Antiprotozoal activity of dicationic 3,5-diphenylisoxazoles, their prodrugs and aza-analogues

Article abstract of DOI:10.1016/j.bmc.2013.10.050

Fifty novel prodrugs and aza-analogues of 3,5-bis(4-amidinophenyl)isoxazole and its derivatives were prepared. Eighteen of the 24 aza-analogues exhibited IC₅₀ values below 25 nM against Trypanosoma brucei rhodesiense or Plasmodium falciparum. S

Full text of DOI:10.1016/j.bmc.2013.10.050

Bioorganic & Medicinal Chemistry 22 (2014) 559–576
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiprotozoal activity of dicationic 3,5-diphenylisoxazoles, their
prodrugs and aza-analogues
Donald A. Patrick a, Stanislav A. Bakunov a, Svetlana M. Bakunova a, Tanja Wenzler b,c, Reto Brun b,c
,
Richard R. Tidwell a,
⇑
a University of North Carolina, Pathology & Laboratory Medicine, 805 Brinkhous-Bullitt Bldg, CB 7525, Chapel Hill, NC 27599-7525, USA
b Swiss Tropical and Public Health Institute, Medical Parasitology & Infection Biology, Socinstrasse 57, 4051 Basel, Switzerland
c University of Basel, Petersplatz 1, 4003 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Fifty novel prodrugs and aza-analogues of 3,5-bis(4-amidinophenyl)isoxazole and its derivatives were
prepared. Eighteen of the 24 aza-analogues exhibited IC50 values below 25 nM against Trypanosoma bru-
cei rhodesiense or Plasmodium falciparum. Six compounds had antitrypanosomal IC50 values below 10 nM.
Twelve analogues showed similar antiplasmodial activities, including three with sub-nanomolar poten-
cies. Forty-four diamidines (including 16 aza-analogues) and the 26 prodrugs were evaluated for efficacy
in mice infected with T. b. rhodesiense STIB900. Six diamidines cured 4/4 mice at daily 5 mg/kg intraperi-
toneal doses for 4 days, giving results far superior to pentamidine and furamidine. One prodrug attained
3/4 cures at daily 25 mg/kg oral doses for 4 days.
Received 27 August 2013
Revised 21 October 2013
Accepted 29 October 2013
Available online 9 November 2013
Keywords:
Diarylisoxazole
Diamidine
Ó 2013 Published by Elsevier Ltd.
Prodrug
Antiprotozoal
Antitrypanosomal
1. Introduction
estimated 219 million cases and 660,000 deaths due to malaria in
2010.5,6 The growing problem of resistance to drugs including
The need for new drugs to treat the protoazoan diseases human
African trypanosomiasis (HAT) and malaria persists. Despite the
decreased number of new cases of HAT reported in recent years,1
an estimated 70 million people are at risk of contracting this dis-
ease.2 Infections of Trypanosoma brucei gambiense account for
95% of the cases of HAT, and the remaining cases are due to an
infection of Trypanosoma brucei rhodesiense. Few treatments are
available, and most of them were introduced prior to 1950. Early
stage HAT, in which the parasites are concentrated in the hem-
olymphatic system, is treated by pentamidine (Fig. 1) and suramin.
The late stage disease, in which the parasites have entered the cen-
tral nervous system (CNS) is treated with melarsoprol and eflorni-
thine (licensed in 1990). All of these drugs require parenteral
administration, are prone to resistance, and are associated with
various toxicities, the most serious being a potentially fatal
encephalopathy associated with melarsoprol.3,4 A combination
therapy of the oral drug nifurtimox and intravenous eflornithine
was licensed in 2009.1,3
chloroquine, sulfadoxine–pyrimethamine, mefloquine, artemisinin
has created a need for new treatments.3,7,8
Diamidines, which are positively charged at physiological pH,
generally exhibit poor oral bioavailability.9 Furamidine10 (Fig. 1,
R = H) is a conformationally restricted analogue of pentamidine.
Its orally effective prodrug pafuramidine11 (R = OCH3), which
undergoes biotransformation via distinct O-demethylation and
N-dehdyroxlation steps,12,13 advanced to Phase III clinical trials
against early stage HAT and Phase II trials against malaria.9,14-17
The compound exhibited nephro- and hepatotoxicity in a recent
expanded phase I trial,9 resulting in a suspension of phase III
trials.9,16,17 In the search for safer and at least equally effective
drugs, structural modifications of furamidine have been exten-
sively studied. Such alterations have included replacing phenyl
rings with pyridyl rings18,19 and the replacement of furan with
benzene,19 other heterocycles,20–23,10,24–26 or an oxymethylene
bridge.27
The trypanocidal properties of 3,5-bis(4-amidinophenyl)isoxaz-
ole (1, Table 1) were reported in the 1970s,23 and a series of ana-
logues was prepared during the last decade.28 These compounds
were highly active against T. b. rhodesiense and P. falciparum
in vitro, with 35 out of the 41 dicationic compounds exhibiting
IC50 values of 50 nM or below against at least one of the two
parasites.28 The present work is a continuation of this endeavor.
Malaria is caused by several Plasmodium species, the most fatal
form due to Plasmodium falciparum. The World Health Organization
(WHO) estimated over 3 billion people to be at risk in 2011, and an
⇑
Corresponding author.
E-mail address: Tidwell@med.unc.edu (R.R. Tidwell).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.10.050

560
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
O
O
H2N
NH2
O
NH2
NR
H2N
RN
NH
NH
Pentamidine
R = H, Furamidine (DB75)
R = OCH3, Pafuramidine (DB289)
Figure 1. Structures of 1,5-bis(4-amidinophenoxy)pentane (pentamidine), 2,5-bis(4-amidinophenyl)furan (furamidine), and 2,5-bis(4-N0-methoxyamidinophenyl)furan
(pafuramidine).
Prodrug derivatives and aza-analogues have now been prepared
and evaluated against these two parasites in vitro. We now report
the efficacies of existing and novel diarylisoxazole derivatives
against T. b. rhodesiense in vivo.
The aza- and diaza-analogues 29–52 (Scheme 2) were prepared
by adaptation of one of the two general pathways employed for the
diphenylisoxazole diamidines.28 Pyridyl halo-nitriles 54a,d were
prepared by dehydration of the corresponding nicotinamides,32,33
while the other aryl halides 54b,c,e,f were commercially available.
The 2-pyridyl chlorides 54a,b and the 3-pyridyl and phenyl bro-
mides 54c–f underwent Sonogashira couplings by the method of
Roesch34 but using the more economical 2-methylbut-3-yn-2-ol
(mebynol)35 in place of (trimethylsilyl)acetylene, to give novel
intermediates 55a–d and known intermediates 55e–f,36,28 which
underwent deprotection using a catalytic amount of sodium hy-
dride in refluxing toluene35 to the known cyanoaryl acetylenes
56a–f.37–40 Pyridyl acetylenes 56a–d have been previously
prepared by desilylation of the corresponding 2-(trimethylsilyl)
ethynylarenes.39,40 Aldehdye 57a was prepared by reaction of 6-
methylnicotinonitrile with DMF-DMA41 followed by periodate
cleavage39 of the enamine derivative. Aldehyde 57b was prepared
by manganese dioxide oxidation of the corresponding hydroxy-
methyl compound.42 The cyano-substituted nicotinaldehyde deriv-
atives 57c,d were prepared by treatment of the corresponding
bromides with copper(I) cyanide in DMF.43,44 The aldehydes
57a–f were treated with hydroxylamine to give oximes 58a,45b–
d,e,46f.47 The oximes were chlorinated at the 10-position using N-
chlorosuccinimide in DMF48 to give synthons 59a,43b–d,e–f,24
which immediately underwent cycloaddition reactions with acety-
lenes 56a–f in the presence of bis(tributyltin) oxide49 to give the
desired bis(cyanoaryl)isoxazoles: 2-pyridyl derivatives 60–71 and
3-pyridyl derivatives 72–83. The dinitriles were reacted with
hydroxylamine in DMSO,29 and the crude diamidoximes under-
went O-acetylation in situ, followed by catalytic hydrogenation,28
to give the corresponding bis(amidinoaryl)isoxazoles, 2-pyridyl
analogues 29–40 and 3-pyridyl analogues 41–52, which were puri-
fied by preparative HPLC and isolated as their hydrochloride salts.
2. Chemistry
Structures of known diphenylisoxazole diamidines 1–2828 se-
lected for in vivo testing are shown in Table 1. Diamidine 1 and
its regioisomers 9, 16, and 24 are the four ‘parent’ molecules from
which all other structures are derived by introduction of substitu-
ents on the phenyl rings or the amidine nitrogen atoms, or by
insertion of nitrogen atoms into the outer aromatic rings. The ami-
doxime prodrug derivatives 1a, 3a–4a, 7a–9a, 11a–13a, 15a–16a,
18a, 21a, 24a, and 28a were prepared by treatment of the corre-
sponding dinitriles 53a–o28 with hydroxylamine29 (Scheme 1).
The methamidoxime prodrugs 1b, 3b, 4b, 7b, 9b, 11–13b, 15–
16b, and 21b were generated by O-methylation of the correspond-
ing amidoximes by with dimethyl sulfate18,29,30 or iodomethane31
using appropriate bases and solvents.
Table 1
Structures of 3,5-diphenylisoxazole diamidinesa
O
N
NH
HN
m
m
3
5
p
NH2
H2N
p
R1
R2
Compd
5-Phenyl
R1
H
NO2
Cl
OMe
H
H
H
OMe
H
NO2
Cl
OMe
H
H
H
H
Cl
OMe
H
H
R2
3-Phenyl
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
meta
H
H
H
H
NO2
Cl
OMe
OMe
H
H
H
H
NO2
Cl
OMe
H
H
H
NO2
Cl
para
para
para
para
para
para
para
para
meta
meta
meta
meta
meta
meta
meta
para
para
para
para
para
para
para
para
meta
meta
meta
meta
meta
3. Biological results
3.1. In vitro data
The activities of the novel diamidines 29–52 against T. b. rhodes-
iense STIB900 and chloroquine-resistant P. falciparum K1, as well as
their cytotoxicities to L6 rat myoblast cells50 are shown in Table 2.
Previously published data for parent diamidines 1, 9, 16, and 2428
are included for sake of comparison. Selectivity indices (SI, the ra-
tio of cytotoxic to antiprotozoal IC50 values) were determined for
both parasites. Standard drugs included pentamidine and furami-
dine (against both parasites), melarsoprol (against T. b. rhodes-
iense), chloroquine and artemisinin (against P. falciparum), and
podophyllotoxin (against L6 cells). The prodrugs, which require
metabolic activation,15 were inactive in vitro, exhibiting IC50 values
H
OMe
NO2
OMe
H
H
H
OMe
OMe
H
Cl
OMe
H
above 5 lM against T. b. rhodesiense and IC50 values above 1 lM
against P. falciparum (data not shown).
Cl
OMe
3.1.1. Activities against T. b. rhodesiense
OMe
In the previous report, compound 16 (IC50 = 3.5 nM) was the
most potent of the four parent diamidines, followed by regioisom-
ers 1 (IC50 = 5.1 nM), 9 (IC50 = 6.3 nM), and 24 (IC50 = 29 nM). Thus
a
Syntheses were reported previously, with all compounds isolated as their
dihydrochloride salts.28

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
561
O
N
HO
O
N
m
m
NC
p
OH
N
N
a
CN
3
5
p
H2N
NH2
R1
R2
R1
R2
53a-o
1a 3a, 4a 7a 8a 9a 11a 12a
,
,
,
,
,
,
,
13a, 15a, 16a, 18a, 21a, 24a, 28a
5-Aryl
para
para
para
para
para
para
para
para
R1
R2
H
3-Aryl
para
para
para
H
53a (for 1a, 1b)
53b (for 3a, 3b
53c (for 4a, 4b
53d (for 7a, 7b
53e (for 8a)
Cl
H
OMe
H
H
b or c
OMe para
OMe para
OMe
H
H
meta
meta
meta
meta
53f (for 9a, 9b)
53g (for 11a, 11b)
53h (for 12a, 12b)
MeO
O
N
OMe
N
N
Cl
H
OMe
H
H
H2N
NH2
53i (for 13a, 13b) para
NO2
R1
R2
53j (for 15a, 15b) para
H
H
OMe meta
meta
meta
H
H
para
para
53k (for 16a, 16b)
53l (for 18a)
1b 3b 4b 7b 9b 11b 12b 13b 15b 16b 21b
,
,
,
,
,
,
,
,
,
,
OMe
H
53m (for 21a, 21b) meta
OMe para
53n (for 24a)
53o (for 28a)
meta
meta
H
H
meta
OMe
OMe meta
Scheme 1. Synthesis of amidoxime and methamidoxime derivatives of 3,5-bis(amidinophenyl)isoxazoles (isolated as their hydrochloride salts). Reagents and conditions: (a)
NH2OHꢀHCl, t-BuOK, DMSO, 25 °C, overnight, 12–95%; (b) Me2SO4, aq NaOH, dioxane or DMF, 0–25 °C, overnight, 13–18%; (c) MeI, NaH/DMF or t-BuOK/DMSO, 25 °C,
overnight, 15–56%.
R
m
NC
m
NC
p
a
c
b
CN
a
b
OH
NC
b
54a-59a
p-CN CH
N
p
b
a
b
a
a
54b-59b m-CN CH
N
CH
CH
54a-b : R = Cl
55a-f
56a-f
54c-59c p-CN
54d-59d
N
N
54c-f
: R = Br
m-CN
Cl
54e-59e p-CN CH CH
54f-59f m-CN CH CH
OHC
m
CN
p
m
d
HON
m
CN
p
CN
HON
b
b
p
a
a
b
a
57a-f
58a-f
59a-f
O
N
O N
NH
m
m
HN m
m
f, g
e
56a-f + 59a-f
CN
NC
3
3
5
5
NH2
p
p
p
p
H2N
b
c
b
c
a
a
d
d
60-83
29-52
Scheme 2. Preparation of 3,5-bis(amidinoaryl)isoxazoles 29–52 (isolated as their hydrochloride salts). Reagents and conditions: (a) 2-methylbut-3-yn-2-ol (mebynol),
PdCl2(PPh3)2 (2 mol %), CuI (2 mol %), Et3N, 60 °C, 3 h, 77–100%; (b) NaH (15 mol %), toluene, reflux (Dean–Stark trap), 0.75–1.5 h, 59–76%; (c) NH2OHꢀHCl, aq EtOH, 25 °C, 1–
2 h, 72–96%; (d) NCS, DMF, 0–25 °C, overnight, 96–100%; (e) bis(tributyltin) oxide (0.5 equiv), CH2Cl2. 25 °C, overnight, 50–81%; (f) NH2OHꢀHCl, t-BuOK, DMSO, 25 °C,
overnight, 86–100%; (g) Ac2O, AcOH, 25 °C, 0.5–18 h and then H2 (60 psi) 10% Pd/C, AcOH, 25 °C, 3–72 h. Structures of dinitriles 60–83 correspond to those of diamidines 29–
52, respectively, as shown in Table 2.
at least one amidine moiety para to the central ring was required
for optimal potency in the absence of additional substituents on
the aromatic rings.28
The 3-pyridyl analogues (a or d = N), in general, showed higher
antitrypanosomal potencies but decreased selectivities relative to
their 2-pyridyl congeners. Compounds 41–43 showed potencies
at least comparable to parent molecule 1, with analogue 41 show-
ing slightly enhanced potency and selectivity. Analogues 44–46
had IC50 values below 25 nM, but only 44 (IC50 = 3.7 nM) was more
potent than parent molecule 9, and all three compounds were less
selective than the parent. Compound 48 (IC50 = 1.8 nM, SI = 1,120)
was the most potent overall and the only 3-pyridyl derivative hav-
ing a selectivity index above 1000, being slightly more potent but
less selective than parent molecule 16. Congeners 47 and 49 had
IC50 values below 20 nM but were less potent than parent molecule
16. Derivatives 51–52, the 3-pyridyl analogues of diamidine 24,
were all significantly less potent than the parent molecule. The
3-phenyl-5-(pyridin-3-yl) derivatives 41 and 44 were more potent
than their 5-phenyl-3-(pyridin-3-yl) counterparts 42 and 45,
respectively, while the converse was true for analogues 47
and 48, showing a trend similar that observed with the 2-pyridyl
derivatives. By contrast, the decreased potencies of di(pyridin-3-
yl) analogues 43, 46, 49, and relative to the corresponding pairs
of phenyl-(pyridin-3-yl) derivatives was less pronounced.
Fifteen of the 24 aza-analogues were highly active against T. b.
rhodesiense, with IC50 values below 100 nM. Six analogues (32,
41–44 and 48) exhibited IC50 values below 10 nM. Six derivatives
(29, 36, 45–47, and 49) had IC50 values below 25 nM, while three
analogues (30, 33, and 35) had IC50 values between 50 and 65 nM.
Compound 32 (IC50 = 7.2 nM, SI = 15, 900) was the most potent
and most highly selective of the 2-pyridyl analogues (b or c = N),
showing comparable potency and enhanced selectivity compared
to the corresponding diphenyl diamidine. Analogues 29 and 36
each had IC50 values below 15 nM, but were less potent than
parent molecules
1 and 16, respectively. The 3-phenyl-5-
(pyridin-2-yl) derivatives 29 and 32 were more potent than their
5-phenyl-3-(pyridin-2-yl) counterparts 30 and 33, respectively,
while the converse was true for analogues 35 and 36. The most
greatly diminished potencies were observed among the derivatives
of amidine 24 (congeners 38–40). In all cases the di(pyridin-2-yl)
analogues 31, 34, 37, and 40 were less potent than either of their
mono-pyridyl counterparts.

