Synthesis and antiprotozoal activities of benzyl phenyl ether diamidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.06.033

Sixty-two cationic benzyl phenyl ether derivatives (36 amidines and 26 prodrugs) were prepared and assayed for activities in vitro and in vivo against Trypanosoma brucei rhodesiense (STIB900), and in vitro against Plasmodium falciparum (K1) and Leishmania

Full text of DOI:10.1016/j.ejmech.2013.06.033

European Journal of Medicinal Chemistry 67 (2013) 310e324
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiprotozoal activities of benzyl phenyl ether
diamidine derivatives
Donald A. Patrick a, Stanislav A. Bakunov a, Svetlana M. Bakunova a, Susan Kilgore Jones a,
,
Tanja Wenzler b c, Todd Barszcz d, Arvind Kumar e, David W. Boykin e, Karl A. Werbovetz d,
,
,
*
Reto Brun b c, 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 and Public Health Institute, Basel, CH 4051, Switzerland
c University of Basel, Basel, CH 4003, Switzerland
d Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
e Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303-3083, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixty-two cationic benzyl phenyl ether derivatives (36 amidines and 26 prodrugs) were prepared and
assayed for activities in vitro and in vivo against Trypanosoma brucei rhodesiense (STIB900), and in vitro
against Plasmodium falciparum (K1) and Leishmania donovani axenic amastigotes. 3-Amidinobenzyl 4-
amidino-2-iodo-6-methoxyphenyl ether dihydrochloride (55, IC50 ¼ 3.0 nM) and seven other com-
pounds exhibited IC50 values below 10 nM against T. b. rhodesiense in vitro. The 2-bromo-4,40-diamidino
analogue 19 (IC50 ¼ 4.0 nM) and 12 other analogues were more potent than pentamidine (IC50 ¼ 46 nM)
Received 28 March 2013
Received in revised form
10 June 2013
Accepted 12 June 2013
Available online 25 June 2013
against P. falciparum. The 30,4-diamidino-2,6-diiodo analogue 49 (IC50 ¼ 1.4
mM) and two other com-
Keywords:
Diamidines
Prodrugs
Benzyl phenyl ether derivatives
Antiprotozoal
pounds were more effective than pentamidine (IC50 ¼ 1.8
m
M) against L. donovani. A prodrug, 30,4-bis(N00-
methoxy)amidino-2-bromo derivative 38, was the most efficacious against trypanosome infected mice,
attaining 4/4 cures in four daily 25 mg/kg oral doses, and the 2-chloro-4,40-diamidine 18 cured 3/4 mice
in four daily 5 mg/kg intraperitoneal doses.
Antitrypanosomal
Antiplasmodial
Antileishmanial
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
estimates, new reported cases of HAT had dropped below 10,000 in
2009 and total estimated cases numbered around 30,000 in 2012
Human African trypanosomiasis (HAT), malaria, and leishma-
niasisdall infections due to protozoan parasites transmitted by
insect bitesdcontinue to impact some of the poorest areas of the
world [1e3]. HAT, which occurs only in sub-Saharan Africa, was
largely controlled by the 1960s, but re-emerged in the 1980s due to
limited resources to combat the disease and years of conflict in the
most affected countries. An estimated 300,000 people were infec-
ted in 2002 [4]. According to World Health Organization (WHO)
[3]. These figures, substantially lower than those in recent years
[3,5,6], are the result in advances and surveillance and treatment
[3]. In the first (or early) stage of HAT, the parasites multiply in the
blood and lymphatic systems. If untreated, the disease progresses to
the second (or late) stage in which parasites invade the central
nervous system (CNS). Late stage HAT leads to body wasting,
neurological disorders, coma, and death. Two forms of HAT exist. A
chronic infection of Trypanosoma brucei gambiense, endemic to
western and central Africa, accounts for over 95% of cases [3]. These
patients may be asymptomatic for months or even years, and the
disease is often in the CNS stage when symptoms first occur [7]. An
acute infection of Trypanosoma brucei rhodesiense, endemic to
eastern and southern Africa, causes the remainder of cases [3]. The
acute infection has a rapid onset and is fatal within six months in
80% of cases if not treated. T. b. rhodesiense also infects cattle, in
contrast toT. b. gambiense, which has no known animal reservoir [8].
Abbreviations: ATMS, artemisinin; BPE, benzyl phenyl ether; CQ, chloroquine;
FMD, furamidine; HAT, human African trypanosomiasis; IC50, 50% inhibition con-
centration; MLSR, melarsoprol; PMD, pentamidine; PPT, podophyllotoxin; SI,
selectivity index; WHO, World Health Organization.
* Corresponding author. Tel.: þ1 919 966 4294; fax: þ1 919 966 0704.
E-mail address: Tidwell@med.unc.edu (R.R. Tidwell).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.033

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
311
The most fatal form of malaria is caused by Plasmodium falcip-
arum. The WHO estimates 660,000 deaths per year and 210 million
cases (mainly young children in sub-Saharan Africa) in 2010, as well
as a tremendous economic impact [2,9,10]. Leishmaniasis is more
widespread geographically and ranges in severity from the most
common cutaneous form to the potentially fatal visceral form. An
epidemiological study led by the WHO estimates that 0.2e
0.4 million and 0.7e1.2 million new cases of visceral and cutaneous
leishmaniasis, respectively, are believed to occur annually [11],
with 90% of the visceral disease in Bangladesh, Brazil, India, Nepal
and Sudan [1]. Cases of patients infected with both visceral leish-
maniasis and HIV have also been reported [12e19].
The need for well-tolerated, orally active and economically
feasible drug therapies against these diseases persists. Pentamidine
(Fig. 1) and suramin (a polysulfonated naphthyl urea), used against
first stage HAT, and melarsoprol (an organoarsenic compound),
active against second stage HAT, were introduced prior to 1950,
require parenteral administration, and have adverse effects. Pent-
amidine is better tolerated than the other drugs. Severe side effects
include anaphylactic shocks, neurotoxicity, and renal failure for
suramin and a potentially fatal encephalopathy for melarsoprol.
Increasing treatment failures have also been reported for mel-
arsoprol [4,5]. Eflornithine, a safer alternative available since 1990,
is used only against second stage T. b. gambiense infections, must be
administered in high doses over long periods, and relapses have
occurred [4,20]. A combination of eflornithine and nifurtimox (an
orally effective drug used against Trypanosoma cruzi) was intro-
duced in 2009 against second stage T. b. gambiense [3].
A growing problem with treatment of malaria has been drug
resistance, especially to inexpensive and widely used drugs such as
chloroquine and sulfadoxineepyrimethamine [2,21]. Thus more
expensive drugs or drug combinations (especially with artemisi-
nins) must be used [22,23]. Various antimalarial drugs which are
currently in clinical use or under development are chemically
related or have similar mechanisms of action (or resistance)
[24,25].
Treatment options for visceral leishmaniasis continue to be
limited. Available drugs (all toxic) include the antiquated penta-
valent antimonials sodium stibogluconate and meglumine anti-
moniate [26e28], amphtotericin B (including lipid formulations)
[27], and paromomycin sulphate [27,28], all of which are adminis-
tered parenterally. Miltefosine, an orally active drug, has been used
both alone and in combination with amphtotericin B [29]. Pent-
amidine was once used as an alternative treatment for visceral
leishmaniasis in cases of antimony failure, but its use against
leishmaniasis is now limited to treatment of some forms of cuta-
neous leishmaniasis in South America [30].
expanded phase I trial [35], resulting in a suspension of phase III
trials [36].
Considerable effort has been made in this lab to prepare more
efficacious and less toxic analogues of pentamidine and furamidine
by alterations of the portion of the molecule between the amidine
moieties [24,25,37e42]. The trypanocidal activity of two benzyl
phenyl ether diamidines was reported in the 1940s [43]. A library of
mono- and di-amidine derivatives of benzyl phenyl ether was
prepared for other purposes later in this lab [44,45]. The present
work describes the synthesis of a number of novel benzyl phenyl
ether diamidine derivatives, including prodrugs, and the biological
evaluation of these and known analogues against trypanosomes
and two other parasites.
2. Chemistry
Sixty-two cationic benzyl phenyl ether (BPE) derivatives were
prepared in this laboratory (Table 1). The compounds are struc-
turally related to pentamidine (Fig. 1) but have oxymethylene
rather than pentylenedioxy linkers bridging the two aromatic rings.
The set includes 13 monoamidines, 22 simple diamidines, a di-N-
isopropylamidine, as well as 26 N00-hydroxyamidine (amidoxime)
and N00-methoxyamidine (methamidoxime) prodrug derivatives.
The syntheses of monoamidines 1 [46], 2e3 [45], 4 [47], 5
[45,46,48], 6, and 7e13 [45] have been reported previously, as have
those of diamidines 14 [43,44,46], 18 [44], 19 [44], 22 [44,45], 25e
27, 31, 33, 36, 39, 43, 46, and 49 [44], 61 [43e45], and 62 [44,46].
Activity against Trypanosoma evansi has been reported for ana-
logues 52 and 55 although their syntheses have yet to be reported
[49]. All novel final compounds are characterized by melting point,
1H NMR spectra, low resolution EIS mass spectra, HPLC, and
elemental analysis. Intermediates are characterized to the same
extent but without mass spectra. 13C NMR spectra are not included
due to the relative simplicity of structures (which are readily
determined by 1H NMR spectra alone) and the high reliability of the
synthetic procedures (where confirmation of expected structures
was required rather than total elucidation of unexpected struc-
tures). Due to the high polarity of these compounds, reverse phase
HPLC retention times are provided in lieu of normal phase TLC Rf
values.
The preparation of nitrile precursors of novel target com-
pounds is depicted in Scheme 1. All starting materials 63e76 were
commercially available except for 64e68, which were prepared by
halogenation [44,50] of 4-cyanophenol (63). Williamson ether
syntheses involving cyanophenols 63e71 and cyanobenzyl bro-
mides 75 and 76 gave dicyano benzyl phenyl ethers 77e87. Similar
reactions involving 4-hydroxybenzaldehydes 72e74 gave cyano-
aldehyde intermediates 88e91. The latter were treated with hy-
droxylamine to give cyano-oxime intermediates 92e95, which
underwent dehydration to dinitriles 96e99 using acetic
anhydride.
Furamidine (DB75, Fig. 1) [31] is a conformationally restricted
analogue of pentamidine. While positively charged molecules have
poor oral bioavailability, their N-hydroxy and N-methoxy de-
rivatives with lower pKa values have shown potential as orally
active prodrugs [32]. Pafuramidine (DB289), a methamodoxime
prodrug of furamidine, advanced to phase III clinical trials against
early stage HAT and phase II trials against malaria [33,34]. The
compound exhibited nephro- and hepato-toxicity in a recent
Novel amidines 28, 32, 52, 52, 55, and 58 and N-isopropylamidine
15 were prepared by Pinner syntheses from the corresponding ni-
triles (Scheme 2), using methodology similar to that used to prepare
the known amidines [43,44,46e48]. The amidoxime prodrugs 16, 20,
Fig. 1. Structures of 1,5-bis(4-amidinophenoxy)pentane (pentamidine), 2,5-bis(4-amidinophenyl)furan (furamidine, DB75), and 2,5-bis[4-(N0-methoxyamidino)phenyl]furan
(pafuramidine, DB289).

