Synthesis and activity of azaterphenyl diamidines against Trypanosoma brucei rhodesiense and Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2009.07.080

A series of azaterphenyl diamidines has been synthesized and evaluated for in vitro antiprotozoal activity against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) and in vivo efficacy in the STIB900 acute mouse model for T

Full text of DOI:10.1016/j.bmc.2009.07.080

Bioorganic & Medicinal Chemistry 17 (2009) 6651–6658
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and activity of azaterphenyl diamidines against Trypanosoma brucei
rhodesiense and Plasmodium falciparum
Laixing Hu a, Reem K. Arafa a, Mohamed A. Ismail a, , Alpa Patel a, Manoj Munde a, W. David Wilson a,
Tanja Wenzler b, Reto Brun b, David W. Boykin a,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
b Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of azaterphenyl diamidines has been synthesized and evaluated for in vitro antiprotozoal activity
against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) and in vivo efficacy
in the STIB900 acute mouse model for T. b. r. Six of the 13 compounds showed IC50 values less than 7 nM
against T. b. r. Twelve of those exhibited IC50 values less than 6 nM against P. f. and six of those showed
IC50 values 60.6 nM, which are more than 25-fold as potent as furamidine. Moreover, two of them
showed more than 40-fold selectivity for P. f. versus T. b. r. Three compounds 15b, 19d and 19e exhibited
in vivo efficacy against T. b. r. much superior to furamidine, and equivalent to or better than azafuram-
idine. The antiparasitic activity of these diamidines depends on the ring nitrogen atom(s) location relative
to the amidine groups and generally correlates with DNA binding affinity.
Received 2 July 2009
Revised 24 July 2009
Accepted 26 July 2009
Available online 7 August 2009
Keywords:
Diamidines
Antiprotozoal agents
DNA binding affinity
Azaterphenyl
Ó 2009 Published by Elsevier Ltd.
1. Introduction
azafuramidine 3b was found to be quite effective against 2nd stage
HAT.5 Although diamidines have been used therapeutically since
Human African trypanosomiasis (HAT) and malaria, which are
caused by the protozoan parasites Trypanosoma brucei and Plasmo-
dium sp., infect millions of people in large parts of the world each
year.1 Numerous aromatic or heterocyclic diamidines exhibit po-
tent antiprotozoal activity against HAT and malaria.2 However,
pentamidine 1 (Fig. 1) is the only one of this class which has seen
significant human clinical use and it has been used to treat 1st
stage HAT for over half a century.2 Furamidine 2a, a diphenyl furan
diamidine analogue, has been shown to be more potent and less
toxic than pentamidine in murine models of trypanosomiasis.3
The oral prodrug of furamidine 2b (pafuramidine) showed promis-
ing results in Phase I and II clinical trials against both HAT and ma-
laria.1–3 Unfortunately, in an additional safety study of
pafuramidine parallelling the Phase III trials, liver and kidney tox-
icities in some volunteers were found and the development of
pafuramidine was suspended.3 Introduction of an N-atom into
one of phenyl ring of furamidine resulted in an aza-analogue of
furamidine 3a, which exhibited more potent in vivo antitrypanos-
omal activity than pentamidine and furamidine, although all three
have similar in vitro activities.4 The methoxyamidine prodrug of
the 1950s, the antiparasitic mode of action of such diamidines is
not well understood. A long-hypothesized mechanism of action ar-
ose from their binding to the minor groove of DNA at AT rich sites
in the nucleus or kinetoplast,6 which has been suggested to inter-
fere with DNA-associated enzymes, such as topoisomerase II, and
possibly direct inhibition of transcription.7 Recent investigations
have shown that both furamidine 2a and azafuramidine 3a accu-
mulated within trypanosomes at millimolar concentration, with
intracellular concentrations over 15,000-fold higher than mouse
plasma concentrations.8 Although the results of this study showed
that the extent of accumulation is not directly correlated with kill-
ing of the trypanosomes, the selective concentration of diamidines
in parasite mitochondria (kinetoplast) appears to represent a piv-
otal step in their antiparasitic activity.8
The previous design of DNA minor groove binding diamidine
analogues has focused on crescent shaped molecules which closely
fit the curvature of the groove, such as pentamidine, furamidine
and their analogues.2,9 Recently, linear dicationic diamidines
CGP40215A 4 and a benzimidazole diamidine 5 have shown strong
DNA minor groove binding and potent antiparasitic activity.10 Both
compounds were found to simulate the curved structure of DNA
minor groove by incorporation of a water molecule into the recog-
nition complex with DNA.10 Based on the discovery of this new
binding mode, we recently prepared a series of linear terphenyl
diamidines which showed significant DNA minor groove binding
* Corresponding author. Tel.: +1 404 413 5498; fax: +1 404 413 5505.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
Present address: Department of Chemistry, College of Science, King Faisal
University, PO Box 380, Hofuf 31982, Saudi Arabia.
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.07.080

6652
L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
Figure 1. Aromatic or heterocyclic diamidine antiprotozoal agents.
affinity and low nanomolar antiprotozoal activity against Trypano-
soma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.).11
The parent terphenyl compound 6a is more effective than furami-
dine in an acute mouse model for T. b. r. STIB 900. Due to the prom-
ising results found in the furan diamidine system, we first
synthesized three aza-analogues 6b–d of terphenyl diamidine 6a
by introduction of a nitrogen atom into the central phenyl ring
or both of the terminal phenyl rings.11 Both aza-diamidine com-
pounds 6b–c in which a nitrogen atom has been placed in the cen-
tral phenyl ring or meta to both of the amidine groups in the
terminal phenyl rings are more effective than furamidine in an
acute mouse model for T. b. r. STIB 900. The diaza-analogue 6d is
of particular interest because it exhibits very strong activity (IC50
= 0.5 nM) against P. f. and at the same time shows a 32-fold selec-
tivity for P. f. compared to antitrypanosomal activity.11 More inter-
estingly, 6d binds specifically to a GC-rich sequence more strongly
than to the usual AT recognition, which is the first non-polyamide,
synthetic compound to specifically recognize a DNA sequence with
a majority of GC base pairs.12 Therefore, we prepared additional
analogues of this series of azaterphenyl diamidines in order to
investigate structure–activity relationships (SAR) and their DNA
binding profiles. More recently, we have reported that this series
of terphenyl and azaterphenyl diamidines exhibit potent in vitro
antileishmanial activity.13 The results indicate that the antileish-
manial activity of these dications strongly depends on the ring
N-atom position relative to the amidine groups and correlates with
the DNA minor groove binding affinity. Here, we describe their
synthesis, in vitro activities against T. b. r. and P. f. and in vivo effi-
cacy in the T. b. r. STIB 900 acute mouse model.
