Synthesis and pharmacological evaluation of mono-arylimidamides as antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2016.03.082

Arylimidamide (AIA) compounds containing two pyridylimidamide terminal groups (bis-AIAs) possess outstanding in vitro antileishmanial activity, and the frontrunner bis-AIA DB766 (2,5-bis[2-(2-isopropoxy)-4-(2-pyridylimino)aminophenyl]furan) is active in visceral leishmaniasis models when given orally. Eighteen compounds containing a single pyridylimidamide terminal group (mono-AIAs) were synthesized and evaluated for their antileishmanial potential. Six of these compounds exhibited sub-micromolar potency against both intracellular Leishmania donovani and Leishmania amazonensis amastigotes, and three of these compounds also displayed selectivity indexes of 25 or greater for the parasites compared to a J774 macrophage cell line. When given orally at a dose of 100 mg/kg/day for five days, compound 1b (N-(3-isopropoxy-4-(5-phenylfuran-2-yl)phenyl)picolinimidamide methanesulfonate) reduced liver parasitemia by 46% in L. donovani-infected mice. Mono-AIAs are thus a new class of candidate molecules for antileishmanial drug development.

Full text of DOI:10.1016/j.bmcl.2016.03.082

Accepted Manuscript
Synthesis and Pharmacological Evaluation of Mono-arylimidamides as Anti-
leishmanial Agents
Xiaohua Zhu, Abdelbasset A. Farahat, Meena Mattamana, April Joice, Trupti
Pandharkar, Elizabeth Holt, Moloy Banerjee, Jamie L. Gragg, Laixing Hu,
Arvind Kumar, Sihyung Yang, Michael Zhuo Wang, David W. Boykin, Karl A.
Werbovetz
PII:
S0960-894X(16)30314-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.082
BMCL 23725
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 February 2016
22 March 2016
24 March 2016
Please cite this article as: Zhu, X., Farahat, A.A., Mattamana, M., Joice, A., Pandharkar, T., Holt, E., Banerjee, M.,
Gragg, J.L., Hu, L., Kumar, A., Yang, S., Wang, M.Z., Boykin, D.W., Werbovetz, K.A., Synthesis and
Pharmacological Evaluation of Mono-arylimidamides as Antileishmanial Agents, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.082
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Synthesis and Pharmacological Evaluation of Mono-arylimidamides as Antileishmanial
Agents
Xiaohua Zhua, Abdelbasset A. Farahatb,c, Meena Mattamanaa, April Joicea, Trupti Pandharkara,
Elizabeth Holtb, Moloy Banerjeeb , Jamie L. Graggb, Laixing Hub,d, Arvind Kumarb, Sihyung
Yange, Michael Zhuo Wange, David W. Boykinb, and Karl A. Werbovetza,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, OH 43210, USA
bDepartment of Chemistry, Georgia State University, Atlanta, GA 30302, USA
cDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
dInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
eDepartment of Pharmaceutical Chemistry, School of Pharmacy, University of Kansas,
Lawrence, KS 66047
*Corresponding author. Tel.: 1-614-292-5499; fax: 1-614-292-2435.
Email address: werbovetz.1@osu.edu (K. A. Werbovetz)
Keywords:
Leishmaniasis
Arylimidamide
DB766
Drug discovery

Abstract
Arylimidamide (AIA) compounds containing two pyridylimidamide terminal groups (bis-AIAs)
possess outstanding in vitro antileishmanial activity, and the frontrunner bis-AIA DB766 (2,5-
bis[2-(2-isopropoxy)-4-(2-pyridylimino)aminophenyl]furan) is active in visceral leishmaniasis
models when given orally. Eighteen compounds containing a single pyridylimidamide terminal
group (mono-AIAs) were synthesized and evaluated for their antileishmanial potential. Six of
these compounds exhibited sub-micromolar potency against both intracellular Leishmania
donovani and Leishmania amazonensis amastigotes, and three of these compounds also
displayed selectivity indexes of 25 or greater for the parasites compared to a J774 macrophage
cell line. When given orally at a dose of 100 mg/kg/day for five days, compound 1b (N-(3-
isopropoxy-4-(5-phenylfuran-2-yl)phenyl)picolinimidamide methanesulfonate) reduced liver
parasitemia by 46% in L. donovani-infected mice. Mono-AIAs are thus a new class of candidate
molecules for antileishmanial drug development.

