Novel Catechol Derivatives of Arylimidamides as Antileishmanial Agents

Article abstract of DOI:10.1002/cbdv.201800228

Two novel bis-arylimidamide derivatives with terminal catechol moieties (9a and 10a) and two parent compounds with terminal phenyl groups (DB613 and DB884) were synthesized as dihydrobromide salts (9b and 10b). The designed compounds were hybrid molecules

Full text of DOI:10.1002/cbdv.201800228

Article Type: Full Paper
a
a
b
,a
,c
Foroogh Rezaei, Lotfollah Saghaei, Razieh Sabet, Afshin Fassihi,* Gholamreza Hatam*
a
Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan,
Iran
E-mail: fassihi@pharm.mui.ac.ir (A. Fassihi)
b
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran
c
Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
E-mail: hatamghr@sums.ac.ir (G. Hatam)
Two novel bis-arylimidamide derivatives with terminal catechol moieties (9a and 10a) and two
parent compounds with terminal phenyl groups (DB613 and DB884) were synthesized as
dihydrobromide salts (9b and 10b). The designed compounds were hybrid molecules consisting of a
catechol functionality embedded in an arylimidamide moiety. All compounds were examined for in
vitro antiparasitic activity upon promastigotes of Leishmania major and L. infantum as well as axenic
amastigotes of L. major. It was shown that conversion of terminal phenyl groups into catechol
moieties resulted in more than 10-fold improvement in potency, coupled with lower cytotoxicity
against fibroblast cells, compared to the corresponding parent compounds. The furan-containing
analog 9a exhibited the highest activity with submicromolar IC values, ranging from 0.29 to 0.36
50
µM, which is comparable in efficacy to the reference drug amphotericin B (IC 0.28-0.33 µM). The
50
results justify further study of this class of compounds. It seems that the combination of catechol
chelating groups with potent antiparasitic agents could improve the efficacy by presenting novel
hybrid compounds.
Keywords: Bis-arylimidamides; Catechol; Antileishmanial agents, Hybrid compounds
1
. Introduction
[
1]
According to the World Health Organization (WHO), leishmaniases which are a group of diseases caused by protozoan parasites from
more than 20 Leishmania species, have affected 12 million people worldwide. It is also estimated that 700,000–1,000,000 new cases and
2
0,000-30,000 deaths have been attributed annually to this neglected tropical disease. The bite of infected female phlebotomine sandflies
transmits each of the three main forms of leishmaniasis. These forms are visceral (VL), also known as kala-azar which is the most serious
form of the disease, cutaneous (CL), the most common form, and mucocutaneous leishmaniasis (MCL).
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201800228
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[
2]
Since no effective vaccine has been approved yet, and the medications used for leishmaniasis
have different limitations such as drug resistance and toxicity, there is an urgent need to develop
[
3, 4]
new efficient drugs.
The diamidine pentamidine, as a first-line drug for CL and as a second-line
[
5, 6]
agent for VL,
is one of the most widely used antiparasitic agents to treat antimony-resistant
Aromatic diamidines have also exhibited promising antiprotozoal activity. In the
[
7, 8]
leishmaniasis.
structure of these scaffolds, the alkyl chain between the two benzene rings bearing the amidine
moieties in pentamidine is replaced with a five membered-ring heterocycle, furan in one of the
[
9]
strongest analogs (DB75). The suggested mechanism of antiparasitic action for this class of
[
10]
compounds is binding to the kinetoplast DNA (kDNA) minor groove. In another modification in the
way of attachment of the amidine moieties to the aromatic ring, reverse amidines, later called
arylimidamides (AIAs), were introduced. The mode of action of members of this scaffold, such as
[
11]
[12]
DB613 and DB884 , which exhibited improved potency against several parasites seems to be
[
13, 14]
more complex and requires more investigations.
compounds are illustrated in Figure 1.
The structures of all above-mentioned
Studies on the antileishmanial activity of a variety of natural products have proven some of
[
15-19]
them as appreciable antileishmanial agents.