Scheme 2 shows the syntheses of the diamidines 10a–c with the
two nitrogen atoms in the central aryl ring (pyrazinyl, pyridazinyl
and pyrimidnyl ring) as well as pyridinyl rings as both the terminal
units. Scheme 3 outlines the approach used to prepare the com-
pounds 19a–g with phenyl or pyridyl ring in the central ring and
nitrogen atom(s) in one terminal aryl ring. The bis-nitriles 18a–g
were obtained from 4-bromophenylboronic acid 16a or 2-chloro-
pyridine-5-boronic acid 16b in two step Suzuki coupling reactions.
2.2. Biology
The results for the evaluation of the 13 azaterphenyl diamidine
analogues against T. b. r. STIB900 and P. f. K116 and their DNA bind-
ing affinities13 are shown in Table 1. For comparative and SAR pur-
poses, the analogous data for pentamidine 1, furamidine 2a,
azafuramidine 3a and the terphenyl analogues 6a–d are also in-
cluded in Table 1. Six of the 13 compounds showed antitrypanos-
omal IC50 values 67 nM, comparable to that of pentamidine and
furamidine. Another six compounds exhibited IC50 values between
7 and 32 nM, and only one compound 10c gave poor antitrypanos-
omal activity (IC50 > 7 lM). More interestingly, these azaterphenyl
dications exhibited very potent activities against P. f. in vitro. Six of
the 13 compounds showed IC50 values 60.6 nM, which are over 25-
fold more potent than furamidine (IC50 = 15.5 nM). Another six of
the compounds exhibited IC50 values between 1.1 and 5.7 nM,
and only one compound 10c gave moderate antimalaria activity
(IC50 = 65 nM). Two of the most active compounds 15a and 15c
showed more than 40-fold selectivity for P. f. compared to T. b. r.
Compound 15c was 106 times more active against P. f.
(IC50 = 0.3 nM) than against T. b. r. (IC50 = 32 nM). Compound 15c
is one of the most potent compounds against P. f. K1 in the STI
in vitro screen.
Our previous study has shown that the number and location of
nitrogen atoms has a significant impact on the antileishmanial
activity for these linear rigid-rod systems.13 The results from the
current study of these azaterphenyl compounds against T. b. r.
and P. f. are generally consistent with that observed in the antile-
ishmanial study. Compounds 10a and 19a, in which a nitrogen
atom has been replaced ortho to one or both of the amidine groups,
showed a five- and two-fold increase in potency compared to the
parent terphenyl diamidine 6a against T. b. r. and a three- and four-
fold increased potency against P. f. The two isomeric compounds
6b and 19b, in which the nitrogen atom is meta to the amidine,
exhibited a significant loss in potency against both T. b. r. and P.
f., compared to 10a and 19a. Introduction of two nitrogen atoms
2. Results and discussion
2.1. Chemistry
The syntheses of the azaterphenyl diamidines begins with Su-
zuki coupling of the appropriate aryl halides with the correspond-
ing aryl boronic acids or esters to yield the azaterphenyl bis-
nitriles (Schemes 1–3).11 The bis-nitriles were converted to the
diamidines by the action of lithium trimethylsilylamide
[LiN(TMS)2] in THF. As illustrated in Scheme 1, azaterphenyl diami-
dine analogues 10a–c with nitrogen atom(s) in both of the terminal
aryl rings (pyridyl, pyrimidinyl and pyrazinyl ring) were prepared
from 5-bromo-2-pyridinecarbonitrile 7a, 5-bromo-2-pyrimidine-
carbonitrile 7b and 5-chloro-2-pyrazinecarbonitrile 7c14 by
coupling with 1,4-phenylenebisboronic acid
8 in two steps.

L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
6653
Scheme 1. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 °C; (ii) (a) LiN(TMS)2, THF; (b) HCl (gas), EtOH.
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 °C; (ii) (a) LiN(TMS)2, THF; (b) HCl (gas), EtOH.
Scheme 3. Reagents and conditions: (i) Pd(pph3)4, NaCO3, toluene. 80 °C; (ii) 4-cyanophenylboronic acid, Pd(PPh3)4, NaCO3, toluene, 80 °C; (iii) (a) LiN(TMS)2, THF; (b) HCl
(gas), EtOH.
ortho to one amidine, 19c, resulted in a modest reduction of activ-
ity against both T. b. r. and P. f. compared to 19a. Interestingly, if
two nitrogen atoms are introduced ortho to both of the amidine
groups, 10b, a six-fold reduction in activity against T. b. r. was ob-
served, conversely a slight enhancement in activity against P. f. was
seen. For compound 10c, in which one of the nitrogen atoms is
meta and another is ortho to both amidine units, a significant
reduction in activity against both T. b. r. and P. f. was seen. Intro-
duction of one nitrogen atom in the central ring (6c) led to a two-
fold increase in activity against T. b. r., however, a seven-fold
reduction in activity against P. f., compared to the parent terphenyl
compound 6a.11 In contrast, introduction of two nitrogen atoms in
the central ring (6d, 15a–c) resulted in a more than two-fold
reduction in activity against T. b. r., however, a three-fold increase
in activity against P. f., except 15b which showed comparable
activity, compared to the parent terphenyl compound 6a. Introduc-
tion of one nitrogen atom in the central ring (6d) in compound 19a
with a nitrogen atom in one terminal aryl ring, led to moderate

6654
L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
Table 1
DNA affinities and antiprotozoan activity for azaterphenyl diamidines
c
Code
Aryl type
A
B
D
M
G
Q
D
Tma (°C)
T. b. r.b IC50 (nM)
P. f.b IC50 (nM)
Selectivity
Cytotoxicityd IC50 (nM)
1
2a
3a
6a
6b
6c
6d
10a
10b
10c
15a
15b
15c
19a
19b
19c
19d
19e
19f
19g
/
/
/
I
I
I
I
I
I
I
I
I
/
/
/
CH
CH
N
/
/
/
/
/
/
/
/
/
CH
N
/
/
/
CH
CH
CH
CH
N
/
/
/
CH
CH
CH
CH
CH
N
12.6
25
19.3
17.1
9.2
18.7
8.1
17
12.8
3.9
8.0
16.9
6.8
19.5
11.7
16.0
15.2
13.8
14.9
12.1
2.2
4.5
6.5
5
47
2
16
1
6
7131
18
14
32
3
11
4
6
46.4
15.5
6.5
1.4
10.1
10
0.9
0.4
0.6
65.4
0.4
1.3
0.3
0.3
1.3
0.6
1.1
2.5
1.5
5.7
0.05
0.3
1.0
3.6
4.7
0.2
17.8
2.5
10.0
109.0
45.0
10.8
106.7
10
8.5
6.7
5.5
2.8
8.0
2.5
2100
6400
77,900
22,100
25,600
49,900
40,900
1200
CH
CH
CH
CH
CH
CH
CH
N
N
CH
/
/
/
/
/
/
/
CH
CH
CH
N
CH
CH
CH
N
CH
N
/
/
/
/
/
/
/
CH
CH
CH
CH
N
CH
CH
CH
CH
N
CH
CH
N
CH
N
N
CH
CH
CH
CH
N
N
2200
CH
CH
CH
N
N
CH
N
N
CH
N
N
46,500
42,500
31,300
5300
2800
26,700
4700
19,900
28,300
6400
CH
CH
CH
CH
CH
N
CH
CH
N
I
N
II
II
II
II
II
II
II
CH
CH
CH
N
N
N
7
12
14
N
CH
N
93,400
a
Increase in thermal melting of poly(dA–dT)2; see Refs. 11 and 13.
