Synthesis and antiprotozoal activity of dicationic m-terphenyl and 1,3-dipyridylbenzene derivatives

Article abstract of DOI:10.1021/jm400508e

4,4″-Diamidino-m-terphenyl (1) and 36 analogues were prepared and assayed in vitro against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium falciparum, and Leishmania amazonensis. Twenty-three compounds were highly active against T. b. rhodesiense or P. falciparum. Most noteworthy were amidines 1, 10, and 11 with IC₅₀ of 4 nM against T. b. rhodesiense, and dimethyltetrahydropyrimidinyl analogues 4 and 9 with IC₅₀ values of ≤ 3 nM against P. falciparum. Bis-pyridylimidamide derivative 31 was 25 times more potent than benznidazole against T. cruzi and slightly more potent than amphotericin B against L. amazonensis. Terphenyldiamidine 1 and dipyridylbenzene analogues 23 and 25 each cured 4/4 mice infected with T. b. rhodesiense STIB900 with four daily 5 mg/kg intraperitoneal doses, as well as with single doses of ≤10 mg/kg. Derivatives 5 and 28 (prodrugs of 1 and 25) each cured 3/4 mice with four daily 25 mg/kg oral doses.

Full text of DOI:10.1021/jm400508e

Article
pubs.acs.org/jmc
Synthesis and Antiprotozoal Activity of Dicationic m‑Terphenyl and
1,3-Dipyridylbenzene Derivatives
Donald A. Patrick,† Mohamed A. Ismail,‡,§ Reem K. Arafa,‡,∥ Tanja Wenzler,⊥,# Xiaohua Zhu,∞
Trupti Pandharkar,∞ Susan Kilgore Jones,† Karl A. Werbovetz,∞ Reto Brun,⊥,# David W. Boykin,‡
and Richard R. Tidwell*,†
†Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina, Chapel Hill, North Carolina
27599-7525, United States
‡Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30303-3083,
United States
§Department of Chemistry, College of Science, King Faisal University, Hofuf 31982, Saudi Arabia
∥Department of Medicinal and Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
⊥Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland
#University of Basel, Basel, Switzerland
∞Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
ABSTRACT: 4,4″-Diamidino-m-terphenyl (1) and 36 analogues were prepared and assayed in vitro against Trypanosoma brucei
rhodesiense, Trypanosoma cruzi, Plasmodium falciparum, and Leishmania amazonensis. Twenty-three compounds were highly active
against T. b. rhodesiense or P. falciparum. Most noteworthy were amidines 1, 10, and 11 with IC50 of 4 nM against T. b. rhodesiense,
and dimethyltetrahydropyrimidinyl analogues 4 and 9 with IC50 values of ≤ 3 nM against P. falciparum. Bis-pyridylimidamide
derivative 31 was 25 times more potent than benznidazole against T. cruzi and slightly more potent than amphotericin B against
L. amazonensis. Terphenyldiamidine 1 and dipyridylbenzene analogues 23 and 25 each cured 4/4 mice infected with T. b.
rhodesiense STIB900 with four daily 5 mg/kg intraperitoneal doses, as well as with single doses of ≤10 mg/kg. Derivatives 5 and
28 (prodrugs of 1 and 25) each cured 3/4 mice with four daily 25 mg/kg oral doses.
to Trypanosoma brucei gambiense, prevalent in western and
central Africa, accounts for about 95% of reported cases. The
remaining cases arise from an acute infection of T. brucei
rhodesiense, prevalent in eastern and southern Africa.8
American trypanosomiasis, or Chagas disease, is caused by
Trypanosoma cruzi. An estimated 10 million people were
infected in 2010, and an estimated 10 000 died in 2008 because
of the disease. Originally endemic to Latin America, the disease
has now spread to other continents. It progresses from an acute
phase lasting about 2 months after infection, in which the
INTRODUCTION
■
The vector borne protozoal diseases trypanosomiasis, malaria,
and leishmaniasis continue to affect some of the poorest areas
of the world.1−5 Human African trypanosomiasis (HAT), which
occurs in over 20 sub-Saharan African countries, was largely
controlled by the 1960s but has since re-emerged. After
continued control efforts, fewer than 10 000 new cases were
reported in 2009 for the first time in 50 years, and the World
Health Organization (WHO) estimates that 30 000 actual cases
currently exist (vs 300 000 cases in 1988).1,6 The disease
progresses from an early hemolymphatic stage to a late central
nervous system (CNS) stage and has a 100% mortality rate if
not treated.7 Two forms of HAT exist. A chronic infection due
Received: April 8, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structures of 1,5-bis(4-amidinophenoxy)pentane (pentamidine), 2,5-bis(4-amidinophenyl)furan (furamidine), and 2,5-bis(4-N′-
methoxyamidinophenyl)furan (pafuramidine).
parasites are concentrated in the blood, to a chronic phase, in
which the parasites are hidden in the heart and digestive
muscle. Myocardial damage can lead to death.4
Malaria is concentrated in sub-Saharan Africa but is also
endemic to other continents, affecting approximately 100
countries. The WHO has estimated 216 million cases of malaria
in 2010 and 655 000 deaths.2 Most of the deaths occur in
Africa, primarily among young children. Four Plasmodium
species are recognized as human pathogens. Among these, P.
falciparum causes the most deadly infection.2,7
Pentavalent antimonial compounds, including sodium
stibogluconate and meglumine antimoniate, introduced in the
1940s, continue to be the drugs of choice against VL in most
endemic countries despite their toxicity. High rates of
treatment failures have been reported in Bihar, India, since
the 1980s.23 Antimony resistant VL was treated with
pentamidine in the past, but the efficacy of pentamidine against
VL has decreased,24 and the use of pentamidine against
leishmaniasis is now restricted to some forms of cutaneous
disease that occur in South America.25 Amphotericin B is an
effective option in the treatment of VL,26 but the less toxic
liposomal formulations of the drug are limited by their expense.
The aminoglycoside paromomycin has shown high efficacy in
India and low toxicity, but a 3-week course of daily injections is
required.27 Miltefosine is orally active but is teratogenic,
nephrotoxic, and hepatotoxic.28 Various miltefosine combina-
tion therapies have been under recent investigations.8,23,28
Furamidine29 (DB75, Figure 1) is a conformationally
restricted analogue of pentamidine. These positively charged
molecules have poor oral bioavailability; however, their N′-
hydroxy and N′-methoxy derivatives with lower pKa values have
shown potential as orally active prodrugs.30 Pafuramidine
(DB289, Figure 1),31 the methamidoxime prodrug of
furamidine, advanced to phase III clinical trials against early
stage HAT and phase II trials against malaria.32,33 The
compound exhibited nephro- and hepatotoxicity in a recent
expanded phase I trial,34 resulting in a suspension of phase III
trials.35
Numerous analogues of pentamidine and furamidine,
including those with modifications of both the central and
outer rings, have been prepared and assayed against various
organisms,36−56 with the intent of finding comparably effica-
cious but less toxic drug candidates. A number of
arylimidamides (AIAs), or “reversed” amidines, have been
prepared and tested primarily against T. cruzi or Leishmania
species.57−60 A few diamidine derivatives of o-, m-, and p-
terphenyl were originally prepared as serine protease
inhibitors.61 More recently, a larger number of dicationic p-
terphenyls and their aza-analogues have been prepared as
antiprotozoals.62−64 Dicationic m-terphenyl derivatives have
remained virtually unexplored, with respect to both the number
of molecules synthesized and their potential antiprotozoal
activities. While the p-terphenyls are linear in shape, the m-
terphenyls are more conformationally similar to furamidine.
The present work describes the synthesis of a rather diverse
group of dicationic m-terphenyl derivatives and their biological
evaluation against four parasites.
Infections due to various species of Leishmania affect nearly
12 million people in 88 countries. The diseases range in severity
from a spontaneously healing cutaneous form due to L.
mexicana to a life-threatening visceral form, 90% of which
occurs in Bangladesh, India, Nepal, and Sudan (due to L.
donovani) or in Brazil (due to L. chagasi).5,8 Cases of patients
infected with both visceral leishmaniasis (VL) and human
immunodeficiency virus (HIV) continue to be reported.9−16
The need for safe, orally active, and economical drugs against
HAT persists. Available therapies are scarce, antiquated, toxic,
prone to resistance, and require parenteral (usually intra-
venous) administration.1,6,7,17−19 Suramin, a polysulfonated
naphthylurea, and pentamidine (Figure 1), an aromatic
diamidine, are the two drugs used to treat early stage HAT.
Late-stage therapies are generally more problematic. The
organoarsenical melarsoprol is associated with an encephalop-
athy leading to death in about 5% of the cases.7,20 Eflornithine
is the only new anti-HAT drug introduced in the past 60 years.
Although better tolerated than melarsoprol, eflornithine is
ineffective against T. b. rhodesiense, and high doses must be
given intravenously four times daily over long periods.6
A
combination of eflornithine and nifurtimox (nifurtimox−
eflornithine combination therapy, NECT) was licensed in
2009.1 It is easier to administer and has a shorter treatment
duration than the eflornithine monotherapy and is currently the
best choice to treat late stage patients with T. b. gambiense
infections.
Drugs of choice against Chagas disease are benznidazole and
nifurtimox, both of which are given orally. Both drugs are
highly effective in curing the disease if treatments begins at the
onset of the acute stage, but the efficacy of each diminishes with
delayed initiation of treatment.4,7 Severe side effects have also
been reported for both drugs.21 No drugs are currently
approved for the chronic phase of the disease, but at least
two drug candidates are currently in clinical trials.4,7
Although a number of economical orally active antimalarial
drugs are available, drug resistance is a growing problem.22
Resistance to chloroquine, observed in Thailand in the 1960s,
has been followed by resistance in other locations and to other
drugs, including sulfadoxine−pyrimethamine and mefloquine.
Resistance to artemisinin led to the development of
artemisinin-based combination therapy, but resistance to at
least one combination, artesunate−mefloquine, has also been
reported.7
CHEMISTRY
■
A total of 37 cationically substituted m-terphenyl analogues
were prepared (Table 1). This group consists of 29 diamidine
derivatives (1−29), six bis-arylimidamides (bis-AIAs, 30−35),
and two mono-AIAs (36 and 37). The amidine group includes
16 simple amidines (1, 7−8, 10−13, 19−26, and 29), seven N-
B
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 1. Structures of Cationic m-Terphenyl and 1,3-Dipyridylbenzene Derivatives 1−37
compd
R1
4-Am
R2
4″-Am
R3
R4
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
4-i-PrAm
4-Im
4″-i-PrAm
4″-Im
4″-DMTHP
4″-AmOH
4″-AmOMe
4″-Am
4″-Am
4″-DMTHP
4″-Am
4″-Am
4″-Am
3″-Am
3″-i-PrAm
3″-Im
3″-DMTHP
3″-AmOH
3″-AmOMe
3″-Am
3″-Am
4″-Am
4″-Am
6″-Am
5″-Am
5″-Am
H
3
H
4
4-DMTHP
4-AmOH
4-AmOMe
4-Am
H
5
H
6
H
7
Cl
8
4-Am
NO2
NO2
NH2
OH
OMe
H
9
4-DMTHP
4-Am
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
4-Am
4-Am
3-Am
3-i-PrAm
3-Im
H
H
3-DMTHP
3-AmOH
3-AmOMe
3-Am
H
H
H
Cl
3-Am
OMe
H
3-Am
3-Am
Cl
6-Am
5-Am
5-Am
5-Am
5″-Am
5-AmOMe
5-AmDMAA
4-Am
5″-AmOMe
5″-AmDMAA
4″-Am
3″-PhIA
3″-PyIA
4″-PhIA
4″-PyIA
4″-PhIA
4″-PyIA
H
3-PhIA
3-PyIA
3-PhIA
3-PyIA
3-PhIA
3-PyIA
4-PyIA
5-PyIA
Cl
Cl
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
O-i-Pr
O-i-Pr
H
a
All compounds were isolated as hydrochloride salts except 26 (acetate salt) and 28 (free base).
alkyl and N,N′-alkylene derivatives (2−4, 9, and 14−16), and
six prodrugs (5, 6, 17, 18, 27, and 28). Structural variations
include the orientation of the cationic groups, substituents on
the amidine nitrogen atoms and/or on the central aromatic
ring, and the insertion of nitrogen atoms in the outer aromatic
rings. Compounds 1−22 and 30−37 have m-terphenyl nuclei,
C
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Symmetric m-Terphenylamidine Derivatives and Prodrugs
a
Reagents and conditions: (a) 3- or 4-cyanophenylboronic acid, Pd(PPh3)4, aq Na2CO3, DME, reflux (52−81%); (b) EtOH, HCl, 1,4-dioxane, and
then NH3 or amine, EtOH; (c) NH2OH·HCl, t-BuOK, DMF; (d) Ac2O, AcOH, and then H2, 10% Pd/C, EtOH, AcOH; (e) H2, 10% Pd/C, EtOH;
(f) CH3I, t-BuOK, DMSO; (g) (CH3)2SO4, aq NaOH, dioxane.
a
Scheme 2. Synthesis of Asymmetric m-Terphenyldiamidines
a
Reagents and conditions: (a) 4-cyanophenylboronic acid, Pd(PPh3)4, aq Na2CO3, DME; (b) (3-cyanophenyl)boronic acid, Pd(PPh3)4, aq Na2CO3,
DME; (c) EtOH, HCl, 1,4-dioxane and then NH3, EtOH; (d) LiN(TMS)2, THF.
while analogues 23−24 and 25−29 have 1,3-bis(pyridin-3-
yl)benzene and 1,3-bis(pyridin-2-yl)benzene scaffolds, respec-
tively. All 37 compounds are novel except 13, which was
previously reported but as a different salt.61 The activities of
analogues 7, 19, 23, and 24 against T. cruzi have been
reported,65,66 although details of their syntheses have yet to be
reported. All target compounds were isolated as their
hydrochloride salts except acetate salt 26 (an alternative salt
form of 25) and free base 28.
starting materials 38, 39, and 41 were commercially available.
Nitro analogue 4067 was prepared by deamination of 2,6-
dibromo-4-nitroaniline. Methoxy derivative 42 was prepared by
bromination68−deamination67 of p-anisidine. Double Suzuki
couplings involving dibromobenzenes 38−42 and 2−2.5 equiv
of 3- or (4-cyanophenyl)boronic acid catalyzed by tetrakis-
(triphenylphosphine)palladium(0) readily gave terphenyldini-
triles 43−50 in yields of 52−81%. The nitriles (except 46) were
then subjected to modified Pinner reaction conditions (ethanol,
HCl gas, dioxane cosolvent)50,51 to generate the corresponding
imidate esters, which were then reacted immediately with
The syntheses of the symmetric m-terphenylamidines and
prodrugs 1−20 are depicted in Scheme 1. Dibromobenzene
D
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of 1,3-Dipyridylbenzeneamidines and Prodrugs
a
Reagents and conditions: (a) bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMSO; (b) 1,3-diiodobenzene, Pd(PPh3)4, Ag2CO3, THF; (c)
NH2OH·HCl, t-BuOK, DMSO; (d) Ac2O, AcOH, and then H2, 10% Pd/C; (e) 1,3-benzenediboronic acid, Pd(PPh3)4, Ag2CO3, THF; (f) Me2SO4,
LiOH·H2O, DMF; (g) dimethylaminoacetyl chloride·HCl, K2CO3, DMF.
ammonia (for simple amidines 1, 7, 8, 12, 13, 19, and 20),
isopropylamine (for N-isopropylamidines 2 and 14), ethyl-
enediamine (for imidazolines 3 and 15), or 2,2-dimethylpro-
pane-1,3-diamine (for 5,5-dimethyl-1,4,5,6-tetrahydropyrimi-
din-2-yl (DMTHP) derivatives 4, 9, and 16). Catalytic
hydrogenation of nitro-substituted diamidine 8 gave the
amino analogue 10. Conversion of nitrile 46 to its amidoxime
derivative using excess hydroxylamine in DMSO,69 followed by
O-acylation and catalytic hydrogenation,50 gave 5′-hydroxydia-
midine 11. Amidoxime prodrugs 5 and 17 were prepared by
similar treatment of nitriles 43 and 48 with hydroxylamine.69
The amidoxime bases were converted to their hydrochloride
salts for biological testing or underwent O-methylation to
methamidoxime derivatives 6 and 18, which were also
converted to their hydrochloride salts.
Syntheses of asymmetric m-terphenyldiamidines 21 and 22
(Scheme 2) were similar to those of their symmetric
counterparts, but their dinitrile precursors were prepared by
successive single Suzuki couplings. High selectivity for single
over double coupling has resulted from the use of
bromoiodobenzene rather than dibromobenzene starting
materials.70 The coupling between bromoiodobenzene 51 and
(4-cyanophenyl)boronic acid gave biphenyl 53 in 71% isolated
yield. An analogous Suzuki coupling involving bromoiodide 52
(prepared by iodination−deamination of 4-bromo-2-chloroani-
line)71,72 gave biphenyl 54 in 59% yield. Successive couplings of
bromobiphenyls 53 and 54 with (3-cyanophenyl)boronic acid
gave the desired asymmetric terphenyldinitriles 55 and 56
(83% and 74% yields). A Pinner synthesis employing dinitrile
55 gave asymmetric diamidine 21. Similar strategy using chloro
analogue 56 failed, presumably because of the extremely low
solubility of the nitrile in the reaction medium. Diamidine 22
was successfully prepared from dinitrile 56 using lithium
bis(trimethylsilyl)amide in THF.64
The preparation of dipyridylbenzene derivatives 23−29 is
shown in Scheme 3. 3-Bromopyridyl starting materials 57 (also
commercially available) and 58 were prepared by treatment of
5-bromo-N-(tert-butyl)picolinamide73 or 5- bromonicotinamide
with phosphorus oxychloride.73 The reactions of compounds
57 and 58 with bis(pinacolato)diboron and catalytic
PdCl2(dppf)74 gave boronate esters 59 and 60, which
underwent Suzuki coupling with 1,3-diiodobenzene under
anhydrous conditions (Pd(PPh3)4, Ag2CO3, THF)75 to give
1,3-di(pyridin-3-yl)benzenedinitriles 61 and 62 (60% and 69%
yields). Attempted Pinner syntheses of amidines 23 and 24
from dipyridylbenzenenitriles 61 and 62 were unsuccessful.
The nitriles were treated with excess hydroxylamine in DMSO
to give amidoxime intermediates 63 and 64. The amidoximes
were treated with acetic anhydride in acetic acid to generate the
N′-acetoxy derivatives in situ, which were hydrogenated at 60
psi over 10% Pd/C in acetic acid.69 The use of neat acetic acid
in place of acetic acid−ethanol69 proved to be necessary for
selective cleavage of the oxygen−nitrogen bond (amidine
deprotection) over the oxygen−carbon bond (reversion to the
amidoxime).
Similar strategy was employed for the syntheses of the
di(pyridin-2-yl)benzene analogues except for the reversal of
functional groups for the Suzuki couplings. 2-Bromopyridylni-
triles 65 and 66 were prepared by dechlorobromination of 6-
chloronicotinonitrile76 or 2-chloro-4-cyanopyridine using phos-
E
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of m-Terphenyl-bis-arylimidamides (Bis-AIAs)
a
Reagents and conditions: (a) 3-nitrophenylboronic acid, Pd(PPh3)4, aq Na2CO3, DME, reflux; (b) SnCl2·2H2O, EtOH; (c) Et3N (for 77 only),
benzonitrile or 2-cyanopyridine, NaN(TMS)2, THF; (d) 4-nitrophenylboronic acid pinacol ester, Pd(PPh3)4, aq Na2CO3, DME; (e) H2, 60 psi, 10%
Pd/C, EtOH; (f) Fe, NH4Cl, aq EtOH.
a
Scheme 5. Synthesis of m-Terphenyl Monoarylimidamides (Mono-AIAs)
a
Reagents and conditions: (a) phenylboronic acid, Pd(PPh3)4, aq Na2CO3, DME; (b) NBS, THF; (c) NaNO2, H2SO4, EtOH; (d) BuLi, THF and
then triisopropyl borate; (e) (CH3)2CHI, Cs2CO3, K2CO3, DMF; (f) 86 or 87, Pd(PPh3)4, aq Na2CO3, DME; (g) SnCl2·2H2O, EtOH; (h)
benzonitrile or 2-cyanopyridine, NaN(TMS)2, THF.