312
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
Table 1
Structures, in vitro antiprotozoal activities and selectivity indices of cationic benzyl phenyl ether derivatives 1e62.a
Compd
R1
R2
R3
R4
T. b. rhodesienseb
P. falciparumd
IC50 M)
L. donovanif
IC50 M)
Cytotoxicityh
IC50 (mM)
c
e
g
IC50
(
m
M)
SIT
(
m
SIP
(
m
SIL
1
2
3
4
5
6
7
8
4-Am
4-Am
4-Am
3-Am
3-Am
H
4-NO2
4-Cl
4-iPr
4-OMe
4-NH2
3-NO2
3-NO2
H
I
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
I
OMe
OMe
OMe
H
H
H
H
H
H
4-NO2
3-NO2
H
3-NO2
4-Am
4-Am
4-Am
4-Am
4-Am
4-Am
4-Am
3-Am
4-Am
4-iPrAm
4-AmOH
4-AmOMe
4-Am
1.84
1.9
2.66
9.32
69
6
3
17
164
45
18
8
4
36
>3.56
0.951
0.296
>3.81
>3.16
>3.56
>3.07
>3.36
2.37
>3.42
2.56
>3.25
>3.07
0.078
0.02
<36
11
26
<41
<25
<47
<26
<6
127
10.7
7.72
156
78.9
167
79.2
20.7
5.36
143
>271
57.8
104
6.96
>144
8.28
>224
3.2
>50.0
34.3
<1
<1
0.481
3.71
4.41
2.71
1.29
3.95
3
2.12
7.28
0.091
1.35
>238
>224
0.022
0.016
24
18.8
0.017
25.8
50.4
0.024
0.017
0.122
0.11
161
9
3
49.5
<1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
PMDj
FMDk
MLSPl
CQm
<42
>106
<18
<34
89
>7369
<1
<39
552
1248
<1
>90
27
14
4-Am
77
8.77
26.6
>100
>100
2.89
<14
>5
<1
2
1
2
<1
<1
3
4-iPrAm
4-AmOH
4-AmOMe
4-Am
>107
<1
9.49
5.78
H
NDi
147
322
<1
Cl
Br
Br
Br
I
0.006
0.004
>11.1
>10.2
0.006
5.29
>10.3
0.014
0.025
0.019
0.177
11.1
>10.6
0.035
0.018
0.051
>11.2
>10.2
0.052
5.8
4-Am
4-Am
2.02
4.99
6.6
101
5.3
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-Am
4-Am
4-Am
4-AmOH
4-AmOMe
4-Am
4-Am
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-Am
4-Am
4-Am
4-AmOH
4-AmOMe
3-Am
3-Am
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
>100
>100
1.94
5
312
<1
<10
898
3
I
I
>100
<1
18.1
86.6
28.5
21.5
13.8
209
56.8
>190
5.93
10.8
13.9
2.26
89.6
15.7
4.39
99.4
6.06
4.73
81
85.4
0.57
14.4
108
4.7
26.8
54.7
19.3
15.8
27.5
21.6
28.9
70.5
18.6
32.1
81.4
96
2
<8
Cl
Br
I
OMe
OMe
OMe
H
NO2
Cl
Cl
Cl
Br
Br
Br
I
1169
1263
1169
1910
<1
2016
849
746
1183
5.11
NDi
168
592
275
<1
<9
302
<1
<10
477
<1
13
2764
3
1
18
39
4
8
6.4
4.09
1.6
>100
34.1
100
4
5
9
<2
1.67
>2
129
>1
0.024
0.034
0.007
49.2
26.1
0.005
14.7
79.1
0.005
50.2
34.7
0.019
0.01
26.4
60.1
0.004
6.89
69.7
0.003
13.6
26.3
0.005
87.9
81.5
0.003
38.7
75.1
0.183
157
250
324
1900
<1
3
3407
<1
1
1188
<1
2
4403
57
<1
2
1270
4
<1
5685
1
1
4073
<1
<1
6210
<1
1
524
<1
>1
694
248
16,643
2000
1222
11.3
2.91
<1
5
<1
<1
8
<1
17
5
<1
<1
3
<1
<1
<2
1
<1
<1
14
2
<1
<2
<1
<1
3
<1
<1
<1
<1
<2
<1
>100
>100
1.86
>100
5.66
H
H
H
H
H
H
H
Cl
Cl
Cl
Br
Br
Br
I
>9.96
0.013
7.31
1.27
I
I
>50.0
>100
28.6
6.3
OMe
Cl
Cl
Cl
Br
Br
Br
I
I
I
Br
Br
Br
I
0.031
0.185
11.5
5.91
0.12
4-Am
3-Am
6.51
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
4-Am
4-AmOH
4-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
3-Am
3-AmOH
3-AmOMe
4-Am
>100
>50.0
3.37
61.1
>50.0
1.39
6.63
44.2
11.4
>100
>100
6.01
>94.5
>100
>100
>100
>100
42.1
7.56
6.79
0.0664
7.38
>7.10
0.0289
>10.4
3.35
0.0245
>9.45
1.12
0.231
>11.5
>10.43
0.195
0.121
0.0464
0.0155
291
2.14
<4
747
<3
21
760
<3
I
I
OMe
OMe
OMe
OMe
OMe
OCH3
OMe
OMe
OMe
H
I
I
73
OMe
OMe
OMe
H
416
<6
70.7
>188
19.3
24
46.6
6.4
109
NDi
99
0.028
0.097
0.003
0.003
0.006
3-Am
H
H
3-Am
198
1004
413
1.84
25
7.78
117
0.201

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
313
Table 1 (continued )
Compd
R1
R2
R3
R4
T. b. rhodesienseb
P. falciparumd
IC50 M)
0.0041
L. donovanif
IC50 M)
Cytotoxicityh
c
e
g
IC50
(m
M)
SIT
(
m
SIP
(
m
SIL
IC50 (mM)
ATMSn
PPTo
>319
0.019
a
All compounds were isolated as hydrochloride salts.
Trypanosoma brucei rhodesiense (STIB900) [75]. Average of duplicate determinations.
Selectivity index for T. b. rhodesiense expressed as the ratio IC50 (L6)/IC50 (T. b. rhodesiense).
Plasmodium falciparum (K1, chloroquine resistant) [75]. Average of duplicate determinations.
Selectivity index for P. falciparum expressed as the ratio IC50 (L6)/IC50 (P. falciparum).
Leishmania donovani (MHOM/SD/62/IS-CL2D) axenic amastigotes [76,77]. Average of at least two determinations.
Selectivity index for L. donovani expressed as the ratio IC50 (L6)/IC50 (L. donovani).
Cytotoxicity to L6 rat myoblast cells [75]. Average of duplicate determinations.
Value not determinable.
Pentamidine.
b
c
d
e
f
g
h
i
j
k
l
Furamidine.
Melarsoprol.
Chloroquine.
Artemisinin.
m
n
o
Podophyllotoxin.
23, 29, 34, 37, 40, 44, 47, 50, 53, 56, and 59 were prepared from the
corresponding nitriles using hydroxylamine hydrochloride and po-
tassium tert-butoxide in DMSO (Scheme 3) [51] These compounds
underwent O-methylation using dimethyl sulphate [52] or
iodomethane with an appropriate base and solvent to give meth-
amidoximes 17, 21, 24, 30, 35, 38, 41, 45, 48, 51, 54, 57, and 60. All
target compounds were characterized and submitted for biological
assays as the hydrochloride salts.
Scheme 1. Synthesis of dicyano benzyl phenyl ether derivatives. Reagents and conditions: (a) NaI, aq. NaOCl, MeOH; (b) KClO3, aq. HCl; (c); KClO3, aq. HBr; (d) K2CO3, DMF; (e)
NH2OH$HCl, aq. EtOH, then aq. NaOH; (f) Ac2O.

314
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
Scheme 2. Synthesis of benzyl phenyl ether diamidines. Reagents and conditions: (a) EtOH, HCl gas, 1,4-dioxane; (b) NH3 or isopropylamine, EtOH.
3. Biological results and discussions
antiprotozoal IC50 values) [53] are shown for each of the three
parasites. Antiprotozoal and cytotoxicity IC50 values are compared
to those of pentamidine and furamidine. Other standard drugs
include melarsoprol (against T. b. rhodesiense), chloroquine and
artemisinin (against P. falciparum), and podophyllotoxin (against L6
cells).
3.1. In vitro potencies, selectivities, and structureeactivity
relationships
In vitro potencies of the target compounds against T. b. rhode-
siense strain STIB900, P. falciparum strain K1 (chloroquine resis-
tant), and Leishmania donovani MHOM/SD/62/IS-CL2D axenic
amastigotes are shown in Table 1, along with their toxicities to rat
L6 myoblast cells. Selectivity indices (SIs, the ratios of cytotoxic to
3.1.1. Antitrypanosomal activities
Eighteen of the 22 simple diamidines exhibited IC50 values at or
below 35 nM against the trypanosome, and eight of these
Scheme 3. Synthesis of prodrug derivatives of benzyl phenyl ether diamidines. Reagents and conditions: (a) NH2OH$HCl, t-BuOK, DMSO, (b) HCl, appropriate solvent; (c) Me2SO4,
2 M NaOH, 1,4-dioxane or DMF; (d) MeI, t-BuOK, DMSO; (e) MeI, NaH, DMF.

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
315
compounds had IC50 values at or below 10 nM. Analogues 55
(IC50 ¼ 3 nM, SI ¼ 6200) and 49 (IC50 ¼ 3 nM, SI ¼ 5700) were the
most potent and most selective. Six other derivatives (33, 36, 39, 43,
46, 49, and 52) had IC50 values below 10 nM, and all but analogue
43 (IC50 ¼ 10 nM, SI ¼ 57) had selectivity indices above between
1150 and 4100. Ten congeners (18, 19, 22, 25e27, 31, 32, 42, and 61)
had IC50 values between 10 and 35 nM. Four of these compounds
had selectivity indices above 1000; analogue 42 (IC50 ¼ 19 nm,
SI ¼ 4400) was the most selective, while derivatives 25e27 had
selectivity indices around 1200. Diamidines 14 and 62 had IC50
values between 90 and 100 nM, while congeners 28 and 58 were
less potent with IC50 values between 100 and 200 nM. Analogue 28
was the only one for these four molecules having a selectivity index
above 1000. Potencies and selectivities for the trypanosome
correlated well. Compounds 55 and 49, the two most potent mol-
ecules, were also the two most selective. A total of 11 diamidines
(25e27, 33, 36, 39, 42, 46, 49, 52, and 55) had IC50 values below
25 nM as well as SIs above 1000. The monoamidines 1e13 were all
less potent than the diamidines, consistent with previous results
10 diamidines had IC50 values below 250 nM. The most active were
the bromo derivative 19 (IC50 ¼ 4 nM, SI ¼ 1250), followed by
chloro and iodo analogues 18 and 22, each having IC50 value of 6 nM
and lower selectivity indices. Derivatives 15, 25, 27, 32, and 39
exhibited IC50 values between 10 and 20 nM and selectivity indices
ranging from 480 (for 39) to greater than 7400 (for 15). Analogues
26, 31, 42, 52, and 55 had IC50 values between 20 and 35 nM and
selectivity indices between 170 (for 31) and 2760 (for 42). Another
10 compounds (14, 28, 33, 36, 43, 46, 49, 58, 61, and 62) had IC50
values between 50 and 250 nM. The selectivity indices for these
compounds ranged between 3 (for 43) and 1180 (for 28). The
monocations 1e13 were less potent than the dications, with only
congeners 2 and 3 having IC50 values above 1 mM.
Derivative 15 was the most selective for the malaria parasite,
with a selectivity index above 7400. Compounds 25 and 42 had
selectivity indices above 2000, while congeners 19 (the most
potent) and 28 had selectivity indices above 1000. Seven com-
pounds (18, 22, 26, 27, 32, 52, and 55) had selectivity indices be-
tween 500 and 1000. Eleven compounds (15, 18, 19, 22, 25e27, 32,
42, 52, and 55) had IC50 values below 50 nM and selectivity indices
above 500.
[25]. Only one monoamidine (5) had an IC50 value below 1 mM.
Potencies of the diamidines were influenced by the positions of
the amidine groups, as exemplified by regioisomers 14, 31, 61, and
62 which lack additional substituents on the aromatic rings. The
30,4-diamidine 31 (where 30 denotes a 3-amidinobenzyl group) was
the most potent (IC50 ¼ 24 nM). The 3,40-diamidine 61, was similar
in potency (IC50 ¼ 28 nM) but was the most selective of the four
isomers (SI z700). The 4,40- and 3,30-diamdines 14 and 62 were
less potent, with IC50 values of 91 and 97 nM, respectively.
Six of the eight derivatives of 14 bearing aromatic of N-alkyl
substituents showed enhanced potency and selectivity relative to
the parent molecule. The six halogenated derivatives (18, 19, 22,
and 25e27) were all more potent than diamidine 14, with IC50
values between 12 nM for diiodo analogue 25 and 24 nM for
dichloro derivative 27. The dihalogenated analogues 25e27 all had
selectivity indices around 1200, while their monohalogenated
counterparts 18, 19, and 22 had lower selectivity indices (between
145 and 325). The dimethoxy analogue 28 (IC50 ¼ 110 nM) was
slightly less potent than the parent molecule, but has the highest
selectivity index (around 1900) of any derivative of diamidine 14.
The di-N-isopropylamidine 15 is nearly 15 times less potent than
the parent molecule, consistent with previous findings [24,25,38e
42].
Eight of the 11 derivatives of diamidine 31 bearing aromatic
substituents exhibited both enhanced potency and selectivity
relative to the parent molecule. These 11 molecules include the
eight most potent analogues overall (33, 36, 39, 43, 46, 49, 52, and
55). The iodo-methoxy iodo (55) and diiodo (49) derivatives, each
with an IC50 value of 3 nM and selectivity index around 6000 were
the two most potent and most selective compounds. The chloro
(33), bromo (36), iodo (39), dibromo (46), and bromo-methoxy (52)
analogues have IC50 values between 4 and 7 nM and selectivity
indices between 1100 and 4100. Dichloro derivative 43
(IC50 ¼ 10 nM, SI ¼ 57) was the one of the eight most potent
compounds with a selectivity index below 1000. Methoxy analogue
42 (IC50 ¼ 19 nM, SI ¼ 4400) was slightly more potent than 31 and
showed the third highest selectivity for the parasite. Only the nitro
(32) and dimethoxy (58) derivatives are less potent than parent
molecule 31. Thus the potencies of both 14 and 31 were enhanced
by halogen substituents and diminished by the presence of two
methoxy groups.
Two cationic groups were also required for antiplasmodial ac-
tivity. Diamidine 31 was the most potent of the four regioisomeric
diamidines lacking additional aromatic substituents, followed by
14, 62, and 61 (IC50 ¼ 35, 85, 120, and 195 nM, respectively). Isomer
62 showed the highest selectivity of the four molecules (SI ¼ 200).
Seven of the eight derivatives of 14 bearing substituents on the
amidine nitrogen atoms or the aromatic rings were more potent
and more selective for the parasite than the parent molecule. The
di-N-isopropylamidine derivative 15 (IC50 ¼ 20 nM, SI > 7400) was
four times more potent than 14 and showed the highest selectivity
overall for this parasite. Other examples of enhanced potencies
resulting from alkylation of the amidine nitrogen atoms have also
been reported [24,25,38e42]. The introduction of aromatic sub-
stituents ortho to the oxygen atom also enhanced the efficacy of 14.
The bromo (18, IC50 ¼ 4 nM, SI ¼ 1250), chloro (19, IC50 ¼ 6 nM,
SI ¼ 550), and iodo (22, IC50 ¼ 6 nM, SI ¼ 900) derivatives were the
three most potent, both in this series and overall. The dichloro (25,
IC50 ¼ 14 nM, SI ¼ 2020), diiodo (27, IC50 ¼ 19 nM, SI ¼ 750), and
dibromo 26 (25, IC50 ¼ 25 nM, SI ¼ 850) analogues were also more
potent and more selective than the parent. Only the dimethoxy
congener 28 (IC50 ¼ 180 nM, SI ¼ 1180) was less potent than parent
molecule 14.
The introduction of aromatic substituents had a more varied
effect upon the efficacy of isomer 31. Five derivatives were more
potent and more selective than the parent molecule. The iodo de-
rivative 39 (IC50 ¼ 13 nM, SI ¼ 480), was the most potent analogue
of 31 and third most potent overall. The nitro (32, IC50 ¼ 18 nM,
SI ¼ 590), iodo-methoxy (55, IC50 ¼ 25 nM, SI ¼ 760), bromo-
methoxy (52, IC50 ¼ 29 nM, SI ¼ 750), and methoxy (42,
IC50 ¼ 31 nM, SI ¼ 2760) congeners were more potent and more
selective than 31. Six analogues, the chloro (33), bromo (36), diiodo
(49), dibromo (46), dichloro (43), and dimethoxy (58) derivatives
were less potent than parent molecule 31, with IC50 values ranging
from 51 nM (for 33) to 231 nM (for 58). Compounds 33, 36, and 49
had IC50 values between 50 and 70 nM and selectivity indices
around 300, while analogue 58 had a selectivity index above 400.
Diamidine 31 was the most potent of the four regioisomers
against both P. falciparum and the trypanosome, although the other
three isomers 14, 61, and 62 showed different orders of potency
against the two parasites. The only di-N-alkylamidine derivative
(15) in this study showed diminished antitrypanosomal and
enhanced antiplasmodial potencies relative to the corresponding
diamidine. All six mono- and di-halogenated derivatives (18, 19, 22,
25e27) showed enhanced potencies, while the dimethoxy
3.1.2. Antiplasmodial activities
Thirteen of the 23 diamidines (including the di-N-iso-
propylamidine 15) were more potent against P. falciparum than
pentamidine, exhibiting IC50 values below 50 nM, while the other

