. Found: C, 80.01; H, 4.72; N, 7.09.
Yield, 0.25 g (24%): mp 187–194 ꢁC (dec.); 1H NMR
d 9.66
(d, J ¼ 8.1 Hz, 1.8H), 9.52 (br s, 2H), 9.19 (br s, 2H), 7.89 (s, 2H), 7.85
(d, J ¼ 7.5 Hz, 2H), 7.80 (d, J ¼ 9.1 Hz, 2H), 7.72 (d, J ¼ 7.5 Hz, 2H),
7.66 (t, J ¼ 7.5 Hz, 2H), 7.41 (s, 2H), 7.11 (dd, J ¼ 8.6 and 2.0 Hz, 2H),

D.A. Patrick et al. / European Journal of Medicinal Chemistry 44 (2009) 3543–3551
3551
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