The T. b. r. (Trypanosoma brucei rhodesiense) strain was STIB900, and the P. f. (Plasmodium falciparum) strain was K1. The Values were average of duplicate determinations;
b
see Ref. 16.
c
Selectivity was the ratio [IC50 (T. b. r.)/IC50 (P. f.)].
Cytotoxicity was evaluated using cultured L6 rat myoblast cells; see Refs. 13 and 17.
d
Table 2
reduction in activity against T. b. r. and P. f. However, it is notewor-
thy that introduction of two nitrogen atoms in the central ring (6e
and 6f) in the compounds 19b and 19c showed no apparent effect
on either antitrypanosomal or antiplasmodial activity.
In vitro and in vivo anti-trypanosomal activity of azaterphenyl diamidines in the
STIB900 mouse modela
Code T. b. r. IC50 (nM) Dosageb (ip, mg/kg) Cures
Survival (days)
c
d
The
DTm values of these linear azaterphenyl diamidines, which
1
2.2
4.5
6.5
20
5
20
5
20
5
20
5
5
5
5
5
NA
5
5
5
5
5
5
5
5
2/4
1/4
3/4
1/4
4/4
3/4
2/4
2/4
2/4
0/4
0/4
1/4
>57.5
>38
>57.75
>46
range from high values of 17–19 °C to low ones of 4–6 °C, are lower
2a
3a
than that of crescent shape molecules e.g. furamidine
(D
Tm = 25 °C).13 Our previous study found that the compounds
>60
which exhibit the higher
DTm values showed the higher antileish-
>54.5
>47.75
>60
>42
23.5
manial activity and the weaker binding compounds show low
activity.13 A similar trend is found for T. b. r., but not for P. f. The
potent antitrypanosomal compounds, which showed IC50 values
less than 7 nM, exhibited higher
On the other hand, the less potent antitrypanosomal compounds,
which showed IC50 values of more than 18 nM, exhibited lower
Tm values (less than 10 °C). These results are generally consistent
with the observed relationship between DNA binding affinities and
antileishmanial activity of this series azaterphenyl diamidines.
6a
6b
6c
6d
5
47
2
16
1
6
7131
18
14
32
3
11
4
6
DTm values (12 °C or greater).
10a
10b
10c
15a
15b
15c
19a
19b
19c
19d
19e
19f
19g
>51.5
>43.25
0/4
3/4
0/4
0/4
1/4
1/4
3/4
4/4
0/4
1/4
36.5
>49.25
34
>48.75
>56.0
>46.25
>60.0
>60.0
>43.25
>31.75
D
However, the correlation between
activity is complex. For example, the potent compounds 6d, 15a
Tm values of
DTm values and antiplasmodial
and 15c which IC50 values of 60.9 nM showed
D
7
12
14
6.8–8.1 °C. In contrast, the potent compounds 10a, 10b, 19a and
19c with IC50 values of 60.6 nM showed significantly higher
5
5
D
Tm values of 12.8–19.5 °C. Clearly, other factors such as trans-
a
b
c
See Ref. 16 for details of STIB900 mouse model.
Dosage was for four days; ip, intraperitoneal.
Number of mice that survive and are parasite free for 60 days.
port, induced changes in DNA topology or protein–DNA complex
inhibition, are important in determining the antiplasmodial activ-
ity of these linear-rod molecules. It is perhaps also significant that
diamidine compounds localize only in the nuclear DNA of plasmo-
dia15 but they are in both the mitochondrial kinetoplast DNA and
in the nucleus in trypanosomes.8c
d
Average days of survive; untreated control expires between day 7 and 9 post-
infection.
Given the promising in vitro T. b. r. activity of these analogues,
we have evaluated them in the stringent STIB900 acute mouse
model for T. b. r.16 The results are shown in Table 2 and for compar-
ative purposes the in vivo data for pentamidine 1, furamidine 2a,
azafuramidine 3a and the terphenyl analogues 6a–d in the same
model are also presented. On intraperitoneal dosing all of the dica-
tions showed a significant increase in survival time for the treated
animals compared to untreated controls. Three of the thirteen

L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
6655
compounds, 15b, 19d and 19e, gave superior and four compounds,
10b, 19b, 19c and 19g, identical results to that for furamidine in
this model at the dose of 5 mg/kg. Five compounds were not able
to cure any mice although they extended the survival significantly.
Two compounds 15b and 19d were as effective as the azafuram-
dine 3a giving 3/4 cures at 5 mg/kg. The best result obtained was
for compound 19e, which showed 4/4 cures at a dose of 5 mg/kg.
It is noteworthy that although the compounds 6b and 19b, in
which the nitrogen atom is meta to the amidine, exhibited a signif-
icant loss of in vitro potency against T. b. r. compared to their iso-
meric compounds 10a and 19a, in which the nitrogen atom is ortho
to the amidine, the in vivo efficacy of 6b and 19b (2/4 and 1/4 cure,
respectively) was superior to the ortho isomers 10a and 19a (0/4
cure). The pyridinyl analogue 19e was also more effective than
the ortho isomer 19d in this mouse model, even though these
two isomers exhibited almost similar in vitro antitrypanosomal
activity. These results are consistent with those observed for the
azafuramidine system.4 Since previous study of the bis-amidox-
imes and bis-O-methylamidoximes prodrugs of the terphenyl
diamidine analogues showed poor bioconversion and were not
effective on oral administration,11 we did not prepare their bis-
amidoximes and bis-O-methylamidoximes prodrugs during this
study. However, further studies to develop novel orally effective
prodrugs of this highly active series of azaterphenyl diamidines
are underway.