316
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
Table 2
derivative 28 showed diminished potencies, against both parasites
relative to the parent molecule 14. The iodo (39), methoxy (42),
bromo-methoxy (52) and iodo-methoxy (55) derivatives showed
enhanced potencies, while dimethoxy analogue 58 showed
diminished potencies, against both parasites relative to the parent
molecule 31. The nitro (32) derivative of diamidine 31 showed
diminished antitrypanosomal but enhanced antiplasmodial po-
tencies relative to the parent molecule. These effects were reversed
for the chloro (33), bromo (36), dibromo (46) and diiodo (49)
derivatives.
In vivo efficacies of select benzyl phenyl ethers in T. b. rhodesiense infected mice.a
Compd
Doseb (mg/kg)
Routec
Cured/Infectedd
MSDe (days)
14
16
4 ꢀ 5
i.p.
0/4
2/4
1/4
0/4
3/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
1/4
0/4
4/4
1/4
0/4
0/4
0/4
0/4
3/4
0/4
0/4
4/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
4/4
1/4
1/4
22.5
4 ꢀ 100
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
p.o.
p.o.
p.o.
i.p.
>46.25
>31.7
13.25
>51.0
29
28.75
>32.5
>41.5
29.75
29.75
30.25
30
24.5
15
14.5
11.75
25.25
23.25
24.5
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
4 ꢀ 5
i.p.
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
p.o.
p.o.
i.p.
p.o.
p.o.
i.p.
i.p.
i.p.
i.p.
p.o.
p.o.
i.p.
i.p.
i.p.
p.o.
p.o.
i.p.
p.o.
p.o.
i.p.
p.o.
p.o.
i.p.
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
3.1.3. Antileishmanial activities
Fewer BPE derivatives were active against L. donovani compared
to the other parasites. Diamidines 22, 27, 36, 39, and 49 exhibited
4 ꢀ 5
4 ꢀ 5
IC50 values below 2
m
M (compared to pentamidine, IC50 ¼ 1.8
mM,
4 ꢀ 5
SI ¼ 25), while congeners 8, 19, 26, 33, and 46 had IC50 values below
4 ꢀ 25
4 ꢀ 25
4 ꢀ 20
4 ꢀ 5
5
m
M. Analogue 49 (IC50 ¼ 1.4 M, SI ¼ 14), the second most potent
m
and one of three derivatives more potent than pentamidine, was
the most promising as it was the only compound having a selec-
tivity index above 10.
4 ꢀ 5
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
>39
The structural effects upon antileishmanial activities were more
similar to those upon antitrypanosomal activities than anti-
plasmodial activities. The 10 compounds most potent (in
decreasing order) against L. donovani (39, 49, 27, 36, 22, 19, 18, 33,
46, and 26) were all halogenated derivatives of 14 or 31. Seven of
these derivatives (all but 18, 22, and 26) were also seven of the 10
analogues most efficacious against T. b. rhodesiense.
29.25
>37.5
12.75
>60.0
>31.25
20
22.25
22
27.5
>53.25
21.75
>35.25
>60.0
20.75
>31.5
21.25
5.75
19.75
>45.75
31.75
>52.25
35.75
20.5
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
4 ꢀ 5
i.p.
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
p.o.
p.o.
i.p.
p.o.
p.o.
p.o.
i.p.
p.o.
p.o.
i.p.
p.o.
p.o.
i.p.
p.o.
p.o
i.p.
p.o.
p.o.
i.p.
3.2. In vivo antitrypanosomal efficacies
Twenty-two diamidines and 26 prodrugs were tested in mice
infected with T. b. rhodesiense strain STIB900 (Table 2). Unless
stated otherwise, the test compounds were administered in daily
doses for four days at 5 mg/kg intraperitoneally (i.p.) for diamidines
and 25 mg/kg orally (p.o.) for prodrugs. This model is highly
stringent for the acute stage of HAT, and the low doses applied
allowed the distinction of activities among the highly trypanocidal
compounds. Pentamidine and furamidine attained 1/4 cures at
4 ꢀ 5 mg/kg i.p, and any compound showing a higher cure rate is
considered to have enhanced activity in vivo. Mice that survived for
60 days after infection without showing a parasitaemia relapse
were considered as cured. Untreated control animals died within 10
days post infection due to a high parasitaemia load.
Eleven of the test compounds, seven prodrugs and four amidines,
demonstrated in vivo efficacy at least comparable to pentamidine or
furamidine, and four of these compounds showed enhanced effi-
cacy. Three prodrugs exhibited excellent in vivo activity. The
methamidoxime prodrug 38 was the most effective overall in the
in vivo studies, curing 4/4 infected mice and showing oral efficacy
comparable to that of pafuramidine [54]. The amidoxime prodrug
44 and methamidoxime 54 cured 3/4 and 2/4 infected mice,
respectively, each with a mean survival rate exceeding 52 days. Four
other prodrugs, methamidoximes 21 and 57 and amidoximes 16 and
34, each cured 1/4 infected mice, with mean survivals between
greater than 32 days (for 21) and greater than 46 days (for 16,
compared to 10 days or less for untreated animals). While ami-
doxime 47 cured 4/4 mice at 4 ꢀ 100 mg/kg p.o., it attained no cures
at the lower 25 mg/kg dose. Similarly, amidoxime 16 attained 2/4
cures at the higher dose and 1/4 cures at the lower dose.
4 ꢀ 100
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
40.25
16.25
16.5
15
19
19.8
>60
>46
>38
4 ꢀ 25
4 ꢀ 25
4 ꢀ 5
4 ꢀ 20
4 ꢀ 2
i.p.
i.p.
i.p.
i.p.
MLSPf
FMDg
PMDh
4 ꢀ 5
4 ꢀ 5
a
Female NMRI mice infected with Trypanosoma brucei rhodesiense (STIB900) [54].
Single daily doses for four days.
Route of administration: intraperitoneal (i.p.) or oral (p.o.).
Mice that survived for 60 days after infection without showing a parasitaemia
b
c
d
relapse.
e
Mean survival days post infection of mice.
f
g
h
Melarsprol.
Furamidine.
Pentamidine.
Overall, ten instances were observed in which a diamidine and/
or a prodrug derivative thereof demonstrated in vivo efficacy. In
only one instance (amidine 36 and methamidoxime 38), were both
a diamidine and a corresponding prodrug curative. In this partic-
ular case, the prodrug was more efficacious at the 4 ꢀ 25 mg/kg oral
regimen than the corresponding amidine at the 4 ꢀ 5 mg/kg
intraperitoneal doses. In no instances did both prodrugs of the
same diamidine demonstrate efficacy in vivo.
Four diamidines gave cure rates greater than or comparable to
pentamidine or furamidine at the same regimens. Diamidine 18
showed enhanced efficacy, curing 3/4 mice with a greater than 51
day average survival rate, while diamidines 22, 36, and 39 each
attained 1/4 cures with mean survival rates ranging between
greater than 31 and greater than 41 days.