With well over one million new cases and 20,000-40,000 deaths annually,1 leishmaniasis is the
second most prevalent infectious disease transmitted by a vector in terms of morbidity and
mortality.2 Leishmaniasis is a spectrum of disease caused by infection with approximately
twenty species of Leishmania protozoa. Among the major clinical forms, visceral leishmaniasis
(VL) is the most severe and is fatal if left untreated, while cutaneous leishmaniasis (CL) is
typically self-healing. Nevertheless, drugs are frequently used to treat CL to limit scarring from
cutaneous lesions, to speed the time to cure, and to prevent the development of the potentially
disfiguring mucocutaneous leishmaniasis manifestation. The current drugs against leishmaniasis
include pentavalent antimonials, amphotericin B (AmB), miltefosine and paromomycin, but they
all suffer from one or more weaknesses, such as parasite resistance, toxicity, high cost, and an
inconvenient route of administration.2 New oral antileishmanial drugs are thus needed to
overcome these issues and provide better treatment options against Leishmania infections.
We have demonstrated that bis-arylimidamides (bis-AIAs), a series of compounds containing
two pyridylimidamide terminal groups, possess excellent in vitro and promising in vivo
antileishmanial activity. In the AIAs, the imino group is bound to an anilino nitrogen atom,
lowering the pKa of the amidine and increasing the lipophilicity of the molecule compared to a
dicationic diamidine compound such as pentamidine.3 DB766 and DB1960, the hydrochloride
and mesylate salts of 2,5-bis[2-(2-isopropoxy)-4-(2-pyridylimino)aminophenyl]furan,
respectively, display IC50 values similar to AmB against both intracellular L. donovani and
intracellular L. amazonenesis and provide dose-dependent reduction of liver parasitemia in a
mouse model of visceral leishmaniasis when administered orally.3, 4 Unfortunately, DB766 and
DB1960 lack the therapeutic window required for advancement as clinical monotherapy
candidates against visceral leishmaniasis.4
As part of a broader study, we previously reported two terphenyl AIAs containing a single
pyridylimidamide group (mono-AIAs) that displayed promising activity against intracellular L.
amazonensis.5 We hypothesize that lower molecular weight mono-AIAs may have improved
pharmacokinetic properties and could display decreased toxicity to animals compared to bis-
AIAs. The synthesis and evaluation of such a group of molecules is reported here to test this
hypothesis.
Most of the mono-AIAs investigated were synthesized as outlined in Scheme 1 using
methodology similar to that employed previously for making bis-AIAs.6-8 The first step involved
Stille coupling between tributyl(furan-2-yl)stannane and various substituted 4-
bromonitrobenzenes. Bromination of the 2-(4-nitrophenyl)furan analogues with NBS at room
temperature yielded the corresponding 5-bromo-2-(4-nitrophenyl)furans. Subsequent reaction of
the 5-bromofurans under either Stille or Suzuki conditions provided the 2-(4-nitrophenyl)-5-
arylfuran intermediates. Catalytic hydrogenation of the nitro group of the diarylfurans gave the
corresponding amino analogues. Finally, reaction of the amino diarylfurans with either 2-pyridyl
or 2-pyrimidyl thioimidate analogues produced, after salt formation, the mono-AIAs 1a-p. Most
target compounds were prepared as the more soluble mesylate salt.4 Hydrochloride salts were
made for 1a and 1d because of the hygroscopic nature of the corresponding mesylates.

The synthesis of triazole 2 employed the Huisgen cycloaddition process under typical “Click
chemistry” conditions to yield the key nitro intermediate 4-(2-isopropoxy-4-nitrophenyl)-1-(4-
methoxyphenyl)-1H-1,2,3-triazole as shown in Scheme 2.9 The subsequent steps are similar to
those of Scheme 1 but the reduction of the nitro group to the amine in this case was achieved
using Raney-nickel and hydrazine.10
The synthesis of the oxazole 3 is presented in Scheme 3 and employed (p-toluenesulfonyl)methyl
isocyanide chemistry for the formation of 5-(2-isopropoxy-4-nitrophenyl)oxazole.11 The latter
compound was converted into 2-phenyl-5-(2-isopropoxy-4-nitrophenyl)oxazole by Pd/Cu
catalyzed arylation with iodobenzene.12, 13 The conversion of the nitro analogue into the mono-
AIA 3 followed methodology previously described in Scheme 1.