In summary, we have prepared a series of azaterphenyl diami-
dine analogues that exhibit potent in vitro activity against both T.
b. r. and P. f., and showed promising activity on intraperitoneal
administration in the T. b. r. STIB900 acute mouse model. Six of
the 13 compounds showed IC50 values 60.6 nM against P. f.,
and two of them showed more than 40-fold selectivity for P. f.
versus T. b. r. Three compounds 15b, 19d and 19e exhibited
in vivo efficacy (3/4 or 4/4 cure at 5 mg/kg dosage, ip) in the
stringent STIB900 model much superior to that of furamidine,
and comparable to or better than azafuramidine. SAR information
shows that the number and location of nitrogen atoms has a sig-
nificant effect on antiparasitic activity. This series of azaterphenyl
diamidines merit further investigation as novel potent antipara-
sitic agents.
3.2.1. 1,4-Bis-(20-cyanopyridin-50-yl)phenylene (9a)
To a stirred solution of 5-bromo-2-pyridinecarbonitrile 7a
(6.60 g, 36.0 mmol), and tetrakis(triphenylphosphine) palladium
(1.80 g, 1.56 mmol) in toluene (100 mL) under a nitrogen atmo-
sphere was added 50 mL of a 2 M aqueous solution of Na2CO3 fol-
lowed by 1,4-phenylenebisboronic acid 8 (3.90 g, 24.0 mmol) in
50 mL of methanol. The vigorously stirred mixture was heat at
80 °C overnight. After cooling, the solution was filtered, and the
precipitate was washed with toluene, water and ether, to afford
the title compound as a white solid 9a (4.70 g, 92% yield);
mp > 300 °C. 1H NMR (DMSO-d6):
d 7.99 (s, 4H), 8.08 (d,
J = 8.0 Hz, 2H), 8.38 (dd, J = 2.0, 8.0 Hz, 2H), 9.14 (d, J = 2.0 Hz,
2H). Anal. Calcd for C18H10N4–0.3H2O: C, 75.14; H, 3.71; N, 19.47.
Found: C, 75.37; H, 3.51; N, 19.15.
3.2.2. 1,4-Bis-(20-amidinopyridin-50-yl)-phenylene
hydrochloride salt (10a)
The dinitrile 9a (570 mg, 2.0 mmol), suspended in freshly dis-
tilled THF (30 mL), was treated with lithium trimethylsilylamide
(1 M solution in THF, 15 mL, 15.0 mmol), and the reaction was al-
lowed to stir overnight at room temperature. The reaction mixture
was then cooled to 0 °C and HCl saturated ethanol (15 mL) was
added, whereupon a precipitate started forming. The mixture
was left to stir overnight, after which it was diluted with ether,
and the resultant solid was collected by filtration. The diamidine
was purified by neutralization with 1 N NaOH followed by filtra-
tion of the resultant solid and washing with water (3ꢀ). Finally,
the dried free base was stirred with ethanolic HCl overnight and di-
luted with ether, and the solid which formed was filtered and dried
to give diamidine salt 10a in 68% yield; mp >300 °C. 1H NMR
(DMSO-d6): d 8.11 (s, 4H), 8.47 (d, J = 8.4 Hz, 2H), 8.60 (dd,
J = 2.0, 8.4 Hz, 2H), 9.24 (d, J = 2.0 Hz, 2H), 9.41 (s, 4H), 9.66 (s,
4H). 13C NMR (DMSO-d6): d 161.6, 147.4, 142.5, 138.5, 135.8,
135.4, 127.8, 123.1. HRMS: m/z 317.1517 (M+1) (calculated for
C18H17N6, 317.1515). Anal. Calcd for C18H16N6–2.0HCl–0.3H2O: C,
54.78; H, 4.75; N, 21.29. Found: C, 55.03; H, 4.60; N, 21.01.
3.2.3. 1,4-Bis-(20-amidinopyrimidin-50-yl)-phenylene
hydrochloride salt (10b)
The same procedure described for 1,4-bis-(20-cyanopyridin-50-
yl)phenylene 9a was used by employing 5-bromo-2-pyrimidine-
carbonitrile 7b and 1,4-phenylenebisboronic acid 8 to furnish
1,4-bis-(20-cyanopyrimidin-50-yl)-phenylene 9b in 69% yield; mp
>300 °C.1H NMR (DMSO-d6): d 8.16 (s, 4H), 9.50 (s, 4H). The com-
pound was used directly in the next step.
3. Experimental
3.1. Biology
3.1.1. Efficacy evaluation
The same procedure described for the preparation of 10a was
used starting with the dinitrile 9b; 68% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.24 (s, 4H), 9.57 (s, 4H), 9.60 (s, 4H), 9.76 (s,
4H). HRMS: m/z 319.1418 (M+1) (calculated for C16H15N8,
319.1420). Anal. Calcd for C16H14N8–2.0HCl–2.5H2O: C, 44.05; H,
4.83; N, 25.68. Found: C, 44.39; H, 4.55; N, 25.28.
In vitro assays with T. b. r. STIB900 and P. f. K1 strain as well as
the efficacy study in an acute mouse model for T. b. r. STIB900 were
carried out as previously reported.16
3.2. Chemistry
3.2.4. 1,4-Bis-(20-cyanopyrazin-50-yl)phenylene (9c)
Melting points were determined on a Mel-Temp 3.0 melting
point apparatus, and are uncorrected. TLC analysis was carried
out on Silica Gel 60 F254 precoated aluminum sheets using UV
light for detection. 1H and 13C NMR spectra were recorded on a
Varian Unity Plus 300 MHz or Bruker 400 MHz spectrometer using
indicated solvents. Mass spectra was obtained from the Georgia
State University Mass Spectrometry Laboratory, Atlanta, GA. Ele-
mental analysis were performed by Atlantic Microlab Inc., Nor-
cross, GA, and are within 0.4 of the theoretical values. The
compounds reported as salts frequently analyzed correctly for frac-
tional moles of water and/or ethanol of solvation. In each case, pro-
ton NMR showed the presence of the indicated solvents. All
chemicals and solvents were purchase from Aldrich Chemical Co.,
VWR International or Frontier Scientific.
The same procedure described for 1,4-bis-(20-cyanopyridin-50-
yl)phenylene 9a was used by employing 5-chloro-2-pyrazinecar-
bonitrile 7c14 and 1,4-phenylenebisboronic acid 8 to furnish the ti-
tle compound 9c in 74% yield; mp >300 °C. 1H NMR (DMSO-d6): d
8.39 (s, 4H), 9.22 (s, 2H), 9.69 (s, 2H). 13C NMR (DMSO-d6): d
151.1, 147.2, 145.9, 136.2, 129.1, 128.0, 116.1. HRMS: m/z
285.0886 (M+1) (calculated for C16H9N6, 285.0889).