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
317
Correlations between the in vitro and in vivo antitrypanosomal
activities may be observed. In nine of 10 instances in which an
amidine and/or one of its prodrugs demonstrated efficacy in vivo,
the amidine species exhibited an IC50 value less than 25 nM in vitro.
The exception was amidine 14 (IC50 ¼ 91 nM), the active species of
amidoxime 16. In regard to the eight compounds most potent
in vitro (IC50 values at or below 10 nM), seven of these and/or a
prodrug thereof demonstrated in vivo efficacy comparable to or
greater than that of pentamidine or furamidine. None of the three
most potent compounds in vitro (55, 49, and 46) demonstrated
efficacy in vivo at 4 ꢀ 5 mg/kg i.p., but methamidoxime 57 (a
prodrug of 55) cured 1/4 infected mice. Amidoxime 47, a prodrug of
46, cured 4/4 mice at the higher 4 ꢀ 100 mg/kg p.o. regimen, but
attained no cures at the lower 25 mg/kg p.o. doses at which the
other compounds were tested. Amidines 36 and 39 (IC50 ¼ 5 nM)
each cured 1/4 mice while 38, the methamidoxime prodrug of 36,
effected 4/4 cures. Amidines 52, 33, and 43 (IC50 values between 5
and 10 nM) attained no cures, but their prodrugs methamidoxime
54 and amidoximes 34 and 44 cured 2/4 mice, 1/4 mice, and 3/4
mice, respectively. Amidoxime 21, a prodrug of 19 (IC50 ¼ 16 nM)
and amidine 22 (IC50 ¼ 17 nM) each effected 1/4 cures. Compound
18 (IC50 ¼ 22 nM) was the most efficacious of the amidines in vitro,
curing 3/4 mice.
pentamidine [70,72], while nephrotoxicity resulted in the cancel-
lation of human clinical trials with pafuramidine [54]. An obvious
approach to decreasing host toxicity is to produce more potent
compounds against the targeted organisms. However, even the
most potent diamidine compounds must be subjected to a battery
of toxicity tests. The very potent activity of these molecules against
pathogenic protozoan organisms warrants continued investigation
of this class of compounds.
4. Conclusions
The dicationic BPE derivatives have shown promising activities
against T.
b rhodesiense (in vitro and in vivo) and against
P. falciparum in vitro. Regarding antitrypanosomal activity, the most
noteworthy was the methamidoxime prodrug 38, which attained 4/
4 cures in a highly stringent murine modeldcomparable results to
those of the former clinical trial candidate pafuramidine in the
same assay [54]. This compound is certainly worthy of further
evaluation for efficacy and toxicity in other animal models. Several
compounds (for example, analogues 15, 19, and 25) showed both
high potencies against P. falciparum and high selectivities for
parasite, and thus warrant evaluation for in vivo efficacy and
toxicity.
Seven of the 11 compounds demonstrating activity against T. b.
rhodesiense in vivo are derivatives of diamidine 31 (having one
cationic group meta to the methylene group and the other para to
the oxygen atom), while the other four are derivatives of 14 (having
both groups in a para orientation). Four of the active compounds
are amidines, three are amidoximes, and four are methamidoximes.
Ten of the 11 compounds bear at least one halogen substituent. This
group includes seven monohalogenated analogues (two chloro,
three bromo and two iodo derivatives), one dichloro derivative, and
two mixed substituent analogues (one bromo-methoxy and one
iodo-methoxy derivative.) Regarding the four compounds having
enhanced efficacy in vivo, the three prodrugs (bromo dimethami-
doxime 38, dichloro diamidoxime 44, and bromo-methoxy dime-
thamidoxime 54) are derivatives of diamidine 31, while chloro
diamidine 18 is a derivative of 14.
5. Experimental
5.1. Biological protocols
In vitro antiprotozoal activities and cytotoxicities against T. b.
rhodesiense (STIB900) [75], chloroquine resistant P. falciparum (K1)
[75], L. donovani axenic amastigotes [76,77], and L6 cells (rat
myoblasts) [75] were measured following established protocols.
In vivo efficacy has been determined in the acute mouse model
with T. b. rhodesiense strain STIB900 infection as previously re-
ported [54]. All protocols and procedures of the mouse models
were reviewed and approved by the local veterinary authorities of
Canton Basel Stadt, Switzerland.
5.2. Chemistry
3.3. Discussion
Uncorrected melting points were measured on a Thomase
Hoover Capillary melting point apparatus. 1H NMR spectra were
recorded on a Varian Gemini 2000 (300 MHz). Unless stated
otherwise, spectra were recorded in DMSO-d6 (with 0.05% TMS) at
300 MHz. 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 neutralized as necessary with
acidic or basic washes, washed with saturated NaCl solution and
dried over Na2SO4 or MgSO4 before being evaporated under
reduced pressure. Normal phase gravity and flash column chro-
matography were performed using Davisil grade 633, type 60A
silica gel (200e425 mesh). Reverse phase flash chromatography
was performed as previously described [41]. Analytical HPLC
chromatograms were recorded on a HewlettePackard 1090 Series II
or Agilent 1200 chromatograph using a Zorbax Rx C8 column
A large number of mechanisms of antimicrobial activity have
been proposed for the diamidine class of molecules [55e67]. These
positively charged molecules bind to many negatively charged
surfaces and receptors, and accordingly have numerous potential
sites of action. The antimicrobial action of these compounds
probably occurs at multiple sites, and the primary site of action may
vary from organism to organism. This is a huge problem with re-
gard to drug design, but is advantageous with regard to the po-
tential development of drug resistance. It has been suggested that
the highly selective toxicity of many of the compounds in this class
towards pathogenic organisms over mammalian cells is due to
active transport mechanisms. For example, trypanosomes have a
number of active transport systems that allow cationic molecules to
be concentrated in the parasites at levels over 1000 times higher
than in mammalian cells [63]. Thus, the only way to optimize these
compounds for specific targets is via focused parallel synthesis and
in vitro testing or focussing on the active transport systems. Un-
fortunately, not enough is known with regard to the specificities of
the transporters to aid in the drug design process.
(4.6 ꢀ 75 mm, 3.5
mm) and UV photodiode array detection at 230,
254, 265, 290, and 320 nm. Wavelengths reported are those at
which the strongest signals of the major products were observed.
Mobile phases consisted of mixtures of CH3CN (0e75%) 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. In Method A, the concentration of
Because of the propensity of dications to bind to numerous sites,
the toxicity of these molecules is always a concern with regard to
drug design [54,68e74]. The nature of the toxicity does not appear
class related, but may vary from compound to compound. For
instance, hypoglycemia is the major concern with regard to

318
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
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. In Method B,
the concentration of CH3CN was increased linearly from 22.5 to 75%
over 10 min, then maintained for 2 min. Method C was similar but
employed an Agilent 1100 instrument, a Zorbax SB C8 column
J ¼ 7.7 and 2.2 Hz, 1H), 7.97 (s, 1H), 7.84 (d, J ¼ 7.7 Hz, 2H), 7.71 (d,
J ¼ 9.3 Hz, 1H), 7.69 (t, J ¼ 7.7 Hz, 1H), 5.55 (s, 2H); MS m/z 314.2
([M þ 1]þ of free base); HPLC (Method A) tR 3.27 min (100 area % at
254 nm). Anal. (C15H15N5O3$2HCl$0.9H2O) C, H, N, Cl.
(3.0 ꢀ 100 mm, 3.5
m
m), a flow rate of 0.6 mL/min, and a concen-
5.2.2.3. 3-Amidinobenzyl 4-amidino-2-methoxyphenyl ether dihydro-
tration of CH3CN increasing from 40 to 75% over 10 min. Preparative
reverse phase HPLC was performed on a Varian ProStar Chroma-
tography Workstation configured with two PS-215 pumps fitted
chloride (42). Yield, 1.36 g (63%): mp > 216 ꢁC dec; 1H NMR
d 9.60e
9.14 (m, 8H), 7.96 (s,1H), 7.82 (m, 2H), 7.66 (t, J ¼ 7.7 Hz, 1H), 7.54 (m,
2H), 7.30 (d, J ¼ 8.8 Hz, 1H), 5.31 (s, 2H); MS m/z 299.3 ([M þ 1]þ of
free base); HPLC (Method A) tR 3.30 min (100 area % at 230 nm). Anal.
(C16H18N4O2$2HCl$0.8H2O) C, H, N, Cl.
ꢀ
with 50 mL pump heads, a Dynamax Microsorb C18 (60 A) column
(41.4 ꢀ 25 cm, 8
mm), PS-320 variable wavelength UVeVis detector,
and a PS-701 fraction collector. Mobile phases consisted of mixtures
of CH3CN (0e75%) 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
analysed for purity by analytical HPLC as described above. Pooled
purified fractions were evaporated under reduced pressure,
reconstituted in water, and lyophilized on a VirTis BenchTop 2K or
6K lyophilizer. Low resolution ESI mass spectra were recorded on
an Agilent Technologies 1100 Series LC/MSD Trap mass spectrom-
eter. Elemental analyses were performed by Atlantic Microlab,
Norcross, GA, and, unless stated otherwise, were within ꢂ0.4% of
calculated values.
5.2.2.4. 3-Amidinobenzyl 4-amidino-2-bromo-6-methoxyphenyl ether
dihydrochloride (52). Yield 1.32 g (71%): mp > 218 ꢁC dec; 1H NMR
d
9.50e9.35 (m, 8H), 7.98 (m, 1H), 7.90 (dm, J ¼ 7.7 Hz,1H), 7.84 (dm,
J ¼ 8.2 Hz, 1H), 7.48 (dm, J ¼ 1.6 Hz, 1H), 7.67 (m, 2H), 5.20 (s, 2H),
4.00 (s, 3H); MS m/z 377.2 ([M þ 1]þ of free base), 378.9 ([M þ 3]þ);
HPLC (Method A) tR 4.31 min (100 area % at 254 nm). Anal.
(C16H17BrN4O2$2HCl$H2O) C, H, N, Br, Cl.
5.2.2.5. 3-Amidinobenzyl 4-amidino-2-iodo-6-methoxyphenyl ether
dihydrochloride (55). Yield, 1.40 g (77%): mp 24e250 ꢁC dec; 1H
NMR d 9.53 (br s, 2H), 9.51 (br s, 2H), 9.33 (br s, 2H), 9.29 (br s, 2H),
8.00 (m, 1H), 7.93 (m, 2H), 7.84 (dm, J ¼ 8.2 Hz, 1H), 7.69 (m, 2H),
5.17 (s, 2H), 3.98 (s, 3H); MS m/z 425.2 ([M þ 1]þ of free base);
HPLC (Method A) tR 4.60 min (100 area % at 254 nm). Anal.
(C16H17IN4O2$2HCl) C, H, N, Cl, I.
5.2.1. 4-N-Isopropylamidinobenzyl 4-N-isopropylamidinophenyl
ether dihydrochloride (15)
Dinitrile 77 (1.41 g, 6.02 mmol) was added to a mixture of
anhydrous EtOH (5 mL) and 1,4-dioxane (45 mL) 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 iceesalt bath. The reaction mixture was then
sealed, slowly warmed to ambient temperature, and stirred until
the nitrile was no longer detectable (5 days). The reaction mixture
was diluted with ether, and crude imidate ester (2.48 g, 96%) was
filtered off, dried, and reacted immediately with isopropylamine
(5.0 mL, 58 mmol) in anhydrous ethanol (40 mL). The mixture was
stirred overnight and diluted with ether. The crude product was
filtered off and recrystallized from aqueous HCl to give a white solid
5.2.2.6. 3-Amidinobenzyl 4-amidino-2,6-dimethoxyphenyl ether dihy-
drochloride (58). The crude product precipitated upon concentra-
tion of the reaction mixture. Yield (after two recrystallizations),
0.771 g (38%): mp > 160 ꢁC dec; 1H NMR
d 9.50 (br s, 2H), 9.47 (br s,
2H), 9.29 (br s, 2H), 9.22 (br 2, 2H), 7.94 (s, 1H), 7.82 (m, 2H), 7.64 (t,
J ¼ 7.7 Hz, 1H), 7.31 (s, 2H), 5.11 (s, 2H), 3.91 (s, 3H); MS m/z 329.1
([M þ 1]þ of free base); HPLC (Method A) tR 3.74 min (100 area % at
265 nm). Anal. (C17H20N4O3$2HCl$1.2H2O) C, H, N, Cl.
5.2.3. General procedure for amidoxime prodrugs 16, 20, 23, 29, 34,
37, 40, 44, 47, 50, 53, 56, and 59
(1.65 g, 64%): mp 312e314 ꢁC (dec); 1H NMR
d
9.67 (d, J ¼ 8.2 Hz,
Potassium tert-butoxide (6.5e10 equiv) was added to a stirred
solution of hydroxylamine hydrochloride (7e10 equiv) in DMSO.
After 30 min the nitrile (1 equiv) was added, and the mixture was
stirred until starting material was no longer detectable (1e3 days).
The reaction mixture was poured over ice-water, and the precipi-
tated amidoxime base was filtered off. An aliquot of the free base
was converted to the hydrochloride salt as stated below.
1H), 9.55 (s, 1H), 9.48 (d, J ¼ 8.1 Hz, 1H), 9.39 (s, 1H), 9.23 (s, 1H),
9.04 (s, 1H), 7.71 (t, J ¼ 9.3 Hz, 4H), 7.67 (d, J ¼ 8.2 Hz, 2H), 7.23
(d, J ¼ 8.9 Hz, 2H), 5.37 (s, 2H), 4.09 (m, 2H), 1.27 (d, J ¼ 6.4 Hz), 1.26
(d, J ¼ 6.4 Hz); MS m/z 353.2 ([M þ 1]þ of free base); HPLC
(Method A) tR 5.25 min (98.1 area
(C21H28N4O$2HCl$0.75H2O) C, H, N, Cl.
% at 254 nm). Anal.
5.2.2. General procedure for diamidines 28, 32, 42, 52, 55, and 58
Procedures were similar to that above, except that the imidate
esters were treated with ethanolic ammonia. Crude products were
precipitated from the reaction mixtures, either directly or after
dilution with ether. All crude products were recrystallized from 1 M
HCl/ethanol.
5.2.3.1. 4-N0-Hydroxyamidinobenzyl 4-N0-hydroxyamidinophenyl ether
dihydrochloride (16). Yield (free base), 2.90 g (96%): mp 203e206 ꢁC;
1H NMR
d
9.65 (s, 1H), 9.48 (s, 1H), 7.68 (d, J ¼ 8.4 Hz, 2H), 7.60 (d,
J ¼ 8.9 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.00 (d, J ¼ 8.9 Hz, 2H), 5.83 (br
s, 2H), 5.76 (br s, 2H), 5.15 (s, 2H). Anal. (C15H16N4O3) C, H, N.
An aliquot of the free base (0.756 g, 2.52 mmol) was recrystal-
lized from ethanolic HCl/ether to give the hydrochloride salt as a
5.2.2.1. 4-Amidinobenzyl 4-amidino-2,6-dimethoxyphenyl ether dihy-
white solid (0.912 g, 97%): mp 204e205 ꢁC; 1H NMR
d 11.31 (br s,
drochloride (28). Yield (after two recrystallizations), 0.744 g (49%):
1H), 11.20 (br s, 1H), 9.06 (br s, 4H), 7.76 (m, 4H), 7.67 (d, J ¼ 8.4 Hz,
2H), 7.23 (d, J ¼ 8.8 Hz, 2H), 5.37 (s, 2H); MS m/z 301.1 ([M þ 1]þ of
free base); HPLC (Method A) tR 4.66 min (98.9 area % at 254 nm).
Anal. (C15H16N4O3$2HCl$0.8H2O) C, H, N, Cl.
mp 293e294 ꢁC dec; 1H NMR
d 9.29 (br s, 2H), 9.44 (br s, 2H), 9.25 (br
s, 2H), 9.21 (br s, 2H), 7.87 (d, J ¼ 7.7 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz, 2H),
7.31 (s, 2H), 5.14 (s, 2H), 3.90 (s, 6H); MS m/z 329.1 ([M þ 1]þ of free
base); HPLC (Method A) tR 3.68 min (100 area % at 254 nm). Anal.
(C17H20N4O3$2HCl) C, H, N, Cl.
5.2.3.2. 4-N0-Hydroxyamidinobenzyl 2-bromo-4-N0-hydroxyamidino-
phenyl ether dihydrochloride (20). Yield (free base), 3.58 g (94%). An
aliquot (1.58 g, 4.17 mmol) was recrystallized from 1 M HCl/ethanol
to give the hydrochloride salt as a white solid (1.80 g, 95%): mp 237e
5.2.2.2. 3-Amidinobenzyl 4-amidino-2-nitrophenyl ether dihydrochl-
oride (32). Yield, 0.831 g (62%): mp 293 ꢁC; 1H NMR
d
9.60 (br s,
2H), 9.54 (br s, 2H), 9.35 (br s, 4H), 8.51 (d, J ¼ 2.2 Hz, 1H), 8.20 (dd,
239 ꢁC dec; 1H NMR
d 11.25 (br s, 2H), 9.06 (br s, 4H), 8.07 (d,