F
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Antiprotozoal Activities, Cytotoxicities, and Selectivity Indices of Compounds 1−37 in Vitro
a
b
c
d
T. b. rhodesiense
f
T. cruzi
f
P. falciparum
f
L. amazonensis
j
g
h
i
k
e
f
compd
IC50 (μM)
SITb
IC50 (μM)
SITc
IC50 (μM)
SIP
IC50 (μM)
SIL
cytotox IC50 (μM)
1
0.004
0.047
0.068
0.140
10.9
8494
2157
37
71.6
>150
18.9
151
<1
<1
<1
<1
0.024
0.016
0.073
0.002
>10
1279
6583
35
31.2
102
2
3
2.55
122
4
871
>14
4
49347
l
l
5
>150
92.6
65.5
57.2
>150
149
ND
<1
<1
>2
ND
>1
<1
<1
4
ND
>150
19.2
21.9
>150
>150
>150
>150
33.0
125
6
4.44
>10
<2
7
0.031
0.020
0.692
0.004
0.004
0.005
0.055
0.617
1.60
700
0.039
0.018
0.003
0.136
0.038
0.048
0.038
0.031
0.619
0.006
8.23
561
8
>7481
>217
>39791
>37500
7266
2268
>243
20
>8376
>46781
>1103
>3947
693
l
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
>150
59.1
35.0
141
3319
>4194
51
>1
2
>150
31.7
56.6
5.60
20.3
30.9
137
20.4
45.4
11.2
24.1
50.2
55.9
27.1
46.6
43.4
>150
>150
>150
117
0.809
2.55
70
1
9498
<1
2
<1
<1
<1
2
11.3
2
9.31
2
0.044
0.029
0.052
0.097
0.006
0.527
0.017
0.022
26.3
709
4695
544
130
3720
>285
>8824
>6856
>6
0.033
0.028
0.015
0.010
0.015
0.058
0.040
0.045
3.56
944
4964
1947
1248
1485
>2577
>3763
>3339
>42
129
1
28.5
12.7
22.3
>150
>150
>150
>150
62.9
>150
4.10
2.50
2.52
2.72
2.27
2.64
9.69
3.77
46.6
6.40
5.12
>150
76.5
150
<1
<1
ND
ND
ND
>1
<1
ND
2
l
l
l
9.84
6
72.3
>150
2.09
0.053
2.14
0.388
2.04
1.54
4.18
2.98
0.486
0.077
0.071
0.006
0.038
0.208
0.039
0.219
>10
l
1.17
>128
8
>1939
58
0.533
0.019
0.635
1.94
2.43
2
133
4
47
1
450
0.095
0.907
0.123
1.02
26
3
66
1
7
13
22
2
0.178
0.109
32.4
13
1
58
24
2
12
0.211
0.933
1.88
13
10
2
<1
2
<1
12.3
<1
1
3.075
0.046
0.014
1
m
PMD
0.003
0.003
0.004
11436
2000
1275
1004
464
n
FMD
23.3
1.30
<1
o
MSP
p
BNZ
115
q
CQ
0.125
0.004
612
r
ATM
34884
s
AMB
0.124
t
PPT
0.017
a
b
c
Trypanosoma brucei rhodesiense (STIB900).83 Trypanosoma cruzi Tulahuen C2C4 Lac Z.83 Plasmodium falciparum (K1, chloroquine resistant).83
d
e
f
Leishmania amazonensis infected CD-1 mouse intracellular amastigotes.82 Cytotoxicity to L6 rat myoblast cells.83 The IC50 values are the mean of
g
two independent assays. Individual values differed by less than 50% of the mean value. Selectivity index for T. b. rhodesiense expressed as the ratio
IC50(L6 cells)/IC50(T. b. rhodesiense). Selectivity index for T. cruzi expressed as the ratio IC50(L6 cells)/IC50(T. b. cruzi). Selectivity index for P.
falciparum expressed as the ratio IC50(L6 cells)/IC50(P. falciparum). The IC50 values are the mean of three independent assays. The standard
h
i
j
k
deviations varied from the mean values by 8−64%. Selectivity index for L. amazonensis expressed as the ratio IC50(L6 cells)/IC50(L. amazonensis).
l
m
n
o
p
q
r
s
t
Value not determinable. Pentamidine. Furamidine. Melarsoprol. Benznidazole. Chloroquine. Artemisinin. Amphotericin-B. Podophyllotox-
in.
phorus tribromide.77 Various attempts at preparing boronic
acids or esters from 65, 66, or their 2-chloro precursors were
unsuccessful. Suzuki couplings of 2-bromopyridines 65 and 66
with 1,3-phenyldiboronic acid under anhydrous conditions
(Pd(PPh3)4, Ag2CO3, THF)75 gave 1,3-di(pyridin-2-yl)-
benzenedinitriles 67 and 68 (63% and 87% yields). Compound
67 was also prepared in 91% yield by the coupling of 6-
chloronicotinonitrile76 and the diboronic acid in toluene/2 M
aqueous Na2CO3.47 Nitriles 67 and 68 were reacted with excess
hydroxylamine in DMSO to give amidoxime intermediates 69
G
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and 70, which underwent O-acetylation followed by catalytic
hydrogenation to give amidine hydrochlorides 25 and 29. The
former compound was also isolated as acetate salt 26. Amidine
25 was also prepared directly from nitrile 67 using lithium
bis(trimethylsilyl)amide in THF.64 The prodrugs, methamidox-
ime 27 (hydrochloride salt) and dimethylaminoacetoxyamidine
28 (free base), were prepared by O-methylation or O-acylation
of amidoxime 69. Compound 27 was also prepared as the
hydrochloride salt (59% yield) directly from nitrile 67 using
methoxylamine hydrochloride in the presence of triethylamine
and thioglycolic acid.78
The syntheses of bis-AIAs 30−35 are depicted in Scheme 4.
A double Suzuki coupling between dibromobenzene 39 and 3-
nitrophenylboronic acid gave the symmetric dinitroterphenyl
71 in 76% isolated yield. Reduction of 71 with tin(II) chloride
dihydrate in ethanol gave the diamine 72, which was reacted
with benzonitrile or 2-cyanopyridine in the presence of sodium
bis(trimethylsilyl)amide in THF79 to give symmetric bis-
phenylimidamide 30 and the corresponding di(pyridin-2-yl)
derivative 31. Similar strategy was employed for the asymmetric
analogues 32−35 except for the requirement of successive
single Suzuki couplings. Bromoiodobenzenes 51 and 5271,72
were coupled with 4-nitrophenylboronic acid pinacol ester to
give bromobiphenyls 73 and 74, which in turn were coupled
with 3-nitrophenylboronic acid to give terphenyls 75 and 76
(52% and 55% yields overall). Hydrogenation of dinitroter-
phenyl 75 over 10% Pd in ethanol gave diamine 77 (72% yield
as the HCl salt). The chloro-substituted dinitro analogue 76
was reduced to diamine 78 (69% yield) using iron powder and
ammonium chloride in aqueous ethanol. Diamines 77 and 78
were reacted with benzonitrile or 2-cyanopyridine in the
presence of sodium bis(trimethylsilyl)amide in THF79 to give
bis-AIAs 32−35 (34−64% yields).
include pentamidine and furamidine (against T. b. rhodesiense
and P. falciparum), melarsoprol (against T. b. rhodesiense),
benznidazole (against T. cruzi), chloroquine and artemisinin
(against P. falciparum), amphotericin B (against L. amazonen-
sis), and podophyllotoxin (against L6 cells).
T. b. rhodesiense. A total of 17 compounds were highly
active with IC50 values below 100 nM, and 11 of these
analogues had selectivity indices above 2000 (more selective
than furamidine and melarsoprol). Five congeners exhibited
IC50 values below 10 nM (comparable in potency to the three
standard drugs). The most potent were lead compound 1 and
derivatives 10 and 11, all exhibiting IC50 values of 4 nM and
selectivity indices above 8000, followed by analogues 12 (IC50
= 5 nM, SI = 7270) and 23 (IC50 = 6 nM, SI = 3720). Eight
congeners (2, 7, 8, 19, 20, 25, 26, and 31) exhibited IC50 values
between 10 and 50 nM, of which analogues 8, 25, and 26 had
selectivity indices above 6000 and derivatives 2 and 20 had
selectivity indices above 2000. Four derivatives (3, 13, 21, and
22) showed IC50 values between 50 and 100 nM, of which
congener 13 had a selectivity index greater than 2000. Another
nine compounds (4, 9, 14, 16, 24, 30, 32, 34, and 35) exhibited
IC50 values between 100 nM and 1.0 μM.
The group of 17 most highly active compounds with IC50
values below 100 nM included 14 simple diamidines, N-
isopropylamidine 2, imidazoline 3, and AIA derivative 31. The
diamidines included all 11 of the terphenyldiamidines (1, 7, 8,
10−13, and 19−22) and three of the five dipyridyl diamidines
23, 25, and 26.
Among the terphenylamidine derivatives 1−22, optimal
potency and selectivity were observed when both cationic
groups were oriented para to the central ring. Lead compound
1 (IC50 = 4 nM, SI ≈ 8500), was over 10 times more potent
and over 4 times more selective than its regioisomer 13 (IC50
=
The syntheses of mono-AIAs 36 and 37 (Scheme 5) began
with the Suzuki coupling of bromoaniline 79 and phenyl-
boronic acid to give biphenylamine 80 (70%). This
intermediate underwent bromination to 81 (N-bromosuccini-
mide in THF, 83%), followed by deamination to bromobi-
phenyl 82 (sodium nitrite and sulfuric acid in ethanol, 86%).
The latter underwent lithiation followed by quenching with
triisopropyl borate to give crude boronic acid 83. This
intermediate underwent Suzuki couplings with bromoisopro-
poxynitrobenzenes 8680 and 8781 (prepared by Williamson
ether syntheses from the corresponding phenols 84 and 8581)
to give nitroterphenyls 88 (82%) and 89 (91%). The nitro
compounds were reduced to the corresponding amines 90
(83%) and 91 (94%) using tin(II) chloride dihydrate in
ethanol. The amines were reacted with 2-cyanopyridine in the
presence of sodium bis(trimethylsilyl)amide in THF to give
mono-AIAs 36 (41%) and 37 (50%) as their HCl salts.
55 nM), with both amidine functions situated meta to the
central ring. The asymmetric 3,4″-diamidine 21 was similar in
potency (IC50 = 52 nM) to 13 but about 4 times less selective.
The introduction of substituents on the amidine nitrogen
atoms resulted in diminished activity. The N-isopropylamidines
2 and 14 were more than 10 times less potent than the
corresponding simple amidines 1 and 13. The corresponding
imidazoline derivatives 3 and 15 showed a further 2-fold
decrease in potency. The DMTHP derivatives 4, 9, and 16
showed varied degrees of decreased potencies relative to the
corresponding amidines 1, 8, and 13. The prodrugs 5, 6, 17,
and 18 were inactive in vitro, as expected.30,33
The introduction of substituents on the central ring had
varied effects upon activity, depending upon the orientation of
the cationic groups. The potency of lead compound 1 was
retained in its amino and hydroxy derivatives 10 and 11, and
these two analogues also had the highest selectivity indices
(above 37 500). The methoxy derivative 12 was similar in both
potency and selectivity to parent molecule 1, while methoxy
analogue 20 was slightly more potent and selective than its
parent molecule 13. None of the chloro analogues 7, 19, and 22
showed advantages of both potency and selectivity compared to
parent molecules 1, 13, and 21, respectively. The nitro
analogue 8 was quite potent (IC50 = 20 nM), albeit 5 times
less potent than parent amidine 1, and showed at least
comparable selectivity.
IN VITRO ANTIPROTOZOAL ACTIVITIES,
SELECTIVITIES, AND STRUCTURE−ACTIVITY
RELATIONSHIPS
■
In vitro activities of target compounds against T. b. rhodesiense
strain STIB900, the intracellular amastigote form of the T. cruzi
Tulahuen strain C2C4, the chloroquine resistant P. falciparum
strain K1, and L. amazonensis intracellular amastigotes were
measured following established protocols,82,83 and the results
are shown in Table 2. Cytotoxicities against L6 rat myoblast
cells83 were determined to calculate the selectivity indices (the
ratios of cytotoxic IC50 values to antiprotozoal IC50 values) of
each compound for each of the four parasites. Standard drugs
The orientation of both amidine functions para to the central
ring was also required for optimal potency among the
regioisomeric dipyridylbenzenediamidines 23−25 and 29.
The points of attachment of the pyridine rings proved to be
H
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
less crucial. Analogues 23 (IC50 = 6 μM, SI ≈ 3700) and 25
(IC50 = 17 μM, SI > 8800), with both cationic groups
positioned para to the central ring, were highly potent and
highly selective for the parasite, and their potencies varied by
less than 3-fold. Their regioisomers with both amidine moieties
situated meta to the central ring were over 50 times less potent
(23 vs 24, 25 vs 29). The hydrochloride salt 25 and acetate salt
26 (different salts of the same diamidine base) gave similar
results in the in vitro assays.
The AIA derivatives 30−37 were, as a whole, less potent and
less selective compared to the amidine derivatives 1−29. Even
though the bis-pyridylimidamide 31 was the sixth most potent
overall (IC50 = 19 nM), it was the least selective (SI = 133) of
the 13 compounds with IC50 values below 50 nM. None of the
other AIA derivatives had IC50 values below 100 nM.
T. cruzi. The bis-pyridylimidamides 31, 33, and 35 were
most potent against the intracellular amastigote form of the T.
cruzi Tulahuen strain. The order of decreasing potencies
continued with the bis-phenylimidamide, monopyridylimida-
mide, and diamidine derivatives. The pyridylimidamide 31
(IC50 = 53, SI = 47) showed the most promising results, being
about 25 times more potent than benznidazole (IC50 = 1.30
μM, SI = 115). The other pyridyl analogues 33 (IC50 = 388
nM) and 35 (IC50 = 1.54 μM) showed potencies greater than
or comparable to benznidazole but selectivity indices below 10.
Compound 31 merits further investigation, as selectivity indices
above 10 are a common cutoff value for in vivo testing against
T. cruzi. None of the other compounds exhibited IC50 values
below 2 μM.
P. falciparum. Twenty-one compounds exhibited IC50
values below 50 nM (compared to IC50 = 46 nM for
pentamidine) against the chloroquine resistant P. falciparum
strain K1. The two most potent derivatives 4 and 9, with IC50
values of 2 and 3 nM, respectively, and each having a selectivity
index above 45 000, were more potent and selective for the
parasite than artemisinin (IC50 = 4 nM, SI = 34 900).
Analogues 16 and 31 each had IC50 values of 6 nM, while
congener 22 had an IC50 value of 10 nM and a selectivity index
of 1250. Only analogue 16 had a selectivity index below 1000.
Four derivatives (2, 8, 21, and 23) exhibited IC50 values
between 10 and 20 nM. All had selectivity indices above 1000,
with those of 2 and 8 being above 5000. Twelve compounds (1,
7, 11−14, 19, 20, 25, 26, 32, and 34) had IC50 values between
20 and 50 nM, of which seven analogues (1, 11, 13, 14, 20, 25,
and 26) had selectivity indices above 1000. Four derivatives (3,
24, 29, and 30) were less potent, with IC50 values between 50
and 100 nM. Overall, 15 compounds (1, 2, 4, 8, 9, 11, 13, 14,
16, 20−23, 25, and 26) exhibited IC50 values below 50 nM and
selectivity indices above 1000.
The DMTHP derivatives 4, 9, and 16 (IC50 values of 2, 3,
and 6 nM, respectively, and SI > 45 000 for 4 and 9) were the
most potent terphenyl N-alkyldiamidines, accounting for three
of the four most potent and the three most highly selective
analogues overall. They were also highly specific for this
parasite, being 70−230 times more potent against P. falciparum
compared to T. b. rhodesiense. The N-isopropylamidines 2 and
14 (IC50 of 16 and 31 nM, respectively, and SI > 4000 for each)
also exhibited enhanced potencies and selectivities relative to
corresponding amidines 1 and 13 and were the eighth and
twelfth most potent overall. The imidazoline derivatives 3 and
15 (IC50 of 73 and 620 nM, respectively) were both less potent
and less selective than the corresponding amidines 1 and 13.
Thus, diamidine 1 and its N-substituted derivatives 2−4 were
all more potent than the corresponding 3,3″-isomers 13−16.
The introduction of substituents on the central ring had
varied effects upon activity, depending on the nature of the
substituent and the position of the cationic groups. Both the
potency and selectivity of the 4,4″-diamidine 1 were diminished
by the presence of chloro, amino, and methoxy substituents on
the central ring (derivatives 7, 10, and 12). The nitro analogue
8 was slightly more potent than 1 with significantly enhanced
selectivity. The potency of 3′3″-diamidine 13 was enhanced by
a chloro or methoxy group on the central ring (analogues 19
and 20), but selectivity was enhanced only in the latter
instance. The potency of asymmetric diamidine 21 was
enhanced, but its selectivity was diminished, by a chlorine
atom on the central ring (derivative 22).
The order of potencies of the four dipyridylbenzene
diamidines was the same as that against T. b. rhodesiense.
Analogues 23 (IC50 = 15 nM) and 25 (IC50 = 40 nM), each
having both amidine groups positioned para to the central ring,
were more potent than their respective counterparts 24 (IC50
=
58 nM) and 29 (IC50 = 77 nM) having two meta-amidine
functions. Selectivity indices ranged from around 1500 for 23 to
greater than 3500 for 25. Acetate salt 26, an alternative salt
form of 25, gave results similar to those of hydrochloride salt
25. The difference in IC50 values between the most potent (23)
and the least potent (29) analogues was just over 5-fold,
compared to a nearly 200-fold differences in their IC50 values
against T. b. rhodesiense.
The AIA derivatives, as a whole, were less potent and less
selective compared to the amidine derivatives. The symmetric
bis-pyridylimidamide 31 (IC50 = 6 nM, SI = 450) was the most
potent AIA derivative and most selective for this parasite,
followed by the asymmetric bis-phenylimidamides 32 and 34,
with IC50 values around 40 nM but selectivity indices below
100. These three compounds had the three lowest selectivity
indices among the 21 compounds with IC50 values below 50
nM, despite the fact that analogue 31 was the third most potent
overall. Congener 30 was the only other AIA derivative with an
IC50 value below 100 nM.
The group of 21 compounds with IC50 values below 50 nM
includes 13 simple amidines (10 terphenyl and three
dipyridylbenzene derivatives), five N-alkylamidines, and three
AIA analogues. In contrast to the structural requirements for
antitrypanosomal activity, antiplasmodial potency was less
dependent on the positions of the cationic groups and was
enhanced by alkylation of the amidine nitrogen atoms. The
regioisomeric diamidines 1, 13, and 21 lacking central ring
substituents showed rather small differences in potency (IC50
values of 24, 38, and 15 nM, respectively) but with activity
favored by at least one cation in the para position. The chloro
analogue 22 (a derivative of 21) was the most potent of the
terphenyl simple diamidines (IC50 = 10 nM).
L. amazonensis. Given that other AIAs have shown
outstanding in vitro and promising in vivo antileishmanial
activity in past work,57,59,60,84 compounds 30−37 were assayed
against L. amazonensis in the intracellular amastigote model.82
The bis-pyridylamidamides 31, 33, and 35 were the three most
potent compounds (in descending order). Analogue 31 (IC50
95 nM) was slightly more potent than amphotericin B (IC50
=
=
124 nM), while congener 33 (IC50 = 123 nM) showed
comparable potency and derivative 35 (IC50 = 211 nM)
showed lower potency. The IC50 values for the five mono- and
bis-phenylimidamides ranged between 907 nM and 2.43 μM for
I
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
bis-AIAs 32 and 30, respectively. Thus, pyridylimidamide 31
and the corresponding phenyl analogue 30 were the most and
least potent. For the relatively small number of compounds
tested, the antileishmanial data are somewhat similar to anti-T.
cruzi data. Bis-pyridylimidamides 31, 33, and 35 were the three
most potent compounds (in descending order) against both
parasites. However, the orders of activity of the phenyl-
imidamides differed, without the clear distinction of potencies
between mono- and bis-phenylimidamides, as observed against
T. cruzi.
idine and furamidine attained 1/4 cures at 4 × 5 mg/kg ip, and
any compound attaining a higher cure rate is considered to
have enhanced activity in vivo.
Three diamidines, lead compound 1 and dipyridyl analogues
23 and 25, each cured 4/4 infected mice at the 4 × 5 mg/kg ip
regimen, exhibiting excellent cure rates comparable to that of
melarsoprol. These three compounds also attained 4/4 cures in
single doses of 10 mg/kg for diamidines 1 and 25 and 5 mg/kg
for analogue 23. Acetate salt 26, a different salt form of 25, also
effected 4/4 cures at the 4 × 5 mg/kg regimen. Diamidines 12
and 11 cured 2/4 and 1/4 infected mice, respectively. Prodrugs
of diamidines 1 and 25 demonstrated efficacy with the 4 × 25
mg/kg oral regimen. Compound 5, the amidoxime derivative of
1, cured 3/4 mice, while methamidoxime derivative 6 attained
1/4 cures. Compound 28, the dimethylaminoacetoxy derivative
of dipyridyl analogue 25, cured 3/4 mice, and the
methamidoxime derivative 27 attained 2/4 cures. Mean survival
rates exceeded 50 days for prodrugs 5, 27, and 28..