562
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
Table 2
Aza-analogues of 3,5-diphenylisoxazole diamidines and their antiprotozoal activities in vitro
N
O
NH
HN
m
m
3
5
NH2
p
p
H2N
c
b
a
d
Compd
5-Aryl
a
b
c
d
3-Aryl
T. b. rhodesiensea
P. falciparumb
Cytotox.c
d
e
d
f
d
IC50 (nM)
SIT
IC50 (nM)
SIp
IC50
(l
M)
1
9
16
24
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
para
para
meta
meta
para
para
para
para
para
para
meta
meta
meta
meta
meta
meta
para
para
para
para
para
para
meta
meta
meta
meta
meta
meta
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
N
N
CH
N
N
CH
N
N
CH
N
CH
CH
CH
CH
N
CH
N
N
CH
N
N
CH
N
N
CH
CH
CH
CH
CH
N
N
CH
N
N
CH
N
N
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
CH
N
N
CH
N
N
CH
N
para
meta
para
meta
para
para
para
meta
meta
meta
para
para
para
meta
meta
meta
para
para
para
meta
meta
meta
para
para
para
meta
meta
meta
5.1g
6.3g
3.5g
29.0g
12.1
50.5
596
7.2
52.8
1670
64.3
13.6
3380
183
492
5710
2.7
418
22.6g
57.8g
2.5g
9.2g
9.6
16.7
134
4.1
5.0
7.4
58.0
7.0
142
024.9
15.0
327
1.7
0.4
0.4
1.1
3.7
94
417
2.1g
3822
8923
5266
2972
915
24.1g
31.2g
153g
35.9
46.2
>180
115
12492
16634
3749
2773
>1348
27,840
14,250
>2432
2573
17,363
>1265
>7226
8696
>550
1051
6332
6653
1291
1168
1403
231
302
15897
1350
>108
2323
8897
>53
>986
265
>32
643
423
398
379
270
206
302
1125
111
964
>309
>29
11436
2000
1275
71.2
>180
149
121
>180
>180
130
>180
1.76
2.49
2.46
1.4
4.32
4.91
4.37
2.08
2.11
>180
>180
>180
46.6
6.4
CH
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
5.9
6.2
3.7
16.0
23.9
14.4
1.8
19.1
207
583
6120
3.0
3.5
18.9
0.5
3801
440
4.8
21.7
18.0
144
46.0
14.0
9167
>10,000
>1250
1004
464
N
PMDh
FMDi
MSPj
CQk
ATMl
PPTm
3.0
4.0
5.12
76.5
>180
125
4.0
612
34,884
0.017
a
b
c
Trypanosoma brucei rhodesiense (STIB900).50
Plasmodium falciparum (K1, chloroquine resistant).50
Cytotoxicity to L6 rat myoblast cells.50
The IC50 values are the mean of two independent assays. Coefficients of variation were less than 50%.
Selectivity index for T. b. rhodesiense expressed as the ratio IC50 (L6 cells)/ IC50 (T. b. rhodesiense).
d
e
f
Selectivity index for P. falciparum expressed as the ratio IC50 (L6 cells)/IC50 (P. falciparum).
g
h
i
Data published previously.28
Pentamidine.
Furamidine.
Melarsoprol.
Chloroquine.
Artemisinin.
j
k
l
m
Podophyllotoxin.
3.1.2. Activities against P. falciparum
more than 10 times more potent and more than 30 times more
selective than parent diamidine 9. Analogues 29 and 30 (IC50 val-
ues of 10 and 17 nM, respectively), were more potent and at least
30 times more selective than parent molecule 1. Derivative 36
(IC50 = 7 nM) was slightly less potent than its parent 16. Deriva-
tives 38 and 39 had IC50 values below 25 nM but were less potent
than parent molecule 24. The di(pyridin-2-yl) derivatives 31, 34,
37, and 40 were all less potent than the corresponding mono-pyr-
idyl analogues (e.g., 31 vs 29 and 30).
The 3-pyridyl analogues showed higher potencies. Derivatives
41–43, all with IC50 values below 2 nM, were more potent and
selective than parent molecule 1. Especially noteworthy were ana-
logues 42 and 43 (IC50 = 0.4 nM and SI >6000 for each), which were
over 50 times more potent and more selective. Analogues 44–46,
with IC50 values below 5 nM and selectivity indices above 1000,
were 15–50 times more potent and about three times more selec-
In the previous report, compound 16 (IC50 = 2.5 nM) was the
most potent of the four parent diamidines, followed by regioisom-
ers 24 (IC50 = 9.2 nM), 1 (IC50 = 23 nM), and 9 (IC50 = 58 nM). Thus
at least one amidine moiety meta to the central ring was required
for optimal potency in the absence of additional aromatic
substituents.28
The in vitro antiplasmodial activities of the pyridyl analogues
were quite promising. Eighteen compounds were highly potent
against this parasite, all having IC50 values below 25 nM. This
group includes three derivatives (42, 43, and 48) with IC50 values
below 1 nM, nine analogues (29, 32, 33, 36, 41, 44–46, and 49) with
IC50 values below 10 nM, and six compounds (30, 40, 41, 49, 50,
and 51) with IC50 values between 15 and 25 nM.
Compounds 32 (IC50 = 4.1 nM, SI = 27,800) and 33 (IC50 = 5 nM,
SI = 14,300) were the most potent 2-pyridyl analogues, both being

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
563
tive than parent diamidine 9. Derivative 48 (IC50 = 0.5 nM,
SI = 3801) was five times more potent but somewhat less selective
than parent molecule 16, while congeners 47 and 49 (IC50 values of
19 and 4.8 nM, respectively) were less potent and far less selective
than the parent. Analogues 50 and 51 had IC50 values below 25 nM
but were less potent than parent diamidine 24. Derivative 52
(IC50 = 144 nM) was the only 3-pyridyl analogue with an IC50 value
above 25 nM. Three of the four di(pyridin-3-yl) analogues (43, 46
and 49) were highly potent (IC50 values below 5 nM), in contrast
to their di(pyridin-2-yl) counterparts 31, 34, and 37, respectively.
In summary, optimal potency against P. falciparum required a 6-
amidinopyridin-3-yl moiety at position 3 on the isoxazole ring, as
seen in compounds 42, 43, and 48.
2 attained 3/4 cures, while methoxy and nitro analogues 4 and 5
each attained 1/4 cures at the 5 mg/kg intraperitoneal doses. Thus
the placement of a nitro group on the 5-phenyl ring was more
advantageous than on the 3-phenyl ring (2 vs 5). The efficacy of
1 was retained in the presence of a methoxy group either on the
3-phenyl ring or on both outer rings (derivatives 7 and 8) but
diminished in the presence of a methoxy group on the 5-phenyl
ring (analogue 4). Prodrugs of two of the most potent compounds
were also active. Amidoxime 1a cured 1/4 mice at the 100 mg/kg
oral regimen. The corresponding methamidoxime 1b was more
efficacious, attaining 2/4 cures at the lower 25 mg/kg regimen.
Amidoxime 7a cured 1/4 mice under similar conditions. By con-
trast, methamidoxime 7b attained no cures. Thus diamidine 1,
and eight diphenyl analogues (five amidines and three prodrugs)
were effective in vivo. All five pyridyl analogues of 1 tested
in vivo proved to be effective. The most efficacious was the di(pyri-
din-3-yl) analogue 43, which cured 3/4 infected mice. Both the 5-
and 3-(pyridin-2-yl) analogues 29 and 30, attained 2/4 cures, as did
the 5-(pyridin-3-yl) analogue 41. The 3-(pyridin-3-yl) analogue 42
was also effective, curing 1/4 mice.
Diamidine 16, the second most efficacious parent diamidine,
and its 19 derivatives accounted for 10 of the 29 effective com-
pounds. The parent molecule cured 2/4 infected mice at the
5 mg/kg intraperitoneal doses. The methoxy-nitro and dimethoxy
analogues 22 and 23 showed excellent efficacy, each attaining 4/
4 cures under the same conditions. The mono-methoxy derivative
18 bearing a methoxy group on the 5-phenyl ring cured 3/4 mice
under the same conditions, while its regioisomer 21 with a meth-
oxy group on the 3-phenyl ring attained no cures. The amidoxime
prodrug 16a attained 2/4 cures at the higher 100 mg/kg oral doses
but was not tested at the lower oral doses, while the corresponding
methamidoxime 16b cured no mice at the 25 mg/kg regimen.
Although the methoxy-substitute amidine 21 attained no cures,
its methamidoxime prodrug 21b was the most efficacious prodrug
in the study, curing 3/4 mice at the 25 mg/kg oral regimen. These
results reflect those of furamidine (1/4 cures) and pafuramidine
(4/4 cures) under similar conditions.51 Thus diamidine 16 and five
diphenylisoxazole derivatives, including two oral prodrugs, dem-
onstrated efficacy in vivo. Four of its five pyridyl analogues tested
were also effective. The 3-(pyridin-3-yl) analogue 48 was the most
effective pyridyl derivative overall, curing 4/4 infected mice. The
3-(pyridin-2-yl) derivative 36 cured 3/4 infected mice, as did
the 3,5-di(pyridin-3-yl) analogue 49. The 5-(pyridin-3-yl) analogue
47 attained 2/4 cures.
Diamidine 9 cured no mice at the 20 mg/kg regimen, and its 21
derivatives accounted for only four of the 29 efficacious com-
pounds. The chloro (11) and methoxy (15) analogues each attained
4/4 cures at the 4 ꢁ 20 mg/kg intraperitoneal regimen, but attained
only 1/4 and 2/4 cures, respectively, at the lower 5 mg/kg doses.
Thus activity was enhanced by a methoxy group on the 3-phenyl
but not the 5-phenyl ring (15 vs 12). Methamidoxime prodrug
15b cured 1/4 mice at the 4 ꢁ 25 mg/kg oral dose. Of the five pyr-
idyl analogues tested, only the 3-(pyridin-3-yl) derivative 45 was
effective, attaining 1/4 cures.
Although diamidine 24 cured 3/4 infected mice at the high
20 mg/kg intraperitoneal doses, it attained no cures at the lower
5 mg/kg regimen. Only its methoxy derivative 26 (with the substi-
tuent on the 5-phenyl ring) demonstrated in vivo efficacy at the
low doses, attaining 1/4 cures. Its dimethoxy analogue 28 cured
no mice but with significantly prolonged survivals compared to
untreated controls. Compound 50, its only pyridyl analogue tested
in vivo, attained no cures.
3.2. In vivo data
Seventy compounds (including 26 prodrugs) were tested in
mice infected with the T. b. rhodesiense strain STIB900 (Table 3).51
Mice that survived for 60 days after infection without showing a
parasitemia relapse were considered as cured. Initially, the diami-
dines (which are generally regarded as having poor bioavailability
due to their positive charges at physiological pH)9 were given
intraperitoneally (single daily dose of 20 mg/kg for 4 consecutive
days, or 4 ꢁ 20 mg/kg/day) while the prodrugs were given orally
(single daily doses of 100 mg/kg for 4 consecutive days, or 4 ꢁ
100 mg/kg/day). However, doses were decreased to 4 ꢁ 5 mg/kg/
day for the amidines and 4 ꢁ 25 mg/kg/day for the prodrugs (ex-
cept for amidoximes 1a and 16a) due to the number of compounds
that were effective at the higher doses. This model is highly strin-
gent for the acute stage of HAT, and the low doses applied allowed
for the distinction of activities among the highly trypanocidal com-
pounds. The primary endpoint is cures. Additionally, to distinguish
activity of weaker compounds, the mean relapse day (MSD) was gi-
ven previously. Due to changes in the protocol the secondary end-
point MSD has been changed to mean relapse day (MSD) in recent
experiments. Pentamidine 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.
Six diamidines (diphenyl derivatives 1, 7, 8, 22, and 23, and pyr-
idyl analogue 48) cured 4/4 infected mice at the 4 ꢁ 5 mg/kg intra-
peritoneal regimen. Five diamidines (diphenyl derivatives 2 and
18, and pyridyl analogues 36, 43, and 49) attained 3/4 cures at
the same dose. Six diamidines (diphenyl derivatives 15 and 16,
and pyridyl analogues 29, 30, 41, and 47) each cured 2/4 infected
mice under the same conditions. Finally, six diamidines (diphenyl
derivatives 4, 5, 11, and 26, and pyridyl analogues 42 and 45) at-
tained 1/4 cures. Compounds 11, 15, and 18 each attained 4/4 cures
at the 20 mg/kg regimen, but their cure rates were lower (as stated
above) at the 5 mg/kg doses. Similarly, derivatives 4, 16, and 24
each cured 3/4 mice at the higher doses. Congeners 4 and 16 at-
tained lower cure rates, as noted above, at the lower dose, but com-
pound 24 cured no mice at the 5 mg/kg regimen. Thus, 23
diamidines were at least as effective as pentamidine or furamidine
under the same conditions in this highly stringent model. Six of the
26 prodrugs tested demonstrated oral efficacy. Methamidoxime
21b was the most effective prodrug, attaining 3/4 cures at 4 ꢁ
25 mg/kg oral regimen, followed by methamidoxime 1b, which
cured 2/4 mice. Amidoxime 7a and methamidoxime 15b each
cured 1/4 mice at the 4 ꢁ 25 mg doses. Amidoximes 1a and 16a at-
tained 1/4 and 2/4 cures, respectively, at the 100 mg/kg regimen
but were not tested at the lower doses.
The lead compound 1 and its 21 derivatives accounted for 14 of
the 29 compounds (23 amidines and six prodrugs) that were effec-
tive in vivo. Diamidine 1 and its methoxy and dimethoxy ana-
logues 7 and 8 each attained 4/4 cures, accounting for three of
the six compounds with this excellent cure rate. Nitro derivative
The in vivo efficacies of the diamidines are defined primarily by
the positions of the cations. The most crucial structural require-
ment was a p-amidino group on the 3-aryl ring. Parent molecules
1 and 16 and their derivatives, which bear this motif, accounted

564
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
Table 3
In vivo efficacies against T. b. rhodesiense STIB900a
Compd
Doseb (mg/kg)
Routec
Cured/infectedd
MRDe (days)
—
MSDf (days)
1
4 ꢁ 5
ip
4/4
1/4
2/4
3/4
0/4
0/4
0/4
3/4
1/4
0/4
0/4
1/4
0/4
4/4
1/4
0/4
4/4
0/4
0/4
0/4
0/4
0/4
4/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
4/4
2/4
0/4
1/4
3/4
2/4
2/4
0/4
0/4
4/4
3/4
0/4
0/4
0/4
0/4
0/4
3/4
4/4
4/4
3/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
2/4
2/4
0/4
0/4
0/4
3/4
2/4
1/4
3/4
0/4
1/4
0/4
2/4
4/4
>60
1a
1b
2
4 ꢁ 100
4 ꢁ 25
4 ꢁ 5
p.o.
p.o.
ip
>40.5
>42.25
>53.75
>31
>36.5
39
>50.25
>48.75
26.25
15.75
>51.25
28
>60
>42
27
>60
12
29.25
20
8
NA
>60
NA
8.5
14
NA
8
7.25
29.75
7.25
13.5
NA
3
4 ꢁ 5
ip
3a
3b
4
4 ꢁ 25
4 ꢁ 25
4 ꢁ 20
4 ꢁ 5
p.o.
p.o.
ip
ip
4a
4b
5
6
7
7a
7b
8
8a
9
9a
9b
10
11
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
p.o.
p.o.
ip
ip
ip
p.o.
p.o.
ip
p.o.
ip
p.o.
p.o.
ip
4 ꢁ 5
4 ꢁ 5
—
—
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
4 ꢁ 25
4 ꢁ 20
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
12.25
—
16
4 ꢁ 20
4 ꢁ 5
ip
ip
11a
11b
12
12a
12b
13
13a
13b
14
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
p.o.
p.o.
ip
p.o.
p.o.
ip
p.o.
p.o.
ip
ip
ip
p.o.
p.o.
ip
18
17
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
7.75
—
15
4 ꢁ 20
4 ꢁ 5
>60
>60
15a
15b
16
4 ꢁ 25
4 ꢁ 25
4 ꢁ 20
4 ꢁ 5
18.25
>37.25
>51.25
>49.25
>43.5
31.25
NA
>60
>60
25.75
NA
24
NA
17.5
>50
>60
>60
>58
21.75
7.5
25
NA
ip
16a
16b
17
4 ꢁ 100
4 ꢁ 25
4 ꢁ 5
p.o.
p.o.
ip
ip
ip
p.o.
ip
ip
8.75
—
18
4 ꢁ 20
4 ꢁ 5
18a
19
20
4 ꢁ 25
4 ꢁ 5
13
4 ꢁ 5
21
4 ꢁ 5
ip
36.25
21a
21b
22
23
24
4 ꢁ 25
4 ꢁ 25
4 ꢁ 5
p.o.
p.o.
ip
ip
ip
ip
p.o.
ip
ip
ip
ip
p.o.
ip
ip
ip
ip
ip
ip
ip
—
—
4 ꢁ 5
4 ꢁ 20
4 ꢁ 5
24a
25
26
27
28
28a
29
30
32
33
35
36
41
42
43
44
45
46
47
48
4 ꢁ 25
4 ꢁ 5
4 ꢁ 5
22.3
26
4 ꢁ 5
22.25
>50
6
>44
>52.75
>38.75
4 ꢁ 5
4 ꢁ 25
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
15.5
53
4 ꢁ 5
28.5
>60
>57.25
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
ip
ip
ip
ip
ip
ip
ip
25.7
24
4 ꢁ 5
4 ꢁ 5
>41.75
4 ꢁ 5
15
25.25
4 ꢁ 5
4 ꢁ 5
>54.5
>60
4 ꢁ 5
—

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
565
Table 3 (continued)
Compd
Doseb (mg/kg)
Routec
Cured/infectedd
MRDe (days)
60
MSDf (days)
49
50
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
4 ꢁ 5
ip
ip
ip
ip
ip
3/4
0/4
4/4
1/4
1/4
>60
21.75
>60
>46
>38
MLSPg
FMDh
PMDi
—
a
Female NMRI mice infected with Trypanosoma brucei rhodesiense (STIB900).51
Single daily doses on 4 consecutive days.
Route of administration: intraperitoneal (ip) or oral (p.o.).
Mice that survived for 60 days after infection without showing a parasitemia relapse were considered as cured.
Mean relapse days post infection.
Mean survival days post infection. All mice were used to calculate the MSD (the relapsed mice and the cured mice (MSD >60 days).
Melarsoprol.
Furamidine.
Pentamidine.
b
c
d
e
f
g
h
i
for 19 of the 23 diamidines (13 diphenyl derivatives and 10 aza-
analogues) that attained at least 1/4 cures at the 5 mg/kg ip regi-
men, as well as for all 11 diamidines which attained at least 3/4
cures. Eight of the 11 efficacious bis(amidinophenyl)isoxzoles
bearing additional aromatic substituents have a methoxy group
on at least one outer ring. The introduction of methoxy groups
on both rings was beneficial, resulting in the retained or enhanced
efficacies of 1 and 16, and prolonged survival relative to 24. The
efficacies of parent molecules 1 and 9, which bear p-amidino
groups on the 5-phenyl ring, were retained or enhanced in the
presence of methoxy groups on the 3-phenyl but not the 5-phenyl
ring. By contrast, the efficacies of 16 and 24, which bear m-amidino
groups on the 5-phenyl ring, were enhanced by the presence of
methoxy groups on the 5-phenyl ring but not the 3-phenyl ring.
Nine of the 10 active aza-analogues were derivatives of parent
molecules 1 and 16, bearing p-amidino groups on the 3-aryl ring.
Seven of these 10 compounds were 3-pyridyl derivatives. Seven
aza-analogues also bear a nitrogen atom in the 3-aryl ring.
pyridyl derivatives 41–43 were similar in potency to their counter-
parts with furan central rings.18
The 2-pyridyl derivatives 29–31 were slightly less potent
against P. falciparum than the corresponding furamidine ana-
logues.18 By contrast, 3-pyridyl analogues 41 and 43 were about
10 times more potent, and 42 was nearly 50 times more potent,
compared to the corresponding furamidine analogues.18
Perhaps the most interesting molecule in this study was aza-
analogue 48, which attained 4/4 cures at 4 ꢁ 5 mg/kg ip against
T. b. rhodesiense in vivo as well being one of three molecules to ex-
hibit sub-nanomolar potency against P. falciparum. A number of
other aza-analogues showed promising antiplasmodial activity
in vitro, as did diphenylisoxazole derivatives28 as well as diami-
dines from other classes.20,52–57,27,19 Their further development as
potential antimalarial drugs has been greatly hindered by the lack
of a suitable animal model for initial studies. Due to the high host
specificities of Plasmodium species, standard rodent models must
use parasites that are non-pathogenic to humans,58 and their rele-
vance to human malaria has been questioned.59,60 For example,
pafuramidine, which is known to be effective against human
malaria,15 was ineffective in the commonly used Plasmodium berg-
hei mouse model, as were furamidine and pentamidine.61 The same
study suggested that Plasmodium vinckei may be a more suitable
surrogate of P. falciparum for testing diamidines and thus may pro-
vide at least a starting point in the evaluation of other classes of
diamidines in vivo.
In conclusion, the dicationic diarylisoxazole derivatives, as a
class, have shown promising activities against the HAT and malaria
parasites. Six diamidines—diphenyldiamidines 1, 7, 8, 22, and 23
and aza-analogue 48—attained 4/4 cures at 4 ꢁ 5 mg/kg ip in the
highly stringent STIB900 mouse model of early stage HAT and
therefore warrant further evaluation against other strains of T. bru-
cei and in other animal models. A number of the novel aza-ana-
logues were highly potent against P. falciparum, including
compounds 42, 43, and 48 with IC50 values below 1 nM merit
in vivo evaluation against Plasmodium parasites, contingent upon
the selection of appropriate animal models.
4. Discussion
The in vivo antitrypanosomal data are promising, as 23 diami-
dines were at least as efficacious as pentamidine and furamidine
in the highly stringent T. b. rhodesiense STIB900 acute mouse mod-
el, including six analogues that attained 4/4 cures and five that at-
tained 3/4 cures at 4 ꢁ 5 mg/kg ip However, the prodrugs gave less
promising results, with only six of the 26 prodrugs demonstrating
oral efficacy on the level of cures at the doses tested. The most effi-
cacious prodrug, methamidoxime 21b, attained 3/4 cures at 4 ꢁ
25 mg/kg p.o., compared to 0/4 for the corresponding diamidine
21 at 4 ꢁ 5 mg/kg ip (analogous to 4/4 cures for pafuramidine vs
1/4 cures for furamidine under the same conditions.)51 Methami-
doximes 1b and 15b were also more efficacious than the corre-
sponding amidoximes 1a and 15a, while amidoximes 7a and 16a
were more effective than the corresponding methamidoximes 7b
and 16b. Methamidoxime 1b attained 2/4 cures, compared to 4/4
cures for pafuramidine under the same conditions,51 although ami-
dine 1 was clearly more effective than furamidine at the 5 mg/kg ip
doses.
5. Experimental
Few other direct comparisons can be made between the activi-
ties of the diarylisoxazole and diarylfuran derivatives. The 2-pyri-
dyl analogue 29 was similar in antitrypanosomal potency
compared to the corresponding analogue of furamidine (IC50 = 7
nM)18 while its regioisomer 30 was about 7 times less potent. Ana-
logues 29 and 30 each attained 2/4 cures at 4 ꢁ 5 mg/kg ip, com-
pared to 3/4 cures for the corresponding furamidine analogue.51
The di(pyridin-2-yl) derivative 31 was nearly 30 times less potent
than the corresponding furamidine analogue (IC50 = 21 nM),18
which also attained 3/4 cures at the 4 ꢁ 5 mg/kg ip dose. The 3-
5.1. Biological protocols
In vitro antiprotozoal activities against T. b. rhodesiense
(STIB900) and P. falciparum (chloroquine resistant K1), and cyto-
toxicities against L6 rat myoblast cells,50 and in vivo activities
against T. b. rhodesiense (STIB900)51 were measured following
established protocols. All protocols and procedures used in the cur-
rent study were reviewed and approved by the local veterinary
authorities of the Canton Basel-Stadt, Switzerland. The in vivo data