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
319
J ¼ 2.1 Hz,1H), 7.80 (d, J ¼ 8.8 Hz,1H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.69 (d,
J ¼ 8.4 Hz, 2H), 7.44 (d, J ¼ 8.8 Hz, 1H), 5.84 (br s, 2H); MS m/z 377.2
([M þ 1]þ of free base), 378.9 ([M þ 3]þ); HPLC (Method A) tR
3.23 min (100 area % at 254 nm). Anal. (C15H15BrN4O3$2HCl) C, H, N,
Cl, Br.
5.2.3.8. 3-N0-Hydroxyamidinobenzyl 2,6-dichloro-4-N0-hydroxyamidin-
ophenyl etherdihydrochloride(44). Yield (free base, recrystallized from
ethanol/water), 2.65 g (90%): mp 198e199 ꢁC; 1H NMR
d 9.88 (s, 1H),
9.66 (s, 1H), 7.86 (m, 1H), 7.79 (s, 2H), 7.68 (dm, J ¼ 7.7 Hz, 1H), 7.54
(dm, J ¼ 7.1 Hz, 1H), 7.43 (t, J ¼ 7.6 Hz, 1H), 5.98 (br s, 2H), 5.83 (br s,
2H), 5.05 (s, 2H). Anal. (C15H14Cl2N4O3) C, H, N.
5.2.3.3. 4-N0-Hydroxyamidinobenzyl 4-N0-hydroxyamidino-2-
An aliquot of the free base (0.502 g, 1.37 mmol) was recrystal-
lized from ethanolic HCl/ether to give the hydrochloride salt as a
iodophenyl ether dihydrochloride (23). Yield (free base), 4.42 g
(104%, crude): mp 205e206 ꢁC; 1H NMR
d
9.66 (s, 1H), 9.58 (s, 1H),
white solid (0.538 g, 89%): mp 184e185 ꢁC; 1H NMR
d 11.32 (br s,
8.08 (d, J ¼ 2.1 Hz, 1H), 7.70 (d, J ¼ 8.3 Hz, 2H), 7.65 (dd, J ¼ 8.6 and
2.1 Hz, 1H), 7.49 (d, J ¼ 8.3 Hz, 2H), 7.08 (d, J ¼ 8.7 Hz, 1H), 5.84 (br s,
2H), 5.82 (br s, 2H), 5.25 (s, 2H). Anal. (C15H16N4O3) C, H, N.
An aliquot of the free base (0.508 g, 1.19 mmol) was recrystal-
lized from ethanolic HCl/ether to give the hydrochloride salt as a
2H), 9.15 (br s, 2H), 8.54 (br s, 2H), 7.98 (s, 2H), 7.90 (m, 2H),
7.77 (dm, J ¼ 7.8 Hz, 1H), 7.68 (t, J ¼ 7.1 Hz, 1H), 5.18 (s, 2H); MS m/z
369.0 ([M þ 1]þ of free base); HPLC (Method A) tR 6.82 min (100
area % at 230 nm). Anal. (C15H14Cl2N4O3$2HCl) C, H, N, Cl.
white solid (0.587 g, 90%): mp 230e235 ꢁC dec; 1H NMR
d
11.28 (br
5.2.3.9. 3-N0-Hydroxyamidinobenzyl 2,6-dibromo-4-N0-hydroxyami-
s,1H),11.22 (br s,1H), 9.04 (br s, 4H), 8.20 (d, J ¼ 2.3 Hz,1H), 7.80 (m,
3H), 7.70 (d, J ¼ 8.5 Hz, 2H), 7.25 (d, J ¼ 8.4 Hz, 1H), 5.44 (s, 2H); MS
m/z 427.0 ([M þ 1]þ of free base); HPLC (Method A) tR 5.03 min (100
area % at 230 nm). Anal. (C15H15IN4O3$2HCl) C, H, N, Cl.
dinophenyl ether dihydrochloride (47). Yield (HCl salt, recrystallized
from 1 M HCl/ethanol), 2.04 g (87%): mp 188 ꢁC dec; 1H NMR
d 11.35
(br s, 2H), 9.20 (br s, 2H), 8.85 (br s, 2H), 8.15 (s, 2H), 7.93 (m, 2H),
7.78 (dm, J ¼ 7.7 Hz),7.69 (t, J ¼ 8.0 Hz, 1H), 5.14 (s, 3H); MS m/z
456.9 ([M þ 1]þ of free base), 458.9 ([M þ 3]þ), 460.8 ([M þ 5]þ);
HPLC (Method A) tR 7.23 min (100 area % at 254 nm). Anal.
(C15H14Br2N4O3$2HCl) C, H, N, Br, Cl.
5.2.3.4. 4-N0-Hydroxyamidinobenzyl 2,6-dimethoxy-4-N0-hydroxyami-
dinophenyl ether dihydrochloride (29). Yield (free base), 4.00 g (93%).
An aliquot (1.00 g, 3.24 mmol) was recrystallized from 1 M HCl/
ethanol to give the hydrochloride salt as a white solid (1.09 g, 90%):
5.2.3.10. 3-N0-Hydroxyamidinobenzyl 4-N0-hydroxyamidino-2,6-diio-
dophenyl ether dihydrochloride (50). Yield (free base, recrystallized
from toluene/ethanol), 3.08 g (90%).
mp 230e231 ꢁC dec; 1H NMR
d 11.30 (br 2, 2H), 9.06 (br s, 4H), 7.76
(d, J ¼ 8.2 Hz, 2H), 7.66 (d, J ¼ 8.2 Hz, 2H), 7.21 (s, 2H), 5.11 (s, 2H),
3.88 (s, 6H); MS m/z 361.0 ([M þ 1]þ of free base); HPLC (Method A)
tR 4.21 min (100 area % at 254 nm). Anal. (C17H20N4O5$2HCl$0.9H2O)
C, H, N, Cl.
The hydrochloride salt was precipitated from a solution of an
aliquot of the base (1.02 g, 1.85 mmol) in dioxane (30 mL) by the
addition of 4 M HCl/dioxane (3 mL) to give a white solid (1.04 g,
90%): mp > 180 ꢁC dec; 1H NMR
d 11.30 (br s, 2H), 9.06 (br s, 4H), 8.24
5.2.3.5. 3-N0-Hydroxyamidinobenzyl 2-chloro-4-N0-hydroxyamidino-
phenyl ether dihydrochloride (34). Yield (free base), 3.58 g (94%).
An aliquot (1.20 g, 3.58 mmol) was purified by C18 reverse phase
(s, 2H), 7.96 (d, J ¼ 7.8 Hz, 1H), 7.95 (d, J ¼ 2.1 Hz, 1H), 7.76 (dd, J ¼ 7.8
and 2.1 Hz, 2H), 7.72 (dd, J ¼ 7.8 and 7.8 Hz, 1H), 5.09 (br s, 2H); MS
m/z 553.0 ([M þ 1]þ of free base); HPLC (Method B) tR 2.31 min (100
area % at 254 nm). Anal. (C15H14I2N4O3$2HCl$0.6H2O) C, H, N, Cl.
flash to give a solid (1.08, 82%): mp 130 ꢁC dec; 1H NMR
d 11.41
(br s, 1H), 11.34 (br s, 1H), 9.13 (br s, 4H), 7.96 (d, J ¼ 1.6 Hz, 1H),
7.87 (s, 1H), 7.78 (m, 3H), 7.66 (t, J ¼ 7.4 Hz), 7.49 (d, J ¼ 8.8 Hz,
1H); MS m/z 335.0 ([M þ 1]þ of free base), 337.0 ([M þ 3]þ); HPLC
5.2.3.11. 3-N0-Hydroxyamidinobenzyl
dino-6-methoxy phenyl ether dihydrochloride (53). Yield (free base,
recrystallized from toluene/ethanol), 2.95 g (72%).
2-bromo-4-N0-hydroxyami-
(Method A) tR 4.69 min (100 area
(C15H15ClN4O3$2HCl$2.2H2O) C, H, N, Cl.
% at 230 nm). Anal.
The hydrochloride salt was precipitated from a solution of an
aliquot of the base (1.00 g, 2.44 mmol) in dioxane (80 mL) by the
addition of 4 M HCl/dioxane (3 mL) to give a grey solid (1.11 g, 94%):
5.2.3.6. 3-N0-Hydroxyamidinobenzyl 2-bromo-4-N0-hydroxyamidino-
phenyl ether dihydrochloride (37). The amidoxime base was recrys-
tallized from 1 M HCl/acetonitrile to give the hydrochloride salt
mp 235e236 ꢁC dec; 1H NMR
d 11.32 (br s, 2H), 9.09 (br s, 2H), 8.98
(br s, 2H), 7.86 (m, 2H), 7.73 (d, J ¼ 7.7 Hz, 1H); 7.67 (d, J ¼ 1.6 Hz, 2),
7.65 (t, J ¼ 7.7 Hz, 1H), 7.59 (d, J ¼ 1.6 Hz, 1H), 5.17 (s, 2H), 3.98 (s,
3H); MS m/z 409.4 ([M þ 1]þ of free base), 410.9 ([M þ 3]þ); HPLC
(3.07 g, 68%): mp > 170 ꢁC dec; 1H NMR
d 11.36 (br s,1H),11.29 (br s 2,
1H), 9.08 (br 2, 4H), 8.07 (d, J ¼ 2.2 Hz,1H), 7.87 (s, 1H), 7.80 (M, 2H),
7.73 (d, J ¼ 7.7 Hz, 1H), 7.66 (t, J ¼ 7.7 Hz, 1H), 7.44 (d, J ¼ 8.8 Hz, 1H),
5.42 (s, 2H); MS m/z 379.0 ([M þ 1]þ of free base), 380.9 ([M þ 3]þ);
HPLC (Method A) tR 5.24 min (100 area % at 254 nm). Anal.
(C15H15BrN4O3$2HCl$0.5H2O) C, H, N, Br, Cl.
(Method A) tR 5.83 min (100 area
(C16H17BrN4O4$2HCl) C, H, N, Br, Cl.
% at 254 nm). Anal.
5.2.3.12. 3-N0-Hydroxyamidinobenzyl 4-N0-hydroxyamidino-2-iodo-
6-methoxyphenyl ether dihydrochloride (56). Yield (free base,
recrystallized from toluene/ethanol), 4.00 g (88%).
5.2.3.7. 3-N0-Hydroxyamidinobenzyl 4-N0-hydroxyamidino-2-
iodophenyl ether dihydrochloride (40). Yield (free base, recrystallized
The hydrochloride salt was precipitated from a solution of an
aliquot of the base (1.00 g, 2.18 mmol) in dioxane (80 mL) by the
addition of 4 M HCl/dioxane (3 mL) to give a grey solid (1.00 g, 84%):
from ethanol/water), 3.04 g (89%): mp > 125 ꢁC dec; 1H NMR
d 9.64
(s, 1H), 9.55 (s, 1H), 8.08 (d, J ¼ 2.1 Hz), 7.79 (s, 1H), 7.67 (m, 2H), 7.51
(d, J ¼ 7.71 Hz,1H), 7.42 (t, J ¼ 7.6 Hz,1H), 7.08 (d, J ¼ 8.7 Hz,1H), 5.80
(br s, 2H), 5.78 (br s, 2H), 5.23 (s, 2H). Anal. (C15H15IN4O3$H2O)
C, H, N.
mp 235e236 ꢁC dec; 1H NMR
d 11.32 (br s, 2H), 9.08 (br s, 2H), 9.00 (br
s, 2H), 7.90 (s,1H), 7.89(d, J ¼ 8.7 Hz,1H), 7.79 (d, J ¼ 1.6 Hz,1H), 7.74 (d,
J¼ 8.2Hz,1H), 7.65(t, J¼ 7.7Hz,1H), 7.57 (d, J¼ 1.6Hz,1H), 5.13(2,2H),
3.96 (s, 3H); MS m/z 457.1 ([M þ 1]þ of free base); HPLC (Method A) tR
6.20 min (100 area % at 254 nm). Anal. (C16H17IN4O4$2HCl) C, H, N, Cl, I.
An aliquot of the free base (0.510 g, 1.20 mmol) was recrystal-
lized from aqueous HCl to give the hydrochloride salt as a white
solid (0.320 g, 54%): mp 170e173 ꢁC dec; 1H NMR
d 11.30 (br s, 1H),
11.20 (br s, 1H), 8.97 (br s, 4H), 8.20 (d, J ¼ 2.3 Hz, 1H), 7.89 (s, 1H),
5.2.3.13. 3-N0-Hydroxyamidinobenzyl 2,6-dimethoxy-4-N0-hydroxya-
midinophenyl ether dihydrochloride (59). Yield (free base), 4.17 g
(97%).
An aliquot (1.17 g, 3.24 mmol) was recrystallized from 1 M HCl/
ethanol to give the hydrochloride salt as a white solid (1.24 g, 88%):
7.77 (m, 2H), 7.70 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 1H), 5.40 (s, 2H); MS
m/z 427.0 ([M
þ
1]þ of free base); HPLC (Method A) tR
5.62 min (100 area % at 230 nm). Anal. (C15H14IN4O3$2HCl$1.8H2O)
C, H, N, Cl.