IN VIVO ACTIVITIES AND STRUCTURE−ACTIVITY
■
RELATIONSHIPS
T. b. rhodesiense. Twenty compounds (including six
prodrugs) were tested in mice infected with the T. b. rhodesiense
strain STIB900 (Table 3).85 The amidines and N-alkylamidines
Table 3. In Vivo Antiprotozoal Activity of Select m-
a
Terphenyl and Dipyridylbenzene Derivatives
Two simple amidine moieties oriented para to the central
ring, regardless of the presence or position of nitrogen atoms in
the outer rings, were required for maximum efficacy in vivo
(compounds 1, 23, and 25). Among the terphenyl analogues,
the introduction of alkyl substituents on the amidine nitrogen
(1 vs 2, 1 vs 3) or meta orientations of one or both amidine
groups (1 vs 13, 1 vs 21, 12 vs 20) resulted in loss of efficacy.
In these respects, a high correlation exists between potencies in
vitro and efficacies in vivo. All analogues of diamidine 1 with
substituents on the central ring were less effective in vivo.
Derivatives 10−12 had potencies in vitro similar to that of the
parent molecule, but the methoxy (12), hydroxy (11), and
amino (10) analogues attained 2/4, 1/4, and 0/4 cures,
respectively, but all with significantly prolonged mean survival
times (exceeding 35 days) compared to untreated mice (10
days or less). The chloro and nitro derivatives 7 and 8, which
were less potent in vivo, cured no mice but with less prolonged
survival times. In cases where prodrugs exist, the prodrugs
reflected the efficacies of the parent amidines. Prodrugs of the
highly effective amidines 1 and 25 demonstrated oral efficacy,
as stated above. Neither amidine 13 nor its prodrugs (17 and
18) were effective in vivo.
L. donovani. On the basis of their in vitro antileishmanial
efficacy and selectivity, compounds 31, 33, and 35 were
selected for in vivo evaluation. These molecules were first
tested in groups of two uninfected BALB/c mice at five daily
doses of 30 mg/kg intraperitoneally to ensure that they were
well tolerated in the animals prior to evaluation of in vivo
antileishmanial efficacy in larger groups. Compounds 31 and 33
were toxic at this dose, with the animals displaying tremors and
hyperactivity after the second and first doses, respectively. Mice
were euthanized after these adverse effects were observed, and
compounds 31 and 33 were not tested further in vivo. After no
overt signs of toxicity were observed when compound 35 was
given at 5 × 30 mg/kg ip, an in vivo antileishmanial efficacy
test82 was carried out with this compound. Unfortunately, only
b
c
d
e
compd
dose (mg/kg)
route
cured/infected
MSD (days)
1
4 × 5
1 × 10
4 × 5
4 × 5
4 × 25
4 × 25
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 5
4 × 25
4 × 25
4 × 5
4 × 5
4 × 5
1 × 5
4 × 5
1 × 10
4 × 5
4 × 25
4 × 25
4 × 5
4 × 5
4 × 5
ip
ip
ip
ip
po
po
ip
ip
ip
ip
ip
ip
po
po
ip
ip
ip
ip
ip
ip
ip
po
po
ip
ip
ip
4/4
4/4
0/4
0/4
3/4
1/4
0/4
0/4
0/4
1/4
2/4
0/4
0/4
0/4
0/4
0/4
4/4
4/4
4/4
4/4
4/4
2/4
3/4
4/4
1/4
1/4
>60
>60
2
13.5
9.25
>51.25
>34.25
15.25
20
3
5
6
7
8
10
11
12
13
17
18
20
21
23
>35
>41.25
>38
18.5
7.25
17.75
10
34.75
>60
>60
25
>60
>60
26
27
28
>60
>53.75
>52
f
MLSP
>60
g
FMD
>46
h
PMD
>38
a
Female NMRI mice infected with Trypanosoma brucei rhodesiense
(STIB900).85 Administration was once per day on 4 consecutive days
b
c
for all compounds, and single doses for select compounds. Route of
administration: intraperitoneal (ip) or oral (po). Mice that survived
for 60 days after infection without showing a parasitemia relapse were
considered as cured. Mean survival days after infection. All mice were
d
e
used to calculate the MSD (the relapsed mice and the cured mice,
f
g
h
where MSD > 60 days). Melarsoprol. Furamidine. Pentamidine.
were given intraperitoneally (four daily doses of 5 mg/kg),
while the prodrugs were given orally (four daily doses of 25
mg/kg). Mice that survived for 60 days after infection without
showing a parasitemia relapse were considered as cured. This
model is highly stringent, mimicking the acute stage of HAT,
and the low doses applied allowed for the distinction of
activities among the highly trypanocidal compounds. Pentam-
23
11% inhibition of liver parasitemia (mean
standard
deviation, n = 4) was observed when L. donovani-infected
BALB/c mice were given compound 35 at 5 × 30 mg/kg ip. In
the same experiment, miltefosine treatment of infected BALB/c
mice at 5 × 10 mg/kg orally resulted in 96 2% inhibition of
liver parasitemia (n = 4).
J
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Summary of antiprotozoal SARs of m-terphenyl derivatives.
nitrogen atoms, especially in the DMTHP derivatives and to a
lesser extent in the N-isopropyl analogues. The imidazoline
derivatives, however, were less potent than the corresponding
amidines. In other examples from this lab, the effect of N-
alkylation has varied from one class of compounds to
another.36−38,50,51 The effects of substituents on the central
ring varied with the nature of the substituents and the
orientation of the cationic groups. Only one dipyridyl analogue,
the bis(6-amidinopyridin-3-yl) derivative 23, was more potent
than the corresponding terphenyldiamidine.
The bis-pyridylimidamide 31 was unique in demonstrating
activity against all four parasites in vitro. Despite its high
potencies against T. b. rhodesiense and P. falciparum, it was
much less selective for these parasites over mammalian cells
compared to the amidine derivatives. This compound, having
both cations in the meta position and a chlorine atom attached
to the central ring, was also the most active against T. cruzi and
L. amazonensis. The SAR of these compounds against T. cruzi
was rather clearly defined: the bis-pyridylimidamides were the
most potent, followed by the bis-phenylamidamides, the mono-
AIAs, and the amidine derivatives. The bis-pyridylimidamides
were also the most active against L. amazonensis; however, the
IC50 values of the less potent bis-phenylamidamides and mono-
AIAs overlapped, and the amidine derivatives were not tested in
this model.
A large number of mechanisms of antimicrobial activity have
been proposed for the diamidine class of molecules.89−101
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 regard to drug design but is advantageous
with regard to the potential development of drug resistance. It
has been suggested that the highly selective toxicity of many of
the compounds in this class toward pathogenic organisms over
DISCUSSION
■
The m-terphenyl derivatives exhibited complementary SAR
profiles against the four parasites, as summarized in Figure 2. In
general, the amidine derivatives were more active against T. b.
rhodesiense and P. falciparum, while the AIAs showed more
promising results against the other parasites. The orientation of
both amidine moieties para to the central ring, regardless of the
presence or position of nitrogen atoms in the outer rings, was
required for optimal activity against T. b. rhodesiense. Altered
positions of the amidine groups, alkylation of the amidine
nitrogen atoms, and substituents on the central ring all resulted
in diminished activity either in vitro or in vivo. A general trend
of diminished activities of N-alkyl analogues has been
observed,37,38,40,41,50,51,86,87 but the effects of substituents on
the central or outer rings have varied from one class of
compounds to another.29,38,51 The insertion of nitrogen atoms
into phenyl rings has resulted in retained or enhanced activity
in the diphenylfuran and p-terphenyl analogues.46,62,64,85 The
replacement of a central furan ring with 1,3-phenylene was
clearly beneficial with respect to the diamidine analogues but
not for the prodrugs. Compound 1 attained 4/4 cures in the
STIB900 acute model, compared to 1/4 cures for furamidine.
Its similarly efficacious dipyridyl analogues 23 and 25 were also
more active in vivo than the corresponding dipyridyl derivatives
of furamidine.46,85 Pafuramidine was more efficacious than the
corresponding amidoxime88 and has been one of few prodrugs
to attain 4/4 cures in the STIB900 model at four daily 25 mg/
kg oral doses.85 By contrast, methamidoxime 6 (a prodrug of 1)
cured 1/4 mice under the same conditions, while the
corresponding amidoxime 5 attained 3/4 cures. Methamidox-
ime 27 (a prodrug of 25) and the di(pyridin-2-yl) analogue of
pafuramidine85 each cured 2/4 mice. Another prodrug of 25,
dimethylaminoacetoxy derivative 28, attained 3/4 cures.
Antiplasmodial activity was less dependent upon the position
of the cationic groups (although still favored by at least one
para cation) and was enhanced by alkylation of the amidine
K
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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.97 Thus, the only way to optimize these compounds for
specific targets is via focused parallel synthesis and in vitro
testing or focusing on the active transport systems. Unfortu-
nately, not enough is known with regard to the specificities of
the transporters to aid in the drug design process.
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.85,102−108 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 pentamidine,104,106 while nephrotoxicity resulted
in the cancellation of human clinical trials with pafuramine.85
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.
class, did not show promising results against T. cruzi, AIA
derivative 31 proved to be 25 times more potent than the
clinical standard benznidazole and thus warrants further
evaluation against this parasite.
EXPERIMENTAL SECTION
■
General Experimental Methods. In vitro antiprotozoal activities
against T. b. rhodesiense (STIB900), T. cruzi (Tulahuen C2C4), P.
falciparum (chloroquine resistant K1),83 and L. amazonensis intra-
cellular amastigotes,82 cytotoxicities against L6 rat myoblast cells,83
and in vivo activities against T. b. rhodesiense (STIB900)85 and L.
donovani (LV82)82 were measured following established protocols. All
protocols and procedures used in the current study were reviewed and
approved by the local veterinary authorities of the Canton Basel-Stadt,
Switzerland, or the Institutional Animal Care and Use Committee at
The Ohio State University. The data in the T. b. rhodesiense model
were generated at the time when the determination of survival was still
accepted by the authorities.
Uncorrected melting points were measured on a Thomas-Hoover
1
capillary melting point apparatus. H NMR spectra were recorded in
DMSO-d6 on a Varian Gemini 2000 or Unity Plus 300 MHz
spectrometer or a Varian Inova 400 MHz spectrometer. Spectra were
recorded at 300 MHz unless the higher field strength is specified. 13C
NMR spectra were recorded in DMSO-d6 on a Varian Unity Plus
operating at 75 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-seal 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
MgSO4 or Na2SO4 before being evaporated under reduced pressure.
Normal phase gravity and flash column chromatography were
performed using Davisil grade 633, type 60A silica gel (200−425
mesh). Reverse phase flash chromatography was performed as
previously described.41 Analytical HPLC chromatograms were
recorded on a Hewlett-Packard 1090 series II or Agilent 1200
chromatograph using a Zorbax Rx C8 column (4.6 mm × 75 mm, 3.5
μm) maintained at 40 °C and UV photodiode array detection at 230,
254, 265, 290, and 320 nm. Area % values are reported at the
wavelengths where the strongest signals of the products were
observed. Mobile phases consisted of mixtures of acetonitrile (0−
75%) or methanol (0−95%) in water containing formic acid (80 mM),
ammonium formate (20 mM), and triethylamine (15 mM). Samples
were eluted at appropriate gradients at a flow rate of 1.5 mL/min.
Similar analyses were performed on a Hewlett-Packard 1100 system
using a Zorbax SB C8 column (3.0 mm × 100 mm, 3.5 μm), eluting at
0.6 mL/min. Preparative reverse phase HPLC was performed on a
Varian ProStar chromatography workstation configured with two PS-
215 pumps fitted with 50 mL pump heads, a Dynamax Microsorb C18
(60 Å) column (41.4 cm × 25 cm, 8 μm), PS-320 variable wavelength
UV−vis detector, and a PS-701 fraction collector. Mobile phases
consisted of mixtures of acetonitrile (0−75%) or methanol (0−95%)
in water containing formic acid (40 mM) and ammonium formate (10
mM). Flow rates were maintained at 40 mL/min. Select fractions were
analyzed for purity by analytical HPLC as described above. Pooled
purified fractions were evaporated under reduced pressure, recon-
stituted 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 spectrometer or
a VG analytical 70-SE spectrometer. Elemental analyses were
performed by Atlantic Microlab, Norcross, GA, and, unless stated
otherwise, were within 0.4% of calculated values. CHN analyses were
performed by combustion using a Perkin-Elmer 2400 or Carlo Erba
1108 automatic analyzer. Chlorine analyses were performed by flask
combustion followed by ion chromatography. The compounds
reported as salts were frequently analyzed correctly for fractional
A number of the m-terphenyl analogues, as well as diamidine
derivatives of other classes of compounds,36−41,50,51 have
shown high potencies against P. falciparum in vitro. However,
the lack of a reliable animal model has been a major obstacle in
their further development against malaria. Standard rodent
models, which must use Plasmodium species that are non-
pathogenic to humans due to the high specificity of P.
falciparum for human erythrocytes, do not necessarily predict
the efficacy of a given drug in human malaria,109 and their
relevance to human malaria has been questioned.110,111 For
example, pafuramidine, which is known to be effective in
human malaria,33 was inactive in a commonly used P. berghei
mouse model,112 as were pentamidine and other analogues of
furamidine.29,56,112 Murine P. falciparum models employing
immunodeficent mice engrafted with human erythrocytes or
hepatocytes (humanized mice)110,112−114 should more accu-
rately predict the efficacies of new therapies in human malaria,
although they are generally more costly and more difficult to
handle than standard rodent models.114 These models may be
useful in determining which standard rodent model offers
better correlation with P. falciparum for given compounds; for
example, one study suggested that P. vinckei may be a better
surrogate than P. berghei for diamidines.112 These findings may
provide at least a starting point in the further evaluation of
other diamidines.
CONCLUSIONS
■
This communication describes several novel diamidine
derivatives that show excellent in vitro potency against T. b.
rhodesiense, are curative in a mouse model of early stage HAT,
and have the potential for reduced toxicity when compared to
the prototype molecule furamidine. Most outstanding were
diamidines 1, 23, and 25, which attained 4/4 cures not only in
four daily intraperitoneal doses of 5 mg/kg but also in single
doses of 10 mg/kg or lower. Prodrugs of compounds 1 and 25
(derivatives 5 and 28) also attained 3/4 cure rates in this model
with four daily 25 mg/kg oral doses. Further evaluations of
these compounds against other T. brucei strains and a mouse
model of late stage HAT are in progress. A number of
compounds, most significantly the DMTHP derivatives 4, 9,
and 16, showed promising antiplasmodial potencies in vitro and
merit in vivo evaluation, contingent upon the selection of
appropriate animal models. Although these compounds, as a
1
moles of water and/or other solvents; in each case H NMR spectra
L
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were consistent with the analysis. The purity of all final compounds
was determined to be ≥95% by combustion analysis.
ice. The precipitated crude product was chromatographed on silica,
eluting with CHCl3/MeOH (20:1) and recrystallized twice from
MeOH−H2O to give white crystals (532 mg, 32%): mp 141−142 °C;
1H NMR δ 8.04 (s, 1H), 7.96 (d, J = 8.3 Hz, 4H), 7.86 (d, J = 8.4 Hz,
4H), 7.79 (dd, J = 7.7 and 1.6 Hz, 2H), 7.03 (t, J = 8.1 Hz, 2H), 3.48
(s, 6H). Anal. (C22H22N4O2) C, H, N.
The base was converted to the HCl salt using aqueous HCl to give a
white solid (570 mg, 87% from salt conversion, 28% from the
amidoxime): mp 192 °C (dec); 1H NMR δ 8.08 (m,1H), 8.01 (d, J =
8.5 Hz, 4H), 7.46 (d, J = 8.5 Hz, 4H), 7.82 (dd, J = 7.6 and 1.6 Hz,
2H), 7.65 (t, J = 7.7 Hz, 1H), 3.87 (s, 6H); ESI MS m/z 375.5 ([M +
H ] + o f f r e e b a s e ) ; H P L C 9 9 a r e a % . A n a l .
(C22H22N4O2·1.95HCl·1.3H2O) C, H, N.
4,4″-Diamidino-5′-chloro-m-terphenyl Dihydrochloride (7).
7 was prepared from nitrile 44 (1.03 g, 3.28 mmol). The product was
purified by reverse phase flash chromatography and converted to the
HCl salt using aqueous HCl to give a solid (532 mg, 53%): mp >250
°C (dec); 1H NMR δ 9.52 (br s, 4H), 9.27 (br s, 4H), 8.16 (d, J = 8.5
Hz, 4H), 8.12 (t, J = 1.5 Hz, 1H), 8.00 (d, J = 8.5 Hz, 4H), 7.96 (d, J =
1.5 Hz, 2H); ESI MS m/z 349.2 ([M + H]+ of free base); HPLC 99.4
area %. Anal. (C20H17ClN4·2HCl·2.3H2O) C, H, N, Cl.
4,4″-Diamidino-5′-nitro-m-terphenyl Dihydrochloride (8). 8
was prepared from nitrile 45 (1.64 g, 5.03 mmol). The product was
purified by reverse phase flash chromatography and converted to the
HCl salt using aqueous HCl to give a solid (929 mg, 42%): mp >350
°C (dec); 1H NMR δ 9.58 (br s, 4H), 9.34 (br s, 4H), 8.62 (d, J = 1.5
Hz, 2H), 8.59 (t, J = 1.6 Hz, 1H), 8.25 (d, J = 8.6 Hz, 4H), 8.05 (d, J =
8.5 Hz, 4H); ESI MS m/z 360.1 ([M + H]+ of free base); HPLC 100
area %. Anal. (C20H17N5O2·2.2HCl·1.9H2O) C, H, N, Cl.
4,4″-Bis[(5,5-dimethyl)-1,4,5,6-tetrahydropyrimidin-2-yl]-5′-
nitro-m-terphenyl Dihydrochloride (9). 9 was prepared analo-
gously to 4 from nitrile 45 (1.66 g, 5.11 mmol), but the imidate
intermediate was prepared in neat ethanolic HCl. The product was
purified by preparative HPLC and converted to the HCl salt using
ethanol HCl and ether to give an off-white solid (1.37g, 47%): mp
>389 °C (dec); 1H NMR δ 10.70 (br s, 4H), 8.61 (m, 3H), 8.27 (d, J
= 8.5 Hz, 4H), 8.01 (d, J = 8.6 Hz, 4H), 3.25 (s, 8H), 1.08 (s, 12H);
ESI MS m/z 496.4 ([M + H]+ of free base); HPLC 99.0 area %. Anal.
(C30H33N5O2·2HCl·H2O) C, H, N, Cl.
General Procedure for Amidines 1−4, 7−9, 12−16, and 19−
21. 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 three-neck flask equipped with a gas inlet tube, a thermometer, and
a drying tube. The reaction mixture was then sealed and stirred at
ambient temperature until the nitrile was no longer detectable. The
reaction mixture was diluted with ether. The crude imidate was filtered
off, dried under high vacuum over KOH, and then reacted immediately
with an excess of ammonia or the appropriate amine in EtOH. The
reaction mixture was diluted with ether, and the crude product was
filtered off. The product was purified by direct recrystallization or by
reverse phase HPLC or flash chromatography followed by conversion
to the dihydrochloride salt using aqueous or ethanolic HCl.
4,4″-Diamidino-m-terphenyl Dihydrochloride (1). 1 was
prepared from nitrile 43 (0.56 g, 2.01 mmol) with ammonium
carbonate used in place of ammonia. After purification by preparative
HPLC, the product was recrystallized from ethanolic HCl−ether to
give a white solid (346 mg, 44%): mp >350 °C; 1H NMR δ 9.50 (br s,
3H), 9.24 (br s, 3H), 8.14 (t, J = 1.6 Hz, 1H), 8.10 (d, J = 8.7 Hz, 4H),
7.99 (d, J = 8.2 Hz, 4H), 7.87 (dd, J = 7.7 and 1.6 Hz, 2H), 7.68 (t, J =
7.7 Hz, 1H); ESI MS m/z 315.1 ([M + H]+ of free base); HPLC 100
area %. Anal. (C20H18N4·2HCl·0.7H2O) C, H, N, Cl.