3.2.5. 1,4-Bis-(20-amidinopyrazin-50-yl)-phenylene
hydrochloride salt (10c)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 9c; 55% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.64 (s, 4H), 9.47 (s, 2H), 9.71 (s, 4H), 9.78 (s,

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L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
2H), 9.92 (s, 4H). HRMS: m/z 319.1419 (M+1) (calculated for
C16H15N8, 319.1420). Anal. Calcd for C16H14N8–2.0HCl–0.65H2O:
C, 47.69; H, 4.33; N, 27.81. Found: C, 48.04; H, 4.18; N, 27.52.
umn chromatography (EtOAc/hexane, 80:20); yield 82%; mp 138–
140 °C. 1H NMR (DMSO-d6): d 7.73 (d, J = 8.8 Hz, 2H), 7.88 (d,
J = 8.8 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 8.34 (dd, J = 2.4, 8.0 Hz,
1H), 9.08 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6): d 149.2, 137.8,
135.4, 134.5, 132.2, 131.4, 129.5, 129.1, 123.2, 117.6. HRMS: m/z
258.9866 (M+1) (calculated for C12H8N2Br, 258.9871).
3.2.6. 2,5-Bis-(40-cyanophenyl)-pyrazine (14a)
The same procedure described for 1,4-bis-(20-cyanopyridin-50-
yl)phenylene 9a was used by employing 2,5-dibromopyrazine
11a18 and 4-cyanophenylboronic acid 12 to furnish the title com-
pound 14a in 52% yield; mp >300 °C. 1H NMR (DMSO-d6): d 7.99
(d, J = 8.4 Hz, 4H), 8.39 (d, J = 8.4 Hz, 4H), 9.42 (s, 2H). Anal. Calcd
for C18H10N4–0.3H2O: C, 75.14; H, 3.71; N, 19.47. Found: C,
75.26; H, 3.47; N, 19.70.
3.2.13. Phenyl[1,10]phenyl[40,500]pyridinyl-4,200-bis-carbonitrile
(18a)
The nitrile 17a and 4-cyanophenylboronic acid were reacted
under the above-mentioned Suzuki coupling conditions to give
the target dinitrile 18a; yield 84%; mp >300 °C. 1H NMR (DMSO-
d6): d 7.90–7.97 (m, 8H), 8.08 (d, J = 8.4 Hz, 1H). 8.38 (dd, J = 2.0,
8.4 Hz, 1H), 9.13 (d, J = 2.0 Hz, 1H). Anal. Calcd for C19H11N3–
0.3H2O: C, 79.59; H, 4.08; N, 14.66. Found: C, 79.65; H, 3.91; N,
14.43.
3.2.7. 2,5-Bis-(40-amidinophenyl)-pyrazine hydrochloride salt
(15a)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 14a; 76% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.05 (d, J = 8.4 Hz, 4H), 8.45 (d, J = 8.4 Hz, 4H),
9.37 (s, 4H), 9.53 (s, 2H), 9.58 (s, 4H). 13C NMR (DMSO-d6): d
165.1, 149.0, 142.1, 140.2, 129.2, 129.0, 127.0. Anal. Calcd for
C18H16N6–2.0HCl–0.55H2O: C, 54.16; H, 4.82; N, 21.05. Found: C,
54.41; H, 4.75; N, 20.71.
3.2.14. Phenyl[1,10]phenyl[40,500]pyridinyl-4,200-bis-amidine
hydrochloride salt (19a)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18a; 65% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.97 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H),
8.04 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.44 (d, J = 8.4 Hz,
1H), 8.58 (dd, J = 2.0, 8.4 Hz, 1H), 9.16 (s, 2H), 9.22 (d, J = 2.0 Hz,
1H), 9.40 (s, 2H), 9.44 (s, 2H), 9.64 (s, 2H). HRMS: m/z 316.1551
(M+1) (calculated for C19H18N5, 316.1562). Anal. Calcd for
C19H17N5–2.0HCl–0.6H2O: C, 57.18; H, 5.10; N, 17.55. Found: C,
57.46; H, 5.08; N, 17.25.
3.2.8. 2,5-Bis-(40-cyanophenyl)-pyridazine (14b)
The same procedure described for 1,4-bis-(20-cyanopyridin-50-
yl)phenylene 9a was used employing 3,6-dichloropyridazine 11b
and 4-cyanophenylboronic acid 12 to furnish the title compound
14b in 80% yield; mp >300 °C. 1H NMR (DMSO-d6): d 8.05 (d,
J = 8.4 Hz, 4H), 8.43 (d, J = 8.4 Hz, 4H), 8.51 (s, 2H). MS: m/z 282
(M+1). Anal. Calcd for C18H10N4–0.25H2O: C, 75.38; H, 3.69; N,
19.54. Found: C, 75.11; H, 3.56; N, 19.75.
3.2.15. 2-(40-Bromophenyl)-5-pyridinecarbonitrile (17b)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used by employing 2-bromo-5-cyanopyr-
idine 7d and 4-bromophenylboronic acid 16a to furnish the title
compound 17b in 78% yield; mp 157–159 °C. 1H NMR (DMSO-
d6): d 7.74 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.8 Hz, 2H), 8.22 (d,
J = 8.4 Hz, 1H), 8.40 (dd, J = 2.4, 8.4 Hz, 1H), 9.09 (d, J = 2.4 Hz,
1H). 13C NMR (DMSO-d6): d 157.9, 152.6, 141.1, 136.1, 132.0,
129.2, 124.4, 120.2, 117.2, 107.7. HRMS: m/z 258.9871 (M+1) (cal-
culated for C12H8N2Br, 258.9871).
3.2.9. 2,5-Bis-(40-amidinophenyl)-pyridazine hydrochloride salt
(15b)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 14b; 72% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.61 (d, J = 8.4 Hz, 4H), 8.06 (d, J = 8.4 Hz, 4H),
8.13 (s, 2H), 8.83 (s, 4H), 9.10 (s, 4H). Anal. Calcd for C18H16N6-
2.5HCl-0.5EtOH: C, 53.00; H, 5.03; N, 19.52. Found: C, 53.27; H,
4.85; N, 19.38.
3.2.16. Phenyl[1,10]phenyl[40,200]pyridinyl-4,500-bis-carbonitrile
(18b)
3.2.10. 2,5-Bis-(20-cyanopyridin-50-yl)-pyrimidine (14c)
The same procedure described for 1,4-bis-(20-cyanopyridin-50-
yl)phenylene 9a was used employing 5-bromo-2-chloropyrimidine
11c and 2-cyanopyridine-5-boronic acid pinacol ester 13 to furnish
the title compound 14c in 78% yield; mp >300 °C. 1H NMR (DMSO-
d6):): d 8.24 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.61 (dd,
J = 2.0, 8.4 Hz, 1H), 8.93 (dd, J = 2.0, 8.4 Hz, 1H), 9.32 (d, J = 2.0 Hz,
1H), 9.50 (s, 2H), 9.68 (d, J = 2.0 Hz, 1H). HRMS: m/z 285.0884
(M+1) (calculated for C16H9N6, 285.0875).