566
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
employing mean survival days were generated at the time when
that endpoint determination was still accepted by the authorities.
Since the experiments were performed over a longer period, two
different protocols were used in the current studies according to
previous or recent guidelines of the local veterinary authorities.
According to the recent guidelines, animals were euthanized after
reappearance (relapse) of the parasitemia. Therefore, the mean
survival day (MDS) has not been assessed but was replaced by
mean relapse day (MRD).
5.2.1.1. 3,5-Bis[4-(N0-hydroxy)amidinophenyl]isoxazole dihy-
drochloride (1a).
Free base, yield 1.16 g (103%, wet). 1H
NMR d 9.87 (s, 1H), 9.83 (s, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.93 (d,
J = 8.9 Hz, 2H), 7.88 (d, J = 7.9 Hz, 2H), 7.86 (d, J = 8.7 Hz, 2H), 7.70
(s, 1H), 5.97 (br s, 2H), 5.96 (br s, 2H). HPLC 98.8 area% (265 nm).
The amidoxime base was converted to the HCl salt using etha-
nolic HCl, yield 924 mg (72% from salt conversion), mp 308–310°
dec. 1H NMR d 11.38 (br s, 2H), 9.08 (br s, 4H), 8.15 (d, J = 8.5 Hz,
2H), 8.13 (d, J = 8.5 Hz, 2H), 8.01 (s, 1H), 7.97 (d, J = 8.8 Hz, 2H),
7.95 (d, J = 8.5 Hz, 2H). ESI MS m/z 338.1 ([M+1]+ of free base),
169.8 ([(M+2)/2]+). HPLC 96.3 area% (265 nm). Anal. Calcd for C17-
H15N5O3ꢀ2HClꢀ0.4H2O: C, 48.91; H, 4.30; N, 16.78; Cl, 16.98. Found:
C, 49.01; H, 4.40; N, 16.58; Cl, 16.83.
5.2. Chemistry
Uncorrected melting points were measured on a Thomas–Hoo-
ver Capillary melting point apparatus. 1H NMR spectra were re-
corded in DMSO-d6 on
a
Varian Gemini 2000 (300 MHz)
5.2.1.2.
hydroxy)amidinophenyl]isoxazole dihydrochloride (3a).
5-[2-Chloro-4-(N0-hydroxy)amidinophenyl]-3-[4-(N0-
The
spectrometer. Anhydrous solvents were purchased from Aldrich
Chemical Co., Milwaukee, WI, in Sure-sealÒ containers and were
used without further purification. Reaction mixtures were moni-
tored 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). Analytical HPLC chromatograms were recorded on a
Hewlett–Packard 1090 Series II or Agilent 1200 chromatograph
product was purified by preparative HPLC and converted to the free
base using ethanolic HCl and ether. Yield 333 mg (12%), mp 295-
300 °C dec. 1H NMR d 11.40 (br s, 1H), 9.19 (br s, 1H), 8.72 (br s,
1H), 8.24 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 8.2 Hz, 1H), 8.11 (d,
J = 1.7 Hz, 1H), 7.95 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.92 (dd,
J1 = 8.2 Hz, J2 = 1.8 Hz, 1H). ESI MS m/z 372.8 ([M+1]+ of free base).
HPLC 96.8 area% (265 nm). Anal. Calcd for C17H14ClN5O3ꢀ2HCl: C,
45.91; H, 3.63; N, 15.75; Cl, 23.92. Found: C, 45.74; H, 3.58; N,
15.38; Cl, 23.81.
using a Zorbax Rx C8 column (4.6 ꢁ 75 mm, 3.5
lm) maintained
5.2.1.3.
amidino-2-methoxyphenyl]isoxazole dihydrochloride (4a).
3-[4-(N0-Hydroxy)amidinophenyl]-5-[4-(N0-hydroxy)
The
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%)in water con-
taining formic acid (80 mM), ammonium formate (20 mM) and
triethylamine (15 mM). Samples were eluted at appropriate gradi-
ents 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
crude free base was purified by suspension in hot ethanol, yield,
1
3.49 g (95%), mp 219–220 °C dec. H NMR d 9.87 (2, 1H), 9.81 (s, 1H),
7.98 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H),
7.52–7.46 (m, 3H), 6.00 (br s, 2H), 5.94 (br s, 2H), 4.04 (s, 3H). HPLC
95.8 area% (265 nm). Anal. Calcd for C18H17N5O4ꢀ0.7H2O: C, 56.90; H,
4.88; N, 18.43. Found: C, 56.91; H, 4.57; N, 18.19.
An aliquot of the amidoxime base was converted to the HCl salt
using ethanolic HCl and ether. Yield 871 mg (97% from salt conver-
sion), mp >250 °C dec. 1H NMR d 11.49 (br s, 1H), 9.22 (br s, 3H),
8.34 (d, J = 8.4 Hz, 2H), 8.23 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 8.4 Hz,
2H), 7.85 (s, 1H), 7.79 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 4.25 (s, 3H).
ESI MS m/z 368.4 ([M+1]+ of free base), 184.9 ([(M+2)/2]+). HPLC
97.1 area% (265 nm). Anal. Calcd for C18H17N5O4ꢀ2HClꢀ1.5H2O: C,
42.26; H, 4.75; N, 14.99; Cl, 15.17. Found: C, 46.20; H, 4.57; N,
14.73; Cl, 15.18.
(3.0 ꢁ 100 mm, 3.5
lm) 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 ꢁ 25 cm, 8
lm), PS-320 variable wavelength UV–Vis detec-
tor, and a PS-701 fraction collector. Mobile phases consisted of
mixtures of acetonitrile (0–75%) in water containing formic acid
(40 mM) and ammonium formate (10 mM). For compounds 55,
57, and 58, 0.1% CF3CO2H was used in place of the formate buffer.
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, and
reconstituted with water and lyophilized (3 cycles) using 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. Elemental analyses were performed by
Atlantic Microlab, Norcross, GA, and, unless stated otherwise,
were within 0.4% of calculated values. The compounds reported
as salts frequently analyzed correctly for fractional moles of water
and/or other solvents; in each case 1H NMR spectra was consis-
tent with the analysis.
5.2.1.4.
amidino-2-methoxyphenyl]isoxazole dihydrochloride (7a).
5-[4-(N0-Hydroxy)amidinophenyl]-3-[4-(N0-hydroxy)
The
crude free base was purified by suspension in hot ethanol, yield
3.67 g (100%), mp 213–216 °C dec. 1H NMR d.9.85 (s, 1H), 9.82 (s, 1H),
7.95 (d, J = 8.5 Hz, 2H), 8.36 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.1 Hz, 1H),
7.49 (s, 1H), 7.46 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 5.98 (br s, 2H), 5.96
(br s, 2H), 3.96 (s, 3H).
An aliquot of the free base underwent further purification by
preparative HPLC followed by conversion to the HCl salt using eth-
anolic HCl and ether. Yield 1.05 g (79% from free base), mp 242–
244 °C. 1H NMR d 11.34 (br s, 1 H), 9.00 (br s, 3H), 8.18 (d,
J = 8.5 Hz, 2H), 8.00 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H),
7.71 (s, 1H), 7.64 (d, J = 1.2 Hz, 1H), 7.48 (dd, J1 = 8.0 Hz,
J2 = 1.5 Hz, 1H), 4.04 (s, 3H). ESI MS m/z 368.1 ([M+1]+ of free base),
184.5 ([(M+2)/2]+). HPLC 100 area% (265 nm). Anal. Calcd for C18-
H17N5Oꢀ2HClꢀ1.5H2O: C, 46.09; H, 4.77; N, 14.93; Cl, 15.11. Found:
C, 46.24; H, 4.75; N, 14.94; Cl, 15.00.
5.2.1. General procedure for amidoximes 1a, 3a, 4a, 7a, 8a, 9a,
11a, 12a, 13a, 15a, 16a, 18a, 21a, 24a, and 28a
Potassium tert-butoxide (10 equiv) was added to a stirred solu-
tion of hydroxylamine hydrochloride (10 equiv) in DMSO. After
30 min the dinitrile (1 equiv) was added, and the mixture was stir-
red overnight. The reaction mixture was poured over ice, and ami-
doxime base was filtered off and dried (high vacuum, P2O5). The
base was converted to the hydrochloride salt using either aqueous
or ethanolic HCl and other solvents as stated.
5.2.1.5. 3,5-Bis[4-(N0-hydroxy)amidino-2-methoxyphenyl]isox-
azole dihydrochloride (8a).
After stirring for 10 days, the
reaction mixture was filtered before work-up. Free base yield
3.14 g (79%), mp 203–204 °C. 1H NMR d 9.87 (br s, 1H), 9.82 (br s,

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
567
1H), 7.89 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.51 (d,
J = 1.3 Hz, 1H), 7.49–7.46 (m, 2H), 7.42 (dd, J1 = 8.15 H, J2 = 1.5 Hz,
1), 7.27 (s, 1H), 6.00 (br s, 2H), 5.98 (br s, 2H), 4.02 (s, 3H), 3.95 (s,
3H). HPLC 98.0 area% (265 nm). Anal. Calcd for C19H19N5O5ꢀ0.6H2O:
C, 55.91; H, 4.99; N, 17.16. Found: C, 56.18; H, 4.98; N, 16.87.
An aliquot of the free base was converted to the HCl salt using
ethanolic HCl and ether. Yield 887 mg (94% from free base), mp
>250 °C dec. 1H NMR d 11.33 (br s, 1H), 9.12 (br s, 3H), 8.10 (d,
J = 8.0 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.67–7.62 (m, 2H), 7.52
(dm, J = 8.2 Hz, 1H), 7.48 (dm, J = 8.0 Hz, 1H), 7.43 (s, 1H), 4.10 (s,
3H), 4.03 (s, 3H). ESI MS m/z 398.3 ([M+1]+ of free base), 199.6
([(M+2)/2]+). HPLC 99.0 area% (265 nm). Anal. Calcd for C19H19N5-
O5ꢀ2HClꢀ0.5H2O: C, 47.61; H, 4.63; N, 14.61; Cl, 14.79. Found: C,
57.64; H, 4.51; N, 14.51; Cl, 14.81.
9.17 (br s, 2H), 8.29 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 1.9 Hz, 1H),
8.16–8.13 (m, 3H), 7.96 (d, J = 8.7 Hz, 1H), 7.76 (s, 1H). ESI MS m/
z 383.1 ([M+1]+ of free base). HPLC 100 area% (290 nm). Anal. Calcd
for C17H14N6O5ꢀ1.9HClꢀ0.5H2Oꢀ0.8C2H5OH: C, 44.91; H, 4.40; N,
16.89; Cl, 13.54. Found: C, 44.67; H, 4.22; N, 17.17; Cl, 13.25.
5.2.1.10. 3-[3-(N0-Hydroxy)amidino-2-methoxyphenyl[-5-[4-(N0-
hydroxy)amidinophenyl]isoxazole dihydrochloride (15a).
Free
base yield 3.42 g (114%, wet). 1H NMR d 9.82 (s, 1H), 9.57 (2, 1H), 8.13
(d, J = 2.3 Hz, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.81
(dd, J1 = 8.7 Hz, J2 = 2.3 Hz, 1H), 7.44 (s, 1H), 7.23 (d, J = 8.9 Hz, 1H),
5.93 (br s, 2H), 5.86 (br s, 2H), 3.94 (s, 3H). HPLC 100 area% (265 nM).
An aliquot of the amidoxime base was converted to the HCl salt
using ethanolic HCl and ether, followed by recrystallization from a mix-
ture of methanol and aqueous HCl. Yield 686 mg (54%), mp >250 °C dec.
1H NMR d 11.22 (br s, 1), 9.10 (br 2 (3H), 8.21 (d, J = 2.4 Hz, 1H), 8.17 (d,
J = 8.3 H, 2H), 7.96–7.92 (m, 3H), 7.72 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H),
4.04 (s, 3H). ESI MS m/z 368.3([M+1]+ of free base). HPLC 98.6 area%
(265 nM). Anal. Calcd for C18H17N5O4ꢀ2HClꢀ1.3H2O: C, 44.62; H, 4.70;
N, 15.10; Cl, 15.29. Found: C, 44.76; H, 4.82; N, 14.86; Cl, 15.28.
5.2.1.6.
amidinophenyl]isoxazole dihydrochloride (9a).
3-[3-(N0-Hydroxy)amidinophenyl]-5-[4-(N0-hydroxy)
The crude
product was purified by preparative HPLC and converted to the
HCl salt using ethanolic HCl and ether. Yield 1.42 g (18%), mp
187 °C. 1H NMR d 11.34 (br s, 1 H), 9.09 (br s, 3H), 8.36 (s, 1H),
8.23 (d, J = 7.9 H, 1H), 8.12 (d, J = 8.5 Hz, 2H), 7.98 (2, 1H), 7.97 d
(9.1 Hz, 2H), 7.92 (d, J = 8.0 Hz), 7.80 (t, J = 7.8 Hz, 1H). ESI MS m/
z 338.4([M+1]+ of free base). HPLC 100 area% (290 nm). Anal. Calcd
for C17H15N5O3ꢀ2HClꢀ1.75H2O: C, 46.22; H, 4.68; N, 15.85; Cl, 16.05.
Found: C, 46.33; H, 4.58; N, 15.76; Cl, 16.11.
5.2.1.11.
3-[4-(N0-Hydroxy)amidinophenyl]-5-[3-(N0-hydroxy)
amidinophenyl]isoxazole dihydrochloride (16a).
Free base
yield 1.58 g (97%).
An aliquot of the free base was converted to the HCl salt using
aqueous HCl and EtOH. Yield 816 mg (85% from free base), mp
268–271 °C. 1H NMR (300 MHz, DMSO-d6) d 11.44 (s, 2H), 9.17
(s, 4H), 8.38 (t, J = 1.7 Hz, 1H), 8.23 (dt, J = 7.8, 1.4 Hz, 1H), 8.13
(d, J = 8.4 Hz, 2H), 8.01–7.86 (m, 4H), 7.82 (t, J = 7.8 Hz, 1H). ESI
MS m/z 338.1 ([M+1]+ of free base). HPLC 100 area% (265 nm). Anal.
Calcd for C17H15N5O3ꢀ2HClꢀ0.7H2O: C, 48.29; H, 4.39; N, 16.56; Cl,
16.77. Found: C, 48.31; H, 4.29; N, 16.49; Cl, 16.78.
5.2.1.7.
hydroxy)amidinophenyl]isoxazole dihydrochloride (11a).
5-[2-Chloro-4-(N0-hydroxy)amidinophenyl]-3-[3-(N0-
The
crude product was purified by preparative HPLC and converted to
the HCl salt using aqueous HCl. Yield 513 mg (19%), mp 257–260 °C
dec. 1H NMR d.11.39 (br s, 1H), 9.22 (br s, 2H), 8.41 (m, 1H), 8.32
(dm, J = 7.9 Hz, 1H), 8.17 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 1.7 Hz, 1H),
7.96 (s, 1H), 7.94–7.91 (m, 2H), 7.80 (t, J = 7.7 Hz, 1H). ESI MS m/z
372.0 ([M+1]+ of free base). HPLC 100 area% (254 nm). Anal. Calcd
for C17H14ClN5O3ꢀ2HClꢀ0.2H2O: C, 45.55; H, 3.69; N, 15.62; Cl, 23.72.
Found: C, 45.42; H, 3.73; N, 15.63; Cl, 23.97.
5.2.1.12.
hydroxy)amidino-2-methoxyphenyl]isoxazole dihydrochloride
(18a). The free base was converted to the HCl salt using 1 M
3-[4-(N0-Hydroxy)amidinophenyl]-5-[3-(N0-
HCl and EtOH. Yield 1.06 g (60%), mp >280° dec. 1H NMR d 11.34
(s, 1H), 9.12 (s, 4H), 8.34 (d, J = 2.3 Hz, 1H), 8.22 (d, J = 8.5 Hz,
2H), 8.02–7.89 (m, 3H), 7.70 (s, 1H), 7.50 (d, J = 9.0 Hz, 1H), 4.12
(s, 3H). ESI MS m/z 368.1 ([M+1]+ of free base). HPLC 100 area%
(254 nm). Anal. Calcd for C18H17N5O4ꢀ2HClꢀH2O: C, 47.17; H, 4.62;
N, 15.28; Cl, 15.47. Found: C, 47.27; H, 4.67; N, 15.10; Cl, 15.22.
5.2.1.8.
hydroxy)amidino-2-methoxyphenyl]isoxazole dihydrochloride
3-[3-(N0-Hydroxy)amidinophenyl]-5-[4-(N0-
(12a).
The crude product was purified by preparative HPLC
as the free base, yield 1.29 (58%), mp 203–206 °C dec. 1H NMR d
9.85 (s, 1H), 9.75 (s, 1H), 8.22 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.90
(d, J = 8.2 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H),
7.52 (s, 1H), 7.48 (dd, J1 = 8.2 Hz, J2 = 1.3 Hz, 1H), 7.42 (s, 1H),
5.97 (br s, 4H), 4.05 (s, 3H).
5.2.1.13. 3-[4-(N0-Hydroxy)amidino-2-methoxyphenyl]-5-[3-(N0-
hydroxy)amidinophenyl]isoxazole dihydrochloride (21a).
The
An aliquot of the free base was converted to the HCl salt using
aqueous HCl, yield 311 mg (84% from free base), mp 248–250 °C.
1H NMR d 11.36 (br s), 9.14 (br s, 4H), 8.40 (s, 1H), 8.30 (d,
J = 8.0 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H),
7.78 (t, J = 7.8 Hz, 1H), 7.75 (s, 1H), 7.68 (s, 1H), 7.53 (d,
J = 8.2 Hz, 1H), 4.14 (s, 3H). ESI MS m/z 735.3 ([2 M+1]+ of free
base), 368.1 ([M+1]+). HPLC 100 area% (230 nm). Anal. Calcd for
free base was converted to the HCl salt using 1 M HCl and EtOH. Yield
1.15 g (79%), mp >210° dec. 1H NMR d 11.39 (s, 2H), 9.10 (s, 4H), 8.40
(d, J = 2.0 Hz, 1H), 8.26 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.90
(d, J = 7.6 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.67 (d, J = 13.6 Hz, 2H), 7.48
(dd, J = 8.1, 1.6 Hz, 1H), 4.05 (s, 3H). ESI MS m/z 368.1 ([M+1]+ of free
base). HPLC 100 area% (265 nm). Anal. Calcd for
C18H17N5O4-
ꢀ2HClꢀ0.9H2O: C, 47.36; H, 4.59; N, 15.34; Cl, 15.53. Found: C, 47.52;
C
18H17N5O4ꢀ2HClꢀ0.7H2O: C, 47.74; H, 4.54; N, 15.46; Cl, 15.66.
H, 4.56; N, 15.27; Cl, 15.36.
Found: C, 47.71; H, 4.49; N, 15.30; Cl, 15.88.
5.2.1.14. 3,5-Bis[3-(N0-hydroxy)amidinophenyl]isoxazole dihy-
5.2.1.9. 3-[3-(N0-Hydroxy)amidino-2-nitrophenyl]-5-[4-(N0-hydroxy)
drochloride (24a).
The free base was converted to the HCl
amidinophenyl]isoxazole dihydrochloride (13a).
The free base
salt using 1 M HCl and EtOH. Yield 1.60 g (80%), mp 218 °C dec.
1H NMR d 9.10 (br s, 3H), 8.38 (s, 2H), 8.28–8.17 (m, 2H), 7.95–
7.88 (m, 3H), 7.87–7.77 (m, 2H). ESI MS m/z 338.3 ([M+1]+ of free
base). HPLC 100 area% (254 nm). Anal. Calcd for C17H15N5O3-
ꢀ2HClꢀ1.5H2O: C, 46.69; H, 4.61; N, 16.02; Cl, 16.22. Found: C,
46.90; H, 4.58; N, 15.76; Cl, 15.89.
was purified by suspension in hot ethanol, yield 3.35 g (95%), mp 231–
232 °C. 1H NMR d 10.18 (s, 1H), 9.88 (s, 1H), 8.16 (d, J = 8.5 Hz, 1H),
8.10 (d, J = 1.8 Hz, 1H), 8.07 (dd, J1 = 8.5 Hz, J2 = 1.9 Hz, 1H), 7.94 (d,
J = 8.6 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.46 (s, 1H), 6.17 (s, 2H), 5.97 (s,
2H). HPLC 100 area% (290 nm). Anal. Calcd for C17H14N6O5ꢀ0.25H2O: C,
52.78; H, 3.78; N, 21.72. Found: C, 52.93; H, 3.58; N, 21.52.
An aliquot of the amidoxime base was converted to the HCl salt
using ethanolic HCl and ether, yield 896 mg (100% from free base),
mp 187–190 °C dec. 1H NMR d 11.39 (br s, 1H), 11.10 (br s, 1H),
5.2.1.15. 3,5-Bis[3-(N0-hydroxy)amidino-2-methoxyphenyl]isox-
azole dihydrochloride (28a).
The free base was converted to
the HCl salt aqueous HCl. Yield 710 mg (61%), mp 130 °C dec. 1H