4,4″-Bis(N-isopropyl)amidino-m-terphenyl Dihydrochloride
(2). 2 was prepared from nitrile 43 and isopropylamine. The product
was purified by reverse phase flash chromatography and recrystallized
from aqueous HCl to give a solid (690 mg, 61%): mp 253−254 °C; 1H
NMR δ 9.69 (d, J = 8.2 Hz, 2H), 9.57 (br s, 2 H), 9.21 (br s, 2H), 8.07
(m, 5H), 7.86 (m, 6H), 7.69 (t, J = 7.8 Hz, 1H), 4.13 (m, 2H), 1.31 (d,
J = 6.4 Hz, 12H); ESI MS m/z 399.2 ([M + H]+ of free base); HPLC
100 area %. Anal. (C26H30N4·2HCl·1.4H2O) C, H, N, Cl.
4,4″-Bis(imidazolin-2-yl)-m-terphenyl Dihydrochloride (3). 3
was prepared from nitrile 43 and ethylenediamine. The crude
precipitated product was recrystallized from aqueous HCl to give a
white solid (1.26g, 79%): mp >250 °C (dec); 1H NMR δ 10.90 (br s,
3H), 8.22 (m, 5H), 8.16 (d, J = 8.7 Hz, 4H), 7.91 (dd, J = 7.8 and 1.7
Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 4.04 (s, 8H); ESI MS m/z 367.2
([M
+
H]+ of free base); HPLC 99.0 area %. Anal.
(C24H22N4·2HCl·2.8H2O) C, H, N, Cl.
4,4″-Diamidino-5′-amino-m-terphenyl Dihydrochloride (10).
Nitro compound 8 (0.536 g, 1.24 mmol) was hydrogenated for 3 h at
60 psi over 10% Pd/C (90 mg, 0.055 mmol) in EtOH/H2O (2:1, 150
mL). The reaction mixture was filtered through Celite and evaporated
to a white solid. The product was recrystallized from EtOH−H2O to
4,4″-Bis[(5,5-dimethyl)-1,4,5,6-tetrahydropyrimidin-2-yl]-m-
terphenyl Dihydrochloride (4). 4 was prepared from nitrile 43 (961
mg, 3.43 mmol) and 2,2-dimethylpropane-1,3-diamine. The product
was recrystallized from aqueous HCl to give white granules (1.33 g,
74%): mp >350 °C (dec); 1H NMR δ 10.39 br s, 3H), 8.14 (t, J = 1.7
Hz, 1H), 8.11 (d, J = 8.6 Hz, 4H), 7.97 (d, J = 8.6 Hz, 4H), 7.87 (dd, J
= 7.7 and 1.6 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 3.24 (s, 8H), 1.07 (s,
12H); ESI MS m/z 451.2 ([M + H]+ of free base); HPLC 99.0 area %.
Anal. (C30H34N4·2HCl·1.25H2O) C, H, N, Cl.
1
give white crystals (401 mg, 80%): mp >250 °C (dec); H NMR δ
9.48 (br s, 4H), 9.25 (br s, 4H), 7.97 (d, J = 9.1 Hz, 4H), 7.93 (d, J =
9.1 Hz, 4H), 7.22 (t, J = 1.5 Hz, 1H), 7.01 (t, J = 1.5 Hz, 2H), 5.53 (br
s, 2H); ESI MS m/z 330.2 ([M + H]+ of free base); HPLC 100 area %.
Anal. (C20H19N5·2HCl·2.7H2O) C, H, N, Cl.
4,4″-Bis(N′-hydroxy)amidino-m-terphenyl Dihydrochloride
(5). Potassium tert-butoxide (8.42 g, 75.0 mmol) was added to a
stirred solution of hydroxylamine hydrochloride (5.25 g, 75.6 mmol)
in dry DMSO (45 mL). After 30 min nitrile 43 (2.11 g, 7.52 mmol)
was added, and the mixture was stirred overnight. The reaction
mixture was poured over ice−water, and the precipitated amidoxime
4,4″-Diamidino-5′-hydroxy-m-terphenyl Dihydrochloride
(11). Dinitrile 46 (0.77 g, 2.6 mmol) was converted to 4,4″-bis(N′-
hydroxy)amidino-5′-hydroxy m-terphenyl (free base) in 99% yield
following the procedure for compound 5: mp 160−163 °C; 1H NMR
δ 9.68 (br s, 3H), 7.76 (d, J = 9 Hz, 4H), 7.69 (d, J = 9 Hz, 4H), 7.37
(s, 1H), 7.05 (s, 2H), 5.84 (br s, 4H); 13C NMR δ 158.4, 151.5, 141.5,
141.0, 131.6, 126.6, 126.2, 116.2, 112.9; MS (ESI) m/z 363 ([M +
H]+).
To a solution of the diamidoxime (0.36 g, 1 mmol) in glacial acetic
acid (10 mL) was slowly added acetic anhydride (0.35 mL). After
stirring overnight, the solvent was evaporated to dryness under
reduced pressure and the oily residue obtained was used without
further purification, where it was dissolved in absolute ethanol (25
mL) and glacial AcOH (10 mL) followed by addition of 10%
palladium on carbon (80 mg). The mixture was hydrogenated on a
Parr apparatus at 50 psi for 4 h at room temperature. The mixture was
filtered through a filter aid and the filter pad washed with water. The
filtrate was evaporated under reduced pressure and the residue was
collected and washed with ether to give 11 as an acetate salt. The
diamidine was purified by neutralization with Na2CO3 followed by
filtration of the resultant solid and washing with water (3×). Finally,
1
base was filtered off (2.05 g, 79%): mp >215 °C (dec); H NMR δ
9.71 (br s, 2H), 7.98 (s, 1H), 7.80 (s, 8H), 7.71 (d, J = 7.3 Hz, 2H),
7.57 (t, J = 7.7 Hz, 1H), 5.89 (br s, 4 H); HPLC 100 area %. Anal.
(C20H18N4O2·0.1H2O) C, H, N.
An aliquot of the base (478 mg, 1.38 mmol) was recrystallized from
aqueous HCl to give a white solid (221 mg, 38%): mp >260° (dec);
1H NMR δ 11.33 (br s, 2H), 9.16 (br s, 4H), 8.11 (s, 1H), 8.08 (d, J =
8.5 Hz, 4H), 7.87 (m, 6H), 7.67 (t, J = 7.7 Hz, 1H); ESI MS m/z
347.2 ([M + H]+ of free base); HPLC 99 area %. Anal.
(C20H18N4O2·2HCl) C, H, N, Cl.
4,4″-Bis(N′-methoxy)amidino-m-terphenyl Dihydrochloride
(6). A mixture of amidoxime base 5 (1.56 g, 4.49 mmol) and
potassium tert-butoxide (1.07 g, 9.53 mmol) in DMSO (20 mL) was
stirred for 2 h followed by the addition of iodomethane (0.9 mL, 14
mmol). The reaction mixture was stirred overnight and poured over
M
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the free base was stirred with ethanolic HCl overnight, diluted with
4H), 8.33 (s, 1H), 8.21 (s, 2H), 8.14 (d, J = 7.5 Hz, 2H), 7.87 (dd, J =
7.7 and 1.6 Hz, 2H), 7.78 (dm, J = 7.8 Hz, 2H), 7.73 (t, J = 7.7 Hz,
2H), 7.67 (t, J = 7.8 Hz, 1H); ESI MS m/z 347.2 ([M + H]+ of free
base); HPLC 100 area %. Anal. (C20H18N4O2·2HCl·0.7H2O·-
0.8EtOH) C, H, N, Cl.
3,3″-Bis(N′-methoxy)amidino-m-terphenyl Dihydrochloride
(18). 18 was prepared from amidoxime base 17 either by the method
employed for compound 6 or using dimethyl sulfate and aqueous
NaOH in dioxane. The combined products from multiple experiments
were chromatographed on silica, eluting with hexanes/EtOAc (2:1)
and converted to the HCl salt using ethanolic HCl−ether to give a
white solid (1.69 g, 88% from salt conversion, 21% from amidoxime):
mp 235−237 °C (dec); 1H NMR δ 8.78 (br s, 2H), 8.34 (s, 1H), 8.24
(s, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.85 (dd, J = 7.7 and 1.6 Hz, 2H),
7.80 (d, J = 8.0 Hz, 2H), 7.67 (m, 3H), 3.89 (s, 6H); ESI MS m/z
375.2 ([M + H]+ of free base); HPLC 98.4 area %. Anal.
(C22H22N4O2·2HCl·0.3H2O) C, H, N, Cl.
ether, and the solid formed was filtered and dried to give the diamidine
1
hydrochloride salt 11 (77%): mp >300 °C; H NMR δ 10.12 (br s,
1H), 9.48 (br s, 4H), 9.26 (br s, 4H), 7.98 (d, J = 9 Hz, 4H), 7.95 (d, J
= 9 Hz, 4H), 7.52 (s, 1H), 7.23 (s, 2H); 13C NMR (DMSO-d6) δ
165.19, 158.7, 145.1, 140.6, 128.8, 127.3, 126.9, 116.8, 114.2. HRMS
calcd for C20H19N4O m/z 331.1559 ([M + H]+ for free base);
observed 331.1564. Anal. (C20H18N4O·2.0HCl·0.8H2O·0.5EtOH) C,
H, N.
4,4″-Diamidino-5′-methoxy-m-terphenyl Dihydrochloride
(12). 12 was prepared from nitrile 47 (1.01 g, 3.22 mmol). The
product was purified by reverse phase flash chromatography and
converted to the HCl salt using aqueous HCl to give a gray solid (960
mg, 71%): mp >225 °C (dec); 1H NMR δ 9.51 (br s, 4H), 9.28 (br s,
4H), 8.11 (d, J = 8.5 Hz, 4H), 7.99 (d, J = 8.5 Hz, 4H), 7.71 (m, 1H),
7.40 (d, J = 1.3 Hz, 2H), 3.95 (s, 3H); ESI MS m/z 345.2 ([M + H]+
of free base); HPLC 98.4 area %. Anal. (C21H20N4O·2HCl·1.6H2O) C,
H, N, Cl.
3,3″-Diamidino-m-terphenyl Dihydrochloride (13). 13 was
prepared from nitrile 48 (1.01 g, 3.60 mmol). The product was
purified by preparative HPLC and converted to the HCl salt using
aqueous HCl to give a solid (716 mg, 51%): mp >350 °C; 1H NMR δ
9.69 (br s, 4H), 9.34 (br s, 4H), 8.48 (s, 1H), (8.37, s, 2H), 8.18 (d, J
= 8.0 Hz, 2H), 7.88 (d, J = 7.8 Hz, 4H), 7.75 (t, J = 7.8 Hz, 2H), 7.67
(t, J = 2.8 Hz, 1H); ESI MS m/z 315.3 ([M + H]+ of free base);
HPLC 100 area %. Anal. (C20H18N4·2HCl·0.6H2O) C, H, N, Cl.
3,3″-Bis(N-isopropyl)amidino-m-terphenyl Dihydrochloride
(14). An aliquot (0.95 g 2.13 mmol) of the imidate intermediate
prepared from nitrile 48 was reacted with isopropylamine and purified
analogously to 2 as a white solid (681 mg, 68%): mp >240 °C (dec);
1H NMR δ 9.83 (d, J = 8.0 Hz, 2H), 9.68 (br s, 2H), 9.22 (br s, 2H),
3,3″-Diamidino-5′-chloro-m-terphenyl Dihydrochloride
(19). 19 was prepared from nitrile 49 (973 mg, 3.09 mmol). The
product was purified by reverse phase flash chromatography and
converted to the HCl salt using aqueous HCl to give a white solid
1
(469 mg, 26%): mp >350 °C; H NMR δ 9.65 (br s, 4H), 9.32 (br s,
4H), 8.42 (s, 1H), 8.36 (s, 2H), 8.23 (d, J = 7.9 Hz, 2H), 7.99 (d, J =
1.4 Hz, 2H), 7.91 (d, J = 7.9 Hz, 2H), 7.76 (t, J = 7.8 Hz, 2H); ESI MS
m/z 349.2 ([M + H]+ of free base); HPLC 100 area %. Anal.
(C20H17ClN4·2HCl·0.75H2O) C, H, N, Cl.
3,3″-Diamidino-5′-methoxy-m-terphenyl Dihydrochloride
(20). 20 was prepared from nitrile 50 (1.00 g, 3.23 mmol). The
product was purified by reverse phase flash chromatography and
converted to the HCl salt using aqueous HCl to give a light gray solid
1
(990 mg, 73%): mp >325 °C; H NMR δ 9.64 (br s, 4H), 9.27 (br s,
4H), 8.32 (s, 2H), 8.18 (d, J = 7.8 Hz, 2H), 7.97 (s, 1H), 7.87 (d, J =
7.9 Hz, 2H), 7.74 (t, J = 7.8 Hz, 2H), 7.44 (d, J = 1.2 Hz, 2H), 3.95 (s,
3H); ESI MS m/z 345.2 ([M + H]+ of free base); HPLC 100 area %.
Anal. (C21H20N4O·2HCl·1.3H2O) C, H, N, Cl.
8.50 (s, 1H), (8.23, s, 2H), 8.13 (d, J = 7.4 Hz, 2H), 7.86 (dd, J = 7.5
and 1.5 Hz, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.73 (t, J = 7.6 Hz, 2H),
7.67 (t, J = 7.7 Hz, 1H), 4.13 (m, 1H), 1.33 (d, J = 6.4 Hz, 12H); ESI
MS m/z 398.3 ([M + H]+ of free base); HPLC 98.7 area %. Anal.
(C26H30N4·2HCl·1.2H2O) C, H, N, Cl.
3,3″-Bis(imidazolin-2-yl)-m-terphenyl Dihydrochloride (15).
15 was prepared from an aliquot of the imidate above (0.99 g, 2.22
mmol) and ethylenediamine. The product was purified by reverse
phase flash chromatography and then converted to the HCl salt using
ethanolic HCl−ether to give a white solid (775 mg, 79%): mp 325−
327 °C (dec); 1H NMR δ 11.04 (br s, 3H), 8.75 (s, 2H), 8.43 (s, 1H),
8.26 (d, J = 8.0 Hz, 2H), 8.08 (d, J = 7.9 Hz, 2H), 7.90 (dd, J = 7.5 and
1.5 Hz, 2H), 7.78 (t, J = 7.9 Hz, 2H), 7.67 (t, J = 7.8 Hz, 1H), 4.05 (s,
8H); ESI MS m/z 367.4 ([M + H]+ of free base); HPLC 99.5 area %.
Anal. (C24H22N4·2HCl·1.6H2O) C, H, N, Cl.
3,4″-Diamidino-m-terphenyl Dihydrochloride (21). 21 was
prepared from nitrile 55 (1.00 g, 3.58 mmol). The crude product was
recrystallized from aqueous HCl to give a white solid (1.18 g, 85%):
1
mp >220 °C (dec); H NMR δ 9.61 (br s, 2H), 9.52 (br s, 2H), 9.32
(br s, 2H), 9.27 (br s, 2H), 8.29 (m, 1H), 8.23 (m, 1H), 8.18 (dm, J =
8.0 Hz, 1H), 8.12 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H), 7.89
(m,3H), 7.75 (t, J = 7.8 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H); ESI MS m/z
315.1 ([M + H]+ of free base); HPLC 97.5 area %. Anal.
(C20H18N4·2HCl·1.8H2O) C, H, N, Cl.
3,4″-Diamidino-5′-chloro-m-terphenyl Dihydrochloride
(22). Lithium bis(trimethylsilyl)amide (1 M solution in THF, 10
mL, 10 mmol) was added dropwise to a suspension of nitrile (1.00 g,
3.18 mmol) in dry THF (15 mL). The mixture was stirred overnight
and then cooled to 0 °C (ice−salt bath) before the slow addition of
saturated ethanolic HCl. After 2 h the stirred solution was diluted with
ether to precipitate the crude product, which was purified by
preparative HPLC. Conversion to the HCl salt using EtOH−aqueous
HCl followed by recrystallization from water−acetone gave a white
3,3″-Bis[(5,5-dimethyl)-1,4,5,6-tetrahydropyrimidin-2-yl]-m-
terphenyl Dihydrochloride (16). 16 was prepared from nitrile 48
(1.02 g, 3.64 mmol) and 2,2-dimethylpropane-1,3-diamine. After
purification by preparative HPLC, the crude HCl salt (prepared using
aqueous HCl) was dissolved in water and treated with aqueous NaOH
to precipitate the free base. This was converted to the HCl salt using a
mixture of ethanolic HCl, dioxane, and ether to give a white solid (671
1
1
mg, 35%): mp >335 °C (dec); H NMR δ 10.51 (br s, 4H), 8.58 (s,
powder (750 mg, 56%): mp >260 °C (dec); H NMR δ 9.50 (br s,
1H), 8.37 (s, 2H), 8.16 (d, J = 8.0 Hz, 2H), 7.88 (dd, J = 8.7 and 1.6
Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.66 (t, J =
7.8 Hz, 1H), 3.52 (s, 8H), 1.07 (s, 12H); ESI MS m/z 451.3 ([M +
H ] + o f f r e e b a s e ) ; H P L C 1 0 0 a r e a % . A n a l .
(C30H34N4·2.3HCl·0.55H2O) C, H, N, Cl.
4H), 9.27 (br s, 4H), 8.30 (m, 1H), 8.23 (dm, J = 8.1 Hz, 1H), 8.20 (t,
J = 1.5 Hz, 1H), 8.17 (d, J = 8.5 Hz, 2H), 8.00 (m, 3H), 7.95 (t, J = 1.7
Hz, 1H), 7.90 (dm, J = 8.3 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H); ESI MS
m/z 349.1 ([M + H]+ of free base); HPLC 100 area %. Anal.
(C20H17ClN4·2HCl·1.75H2O) C, H, N, Cl.
3,3″-Bis(N′-hydroxy)amidino-m-terphenyl Dihydrochloride
(17). 17 was prepared following the procedure for amidoxime 5
from nitrile 48 (2.81 g, 10.0 mmol). The amidoxime base was
recrystallized from EtOH to give white crystals (3.57 g, 103%): mp
212−213 °C; 1H NMR δ 9.70 (br s, 2H), 8.03 (t, J = 1.6 Hz, 2H), 8.01
(t, J = 1.5 Hz, 1H), 7.78 (dm, J = 7.7 Hz, 2H), 7.73 (m, 2H), 7.71 (m,
2H), 7.60 (dd, J = 8.4 and 7.9 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 5.98
(br s, 4H). Anal. (C20H18N4O2·0.9EtOH.) C, H, N.
General Procedure for 1,3-Dipyridylbenzenediamidines 23−
25 and 29. A solution of the amidoxime (3−5 mmol) in acetic acid
was treated with acetic anhydride (5 mL), giving a precipitate. The
mixture was transferred to a hydrogenation bottle, and more AcOH
and 10% Pd/C (10−20 mol %) were added. The mixture was
hydrogenated at 60 psi until completion and filtered through Celite.
The product was purified by preparative HPLC and converted to the
HCl salt using aqueous HCl unless stated otherwise.
An aliquot of the base (511 mg, 1.27 mmol) was converted to the
HCl salt using ethanolic HCl−ether to give a white solid (546 mg,
1,3-Bis(6-amidinopyridin-3-yl)benzene Dihydrochloride
(23). 23 was prepared from amidoxime 63 (1.28 g, 3.65 mmol) as a
1
1
88%): mp >110 °C (dec); H NMR δ 11.32 (br s, 2H), 9.14 (br s,
white solid (668 mg, 47%): mp 342−343 °C; H NMR δ 9.71 (br s,
N
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4H), 9.50 (br s, 4H), 9.31 (d, J = 2.2 Hz, 2H), 8.68 (dd, J = 8.4 and 2.3
Hz, 2H), 8.52 (d, J = 8.4 Hz, 2H), 8.38 (m, 1H), 8.05 (dd, J = 7.8 and
1.7 Hz, 2H), 7.75 (t, J = 7.8 Hz, 1H); ESI MS m/z 317.1 ([M + H]+ of
free base); HPLC 100 area %. Anal. (C18H16N6·2HCl·1.5H2O) C, H,
N, Cl.
3,3″-Bis(benzimidoylamino)-5′-chloro-m-terphenyl Dihy-
drochloride (30). A solution of 5′-chloro-3,3″-diamino-m-terphenyl
(72) in THF (30 mL) was added dropwise to a mixture of sodium
bis(trimethylsilyl)amide (2 M solution in THF, 6.0 mL, 12.0 mmol)
and THF (6.0 mL). The mixture was stirred for 20 min and then split
into two portions.