The nitrile 17b and 4-cyanophenylboronic acid were reacted
under the above-mentioned Suzuki coupling conditions to give
the target dinitrile 18b; yield 94%; mp >300 °C. 1H NMR (DMSO-
d6): d 7.68 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.86 (d,
J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.0 Hz, 1H).
8.42 (dd, J = 2.0, 8.0 Hz, 1H), 9.17 (d, J = 2.0 Hz, 1H). HRMS: m/z
282.1038 (M+1) (calculated for C19H12N3, 282.1031).
3.2.17. Phenyl[1,10]phenyl[40,200]pyridinyl-4,500-bis-amidine
hydrochloride salt (19b)
3.2.11. 2,5-Bis-(20-amidinopyridin-50-yl)-pyrimidine hydrochlo-
ride salt (15c)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18b; 75% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.98 (d, J = 8.4 Hz, 4H), 8.04 (d, J = 8.4 Hz, 2H),
8.06 (d, J = 8.4 Hz, 2H), 8.33–8.37 (m, 4H), 9.11 (s, 1H), 9.26 (s,
2H), 9.38 (s, 2H), 9.49 (s, 2H), 9.66 (s, 2H). 13C NMR (DMSO-d6): d
165.2, 163.8, 159.3, 149.0, 144.2, 140.0, 137.5, 137.1, 128.9,
127.9, 127.7, 127.6, 127.1, 123.0, 119.9. Anal. Calcd for C19H17N5–
2.0HCl–0.9H2O: C, 56.42; H, 5.18; N, 17.30. Found: C, 56.74; H,
4.94; N, 16.90.
The same procedure described for the preparation of 10a was
used starting with the dinitrile 14c; 67% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.51 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 8.4 Hz, 1H),
8.77 (dd, J = 2.0, 8.4 Hz, 1H), 9.08 (dd, J = 2.0, 8.4 Hz, 1H), 9.40 (d,
J = 2.0 Hz, 1H), 9.47 (s, 4H), 9.60 (s, 2H), 9.72 (s, 2H), 9.75 (s, 2H),
9.76 (d, J = 2.0 Hz, 1H). HRMS: m/z 319.1425 (M+1) (calculated for
C16H15N8, 319.1420). Anal. Calcd for C16H14N8–2.0HCl–0.8H2O: C,
47.37; H, 4.37; N, 27.62. Found: C, 47.47; H, 4.14; N, 27.32.
3.2.12. 5-(40-Bromophenyl)-2-pyridinecarbonitrile (17a)
5-Bromo-2-cyanopyridine 7a and 4-bromophenylboronic acid
16a were reacted under the above-mentioned Suzuki coupling
conditions to give the target nitrile 17a, which was purified by col-
3.2.18. Phenyl[1,10]phenyl[40,500]pyrimidinyl-4,200-bis-
carbonitrile (18c)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyr-

L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
6657
imidine 7b and 4- bromophenylboronic acid 16a to furnish 5-(40-
Bromophenyl)-2-pyrimidinecarbonitrile 17c in 58% yield; mp
202–204 °C. 1H NMR (DMSO-d6): d 7.79 (d, J = 8.4 Hz, 2H), 7.88
(d, J = 8.4 Hz, 2H), 9.39 (s, 2H). The compound was used directly
in the next step. The above nitrile 17c and 4-cyanophenylboronic
acid were reacted under the above-mentioned Suzuki coupling
conditions to give the target dinitrile 18c; yield 74%; mp >300 °C.
1H NMR (DMSO-d6): d 7.95–8.01 (m, 6H), 8.08 (d, J = 8.4 Hz, 2H),
9.47 (s, 2H). HRMS: m/z 283.0983 (M+1) (calculated for C18H11N4,
283.0984).
(dd, J = 2.0, 8.4 Hz, 1H), 8.55 (dd, J = 2.4, 8.4 Hz, 1H), 9.12 (d,
J = 2.0 Hz, 1H), 9.15 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6): d
155.8, 152.7, 152.0, 148.7, 141.3, 138.1, 132.0, 124.6, 120.8,
117.0, 108.4. HRMS: m/z 216.0323 (M+1) (calculated for C11H7N3Cl,
216.0329).
3.2.24. Phenyl[1,20]pyridinyl[50,200]pyridinyl-4,500-bis-carbo-
nitrile (18e)
The nitrile 17e and 4-cyanophenylboronic acid were reacted
under the above-mentioned Suzuki coupling conditions to give
the target dinitrile 18e; yield 76%; mp >300 °C. 1H NMR (DMSO-
d6): d 8.00 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.4 Hz, 1H), 8.39 (d,
J = 8.4 Hz, 3H), 8.48 (dd, J = 2.0, 8.4 Hz, 1H), 8.68 (dd, J = 2.0,
8.4 Hz, 1H), 9.16 (d, J = 2.0 Hz, 1H), 9.48 (d, J = 2.0 Hz, 1H). HRMS:
m/z 283.0976 (M+1) (calculated for C18H11N4, 283.0984).
3.2.19. Phenyl[1,10]phenyl[40,500]pyrimidinyl-4,200-bis-amidine
hydrochloride salt (19c)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18c; 75% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.97 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H),
8.06 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 8.4 Hz, 2H), 9.13 (s, 2H), 9.44
(s, 2H), 9.54 (s, 2H), 9.57 (s, 2H), 9.76 (s, 2H). 13C NMR (DMSO-
d6): d 165.1, 159.5, 155.6, 151.6, 144.0, 139.7, 135.0, 132.5, 128.9,
128.4, 128.0, 127.3, 127.2. Anal. Calcd for C18H16N6–2.0HCl–
0.6H2O: C, 54.04; H, 4.84; N, 21.00. Found: C, 54.29; H, 4.68; N,
20.86.
3.2.25. Phenyl[1,20]pyridinyl[50,200]pyridinyl-4,500-bis-amidine
hydrochloride salt (19e)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18e; 57% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.00 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H),
8.42–8.44 (m, 4H), 8.71 (dd, J = 2.0, 8.4 Hz, 1H), 9.14 (d, J = 2.0 Hz,
1H), 9.21 (s, 2H), 9.35 (s, 2H), 9.48 (s, 2H), 9.52 (d, J = 2.0 Hz, 1H),
9.65 (s, 2H). 13C NMR (DMSO-d6): d 165.2, 163.7, 157.4, 155.4,
149.2, 148.5, 142.6, 137.7, 136.0, 132.3, 128.8, 128.7, 127.1,
123.7, 121.3, 120.4. Anal. Calcd for C18H16N6–2.5HCl–1.5H2O: C,
49.75; H, 4.99; N, 19.34. Found: C, 50.03; H, 4.73; N, 19.25.
