Azaterphenyl diamidines as antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2007.10.091

Eighteen diamidino azaterphenyls and analogues were evaluated as anti-leishmanials; nine of the compounds gave IC₅₀ values less than 1 μM, five exhibited values less than 0.40 μM, and two gave values less than 0.10 μM in a Leishmania donovani a

Full text of DOI:10.1016/j.bmcl.2007.10.091

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 247–251
Azaterphenyl diamidines as antileishmanial agents
Laixing Hu,a Reem K. Arafa,a Mohamed A. Ismail,a Tanja Wenzler,b Reto Brun,b
Manoj Munde,a W. David Wilson,a Sandra Nzimiro,c Serene Samyesudhas,c
Karl A. Werbovetzc,* and David W. Boykina,*
aDepartment of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA
bParasite Chemotherapy, Swiss Tropical Institute, Basel, CH4002, Switzerland
cDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 21 September 2007; revised 23 October 2007; accepted 25 October 2007
Available online 30 October 2007
Abstract—Eighteen diamidino azaterphenyls and analogues were evaluated as anti-leishmanials; nine of the compounds gave IC50
values less than 1 lM, five exhibited values less than 0.40 lM, and two gave values less than 0.10 lM in a Leishmania donovani axe-
nic amastigote assay. The activity of the diamidines strongly depends on the ring N-atom location relative to the amidine groups and
correlates with DNA affinity. Transmission electron microscopy studies showed a dramatic dilation of the mitochondrion and evi-
dence of disintegration of the kinetoplast of the amastigotes.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Over 20 species of the Leishmania parasite contribute to
human leishmanial disease.1 The disease is transmitted
by the bite of more than 30 different species of sand
files.1 The Leishmania parasite is broadly distributed in
humans and animals, and recent estimates suggest that
two million new human cases occur annually.2 The dis-
ease is found in the Far East, southern Europe, and in
both Americas including the United States.3 The clinical
manifestations of the disease can be generally classified
into three forms: cutaneous, mucosal, and visceral.4
The drugs currently in use, pentavalent antimonials,
paromomycin, amphotericin, miltefosine, and pentami-
dine, all suffer from one or more of the following defi-
ciencies: significant toxicity, variable efficacy, lack of
oral bioavailability, extensive courses of parenteral
administration, and problems of cost and supply.1,2,5
Moreover, there is increasing evidence that the fre-
quency and extent of use of these drugs is leading to
selection of drug-resistant parasites.6 The need for more
effective drugs with desirable pharmacokinetic proper-
ties to treat human leishmanial disease is evident.2
Dicationic molecules, of which pentamidine (I; Fig. 1) is
the best known member of the diamidine class, were first
reported to have significant antiprotozoan activity in the
1930s.7 Despite numerous studies of various structural
classes of dications, pentamidine is the only compound
which has seen significant human use. The primary use
of pentamidine against human leishmanial disease is to
treat antimony-resistant leishmaniasis.1 The diamidine
furamidine (IIa) and its analogues have significant activ-
ity against various parasites including Leishmania.7a
Pafuramidine (IIb), an orally effective prodrug of furam-
idine, is currently in Phase III trials against both Human
African Trypanosomiasis (HAT) and Pneumocystis cari-
nii pneumonia.8 Compound I causes swelling of the
mitochondrion and destruction of mitochondrial (kine-
toplast) DNA in both African trypanosomes9 and Leish-
mania,10 and
I also decreases the mitochondrial
membrane potential in Leishmania donovani promastig-
otes.11 It has also been suggested that these dicationic
molecules, including pentamidine, act by binding in
the minor groove of DNA at AT rich sites.7a It has been
hypothesized, based on considerable evidence, that the
minor groove binding leads to inhibition of DNA
dependent enzymes or possibly direct inhibition of tran-
scription.7a,12 Although the lethal events have not yet
been determined, mitochondrial disruption and interfer-
ence with kinetoplast DNA processing may together re-
sult in the death of diamidine-treated parasites.
Keywords: Leishmania; Diamidines; Azaterphenyl; DNA binding; S-
uzuki coupling.
*
Corresponding authors. Tel.: +1 614 292 5499 (K.A.W.); tel.: +1 404
413 5498; fax: +1 404 413 5505 (D.W.B.); e-mail addresses:
werbovetz.1@osu.edu; dboykin@gsu.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.091