1,3-Bis(5-amidinopyridin-3-yl)benzene Trihydrochloride
(24). 24 was prepared from amidoxime 64 (1.04 g, 2.97 mmol) as a
white solid (542 mg, 42%): mp >300 °C; 1H NMR δ 9.79 (br s, 4H),
9.42 (br s, 4H), 9.38 (d, J = 2.1 Hz, 2H), 9.04 (d, J = 2.1 Hz, 2H), 8.78
(t, J = 2.1 Hz, 2H), 8.57 (m, 1H), 8.04 (dd, J = 7.7 and 1.7 Hz, 2H),
7.56 (t, J = 7.8 Hz, 1H); ESI MS m/z 317.1 ([M + H]+ of free base);
HPLC 97.5 area %. Anal. (C18H18N6·2.8HC·0.8H2O) C, H, N, Cl.
1,3-Bis(5-amidinopyridin-2-yl)benzene Trihydrochloride
(25). 25 was prepared from amidoxime 69 (1.17 g, 3.35 mmol).
The product was converted to the HCl salt using ethanolic HCl−ether
to give a white solid (992 mg, 76%): mp >350 °C; 1H NMR δ 9.71 (br
s, 4H), 9.41 (br s, 4H), 9.15 (t, J = 1.1 Hz, 2H), 9.01 (s, 1H), 8.44 (d, J
= 8.5 Hz, 2H), 8.40 (dd, J = 8.7 and 2.0 Hz, 2H), 8.37 (dd, J = 7.8 and
1.8 Hz, 2H), 7.75 (t, J = 7.8 Hz, 1H); ESI MS m/z 317.1 ([M + H]+ of
free base); HPLC 100 area %. Anal. (C18H16N6·3HCl·0.6H2O) C, H,
N, Cl.
To the first aliquot was added a solution of benzonitrile (0.75 mL,
7.3 mmol), and the mixture was stirred for 3 days. More sodium
bis(trimethylsilyl)amide solution (3.0 mL, 6.0 mmol) was added, and
the reaction mixture was stirred an additional 3 h before being poured
over ice and extracted into CH2Cl2. The product was purified by
preparative HPLC and lyophilized. A solution of the lyophilized
product in EtOH, water, and 1 N HCl was evaporated, dissolved in hot
water, filtered, and lyophilized to give the HCl salt as a white powder
(682 mg, 42%): mp >200 °C (dec); 1H NMR (400 MHz) δ 11.72 (br
s, 2H), 9.95 (br s, 2H), 9.18 (br s, 2H), 8.07 (s, 1H), 8.02 (s, 2H), 7.97
(m, 6H), 7.89 (s, 2H), 7.80 (t, J = 7.4 Hz, 2H), 7.70 (m, 6H), 7.53 (d,
J = 7.9 Hz, 2H); ESI MS m/z 501.3 ([M + H]+ of free base); HPLC
98.8 area %. Anal. (C32H25ClN4·2HCl·1.5H2O) C, H, N, Cl.
3,3″-Bis(picolimidoylamino)-5′-chloro-m-terphenyl Dihy-
drochloride (31). 31 was prepared analogously to 30 using the
second aliquot from above and 2-cyanopyridine (0.89 g, 8.55 mmol) in
place of benzonitrile to give an off-white powder (661 mg, 41%): mp
1,3-Bis(5-amidinopyridin-2-yl)benzene Diacetate 26). 26 was
prepared and chromatographed analogously to 25 from amidoxime 69
(700 mg, 2.01 mmol). The product was converted to the free base
using aqueous NaOH. A mixture of the free base in H2O (20 mL) and
AcOH (2 mL) was heated and filtered, and the filtrate was lyophilized
to give the acetate salt (319 mg, 36%): mp >227−230 °C; 1H NMR δ
10.24 (br s, 8H), 9.10 (d, J = 1.4 Hz, 2H), 8.96 (t, J = 1.5 Hz 1H), 8.33
(m. 6H), 7.73 (t, J = 7.8 Hz, 1H), 1.79 (s, 6H); ESI MS m/z 317.1
1
>170 °C; H NMR (400 MHz) δ 11.94 (br s, 2H), 10.19 (br s, 2H),
9.46 (br s, 2H), 8.91 (d, J = 4.0 Hz, 2H), 8.55 (d, J = 7.9 Hz, 2H), 8.24
(t, J = 7.6 Hz, 2H), 8.08 (s, 1H), 8.02 (s, 2H), 7.96 (d, J = 7.7 Hz,
2H), 7.87 (m, 4H), 7.70 (t, J = 7.8 Hz, 2H), 7.54 (d, J = 7.7 Hz, 2H);
ESI MS m/z 503.3 ([M + H]+ of free base); HPLC 98.1 area %. Anal.
(C30H23ClN6·2HCl·2H2O) C, H, N, Cl.
+
H]+ of free base); HPLC 100 area %. Anal.
3,4″-Bis(benzimidoylamino)-m-terphenyl Dihydrochloride
(32). Triethylamine (1.5 mL, 10.8 mmol) was added to a stirred
suspension of 3,4″-diamino-m-terphenyl dihydrochloride (77, 1.01 g,
3.03 mmol) in THF (50 mL). After 1.5 h benzonitrile (1.0 mL, 9.80
mmol) was added, followed by the slow addition of sodium
bis(trimethylsilyl)amide (2 M solution in THF, 8.0 mL, 16.0 mmol).
The reaction mixture was stirred for 3 days, poured over ice, and
extracted into CHCl3. The product was purified by preparative HPLC
and lyophilized. A solution of the lyophilized product in EtOH and 1
N HCl was evaporated, dissolved in hot water, filtered, and lyophilized
to give the HCl salt as a white powder (558 mg, 34%): mp 216 °C; 1H
NMR (400 MHz) δ 11.70 (br s, 2H), 9.92 (br s, 2H), 9.15 (br s, 2H),
8.07 (s, 1H), 7.98 (m, 7H), 7.91 (d, J = 7.3 Hz, 1H), 7.79 (m, 4H),
7.70 (m, 6H), 7.61 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 7.3 Hz, 1H); ESI
MS m/z 467.6 ([M + H]+ of free base); HPLC 100 area %. Anal.
(C32H26N4·2HCl·1.5H2O) C, H, N, Cl.
3,4″-Bis(picolimidoylamino)-m-terphenyl Dihydrochloride
(33). 33 was prepared analogously to 32 with 2-cyanopyridine used
in place of benzonitrile as a yellow powder (1.04 g, 64%): mp 190 °C;
1H NMR (400 MHz) δ 11.96 (br s, 1H), 11.95 (br s, 1H), 10.17 (br s,
1H), 10.15 (br s, 1H), 9.45 (br s, 1H), 9.39 (br s, 1H), 8.91 (s, 2H),
8.56 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.1 Hz, 1H), 8.24 (m, 2H), 8.08
(s, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.97 (s, 1H), 7.92 (d, J = 7.7 Hz,
1H), 7.87 (m, 2H), 7.80 (m, 2H), 7.71 (t, J = 7.9 Hz, 1H), 7.76 (t, J =
7.8 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 7.8 Hz, 1H); ESI
MS m/z 469.6 ([M + H]+ of free base); HPLC 98.9 area %. Anal.
(C30H24N6·2HCl·1.9H2O) C, H, N, Cl.
3,4″-Bis(benzimidoylamino)-5′-chloro-m-terphenyl Dihy-
drochloride (34). To a solution of 5″-chloro-3,4″-diamino-m-
terphenyl (78, 1.01 g, 3.42 mmol) and benzonitrile (1.10 g, 10.7
mmol) in THF (40 mL) was slowly added sodium bis(trimethylsilyl)-
amide (2 M solution in THF, 7.0 mL, 14.0 mmol). The mixture was
stirred for 3 days. Workup and purification were analogous to 32. The
final product was recrystallized from hot water to give a gray powder
(954 mg, 49%): mp >200 °C (dec); 1H NMR (400 MHz) δ 11.71 (br
s, 2H), 9.95 (br s, 2H), 9.19 (br s, 1H), 9.17 (br s, 1H), 8.06 (m, 4H),
7.98 (m, 5H), 7.87 (d, J = 8.4 Hz, 2H), 7.80 (t, J = 7.3 Hz, 2H), 7.70
(m, 5H), 7.63 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 7.7 Hz, 1H); ESI MS
m/z 501.2 ([M + H]+ of free base); HPLC 98.7 area %. Anal.
(C32H25ClN4·2HCl·2.4H2O) C, H, N, Cl.
([M
(C18H18N6·2.25CH3CO2H·2H2O) C, H, N, Cl.
1,3-Bis-(5-N′-methoxyamidinopyridin-2-yl)benzene Tetra-
hydrochloride 27. To a suspension of diamidoxime 69 (348 mg,
1.00 mmol) in DMF (10 mL) was added LiOH·H2O (252 mg, 6.01
mmol, in 3 mL of H2O) which was followed by dimethyl sulfate (630
mg, 4.99 mmol). The reaction mixture was stirred overnight, after
which it was poured onto ice−water and the precipitate was filtered,
washed with water, and dried to give the free base of 27 in 77% yield:
1
mp 157−158 °C; H NMR δ 8.96 (s, 2H), 8.84 (s, 1H), 8.09−8.22
(m, 6H), 7.63 (t, J = 7.8 Hz, 1H), 6.30 (br s, 4H), 3.78 (s, 6H); 13C
NMR δ 156.1, 149.0, 146.8, 138.6, 134.3, 129.4, 127.5, 127.1, 124.7,
119.8, 60.7.
The free base (200 mg, 0.53 nM) was converted to the
tetrahydrochloride salt by stirring overnight in ethanol saturated
with HCl(g) to yield a pale yellow solid (205 mg, 69%): mp 196−
197.5 °C; MS m/z 377 ([M + H]+ of free base). Anal.
(C20H20N6O2·4.0 HCl·1.1H2O·0.5EtOH) C, H, N.
1,3-Bis[(5-[N′-dimethylaminoacetoxy]amidino)pyridin-2-yl]-
benzene (28). To a stirred solution of diamidoxime 69 (348 mg, 1.00
mmol) and K2CO3 (966 mg, 7 mmol) in DMF (10 mL) was added
dimethylaminoacetyl chloride hydrochloride (632 mg, 4 mmol). The
reaction mixture was stirred overnight at room temperature and then
poured onto brine solution. The precipitate was filtered, recrystallized
1
from methanol to furnish 28 in a 79% yield: mp 156−157.5 °C; H
NMR (DMSO-d6) δ 9.02 (s, 2H), 8.90 (s, 1H), 8.19−8.25 (m, 6H),
7.66 (m, 1H), 7.05 (s, 4H), 3.39 (s, 4H), 2.30 (s, 12H); 13C NMR
(DMSO-d6) δ 167.9, 157.2, 154.8, 147.6, 138.4, 135.6, 129.5, 127.9,
126.3, 125.0, 119.9, 58.2, 44.5; MS m/z 519 ([M + H]+ of free base).
HRMS calcd for C26H31N8O4 [M + H]+ 519.2468; observed 519.2457.
Anal. (C26H30N8O4·1.25H2O) C, H, N.
1,3-Bis(4-amidinopyryidin-2-yl)benzene Dihydrochloride
(29). 29 was prepared by the general procedure from amidoxime 70
(2.19 g,5.34 mmol). The product was converted to the HCl salt using
ethanolic HCl with a trace of water to give a pale pink solid (1.22 g,
1
59%): mp 309−310 °C; H NMR δ 9.92 (br s, 4H), 9.59 (br s, 4H),
9.14 (s,1H), 9.00 (d, J = 5.1 Hz, 2H), 8.64 (s, 2H), 8.35 (dd, J = 8.7
and 1.7 Hz, 2H), 7.84 (dd, J = 5.1 and 1.6 Hz, 2H), 7.75 (t, J = 7.8 Hz,
1H); ESI MS m/z 317.1 ([M + H]+ of free base); HPLC 97.7 area %.
Anal. (C18H16N6·2.4HCl·1.1H2O) C, H, N, Cl.
O
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3,4″-Bis(picolimidoylamino)-5′-chloro-m-terphenyl Dihy-
drochloride (35). 35 was prepared analogously to 34 with 2-
cyanopyridine used in place of benzonitrile. The HCl salt was prepared
using ethanolic HCl to give a yellow powder (668 mg, 34%): mp >190
(d, J = 1.5 Hz, 2H), 8.55 (t, J = 1.5 Hz, 1H), 8.18 (d, J = 8.3 Hz, 4H),
8.03 (d, J = 8.3 Hz, 4H). Anal. (C20H11N3O2·0.1H2O) C, H, N.
4,4″-Dicyano-5′-hydroxy-m-terphenyl (46). To a stirred
solution of 3,5-dibromophenol (41, 1.25 g, 5.0 mmol) and
tetrakis(triphenylphosphine)palladium (200 mg) in toluene (10 mL)
under a nitrogen atmosphere was added 5 mL of a 2 M aqueous
solution of Na2CO3 followed by 4-cyanophenylboronic acid (0.37 g,
2.5 mmol) in methanol (5 mL). The vigorously stirred mixture was
warmed to 80 °C for 12 h. The solvent was evaporated, and the
precipitate was partitioned between methylene chloride (200 mL) and
2 M aqueous Na2CO3 (15 mL) containing 3 mL of concentrated
ammonia. The organic layer was dried and then concentrated to
dryness under reduced pressure to afford 46 as a white solid in 76%
1
°C (dec); H NMR (400 MHz) δ 11.94 (br s, 2H), 10.18 (br s, 1H),
10.16 (br s, 1H), 9.47 (br s, 1H), 9.41 (br s, 1H), 8.92 (m, 2H), 8.54
(d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.24 (m, 2H), 8.06 (t, J =
7.5 Hz, 4H), 7.97 (d, J = 7.9 Hz, 1H), 7.89 (m, 2H), 7.86 (m, 2H),
7.71 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 7.9 Hz,
1H); ESI MS m/z 503.5 ([M + H]+ of free base); HPLC 98.9 area %.
Anal. (C30H23ClN6·2.5HCl·1.5H2O) C, H, N, Cl.
2-Isopropoxy-4-(picolimidoylamino)-5′-chloro-m-terphenyl
Dihydrochloride (36). 36 was prepared analogously to 34 from 4-
amino-5′-chloro-2-isopropoxy-m-terphenyl (90, 1.20 g, 3.55 mmol), 2-
cyanopyridine (0.67 g, 6.44 mmol), and sodium bis(trimethylsilyl)-
amide (2 M solution in THF, 3.0 mL, 6.0 mmol) in THF (60 mL).
The product was converted to the HCl salt using ethanolic HCl and
1
yield: mp 290−292 °C; H NMR δ 10.00 (br s, 1H), 7.92−7.94 (m,
8H), 7.58 (s, 1H), 7.15 (s, 2H); 13C NMR δ 158.5, 144.4, 140.5, 132.8,
127.8, 118.8, 116.9, 114.2, 110.3.
4,4″-Dicyano-5′-methoxy-m-terphenyl (47). 47 was prepared
from dibromobenzene 42 (1.60 g, 6.03 mmol) and 4-cyanophenylbor-
onic acid (2.21g, 15.0 mmol). Purification by column chromatography,
eluting with C2HCl2/hexanes (4:1) followed by recrystallization from
CH3CN−H2O, gave a solid (1.51 g, 81%): mp 239−241 °C; 1H NMR
δ 8.04 (d, J = 8.6 Hz, 4H), 7.95 (d, J = 8.5 Hz, 4H), 7.66 (t, J = 1.5 Hz,
1H), 7.37 (d, J = 1.5 Hz, 2H), 3.93 (s, 3H). Anal. (C21H14N2O2) C, H,
N.
1
ether to give a solid (699 mg, 41%): mp >154 °C (dec); H NMR
(400 MHz) δ 11.88 (br s, 1H), 10.14 (br s, 1H), 9.38 (br s, 1H), 8.91
(d, J = 4.3 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.24 (td, J = 7.8 and 1.4
Hz, 1H), 7.87 (td, J = 7.4 and 4.8 Hz, 1H), 7.76 (m, 2H), 7.74 (s, 1H),
7.71 (d, J = 1.7 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.61 (t, J = 1.5 Hz,
1H), 7.52 (t, J = 7.5 Hz, 2H), 7.43 (tt, J = 7.3 and 2.1 Hz, 1H), 7.32 (s,
1H), 7.15 (dd, J = 8.1 and 1.4 Hz, 1H), 4.71 (septet, J = 6.0 H, 1H),
1.31 (d, J = 6.0 Hz, 6H); ESI MS m/z 442.8 ([M + H]+ of free base);
HPLC 100 area %. Anal. (C27H24ClN3O·2HCl·0.25H2O) C, H, N, Cl.
2-Isopropoxy-5-(picolimidoylamino)-5′-chloro-m-terphenyl
Dihydrochloride (37). 37 was prepared analogously to 36 from 5-
amino-5′-chloro-2-isopropoxy-m-terphenyl (91, 1.42 g, 4.20 mmol) to
3,3″-Dicyano-m-terphenyl (48).61 48 was prepared from 1,3-
dibromobenzene (38, 2.66 g, 11.3 mmol) and 3-cyanophenylboronic
acid (3.75 g, 25.5 mmol). Column chromatography, eluting with
hexanes/EtOAc (3:1) mixtures followed by recrystallization from
CH3CN, gave a white solid (2.38 g, 75%): mp 165−167 °C (lit.61
1
1
164−165°); H NMR δ 8.37 (t, J = 1.5 Hz, 2H), 8.18 (ddd, J = 8.0,
give a solid (1.01 g, 50%): mp 172−177 °C; H NMR (400 MHz) δ
1.9, and 1.1 Hz, 2H), 8.13 (t, J = 1.6 Hz, 1H), 7.87 (dt, J = 7.7 and 1.4
Hz, 2H), 7.82 (dd, J = 8.0 and 1.5 Hz, 2H), 7.71 (t, J = 7.8 Hz, 2H),
7.55 (t, J = 7.6 Hz, 1H). Anal. (C20H12N2) C, H, N.
11.68 (br s, 1H), 10.03 (br s, 1H), 9.26 (br s, 1H), 8.89 (dd, J = 3.9
and 0.8 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.22 (td, J = 7.8 and 1.2 Hz,
1H), 7.85 (m, 2H), 7.75 (t, J = 7 1.6 Hz, 1H), 7.73 (s, 1H), 7.71 (t, J =
1.0 Hz, 1H), 7.67 (t, J = 1.7 Hz, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.51 (t,
J = 7.5 Hz, 2H), 7.43 (m, 2H), 7.33 (d, J = 9.1 Hz, 1H), 4.76 (septet, J
= 6.0 H, 1H), 1.31 (d, J = 6.0 Hz, 6H); ESI MS m/z 442.9 ([M + H]+
of free base); HPLC 99.1 area %. Anal. (C27H24ClN3O·2HCl·0.1H2O)
C, H, N, Cl.
3,3″-Dicyano-5′-chloro-m-terphenyl (49). 49 was prepared
from dibromobenzene 39 (1.63 g, 6.03 mmol) and 3-cyanophenylbor-
onic acid (2.22 g, 15.1 mmol). Purification by column chromatog-
raphy, eluting with CHCl3 followed by recrystallization from DMF,
1
gave a light gray solid (991 mg, 52%): mp 279−277 °C; H NMR δ
8.43 (t, J = 1.6 Hz, 2H), 8.23 (dm, J = 8.0 Hz, 2H), 8.11 (t, J = 1.6 Hz,
1H), 7.93 (d, J = 1.6 Hz, 2H), 7.90 (dt, J = 7.9 and 1.3 Hz, 2H), 7.71
(t, J = 7.9 Hz, 2H). Anal. (C20H11ClN2·0.1H2O) C, H, N.
General Suzuki Coupling Procedure for Cyano-Substituted
Terphenyls and Biphenyls 43−45, 47−50, and 53−56. A
solution of tetrakis(triphenylphosphine)palladium(0) (5−10 mol %)
and the dibromobenzene (1 equiv) in DME (50 mL) was stirred for
15−30 min under Ar. The boronic acid (2.2−2.5 equiv for double
couplings, 1−1.2 equiv for single couplings) was added, followed by
Na2CO3 (10% solution, 75 mL). The mixture was refluxed until
reaction was complete. The cooled reaction mixture was worked up by
extraction into an appropriate solvent. The product was purified by
chromatography on silica gel and recrystallization from an appropriate
solvent.