3.2.20. 5-(20-Chloropyridin-50-yl)-2-pyridinecarbonitrile (17d)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyr-
idine 7a and 2-chloropyridine-5-boronic acid 16b to furnish the
title compound 17d in 98% yield; mp 220–224 °C. 1H NMR
(DMSO-d6): d 8.22 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 8.51
(dd, J = 2.0, 8.4 Hz, 1H), 8.75 (dd, J = 2.0, 8.0 Hz, 1H), 9.19 (d,
J = 2.0 Hz, 1H), 9.48 (d, J = 2.0 Hz, 1H). 13C NMR (DMSO-d6): d
151.1, 149.4, 148.6, 135.9, 135.0, 132.0, 130.6, 129.1, 124.6,
117.5. HRMS: m/z 216.0334 (M+1) (calculated for C11H7N3Cl,
216.0329).
3.2.26. 5-(20-Chloropyridin-50-yl)-2-pyrimidinecarbonitrile (17f)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyr-
imidine 7b and 2-chloropyridine-5-boronic acid 16b to furnish the
title compound 17f in 27% yield; mp 158–160 °C. 1H NMR (DMSO-
d6): d 7.77 (d, J = 8.4 Hz, 2H), 8.41 (dd, J = 2.4, 8.4 Hz, 1H), 8.97 (d,
J = 2.4 Hz, 1H), 9.46 (s, 2H). 13C NMR (DMSO-d6): d 156.5, 151.7,
148.7, 143.1, 138.6, 132.0, 127.8, 124.8, 116.1. HRMS: m/z
217.0288 (M+1) (calculated for C10H6N4Cl, 217.0281).
3.2.21. Phenyl[1,20]pyridinyl[50,500]pyridinyl-4,200-bis-carbo-
nitrile (18d)
The nitrile 17d and 4-cyanophenylboronic acid were reacted
under the above-mentioned Suzuki coupling conditions to give
the target dinitrile 18d; yield 80%; mp 270–272 °C. 1H NM R1H
NMR (DMSO-d6): d 7.99 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H),
8.17 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.39 (dd, J = 2.0,
8.4 Hz, 1H), 8.75 (dd, J = 2.0, 8.4 Hz, 1H), 9.18 (d, J = 1.6 Hz, 1H),
9.52 (d, J = 2.0 Hz, 1H). 13C NMR (DMSO-d6): d 151.6, 148.9,
148.0, 140.5, 136.3, 135.5, 134.9, 133.7, 132.5, 132.3, 128.6,
127.4, 121.4, 118.1, 117.0, 110.9. HRMS: m/z 283.0974 (M+1) (cal-
culated for C18H11N4, 283.0984).
3.2.27. Phenyl[1,20]pyridinyl[50,500]pyrimidinyl-4,200-bis-carbo-
nitrile (18f)
The nitrile 17f and 4-cyanophenylboronic acid were reacted un-
der the above-mentioned Suzuki coupling conditions to give the
target dinitrile 18f; yield 77%; mp 272–274 °C. 1H NMR (DMSO-
d6): d 8.01 (d, J = 8.4 Hz, 2H), 8.34 (d, J = 8.4 Hz, 1H), 8.40 (d,
J = 8.4 Hz, 2H), 8.53 (dd, J = 2.0, 8.4 Hz, 1H), 9.28 (d, J = 2.0 Hz,
1H), 9.55 (s, 2H). 13C NMR (DMSO-d6): d 156.4, 154.9, 148.5,
142.9, 141.8, 136.3, 132.9, 132.5, 127.9, 127.5, 121.3, 118.7,
116.2, 112.0. HRMS: m/z 284.0949 (M+1) (calculated for
C17H10N5, 284.0936).
3.2.22. Phenyl[1,20]pyridinyl[50,500]pyridinyl-4,200-bis-amidine
hydrochloride salt (19d)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18d; 71% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 8.00 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H),
8.43 (d, J = 8.4 Hz, 2H), 8.47–8.51 (m, 2H), 8.66 (dd, J = 2.0, 8.4 Hz,
1H), 9.18 (s, 2H), 9.26 (d, J = 2.0 Hz, 1H), 9.30 (d, J = 2.0 Hz, 1H),
9.42 (s, 2H), 9.46 (s, 2H), 9.66 (s, 2H). HRMS: m/z 317.1502
(M+1) (calculated for C18H17N6, 317.1515). Anal. Calcd for
C18H16N6–2.0HCl–1.0H2O: C, 53.08; H, 4.95; N, 20.63. Found: C,
53.28; H, 4.59; N, 20.34.
3.2.28. Phenyl[1,20]pyridinyl[50,500] pyrimidinyl -4,200-bis-
amidine hydrochloride salt (19f)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18f; 51% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.99 (d, J = 8.4 Hz, 2H), 8.38 (d, J = 8.4 Hz, 1H),
8.44 (d, J = 8.4 Hz, 2H), 8.59 (dd, J = 2.0, 8.4 Hz, 1H), 9.16 (s, 2H),
9.34 (d, J = 2.0 Hz, 1H), 9.47 (s, 2H), 9.60 (s, 2H), 9.62 (s, 2H), 9.79
(s, 2H). 13C NMR (DMSO-d6): d 165.2, 159.5, 155.9, 155.1, 152.0,
148.7, 142.4, 136.5, 132.8, 128.8, 128.8, 128.1, 127.0, 121.2. HRMS:
m/z 318.1464 (M+1) (calculated for C17H16N7, 318.1467). Anal.
Calcd for C17H15N7–2.0HCl–0.6H2O: C, 50.91; H, 4.57; N, 24.45.
Found: C, 50.68; H, 4.49; N, 24.13.
3.2.23. 2-(20-Chloropyridin-50-yl)-5-pyridinecarbonitrile (17e)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used by employing 2-bromo-5-cyanopyr-
idine 7d and 2-chloropyridine-5-boronic acid 16b to furnish the
title compound 17e in 83% yield; mp 198–200 °C. 1H NMR
(DMSO-d6): d 7.68 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.45
3.2.29. 5-(20-Chloropyridin-50-yl)-2-pyrazinecarbonitrile (17g)
The same procedure described for 5-(40-bromophenyl)-2-pyri-
dinecarbonitrile 17a was used employing 2-chloro-5-cyanopyr-

6658
L. Hu et al. / Bioorg. Med. Chem. 17 (2009) 6651–6658
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4. Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F. A.; Wilson, W. D.; Boykin,
D. W. J. Med. Chem. 2003, 46, 4761.
azine 7c and 2-chloropyridine-5-boronic acid 16b to furnish the ti-
tle compound 17g in 47% yield; mp 118–120 °C. 1H NMR (DMSO-
d6): d 7.75 (d, J = 8.4 Hz, 1H), 8.58 (dd, J = 2.4, 8.4 Hz, 1H), 9.18 (d,
J = 2.4 Hz, 1H), 9.25 (s, 1H), 9.65 (s, 1H). 13C NMR (DMSO-d6): d
152.4, 149.1, 148.7, 147.6, 146.0, 138.2, 129.6, 129.1, 124.8,
115.9. HRMS: m/z 217.0273 (M+1) (calculated for C10H6N4Cl,
217.0281).