320
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
mp 228e229 ꢁC dec; 1H NMR
d
11.32 (br s, 2H), 9.08 (br s, 4H), 7.85
3.55 min (100 area % at 265 nm). Anal. (C19H24N4O3$2HCl$0.7H2O) C,
(s, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H), 7.77 (d, J ¼ 8.2 Hz, 1H), 7.61 (t,
J ¼ 8.0 Hz, 1H), 7.22 (s, 2H), 5.08 (s, 2H), 3.90 (s, 6H); MS m/z 361.1
([M þ 1]þ of free base); HPLC (Method A) tR 4.36 min (100 area % at
254 nm). Anal. (C17H20N4O5$2HCl) C, H, N, Cl.
H, N, Cl.
5.2.4.5. 3-N0-Methoxyamidinobenzyl 2-chloro-4-N0-methoxyamidin-
ophenyl ether dihydrochloride (35). The compound was prepared
using the general procedure above, but DMSO was used in place of
dioxane. The column-purified free base was converted to the HCl
salt using 3 M HCl/ethanol, followed by lyophilization to give a solid
5.2.4. General procedure for methamidoxime prodrugs 17, 30, 35,
38, 41, 48, 51, 54, 57, and 60
To a stirred solution of the amidoxime free base in dioxane
was added 2 M NaOH solution. The mixture was cooled to 0 ꢁC,
and a solution of dimethyl sulphate (2.4e3.0 equiv) was added
dropwise. The solution was maintained for 30 min, and then
stirred overnight at ambient temperature. The reaction mixture
was diluted with water and extracted into ethyl acetate. The
crude product was purified by column chromatography (hexanes/
ethyl acetate) and was converted to the hydrochloride salt using
1 M HCl/ethanol.
(0.75 g, 48%): mp 98e100 ꢁC; 1H NMR
d 8.77 (br s, 4H), 7.97 (d,
J ¼ 1.6 Hz,1H), 7.91 (s,1H), 7.73 (m, 3H), 7.62 (t, J ¼ 8.24 Hz,1H), 7.53
(d, J ¼ 8.8 Hz, 1H), 5.40 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H); MS m/z
363.1 ([M þ 1]þ of free base), 365.1 ([M þ 3]þ); HPLC (Method B) tR
4.76 min (100 area % at 230 nm). Anal. (C17H19ClN4O3$2HCl$3H2O)
C, H, N, Cl.
5.2.4.6. 3-N0-Methoxyamidinobenzyl 2-bromo-4-N0-methoxyamidin-
ophenyl ether dihydrochloride (38). Yield, 0.679
g
(34%):
mp > 140 ꢁC dec; 1H NMR
d
8.05 (d, J ¼ 1.6 Hz, 1H), 7.78 (s, 1H),
5.2.4.1. 4-N0-Methoxyamidinobenzyl
4-N0-methoxyamidinophenyl
7.78 (dd, J ¼ 8.8 and 1.6 Hz, 1H), 7.71 (m, 2H), 7.58 (t, J ¼ 7.7 Hz, 1H),
7.39 (d, J ¼ 8.8 Hz, 1H), 5.37 (s,2H), 3.84 (s, 3H), 3.82 (s, 3H); MS m/z
407.2 ([M þ 1]þ of free base), 409.0 ([M þ 3]þ); HPLC (Method B) tR
5.12 min (100 area % at 254 nm). Anal. (C17H19BrN4O3$2HCl$1.2H2O)
C, H, N, Cl.
ether dihydrochloride (17). The compound was prepared by the
general procedure, with DMF was in place of dioxane. The crude
product was precipitated from the reaction mixture by dilution
with water, purified by preparative HPLC, and converted to the HCl
salt using aqueous HCl to obtain a solid (0.423 g, 57%): mp 262 ꢁC
dec; 1H NMR
d
8.95 (br s, 1H), 7.77 (d, J ¼ 8.2 Hz, 2H), 7.76 (d,
5.2.4.7. 3-N0-Methoxyamidinobenzyl 2-iodo-4-N0-methoxyamidino-
phenyl ether dihydrochloride (41). The compound was prepared
using the general procedure above, but DMF was used in place of
dioxane. The crude product was precipitated by pouring the reac-
tion mixture over ice, and was then purified by column chroma-
tography eluting with 5% methanol in chloroform. The purified free
base was converted to the HCl salt using ethanolic HCl/ether to give
J ¼ 9.2Hz,1H), 7.59 (d, J ¼ 8.4 Hz, 2H), 7.23 (d, J ¼ 8.9 Hz, 2H), 5.32 (s,
2H), 3.84 (s, 3H), 3.83 (s, 3H); MS m/z 329.2 ([M þ 1]þ of free base);
HPLC (Method A) tR 8.50 min (100 area % at 254 nm). Anal.
(C17H20N4O3$2HCl) C, H, N, Cl.
5.2.4.2. 4-N0-Methoxyamidinobenzyl 2-bromo-4-N0-methoxyamidino-
phenyl ether dihydrochloride (21). Potassium tert-butoxide (1.15 g,
10.2 mmol) was added to a solution of amidoxime 20 (1.80 g,
4.75 mmol) in DMSO (50 mL). The mixture was stirred at 0 ꢁC for
30 min before the addition of iodomethane (1.0 mL, 16.1 mmol) and
was then stirred overnight at ambient temperature. The reaction
mixture was diluted with water and extracted into ethyl acetate. The
product was purified by flash chromatography to give the free base
as a white solid (1.04 g), which was recrystallized from 1 M HCl/
ethanol to give the HCl salt as a white solid (1.09 g, 48%): mp 229e
a solid (0.090 g, 34%): mp 157e160 ꢁC dec; 1H NMR
d 8.19 (d,
J ¼ 2.2.Hz, 1H), 7.87 (s, 1H), 7.78 (dd, J ¼ 8.6 and 2.2 Hz, 2H), 7.70 (m,
2H), 7.57 (t, J ¼ 7.7 Hz, 1H), 7.26 (d, J ¼ 8.8 Hz, 1H), 5.34 (s, 2H), 3.83
(s, 3H), 3.82 (s, 3H); MS m/z 455.2 ([M þ 1]þ of free base);
HPLC (Method A) tR 9.63 min (100 area % at 254 nm). Anal.
(C17H19IN4O3$2HCl$1.4H2O) C, H, N, Cl.
5.2.4.8. 3-N0-Methoxyamidinobenzyl 2,6-dichloro-4-N0-methoxyami-
dinophenyl ether dihydrochloride (45). Sodium hydride (60%
dispersion in mineral oil, 1.16 g, 28.9 mmol) was added to a so-
lution of amidoxime 44 (5.01 g, 13.6 mmol) in DMF at 0 ꢁC. The
mixture was stirred for 30 min before the addition of iodo-
methane (2.5 mL, 40.2 mmol), and then was stirred overnight at
ambient temperature. The reaction mixture was poured over a
mixture of ice and saturated NaCl solution and extracted into
dichloromethane. The evaporated extract was purified by column
chromatography eluting with chloroform/methanol (20:1). Puri-
fied fractions were recrystallized from water/ethanol to give the
231 ꢁC dec; 1H NMR
d
8.42 (br s, 4H), 8.06 (d, J ¼ 2.2 Hz, 1H), 7.81 (d,
J ¼ 8.4 Hz, 2H), 7.79 (dd, J ¼ 8.8 and 2.2 Hz, 1H), 7.65 (d, J ¼ 8.4 Hz,
2H), 7.35 (d, J ¼ 8.8 Hz, 1H), 5.42 (s, 2H), 3.85 (s, 3H), 3.82 (s, 3H);
MS m/z 407.1 ([M þ 1]þ of free base), 409.0 ([M þ 3]þ); HPLC
(Method B) tR 7.72 min (100 area
% at 254 nm). Anal.
(C17H19BrN4O3$2HCl$0.6H2O) C, H, N, Br, Cl.
5.2.4.3. 4-N0-Methoxyamidinobenzyl 2-iodo-4-N0-methoxyamidino-
phenyl ether dihydrochloride (24). The compound was prepared
following the procedure for 21. The reaction mixture was extracted
into ether, and the crude product was purified by column chro-
matography eluting with 2% methanol in chloroform. The purified
product was converted to the HCl salt using ethanolic HCl/ether to
give a white solid (0.573 g, 21%): mp 220e222 ꢁC dec; 1H NMR
free base (2.99 g, 55%): mp 123e127 ꢁC, 1H NMR
7.79 (s, 2H), 7.67 (dm, J ¼ 8.0 Hz, 1H), 7.57 (dm, J ¼ 7.7 Hz, 1H), 7.44
(t, J ¼ 7.7 Hz, 1H), 6.27 (br s, 2H), 6.10 (br s, 2H), 5.06 (s, 2H), 3.76
(s, 3H), 3.75 (s, 3H). Anal. (C17H18Cl2N4O3) C, H, N.
d 7.82 (m, 1H),
The free base (2.52 g) was dissolved in a mixture of ethanol and
aqueous HCl. After a small amount precipitated from solution, the
mother liquor was concentrated and lyophilized to give the HCl
d
8.51 (br s, 1H), 8.20 (d, J ¼ 2.2 Hz, 1H), 7.82 (m, 3H), 7.65 (d,
J ¼ 8.4 Hz, 2H), 7.24 (d, J ¼ 8.8 Hz, 1H), 5.41 (s, 2H), 3.84 (s, 3H), 3.82
(s, 3H); MS m/z 455.0 ([M þ 1]þ of free base); HPLC (Method C) tR
7.72 min (100 area % at 230 nm). Anal. (C17H19IN4O3$2HCl$0.8H2O)
C, H, N, Cl.
salt (1.62 g, 54%): mp > 125 ꢁC dec; 1H NMR
d 7.92 (m, 1H), 7.87 (s,
2H), 7.84 (dm, J ¼ 7.5 Hz, 1H), 7.78 (dm, J ¼ 8.0 Hz, 1H), 7.64 (t,
J ¼ 7.5 Hz, 1H), 5.13 (s, 2H), 3.87 (s, 3H), 3.79 (s, 3H); MS m/z 379.3
([M þ 1]þ of free base); HPLC (Method B) tR 6.21 min (100 area % at
230 nm). Anal. (C17H18Cl2N4O3$2.05HCl$2H2O) C, H, N, Cl.
5.2.4.4. 4-N0-Methoxyamidinobenzyl
yamidinophenyl ether dihydrochloride (30). Yield, 0.953 (30%):
mp > 170 ꢁC dec; 1H NMR
2,6-dimethoxy-4-N0-methox-
d
8.80 (very br s, 4H), 7.77 (d, J ¼ 7.7 Hz,
5.2.4.9. 3-N0-Methoxyamidinobenzyl 2,6-dibromo-4-N0-methoxyami-
2H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.24 (s, 2H), 5.10 (s, 2H), 3.88 (s, 9H), 3.85
dinophenyl ether dihydrochloride (48). Yield, 0.312
g (37%):
8.04 (s, 2H), 7.94 (s, 1H), 7.86 (d,
(s, 3H); MS m/z 389.1 ([M þ 1]þ of free base); HPLC (Method B) tR
mp > 133 ꢁC dec; 1H NMR
d