Methods employed for the evaluation of the efficacy of mono-AIA target compounds against
intracellular L. amazonensis14 and intracellular L. donovani15 and for toxicity to murine J774
macrophages16 have been described previously. In these assays, the reference compound
amphotericin B displayed IC50 values of 40 ± 3 nM (mean ± standard error, n = 15) and 120 ± 10
nM (mean ± standard error, n = 26) against L. donovani and L. amazonensis, respectively, while
the cytotoxicity standard podophyllotoxin showed an IC50 value of 24 ± 5 nM (mean ± standard
error, n = 15) against J774 macrophages. For ease in illustrating the antileishmanial SAR of this
series of compounds, the central phenyl ring, the linker, and the terminal phenyl ring are referred
to as rings A, B, and C, respectively (Figure 1).
For derivatives bearing substitutions at the 5 position of the furan “B” ring (at the position
occupied by the C ring in Figure 1), we observed that the most active compound was 1b, where
the C ring is a phenyl group. Compound 1b displayed IC50 values of 310 and 130 nM against
intracellular L. donovani and intracellular L. amazonensis, respectively (Table 1). Activity was
maintained when the phenyl ring was replaced with a furan ring (1d). The antileishmanial
potency decreased by 4-20 fold when this position was substituted with other groups, however,
such as hydrogen (1a), 5-pyrimidyl (1c), 3-pyridyl (1e), or 4-pyridyl (1f).

Table 1. In vitro activity of compounds (1a–f) against intracellular Leishmania amastigotes and
murine J774 macrophages.
Compound
R
IC50 (nM)a
Intracellular L.
Intracellular L.
J774 macrophages
amazonensis
donovani
H
1a
1b
2,100 ± 200
130 ± 30
2,400 ± 300
310 ± 10
22,000 ± 4,000
13,000 ± 1,000
1c
1,100 ± 300
250 ± 10
2,100 ± 400
340 ± 50
>20,000
1d
1e
8,500 ± 800
900 ± 230b
1,200 ± 100
>50,000
1f
2,700 ± 200b
2,600 ± 100
22,000 ± 1,000
aThe values are means ± standard errors of at least three independent experiments.
bThe values are the means ± range of two independent experiments.
The addition of a methoxy group or a fluorine atom at position R2 or a methoxy group at position
R1 of phenyl ring C led to decreased antileishmanial potency in both the intracellular L.
amazonensis and L. donovani assays (Table 2). In the series of compounds bearing different
substitutions on phenyl ring “A”, the cyclopentyloxy substituted derivative 1k was the most
active compound, with IC50 values comparable to that of 1b and amphotericin B (Table 3). As
the size of the alkoxy substituent increased from methoxy (1m) to ethoxy (1l) to isopropoxy (1b)
and cyclopentyloxy (1k), antileishmanial activity increased, but switching the position of the

isopropoxy substitution on the “A” ring (1j) resulted in a loss of antiparasitic activity.
Replacement of furan as the “B” ring with other heterocycles (thiophene 1o, triazole 2, or
oxazole 3) lowered the antileishmanial activity, with 3 being intermediate in potency between the
highly active 1b and the moderately active 2 and 1o (Table 4). Replacement of the 2-pyridyl
terminal group with a 2-pyrimidyl group (1p) resulted in a reduction in potency against L.
amazonensis but not against L. donovani (Table 4). In the host cell counterscreen, the mono-
AIAs exhibited IC50 values ranging from 5,300 to >50,000 nM against murine J774
macrophages, resulting in selectivity indexes (IC50 vs J774 macrophages/IC50 vs Leishmania) of
2.4-100 against intracellular L. amazonensis and 3.1-76 against intracellular L. donovani in vitro.
Table 2. In vitro activity of compounds 1b, 1g–i against intracellular Leishmania amastigotes
and murine J774 macrophages
Compound
R1
R2
IC50 (nM)a
Intracellular L.
Intracellular L.
J774 macrophages
amazonensis
donovani
H
H
H
OCH3
F
130 ± 30
820 ± 310
920 ± 190
1,400 ± 200
310 ± 10
900 ± 150
1,100 ± 100
1,300 ± 100
13,000 ± 1,000
6,700 ± 900
1b
1g
1h
1i
H
8,000 ± 100
OCH3
H
5,300 ± 1,000
aThe values are means ± standard errors of at least three independent experiments.