248
L. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 247–251
O
O
NR
O
RN
H2N
HN
NH
NH2
NH2
NH2
I
IIa R = H
IIb R = OMe
Figure 1.
Since the pioneering work of Dickerson and coworkers,
the design of minor groove binders has focused on cres-
cent shaped molecules whose molecular shape comple-
ments the DNA minor groove.13 More recently, linear
dications have been discovered which exhibit high minor
groove binding affinity and show excellent antiparasitic
activity.14 X-ray structures of co-crystals of linear dica-
tions and oligonuleotides revealed that a strategically lo-
cated water molecule in effect provides the curvature
needed to complement the groove.14 Based on the recog-
nition of this new binding mode we recently prepared a
series of linear terphenyl diamidines and found that they
were highly active in vitro against African trypanosomes
and against malaria.15 More recently, we found that the
parent molecule in this series, 4,40-diamidinoterphenyl
4a, was quite active against Leishmania donovoni (L.
d.) in vitro. This communication reports the synthesis
of new aza-terphenyl dications, their DNA affinity and
evaluation against Leishmania.
The syntheses of the azaterphenyl diamidines were
achieved by Suzuki coupling of the appropriate aryl ha-
lides with the corresponding aryl boronic acids or esters
to form the teraryl bis-nitriles.15 The bis-nitriles were
converted to the diamidines by the action of LiN(TMS)2
followed by hydrolysis. Scheme 1 outlines the approach
used to prepare azaterphenyl analogues with nitrogen
atom(s) in one or both of the terminal aryl rings. Scheme
2 shows the synthesis of diamidines with nitrogen
atom(s) in the central aryl ring as well as those in both
the terminal aryl rings. Finally, Scheme 3 provides our
approach to preparation of the compounds with nitro-
gen atom(s) in the central ring and one terminal aryl
ring.
Q
Q
P
Q
(i)
+
(HO)2B
B(OH)2
NC
X
NC
CN
P
M
M
M P
2
3
1 X=Br or Cl
HN
Q
Q
NH
NH2
(ii)
H2N
P
M
M P
4a: M=P=Q=CH;
b: M=Q=CH, P=N;
c: P=Q=CH, M=N;
d: M=CH, P=Q=N;
e: P=CH, M=Q=N.
Scheme 1. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 ꢁC; (ii) a—LiN(TMS)2, THF, rt, overnight; b—HCl (gas), ethanol, rt,
overnight.
(HO)2B
CN
6
or
D
A
D
A
(i)
(ii)
+
Y
X
NC
CN
B
O
O
P
B
P
B
CN
5 X, Y= Br or Cl
8
N
7
HN
D
NH
NH2
H2N
P
A
B
P
9a: A=B=CH, D=CF, P=CH;
b: A=N, B=D=CH, P=CH;
c: B=D=N, A=CH, P=CH;
d: A=B=N, D=CH, P=CH;
e: A=D=N, B=CH, P=CH;
f: A=D=N, B=CH, P=N.
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 ꢁC; (ii) a—LiN(TMS)2, THF, rt, overnight; b—HCl (gas), ethanol, rt,
overnight.

L. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 247–251
249
Q
Q
P
(i)
(iii)
+
X
CN
NC
Y
X
B(OH)2
A
M P
M
A
12
11 Y=Br or Cl
10a: A=CH, X=Br;
b: A=N, X=Cl.
HN
H2N
Q
NH
Q
CN
M P
(ii)
NC
NH2
A
M P
A
13
14a: M=Q=CH, P=N, A=CH;
b: P=Q=CH, M=N, A=CH;
c: M=CH, P=Q=N, A=CH;
d: M=Q=CH, P=N, A=N;
e: P=Q=CH, M=N, A=N;
f: M=CH, P=Q=N, A=N;
g: P=CH, M=Q=N, A=N.
Scheme 3. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 ꢁC; (ii) a—LiN(TMS)2, THF, rt, overnight; b—HCl (gas), ethanol, rt,
overnight; (iii) —4-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, toluene, 80 ꢁC.
Table 1 contains the results obtained for 18 azaterphenyl
diamidines and closely related analogues using an axenic
assay with L. donovani amastigote-like parasites. Nine of
the compounds gave IC50values less than 1 lM, five of
those exhibited IC50 values less than 0.40 lM, and two
of those gave values less than 0.10 lM. These results
represent a marked improvement in activity over that
found in our previous study16 of 58 compounds from di-
verse classes of diamidines which yielded only two com-
pounds with IC50 values less than 1 lM with none in the
sub 0.40 lM range. The number and location of nitro-
gen atoms has a significant impact on the anti-leishman-
ial activity of this linear rigid-rod system. Compounds
14a and 4b in which a nitrogen atom has been placed
ortho to one and both of the amidine groups, respec-
tively, in the parent terphenyl system 4a show a three-
and fourfold increase in activity over the parent and rep-
resent the two most active compounds found in this
study with IC50 values of 0.084 and 0.063 lM, respec-
tively. The two isomeric compounds 14b and 4c, in
which the nitrogen atom is meta to the amidine, show
a 16- and a more than 1500-fold reduction in activity
compared to 14a and 4b. Interestingly, introduction of
two nitrogen atoms ortho to one amidine, 14c, results
in a modest reduction of activity compared to 4b
(IC50 = 0.20 lM), however if two nitrogen atoms are
introduced ortho to both amidine units, 4d, a significant
reduction in activity is observed (IC50 = 1.70 lM).
Introduction of one or more nitrogen atoms in the cen-
tral ring (9b–9e) results in a two- to fourfold reduction in
Table 1. DNA affinities and in vitro antileishmanial activity for azaterphenyl diamidines
HN
Q
NH
HN
Q
P
D
A
Q
NH
or
H2N
NH2
H2N
NH2
A
M P
M
B
M P
II
I
a
Code
Aryl type
A
B
D
M
P
Q
DTm (ꢁC)
L.d.b IC50 (lM)
Cytotoxicityc IC50 (lM)
4a
4b
I
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CF
CH
N
CH
CH
N
CH
N
CH
CH
CH
N
17.1
17
0.27
0.063
>100
1.70
>105
0.34
0.82
0.56
0.72
1.10
1.80
0.084
1.40
0.20
0.51
6.8
22
I
1.2
26
4c
4d
I
CH
N
9.2
I
CH
N
12.8
3.9
2.2
47
4e
9a
I
CH
CH
CH
CH
CH
CH
N
N
I
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
N
15.1
18.7
8.0
6.4
50
9b
9c
I
I
CH
N
43
31
9d
9e
I
N
CH
N
16.9
8.1
I
N
CH
CH
/
41
9f
I
N
N
6.8
5.3
2.8
27
14a
14b
14c
14d
14e
14f
14g
II
II
II
II
II
II
II
CH
CH
CH
N
/
N
19.5
11.7
16.0
15.2
13.8
14.9
12.1
/
/
CH
N
/
/
CH
CH
N
4.7
20
/
/
N
CH
CH
N
N
/
/
CH
N
28
N
/
/
CH
N
2.6
16.0
6.4
93
N
/
/
CH
N
a Increase in thermal melting of poly(dA-dT)2; see Ref. 15.
b See Refs. 16 and 18; IC50 values are means of at least two independent assays.
c Cytotoxicity was evaluated using cultured L6 rat myoblast cells; see Ref. 20.

250
L. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 247–251
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
-5
-5.5
0
5
10
15
20
ΔTm
Figure 2. A plot of biological activity, on an increasing scale of log(1/IC50), versus DNA binding affinity, plotted as DTm, is shown for all of the
compounds in Table 1.
activity compared to the parent terphenyl 4a. A diminu-
tion in activity with central ring nitrogen atom substitu-
tion also occurs when nitrogen atoms are also present
either ortho or meta to the amidine functions (cf. 9f,
14d–14f). Hence, we have found a diamidine system
which is highly active and shows a consistent and dis-
tinctive structure activity relationship in the L. donovani
axenic amastigote assay. Due to their high activity in the
axenic amastigote assay, evaluation of the parent mole-
cule 4a and the most active compound 4b using a Leish-
mania infected macrophage assay was warranted in
order to have a better predictor of the in vivo potential
of this class of compounds. This assay was conducted
using L. amazonensis parasites bearing the stably inte-
grated b-lactamase reporter gene according to the meth-
od of Buckner and Wilson17 with the modification that
J774 murine cultured macrophages were used as the host
cell rather than murine peritoneal macrophages. Neither
4a nor 4b was active in this assay at concentrations be-
low 5 lM, and higher concentrations of both com-
pounds were toxic to macrophages. In contrast, the
antileishmanial drug amphotericin B possessed an IC50
value of 0.046 0.007 lM in this assay (mean stan-
dard error of 13 independent measurements), while the
reversed amidine 2,5-bis[3-methoxy-4-(2-pyridylimi-
no)aminophenyl]furan18 possessed an IC50 value of
0.22 0.05 lM in this assay (mean range of two inde-
pendent experiments). In contrast to reversed amidines,
it would appear that the entry of typical amidines into
the parasitopherous vacuole containing intracellular
Leishmania parasites is poor. Thus, the challenge re-
mains to develop macrophage delivery methods for the
azaterphenyl diamidines.
To qualitatively estimate the DNA binding affinities of
these linear diamidines, we measured melting tempera-
tures for their complexes with poly(dA-dT) and Table
1 reports the DTm values (difference in melting tempera-
ture of free DNA in solution and that of the diamidine-
DNA complex). The DTm range from high values of 17–
19 ꢁC to low ones of 4–6 ꢁC. Although the high values
represent strong DNA affinities for these linear mole-
Figure 3. Ultrastructural effects of 4a on L. donovani axenic amastigotes. Parasites were incubated in the absence (control, panel A) or the presence
(panels B and C) of 0.68 lM 4a and were examined by transmission electron microscopy as described in the text. f, flagellum; k, kinetoplast; m,
mitochondrion; n, nucleus. The scale is the same for all of the micrographs (bar = 500 nm).