5. Ansede, J. H.; Voyksner, R. D.; Ismail, M. A.; Boykin, D. W.; Tidwell, R. R.; Hall, J.
E. Xenobiotica 2005, 35, 211.
6. (a) Boykin, D. W.; Kumar, A.; Spychala, J.; Zhou, M.; Lombardy, R.; Wilson, W.
D.; Dykstra, C. C.; Jones, S. K.; Hall, J. E.; Tidwell, R. R.; Laughton, C.; Nunn, C. M.;
Neidle, S. J. Med. Chem. 1995, 38, 912; (b) Boykin, D. W.; Kumar, A.; Xiao, G.;
Wilson, W. D.; Bender, B. C.; McCurdy, D. R.; Hall, J. E.; Tidwell, R. R. J. Med.
Chem. 1998, 41, 124; (c) Francesconi, I.; Wilson, W. D.; Tanious, F. A.; Hall, J. E.;
Bender, B. C.; Tidwell, R. R.; McCurdy, D.; Boykin, D. W. J. Med. Chem. 1999, 42,
2260; (d) Laughton, C. A.; Tanious, F. A.; Nunn, C. M.; Boykin, D. W.; Wilson, W.
D.; Neidle, S. Biochemistry 1996, 35, 5655; (e) Trent, J. O.; Clark, G. R.; Kumar, A.;
Wilson, W. D.; Boykin, D. W.; Hall, J. E.; Tidwell, R. R.; Blagburn, B. L.; Neidle, S.
J. J. Med. Chem. 1996, 39, 4554.
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Houssier, C.; Bailly, C. J. Am. Chem. Soc. 1998, 120, 10310; (b) Bailly, C.;
Dassonneville, L.; Carrascol, C.; Lucasl, D.; Kumar, A.; Boykin, D. W.; Wilson, W.
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Chem. 1999, 274, 27128; (d) Dykatra, C. C.; McClernon, D. R.; Elwell, L. P.;
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Tidwell, R. R. Mol. Pharmacol. 2006, 70, 1585; (b) Mathis, A. M.; Holman, J. L.;
Sturk, L. M.; Ismail, M. A.; Boykin, D. W.; Tidwell, R. D.; Hall, J. E. Antimicrob.
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Kumar, A.; Francesconi, I.; Anbazhagan, M.; Hu, Q.; Tanious, F. A.; Wanzler, T.;
Saulter, J.; Wilson, W. D.; Brun, R.; Boykin, D. W.; Tidwell, R. D.; Hall, J. E.
Antimicrob. Agents Chemother. 2007, 51, 2801.
3.2.30. Phenyl[1,20]pyridinyl[50,500]pyrazinyl-4,200-bis-carbo-
nitrile (18g)
The nitrile 17g and 4-cyanophenylboronic acid were reacted
under the above-mentioned Suzuki coupling conditions to give
the target dinitrile 18g; yield 70%; mp >300 °C. 1H NMR (DMSO-
d6): d 8.01 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H), 8.39 (d,
J = 8.4 Hz, 2H), 8.68 (dd, J = 2.0, 8.4 Hz, 1H), 9.25 (s, 1H), 9.48 (s,
1H), 9.72 (s, 1H). HRMS: m/z 284.0948 (M+1) (calculated for
C17H10N5, 284.0936).
3.2.31. Phenyl[1,20]pyridinyl[50,500]pyrizinyl-4,200-bis-amidine
hydrochloride salt (19g)
The same procedure described for the preparation of 10a was
used starting with the dinitrile 18g; 75% yield; mp >300 °C. 1H
NMR (DMSO-d6): d 7.80 (d, J = 8.4 Hz, 2H), 8.38 (d, J = 8.4 Hz, 1H),
8.46 (d, J = 8.4 Hz, 2H), 8.97 (dd, J = 2.4, 8.4 Hz, 1H), 9.20 (s, 2H),
9.49 (s, 3H), 9.67 (s, 2H), 9.74 (d, J = 2.0 Hz, 1H), 9.79 (s, 1H), 9.91
(s, 2H). 13C NMR (DMSO-d6): d 165.2, 160.7, 155.7, 148.9, 148.4,
146.5, 142.9, 142.4, 139.4, 136.4, 129.7, 128.8, 128.7, 127.1,
121.1. Anal. Calcd for C17H15N7–2.7HCl–2.0H2O: C, 45.19; H, 4.84;
N, 21.70. Found: C, 45.52; H, 4.71; N, 21.34.
9. Goodsell, D.; Dickerson, R. E. J. Med. Chem. 1986, 29, 727.
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W. D. J. Am. Chem. Soc. 2002, 124, 13680; (b) Nguyen, B.; Hamelberg, D.; Bailly,
C.; Colson, J.; Stenek, J.; Brun, R.; Neidle, S.; Wilson, W. D. Biophys. J. 2004, 86,
1028; (c) Miao, Y.; Lee, M. P. H.; Parkinson, G. N.; Batista-Parra, A.; Ismail, M. A.;
Neidle, S.; Boykin, D. W.; Wilson, D. W. Biochemistry 2005, 44, 14701.
11. Ismail, M. A.; Arafa, R. K.; Brun, R.; Wenzler, T.; Miao, Y.; Wilson, D. W.;
Generaux, C.; Bridges, A.; Hall, J. E.; Boykin, D. W. J. Med. Chem. 2006, 49, 5324.
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Cordonnier, M.-H.; Lansiaux, A.; Bailly, C.; Boykin, D. W.; Wilson, W. D. J. Am.
Chem. Soc. 2007, 129, 13732.
Acknowledgments
This work was supported by an award from the Bill and Melinda
Gates Foundation and by an award NIH AI064200.
13. Hu, L.; Arafa, R. K.; Ismail, M. A.; Wenzler, T.; Brun, R.; Munde, M.; Wilson, W.
D.; Nzimiro, S.; Samyesudhas, S.; Werbovetz, K. A.; Boykin, D. W. Bioorg. Med.
Chem. Lett. 2008, 18, 247.
14. Jampilek, J.; Dolezal, M.; Kunes, J.; Satinsky, D.; Raich, I. Curr. Org. Chem. 2005,
9, 49.
References and notes
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