Proposed mechanisms for these compounds
include inhibition of kDNA synthesis, induction of topoisomerase-II mediated cleavage of kDNA
[
16]
minicircles by luteolin and quercetin, contribution of quercetin as a chelating agent to interfere
[
20-22]
with metal-mediated proliferation of the parasites in leishmanial infection
and production of
[
23]
reactive oxygen species which leads to the leishmania parasite death. A notable structural feature
of many of the natural products is the presence of chelating catechol groups. The role of these
groups in antileishmanial effects has been partly investigated.
Figure 1. Chemical structures of pentamidine, DB75 and two arylimidamides
Given the essentiality of kinetoplast DNA and metal-mediated metabolism pathways in the
proliferation of leishmania, and the highly probable interference of aromatic diamidines and
catechol groups with the above-mentioned pathways, we decided to prepare novel catechol
derivatives of AIAs (9a and 10a). The designed compounds could be regarded as hybrid molecules
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consisting of a catechol functionality which is embedded in an arylimidamide moiety. Thus, they are
supposed to chelate metal cations such as iron which are essential for the parasite and also inhibit
its growth by the mechanisms proposed for AIAs. The structures of these compounds are provided in
Figure 2. For comparison purposes, the dihydrobromide salt of the previously reported
arylimidamides DB613 and DB884, with a general structure close to the designed compounds were
also prepared as 9b and 10b, respectively. The prepared compounds were evaluated for their in vitro
antileishmanial activity against L. major and L. infantum promastigotes, and also L. major axenic
amatigotes.
Figure 2. Chemical structures of the designed compounds
2
. Results and Discussion
2
.1. Chemistry
The desired bis-arylimidamides 9a, 9b, 10a and 10b, were prepared from their corresponding
diamino intermediates 3 and 6 and S-(2-naphthylmethyl)-2-thioimidate
hydrobromides 8a and 8b. The diamino compound 3, with a furan ring in its structure, was obtained
[
11]
in two steps according to the method described previously. This method starts with a Stille-
coupling reaction between 1-bromo-4-nitrobenzene and 2,5-bis(tributylstannyl)furan (1) to furnish
the corresponding 2,5-bis(4-nitrophenyl)furan (2). Subsequent reduction of the dinitro derivative 2
by catalytic hydrogenation produced the expected diamino compound 3 in 90% yield (Scheme 1). For
the preparation of the diamino compound 6, bearing a pyrrole ring in the structure, Paal-Knorr
[
24]
synthesis was applied. The precursor 1,4-diketone was prepared using the aldol condensation of
2
-bromo-4'-nitroacetophenone with 4'-nitroacetophenone in the presence of zinc chloride-
[
25]
diethylamine-t-butanol complex as the condensation agent.
The resulted diketone then yielded
the 2,5-bis(4-nitrophenyl)pyrrole (5) in the Paal-Knorr reaction. The nitro groups in compound 5
were reduced to amino moieties using catalytic hydrogenation in the presence of Pd/C to give the
desired 2,5-bis(4-aminophenyl)pyrrole (6) in 81% yield (Scheme 2).
3 4 2
Scheme 1. Reagents and conditions: a. Pd(PPh ) , reflux in 1,4-dioxane; b. Pd/C (10%), H , EtOH and EtOAc
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2 4 2
Scheme 2. Reagents and conditions: a. ZnCl , benzene, t-BuOH, diethylamine, room temperature; b. reflux in NH OAc, EtOH and HOAc; c. Pd/C (10%), H , EtOH
and EtOAc
As shown in Scheme 3, the thioimidate reagent 8a was prepared by two successive steps. The initial step was the conversion of
[
26]
3
,4-dihydroxybenzonitrile to the corresponding aryl thioamide 7 in the presence of sodium hydrosulfide hydrate and sulfuric acid. The
next reaction for the preparation of 8a was the reaction between 3,4-dihydroxybenzothioamide (7) and 2-(bromomethyl)naphthalene in
CHCl which furnished the desired thioimidate reagent 8a. S-(2-Naphthylmethyl)thiobenzimidate hydrobromide (8b) was prepared from
commercially available thiobenzamide reacting with 2-(bromomethyl)naphthalene in refluxing CHCl
3
[
11]
3
according to literature procedure.
between the diamino compounds 3/6 and 8a/8b resulted in the desired compounds 9a, 9b,
[
11, 27]
A one-pot addition/elimination reaction
0a and 10b in 62-89% yields (Scheme 4).