L. Hu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 247–251
251
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cent shape ones which complement the DNA minor
groove such as furamidine (DTm = 25 ꢁC). In general,
the compounds which exhibit the higher DTm values
show the higher antileishmanial activity and the weaker
binding compounds show low activity. Interestingly,
when the DTm values are plotted versus the L. donovani
IC50 values a rough correlation is observed (Fig. 2). For
example, all compounds with DTm values above 15 ꢁC
have IC50 values lower than 1 lM while all except one
compound with DTm values below 15 have IC50 values
above 1 lM. The compound with the highest DTm has
an activity below 0.10 lM, while the compound with
the lowest DTm has an activity of greater than
100 lM. These results are consistent with kinetoplast
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In an attempt to gain insight into the antileishmanial
mechanism of action of these linear dications, L. dono-
vani axenic amastigotes were incubated in the presence
or absence of 0.68 lM 4a (2.5· the IC50 value) for
24 h at 37 ꢁC, fixed, stained, and examined by transmis-
sion electron microscopy (TEM) by methods described
previously.19 In contrast to controls (Fig. 3A), the
majority of parasites exposed to 4a displayed a dramatic
dilation of the mitochondrion (m) and evidence of a dis-
integrating kinetoplast (k) as shown in Figure 3B and C.
These observations are consistent with the ultrastructur-
al changes observed in Leishmania amazonensis prom-
astigotes exposed to pentamidine.10
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The azaterphenyl diamidines were found to have potent
activity against L. d. axenic amastigotes. A marked
dependence on location of the ring N-atom(s) relative
to the amidine groups was noted. A general correlation
between DTm and IC50 values was observed. TEM stud-
ies showed a dramatic dilation of the mitochondrion
and evidence of disintegration of the kinetoplast of the
amastigotes which is consistent with targeting DNA.
Unfortunately, both 4a and 4b were not effective in a
Leishmania infected macrophage assay suggesting that
these diamidines are not delivered well to the parasites
within the macrophage host cell. Further studies focus-
ing on macrophage delivery are necessary to take advan-
tage of the potent intrinsic antileishmanial activity of
these azaterphenyl diamidines.
16. Brendle, J. J.; Outlaw, A.; Kumar, A.; Boykin, D. W.;
Patrick, D. A.; Tidwell, R. R.; Werbovetz, K. A.
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Acknowledgment
18. Stephens, C. E.; Brun, R.; Salem, M.; Werbovetz, K. A.;
Tanious, F.; Wilson, W. D.; Boykin, D. W. Bioorg. Med.
Chem. Lett. 2003, 13, 2065.
This work was supported by an award from the Bill and
Melinda Gates Foundation.
´
19. Delfın, D. A.; Bhattacharjee, A. K.; Yakovich, A. J.;
Werbovetz, K. A. J. Med. Chem. 2006, 49, 4196.
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Buendia, A.; Kaiser, M.; Brun, R.; Ruiz-Perez, L. M.;
Johansson, N. G.; Gonzalez-Pacanowska, D.; Gilbert, I.
H. J. Med. Chem. 2005, 48, 5942.
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