D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
321
J ¼ 7.7 Hz, 1H), 7.78 (d, J ¼ 8.2 Hz, 1H), 7.64 (t, J ¼ 7.7 Hz, 1H), 5.10 (s,
2H), 3.86 (s, 3H), 3.78 (s,3H); MS m/z 485.2 ([M þ 1]þ of free base),
487.0 ([M þ 3]þ), 488.9 ([M þ 5]þ); HPLC (Method B) tR 6.53 min
J ¼ 8.2 and 1.6 Hz,1H), 7.67 (d, J ¼ 8.2 Hz, 1H), 7.35 (d, J ¼ 8.2 Hz,1H),
5.44 (s, 2H); HPLC (Method B) tR 6.60 min (100 area % at 254 nm).
Anal. (C15H9BrN2O) C, H, N, Br.
(100 area
C, H, N, Cl.
% at 265 nm). Anal. (C17H18Br2N4O3$2HCl$1.3H2O)
5.2.5.3. 4-Cyanobenzyl 4-cyano-2-iodophenyl ether (79). The com-
pound was prepared by the variation from the general procedure
employed for 77, but with a reaction temperature of 50e60 ꢁC, and
the crude product was recrystallized from toluene. Yield, 5.09 g
5.2.4.10. 3-N0-Methoxyamidinobenzyl 2,6-diiodo-4-N0-methoxyami-
dinophenyl ether dihydrochloride (51). After preparation following
the general procedure above and purification by column chro-
matography, the free base was converted to the HCl salt using
dioxane and 4 M HCl/dioxane, followed by recrystallization from
1 M HCl/ethanol to give a white solid (1.42 g, 44%): mp > 150 ꢁC
(93%): mp 196e197 ꢁC; 1H NMR
J ¼ 8.4 Hz, 2H), 7.88 (dd, J ¼ 8.6 and 2.0 Hz, 1H), 7.69 (d, J ¼ 8.3 Hz,
2H), 7.22 (d, J ¼ 8.7 Hz, 1H), 5.43 (s, 2H); HPLC (Method B) tR
7.51 min (98.6 area % at 254 nm). Anal. (C15H9IN2O) C, H, N.
d
8.30 (d, J ¼ 2.0 Hz, 1H), 7.92 (d,
dec; 1H NMR
d
8.22 (s, 2H), 7.96 (s, 1H), 7.91 (d, J ¼ 7.7 Hz, 1H),
7.78 (d, J ¼ 8.2 Hz, 1H), 7.65 (t, J ¼ 7.7 Hz, 1H), 5.16 (s, 2H), 3.87 (s,
5.2.5.4. 3-Cyanobenzyl 4-cyano-2-nitrophenyl ether (80). Yield
(after recrystallization from acetonitrile), 2.17 g (78%): mp 204e
3H), 3.80 (s, 3H); MS m/z 581.0 ([M þ 1]þ of free base); HPLC
(Method B) tR 6.92 min (100 area
%
at 254 nm). Anal.
207 ꢁC; 1H NMR
2.2 Hz, 1H), 7.93 (s, 1H), 7.87 (d, J ¼ 7.7 Hz, 1H), 7.80 (d,
J ¼ 8.2 Hz, 1H), 7.66 (m, 2H), 5.49 (s, 2H); HPLC (Method B) tR
6.12 min (100 area % at 254 nm). Anal. (C15H9N3O3) C, H, N.
d
8.55 (d, J ¼ 2.2 Hz, 1H), 8.19 (dd, J ¼ 8.8 and
(C17H18I2N4O3$2HCl$1.8H2O$0.4C2H5OH) C, H, N, Cl, I.
5.2.4.11. 3-N0-Methoxyamidinobenzyl
2-bromo-6-methoxy-4-N0-
methoxyamidinophenyl ether dihydrochloride (54). Yield, 0.854 g
(38%): mp > 160 ꢁC dec; 1H NMR
d
7.89 (s,1H), 7.79 (d, J ¼ 7.7 Hz,1H),
5.2.5.5. 3-Cyanobenzyl
2-chloro-4-cyanophenyl
ether
(81).
7.74 (d, J ¼ 8.2 Hz, 1H), 7.62 (d, J ¼ 2.2 Hz, 1H), 7.59 (t, J ¼ 7.7 Hz, 1H),
5.12 (s, 2H), 3.95 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H); MS m/z 437.1
([M þ 1]þ of free base), 439.0 ([M þ 3]þ); HPLC (Method B) tR
5.71 min (100 area % at 254 nm). Anal. (C18H21BrN4O4$2HCl$1.4H2O)
C, H, N, Cl, I.
Yield, 4.19 g (96%): mp 122e123 ꢁC; 1H NMR
d
8.07 (s, 1H), 7.94 (s,
1H), 7.93 (s, 1H), 7.83 (m, 3H), 7.66 (m 1H), 7.42 (d, J ¼ 8.8 Hz, 1H),
5.49 (s, 2H); HPLC (Method B) tR 6.88 min (98.1 area % at 254 nm).
Anal. (C15H9ClN2O) C, H, N.
5.2.5.6. 3-Cyanobenzyl
Yield (after recrystallization from acetonitrile), 4.52 g (96%): mp
144e145 ꢁC; 1H NMR
7.90 (dd, J ¼ 8.8 and 2.2 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 1H), 7.84 (d,
J ¼ 8.2 Hz, 1H), 7.66 (t, J ¼ 8.4 Hz, 1H), 7.37 (d, J ¼ 8.8 Hz, 1H), 5.39 (s,
2H); HPLC (Method B) tR 7.2 min (100 area % at 254 nm). Anal.
(C15H9BrN2O) C, H, N.
2-bromo-4-cyanophenyl
ether
(82).
5.2.4.12. 3-N0-Methoxyamidinobenzyl 2-iodo-6-methoxy-4-N0-metho-
xyamidinophenyl ether dihydrochloride (57). Yield, 1.87 g (54%):
d
8.20 (s, d, J ¼ 2.2 Hz, 1H), 7.94 (s, 1H),
mp > 165 ꢁC dec; 1H NMR
d
7.90 (s, 1H), 7.82 (d, J ¼ 7.7 Hz, 1H),
7.78 (d, J ¼ 1.6 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.59 (t, J ¼ 7.7 Hz, 1H),
7.55 (d, J ¼ 1.6 Hz, 1H), 5.09 (s, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 3.84 (s,
3H); MS m/z 485.0 ([M þ 1]þ of free base); HPLC (Method B) tR
5.43 min (100 area % at 265 nm). Anal. (C18H21IN4O4$2HCl$H2O) C, H,
N, Cl, I.
5.2.5.7. 3-Cyanobenzyl 4-cyano-2-iodophenyl ether (83). The com-
pound was prepared and recrystallized as described above for 79.
5.2.4.13. 3-N0-Methoxyamidinobenzyl 2,6-dimethoxy-4-N0-metho-
Yield, 10.4 g (96%): mp 153e154 ꢁC; 1H NMR
1H), 7.96 (s, 1H), 7.89 (dd, J ¼ 8.6 and 2.1 Hz, 1H), 7.84 (m, 2H), 7.66
(t, J ¼ 7.9 Hz, 1H), 7.24 (d, J ¼ 2.0 Hz, 1H), 5.37 (s, 2H); HPLC (Method
B) tR 7.49 min (98.6 area % at 230 nm). Anal. (C15H9IN2O) C, H, N.
d
8.30 (d, J ¼ 2.0 Hz,
xyamidinophenyl ether dihydrochloride (60). Yield, 1.11 g (35%): mp
160e162 ꢁC dec; 1H NMR
d
7.86 (s, 1H), 7.70 (m, 2H), 7.55 (dd, J ¼ 7.7
and 7.7 Hz, 1H), 7.24 (s, 2H), 5.06 (s, 2H), 3.89 (s, 6H), 3.88 (s, 3H),
3.85 (s, 3H); MS m/z 389.2 ([M þ 1]þ of free base); HPLC (Method B)
tR 3.68 min (100 area % at 265 nm). Anal. (C19H24N4O5$2HCl$H2O) C,
H, N, Cl.
5.2.5.8. 3-Cyanobenzyl 4-cyano-2-methoxyphenyl ether (84).
Yield (after recrystallization from acetonitrile), 3.90 g (98%): mp
127e128 ꢁC; 1H NMR
(dm, J ¼ 7.7 Hz, 1H), 7.63 (t, J ¼ 7.7 Hz, 1H), 7.43 (m, 2H), 7.21 (d,
J ¼ 8.8 Hz, 1H), 5.25 (s, 2H); HPLC (Method B) tR 6.03 min (100 area
% at 254 nm). Anal. (C16H12N2O2) C, H, N.
d
7.92 (m, 1H), 7.84 (dm, J ¼ 7.7 Hz, 1H), 7.80
5.2.5. General procedure for dinitriles 77e87
Potassium carbonate (1e1.6 equiv) was added to a stirred so-
lution of the phenol (1 equiv) in DMF. After 30 min the cyanobenzyl
bromide (1e1.2 equiv) was added, and the mixture was stirred until
completion (1e4 days). The reaction mixture was poured over ice
or diluted with water. The precipitated product was filtered off and
recrystallized if needed.
5.2.5.9. 3-Cyanobenzyl 4-cyano-2,6-dichloro-phenyl ether (85).
The compound was prepared following the procedural variations
employed for 77, but at 70e80 ꢁC for 2 h and at ambient temper-
ature for 60 h. Yield (after recrystallization from toluene), 5.52 g
5.2.5.1. 4-Cyanobenzyl 4-cyanophenyl ether (77). The compound
was prepared by the general procedure, but the reaction mixture
was maintained at 110 ꢁC for 30 min before the dropwise addition
of a solution of the benzyl bromide in DMF over 30 min, and for
another 30 min thereafter. Yield, 11.4 g (96%): mp 167e169 ꢁC; 1H
(72%): mp 199e201 ꢁC; 1H NMR
d 8.20 (s, 1H), 7.98 (s, 1H), 7.88 (d,
J ¼ 8.0 Hz, 1H), 7.87 (d, J ¼ 7.8 Hz, 1H), 7.66 (t, J ¼ 7.8 Hz, 1H), 5.20 (s,
2H); HPLC (Method B) tR 7.82 min (98.4 area % at 230 nm). Anal.
(C15H8Cl2N2O) C, H, N.
NMR
d
7.89 (dm, J ¼ 8.4 Hz, 2H), 7.81 (dm, J ¼ 8.9 Hz, 2H), 7.65 (d,
5.2.5.10. 3-Cyanobenzyl 2,6-dibromo-4-cyanophenyl ether (86).
J ¼ 8.4 Hz, 2H), 7.20 (dm, J ¼ 9.1 Hz, 2H), 5.33 (s, 2H); HPLC (Method
B) tR 6.28 min (100 area % at 230 nm). Anal. (C15H10N2O$0.1H2O) C,
H, N.
Yield (after recrystallization from acetonitrile), 3.53 g (90%): mp
205e207 ꢁC; 1H NMR
d 8.36 (s, 2H), 8.00 (m, 1H), 7.9 (m, 2H), 7.67 (t,
J ¼ 7.7 Hz,1H), 5.14 (s, 2H); HPLC (Method B) tR 8.08 min (100 area %
at 254 nm). Anal. (C15H8Br2N2O) C, H, N, Br.
5.2.5.2. 4-Cyanobenzyl 2-bromo-4-cyanophenyl ether (78). Yield
(after recrystallization from acetonitrile), 8.60 g (92%): 191e192 ꢁC;
5.2.5.11. 3-Cyanobenzyl 4-cyano-2,6-diiodophenyl ether (87).
Yield (after recrystallization from acetonitrile), 3.44 g (36%): mp
1H NMR
d
8.21 (d, J ¼ 2.2 Hz, 1H), 7.91 (d, J ¼ 8.2 Hz, 2H), 7.86 (dd,