568
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
NMR d 11.28 (br s, 1H), 9.11 (br 2, 3H), 8.32 (d, J = 2.2 Hz 1H), 8.19
(d, J = 2.2 Hz, 1H), 7.95 (dd, J1 = 8.8 Hz, J2 = 2.2 Hz), 7.48 (t,
J = 8.8 Hz, 2H), 7.25 (s, 1H), 4.10 (s, 3H), 4.02 (s, 3H). ESI MS m/z
398.1 ([M+1]+ of free base). HPLC 98.3 area% (254 nm). Anal. Calcd
for C19H19N5O5ꢀ2HClꢀ1.4H2O: C, 46.05; H, 4.84; N, 14.13; Cl, 14.31.
Found: C, 46.21; H, 4.83; N, 14.03; Cl, 14.20.
HCl gave a solid, yield 412 mg (18%), mp 210–211 °C. 1H NMR d 8.11 (d,
J = 8.4 Hz, 2H), 8.00 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.61 (d,
J = 1.2 Hz, 1H), 7.59 (s, 1H), 7.51 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H), 4.09
(s, 3H), 3.86 (s, 6H). ESI MS m/z 397.0 ([M+1]+ of free base). HPLC
100 area% (320 nm). Anal. Calcd for C20H21N5O4ꢀ2HClꢀ1.3H2O: C,
48.85; H, 5.25; N, 14.24; Cl, 14.42. Found: C, 48.74; H, 5.13; N, 14.11;
Cl, 14.61.
5.2.2. General procedure for methamidoximes 1b, 3b, 4b, 7b, 9b,
11b, 12b, 13b, 15b, 16b, and 21b
5.2.2.4. 3-[2-Methoxy-4-(N0-methoxy)amidinophenyl]-5-[4-(N0-
5.2.2.1. 3,5-Bis[4-(N0-methoxy)amidinophenyl]isoxazole dihy-
methoxy)amidinophenyl]isoxazole dihydrochloride (7b).
Potas-
drochloride (1b).
A solution of amidoxime 1a (free base,
sium tert-butoxide (1.48 g, 12.1 mmol) was added to a stirred solution
of amidoxime 7a (free base, 2.22 g, 6.05 mmol) in DMSO (30 mL). After
2 h CH3I (2.0 mL, 32.1 mmol) was added, and stirring continued over-
night. The reaction mixture was poured over ice–water and extracted
into CH2Cl2. Chromatography on a column of silica eluting with CHCl3/
MeOH (19:1) followed by recrystallization from hexanes/toluene gave
the free base as a light yellow solid, yield (1.15 g, 48%), mp 163–
167 °C. 1H NMR d 7.97 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 3H), 7.81
(d, J = 8.1 Hz, 1H), 7.48 (s, 1H), 7.46 (d, J = 1.5 Hz, 1H), 7.40 (dd, J = 8.0,
1.5 Hz, 1H), 6.24 (s, 5H), 3.96 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H). HPLC
97.0 area% (290 nM). Anal. Calcd for C20H21N5O4ꢀ0.1C6H5CH3: C, 61.45;
H, 5.43; N, 17.31. Found: C, 61.28; H, 5.41; N, 17.12.
The free base was converted to the HCl salt using aqueous HCl,
followed by recrystallizations from ethanolic HCl/CHCl3 and etha-
nolic HCl/ether, yield 730 mg (54% from free base), 207–208 °C.
1H NMR d 8.08 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.1, 1H), 7.92 (d,
J = 8.5, 2H), 7.61 (s, 1H), 7.59 (d, J = 1.5 Hz, 1H), 7.47 (dd, J = 8.1,
1.6 Hz, 1H), 4.01 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H). ESI MS m/z
396.3 ([M+1]+ of free base). HPLC 100 area% (290 nM). Anal. Calcd
for C20H21N5O4ꢀ1.75HClꢀ0.4H2Oꢀ0.3C2H5OH: C, 51.52; H, 5.32; N,
14.58; Cl, 12.92. Found: C, 51.62; H, 5.28; N, 14.55; Cl, 12.68.
2.26 g, 6.69 mmol) in DMF (75 mL) maintained at <0 °C (ice–salt
bath) was treated with 2 M NaOH (25 mL), followed by the drop-
wise addition of Me2SO4 (5.0 mL, 52.8 mmol) in DMF (25 mL).
The ice bath was removed and the reaction mixture was stirred
overnight at room temperature. Dilution of the reaction mixture
with water gave a precipitate (2.05 g, 84%). Purification on a col-
umn of silica eluting with CHCl3/MeOH (20:1) followed by suspen-
sion of the product in hot MeOH give the free base, yield 623 mg
(25%), mp 235–239 °C. 1H NMR d 7.94 (d, J = 8.7 Hz, 4H), 7.86 (d,
J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 6.19 (br s,
2H), 6.17 (br s, 2H), 3.78 (s, 6H). HPLC 98.2 area% (290 nm). Anal.
Calcd for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C,
62.25; H, 5.24; N, 18.99.
Crystallization from ethanol and aqueous HCl gave the HCl
salt, yield 747 mg (74% from free base, 15% overall), mp 242–
243 °C dec. 1H NMR d 8.08 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.2 Hz,
2H),7.95 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 7.91 (s, 1H),
3.87 (s, 3H), 3.86 (s, 3H). ESI MS m/z 366.2([M+1]+ of free base).
HPLC 100 area% (290 nm). Anal. Calcd for C19H19N5O3ꢀ2HCl: C,
52.06; H, 4.83; N, 15.98; Cl, 16.18. Found: C, 52.04; H, 4.91; N,
15.90; Cl, 16.25.
5.2.2.5.
amidinophenyl]isoxazole dihydrochloride (9b).
3-[3-(N0-Methoxy)amidinophenyl]-5-[4-(N0-methoxy)
Reaction
5.2.2.2.
methoxy)amidinophenyl]isoxazole dihydrochloride (3b).
5-[2-Chloro-(4-N0-methoxy)amidinophenyl]-3-[4-(N0-
To
conditions and chromatographic purification were similar to those
employed for 7b above. The free base was recrystallized from
EtOH, yield (1.17 g, 65%), mp 201–202 °C. 1H NMR d 8.21 (t,
J = 1.6 Hz, 1H), 7.95 (d, J = 8.6 Hz, 3H), 7.87 (d, J = 8.5 Hz, 2H),
7.81 (dt, J1 = 8.0 Hz, J2 = 1.3 Hz, 1H), 7.72 (s, 1H), 7.57 (t,
J = 7.8 Hz, 1H), 6.23 (br s, 2H), 6.22, br s, 2H), 3.79 (s, 3H), 3.78
(s, 3H). Anal. Calcd for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17.
Found: C, 62.66; H, 5.30; N, 19.32.
The free base was converted to the HCl salt using aqueous HCl,
yield 1.20 g (86% from free base), mp 180 °C dec. 1H NMR d 8.34 (t,
J = 1.7 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.95
(d, J = 8.6 Hz, 2H), 7.93–7.86 (m, 2H), 7.73 (t, J = 7.8 Hz, 1H), 3.88 (s,
3H), 3.85 (s, 3H). ESI MS m/z 366.2 ([M+1]+ of free base). HPLC 97.0
area% (254 nM). Anal. Calcd for C19H19N5O3ꢀ2HClꢀ2.2H2O: C, 47.75;
H, 5.36; N, 14.65; Cl, 14.84. Found: C, 47.66; H, 5.27; N, 14.41; Cl,
15.10.
a stirred solution of amidoxime 3a (free base, 1.86 g, 5.00 mmol)
in DMF (20 mL) was added NaH (60% dispersion, 0.50 g,
12.5 mmol). After 3 h CH3I (2.0 mL, 32 mmol) was added. After
stirring overnight, the mixture was poured over ice, and the
resulting precipitate (1.88 g, 94%) was filtered off. Purification
on a column of silica, gradient elution from 0–5% MeOH in
CHCl3, followed by crystallization from Et2O/CHCl3 gave the free
base, yield 655 mg (33%), mp 142–144 °C dec. 1H NMR d 8.01 (d,
J = 8.5 Hz, 2H), 8.00 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 1.7 Hz, 1H),
7.85 (dd, J1 = 8.3 Hz, J2 = 1.7 Hz, 1H), 7.84 (d, J = 8.6 Hz, 2H),
7.73 (s, 1H), 6.33 (br s, 2H), 6.21 (br s, 2H), 3.80 (s, 3H), 3.78
(s, 3H). HPLC 95.6 area% (290 nm). Anal. Calcd for C19H18ClN5O3:
C, 57.07; H, 4.54; N, 17.52. Found: C, 56.98; H, 4.48; N, 17.35.
An aliquot was converted to the HCl salt using ethanolic HCl
and ether. Yield 385 mg (93% from free base), mp 232–234 °C
dec. 1H NMR d 8.15 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 1H),
8.01 (d, J = 1.6 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.88 (dd,
J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 7.83 (s, 1H), 3.86 (d, J = 1.2 Hz, 3H),
3.82 (d, J = 1.2 Hz, 3H). ESI MS m/z 400.1 ([M+1]+ of free base),
200.5 ([(M+1)/2]+). HPLC 97.7 area% (290 nm). Anal. Calcd for C19-
H18ClN5O3ꢀ2HClꢀ0.4H2Oꢀ0.3C2H5OH: C, 47.46; H, 4.56; N, 14.26;
Cl, 21.66. Found: C, 47.56; H, 4.33; N, 14.43; Cl, 21.40.
5.2.2.6.
methoxy)amidinophenyl]isoxazole dihydrochloride (11b).
5-[2-Chloro-4-(N0-methoxy)amidinophenyl]-3-[3-(N0-
Reac-
tion conditions and chromatographic purification were similar to those
employed for 7b above. The product was crystallized from CHCl3/ether
to give the free base, yield 736 mg (37%), mp 171173 °C. 1H NMR d
8.24 (t, J = 1.8 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.97 (d, J = 1.6 Hz, 1H),
7.87–7.82 (m, 2H), 7.73 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.33 (br s, 2H),
6.26 (br s, 2H), 3.80 (s, 3H), 3.79 (s, 3H). HPLC 95.3 area% at 265 nm. Anal.
Calcd for C19H18ClN5O3ꢀ0.3H2O: C, 56.31; H, 4.63; N, 17.28. Found: C,
56.22; H, 4.44; N, 17.09.
An aliquot of the free base was converted to the HCl salt using
ethanolic HCl and ether, yield 393 mg (91% from free base), mp
229–230 °C. 1H NMR d 8.37 (t, J = 1.7 Hz, 1H), 8.22 (dm, J = 7.8 Hz,
1H), 8.05 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 1.7 Hz, 1H), 7.96–7.85 (m,
2H), 7.84 (s, 1H), 7.72 (t, J = 7.8 Hz, 1H), 3.87 (s, 3H), 3.82 (s, 3H).
5.2.2.3. 5-[2-Methoxy-4-(N0-methoxy)amidinophenyl]-3-[4-(N0-
methoxy)amidinophenyl]isoxazole dihydrochloride (4b).
Reac-
tion conditions were similar to those for 1c except for the addition of
second portion of Me2SO4 (total of 17 equiv). The reaction mixture
was poured over ice, giving a cloudy mixture that was extracted into
EtOAc. The dried extracts were evaporated to an oil, which was treated
with ethanolic HCl and ether to give a solid (1.15 g, 49%). Purification by
preparative HPLC followed by multiple recrystallizations from aqueous