Table 3. In vitro activity of compounds 1b, 1j–n against intracellular Leishmania amastigotes
and murine J774 macrophages
Compound
R1
R2
IC50 (nM)a
Intracellular L. Intracellular L. J774 macrophages
amazonensis
130 ± 30
donovani
310 ± 10
OCH(CH3)2
H
H
13,000 ± 1,000
13,000 ± 1,000
13,000 ± 3,000
6,600 ± 600
1b
OCH(CH3)2
1,600 ± 300
140 ± 10
1,400 ± 300
170 ± 40
1j
OCH(CH2)4
OCH2CH3
OCH3
H
H
H
H
1k
1l
980 ± 70b
850 ± 170
1,400 ± 300
6,100 ± 1,400
3,700 ± 1,300b
>10,000b
9,000 ± 2,000
>20,000
1m
1n
CF3
aThe values are means ± standard errors of at least three independent experiments.
bThe values are the means ± range of two independent experiments.

Table 4. In vitro activities of compounds 1b, 1o, 1p, 2, and 3 against intracellular Leishmania
amastigotes and murine J774 macrophages.
Compound
X
R1
R2
IC50 (nM)a
Intracellular L. Intracellular
J774
amazonensis
L. donovani
macrophages
2-pyridyl
2-pyridyl
2-pyrimidyl
2-pyridyl
2-pyridyl
H
H
1b
130 ± 30
310 ± 10
13,000 ± 1,000
1o
1p
2
2,300 ± 300b
650 ± 160
2,200 ± 500
440 ± 60
6,900 ± 400
H
12,000 ± 1,000
OCH3
H
2,900 ± 100
590 ± 60
1,600 ± 200
1,000 ± 0
16,000 ± 3,000
24,000 ± 1,000
3
aThe values are means ± standard errors of at least three independent experiments.
bThe value is the mean ± range of two independent experiments.
Among these 18 mono-AIAs, 1b, 1d, and 1k displayed outstanding antileishmanial potency and
good selectivity for intracellular Leishmania compared to J774 murine macrophages (selectivity
indexes ≥ 25), warranting the in vivo evaluation of these derivatives. Compounds 1b, 1d, and 1k
were dissolved in water and administered to healthy BALB/c mice for assessment of their in vivo
toxicity. Each of these compounds was well tolerated when administered by the i.p. route at 30
mg/kg/day for 5 days and were thus evaluated at this dose in a murine model of visceral
leishmaniasis.4 Animals were infected with L. donovani LV82 promastigotes and then treated
with different compounds one week post infection for five consecutive days. These mice were
euthanized two weeks post infection and liver smear slides were prepared for the microscopic
determination of parasitemia. When given at the dose listed above, administration of 1b, 1d, and
1k resulted in 37%, 13%, and 20% suppression of liver parasitemia, respectively, compared to
untreated control groups (Fig. 2A, B). As the most effective of these three compounds when

given i.p., the oral efficacy of 1b was also evaluated in the murine visceral leishmaniasis model.
Compound 1b reduced liver parasitemia by 46% at an oral dose of 100 mg/kg/day for five days
compared to the control group (Fig. 2C). The in vivo antileishmanial efficacy of 1b is thus
similar to that of DB1960 and lower than that of DB766 when the compounds are administered
orally at 100 mg/kg in five daily doses.3, 4 When given to infected mice at a dose of 10
mg/kg/day for five days by either the i.p. or oral route, miltefosine administration resulted in
>90% inhibition of liver parasitemia, consistent with our previous observations.4, 15
A
C
*
B
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
i.p.
*
p.o.
i.p.
99
98
96
*
*
#
*
46
37
37
20
13
Milt
10 mg/kg
1d
30 mg/kg
Miltefosine
10 mg/kg ip
1k
30 mg/kg
1b
Milt
10 mg/kg
1b
100 mg/kg
1b
50 mg/kg
30 mg/kg ip
Figure 2. In vivo efficacy of mono-AIAs in the murine visceral leishmaniasis model. Compounds were
administered i.p. (A and B) or p.o. (C) once daily for 5 days to infected mice. Results are presented as the
percentage inhibition of liver parasitemia. Bars and error bars show the mean and standard deviation,
respectively (n = 4) *: p < 0.01, compared to untreated control, #: p < 0.05, compared to untreated control.
To confirm the exposure and accumulation of 1b in target organs after oral administration,
plasma and tissue concentrations of this compound were determined at 1, 2 and 24 h after a
single oral dose at 100 μmol/kg (or 40 mg/kg) in mice. Compound 1b accumulated in the target
organs such as liver and spleen at higher concentrations (C1h = 12.6 and 4.7 µM, respectively)
than in the plasma (C1h = 1.9 µM) (Figure 3). At 24 h post dose, 1b concentrations decreased
substantially to 0.14 µM in the plasma and 0.6-0.7 µM in the liver and spleen.