3,3″-Dicyano-5′-methoxy-m-terphenyl (50). 50 was prepared
from dibromobenzene 42 (1.33 g, 5.01 mmol) and 3-cyanophenylbor-
onic acid 1.84 g, 12.5 mmol). The product was chromatographed on
silica, eluting with CH2Cl2/hexanes (4:1) and recrystallized from
1
CH3CN−H2O to give a solid (1.32 g, 71%): mp 194−196 °C; H
NMR δ 8.38 (t, J = 1.5 Hz, 2H), 8.19 (dm, J = 8.7 Hz, 2H), 7.87 (dt, J
= 7.8 and 1.3 Hz, 2H), 7.70 (t, J = 1.5 Hz, 1H), 7.69 (t, J = 7.7 Hz,
2H), 7.37 (d, J = 1.5 Hz, 2H), 3.93 (s, 3H). Anal. (C21H14N2O) C, H,
N.
4,4″-Dicyano-m-terphenyl (43). 43 was prepared from 1,3-
dibromobenzene (38, 1.97 g, 8.35 mmol) and 4-cyanophenylboronic
acid (2.61g, 17.8 mmol). Column chromatography by gradient elution
using hexanes/EtOAc mixtures followed by recrystallization from
EtOAc−hexanes gave an ivory colored solid (1.59 g, 68%): mp 199−
203 °C; 1H NMR δ 8.09 (t, J = 1.8 Hz, 1H), 8.04 (d, J = 8.5 Hz, 4H),
8.97 (d, J = 8.4 Hz, 4H), 7.84 (dd, (d, J = 7.7 and 1.5 Hz, 2H), 7.66 (t,
J = 7.8 Hz, 1H); HPLC 100 area %. Anal. (C20H12N2) C, H, N.
4,4″-Dicyano-5′-chloro-m-terphenyl (44). 44 was prepared
from dibromobenzene 39 (1.62g, 6.00 mmol) and 4-cyanophenylbor-
onic acid (2.21 g, 15.0 mmol). Column chromatography, eluting with
CHCl3 followed by recrystallization from toluene containing a trace of
DMF, gave a white solid (1.48 g, 78%): mp 297−301 °C; 1H NMR δ
8.09 (m, 5H), 7.98 (d, J = 8.4 Hz, 4H), 7.92 (d, J = 1.5 Hz, 2H). Anal.
(C20H11ClN2) C, H, N.
3-Bromo-4′-cyanobiphenyl (53). 53 was prepared from
iodobenzene 51 (3.06 g, 10.8 mmol) and 4-cyanophenylboronic acid
(1.50 g, 10.2 mmol). The product was chromatographed on a silica
column, eluting with hexanes/EtOAc (9:1) and recrystallized from
toluene−hexanes to give white crystals (1.86 g, 71%): mp 56−57 °C;
1H NMR δ 7.97 (t, J = 1.8 Hz, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.92 (d, J
= 8.9 Hz, 2H), 7.77 (ddd, J = 7.7, 1.6, and 1.0 Hz, 1H), 7.66 (ddd, J =
8.1, 2.0, and 1.1 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H). Anal. (C13H8BrN)
C, H, N.
3-Bromo-5-chloro-4′-cyanobiphenyl (54). 54 was prepared
from iodobenzene 52 (868 mg, 2.73 mmol) and 4-cyanophenylbor-
onic acid (402 mg, 2.73 mmol). The product was purified on a silica
column, eluting with hexane/EtOAc (9:1) to give a solid (0.47 g,
59%): mp 128−129 °C; 1H NMR δ 7.96 (m, 5H), 7.86 (m, 1H), 7.81
(m, 1H); HPLC 100 area %. Anal. (C13H7BrClN) C, H, N.
3,4″-Dicyano-m-terphenyl (55). 55 was prepared from bromo-
biphenyl 53 (2.59 g, 10.0 mmol) and 3-cyanophenylboronic acid (1.77
g, 12.1 mmol). The product was chromatographed on a silica column,
eluting with CHCl3 and recrystallized from CH3CN−H2O to give
white crystals (2.35 g, 83%): mp 139−142 °C; 1H NMR δ 8.37 (t, J =
4,4″-Dicyano-5′-nitro-m-terphenyl (45). 45 was prepared from
dibromobenzene 40 (1.69 g, 6.01 mmol) and 4-cyanophenylboronic
acid (2.21g, 15.0 mmol). The reaction mixture was poured over ice to
precipitate the crude product, which was recrystallized from DMF−
EtOH to give a solid (1.18 g, 60%): mp 319−320 °C; 1H NMR δ 8.58
P
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.5 Hz, 1H), 8.16 (dm, J = 8.0 Hz, 1H), 8.12 (t, J = 1.8 Hz, 1H), 8.06
(d, J = 8.7 Hz, 2H), 7.97 (d J = 8.7 Hz, 2H), 7.87 (dt, J = 7.8 and 1.3
Hz, 1H), 7.82 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.64 (t, J = 7.7 Hz,
1H). Anal. (C20H12N2) C, H, N.
gray solid (1.07 g, 101%): 1H NMR δ 9.92 (s, 2H), 9.02 (d, J = 2.2 Hz,
2H), 8.90 (d, J = 1.9 Hz, 2H), 8.38 (t, J = 2.0 Hz, 2H), 8.19 (m, 1H),
7.87 (dd, J = 7.8 and 1.5 Hz, 2H), 7.68 (t, J = 8.0 Hz, 1H), 6.14 (br s,
4H). The product was used in the next step without further
purification.
1,3-Bis(5-cyanopyridin-2-yl)benzene (67). 67 was prepared
analogously to 61 from bromopyridine 65 (2.02 g, 11.0 mmol) and
1,3-benzenediboronic acid (912 mg, 5.5 mmol). The product was
purified on a column of silica, eluting with a gradient of 0−2% MeOH
in CHCl3 followed by recrystallization from CH3CN to give a solid
(976 mg, 63%): mp 241−242 °C; 1H NMR δ 9.16 (dd, J = 2.0 and 0.9
Hz, 2H), 8.99 (t, J = 1.9 Hz, 1H), 8.46 (dd, J = 8.4 and 2.1 Hz, 2H),
8.37 (dd, J = 8.3 and 1.0 Hz, 2H), 8.33 (dd, J = 7.9 and 1.8 Hz, 2H),
7.73 (t, J = 7.8 Hz, 1H); HPLC 98.0 area %. Anal. (C18H10N4) C, H,
N.
1,3-Bis(4-cyanopyridin-2-yl)benzene (68). 68 was prepared
analogously to 61 from bromopyridine 66 (4.50 g, 24.6 mmol) and
1,3-benzenediboronic acid (2.00 g, 12.1 mmol). After filtration of the
reaction mixture through Celite, the filtrate was concentrated and
diluted with ether to give a precipitate (2.98 g, 87%): mp 238−239 °C;
1H NMR δ 8.96 (dd, J = 5.0 and 0.9 Hz, 2H), 8.91 (t, J = 1.7 Hz, 1H),
8.69 (t, J = 1.1 Hz, 2H), 8.32 (dd, J = 7.8 and 1.8 Hz, 2H), 7.88 (dd, J
= 5.0 and 1.4 Hz, 2H), 7.71 (t, J = 7.8 Hz, 1H); HPLC 99.0 area %.
Anal. (C10H18N4) C, H, N.
1,3-Bis(5-N′-hydroxyamidinopyryidin-2-yl)benzene (69). 69
was prepared analogously to 63 from nitrile 67 (0.960 g, 3.40 mmol)
to give a light gray solid (1.20 g, 101%): 1H NMR δ 9.92 (s, 2H), 9.01
(dd, J = 1.8 and 1.2 Hz, 2H), 8.87 (t, J = 1.6 Hz, 1H), 8.20 (dd, J = 7.8
and 1.8 Hz, 2H), 8.15 (m, 4H), 7.68 (t, J = 7.8 Hz, 1H), 6.07 (br s,
4H); HPLC 98.5 area %. The product was used in the next step
without further purification.
5′-Chloro-3,4″-dicyano-m-terphenyl (56). 56 was prepared
from 54 (1.20 g, 4.11 mmol) and 3-cyanophenylboronic acid (788
mg, 5.36 mmol). The reaction mixture was diluted with water to
precipitate the product, which was then recrystallized from CH3CN to
give a solid (950 mg, 74%): mp 235−236 °C; 1H NMR δ 8.42 (d, J =
1.6 Hz, 1H), 8.21 (ddd, J = 8.0, 1.8, and 1.1 Hz, 1H), 8.09 (m, 3H),
7.98 (d, J = 8.5 Hz, 2H), 7.94 (t, J = 1.7 Hz, 1H), 7.89 (m, 2H), 7.71
(t, J = 7.8 Hz, 1H); HPLC 97.1 area %. Anal. (C20H11ClN2) C, H, N.
5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
picolinonitrile (59). A mixture of 5-bromopicolinonitrile (57, 2.93 g,
16.0 mmol), bis(pinacolato)diboron (4.50 g, 17.7 mmol),
PdCl2·CH2Cl2 (401 mg, 0.191 mmol), and potassium acetate (4.75
g, 48.4 mmol) in DMSO (45 mL) was heated for 90 °C for 6 h. The
reaction mixture was partitioned between water and toluene and then
filtered through Celite, and the aqueous layer was extracted with
toluene. The dried extracts were evaporated to a solid (3.11 g, 85%)
1
which was used directly in the next step: H NMR δ 8.88 (m, 1H),
8.22 (m, 1H), 8.04 (m, 1H), 1.33 (s, 12H).
5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
nicotinonitrile (60). 60 was prepared analogously to 59 from 5-
bromonicotinonitrile (58, 5.16 g, 28.19 g) to give a solid (6.37 g, 98%)
1
which was used directly in the next step: H NMR δ 9.14 (d, J = 2.1
Hz, 1H), 8.99 (d, J = 1.5 Hz, 1H), 8.40 (t, J = 1.9 Hz,, 1H), 1.33 (s,
12H).
General Suzuki Coupling Procedure for Dipyridylbenzene
Derivatives 61, 62, 67, and 68. A mixture of the appropriate aryl
halide and boronic acid or ester, tetrakis(triphenylphosphine)-
palladium(0) (0.05 equiv), and silver carbonate (2.5 equiv) was
stirred in refluxing THF until reaction was complete (2−3 h). The
reaction mixture was filtered through Celite, and the Celite pad was
rinsed with hot THF. The evaporated filtrate was purified by column
chromatography or by direct recrystallization.
1,3-Bis(6-cyanopyridin-3-yl)benzene (61). 61 was prepared
from 1,3-diodobenzene (2.02 g, 6.12 mmol) and 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinonitrile (59, 3.11 g, 13.5
mol). Column chromatography on silica, eluting with 2% MeOH in
CHCl3 followed by recrystallization of the crude product from
CH3CN−ether, gave a white solid (1.04 g, 60%): mp 233−236 °C; 1H
NMR δ 9.27 (d, J = 2.2 Hz, 2H), 8.53 (dd, J = 8.2 and 2.3 Hz, 2H),
8.30 (t, J = 1.8 Hz, 1H), 8.19 (d, J = 8.1 Hz, 2H), 7.98 (dd, J = 7.8 and
1.8 Hz, 2H), 7.83 (t, J = 7.8 Hz, 1H); HPLC 98.0 area %. Anal.
(C18H10N4) C, H, N.
1,3-Bis(5-cyanopyridin-3-yl)benzene (62). 62 was prepared
from 1,3-diiodobenzene (1.65 g, 5.00 mmol) and boronate ester 60
(2.88 g, 12.5 mmol). The crude product was directly recrystallized
from CH3CN−ether to give a white solid (981 mg, 69%): mp 254−
255 °C; 1H NMR δ 9.37 (d, J = 2.3 Hz, 2H), 9.07 (d, J = 1.9 Hz, 2H),
8.86 (t, J = 2.2 Hz, 2H), 8.30 (t, J = 1.8 Hz, 1H), 7.97 (dd, J = 7.8 and
1.8 Hz, 2H), 7.71 (t, J = 7.8 Hz, 1H); HPLC 98.2 area %. Anal.
(C10H10N4·0.2H2O) C, H, N.
General Procedure for Amidoximes 63, 64, 69, and 70.
Potassium tert-butoxide (10 equiv) was added to a solution of
hydroxylamine hydrochloride (10 equiv) in DMSO (0.5−0.6 mL/
mmol hydroxylamine hydrochloride). After 1 h the nitrile (1 equiv)
was added and the reaction mixture was stirred overnight. The reaction
mixture was poured over ice. The resulting precipitated product was
filtered off and dried.
1,3-Bis(4-N′-hydroxyamidinopyryidin-2-yl)benzene (70). 70
was prepared analogously to 63 from nitrile 68 (1.51 g, 5.34 mmol).
The sticky, filtered precipitate was dissolved in hot ethanol, and the
1
solution was evaporated to a light gray solid (2.19 g, 118%, wet): H
NMR δ 10.12 (s, 2H), 8.88 (t, J = 1.6 Hz, 1H), 8.71 (d, J = 5.1 Hz,
2H), 8.30 (s, 2H), 8.21 (dd, J = 7.8 and 1.7 Hz, 2H), 7.66 (m, 3H),
6.20 (br s, 4H); HPLC 98.0 area %. The product was used in the next
step without further purification.
Nitro-Substituted Terphenyl and Biphenyl Derivatives 71
and 73−76. Compounds 71 and 73−76 were prepared by Suzuki
coupling reaction conditions analogous to those for compounds 43−
45, 47−50, and 53−56.
5′-Chloro-3,3″-dinitro-m-terphenyl (71). 71 was prepared from
dibromide 39 (1.35 g, 5.00 mmol) and 3-nitrophenylboronic acid
(1.85 g, 11.1 mmol). The reaction mixture was poured over ice, and
the precipitated crude product was recrystallized from DMF as a gray
solid (1.35 g, 76%): mp 242−243 °C; 1H NMR δ 8.66 (t, J = 2.0 Hz,
2H), 8.34 (dm, J = 8.5 Hz, 2H), 8.29 (dm, J = 8.2 Hz, 2H), 8.16 (t, J =
1.6 Hz, 1H), 7.97 (d, J = 1.6 Hz, 2H), 7.81 (t, J = 8.0 Hz, 2H). Anal.
(C18H11ClN2O4·0.2H2O) C, H, N.
5′-Chloro-3,3″-diamino-m-terphenyl (72). A mixture of dini-
troterphenyl 71 (2.98 g, 8.40 mmol) and tin(II) chloride dihydrate (3
× 10.0 g portions, 133 mmol) in EtOH (100 mL) was refluxed for 3 h.
The reaction mixture was diluted with water, neutralized by cautious
addition of NaHCO3, and extracted into CH2Cl2. Both layers were
filtered through Celite, and the aqueous layer was extracted with
CH2Cl2. Combined dried extracts were evaporated to a white solid
(2.16 g, 87%), which was recrystallized from EtOH to give a white
solid (1.24 g, 50%): mp 153−154 °C; 1H NMR δ 7.65 (t, J = 1.6 Hz,
1H), 7.53 (d, J = 1.6 Hz, 2H), 7.13 (t, J = 7.8 Hz, 2H), 6.92 (t, J = 1.9
Hz, 2H), 6.86 (dd, J = 7.6 and 0.9 Hz, 2H), 6.62 (ddd, J = 8.0, 2.2, and
0.9 Hz, 2H), 5.20 (br s, 4H); ESI MS m/z 295.7 ([M + H]+). Anal.
(C18H15ClN2·0.1H2O) C, H, N.
1,3-Bis(6-N′-hydroxyamidinopyridin-3-yl)benzene (63). 63
was prepared from nitrile 61 (1.03 g, 3.65 mmol) to give a white
1
solid (1.28 g, 101%): H NMR δ 10.06 (s, 2H), 9.02 (d, J = 2.3 Hz,
2H), 8.26 (dd, J = 8.4 and 2.3 Hz, 2H), 8.14 (t, J = 1.6 Hz, 1H), 7.96
(d, J = 8.4 Hz, 2H), 7.84 (dd, J = 7.7 and 1.6 Hz, 2H), 7.66 (t, J = 7.4
Hz, 1H), 5.91 (br s, 4H); HPLC 97.2 area %. The product was used in
the next step without further purification.
3-Bromo-4′-nitrobiphenyl (73). 73 was prepared from iodoben-
zene 51 (5.76 g, 20.4 mmol) and 4-nitrophenylboronic acid pinacol
ester (5.04 g, 20.2 mmol). The product was purified on a column of
silica, eluting with a gradient of 0−40% EtOAc in hexanes and
recrystallized from EtOAc/hexanes to give yellow crystals (4.60 g,
82%): mp 88 °C; 1H NMR δ 8.31 (d, J = 8.8 Hz, 2H), 8.01 (m, 3H),
1,3-Bis(5-N′-hydroxyamidinopyridin-3-yl)benzene (64). 64
was prepared from nitrile 62 (858 mg, 3.04 mmol) to give a light
Q
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.87 (dm, J = 7.8 Hz, 1H), 7.69 (dm, J = 9.0 Hz, 1H), 7.50 (t, J = 7.9
Hz, 1H); HPLC 98.8 area %. Anal. (C12H8BrN2O) C, H, N.
3-Bromo-5-chloro-4′-nitrobiphenyl (74). 74 was prepared from
iodobenzene 52 (2.55 g, 8.04 mmol) and 4-nitrophenylboronic acid
pinacol ester (2.00 g, 8.04 mmol). The product was purified on a
column of silica, eluting with hexanes/EtOAc (19:1) and recrystallized
from EtOAc−hexane to give white crystals (1.51 g, 60%): mp 147−
148 °C; 1H NMR δ 8.30 (d, J = 8.7 Hz, 2H), 8.06 (d, J = 8.8 Hz, 2H),
8.00 (t, J = 1.6 Hz, 1H), 7.92 (t, J = 1.7 Hz, 1H), 7.84 (t, J = 1.7 Hz,
1H); HPLC 100 area %.
Hz, 2H), 7.59 (m, 1H), 7.41 (tm, J = 7.6 Hz, 2H), 7.30 (tm, J = 7.3
Hz, 1H), 5.54 (s, 2H); HPLC 100 area %. Anal. (C12H9BrClN) C, H,
N.
3-Bromo-5-chlorobiphenyl (82). A solution of 4-amino-3-
bromo-5-chlorobiphenyl (81, 4.51 g, 16.0 mmol) in EtOH (25 mL)
was treated dropwise with H2SO4 (5 mL). Sodium nitrite (2.74 g, 39.7
mmol) was added, and the mixture was refluxed for 30 min. The
reaction mixture was poured over ice and excess NaHCO3 and then
extracted into EtOAc. Column chromatography on silica, eluting with
hexanes, gave a clear oil that solidified upon standing (4.52 g, 86%):
mp 32−33 °C; 1H NMR δ 7.85 (t, J = 1.5 Hz, 1H), 7.77 (t, J = 1.6 Hz,
1H), 7.73 (m, 3H), 7.49 (m, 2H), 7.43 (m, 1H); HPLC 99.5 area %.
Anal. (C12H8BrCl) C, H, N.
(5-Chloro-biphenyl-3-yl)boronic Acid (83). Butyllithium (2.5
M/hexanes, 4.5 mL, 11.25 mmol) was added dropwise over 15 min to
a solution of 3-bromo-5-chlorobiphenyl (82, 2.49 g, 9.31 mmol) in
THF (150 mL) at −78 °C. The mixture was maintained for 1 h before
the dropwise addition of triisopropyl borate (4.5 mL, 19.6 mmol). The
mixture was maintained another hour at −78 °C before being warmed
to ambient temperature. The reaction mixture was quenched with 1 N
HCl (150 mL) and extracted with ether. The dried extract was
evaporated to a white solid (2.12 g, 98%) which was used in the next
step without further purification.
3,4″-Dinitro-m-terphenyl (75). 75 was prepared from bromobi-
phenyl 73 (4.53 g, 16.3 mmol) and 3-nitrophenylboronic acid (2.82 g,
16.9 mmol). Column chromatography, eluting with a gradient from
hexanes/EtOAc (4:1) to neat EtOAc followed by recrystallization
1
from EtOH, gave a white solid (3.27 g, 63%): mp 188−189 °C; H
NMR δ 8.60 (t, J = 2.0 Hz, 1H), 8.34 (dm, J = 8.9 Hz, 2H), 8.28 (m,
2H), 8.17 (t, J = 1.7 Hz, 1H), 8.13 (dm, J = 8.9 Hz, 2H), 7.89 (m, 2H),
7.81 (t, J = 8.0 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H). Anal.
(C18H12N2O4·0.05H2O) C, H, N.