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
569
ESI MS m/z 400.1 ([M+1]+ of free base). HPLC 98.0 area% (265 nM).
Anal. Calcd for C19H18ClN5O3ꢀ2HClꢀ0.5H2Oꢀ0.3C2H5OH: C, 47.50; H,
4.64; N, 14.13; Cl, 21.46. Found: C, 47.81; H, 4.49; N, 14.09; Cl,
21.14.
EtOAc (1:1) to give a white solid (0.75 g). A solution of the free base
in dioxane was was treated with 4 M HCl/dioxane and then diluted
with ether (to give the precipitated HCl salt, yield 450 mg (19%), mp
>182 °C dec. 1H NMR d 8.35 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.08 (d,
J = 8.2 Hz, 2H), 7.96 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 7.9 Hz, 1H), 7.88
(s, 1H), 7.74 (t, J = 7.9 Hz, 1H), 3.87 (d, J = 1.5 Hz, 6H). ESI MS m/z
366.1 ([M+1]+ of free base). HPLC 100 area% (265 nm). Anal. Calcd
for C19H19N9O3ꢀ2HClꢀ1.3H2O: C, 49.42; H, 5.15; N, 15.17; Cl, 15.36.
Found: C, 49.53; H, 5.14; N, 15.09; Cl, 15.12.
5.2.2.7. 5-[2-Methoxy-4-(N0-methoxy)amidinophenyl]-3-[3-(N0-
methoxy)amidinophenyl]isoxazole dihydrochloride (12b).
Reac-
tion conditions and chromatographic purification were similar to those
employed for 7b above. The free base was recrystallized from
EtOH, yield 827 mg (46%), mp 207–208 °C. 1H NMR d 8.21 (t,
J = 1.6 Hz, 1H), 8.00 (dt, J1 = 8.1 Hz, J2 = 1.3 Hz, 1H), 7.91 (d, J = 8.2 Hz,
1H), 7.81 (dt, J1 = 8.3 Hz, J2 = 1.4 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.49
(d, J = 1.3 Hz, 1H), 7.46 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H), 7.46 (s, 1H),
6.25 (br s, 4H), 4.05 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H). Anal. Calcd for C20-
H21N5O4ꢀ0.3H2O: C, 60.75; H, 5.35; N, 17.71. Found: C, 60.64; H, 5.55; N,
17.39.
The free base was converted to the HCl salt using aqueous
HCl, followed by recrystallization from ethanolic HCl and CHCl3,
yield 576 mg (59% from free base), mp 246–247 °C. 1H NMR d
8.40 (t, J = 1.8 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.04 (d,
J = 8.2 Hz, 1H), 7.92 (dm, J = 7.7 Hz, 1H), 3.90–3.88 (m, 3H),
7.74 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.65 (d, J = 1.1 Hz, 1H),
7.53 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H), 4.11 (s, 3H), 3.89 (s, 3H),
3.87 (s, 3H). ESI MS m/z 396.5 ([M+1]+ of free base). HPLC 100
area% (265 nM). Anal. Calcd for C19H18ClN5O3ꢀ2HClꢀ0.75H2O: C,
49.85; H, 5.12; N, 14.53; Cl, 14.72. Found: C, 50.04; H, 5.10; N,
14.32; Cl, 14.91.
5.2.2.11. 3-[2-Methoxy-4-(N0-methoxy)amidino-phenyl]-5-[3-
(N0-methoxy)amidinophenyl]isoxazole
dihydrochloride
Reaction conditions and chromatography were similar
(21b).
to those employed for 16b above. The free base was recrystallized
from 1 N HCl/EtOH to give the dihydrochloride salt, yield 400 mg
(36%), mp 242–243 °C dec. 1H NMR d 8.38 (d, J = 2.2 Hz, 1H), 8.16
(d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H),
7.72 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 1.7 Hz, 1H), 7.63 (s, 1H), 7.49
(dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H), 4.04 (s, 3H), 3.88 (s, 6H). ESI MS
m/z 396.4 ([M+1]+ of free base). HPLC 100 area% (265 nm). Anal.
Calcd for C20H21N5O3ꢀ2HCl: C, 51.29; H, 4.95; N, 14.95; Cl, 15.14.
Found: C, 51.10; H, 5.02; N, 14.67; Cl, 15.16.
5.2.3. General procedure for diamidines 29–52
5.2.3.1. 3-(4-Amidinophenyl)-5-(5-amidinopyridin-2-yl)isoxaz-
ole dihydrochloride (29).
Dinitrile 60 (1.02 g, 3.74 mmol)
was converted to 3-(4-N0-hydroxyamidinophenyl)-5-(5-N0-
hydroxyamidinopyridin-2-yl)isoxazole (free base) following the
general procedure for 1a above. Yield (after suspension in hot
EtOH) 1.10 g (87%), mp >275 °C dec. 1H NMR d.10.07 (s, 1H), 9.85
(s, 1H), 9.04 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.25 (dd, J1 = 8.3 Hz,
J2 = 2.2 Hz, 1H), 8.04 (dd, J1 = 8.2 Hz, J2 = 0.9 Hz, 1H), 8.00 (d,
J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 7.79 (s, 1H), 6.14 (s, 2H),
5.99 (s, 2H). Anal. Calcd for C16H14N6O3ꢀ0.4H2O: C, 55.62; H, 4.32;
N, 24.32. Found: C, 55.95; H, 4.30; N, 24.01.
5.2.2.8.
methoxy)amidinophenyl]isoxazole
(13b). Reaction conditions and chromatographic purification
3-[3-(N0-Methoxy)amidino-2-nitrophenyl]-5-[4-(N0-
dihydrochloride
were similar to those employed for 3b above. The free base was
recrystallized from a mixture of CHCl3, MeOH, and ether, yield
900 mg (23%), mp 201–202 °C. 1H NMR d 8.17 (d, J = 8.5 Hz, 1H),
8.09 (d, J = 1.8 Hz, 1H), 8.06 (dd, J1 = 8.5, Hz, J2 = 1.9 Hz, 1H), 7.95
(d, J = 8.7 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.50 (s, 1H), 6.44 (br s,
2H), 6.23 (br s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). HPLC 98.7 area%
(265 nm). Anal. Calcd for C19H18N6O5ꢀ0.5H2O: C, 54.41; H, 4.57;
N, 20.04. Found: C, 54.39; H, 4.36; N, 20.01.
An aliquot of the free base was converted to the HCl salt using
ethanolic HCl and ether, yield 425 mg (90% from free base), mp
170 °C dec. 1H NMR d 8.20 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 1.8 Hz,
1H), 8.08 (dd, J1 = 8.5, Hz, J2 = 1.9 Hz, 1H), 8.06 (d, J = 8.5 Hz, 2H),
7.93 (d, J = 8.5 Hz, 2H), 7.61 (s, 1H), 3.85 (s, 3H), 3.82 (s, 3H). ESI
MS m/z 411.2 ([M+1]+ of free base). HPLC 100 area% (265 nM). Anal.
Calcd for C19H18N6O5ꢀ1.5HClꢀ1.6H2O: C, 46.20; H, 4.63; N, 17.02; Cl,
10.77. Found: C, 46.09; H, 4.46; N, 16.87; Cl, 10.75.
A stirred solution of amidoxime (1.10 g, 3.25 mmol) in acetic acid
(150 mL) in a 500 mL Parr shaker bottle was treated with acetic
anhydride (5 mL, 53 mmol). The bis(N-acetoxy)amidine derivative
began to precipitate, and the mixture was stirred until acetylation
was complete (1.5 h). The stirring bar was removed, and 10% Pd/C
(500 mg, 0.47 mmol) and acetic acid (100 mL) were added. The mix-
ture was hydrogenated at 60 psi to completion (4 h). The reaction
mixture was filtered through Celite, and the Celite pad was rinsed
with EtOH and H2O. Combined filtrates were evaporated under re-
duced pressure, and the product was purified by preparative HPLC.
The product was converted to the HCl salt using aqueous HCl.
Yield 543 mg (44%), mp >350 °C dec. 1H NMR d 9.77 (s, 2H), 9.56
(s, 2H), 9.48 (s, 2H), 9.32 (s, 2H), 9.17 (dd, J1 = 2.3 Hz, J2 = 0.8 Hz,
1H), 8.49 (dd, J1 = 8.3 Hz, J2 = 2.4 Hz, 1H), 8.28 (dd, J1 = 8.2 Hz,
J2 = 0.8 Hz, 1H), 8.26 (d, J = 8.5 Hz 2H), 8.11 (s, 1H), 8.03 (d,
J1 = 8.5 Hz, 2H). ESI MS m/z 307.1 ([M+1]+ of free base), 154.0
([(M+2)/2]+) HPLC 100 area% (265 nm). Anal. Calcd for C16H14N6-
Oꢀ2.1HClꢀ1.7H2O: C, 46.47; H, 4.75; N, 20.32; Cl, 18.00. Found: C,
46.63; H, 4.77; N, 20.21; Cl, 17.81.
5.2.2.9. 5-[2-Methoxy-3-(N0-methoxy)amidinophenyl]-5-[4-(N0-
methoxy)amidinophenyl]isoxazole dihydrochloride (15b).
Reac-
tion conditions were similar to those employed for 1b above. The free
base was purified on a column of silica eluting with hexanes/EtOAc
(2:3), yield 300 mg (29%). Recrystallization from aqueous HCl gave the
HCl salt, 181 mg (13%), 179–185 °C. 1H NMR d 8.23 (d, J = 2.3 Hz, 1H),
8.06 (d, J = 8.3 Hz, 2H), 7.96 (dd, J1 = 8.9 Hz, J2 = 2.3 Hz, 1H), 7.92 (d,
J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.43 (d, J = 8.9 Hz, 1H), 4.02 (s, 3H), 3.87 (s,
3H), 3.84 (s, 3H). ESI MS m/z 396.2 ([M+1]+ of free base). HPLC 100 area%
(265 nM). Anal. Calcd for C20H21N5O4ꢀ2HClꢀ1.8H2O: C, 47.97; H, 5.35; N,
13.99; Cl, 14.16. Found: C, 47.95; H, 5.28; N, 14.09; Cl, 14.27.
5.2.3.2. 5-(4-Amidinophenyl)-3-(5-amidinopyridin-2-yl)isoxaz-
ole dihydrochloride (30).
Yield of diamidoxime 1.23 g
(93%), mp >275 °C dec. 1H NMR d.10.04 (s, 1H), 9.88 (s, 1H), 9.03
(d, J = 1.7 Hz, 1H), 8.23 (dd, J1 = 8.3 Hz, J2 =2.2 Hz, 1H), 8.09 (d,
J = 8.1 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H),
7.68 (s, 1H), 6.13 (s, 2H), 5.96 (s, 2H). HPLC 100 area% (290 nM).
Anal. Calcd for C16H14N6O3ꢀ0.4H2Oꢀ0.6DMSO: C, 52.65; H, 4.73; N,
21.42. Found: C, 52.48; H, 4.39; N, 21.71.
5.2.2.10. 3-[4-(N0-Methoxy)amidinophenyl]-5-[3-(N0-methoxy)
amidinophenyl]isoxazole dihydrochloride (16b).
Reaction
conditions were similar to those employed for 3b above, but with
dioxane used in place of DMF. Upon completion, the reaction mix-
ture was diluted with water and extracted into EtOAc, and the
product was purified on a column of silica, eluting with hexanes/
The purified diamidine was converted to the HCl salt using eth-
anolic HCl and ether, yield 791 mg (69%), mp >350 °C dec. 1H NMR
d.9.79 (s, 2H), 9.59 (s, 2H), 9.51 (s, 2H), 9.37 (s, 2H), 9.19 (dd,

570
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
J1 = 2.4 Hz, J2 = Hz, 1H), 8.46 (dd, J1 = 8.3 Hz, J2 = 2.3 Hz, 1H), 8.32
(dd, J1 = 8.3 Hz, J2 = 0.9 Hz, 1H), 8.27 (d, J = 8.5 Hz, 2H), 8.05 (d,
J = 8.6 Hz, 2H), 7.99 (s, 1H). ESI MS m/z 307.1 ([M+1]+ of free base),
154.0 ([(M+2)/2]+). HPLC 100 area% (290 nm). Anal. Calcd for C16-
H14N6Oꢀ2.5HClꢀH2O: C, 46.25; H, 4.49; N, 20.23; Cl, 21.33. Found:
C, 46.26; H, 4.41; N, 20.05; Cl, 21.14.
1H), 9.07 (d, J = 5.1 Hz, 1H), 8.51 (dd, J1 = 1.8 Hz, J2 = 0.9 Hz, 1H),
8.48 (dd, J1 = 8.3 Hz, J2 = 2.3 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H), 7.98
(dd, J1 = 5.1 Hz, J2 = 1.8 Hz, 1H), 7.95 (s, 1H). ESI MS m/z 308.6
([M+1]+ of free base), 155.4 ([(M+2)/2]+). HPLC 99.1 area%
(290 nm). Anal. Calcd for C15H13N7Oꢀ2HClꢀ1.5H2O: C, 44.22; H,
4.50; N, 24.06; Cl, 17.40. Found: C, 44.40; H, 4.57; N, 23.82; Cl,
17.69.
5.2.3.3. 3,5-Bis(5-amidinopyridin-2-yl)isoxazole dihydrochlo-
ride (31).
Yield of diamidoxime 4.85 g (117%, wet). 1H NMR
5.2.3.7. 3-(4-Amidinophenyl)-5-(4-amidinopyridin-2-yl)isoxaz-
d. 10.10 (s, 1H), 10.06 (s, 1H), 9.04 (dt, J1 = 2.0 Hz, J2 = 0.9 Hz, 2H),
8.24 (ddd, J1 = 8.3 Hz, J2 = 3.8 Hz, J3 = 2.2 Hz, 2H), 8.12 (dt,
J1 = 8.3 Hz, J2 = 0.8 Hz, 2H), 7.68 (s, 1H), 6.15 (s, 4H).
ole dihydrochloride (35).
Yield of diamidoxime (after sus-
pension in hot EtOH) 762 mg (89%). 1H NMR d 10.23 (s, 1H), 9.83
(s, 1H), 8.76 (dd, J1 = 5.1 Hz, J2 = 0.8 Hz, 1H), 8.28 (dd, J1 = 1.8 Hz,
J2 = 0.8 Hz, 1H), 8.01 (dt, J1 = 8.6 Hz, J2 = 1.9 Hz, 2H), 7.85 (dd,
J = 8.5, 2.0 Hz, 2H), 7.79 (dd, J1 = 5.2 Hz, J2 = 1.7 Hz, 1H), 7.76 (s,
1H), 6.20 (s, 2H), 5.95 (s, 2H). HPLC 96.2 area% (265 nM).
Yield of diamidine 236 mg (28%), mp >320 °C dec. 1H NMR d
9.92 (s, 2H), 9.67 (s, 2H), 9.59 (s, 2H), 9.37 (s, 2H), 9.06 (dd,
J1 = 5.1 Hz, J2 = 0.8 Hz, 1H), 8.50 (dd, J1 = 1.8 Hz, J2 = 0.8 Hz, 1H),
8.24 (d, J = 8.5 Hz, 2H), 8.06 (s, 1H), 8.04 (d, J = 8.5 Hz, 2H), 7.98
(dd, J1 = 5.1 Hz, J2 = 1.7 Hz, 1H). HPLC 100 area% (254 nm). ESI MS
m/z 307.1 ([M+1]+ of free base). Anal. Calcd for C16H14N6-
Oꢀ2HClꢀ1.4H2O: C, 47.51; H, 4.69; N, 20.78; Cl, 17.53. Found: C,
47.42; H, 4.61; N, 20.71; Cl, 17.73.
Yield of diamidine 208 mg (21%), mp >335 °C dec. 1H NMR d.
9.74 (s, 4H), 9.44 (s, 4H), 9.17 (t, J = 2.6 Hz, 2H), 8.46 (td,
J1 = 8.0 Hz, J2 = 2.3 Hz, 2H), 8.43–8.31 (m, 2H), 7.96 (s, 1H). ESI
MS m/z 308.2 ([M+1]+ of free base), 154.5 ([(M+2)/2]+). HPLC 99.5
area% (290 nm). Anal. Calcd for C16H14N6Oꢀ2.6HClꢀ1.3H2O: C,
42.34; H, 4.31; N, 23.04; Cl, 21.66. Found: C, 42.55; H, 4.18; N,
22.91; Cl, 21.65.
5.2.3.4. 3-(3-Amidinophenyl)-5-(5-amidinopyridin-2-yl)isoxaz-
ole dihydrochloride (32).
Yield of diamidoxime 993 mg
(93%), mp 218–220 °C. 1H NMR d 10.07 (s, 1H), 9.78 (s, 1H), 9.04
(dd, J1 = 2.1 Hz, J2 = 0.9 Hz, 1H), 8.28 (t, J = 1.8 Hz, 1H), 8.25 (dd,
J1 = 8.3 Hz, J2 = 2.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.99 (td, J1 =
7.8 Hz, J2 = 1.4 Hz, 1H), 7.90–7.82 (m, 1H), 7.80 (s, 1H), 7.55 (t,
J = 7.8 Hz, 1H), 6.14 (s, 2H), 6.00 (s, 2H). HPLC 100 area%
(230 nM). Anal. Calcd for C16H14N6O3ꢀ0.95H2Oꢀ: C, 54.07; H, 4.51;
N, 23.64. Found: C, 54.13; H, 4.06; N, 23.23.
5.2.3.8. 5-(3-Amidinophenyl)-3-(5-amidinopyridin-2-yl)isoxaz-
ole dihydrochloride (36).
Yield of diamidoxime 1.22 g
(96%), mp 223–224 °C dec. 1H NMR d 10.03 (s, 1H), 9.82 (s, 1H),
9.03 (d, J = 2.1 Hz, 1H), 8.29 (t, J = 1.7 Hz, 1H), 8.23 (dd,
J1 = 8.3 Hz, J2 = 2.2 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.99 (dd,
J1 = 8.0 Hz, J2 = 1.5 Hz, 1H), 7.86 (dd, J1 = 8.1 Hz, J2 = 1.5 Hz, 1H),
7.68 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.13 (s, 2H), 6.04 (s, 2H). HPLC
97.8 area% (254 nM).
Yield of diamidine 619 mg (56%), mp 210 °C dec. 1H NMR d 9.74
(s, 2H), 9.58 (s, 2H), 9.44 (s, 2H), 9.32 (s, 2H), 9.17 (dd, J1 = 2.4 Hz,
J2 = 0.8 Hz, 1H), 8.53–8.43 (m, 2H), 8.35 (dt, J1 = 7.8 Hz, J2 = 1.3 Hz,
1H), 8.28 (dd, J1 = 8.3 Hz, J2 = 0.8 Hz, 1H), 8.08 (s, 1H), 8.01 (dm,
J = 8.3 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H). ESI MS m/z 307.1 ([M+1]+
of free base), 154.0 ([(M+2)/2]+). HPLC 100 area% (230 nm). Anal.
Calcd for C16H14N6Oꢀ1.9HClꢀ2.25H2O: C, 46.18; H, 4.94; N, 20.20;
Cl, 16.19. Found: C, 46.52; H, 4.70; N, 19.84; Cl, 16.38.
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 883 mg (68%), mp 315–
316 °C dec. 1H NMR d 9.75 (s, 2H), 9.60 (s, 2H), 9.45 (s, 2H), 9.35
(s, 2H), 9.18 (dd, J1 = 2.4 Hz, J2 = 0.9 Hz, 1H), 8.52 (t, J = 1.7 Hz,
1H), 8.44 (dd, J1 = 8.3 Hz, J2 = 2.3 Hz, 1H), 8.36 (dm, J = 8.0 Hz,
1H), 8.32 (dd, J1 = 8.3 Hz, J2 = 0.9 Hz, 1H), 8.01 (dt, J1 = 7.9 Hz,
J2 = 1.3 Hz, 1H), 7.91 (s, 1H), 7.84 (t, J = 7.9 Hz, 1H). ESI MS m/z
307.2 ([M+1]+ of free base), 154.0 ([(M+2)/2]+). HPLC 98.3 area%
(230 nm). Anal. Calcd for C16H14N6OꢀHClꢀ1.1H2Oꢀ0.05C2H5OH: C,
48.18; H, 4.65; N, 20.94; Cl, 17.67. Found: C, 48.32; H, 4.46; N,
20.66; Cl, 17.82.
5.2.3.5. 5-(4-Amidinophenyl)-3-(4-amidinopyridin-2-yl)isoxaz-
ole dihydrochloride (33).
Yield of diamidoxime 1.33 g
(98%), mp 247 °C dec. 1H NMR d 10.20 (s, 1H), 9.87 (s, 1H),
8.75 (dt, J1 = 5.6 Hz, J2 = 0.7 Hz, 1H), 8.40–8.32 (m, 1H), 8.00
(dd, J1 = 8.4 Hz, J2 = 1.7 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.80
(dd, J1 = 5.2 Hz, J2 = 1.5 Hz, 1H), 7.65 (d, J = 0.6 Hz, 1H), 6.17 (s,
2H), 5.96 (s, 2H). HPLC 99.5 area% (290 nM). Calcd for
5.2.3.9.
3-(5-Amidinopyridin-2-yl)-5-(4-amidinopyridin-2-yl)
Yield of diamidoxime
C16H14N6O3: C, 56.80; H, 4.17; N, 24.84. Found: C, 56.55; H,
isoxazole dihydrochloride (37).
4.18; N, 24.68.
3.89 g (102%, wet). 1H NMR d 10.25 (s, 1H), 10.05 (s, 1H), 9.04
(dd, J1 = 2.3 Hz, J2 = 0.9 Hz, 1H), 8.75 (dd, J1 = 5.2 Hz, J2 = 0.7 Hz,
1H), 8.36 (dd, J1 = 1.8 Hz, J2 = 0.8 Hz, 1H), 8.24 (dd, J1 = 8.3 Hz,
J2 = 2.2 Hz, 1H), 8.13 (dd, J1 = 8.3 Hz, J2 = 0.9 Hz, 1H), 7.80 (dd,
J1 = 5.2 Hz, J2 = 1.6 Hz, 1H), 7.66 (s, 1H), 6.21 (s, 2H), 6.13 (s, 2H).
Yield of diamidine 315 mg (34%), mp >335 °C dec. 1H NMR d
9.87 (s, 2H), 9.76 (s, 2H), 9.62 (s, 2H), 9.46 (s, 2H), 9.18 (dd,
J1 = 2.3 Hz, J2 = 0.9 Hz, 1H), 9.06 (dd, J1 = 5.1 Hz, J2 = 0.8 Hz, 1H),
8.58 (t, J = 1.2 Hz, 1H), 8.45 (dd, J1 = 8.3 Hz, J2 = 2.3 Hz, 1H), 8.36
(dd, J1 = 8.2 Hz, J2 = 0.9 Hz, 1H), 7.97 (dd, J1 = 5.1 Hz, J2 = 1.7 Hz,
1H), 7.89 (s, 1H). ESI MS m/z 308.2 ([M+1]+ of free base), 154.6
([(M+2)/2]+). HPLC 100area% (254 nm). Anal. Calcd for C16H14N6-
Oꢀ2.3HClꢀ0.9H2O: C, 44.22; H, 4.23; N, 24.07; Cl, 20.02. Found: C,
44.05; H, 4.27; N, 23.84; Cl, 20.23.
Yield of diamidine 759 mg (52%), mp 294–320 °C dec. 1H NMR d
9.86 (s, 2H), 9.60 (s, 2H), 9.58 (s, 2H), 9.34 (s, 2H), 9.06 (d, J = 5.1 Hz,
1H), 8.47 (t, J = 1.1 Hz, 1H), 8.26 (d, J = 8.5 Hz, 2H), 8.04 (d,
J = 8.4 Hz, 2H), 8.00–7.93 (m, 2H). ESI MS m/z 307.6 ([M+1]+ of free
base), 154.9 ([(M+2)/2]+). HPLC 100 area% (290 nm). Anal. Calcd for
C
16H14N6Oꢀ2.4HClꢀ1.8H2O: C, 45.10; H, 4.68; N, 19.73; Cl, 19.97.
Found: C, 45.40; H, 4.70; N, 19.34; Cl, 19.59.
5.2.3.6.
3-(4-Amidinopyridin-2-yl)-5-(5-amidinopyridin-2-yl)
Yield of diamidoxime1.73 g
isoxazole dihydrochloride (34).
(108%, wet), mp 262–263 °C dec. 1H NMR d 10.21 (s, 1H), 10.08
(s, 1H), 9.04 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.76 (d, J = 5.3 Hz,
1H), 8.39 (t, J = 1.2 Hz, 1H), 8.24 (dd, J1 = 8.3 Hz, J2 = 2.2 Hz, 1H),
8.12 (dd, J1 = 8.2 Hz, J2 = 1.0 Hz, 1H), 7.81 (dd, J1= 5.2 Hz,
J2 = 1.7 Hz, 1H), 7.66 (s, 1H), 6.18 (s, 2H), 6.13 (s, 2H). HPLC 97.7
area% (230 nM).
5.2.3.10. 3-(3-Amidinophenyl)-5-(4-amidinopyridin-2-yl)isox-
azole dihydrochloride (38).
Yield of diamidoxime 1.54 g
Yield of diamidine 644 mg (44%), mp 324–325 °C dec. 1H NMR d
9.86 (s, 2H), 9.77 (s, 2H), 9.60 (s, 2H), 9.48 (s, 2H), 9.17 (d, J = 1.7 Hz,
(106%, wet). 1H NMR d 10.23 (s, 1H), 9.77 (s, 1H), 8.76 (dd,
J1 = 5.1 Hz, J2 = 0.8 Hz, 1H), 8.33–8.25 (m, 2H), 7.99 (dt,