1
2 2 4 3
Scheme 3. Reagents and conditions:a. NaSH-xH O, H SO , triethylene glycol; b. 2-(bromomethyl)naphthalene, reflux in CHCl
Scheme 4. Reagents and conditions: MeCN, EtOH, room temperature
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2
.2. Biological evaluation
2
.2.1. In vitro antileishmanial activities
The synthesized compounds 9a, b and 10a, b were tested for their ability to inhibit growth of L.
major and L. infantum promastigotes and L. major axenic amastigotes. Amphotericin B was used as
the reference drug, and the compounds concentrations causing 50% reduction in parasite viability
(
IC ) are presented in Table 1. As can be seen in the table, the two new catechol derivatives 9a and
50
1
0a exhibited much more potent antileishmanial activity than their corresponding furan- and
pyrrole-based analogs 9b and 10b, respectively. Of interest, the compound 9a showed promising
activity in all assays with submicromolar IC values (ranging from 0.29 to 0.36 µM), substantially
50
possessing similar potency to amphotericin B (with IC values from 0.28 to 0.33 µM). The pyrrole-
50
containing catecholic analog 10a also showed good activity with IC values ranging from 4.54 to 7.45
50
µM which demonstrated more than 10-fold increase in inhibitory potency when compared to the
non-catecholic parent compound 10b (with IC values ranging from 64.44 to 81.46 µM). Notably,
50
based on the comparison of parent compounds 9b and 10b, the pyrrole analog 10b showed a
dramatic reduction in antileishmanial activity. It should be noticed that furan analogs found to be
more effective than pyrrole analogs of the same scaffold. However, the IC values of the pyrrole-
50
containing catecholic analog 10a and the furan-based non-catecholic analog 9b showed similar levels
of antileishmanial activities (IC values in the ranges of 4.54-7.45 µM and 4.85-5.56 µM for 10a and
50
9
b, respectively).
2
.2.2. Cytotoxicity
Cytotoxicity on human fibroblast cell line was evaluated for the synthetic compounds 9a, b and 10a,
b (Table 1). The cytotoxic index (CI) was calculated as the ratio of cytotoxicity (TD50 value on
fibroblast cells) to activity (IC value on leishmania promastigotes and axenic amastigotes). This in
50
vitro therapeutic index showed that the furan-based catechol derivative 9a possessed the best
selectivity profile (CI of 33.03, 41.00 and 37.16 for L. infantum promastigotes, L. major
promastigotes and axenic amastigotes, respectively). The least active compound 10b, the pyrrole-
based non-catecholic analog, exhibited the lowest CI (0.06 and 0.05). In general, the catechol
derivatives 9a and 10a, demonstrated higher selective potency in vitro against leishmania cells than
the non-catechol ones 9b and 10b, respectively, as evidenced by their cytotoxic indices.
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Table 1. In vitro antileishmanial activities of bis-arylimidamide analogs
L. infantum promastigotes
L. major
Cell toxicity
Compound
promastigotes
axenic amastigotes
a
TD50 (µM)
Cytotoxic index
Cytotoxic index
TD50/IC50
Cytotoxic index
TD50/IC50
a
a
a
IC50 (µM)
IC50 (µM)
IC50 (µM)
TD50/IC50
9
a
b
11.89 (±0.62)
4.76 (±0.31)
11.35 (±0.49)
3.97 (±0.26)
0.36 (±0.05)
5.23 (±0.35)
5.72 (±0.24)
68.17 (±2.16)
0.28 (±0.03)
33.03
0.91
1.98
0.06
-
0.29 (±0.02)
4.85 (±0.25)
4.54 (±0.19)
64.44 (±3.81)
0.33 (±0.02)
41
0.98
2.50
0.06
-
0.32 (±0.04)
5.56 (±0.29)
7.45 (±0.38)
81.46 (±3.67)
0.30 (±0.06)
37.16
0.86
1.52
0.05
-
9
1
0a
0b
1
b
c
Amphotericin B
NT
a
b
c
The values are means ± SD of three independent experiments.