5′-Chloro-3,4″-dinitro-m-terphenyl (76). 76 was prepared from
bromobiphenyl 74 (2.09 g, 6.68 mmol) and 3-nitrophenylboronic acid
(1.69 g, 8.33 mmol). The reaction mixture was diluted with water. The
resulting precipitate was filtered off, dried, and purified by suspension
in hot ethanol to give a light gray solid (2.18 g, 92%): mp 216−218
°C; 1H NMR δ 8.64 (t, J = 2.0 Hz, 1H), 8.33 (m, 3H), 8.29 (ddd, J =
8.2, 2.2, and 0.9 Hz, 1H), 8.17 (d, J = 8.9 Hz, 2H), 8.14 (t, J = 1.6 Hz,
1H), 7.99 (t, J = 1.7 Hz, 1H), 7.95 (t, J = 1.7 Hz, 1H), 7.81 (t, J = 8.0
Hz, 1H); HPLC 100 area %. Anal. (C18H11ClN2O4·0.5H2).
3,4″-Diamino-m-terphenyl Dihydrochloride (77). A mixture of
dinitroterphenyl 75 (3.00 g, 9.37 mmol) and 10% Pd/C (0.60 g, 0.56
mmol) in EtOH (300 mL) was hydrogenated at 60 psi. The reaction
mixture was filtered through Celite. The filtrate was concentrated to a
small volume, treated with saturated ethanolic HCl, and diluted with
ether to give the hydrochloride salt as a white solid (2.24 g, 72%): mp
>250 °C (dec); 1H NMR δ 10.10 (br s, 4H), 7.89 (m, 1H), 7.85 (d, J
= 8.5 Hz, 2H), 7.77 (dm, J = 7.8 Hz, 1H), 7.72 (m, 2H), 7.61 (m, 3H),
7.43 (d, J = 8.5 Hz, 2H), 7.36 (dm, J = 7.8 Hz, 1H). Anal.
(C18H16N2·2HCl·0.2H2O) C, H, N, Cl.
5″-Chloro-3,4″-diamino-m-terphenyl (78). Iron powder (3.60
g, 64.5 mmol) was added to a mixture of dinitroterphenyl 76 (2.19 g,
6.15 mmol) and ammonium chloride 1.02 g, 19.1 mmol) in refluxing
EtOH/H2O (2:1 mixture, 90 mL), and refluxing was continued for 2.5
h. The reaction mixture was cooled and filtered through Celite. The
filtrate was diluted with water and extracted into ether. The extract was
concentrated to ∼50 mL and treated with saturated ethanolic HCl (3
mL) to precipitate the HCl salt (1.94 g, 85%). The salt was dissolved
in hot water, filtered, and diluted with NaOH solution to precipitate
the free base as a white solid (1.26 g, 69%) which was used directly in
the next step: 1H NMR δ 7.62 (t, J = 1.5 Hz, 1H), 7.51 (t, J = 1.7 Hz,
1H), 7.45 (d, J = 8.6 Hz, 2H), 7.39 (t, J = 1.7 Hz, 1H), 7.12 (t, J = 7.8
Hz, 1H), 6.91 (t, J = 1.9 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.65 (d, (t,
J = 8.5 Hz, 2H), 6.60 (d, J = 7.9 Hz, 1H), 5.34 (br s, 2H), 5.17 (br s,
2H); HPLC 96.3 area %.
5′-Chloro-2-isopropoxy-4-nitro-m-terphenyl (88). 88 was
prepared analogously to compounds 43−50 from bromobenzene 86
(1.35 g, 5.17 mmol) and boronic acid 83 (1.54 g, 6.62 mmol). The
product was purified on a column of silica, eluting with hexanes/
EtOAc (50:1) followed by recrystallization from EtOH to give yellow
1
crystals (1.57 g, 82%): mp 100−101 °C; H NMR δ 7.89 (m, 2H),
7.80 (t, J = 1.8 Hz, 1H), 7.76 (m, 4H), 7.63 (t, J = 1.7 Hz, 1H), 7.50
(tm, J = 7.4 Hz, 2H), 7.42 (tm, J = 7.3 Hz, 1H), 4.87 (septet, J = 6.0
Hz, 1H), 1.30 (d, J = 6.0 Hz, 6H); HPLC 96.2 area %. Anal.
(C21H18ClNO3) C, H, N.
5′-Chloro-2-isopropoxy-5-nitro-m-terphenyl (89). 89 was
prepared analogously to compounds 43−50 from bromobenzene 87
(1.30 g, 5.01 mmol) and boronic acid 83 (1.50 g, 1.29 mmol). The
product was purified on a column of silica, eluting with hexane/EtOAc
1
(17:3) to give a white solid (1.68 g, 91%): mp 111 °C; H NMR δ
8.28 (dd, J = 7.5 and 2.9 Hz, 1H), 8.26 (d, J = 2.9 Hz, 1H), 7.80 (t, J =
1.5 Hz, 1H), 7.77 (d, J = 1.4 Hz, 1H), 7.74 (t, J = 1.8 Hz, 2H), 7.63 (t,
J = 1.7 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.41 (m, 2H), 4.91 (septet, J
= 6.0 Hz, 1H), 1.32 (d, J = 6.0 Hz, 6H); HPLC 100%. Anal.
(C21H18ClNO3) C, H, N.
4-Amino-5′-chloro-2-isopropoxy-m-terphenyl (90). A solu-
tion of nitroterphenyl 88 (1.57 g, 4.26 mmol) in EtOH (75 mL) was
brought to reflux. Tin(II) chloride dihydrate (5.18 g, 29.6 mmol) was
slowly added via the condenser, and refluxing was continued for 1.5 h.
The reaction mixture was portioned between ether and water basified
with 1 N NaOH, and the aqueous layer was extracted twice with ether.
Combined extracts were filtered through Celite. Column chromatog-
raphy on silica, eluting with hexanes/EtOAc (3:2), gave a glass (1.20 g,
4-Amino-3-chlorobiphenyl (80). 80 was prepared analogously to
compound 71 from 4-bromo-2-chloroaniline (79, 5.25 g, 25.4 mmol)
and phenylboronic acid (3.75 g, 30.8 mmol). The product was
chromatographed on a column of silica, eluting with 15−20% EtOAc
in hexanes followed by recrystallization from hexanes to give a solid
1
83%): H NMR δ 7.70 (d, J = 7.2 Hz, 2H), 7.66 (t, J = 1.5 Hz, 1H),
7.48 (m, 4H), 7.40 (t, J = 7.3 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.34
(d, J = 2.0 Hz, 1H), 6.25 (dd, J = 8.2 and 2.0 Hz, 1H), 5.30 (s, 2H),
4.47 (septet, J = 6.1 Hz, 1H), 1.25 (d, J = 6.0 Hz, 6H). The product
was used in the next step without further purification.
1
(3.62 g, 70%): mp 69−70 °C; H NMR δ 7.56 (d, J = 7.3 Hz, 2H),
7.50 (d, J = 2.0 Hz, 1H), 7.38 (m, 3H), 7.25 (t, J = 7.3 Hz, 1H), 6.87
(d, J = 8.4 Hz, 1H), 5.48 (s, 2H); HPLC 100 area %. Anal.
(C12H10ClN) C, H, N.
5-Amino-5′-chloro-2-isopropoxy-m-terphenyl (91). 91 was
prepared analogously to compound 90 from nitroterphenyl 89 (1.64 g,
4.46 mmol). Column chromatography on silica, eluting with hexane/
4-Amino-3-bromo-5-chlorobiphenyl (81). N-Bromosuccini-
mide (3.84 g, 21.6 mmol) was added to a solution of aminobiphenyl
80 (4.33 g, 21.3 mmol) in THF (60 mL) at 0 °C. After 1 h the
reaction mixture was quenched with NaHSO3 solution, neutralized
with NaHCO3 solution, and extracted into EtOAc. Column
chromatography on silica, eluting with hexanes/EtOAc (9:1) followed
by recrystallization from EtOAc−hexanes, gave crystals (5.00 g, 83%):
mp 111−112 °C; 1H NMR δ 7.71 (d, J = 2.1 Hz, 1H), 7.61 (t, J = 2.0
1
EtOAc (2:1), gave a glass (1.42 g, 94%): H NMR δ 7.72 (m, 3H),
7.64 (t, J = 1.8 Hz, 1H), 7.51 (m, 3H), 7.41 (tm, J = 7.3 Hz, 1H), 6.84
(d, J = 8.7 Hz, 1H), 6.69 (d, J = 2.9 Hz, 1H), 6.57 (dd, J = 8.6 and 2.8
Hz, 1H), 4.79 (s, 2H), 4.15 (septet, J = 6.1 Hz, 1H), 1.11 (d, J = 6.0
Hz, 6H); HPLC 97.5 area %. The product was used in the next step
without further purification.
R
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(16) Wolday, D.; Berhe, N.; Akuffo, H.; Desjus, P.; Britton, S.
Emerging leishmania/HIV co-infection in Africa. Med. Microbiol.
Immunol. 2001, 190, 65−67.
(17) Anonymous. Human African trypanosomiasis (sleeping sick-
ness): epidemiological update. Wkly. Epidemiol. Rec. 2006, 81, 71−80.
(18) Croft, S.; Barrett, M.; Urbina, J. Chemotherapy of
trypanosomiases and leishmaniasis. Trends Parasitol. 2005, 21, 508−
512.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 919-966-4294. Fax: 919-966-0704. E-mail: Tidwell@
med.unc.edu.
Notes
The authors declare no competing financial interest.
(19) Delespaux, V.; de Koning, H. P. Drugs and drug resistance in
African trypanosomiasis. Drug Resist. Updates 2007, 10, 30−50.
(20) Pepin, J.; Guern, C.; Milord, F.; Schechter, P. J.
Difluoromethylornithine for arseno-resistant Trypanosoma brucei
gambiense sleeping sickness. Lancet 1987, 2, 1431−1433.
(21) Rodriques, C. J.; de Castro, S. L. A critical review on Chagas
disease chemotherapy. Mem. Inst. Oswaldo Cruz 2002, 97, 3−24.
(22) Murambiwa, P.; Masola, B.; Govender, T.; Mukaratirwa, S.;
Musabayane, C. T. Anti-malarial drug formulations and novel delivery
systems: a review. Acta Trop. 2011, 118, 71−79.
(23) Griensven, J. v.; Balasegaram, M.; Meheus, F.; Avlar, J.; lynen,
L.; Boelaert, M. Combination therapy for visceral leishmaniasis. Lancet
Infect. Dis. 2010, 10, 184−194.
(24) Olliaro, P. L.; Guerin, P. J.; Gerstl, S.; Haaskjold, A. A.;
Rottingen, J.-A.; Sundar, S. Treatment options for visceral
leishmaniasis: a systematic review of clinical studies done in India,
1980−2004. Lancet Infect. Dis. 2005, 5, 763−774.
(25) Control of the Leishmaniasis; WHO Technical Report Series No.
949; World Health Organization: Geneva, Switzerland, 2010.
(26) Sundar, S.; Chakravarty, J.; Agarwal, D.; Rai, M.; Murray, H. W.
Single-dose liposomal amphotericin B for visceral leishmaniasis in
India. N. Eng. J. Med. 2010, 362, 504−512.
(27) Sundar, S.; Jha, T. K.; Thakur, C. P.; Sinha, P. K.; Bhattacharya,
S. K. Injectable paromomycin for visceral leishmaniasis in India. N.
Engl. J. Med. 2007, 356, 2571−2581.
(28) Dorlo, T. P. C.; Balasegaram, M.; Beijnen, J. H.; de Vries, P. J.
Miltefosine: a review of its pharmacology and therapeutic efficacy in
the treatment of leishmaniasis. J. Antimicrob. Chemother. 2012, 67,
2576−2597.
(29) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity of
2,5-bis(4-guanylphenyl)furans. J. Med. Chem. 1977, 20, 531−536.
(30) Werbovetz, K. Diamidines as antitrypanosomal, antileishmanial
and antimalarial agents. Curr. Opin. Invest. Drugs 2006, 7, 147−157.
(31) Boykin, D. W.; Kumar, A.; Hall, J. E.; Bender, B. C.; Tidwell, R.
R. Anti-pneumocystis activity of bisamidoximes and bis-O-alkylami-
doximes prodrugs. Bioorg. Med. Chem. Lett. 1996, 6, 3017−3020.
(32) Yeates, C. DB-289 Immtech International. IDrugs 2003, 6,
1086−1093.
(33) Yeramian, P.; Meshnick, S. R.; Krudsood, S.; Chalermrut, K.;
Silachamroon, U.; Tangpukdee, N.; Allen, J.; Brun, R.; Kwiek, J. J.;
Tidwell, R.; Looareesuwan, S. Efficacy of DB289 in Thai patients with
Plasmodium vivax or acute, uncomplicated Plasmodium falciparum
infections. J. Infect. Dis. 2005, 192, 319−322.
(34) Thuita, J. K.; Karanja, S. M.; Wenzler, T.; Mdachi, R. E.;
Ngotho, J. M.; Kagira, J. M.; Tidwell, R.; Brun, R. Efficacy of the
diamidine DB75 and its prodrug DB289, against murine models of
human African trypanosomiasis. Acta Trop. 2008, 108, 6−10.
(35) Goldsmith, R. B.; Gray, D. R.; Yan, Z.; Generaux, C. N.; Tidwell,
R. R.; Reisner, H. M. Application of monoclonal antibodies to measure
metabolism of an anti-trypanosomal compound in vitro and in vivo. J.
Clin. Lab. Anal. 2010, 24, 187−194.
(36) Bakunov, S. A.; Bakunova, S. M.; Bridges, A. S.; Wenzler, T.;
Barszcz, T.; Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Synthesis and
antiprotozoal properties of pentamidine congeners bearing the
benzofuran motif. J. Med. Chem. 2009, 52, 5763−5767.
(37) Bakunov, S. A.; Bakunova, S. M.; Wenzler, T.; Barszcz, T.;
Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal
activity of cationic 2-phenylbenzofurans. J. Med. Chem. 2008, 51,
6927−6944.
(38) Bakunov, S. A.; Bakunova, S. M.; Wenzler, T.; Ghebru, M.;
Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal
ACKNOWLEDGMENTS
■
This work was supported by the Consortium for Parasitic Drug
Development (CPDD), the Bill and Melinda Gates Founda-
tion, and Medicines for Malaria Venture (MMV).
ABBREVIATIONS USED
■
AIA, arylimidamide; DMTHP, 5,5-dimethyl-1,4,5,6-tetrahydro-
pyrimidin-2-yl; HAT, human African trypanosomiasis; NECT,
nifurtimox−eflornithine combination therapy; pKa, ionization
constant; SI, selectivity index; VL, visceral leishmaniasis; WHO,
World Health Organization
REFERENCES
■
(1) African trypanosomiasis (sleeping sickness). http://www.who.
int/mediacentre/factsheets/fs259/en/ (accessed Mar 28, 2013).
(2) Malaria. http://www.who.int/mediacentre/factsheets/fs094/en/
index.html (accessed Mar 28, 2013).
(3) Leishmaniasis. http://www.who.int/topics/leishmaniasis/en/
(accessed Mar 28, 2013).
(4) Chagas disease (American trypanosomiasis). http://www.who.
int/mediacentre/factsheets/fs340/en/index.html (accessed Apr 26,
2012).
(5) Leishmaniasis. http://www.who.int/leishmaniasis/burden/en/
(accessed Mar 28, 2013).
(6) Legros, D.; Ollivier, G.; Gasteullu-Etchegorry, M.; Paquet, C.;
Burri, C.; Jannin, J.; Buscher, P. Treatment of human African
trypanosomiasispresent situation and needs for research and
development. Lancet Infect. Dis. 2002, 2, 437−440.
(7) Astelbauer, F.; Walochnik, J. Antiprotozoal compounds: state of
the art and new developments. Int. J. Antimicrob. Agents 2011, 38,
118−124.
(8) Cavalli, A.; Bolognesi, M. L. Neglected tropical diseases: multi-
target-directed ligands in the search for novel lead candidates against
Trypanosoma and Leishmania. J. Med. Chem. 2009, 52, 7339−7359.
(9) Alvar, J.; Aparicio, P.; Aseffa, A.; Den, B. M.; Canavate, C.; Dedet,
J.-P.; Gradoni, L.; Ter, H. R.; Lopez-Velez, R.; Moreno, J. The
relationship between leishmaniasis and AIDS: the second 10 years.
Clin. Microbiol. Rev. 2008, 21, 334−359.
(10) Cruz, I.; Nieto, J.; Moreno, J.; Canavate, C.; Desjeux, P.; Alvar, J.
Leishmania/HIV co-infections in the second decade. Indian J. Med.
Res. 2006, 123, 357−388.
(11) Desjeux, P.; Alvar, J. Leishmania/HIV co-infections: epidemi-
ology in Europe. Ann. Trop. Med. Parasitol. 2003, 97 (Suppl. 1), 3−15.
(12) Lachaud, L.; Bourgeois, N.; Plourde, M.; Leprohon, P.; Bastien,
P.; Ouellette, M. Parasite susceptibility to amphotericin B in failures of
treatment for visceral leishmaniasis in patients coinfected with HIV
type 1 and Leishmania infantum. Clin. Infect. Dis. 2009, 48, e16−e22.
(13) Pintado, V.; Lopez-velez, R. HIV-associated viseceral
leishmaniais. Clin. Microbiol. Infect. 2003, 7, 291−300.
(14) Rabello, A.; Orsini, M.; Disch, J. Leishmania/HIV co-infection
in Brazil: an appraisal. Ann. Trop. Med. Parasitol. 2003, 97 (Suppl. 1),
17−28.
(15) Sinha, P. K.; van Griensven, J.; Pandey, K.; Kumar, N.; Verma,
N.; Mahajan, R.; Kumar, P.; Kumar, R.; Das, P.; Mitra, G.; Flevaud, L.;
Ferreyra, C.; Remartinez, D.; Pece, M.; Palma, P. P. Liposomal
amphotericin B for visceral leishmaniasis in human immunodeficiency
virus-coinfected patients: 2-year treatment outcomes in Bihar, India.
Clin. Infect. Dis. 2011, 53, e91−e98.
S
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
activity of cationic 1,4-diphenyl-1H-1,2,3-triazoles. J. Med. Chem. 2010,
53, 254−272.
(39) Bakunova, S. M.; Bakunov, S. A.; Patrick, D. A.; Kumar, E. V. K.
S.; Ohemeng, K. A.; Bridges, A. S.; Wenzler, T.; Barszcz, T.; Jones, S.
K.; Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Structure−activity study
of pentamidine analogues as antiprotozoal agents. J. Med. Chem. 2009,
52, 2016−2035.
(55) Xiao, G.; Kumar, A.; Li, K.; Rigl, C. T.; Bajic, M.; Davis, T. M.;
Boykin, D. W.; Wilson, W. D. Inhibition of the HIV-1 rev-RRE
complex formation by unfused aromatic cations. Bioorg. Med. Chem.
2001, 9, 1097−1113.
(56) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity of
2,5-bis(4-guanylphenyl)thiophenes and -pyrroles. J. Med. Chem. 1977,
20, 1219−1221.
(40) Bakunova, S. M.; Bakunov, S. A.; Wenzler, T.; Barszcz, T.;
Werbovetz, K. A.; Brun, R.; Hall, J. E.; Tidwell, R. R. Synthesis and in
vitro antiprotozoal activity of bisbenzofuran cations. J. Med. Chem.
2007, 50, 5807−5823.
(41) Bakunova, S. M.; Bakunov, S. A.; Wenzler, T.; Barszcz, T.;
Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal
activity of pyridyl analogues of pentamidine. J. Med. Chem. 2009, 52,
4657−4667.
(42) Branowska, D.; Farahat, A. A.; Kumar, A.; Wenzler, T.; Brun, R.;
Liu, Y.; Wilson, W. D.; Boykin, D. W. Synthesis and antiprotozoal
activity of 2,5-bis[amidinoaryl]thiazoles. Bioorg. Med. Chem. 2010, 18,
3551−3558.
(43) Chackal-Catoen, S.; Miao, Y.; Wilson, W. D.; Wenzler, T.; Brun,
R.; Boykin, D. W. Dicationic DNA-targeted antiprotozoal agents:
naphthalene replacement of benzimidazole. Bioorg. Med. Chem. 2006,
14, 7434−7445.
(44) Crowell, A. L.; Stephens, C. E.; Kumar, A.; Boykin, D. W.;
Secor, W. E. Activities of dicationic compounds against Trichomonas
vaginalis. Antimicrob. Agents Chemother. 2004, 48, 3602−3605.
(45) Czarny, A.; Wilson, W. D.; Boykin, D. W. Synthesis of mono-
cationic and dicationic analogs of Hoechst 33258. J. Heterocycl. Chem.
1996, 33, 1393−1397.