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
571
J1 = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.85 (dt, J1 = 7.9 Hz, J2 = 1.4 Hz, 1H),
7.80 (dd, J1 = 5.2 Hz, J2 = 1.7 Hz, 1H), 7.77 (s, 1H), 7.55 (t,
J = 7.8 Hz, 1H), 6.20 (s, 2H), 6.00 (s, 2H). HPLC 96.3 area% (265 nm).
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 679 mg (42%), mp 310–
312 °C. 1H NMR d. 9.87 (s, 2H), 9.61 (s, 2H), 9.60 (s, 2H), 9.35 (s,
2H), 9.06 (d, J = 5.0 Hz, 1H), 8.48 (dt, J1 = 8.4 Hz, J2 = 1.5 Hz, 2H),
8.35 (dm, J = 7.9 Hz, 1H), 8.07–7.98 (m, 2H), 7.96 (dd, J1 = 5.1 Hz,
J2 = 1.7 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H). ESI MS m/z 307.1 ([M+1]+
of free base), 154.0 ([(M+2)/2]+). HPLC 100 area% (265 nm). Anal.
Calcd for C16H14N6Oꢀ2HClꢀ2.2H2O: C, 45.88; H, 4.91; N, 20.06; Cl,
16.93. Found: C, 46.02; H, 4.83; N, 19.97; Cl, 16.90.
(dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.32 (dd, J1 = 8.4 Hz, J2 = 2.2 Hz,
1H), 8.03 (dd, J1 = 8.6 Hz, J2 = 0.9 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H),
7.89 (d, J = 8.6 Hz, 2H), 7.78 (s, 1H). HPLC 94.1 area% (290 nm).
Anal. Calcd for C16H14N6O3ꢀ0.5C2H6OS: C, 54.10; H, 4.54; N,
22.27. Found: C, 54.13; H, 4.32; N, 22.17.
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 608 mg (54%), mp >330 °C
dec. 1H NMR d. 9.79 (s, 2H), 9.61 (s, 3H), 9.59 (s, 1H), 9.37 (s,
2H), 9.35 (d, J = 1.5 Hz, 1H), 8.70 (dd, J1 = 8.3 Hz, J2 = 2.1 Hz, 1H),
8.56 (d, J = 7.9 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 8.15 (s, 1H), 8.07
(d, J = 8.5 Hz, 2H). ESI MS m/z 307.3 ([M+1]+ of free base), 154.3
([(M+2)/2]+). HPLC 100 area% (290 nm). Anal. Calcd for C16H14N6-
Oꢀ2HClꢀ0.4H2Oꢀ0.1C2H5OH: C, 49.76; H, 4.49; N, 21.49; Cl, 18.13.
Found: C, 49.65; H, 4.42; N, 21.28; Cl, 18.18.
5.2.3.11. 5-(3-Amidinophenyl)-3-(4-amidinopyridin-2-yl)isox-
azole trihydrochloride (39).
Yield of diamidoxime 1.42 g
(117%, wet), mp 215–217 °C dec. 1H NMR d 10.20 (d, J = 5.0 Hz,
1H), 9.81 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 8.37 (dd, J1 = 1.7 Hz,
J2 = 0.9 Hz, 1H), 8.29 (t, J = 1.8 Hz, 1H), 7.99 (dt, J1 = 7.9 Hz,
J2 = 1.3 Hz, 1H), 7.86 (dt, J1 = 7.9 Hz, J2 = 1.3 Hz, 1H), 7.80 (dd,
J1 = 5.2 Hz, J2 = 1.7 Hz, 1H), 7.66 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H),
6.17 (s, 2H), 6.02 (s, 2H). HPLC 100 area% (265 nm).
5.2.3.15. 3,5-Bis(6-amidinopyridin-3-yl)isoxazole dihydrochlo-
ride (43).
Yield of diamidoxime 1.20 g (91%), mp >330 °C dec.
1H NMR d.10.19 (s, 1H), 10.14 (s, 1H), 9.11 (ddd, J1 = 7.0 Hz,
J2 = 2.2 Hz, J3 = 0.9 Hz, 2H), 8.32 (ddd, J1 = 8.4 Hz, J2 = 4.5 Hz,
J3 = 2.2 Hz, 2H), 8.05 (ddd, J1 = 8.4 Hz, J2 = 3.4 Hz, J3 = 0.8 Hz, 2H),
7.92 (s, 1H), 5.96 (s, 5H). HPLC 97.2 area% (290 nm). Anal. Calcd
for C15H13N7O3ꢀ0.75H2Oꢀ0.3(CH3)2SO: C, 49.86; H, 4.19; N, 26.11.
Found: C, 49.86; H, 3.86; N, 26.07.
Yield of diamidine 577 mg (38%), mp 315–317 °C dec. 1H NMR d
9.89 (s, 2H), 9.66 (s, 2H), 9.64 (s, 2H), 9.43 (s, 2H), 9.06 (d, J = 5.1 Hz,
1H), 8.53 (t, J = 1.8 Hz, 1H), 8.50–8.44 (m, 1H), 8.35 (d, J = 7.9 Hz,
1H), 8.02 (dm, J = 8.0 Hz, 1H), 7.98 (dd, J1 = 5.1 Hz, J2 = 1.7 Hz,
1H), 7.89 (s, 1H), 7.84 (t, J = 7.9 Hz, 1H). ESI MS m/z 307.1
([M+1]+ of free base), 155.0 ([(M+2)/2]+). HPLC 100 area%
(230 nm). Anal. Calcd for C16H14N6Oꢀ2HClꢀ3.2H2O: C, 43.97; H,
5.21; N, 19.23; Cl, 16.22. Found: C, 44.08; H, 6.15; N, 18.85; Cl,
16.32.
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 277 mg (19%), mp >330 °C dec.
1H NMR d. 9.76 (s, 4H), 9.51 (s, 4H), 9.37 (d, J = 2.1 Hz, 1H), 9.35 (d,
J = 2.3 Hz, 1H), 8.72 (dt, J1 = 8.1 Hz, J2 = 2.2 Hz, 2H), 8.53 (dd,
J1 = 8.5 Hz, J2 = 2.6 Hz, 2H), 8.29 (s, 1H). ESI MS m/z 308.6 ([M+1]+
of free base), 156.1 ([(M+2)/2]+). HPLC 98.9 area% (290 nm). Anal.
Calcd for C15H13N7Oꢀ2HClꢀ0.5H2Oꢀ0.15C2H5OH: C, 46.39; H, 4.30; N,
224.75; Cl, 17.90. Found: C, 46.45; H, 4.24; N, 24.37; Cl, 17.93.
5.2.3.12. 3,5-Bis(4-amidinopyridin-2-yl)isoxazole dihydrochlo-
ride (40).
Yield of diamidoxime 1.50 g (109%, wet), mp 265–
5.2.3.16. 3-(3-Amidinophenyl)-5-(6-amidinopyridin-3-yl)isox-
267 °C dec. 1H NMR d 10.25 (s, 1H), 10.22 (s, 1H), 8.81–8.71 (m,
2H), 8.40 (t, J = 1.1 Hz, 1H), 8.36 (t, J = 0.8 Hz, 1H), 7.81 (ddd,
J1 = 5.0 Hz, J2 = 3.1 Hz, J3 = 1.7 Hz, 2H), 7.65 (s, 1H), 6.27–6.15 (m,
4H). HPLC 98.8 area% (265 nm).
azole dihydrochloride (44).
The crude diamidoxime was sus-
pended in hot EtOH, filtered off, and dried. Yield 994 mg (86%), mp
248–250 °C dec. 1H NMR d.10.18 (s, 1H), 9.78 (s, 1H), 9.13 (dd,
J1 = 2.3 Hz, J2 = 0.9 Hz, 1H), 8.32 (dd, J = 8.4 Hz, J2 = 2.3 Hz, 1H),
8.24 (t, J = 1.7 Hz, 1H), 8.04 (dd, J1 = 8.4 Hz, J2 = 0.9 Hz, 1H), 7.93
(dt, J1 = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.84–7.81(m, 1H), 7.82 (s, 1H),
7.58 (t, J = 7.8 Hz, 1H), 5.99 (s, 2H), 5.96 (s, 2H). Anal. Calcd for C16-
H14N6O3ꢀ0.5H2O: C, 55.33; H, 4.35; N, 24.20. Found: C, 55.33; H,
4.03; N, 23.86.
Yield of diamidine 608 mg (37%), mp >335 °C dec. 1H NMR d
9.92 (s, 2H), 9.89 (s, 2H), 9.68 (s, 2H), 9.64 (s, 2H), 9.06 (dd,
J1 = 5.2 Hz, J2 = 2.9 Hz, 2H), 8.59 (t, J = 1.3 Hz, 1H), 8.55–8.48 (m,
1H), 7.99 (dt, J1 = 5.1 Hz, J2 = 1.4 Hz, 2H), 7.88 (s, 1H). ESI MS m/z
308.6 ([M+1]+ of free base), 155.4 ([(M+2)/2]+). HPLC 97.9 area%
(230 nm). Anal. Calcd for C15H13N7Oꢀ3Clꢀ0.5H2O: C, 42.40; H,
3.84; N, 23.08; Cl, 25.03. Found: C, 42.16; H, 4.10; N, 22.81; Cl,
24.77.
Yield of diamidine 499 mg (48%), mp >320 °C dec. 1H NMR
d.9.78 (s, 2H), 9.63 (s, 2H), 9.55 (s, 2H), 9.36 (dd, J1 = 2.3 Hz,
J2 = 0.9 Hz, 2H), 8.70 (dd, J1 = 8.3 Hz, J2 = 2.2 Hz, 1H), 8.55 (dd,
J1 = 8.4 Hz, J2 = 0.8 Hz, 1H), 8.44 (t, J = 1.7 Hz, 1H), 8.28 (dt,
J1 = 7.8 Hz, J2 = 1.5 Hz, 1H), 8.22 (s, 1H), 8.02 (ddd, J1 = 8.0 Hz,
J2 = 1.9 Hz, J3 = 1.2 Hz, 1H), 7.85 (t, J = 7.8 Hz, 1H). ESI MS m/z
307.4 ([M+1]+ of free base), 154.6 ([(M+2)/2]+). HPLC 99.2 area%
(230 nm). Anal. Calcd for C16H14N6Oꢀ2HClꢀ1.5H2O: C, 47.30; H,
4.71; N, 20.69; Cl, 17.45. Found: C, 47.38; H, 4.90; N, 20.47; Cl,
17.57.
5.2.3.13. 3-(4-Amidinophenyl)-5-(6-amidinopyridin-3-yl)isox-
azole dihydrochloride (41).
Yield of diamidoxime (after sus-
pension in hot EtOH) 1.29 g (97%), mp >300 °C dec. 1H NMR d.10.18
(s, 1H), 9.83 (s, 1H), 9.11 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.30 (dd,
J1 = 8.4 Hz, J2 = 2.2 Hz, 1H), 8.04 (dd, J1= 8.4 Hz, J2 = 0.9 Hz, 1H),
7.94 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 7.84 (s, 1H), 5.96
(s, 4H). Anal. Calcd for C16H14N6O3ꢀ0.75H2O: C, 54.62; H, 4.44; N,
23.89. Found: C, 54.43; H, 4.03; N, 23.60.
5.2.3.17. 5-(4-Amidinophenyl)-3-(5-amidinopyridin-3-yl)isox-
Yield of diamidine 108 mg (8%), mp >350 °C dec. 1H NMR d.9.78
(s, 2H), 9.57 (s, 4H), 9.37 (dd, J1 = 2.1 Hz, J2 = 0.8 Hz, 1H), 9.33 (s,
2H), 8.70 (dd, J1 = 8.3 Hz, J2 = 2.2 Hz, 1H), 8.55 (dd, J1 = 8.3 Hz,
J2 = 0.8 Hz, 1H), 8.21 (s, 1H), 8.18 (d, J = 8.5 Hz, 2H), 8.05 (d,
J = 8.5 Hz, 2H). ESI MS m/z 307.1 ([M+1]+ of free base), 154.0
([(M+2)/2]+). HPLC 99.3 area% (290 nm). Anal. Calcd for C16H14N6-
Oꢀ2HClꢀ0.2H2O: C, 50.20; H, 4.32; N, 21.95; Cl, 18.52. Found: C,
50.22; H, 4.32; N, 21.84; Cl, 18.50.
azole dihydrochloride (45).
Yield of diamidoxime 1.12 g
(106%, wet). 1H NMR d.10.01 (s, 1H), 9.90 (s, 1H), 9.11 (d,
J = 2.1 Hz, 1H), 9.00 (d, J = 2.1 Hz, 1H), 8.54 (t, J = 2.1 Hz, 1H), 7.95
(d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.79 (s, 1H), 6.16 (s,
2H), 6.02 (s, 2H).
Yield of diamidine 388 mg (33%), mp >335 °C dec. 1H NMR d 9.83
(s, 2H), 9.60 (s, 2H), 9.52 (s, 2H), 9.41 (d, J = 2.0 Hz, 1H), 9.37 (s, 2H),
9.15 (d, J = 2.2 Hz, 1H), 8.84 (t, J = 2.2 Hz, 1H), 8.16 (d, J = 8.4 Hz, 2H),
8.12 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H). ESI MS m/z 307.6 ([M+1]+ of free
base), 155.0 ([(M+2)/2]+). HPLC 98.1 area% (290 nm). Anal. Calcd for
5.2.3.14. 5-(4-Amidinophenyl)-3-(6-amidinopyridin-3-yl)isox-
azole dihydrochloride (42).
Yield of diamidoxime 1.08 g
C
16H14N6Oꢀ2HClꢀ1.5H2O: C, 47.30; H, 4.71; N, 20.69; Cl, 17.45.
(92%), mp >330 °C dec. 1H NMR d.10.13 (s, 1H), 9.87 (s, 1H), 9.10
Found: C, 47.56; H, 4.72; N, 20.38; Cl, 17.47.