322
D.A. Patrick et al. / European Journal of Medicinal Chemistry 67 (2013) 310e324
183e184 ꢁC; 1H NMR
J ¼ 7.7 and 2.1 Hz, 1H), 7.90 (dd, J ¼ 7.7 and 2.1 Hz, 1H), 7.70 (dd,
J ¼ 7.7 and 7.7 Hz, 1H), 5.07 (s, 2H); HPLC (Method B) tR 8.52 min
(100 area % at 254 nm). Anal. (C15H8I2N2O) C, H, N, I.
d
8.43 (s, 2H), 8.03 (d, J ¼ 2.1 Hz, 1H), 7.94 (dd,
J ¼ 1.6 Hz, 1H), 5.05 (s, 2H), 3.86 (s, 3H); HPLC (Method B) tR
6.74 min (100 area % at 254 nm). Anal. (C16H13IN2O3) C, H, N, I.
5.2.7.4. 3-Cyanobenzyl 4-[(hydroxyimino)methyl]-2-iodo-6-meth-
oxyphenyl ether (94). Yield (after recrystallization from EtOH),
5.2.6. General procedure for cyano-aldehydes 88e91
The cyano-aldehydes were prepared following the general
procedure described above for dinitriles 77e87. All products were
recrystallized from ethanol.
10.3 g (90%): mp 135e137 ꢁC; 1H NMR
d 11.13 (s, 1H), 8.06 (s, 1H),
7.90 (br s, 1H), 7.78 (m, 2H), 7.59 (dd, J ¼ 8.2 and 7.7 Hz, 1H), 6.93 (s,
2H), 5.00 (s, 2H), 3.79 (s, 6H); HPLC (Method B) tR 5.11 min (100 area
% at 254 nm). Anal. (C17H16N2O4) C, H, N.
5.2.6.1. 4-Cyanobenzyl 2,6-dimethoxy-4-formylphenyl ether (88).
5.2.8. Dinitriles 96e99
The nitriles were prepared from the corresponding aldehydes
92e95 following the representative procedure for nitrile 96.
Yield, 13.7 g (92%): mp 127e128 ꢁC; 1H NMR
d
9.90 (s, 1H), 7.84 (d,
J ¼ 8.2 Hz, 2H), 7.66 (d, J ¼ 8.2 Hz, 2H), 7.28 (s, 2H), 5.10 (s, 2H), 3.87
(s, 6H); HPLC (Method B) tR 6.16 min (100 area % at 254 nm). Anal.
(C17H15NO4) C, H, N.
5.2.8.1. 4-Cyanobenzyl 4-cyano-2,6-dimethoxyphenyl ether (96).
A solution of oxime 92 (8.00 g, 25.6 mmol) in acetic anhydride
(100 mL) was refluxed overnight, cooled, and refrigerated. A
formed precipitate was separated, washed with water, and dried to
give 96 as a beige solid (6.00 g, 80%). A small portion was recrys-
tallized from EtOAc to give an analytical sample: mp 184e186 ꢁC;
5.2.6.2. 3-Cyanobenzyl 2-bromo-4-formyl-6-methoxyphenyl ether
(89). Yield, 4.85 g (93%): mp 115e117 ꢁC; 1H NMR
d 9.90 (s, 1H), 7.94
(br s,1H), 7.84 (dd, J ¼ 7.7 and 1.1 Hz, 2H), 7.79 (d, J ¼ 1.6 Hz,1H), 7.63
(dd, J ¼ 7.7 and 7.7 Hz,1H), 7.57 (d, J ¼ 1.6 Hz,1H), 5.20 (s, 2H), 3.95 (s,
3H); HPLC (Method B) tR 7.35 min (100 area % at 254 nm). Anal.
(C16H12BrNO3$0.1H2O) C, H, N, Br.
1H NMR
d
7.84 (d, J ¼ 8.2 Hz, 2H), 7.64 (d, J ¼ 8.2 Hz, 2H), 7.23 (s, 2H),
5.10 (s, 2H), 3.83 (s, 6H); HPLC (Method B) tR 6.62 min (100 area % at
254 nm). Anal. (C17H14N2O3) C, H, N.
5.2.6.3. 3-Cyanobenzyl 4-formyl-2-iodo-6-methoxyphenyl ether
(90). Yield, 5.72 g (97%): mp 105e107 ꢁC; 1H NMR
d
9.88 (s, 1H),
5.2.8.2. 3-Cyanobenzyl 2-bromo-4-cyano-6-methoxyphenyl ether (97).
7.96 (m, 2H), 7.86 (m, 2H), 7.63 (dd, J ¼ 8.2 and 7.7 Hz, 1H), 7.56 (d,
J ¼ 1.6 Hz, 1H), 5.17 (s, 2H), 3.93 (s, 3H); HPLC (Method B) tR
7.63 min (100 area % at 254 nm). Anal. (C16H12INO3) C, H, N, I.
Yield (after recrystallization from acetonitrile), a beige solid (3.92 g,
93%): mp 149e150 ꢁC; 1H NMR
d 7.93 (br s, 1H), 7.84 (m, 2H), 7.80 (d,
J ¼ 1.1 Hz,1H), 7.65 (d, J ¼ 1.1 Hz,1H), 7.62 (dd, J ¼ 7.7 and 7.7 Hz,1H),
5.17 (s, 2H), 3.91 (s, 3H); HPLC (Method B) tR 7.63 min (100 area % at
254 nm). Anal. (C16H11BrN2O2) C, H, N, Br.
5.2.6.4. 3-Cyanobenzyl 2,6-dimethoxy-4-formylphenyl ether (91).
Yield, 12.9 g (87%): mp 91e92 ꢁC; 1H NMR
d 9.89 (s, 1H), 7.90 (dd,
J ¼ 1.1 and 1.1 Hz,1H), 7.79 (dd, J ¼ 7.7 and 1.1 Hz, 2H), 7.59 (dd, J ¼ 7.7
and 7.7 Hz, 1H), 7.27 (s, 2H), 5.13 (s, 2H), 3.87 (s, 6H); HPLC (Method
B) tR 6.06 min (100 area % at 254 nm). Anal. (C17H15NO4) C, H, N.
5.2.8.3. 3-Cyanobenzyl 4-cyano-2-iodo-6-methoxyphenyl ether (98).
Yield (after recrystallization from acetonitrile), 4.52 g (96%): mp
141e142 ꢁC; 1H NMR
d
7.96 (br s, 1H), 7.89 (d, J ¼ 1.6 Hz, 1H), 7.85
(dd, J ¼ 8.2 and 1.6 Hz, 1H), 7.84 (dd, J ¼ 7.7 and 1.6 Hz, 1H), 7.64 (d,
J ¼ 1.6 Hz, 1H), 7.63 (dd, J ¼ 8.2 and 7.7 Hz, 1H), 5.14 (s, 2H), 3.90 (s,
3H); HPLC (Method B) tR 7.87 min (100 area % at 254 nm). Anal.
(C16H11IN2O2) C, H, N, I.
5.2.7. Oximes 92e95
The oximes were prepared from the corresponding aldehydes
88e91 following the representative procedure for oxime 92.
5.2.7.1. 4-Cyanobenzyl
2,6-dimethoxy-4-[(hydroxyimino)methyl]
5.2.8.4. 3-Cyanobenzyl 4-cyano-2,6-dimethoxyphenyl ether (99).
Yield (after recrystallization from EtOAc), 6.50 g (86%): mp 143e
phenyl ether (92). To a refluxed solution of cyano-aldehyde 88
(11.0 g, 40.0 mmol) in EtOH (100 mL) was added a solution of
NH2OH$HCl (2.83 g, 40.7 mmol) in water (15 mL). The mixture was
refluxed for 5 min followed by addition of a solution of NaOH
(1.48 g, 37.0 mmol) in water (15 mL). After further reflux for 5 min,
the mixture was cooled and refrigerated overnight. A formed pre-
cipitate was separated, washed with water and EtOH, and dried to
give 92 as a creamy solid (10.6 g, 92%): mp 168e169 ꢁC; 1H NMR
145 ꢁC; 1H NMR
d
7.88 (br s, 1H), 7.78 (m, 2H), 7.59 (dd, J ¼ 8.2 and
7.7 Hz, 1H), 7.22 (s, 2H), 5.07 (s, 2H), 3.83 (s, 6H); HPLC (Method B)
tR 6.58 min (100 area % at 254 nm). Anal. (C17H14N2O3) C, H, N.
Acknowledgements
This work was supported by the Consortium for Parasitic Drug
Development (CPPD), the Bill and Melinda Gates Foundation, and
Medicines for Malaria Venture (MMV).
d
11.13 (s, 1H), 8.06 (s, 1H), 7.84 (d, J ¼ 8.2 Hz, 2H), 7.66 (d, J ¼ 8.2 Hz,
2H), 6.93 (s, 2H), 5.02 (s, 2H), 3.79 (s, 6H); HPLC (Method B) tR
5.17 min (100 area % at 254 nm). Anal. (C17H16N2O4) C, H, N.
References
5.2.7.2. 3-Cyanobenzyl 2-bromo-4-[(hydroxyimino)methyl]-6-meth-
oxyphenyl ether (93). Yield (after recrystallization from EtOH),
[1] Leishmaniasis. http://www.who.int/leishmaniasis/burden/en/, 2/15/2013.
[2] Malaria. http://www.who.int/malaria/en/, 2/15/2013.
4.78 g (98%): mp 204e205 ꢁC; 1H NMR
d 11.36 (s, 1H), 8.10 (s, 1H),
[3] African
trypanosomiasis
(sleeping
sickness).
http://www.who.int/
7.93 (br s, 1H), 7.84 (m, 2H), 7.62 (dd, J ¼ 7.7 and 7.7 Hz, 1H), 7.40 (d,
mediacentre/factsheets/fs259/en/index.html, 2/15/2013.
J ¼ 1.6 Hz,1H), 7.32 (d, J ¼ 1.6 Hz,1H), 5.08 (s, 2H), 3.87 (s, 3H); HPLC
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(Method B) tR 6.48 min (100 area
% at 254 nm). Anal.
(C16H13BrN2O3) C, H, N, Br.
5.2.7.3. 3-Cyanobenzyl 4-[(hydroxyimino)methyl]-2-iodo-6-meth-
oxyphenyl ether (94). Yield (after recrystallization from EtOH),
5.28 g (96%): mp 177e178 ꢁC; 1H NMR
d 11.31 (s, 1H), 8.08 (s, 1H),
7.96 (br s, 1H), 7.87 (d, J ¼ 7.7 Hz, 1H), 7.83 (dd, J ¼ 8.2 and 1.6 Hz,
1H), 7.63 (dd, J ¼ 8.2 and 7.7 Hz, 1H), 7.58 (d, J ¼ 1.6 Hz, 1H), 7.33 (d,

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