Figure 3. Concentration of 1b in liver ( ), spleen ( ), kidney ( ), and plasma ( ) after oral
administration at a dose of 100 μmol/kg (or 40 mg/kg) to mice. Bars and error bars represent
means and standard errors of triplicate determinations, respectively.
Our efforts to explore the mechanism of antileishmanial action of the bis-AIAs led to the
observation that DB766 is synergistic with posaconazole against Leishmania donovani in vitro;17
other investigators reported in vitro antileishmanial synergy between posaconazole and the
squalene synthase inhibitor E5700.18 A combination study with 1b and posaconazole was thus
performed against intracellular L. amazonensis amastigotes. While azoles have been used to treat
CL,19, 20 some of these compounds exhibit low potency against certain Leishmania species in
vitro.21, 22 We found this to be true for posaconazole against intracellular L. amazonensis under
the conditions of our assay (IC50 > 10 µM; posaconazole exhibits low solubility at higher
concentrations), making standard isobologram analysis impossible in this case. Instead, the
method described by Peters et al.23 was employed to determine whether posaconazole enhances
the potency of 1b. Fig. 4 demonstrates that this azole augmented the potency of the mono AIA
1b against intracellular L. amazonensis, with 1b displaying an IC50 value that is fivefold lower in
the presence of 8 µM posaconazole compared to the IC50 of this AIA in the absence of the azole.
IC50 values for 1b (DB2002) alone in this experiment and in the presence of 8 µM posaconazole
are 0.26 ± 0.05 and 0.055 ± 0.015 µM, respectively. We are aware that de Macedo-Silva et al.
reported an IC50 value of 1.6 µM for posaconazole against intracellular L. amazonensis.24
Although both assays involved incubating infected murine peritoneal macrophages with
posaconazole for 72 hr, de Macedo-Silva exposed peritoneal macrophages to L. amazonensis for
a shorter time compared to our studies prior to removing extracellular parasites (2 hr vs.
overnight). Our studies with intracellular L. donovani have indicated that the level of
macrophage infection influences the apparent in vitro antileishmanial activity of azoles (A. Joice,
unpublished data). Thus, a likely explanation for the discrepancy between IC50 values for
posaconazole against intracellular L. amazonensis in the two studies is the differences in assay
conditions employed.

Figure 4. In vitro interactions between 1b and posaconazole against intracellular L.
amazonensis. Results are presented as the IC50 of DB2002 alone and in different combinations.
Bars and error bars show the mean and standard deviation of three independent experiments.
In summary, eighteen mono-AIAs were synthesized and evaluated for their antileishmanial
activities. Compounds 1b, 1d and 1k displayed the highest potency against intracellular L.
amazonensis and intracellular L. donovani amastigotes with their IC50 values slightly higher than
those of amphotericin B and DB766. These three mono-AIAs were well tolerated in healthy
mice, indicating that mono-AIAs may be generally less toxic than bis-AIAs.8, 25 Compound 1b
shows moderate in vivo antileishmanial efficacy in a mouse model of visceral leishmaniasis.
Furthermore, posaconazole enhances the activity of this compound against intracellular L.
amazonensis amastigotes, consistent with our observations with the bis-AIA DB766.17 Mono-
AIAs are thus promising antileishmanial leads, and further optimization of this class of
compounds to improve their in vivo efficacy is needed. Given the in vitro activity of the
compound 1b/posaconazole combination, combining a mono AIA with an azole drug could be a
fruitful strategy for the development of antileishmanial therapies.
Acknowledgements
This work was supported in part by the Bill and Melinda Gates Foundation through the
Consortium for Parasitic Drug Development and by the Office of the Assistant Secretary of
Defense for Health Affairs under the Peer Reviewed Medical Research Program through Award
No. W81XWH-14-2-0017.
Supplementary data
Complete characterizations of target compounds 1b, 1d, 1k, 2, and 3 can be found, in the online
version, at …(to be added upon publication).

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Synthesis and Pharmacological Evaluation of Mono-arylimidamides as Antileishmanial
Agents
Xiaohua Zhu, Abdelbasset A. Farahat, Meena Mattamana, April Joice, Trupti Pandharkar,
Elizabeth Holt, Moloy Banerjee, Jamie L. Gragg, Laixing Hu, Arvind Kumar, Sihyung Yang,
Michael Zhuo Wang, David W. Boykin, and Karl A. Werbovetz*