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D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
5.2.3.18.
3-(5-Amidinopyridin-3-yl)-5-(6-amidinopyridin-3-
Yield of diamidoxime
ESI MS m/z 308.1 ([M+1]+ of free base), 154.5 ([(M+2)/2]+). HPLC
100 area% (265 nm). Anal. Calcd for C15H13N7Oꢀ3.05Clꢀ0.05H2O: C,
42.96; H, 3.88; N, 23.38; Cl, 25.78. Found: C, 43.06; H, 3.87; N,
23.18; Cl, 25.78.
yl)isoxazole dihydrochloride (46).
1.14 g (112%, wet). 1H NMR d.10.20 (s, 1H), 10.03 (s, 1H), 9.13
(dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 9.12 (d, J = 2.1 Hz, 1H), 9.01 (d,
J = 2.1 Hz, 1H), 8.55 (t, J = 2.1 Hz, 1H), 8.32 (dd, J1 = 8.4 Hz,
J2 = 2.2 Hz, 1H), 8.06 (dd, J1 = 8.5 Hz, J2 = 0.9 Hz, 1H), 7.94 (s, 1H),
6.20 (s, 2H), 5.98 (s, 2H), 2.54 (s, 2H).
5.2.3.22. 3-(3-Amidinophenyl)-5-(5-amidinopyridin-3-yl)isox-
azole dihydrochloride (50).
An ethanolic solution of the
Yield of diamidine 677 mg (59%), mp >300 °C dec. 1H NMR d
9.84 (s, 2H), 9.81 (s, 2H), 9.60 (s, 2H), 9.54 (s, 2H), 9.41 (d,
J = 2.0 Hz, 1H), 9.35 (dd, J1 = 2.2 Hz, J2 = 0.8 Hz, 1H), 9.16 (d,
J = 2.2 Hz, 1H), 8.84 (t, J = 2.1 Hz, 1H), 8.70 (dd, J1 = 8.3 Hz,
J2 = 2.2 Hz, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.29 (s, 1H). ESI MS m/z
308.6 ([M+1]+ of free base), 155.4 ([(M+2)/2]+). Anal. Calcd for C15-
H13N7OꢀHClꢀH2O: C, 45.24; H, 4.30; N, 24.62; Cl, 17.80. Found: C,
45.08; H, 4.33; N, 24.33; Cl, 17.61.
crude diamidoxime product was gravity filtered, and the filtrate
was evaporated under reduced pressure. The residue was sus-
pended in ether and the solid was filtered off. Yield 746 mg
(91%). 1H NMR d.10.05 (s, 1H), 9.91 (s, 1H), 9.14 (d, J = 2.1 Hz,
1H), 8.99 (d, J = 2.1 Hz, 1H), 8.54 (t, J = 2.1 Hz, 1H), 8.25 (t,
J = 1.7 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.85 (dt, J1 = 8.2 Hz,
J2 = 1.4 Hz, 2H), 7.84 (s, 1H), 7.60 (t, J = 7.8 Hz, 1H), 6.21 (s, 4H).
The purified diamidine product was converted to the HCl salt
using EtOH and aqueous HCl, yield 133 mg (15%), mp >235 °C
dec. 1H NMR d.9.82 (s, 2H), 9.64 (s, 2H), 9.53 (s, 2H), 9.42 (d,
J = 2.0 Hz, 1H), 9.37 (s, 2H), 9.13 (d, J = 2.2 Hz, 1H), 8.81 (t,
J = 2.1 Hz, 1H), 8.43 (t, J = 1.7 Hz, 1H), 8.27 (d, J = 7.7 Hz, 1H), 8.10
(s, 1H), 8.02 (dt, J1 = 8.0 Hz, J2 = 1.4 Hz, 1H), 7.85 (t, J = 7.8 Hz,
1H). ESI MS m/z 307.1 ([M+1]+ of free base), 154.0 ([(M+2)/2]+).
HPLC 99.2 area% (233 nm). Anal. Calcd for C16H14N6Oꢀ2HClꢀ1.5H2O:
C, 47.30; H, 4.71; N, 20.69; Cl, 17.45. Found: C, 47.27; H, 4.60; N,
20.44; Cl, 17.59.
5.2.3.19. 3-(4-Amidinophenyl)-5-(5-amidinopyridin-3-yl)isox-
azole dihydrochloride (47).
The crude diamidoxime product
was suspended in hot EtOH, filtered off, and dried. Yield 1.60 g
(94%). 1H NMR d. 10.03 (s, 1H), 9.83 (s, 1H), 9.13 (d, J = 2.1 Hz,
1H), 8.99 (d, J = 2.1 Hz, 1H), 8.52 (t, J = 2.1 Hz, 1H), 7.94 (d,
J = 8.6 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.84 (s, 1H), 6.17 (s, 2H),
5.95 (s, 2H).
Yield of diamidine 468 mg (36%), mp >350 °C dec. 1H NMR d.
9.82 (s, 2H), 9.58 (s, 2H), 9.53 (s, 2H), 9.43 (d, J = 2.0 Hz, 1H), 9.34
(s, 2H), 9.13 (d, J = 2.2 Hz, 1H), 8.83 (t, J = 2.1 Hz, 1H), 8.16 (d,
J = 8.5 Hz, 2H), 8.11 (s, 1H), 8.05 (d, J = 8.5 Hz, 2H). ESI MS m/z
307.1 ([M+1]+ of free base), 154.0 ([(M+2)/2]+). HPLC 100 area%
(265 nm). Anal. Calcd for C16H14N6Oꢀ2HClꢀ2H2O: C, 46.28; H,
4.85; N, 20.24; Cl, 17.07. Found: C, 46.63; H, 4.69; N, 20.13; Cl,
17.27.
5.2.3.23. 5-(3-Amidinophenyl)-3-(5-amidinopyridin-3-yl)isox-
azole dihydrochloride (51).
Yield of diamidoxime 1.05 g
(113%, wet). 1H NMR d.10.01 (s, 1H), 9.87 (s, 1H), 9.12 (d,
J = 2.0 Hz, 1H), 9.00 (d, J = 2.1 Hz, 1H), 8.55 (t, J = 2.1 Hz, 1H), 8.25
(t, J = 1.7 Hz, 1H), 7.95 (dt, J1 = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.85 (dt,
J1 = 7.9 Hz, J2 = 1.3 Hz, 1H), 7.79 (s, 1H), 7.61 (t, J = 7.8 Hz, 1H),
6.29–6.04 (m, 2H).
5.2.3.20. 5-(3-Amidinophenyl)-3-(6-amidinopyridin-3-yl)isox-
Yield of diamidine 175 mg (17%), mp >245 °C dec. 1H NMR d
9.81 (s, 2H), 9.66 (s, 2H), 9.51 (s, 2H), 9.40 (d, J = 2.0 Hz, 1H), 9.39
(s, 2H), 9.15 (d, J = 2.2 Hz, 1H), 8.81 (t, J = 2.2 Hz, 1H), 8.43 (t,
J = 1.7 Hz, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.04 (s, 1H), 8.01 (d,
J = 8.0 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H). ESI MS m/z 307.6 ([M+1]+
of free base), 155.0 ([(M+2)/2]+). HPLC 97.4 area% (265 nm). Anal.
Calcd for C16H14N6Oꢀ2.5HClꢀ2.2H2O: C, 43.96; H, 4.82; N, 19.23;
Cl, 20.28. Found: C, 44.27; H, 4.59; N, 19.13; Cl, 19.99.
azole dihydrochloride (48).
Yield of diamidoxime 1.60 g
(94%). 1H NMR d.10.14 (d, J = 0.5 Hz, 1H), 9.81 (d, J = 0.5 Hz, 1H),
9.11 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.33 (dd, J1 = 8.4 Hz,
J2 = 2.2 Hz, 1H), 8.24 (t, J = 1.8 Hz, 1H), 8.03 (dd, J1 = 8.4 Hz,
J2 = 0.8 Hz, 1H), 7.93 (dt, J = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.85 (dt,
J1 = 7.8 Hz, J2 = 1.3 Hz, 1H), 7.78 (s, 1H), 7.61 (t, J = 7.8 Hz, 1H),
6.01 (s, 3H), 5.96 (s, 2H), 2.61–2.48 (m, 2H). HPLC 98.6 area%
(254 nm).
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 736 mg (53%), mp 311–
315 °C dec. 1H NMR d 9.79 (s, 2H), 9.67 (s, 2H), 9.57 (s, 2H), 9.40
(s, 2H), 9.34 (dd, J1 = 2.0 Hz, J2 = 0.8 Hz, 1H), 8.70 (dd, J1 = 8.3 Hz,
J2 = 2.1 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.46 (t, J = 1.8 Hz, 1H),
8.28 (d, J = 7.6 Hz, 1H), 8.10 (s, 1H), 8.02 (dm, J = 8.2 Hz, 1H), 7.87
(t, J = 7.9 Hz, 1H). ESI MS m/z 307.1 ([M+1]+ of free base), 154.0
([(M+2)/2]+). HPLC 100 area% (265 nm). Anal. Calcd for C16H14N6-
Oꢀ2HClꢀ1.5H2O: C, 47.30; H, 4.71; N, 20.69; Cl, 17.45. Found: C,
47.23; H, 4.41; N, 20.39; Cl, 17.12.
5.2.3.24. 3,5-Bis(5-amidinopyridin-3-yl)isoxazole dihydrochlo-
ride (52).
(s, 1H), 10.01 (s, 1H), 9.13 (dd, J1 = 7.0 Hz, J2 = 2.1 Hz, 2H), 9.01 (t,
J = 2.3 Hz, 2H), 8.55 (dt, J1 = 4.1 Hz, J2 = 2.1 Hz, 4H), 7.93 (s, 1H),
6.18 (s, 2H), 6.16 (s, 2H).
Yield of diamidoxime 1.18 g (99%). 1H NMR d.10.04
Yield of diamidine 724 mg (54%), mp >235 °C dec. 1H NMR d
9.83 (s, 4H), 9.54 (s, 2H), 9.52 (s, 2H), 9.42 (d, J = 2.0 Hz, 1H), 9.40
(d, J = 2.0 Hz, 1H), 9.16 (d, J = 2.1 Hz, 1H), 9.14 (d, J = 2.2 Hz, 1H),
8.81 (t, J = 2.2 Hz, 2H), 8.16 (s, 1H). ESI MS m/z 308.6 ([M+1]+ of free
base), 155.4 ([(M+2)/2]+). HPLC 98.1 area% (230 nm). Anal. Calcd
for C15H13N7Oꢀ2HClꢀ1.25H2O: C, 44.73; H, 4.38; N, 24.34; Cl,
17.61. Found: C, 44.70; H, 4.27; N, 24.09; Cl, 17.56.
5.2.3.21. 3-(6-Amidinopyridin-3-yl)-5-(5-amidinopyridin-3-yl)
isoxazole trihydrochloride (49).
Yield of diamidoxime
1.35 g (105%, wet). 1H NMR d.10.24 (s, 1H), 10.13 (s, 1H), 9.23 (d,
J1 = 2.1 Hz, 1H), 9.20 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 9.09 (d,
J = 2.1 Hz, 1H), 8.62 (t, J = 2.1 Hz, 1H), 8.42 (dd, J1 = 8.4 Hz,
J2 = 2.3 Hz, 2H), 8.14 (dd, J1 = 8.4 Hz, J2 = 0.9 Hz, 2H), 8.03 (s, 1H),
6.28 (s, 2H), 6.05 (s, 2H), 2.68–2.56 (m, 6H), 0.96 (t, J = 7.3 Hz,
1H), 0.09 (d, J = 0.7 Hz, 2H). HPLC 98.1 area% (265 nm).
The purified diamidine product was converted to the HCl salt
using ethanolic HCl and ether, yield 835 mg (59%), mp >350 °C
dec. 1H NMR d 9.86 (s, 2H), 9.81 (s, 2H), 9.61 (s, 2H), 9.57 (s, 2H),
9.44 (d, J = 2.0 Hz, 1H), 9.34 (dd, J1 = 2.2 Hz, J2 = 0.8 Hz, 1H), 9.15
(d, J = 2.2 Hz, 1H), 8.85 (t, J = 2.1 Hz, 1H), 8.70 (dd, J1 = 8.3 Hz,
J2 = 2.1 Hz, 1H), 8.57 (dd, J1 = 8.3 Hz, J2 = 0.9 Hz, 1H), 8.23 (s, 1H).
5.2.4. General procedure for intermediates 55a–d
A mixture of an aryl halide 54 (25–75 mmol), mebynol (1.2–
1.5 equiv), PdCl2(PPh3)2 (2 mol %), and CuI (2 mol %) in Et3N (75–
100 mL) was a minimum temperature of 60 °C for a minimum of
3 h. The cooled reaction mixture was filtered through silica gel.
The filter pad was rinsed with ethyl acetate. Combined filtrates
were concentrated and chromatographed on silica gel eluting with
hexanes/EtOAc (3:2). Compounds 55c,d which contained small
amounts of the homo-coupling product of mebynol, as evidenced
by the presence of singlets at d 5.56 and 1.37 in 1:6 ratios in their
1H NMR spectra, were used in the next step without further
purification.

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
573
5.2.4.1.
(55a).
6-(3-Hydroxy-3-methylbut-1-yn-1-yl)nicotinonitrile
The product was recrystallized from hexanes–EtOAc,
99.5 area% (230 nm). Anal. Calcd for C8H4N2: C, 74.99; H, 3.15; N,
21.86. Found: C, 75.04; H, 2.97; N, 21.96.
yield 8.01 g (77%) white crystals, mp 143–144 °C. 1H NMR d 8.99
(dt, J1 = 2.2, J2 = 0.7 Hz, 1H), 8.31 (ddd, J1 = 8.2 Hz, J2 = 2.2 Hz,
J3 = 0.5 Hz, 1H), 7.66 (dt, J1 = 8.2 Hz, J2 = 0.7 Hz, 1H), 5.72 (s, 1H),
1.49 (s, 6H). HPLC 100 area% (254 nm). Anal. Calcd for C11H10N2O:
C, 70.95; H, 5.41; N, 15.04. Found: C, 70.63; H, 5.40; N, 14.65.
5.2.6. 6-Formylnicotinonitrile (57a)
A mixture of 6-methylnicotinonitrile (4.20 g, 35.6 mmol) and
DMF-DMA (8.45 g, 70.9 mmol) in DMF (25 mL) was refluxed for
3 days. The cooled reaction mixture was poured over ice to give
(E)-6-[2-(dimethylamino)vinyl]nicotinonitrile as
a
precipitate
5.2.4.2. 2-(3-Hydroxy-3-methylbut-1-yn-1-yl)isonicotinonitrile
(3.75 g, 91%); mp 103–104 °C; 1H NMR d 8.51 (d, J = 2.2 Hz, 1H),
7.77 (d, J = 12.9 Hz, 1H), 7.74 (dd, J1 = 8.5 Hz, J2 = 2.3 Hz, 1H), 6.98
(d, J = 8.5 Hz, 1H), 5.19 (d, J = 12.9 Hz, 1H), 2.94 (s, 6H). Anal. Calcd
for C10H11N3ꢀ0.25H2O: C, 67.58; H, 6.52; N, 23.64. Found: C, 67.55;
H, 6.19; N, 23.66.
(55b).
The product was recrystallized from hexanes–EtOAc,
yield 11.1 g (79%) brown crystals, mp 68–69 °C. 1H NMR d 8.79
(dd, J1 = 5.0 Hz, J2 = 1.0 Hz, 1H), 7.96 (dd, J1 = 1.5 Hz, J2 = 1.0 Hz,
1H), 7.85 (dd, J1 = 5.1 Hz, J2 = 1.5 Hz, 1H), 5.68 (s, 1H), 1.49 (s,
6H). HPLC 98.0 area% (29 nm). Anal. Calcd for C11H10N2ꢀ0.25H2O:
C, 69.27; H, 5.55; N, 14.69. Found: C, 69.32; H, 5.40; N, 14.44.
To a stirred solution of the enamine (6.16 g, 35.6 mmol) in THF
(130 mL) was added a solution of sodium periodate (23.0 g,
107 mmol) in water (130 mL). The mixture was stirred for 2 h
and filtered (Celite). The filtrate was diluted with water and ex-
tracted into CH2Cl2. Extracts were washed with brine, dried
(MgSO4) and evaporated. The product was purified by flash chro-
matography on silica eluting with hexanes/ethyl acetate (7:3) to
give the aldehyde (2.94 g, 63%): mp 91–92 °C; 1H NMR d 10.03
(d, J = 0.7 Hz, 1H), 9.28 (dd, J1 = 2.0 Hz, J2 = 0.9 Hz, 1H), 8.58 (ddd,
J1 = 8.1 Hz, J2 = 2.0 Hz, J3 = 0.7 Hz, 1H), 8.07 (dd, J1 = 8.1 Hz,
J2 = 0.9 Hz, 1H). Anal. Calcd for C7H4N2O: C, 63.64; H, 3.05; N,
21.20. Found: C, 63.57; H, 3.00; N, 21.11.
5.2.4.3.
(55c).
5-(3-Hydroxy-3-methylbut-1-yn-1-yl)picolinonitrile
Yield, 5.15 g (111%, contaminated by mebynol homo-
coupling product). 1H NMR (300 MHz, DMSO-d6) d 8.75 (dd,
J1 = 1.8 Hz, J2 = 1.2 Hz, 1H), 8.06 (t, J = 1.4 Hz, 2H), 5.68 (s, 1H),
5.56 (s, 0H, contaminant), 1.49 (s, 6H), 1.37 (s, 1H, contaminant).
HPLC 100 area% (265 nm). The product was used in the next step
without further purification.
5.2.4.4.
(55d).
5-(3-Hydroxy-3-methylbut-1-yn-1-yl)nicotinonitrile
Yield, 5.66 g (101%, contaminated by mebynol homo-
coupling product). 1H NMR d 8.99 (d, J = 2.0 Hz, 1H), 8.85 (d,
J = 2.1 Hz, 1H), 8.39 (t, J = 2.0 Hz, 1H), 5.65 (s, 1H), 5.56 (s, 0H, con-
taminant), 1.49 (s, 6H), 1.37 (s, 1H, contaminant). HPLC 100 area%
(254 nm). The product was used in the next step without further
purification.
5.2.7. General procedure for oximes 58a–d
A stirred suspension of the aldehyde (16–25 mmol) in EtOH
(80–100 mL) was treated with a solution of NH2OHꢀHCl (1.1 equiv)
in H2O (20–25 mmol) and stirred for 1–2 h. The reaction mixture
was diluted with water. The precipitated product was filtered off,
dried (high vacuum, P205).
5.2.5. General procedure for acetylenes 56a–d
A mixture of the protected acetylene 55 (20–50 mmol) and NaH
(60% dispersion in mineral oil, 0.15 equiv) in toluene in a round
bottom flask fitted with a Dean–Stark trapped capped by a reflux
condenser was refluxed for 0.75–1.5 h. The cooled reaction mixture
was filtered through silica gel. The filtrate was evaporated and
chromatographed on silica gel eluting with hexanes/EtOAc. The
product was recrystallized from hexanes.
5.2.7.1. 6-Formylnicotinonitrile oxime (58a)62
.
Yield 2.46 g
(93%) pale yellow solid, mp 218–219 °C. 1H NMR d 12.21 (s, 1H),
9.03 (dd, J1 = 2.1 Hz, J2 = 0.9 Hz, 1H), 8.31 (dd, J1 = 8.3 Hz,
J2 = 2.1 Hz, 1H), 8.16 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H). HPLC 100 area%
(254 nm).
5.2.7.2. 2-Formylisonicotinonitrile oxime (58b).
Yield
3.46 g (92%) white solid, mp 216–217 °C. 1H NMR d 12.07 (s, 1H),
8.83 (d, J = 5.1 Hz, 1H), 8.15 (s, 1H), 8.13 (s, 1H), 7.85 (dd,
J1 = 5.0 Hz, J2 =1.5 Hz, 1H). HPLC 98.6 area% (254 nm). Anal. Calcd
for C7H5N3O: C, 57.14; H, 3.43; N, 28.58. Found: C, 57.35; H,
3.39; N, 28.76.
5.2.5.1. 6-Ethynylnicotinonitrile (56a)39
.
Combined yield
from two batches 6.81 g (59%) yellow solid, mp 158–159 °C (lit.39
158 °C) 1H NMR d 9.02 (dt, J1 = 1.5 Hz, J2 = 0.7 Hz, 1H), 8.36 (ddd, J1=
8.2 Hz, J2 = 2.2 Hz, J3 = 0.6 Hz, 1H), 7.79 (dt, J1 = 8.2 Hz, J2 = 0.7 Hz,
1H), 4.76 (d, J = 0.6 Hz, 1H). HPLC 100 area% (254). Anal. Calcd
C8H4N2: C, 74.99; H, 3.15; N, 21.86. Found: C, 74.70; H, 3.20; N, 21.82.
5.2.7.3. 5-Formylpicolinonitrile oxime (58c).
Yield (after
column chromatography) 1.69 g (73%) white solid, mp 218–
219 °C. 1H NMR d 12.03 (s, 1H), 8.94 (d, J = 1.5 Hz, 1H), 8.33 (s,
1H), 8.22 (dd, J1 = 8.1 Hz, J2 = 2.1 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H).
HPLC 97.9 area% (254 nm). Anal. Calcd for C7H5N3Oꢀ0.05CH3CO2C2-
H5: C, 57.22; H, 3.33; N, 27.80. Found: C, 57.01; H, 3.57; N, 27.46.
5.2.5.2. 2-Ethynylisonicotinonitrile (56b)40
(76%) yellow crystals mp 123–125 °C. 1H NMR
J1 = 5.1 Hz, J2 = 1.0 Hz, 1H), 8.11 (dd, J1 = 1.6 Hz, J2 = 1.0 Hz, 1H),
7.91 (dd, J1 = 5.1 Hz, J2 = 1.6 Hz, 1H), 4.61 (s, 1H). HPLC 96.3 area%
(230 nm). Anal. Calcd for C8H4N2: C, 74.99; H, 3.15; N, 21.86.
Found: C, 74.82; H, 3.16; N, 21.74.
.
Yield 4.98 g
8.82 (dd,
d
5.2.7.4. 5-Formylnicotinonitrileoxime (58d).
Yield 3.44 g
(96%) white solid, mp 244–245 °C. 1H NMR d 11.90 (s, 1H), 9.04
(d, J = 2.1 Hz, 1H), 9.01 (d, J = 2.0 Hz, 1H), 8.45 (t, J = 2.0 Hz,1H),
8.25 (s, 1H). HPLC 100 area% (254 nm). Anal. Calcd for C7H5N3O:
C, 57.14; H, 3.43; N, 28.58. Found: C, 57.00; H, 3.39; N, 28.32.
5.2.5.3. 5-Ethynylpicolinonitrile (56c)39
.
Yield 2.46 g (77%)
white crystals, mp 123 °C (lit.39 126–126.5 °C. 1H NMR d 8.85 (dt,
J = 1.7 and 0.8 Hz, 1H), 8.19 (ddd, J1 = 8.1 Hz, J2 = 2.1 Hz,
J3 = 0.7 Hz, 1H), 8.09 (dt, J1 = 8.1 Hz, J2 =0.8 Hz, 1H), 4.85 (s, 1H).
HPLC 100 area% (265 nm). Anal. Calcd for C8H4N2: C, 74.99; H,
3.15; N, 21.86. Found: C, 74.91; H, 3.17; N, 21.83.
5.2.8. General procedure for 10-Chloroaryl oximes 59a–d
NCS (1.1 equiv) was added to a mixture of the oxime in DMF
maintained at 0 °C. The mixture was stirred overnight at ambient
temperature, poured over ice, and extracted into ether. Combined
extracts were washed with brine, dried (MgSO4) and evaporated
to a solid. The products were used immediately in the next step
without further purification.
5.2.5.4. 5-Ethynylnicotinonitrile (56d)40
.
The reaction mix-
ture was worked up by extraction into EtOAc. Yield 1.63 g (63%)
yellow crystals, mp 123–125 °C. 1H NMR d 9.04 (d, J = 2.0 Hz, 1H),
8.95 (d, J = 2.1 Hz, 1H), 8.52 (t, J = 2.1 Hz, 1H), 4.69 (s, 1H). HPLC