(46) Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F. A.;
Wilson, W. D.; Boykin, D. W. Synthesis and antiprotozoal activity of
aza-analogues of furmadine. J. Med. Chem. 2003, 46, 4761−4769.
(47) Ismail, M. A.; Brun, R.; Wenzler, T.; Tanious, F. A.; Wilson, W.
D.; Boykin, D. W. Dicationic biphenyl benzimidazole derivatives as
antiprotozoal agents. Bioorg. Med. Chem. 2004, 12, 5405−5413.
(48) Kumar, A.; Zhao, M.; Wilson, W. D.; Boykin, D. W. Dicationic
2,4-diaryl pyrimidines as DNA selective binding agents. Bioorg. Med.
Chem. Lett. 1994, 4, 2913−2918.
(49) Nguyen, B.; Tardy, C.; Bailly, C.; Colson, P.; Houssier, C.;
Kumar, A.; Boykin, D. W.; Wilson, W. D. Influence of compound
structure on affinity, sequence selectivity, and mode of binding to
DNA for unfused aromatic dications related to furamidine. Biopolymers
2002, 63, 281−297.
(50) Patrick, D. A.; Bakunov, S. A.; Bakunova, S. M.; Kumar, E. V. K.
S.; Chen, H.; Jones, S. K.; Wenzler, T.; Barzcz, T.; Werbovetz, K. A.;
Brun, R.; Tidwell, R. R. Synthesis and antiprotozoal activities of
dicationic bis(phenoxymethyl)benzenes, bis(phenoxymethyl)-
naphthalenes, and bis(benzyloxy)naphthalenes. Eur. J. Med. Chem.
2009, 44, 3543−3551.
(57) Stephens, C. E.; Brun, R.; Salem, M. M.; Werbovetz, K. A.;
Tanious, F.; Wilson, W. D.; Boykin, D. W. The activity of diguanidino
and “reversed” diamidino 2,5-diarylfurans versus Trypanosoma cruzi
and Leishmania donovani. Bioorg. Med. Chem. Lett. 2003, 13, 2065−
2069.
(58) Stephens, C. E.; Tanious, F.; Kim, S.; Wilson, W. D.; Schell, W.
A.; Perfect, J. R.; Franzblau, S. G.; Boykin, D. W. Diguanidino and
“reversed” diamidino 2,5-diarylfurans as antimicrobial agents. J. Med.
Chem. 2001, 44, 1741−1748.
(59) Wang, M. Z.; Zhu, X.; Srivastava, A.; Liu, Q.; Sweat, J. M.;
Pandharkar, T.; Stephens, C. E.; Riccio, E.; Parman, T.; Munde, M.;
Mandal, S.; Madhubala, R.; Tidwell, R. R.; Wilson, W. D.; Boykin, D.
W.; Hall, J. E.; Kyle, D. E.; Werbovetz, K. A. Novel arylimidamides for
treatment of visceral leishmaniasis. Antimicrob. Agents Chemother.
2010, 54, 2507−2516.
(60) Zhu, X.; Liu, Q.; Yang, S.; Parman, T.; Green, C. E.; Mirsalis, J.
C.; Soeiro, M. d. N. C.; Souza, E. M. d.; Silva, C. F. d.; Batista, D. d. G.
J.; Stephens, C. E.; Banerjee, M.; Farahat, A. A.; Munde, M.; Wilson,
W. D.; Boykin, D. W.; Wang, M. Z.; Werbovetz, K. A. Evaluation of
arylimidamides DB1955 and DB1960 as candidates against visceral
leishmaniasis and chagas diseasein vivo efficacy, acute toxicity,
pharmacokinetics and toxicology studies. Antimicrob. Agents Chemo-
ther. 2012, 56, 3690−3699.
(61) von der Saal, W.; Engh, R. A.; Eichinger, A.; Gabriel, B.;
Kucznierz, R.; Sauer, J. Syntheses and selective inhibitory activities of
terphenyl-bisamidines for serine proteases. Arch. Pharm. 1996, 329,
73−82.
(62) Hu, L.; Arafa, R. K.; Ismail, M. A.; Patel, A.; Munde, M.; Wilson,
W. D.; Wenzler, T.; Brun, R.; Boykin, D. W. Synthesis and activity of
azaterphenyl diamidines against Trypanosoma brucei rhodesiense and
Plasmodium falciparum. Bioorg. Med. Chem. 2009, 17, 6651−6658.
(63) Hu, L.; Arafa, R. K.; Ismail, M. A.; Wenzler, T.; Brun, R.;
Munde, M.; Wilson, W. D.; Nzimiro, S.; Samyesudhas, S.; Werbovetz,
K. A.; Boykin, D. W. Azaterphenyl diamidines as antileishmanial
agents. Bioorg. Med. Chem. Lett. 2008, 18, 247−251.
(64) Ismail, M. A.; Arafa, R. K.; Brun, R.; Wenzler, T.; Miao, Y.;
Wilson, W. D.; Generaux, C.; Bridges, A.; Hall, J. E.; Boykin, D. W.
Synthesis, DNA affinity, and antiprotozoal activity of linear dications:
terphenyl diamidines and analogues. J. Med. Chem. 2006, 49, 5324−
5332.
(65) Da Silva, C. F.; Da Silva, P. B.; Batista, M. M.; Daliry, A.;
Tidwell, R. R.; Soeiro, M. d. N. C. The biological in vitro effect and
selectivity of aromatic dicationic compounds on Trypanosoma cruzi.
Mem. Inst. Oswaldo Cruz 2010, 105, 239−245.
(51) Patrick, D. A.; Bakunov, S. A.; Bakunova, S. M.; Kumar, E. V. K.
S.; Lombardy, R. J.; Jones, S. K.; Bridges, A. S.; Zhirnov, O.; Hall, J. E.;
Wenzler, T.; Brun, R.; Tidwell, R. R. Synthesis and in vitro
antiprotozoal activities of dicationic 3,5-diphenylisoxazoles. J. Med.
Chem. 2007, 50, 2468−2485.
(52) Patrick, D. A.; Boykin, D. W.; Wilson, W. D.; Tanious, F. A.;
Spychala, J.; Bender, B. C.; Hall, J. E.; Dykstra, C. C.; Ohemeng, K. A.;
Tidwell, R. R. Anti-Pneumocystis carinii pneumonia activity of
dicationic carbazoles. Eur. J. Med. Chem. 1997, 32, 781−793.
(53) Patrick, D. A.; Hall, J. E.; Bender, B. C.; McCurdy, D. R.;
Wilson, W. D.; Tanious, F. A.; Saha, S.; Tidwell, R. R. Synthesis and
anti-Pneumocystis carinii pneumonia activity of novel dicationic
dibenzothiophenes and orally active prodrugs. Eur. J. Med. Chem.
1999, 34, 575−583.
(66) Daliry, A.; Da Silva, P. B.; Da Silva, C. F.; Batista, M. M.; De
Castro, S. L.; Tidwell, R. R.; Soeiro, M. d. N. C. In vitro analyses of the
effect of aromatic diamidines upon Trypanosoma cruzi. J. Antimicrob.
Chemother. 2009, 64, 747−750.
(67) Chanteau, S. H.; Tour, J. M. Synthesis of anthropomorphic
molecules: the NanoPutians. J. Org. Chem. 2003, 68, 8750−8766.
(68) Kajigaeshi, S.; Kakinami, T.; Inoue, K.; Kondo, M.; Nakamura,
H.; Fujikawa, M.; Okamoto, T. Halogenation using quaternary
ammonium polyhalides. VI. Bromination of aromatic amines by use
of benzyltrimethylammonium tribromide. Bull. Chem. Soc. Jpn. 1988,
61, 597−599.
(69) Anbazhagan, M.; Saulter, J. Y.; Hall, J. E.; Boykin, D. W.
Synthesis of metabolites of the prodrug 2,5-bis(4-O-
methoxyamidinophenyl)furan. Heterocycles 2003, 60, 1133−1145.
(70) Sinclair, D. J.; Sherburn, M. S. Single and double Suzuki−
Miyaura couplings with symmetric dihalobenzenes. J. Org. Chem. 2005,
70, 3730−3733.
(54) Wang, S.; Hall, J. E.; Tanious, F. A.; Wilson, W. D.; Patrick, D.
A.; McCurdy, D. R.; Bender, B. C.; Tidwell, R. R. Dicationic
dibenzofuran derivatives as anti-Pneumocystis carinii pneumonia agents:
synthesis, DNA binding affinity, and anti-P. carinii activity in an
immunosuppressed rat model. Eur. J. Med. Chem. 1999, 34, 215−224.
T
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(71) Pelter, M. W.; Pelter, L. S. W.; Colovic, D.; Strug, R. Microscale
synthesis of 1-bromo-3-chloro-5-iodobenzene: an improved deami-
nation of 4-bromo-2-chloro-6-iodoaniline. J. Chem. Educ. 2004, 81,
111−112.
(72) Ault, A.; Kraig, R. 1-Bromo-3-chloro-5-iodobenzene. An eight-
step synthesis from benzene. J. Chem. Educ. 1966, 43, 213−214.
(73) Markevitch, D. Y.; Rapta, M.; Hecker, S. J.; Renau, T. E. An
efficient synthesis of 5-bromopyridine-2-carbonitrile. Synth. Commun.
2003, 33, 3285−3289.
(74) Ishiyama, T.; Murata, M.; Miyaura, N. Palladium(0)-catalyzed
cross-coupling reaction of alkoxydiboron with haloarenes: a direct
procedure for arylboronic esters. J. Org. Chem. 1995, 60, 7508−7510.
(75) Chaumeil, H.; Le Drian, C.; Defoin, A. Efficient synthesis of
substituted terphenyls by Suzuki coupling reaction. Synthesis 2002,
757−760.
(76) Ramachandran, U.; Mital, A.; Bharatam, P. V.; Khanna, S.; Rao,
P. R.; Srinivasan, K.; Kumar, R. C.; Singh, H. P.; Lal Kaul, C.; Raichur,
S.; Chakrabartid, R. Studies on some glitazones having pyridine as the
linker unit. Bioorg. Med. Chem. 2004, 12, 655−662.
(77) Zhang, N.; Thomas, L.; Wu, B. Palladium-catalyzed selective
cross-coupling between 2-bromopyridines and aryl bromides. J. Org.
Chem. 2001, 66, 1500−1502.
(78) Al-Saffar, F.; Berlin, S.; Musil, T.; Sivadasan, S. Process for
Preparing Protected Amidines from Nitriles. WO2006090153A1,
2006.
(79) Lange, J. H. M.; van Stuivenberg, H. H.; Coolen, H. K. A. C.;
Adolfs, T. J. P.; McCreary, A. C.; Keizer, H. G.; Wals, H. C.; Veerman,
W.; Borst, A. J. M.; de Looff, W.; Verveer, P. C.; Kruse, C. G.
Bioisosteric replacements of the pyrazole moiety of rimonabant:
synthesis, biological properties, and molecular modeling investigations
of thiazoles, triazoles, and imidazoles as potent and selective CB1
cannabinoid receptor antagonists. J. Med. Chem. 2005, 48, 1823−1838.
(80) Rix, D.; Clavier, H.; Coutard, Y.; Gulajski, L.; Grela, K.;
Mauduit, M. Activated pyridinium-tagged ruthenium complexes as
efficient catalysts for ring-closing metathesis. J. Organomet. Chem.
2006, 691, 5397−5405.
(81) Michrowska, A.; Bujok, R.; Harutyunyan, S.; Sashuk, V.;
Dolgonos, G.; Grela, K. Nitro-substituted Hoveyda−Grubbs ruthe-
nium carbenes: enhancement of catalyst activity through electronic
activation. J. Am. Chem. Soc. 2004, 126, 9318−9325.
(82) Delfin, D. A.; Morgan, R. E.; Zhu, X.; Werbovetz, K. A. Redox-
active dinitrodiphenylthioethers against Leishmania: synthesis, struc-
ture−activity relationships and mechanism of action studies. Bioorg.
Med. Chem. 2009, 17, 820−829.
(83) Orhan, I.; Sener, B.; Kaiser, M.; Brun, R.; Tasdemir, D.
Inhibitory activity of marine sponge-derived natural products against
parasitic protozoa. Mar. Drugs 2010, 8, 47−58.
(89) Baker, B. R.; Cory, M. Irreversible enzyme inhibitors. CLXIV.
Proteolytic enzymes. 14. Inhibition of guinea pig complement by
meta-substituted benzamidines. J. Med. Chem. 1969, 12, 1049−1052.
(90) Basselin, M.; Badet-Denisot, M.-A.; Lawrence, F.; Robert-Gero,
M. Effects of pentamidine on polyamine level and biosynthesis in wild-
type, pentamidine-treated, and pentamidine-resistant Leishmania. Exp.
Parasitol. 1997, 85, 274−282.
(91) Cory, M.; Tidwell, R. R.; Fairley, T. A. Structure and DNA
binding activity of analogs of 1,5-bis(4-amidinophenoxy)pentane
(pentamidine). J. Med. Chem. 1992, 35, 431−438.
(92) de Koning, H. P. Transporters in African trypanosomes: role in
drug action and resistance. Int. J. Parasitol. 2001, 31, 512−522.
(93) Dykstra, C. C.; Tidwell, R. R. Inhibition of topoisomerases from
Pneumocystis carinii by aromatic dicationic molecules. J. Protozool.
1991, 38, 78S−81S.
(94) Ghosh, A. K.; Bhattacharyya, F. K.; Ghosh, D. K. Leishmania
donovani: amastigote inhibition and mode of action of berberine. Exp.
Parasitol. 1985, 60, 404−413.
(95) Lanteri, C. A.; Trumpower, B. L.; Tidwell, R. R.; Meshnick, S. R.
DB75, a novel trypanocidal agent, disrupts mitochondrial function in
Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 2004, 48,
3968−3974.
(96) Mathis, A. M.; Bridges, A. S.; Ismail, M. A.; Kumar, A.;
Francesconi, I.; Anbazhagan, M.; Hu, Q.; Tanious, F. A.; Wenzler, T.;
Saulter, J.; Wilson, W. D.; Brun, R.; Boykin, D. W.; Tidwell, R. R.;
Hall, J. E. Diphenyl furans and aza analogs: effects of structural
modification on in vitro activity, DNA binding, and accumulation and
distribution in trypanosomes. Antimicrob. Agents Chemother. 2007, 51,
2801−2810.
(97) Moreno, S. N. J. Pentamidine is an uncoupler of oxidative
phosphorylation in rat liver mitochondria. Arch. Biochem. Biophys.
1996, 326, 15−20.
(98) Stead, A. M. W.; Bray, P. G.; Edwards, I. G.; DeKoning, H. P.;
Elford, B. C.; Stocks, P. A.; Ward, S. A. Diamidine compounds:
selective uptake and targeting in Plasmodium falciparum. Mol.
Pharmacol. 2001, 59, 1298−1306.
(99) Tidwell, R. R.; Jones, S. K.; Geratz, J. D.; Ohemeng, K. A.; Cory,
M.; Hall, J. E. Analogs of 1,5-bis(4-amidinophenoxy)pentane
(pentamidine) in the treatment of experimental Pneumocystis carinii
pneumonia. J. Med. Chem. 1990, 33, 1252−1257.
(100) Werbovetz, K. A. Promising therapeutic targets for
antileishmanial drugs. Expert Opin. Ther. Targets 2002, 6, 407−422.
(101) Zhu, W.; Zhang, Y.; Sinko, W.; Hensler, M. E.; Olson, J.;
Molohon, K. J.; Lindert, S.; Cao, R.; Li, K.; Wang, K.; Wang, Y.; Liu,
Y.-L.; Sankovsky, A.; de Oliveira, C. A. F.; Mitchell, D. A.; Nizet, V.;
McCammon, J. A.; Oldfield, E. Antibacterial drug leads targeting
isoprenoid biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 123−
128.
(102) Bibler, M. R.; Chou, T. C.; Toltzis, R. J.; Wade, P. A. Recurrent
ventricular tachycardia due to pentamidine-induced cardiotoxicity.
Chest 1988, 94, 1303−1306.
(103) Biyah, K.; Molimard, M.; Naline, E.; Bazelly, B.; Advenier, C.
Indirect muscarinic receptor activation by pentamidine on airway
smooth muscle. Br. J. Pharmacol. 1996, 119, 1131−1136.
(104) Bouchard, P.; Sai, P.; Reach, G.; Caubarrere, I.; Ganeval, D.;
Assan, R. Diabetes mellitus following pentamidine-induced hypo-
glycemia in humans. Diabetes 1982, 31, 40−45.
(105) Kempin, S. J.; Jackson, C. W.; Edwards, C. C. In vitro
inhibition of platelet function and coagulation by pentamidine
isethionate. Antimicrob. Agents Chemother. 1977, 12, 451−454.
(106) Murphey, S. A.; Josephs, A. S. Acute pancreatitis associated
with pentamidine therapy. Arch. Intern. Med. 1981, 141, 56−58.
(107) Small, C. B.; Harris, C. A.; Friedland, G. H.; Klein, R. S. The
treatment of Pneumocystis carinii pneumonia in the acquired
immunodeficiency syndrome. Arch. Intern. Med. 1985, 145, 837−840.
(108) Western, K. A.; Schultz, M. G. Pentamidine nontoxicity. Ann.
Intern. Med. 1969, 70, 234−234.
(84) Collar, C. J.; Zhu, X.; Werbovetz, K.; Boykin, D. W.; Wilson, W.
D. Molecular factors governing inhibition of arylimidamides against
Leishmania: conservative computational modeling to improve chemo-
therapies. Bioorg. Med. Chem. 2011, 19, 4552−4561.
(85) Wenzler, T.; Boykin, D. W.; Ismail, M. A.; Hall, J. E.; Tidwell, R.
R.; Brun, R. New treatment option for second-stage African sleeping
sickness: in vitro and in vivo efficacy of aza analogs of DB289.
Antimicrob. Agents Chemother. 2009, 53, 4185−4192.
(86) Donkor, I. O.; Assefa, H.; Rattendi, D.; Lane, S.; Vargas, M.;
Goldberg, B.; Bacchi, C. Trypanocidal activity of dicationic
compounds related to pentamidine. Eur. J. Med. Chem. 2001, 36,
531−538.
(87) Donkor, I. O.; Huang, T. L.; Tao, B.; Rattendi, D.; Lane, S.;
Vargas, M.; Goldberg, B.; Bacchi, C. Trypanocidal activity of
conformationally restricted pentamidine congeners. J. Med. Chem.
2003, 46, 1041−1048.
(88) Ansede, J. H.; Anbazhagan, M.; Brun, R.; Easterbrook, J. D.;
Hall, J. E.; Boykin, D. W. O-Alkoxyamidine prodrugs of furamidine: in
vitro transport and microsomal metabolism as indicators of in vivo
efficacy in a mouse model of Trypanosoma brucei rhodesiense infection.
J. Med. Chem. 2004, 47, 4335−4338.
U
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(109) Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S.
Antimalarial drug discovery: efficacy models for compound screening.
Nat. Rev. Drug Discovery 2004, 3, 509−520.
(110) Arnold, L.; Tyagi, R. K.; Meija, P.; Swetman, C.; Gleeson, J.;
Perignon, J.-L.; Druilhe, P. Further improvements of the P. falciparum
humanized mouse model. PLoS One 2011, 6, e18045.
(111) Langhorne, J.; Buffet, P.; Galinski, M.; Good, M.; Harty, J.;
Leroy, D.; Mota, M. M.; Pasini, E.; Renia, L.; Riley, E.; Stins, M.;
Duffy, P. The relevance of non-human primate and rodent malaria
models for humans. Malar. J. 2011, 10, 23.
(112) Angulo-Barturen, I.; Jimenez-Diaz, M. B.; Mulet, T.; Rullas, J.;
Herreros, E.; Ferrer, S.; Jimenez, E.; Mendoza, A.; Regadera, J.;
Rosenthal, P. J.; Bathurst, I.; Pompliano, D. L.; de las Heras, F. G.;
Gargallo-Viola, D. A murine model of falciparum-malaria by in vivo
selection of competent strains in non-myelodepleted mice engrafted
with human erythrocytes. PLoS One 2008, 3, e2252.
(113) Moreno, A.; Badell, E.; Van, R. N.; Druilhe, P. Human malaria
in immunocompromised mice: new in vivo model for chemotherapy
studies. Antimicrob. Agents Chemother. 2001, 45, 1847−1853.
(114) Moreno, A.; Perignon, J. L.; Morosan, S.; Mazier, D.; Benito, A.
Plasmodium falciparum-infected mice: more than a tour de force.
Trends Parasitol. 2007, 23, 254−259.
V
dx.doi.org/10.1021/jm400508e | J. Med. Chem. XXXX, XXX, XXX−XXX