574
D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
5.2.8.1. 10-Chloro-6-formylnicotinonitrile oxime (59a)63
.
Yield
(290 nm). Anal. Calcd for C16H8N4O: C, 70.58; H, 2.96; N, 20.58.
Found: C, 70.41; H, 2.85; N, 20.71.
2.74 g (98%) white solid. 1H NMR d 13.16 (s, 1H), 9.13 (dd, J1 = 2.1 Hz,
J2 = 0.8 Hz, 1H), 8.25 (dd, J1 = 8.4 Hz, J2 = 2.2 Hz, 1H), 7.98 (d, J = 8.0 Hz,
1H.
5.2.9.6. 3-(4-Cyanopyridin-2-yl)-5-(5-cyanopyridin-2-yl)isoxaz-
ole (65).
The title compound was prepared from synthons
5.2.8.2. 10-Chloro-2-formylisonicotinonitrile oxime (59b).
Yield
56a and 59b. Yield 1.30 g (74%), mp 284–285 °C dec. 1H NMR d
9.21 (dd, J1 = 2.2 Hz, J2 = 1.1 Hz, 1H), 9.02 (dd, J1 = 5.1 Hz,
J2 = 0.9 Hz, 1H), 8.62–8.50 (m, 2H), 8.30 (dd, J1= 8.3 Hz, J2 = 1.1 Hz,
1H), 8.06 (dd, J1 = 5.0 Hz, J2 = 1.6 Hz, 1H), 7.92–7.85 (m, 1H). HPLC
98.9 area% (290 nm). Anal. Calcd for C15H7N5Oꢀ0.15H2O: C, 65.29;
H, 2.61; N, 25.38. Found: C, 65.49; H, 2.65; N, 25.02.
4.27 g (100%), mp 195–196 °C dec. 1H NMR d 13.03 (s, 1H), 8.92 (dd,
J1 = 5.0 Hz, J2 = 0.9 Hz, 1H), 8.25 (t, J = 2.2 Hz, 1H), 7.98 (dd,
J1 = 5.0 Hz, J2 = 1.5 Hz, 1H). Anal. Calcd for C7H4ClN3O: C, 46.30; H,
2.22; N, 23.14. Found: C, 46.32; H, 2.23; N, 22.89
5.2.8.3. 10-Chloro-5-formylpicolinonitrile oxime (59c).
Yield
2.00 g (96%). 1H NMR d 13.15 (s, 1H), 9.11 (dd, J1 = 1.5 Hz, J2 = 0.7 Hz,
1H), 8.37 (dd, J1 = 8.2 Hz, J2 = 2.3 Hz, 1H), 8.16 (dd, J1 = 8.2 Hz,
J2 = 0.7 Hz, 1H).
5.2.9.7.
(66).
3-(4-Cyanophenyl)-5-(4-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56b
and 59e. Yield 687 mg (50%), mp 250–251 °C dec. 1H NMR d 9.02
(dt, J1 = 5.0 Hz, J2 = 0.8 Hz, 1H), 8.52 (dt, J1 = 1.6 Hz, J2 = 0.8 Hz,
1H), 8.19 (d, J = 8.0 Hz, 2H), 8.11–8.02 (m, 3H), 8.00 (s, 1H). Anal.
Calcd for C16H8N4O: C, 70.58; H, 2.96; N, 20.58. Found: C, 70.52;
H, 3.00; N, 20.57.
5.2.8.4. 10-Chloro-5-formylnicotinonitrileoxime (59d).
Yield
4.46 g (98%) yellow solid, mp >330 °C dec. 1H NMR d 13.02 (s, 1H),
9.20 (d, J = 2.2 Hz, 1H), 9.13 (d, J = 1.9 Hz, 1H), 8.62 (t, J = 2.1 Hz, 1H).
5.2.9. General procedure for dinitriles 60–83
5.2.9.8.
(67).
5-(3-Cyanophenyl)-3-(5-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56f
Bis(tributyltin) oxide (0.5 equiv) was added to a stirred mixture
of an acetylene 56 (1.1 equiv) and a chlorooxime 59 (1 equiv) in
CH2Cl2, resulting in dissolution of all solids followed by formation
of a precipitate. After stirring overnight, the reaction mixture was
diluted with ether, and the product was filtered off and dried.
and 59a. Yield 944 mg (69%), mp 235–236 °C. 1H NMR d 9.24 (dd,
J1 = 2.1 Hz, J2 = 0.9 Hz, 1H), 8.55–8.50 (m, 2H), 8.32 (dt,
J1 = 8.0 Hz, J2 = 1.4 Hz, 1H), 8.26 (dd, J1 = 8.2 Hz, J2 = 0.9 Hz, 1H),
8.02 (dt, J1 = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.90 (s, 1H), 7.80 (t,
J = 7.9 Hz, 1H). HPLC 100 area% (254 nm). Anal. Calcd for
5.2.9.1.
(60).
3-(4-Cyanophenyl)-5-(5-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56a
C16H8N4O: C, 70.58; H, 2.96; N, 20.58. Found: C, 70.36; H, 2.91;
N, 20.40.
and 59e. Yield 1.05 g (77%), mp 268–270 °C dec. 1H NMR d 9.22 (d,
J = 2.1, 1H), 8.58 (dd, J1 = 8.3 Hz, J2 = 2.1 Hz, 1H), 8.21 (d, J = 8.0 Hz,
1H), 8.20 (d, J = 8.3 Hz, 2H), 8.06 (s, 1H), 8.05 (d, J = 7.0 Hz, 2H). HPLC
96.5 area% (265 nm). Anal. Calcd for C16H8N4Oꢀ0.1H2O: C, 70.12; H,
3.02; N, 20.44. Found: C, 69.97; H, 2.96; N, 20.25.
5.2.9.9. 3-(5-Cyanopyridin-2-yl)-5-(4-cyanopyridin-2-yl)isoxaz-
ole (68).
The title compound was prepared from synthons
56b and 59a. Yield 3.04 g (72%), mp 285–287 °C dec. 1H NMR d
9.24 (dd, J1 = 2.1 Hz, J2 = 0.9 Hz, 1H), 9.02 (dd, J1 = 5.0 Hz,
J2 = 0.9 Hz, 1H), 8.64 (dd, J1 = 1.5 Hz, J2 = 1.0 Hz, 1H), 8.54 (dd,
J1 = 8.3 Hz, J2 = 2.2 Hz, 1H), 8.30 (dd, J1 = 8.2 Hz, J2 = 0.9 Hz, 1H),
8.05 (dd, J1 = 5.0 Hz, J2 = 1.5 Hz, 1H), 7.88 (s, 1H). HPLC 100 area%
(254 nm). Anal. Calcd for C15H7N5Oꢀ0.05H2O: C, 65.72; H, 2.61; N,
25.55. Found: C, 65.64; H, 2.58; N, 25.55.
5.2.9.2.
(61).
5-(4-Cyanophenyl)-3-(5-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56e
and 59a. Yield 952 mg (67%), mp 286–288 °C dec. 1H NMR d 9.23
(dd, J1 = 2.1 Hz, J2 = 0.9 Hz, 1H), 8.53 (dd, J1 = 8.2 Hz, J2 = 2.1 Hz,
1H), 8.26 (dd, J1 = 8.3 Hz, J2 = 0.9 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H),
8.06 (d, J = 8.4 Hz, 2H), 7.94 (s, 1H). HPLC 96.7 area% (265 nm).
Anal. Calcd for C16H8N4Oꢀ0.2H2O: C, 69.66; H, 3.07; N, 20.31.
Found: C, 69.68; H, 2.96; N, 20.36.
5.2.9.10.
(69).
3-(3-Cyanophenyl)-5-(4-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56b
chlorooxime 59f. Yield 1.18 g (72%), mp 226–228 °C dec. 1H NMR
d
9.02 (dd, J1 = 5.0 Hz, J2 = 0.9 Hz, 1H), 8.49 (dd, J1 = 1.5 Hz,
5.2.9.3. 3,5-Bis(5-cyanopyridin-2-yl)isoxazole (62).
The title
J2 = 0.9 Hz, 1H), 8.46 (td, J1 = 1.7 Hz, J2 = 0.6 Hz, 1H), 8.32 (ddd,
J1 = 7.9 Hz, J2 = 1.8, J3 = 1.1 Hz, 1H), 8.06–8.01 (m, 2H), 7.99 (s, 1H),
7.79 (td, J1 = 7.8 Hz, J2 = 0.6 Hz, 1H). Anal. Calcd for C16H8N4O: C,
70.58; H, 2.96; N, 20.58. Found: C, 70.33; H, 2.89; N, 20.58.
compound was prepared from synthons 56a and 59a. Yield 3.33 g
(81%), mp 327 °C dec. 1H NMR d 9.28–9.19 (m, 2H), 8.63–8.50 (m,
2H), 8.32 (dd, J1 = 8.2 Hz, J2 = 4.9 Hz, 2H), 7.93 (s, 1H). Anal. Calcd
for C15H7N5Oꢀ0.1H2O: C, 65.50; H, 2.64; N, 25.46. Found: C,
65.44; H, 2.58; N, 25.36.
5.2.9.11.
(70).
5-(3-Cyanophenyl)-3-(4-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56f
5.2.9.4.
(63).
3-(3-Cyanophenyl)-5-(5-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56a
and 59b. Yield 1.02 g (68%), mp 220 °C dec. 1H NMR d 9.02 (dd,
J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 8.52 (t, J = 1.7 Hz, 1H), 8.48 (t,
J = 1.2 Hz, 1H), 8.31 (dt, J1 = 8.1 Hz, J2 = 1.4 Hz, 1H), 8.06 (dd,
J1 = 5.0 Hz, J2 = 1.5 Hz, 1H), 8.02 (dt, J1 = 7.8 Hz, J2 = 1.4 Hz, 1H),
7.86 (s, 1H), 7.80 (t, J = 7.9 Hz, 1H). HPLC 100 area% (230 nm). Anal.
Calcd for C16H8N4Oꢀ0.1H2O: C, 79.66; H, 3.00; N, 20.31. Found: C,
69.64; H, 3.00; N, 20.07.
and 59f. Yield 899 mg (66%), mp 250–252 °C dec. 1H NMR d 9.22
(dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 8.58 (dd, J1 = 8.3 Hz, J2 = 2.1 Hz,
1H), 8.48 (t, J = 1.7 Hz, 1H), 8.39–8.29 (m, 1H), 8.20 (dd, J1 =
8.3 Hz, J2 = 0.9 Hz, 1H), 8.06 (s, 1H), 8.04 (dt, J1 = 7.8 Hz, J2
= 1.4 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H). HPLC 97.4 area% (290 nm).
Anal. Calcd for C16H8N4Oꢀ0.1H2O: C, 70.12; H, 3.02; N, 20.44.
Found: C, 70.13; H, 2.96; N, 20.43.
5.2.9.12. 3,5-Bis(4-cyanopyridin-2-yl)isoxazole (71).
The ti-
tle compound was prepared from synthons 56b and 59b. Yield
1.13 g (75%), mp 291–293 °C dec. 1H NMR d 9.03 (dd, J1 = 5.0 Hz,
J2 = 3.8 Hz, 2H), 8.63 (s, 1H), 8.54 (s, 1H), 8.05 (td, J1 = 6.3 Hz,
J2 = 5.7 Hz, J3 = 1.7 Hz, 2H), 7.90–7.82 (m, 1H). HPLC 100 area%
(230 nm). Anal. Calcd for C15H7N5Oꢀ0.1H2O: C, 65.07; H, 2.62; N,
25.30. Found: C, 65.11; H, 2.44; N, 25.22.
5.2.9.5.
(64).
5-(4-Cyanophenyl)-3-(4-cyanopyridin-2-yl)isoxazole
The title compound was prepared from synthons 56e
and 59b. Yield 1.11 g (74%), mp 250–251 °C dec. 1H NMR d 9.02
(dd, J1 = 5.0 Hz, J2 = 0.9 Hz, 1H), 8.48 (t, J = 1.2 Hz, 1H), 8.19 (d,
J = 8.4 Hz, 2H), 8.11–8.02 (m, 3H), 7.91 (s, 1H). HPLC 100 area%

D. A. Patrick et al. / Bioorg. Med. Chem. 22 (2014) 559–576
575
5.2.9.13. 3-(4-Cyanophenyl)-5-(6-cyanopyridin-3-yl)isoxazole
5.2.9.21. 3-(6-Cyanopyridin-3-yl)-5-(5-cyanopyridin-3-yl)isox-
(72).
The title compound was prepared from synthons 56c
azole (80).
The title compound was prepared from synthons
and 59e. Yield 1.10 g (81%), mp 291–293 °C dec. 1H NMR d 9.30
(dd, J1 = 2.2 Hz, J2 = 0.8 Hz, 1H), 8.55 (dd, J1 = 8.2 Hz, J2 = 2.2 Hz,
1H), 8.33–8.23 (m, 1H), 8.20–8.02 (m, 5H). Anal Calcd for C16H8N4-
Oꢀ0.1H2O: C, 70.12; H, 3.02; N, 20.44. Found: C, 69.97; H, 2.91; N,
20.34.
56d and 59c. Yield 1.01 g (69%), mp 224–226 °C dec. 1H NMR d
9.39 (d, J = 2.2 Hz, 1H), 9.27 (dd, J1 = 2.2 Hz, J2 = 0.9 Hz, 1H), 9.19
(d, J = 1.9 Hz, 1H), 8.89 (t, J = 2.0 Hz, 1H), 8.54 (dd, J1 = 8.1 Hz,
J2 = 2.2 Hz, 1H), 8.29 (dd, J1 = 8.1 Hz, J2 = 0.8 Hz, 3H), 8.07 (s, 1H).
Material was used in the next step without further purification.
5.2.9.14. 5-(4-Cyanophenyl)-3-(6-cyanopyridin-3-yl)isoxazole
5.2.9.22.
(81).
3-(3-Cyanophenyl)-5-(5-cyanopyridin-3-yl)isoxazole
(73).
The title compound was prepared from synthons 56e
The title compound was prepared from synthons 56d
and 59c. Yield 876 mg (65%), mp 257–258 °C. 1H NMR d 9.28 (dd,
J1 = 2.2 Hz, J2 = 0.8 Hz, 1H), 8.56 (dd, J1 = 8.1 Hz, J2 = 2.2 Hz, 1H),
8.28 (dd, J1 = 8.1 Hz, J2 = 0.9 Hz, 1H), 8.10 (s, 4H), 8.04 (s, 1H). Anal.
Calcd for C16H8N4Oꢀ0.25H2O: C, 69.43; H, 3.10; N, 20.24. Found: C,
69.28; H, 2.96; N, 20.62.
and 59f. Yield 689 mg (50%), mp 217–220 °C. 1H NMR d. 9.38 (d,
J = 2.1 Hz, 1H), 9.18 (d, J = 2.0 Hz, 1H), 8.86 (t, J = 2.1 Hz, 1H), 8.36
(t, J = 1.7 Hz, 1H), 8.25 (dt, J = 7.9, 1.5 Hz, 1H), 8.06 (dt, J = 7.9,
1.4 Hz, 1H), 8.00 (s, 1H), 7.81 (t, J = 7.8 Hz, 1H). Anal. Calcd for
C16H8N4O: C, 70.58; H, 2.96; N, 20.58. Found: C, 70.30; H, 3.00;
N, 20.53.
5.2.9.15. 3,5-Bis(6-Cyanopyridin-3-yl)isoxazole (74).
tle compound was prepared from synthons 56c and 59c. Yield
994 mg (73%), mp 280–285 °C dec. 1H NMR
9.29 (dd,
J1 = 5.8 Hz, J2 = 1.5 Hz, 2H), 8.55 (dd, J1 = 7.8 Hz, J2 = 2.2 Hz, 2H),
8.29 (ddd, J1 = 7.7 Hz, J2 = 2.7 Hz, J3 = 0.9 Hz, 2H), 8.15 (s, 1H). Anal.
Calcd for C15H7N5Oꢀ0.2H2O: C, 65.07; H, 2.69; N, 25.30. Found: C,
64.90; H, 2.68; N, 25.19.
The ti-
5.2.9.23.
(82).
5-(3-Cyanophenyl)-3-(5-cyanopyridin-3-yl)isoxazole
The title compound was prepared from synthons 56f
d
and 59d. Yield 689 mg (50%), mp 221–223 °C dec. 1H NMR d.9.36
(d, J = 2.1 Hz, 1H), 9.20 (d, J = 2.0 Hz, 1H), 8.83 (t, J = 2.1 Hz, 1H),
8.44–8.36 (m, 1H), 8.25–8.19 (m, 1H), 8.05 (dt, J1 = 7.8 Hz,
J2 = 1.3 Hz, 1H), 7.94 (s, 1H), 7.83 (t, J = 7.8 Hz, 1H). Anal. Calcd
for C16H8N4Oꢀ0.5H2O: C, 69.66; H, 3.00; N, 20.31. Found: C,
69.45; H, 2.88; N, 20.41.
5.2.9.16. 3-(3-Cyanophenyl)-5-(6-cyanopyridin-3-yl)isoxazole
(75).
The title compound was prepared from synthons 56c
and 59f. Yield 871 mg (64%), mp 204–205 °C. 1H NMR d 9.28 (dd,
J1 = 2.2, 0.8 Hz, 1H), 8.53 (dd, J1 = 8.2 Hz, J2 = 2.2 Hz, 1H), 8.37 (dt,
J1 = 2.2 Hz, J2 = 1.1 Hz, 1H), 8.32–8.23 (m, 2H), 8.08 (s, 1H), 8.05
(dt, J = 7.8, 1.4 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H). Anal. Calcd for C16H8-
N4Oꢀ0.3H2O: C, 69.21; H, 3.12; N, 20.18. Found: C, 69.54; H, 3.32; N,
17.19.
5.2.9.24. 3,5-Bis(5-cyanopyridin-3-yl)isoxazole (83).
The ti-
tle compound was prepared from synthons 56d and 59d. Yield
985 mg (65%). 1H NMR d.9.37 (dd, J1 = 4.2 Hz, J2 = 2.1 Hz, 2H),
9.20 (dd, J1 = 5.5 Hz, J2 = 2.0 Hz, 2H), 8.86 (dt, J1 = 12.7 Hz,
J2 = 2.1 Hz, 2H), 8.02 (s, 1H). Anal. Calcd for C15H7N5Oꢀ0.1H2O: C,
65.07; H, 2.62; N, 25.30. Found: C, 65.13; H, 2.61; N, 25.6.
Acknowledgments
5.2.9.17. 5-(4-Cyanophenyl)-3-(5-cyanopyridin-3-yl)isoxazole
(76).
The title compound was prepared from synthons 56e
and 59d. Yield 880 mg (64%), mp 276–278 °C. 1H NMR d 9.38 (d,
J = 2.1 Hz, 1H), 9.20 (d, J = 2.0 Hz, 1H), 8.84 (t, J = 2.1 Hz, 1H), 8.09
(s, 4H), 7.99 (s, 1H). Material was used in the next step without fur-
ther purification.
This work was supported by Consortium for Parasitic Drug
Development (CPPD), the Bill and Melinda Gates Foundation, and
Medicines for Malaria Venture (MMV).
Supplementary data
5.2.9.18. 3-(5-Cyanopyridin-3-yl)-5-(6-cyanopyridin-3-yl)isox-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.050.
azole (77).
The title compound was prepared from synthons
56c and 59d. Yield 835 mg (55%), mp 248–229 °C. 1H NMR d 9.38
(d, J = 2.1 Hz, 1H), 9.28 (dt, J1 = 2.2 Hz, J2 = 0.7 Hz, 1H), 9.21 (d,
J = 2.0 Hz, 1H), 8.85 (td, J1 = 2.1 Hz, J2 = 0.6 Hz, 1H), 8.54 (ddd,
J1 = 8.1 Hz, J2 = 2.2 Hz, J3 = 0.6 Hz, 2H), 8.32 (d, J = 0.6 Hz, 0H), 8.30
(dt, J1= 8.2 Hz, J2 = 0.7 Hz, 1H), 8.11 (s, 1H). Anal. Calcd for
References and notes
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ole(78).
3-(4-Cyanophenyl)-5-(5-cyanopyridin-3-yl)isoxaz-
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5.2.9.20. 5-(3-Cyanophenyl)-3-(6-cyanopyridin-3-yl)isoxazole
(79).
The title compound was prepared from synthons 56f
and 59c. Yield 1.08 g (71%), mp 221–222 °C. 1H NMR d 9.27 (dd,
J1 = 2.3 Hz, J2 = 0.9 Hz, 1H), 8.54 (dd, J1 = 8.0 Hz, J2 = Hz, 1H), 8.42
(t, J = 1.6 Hz, 1H), 8.28 (dd, J1 = 8.0, Hz, J2 = 0.8 Hz, 1H), 8.24 (dt,
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