.
Amphotericin B was used as antileishmanial reference
NT = Not Tested
3
. Conclusions
In the present report, two novel arylimidamides 9a and 10a bearing terminal catechol moieties have been synthesized and tested in vitro
against L. major and L. infantum promastigotes along with L. major axenic amastigotes. Also, the dihydrobromide salts of the two
previously reported AIAs, DB613 and DB884 with terminal unsubstituted phenyl groups were prepared as lead compounds 9b and 10b,
respectively. Amphotericin B was used as the reference antileishmanial drug in biological evaluations. The IC50 values represented a
significant improvement in antileishmanial activity of the structures bearing terminal catechol moieties over the non-catecholic
derivatives. The catchol derivatives also exhibited higher therapeutic index than the non-catecholic ones as they were tested for toxicity
against human fibroblast cells. Furthermore, it appears that furan analogs resulted in higher efficacy than pyrrole analogs. The furan-based
catecholic analog 9a possessed the highest activity upon both promastigotes and axenic amastigotes with submicromolar IC50 values and
displayed activities as high as amphotericin B. It must be noted that although the pyrrole analog DB884 has demonstrated higher DNA
[
12]
binding affinity than the furan analog DB613 in the previous studies, the pyrrole derivatives displayed lower efficacies than the furan
[
28]
ones in the present study. Accordingly, although the antileishmanial effect of AIAs is likely to be in part influenced by DNA affinity,
further mechanistic studies need to be done to explore additional modes of action.
As it was mentioned previously, AIAs as a promising class of molecules possess broad-spectrum
antiprotozoal activities. In the current study, the in vitro antileishmanial effects of some novel
derivatives of this chemical entity were evaluated. AIAs have shown considerable efficacies against
other protozoan parasites such as Trypanosoma cruzi and Plasmodium falciparum. Furthermore,
chelating agents have long been proven as effective antimalarial agents. Thus, complementary
studies on the designed compounds to evaluate the possible broad range of antiparasitic activity of
them and their in vivo efficacies is suggested. Since the newly synthesized compounds exhibited
appreciable activity against both promastigotes and axenic amastigotes, they are likely to fulfill the
first requirement of an ideal antiparasitic agent that is declared by WHO, namely combating
different forms of a parasite in its life cycle. Finally, the approach of combining the chelating
catechol moiety to arylimidamides appears to result in appreciable antileishmanial effects and this
strategy deserves to be considered for further investigations.
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4
. Experimental Section
4
.1. Chemistry
All of the reagents were purchased from commercial sources and freshly used after being purified by
standard procedures. Melting points were determined on the Electrothermal 9200 Melting Point
1
13
apparatus and were uncorrected. H-NMR and C-NMR spectra were recorded in DMSO-d on
6
Bruker-Ultrashield 300MHz (for final compounds) and 400MHz (for intermediates) spectrometers
(Germany). All chemical shifts are reported as (δ) values (ppm) against tetramethylsilane as an
internal standard. Electrospray mass spectra (ESI-MS) were obtained in negative and positive ion
mode on an AB SCIEX 3200 QTRAP spectrometer. FT-IR (KBr) spectra were recorded on JASCO 6300
(
(
Japan) apparatus. All chemical reactions were monitored by analytical thin layer chromatography
TLC) on pre-coated silica gel 60 F254 aluminum plates (Merck, Germany). The purity of compounds
was determined by thin layer chromatography using several solvent systems of different polarities.
The structures of intermediates were confirmed by comparing their melting points with literature
1
1
13
and H-NMR spectra. Structural elucidation of all final compounds was performed using H-NMR, C-
NMR, Mass and FT-IR spectroscopy.
4
.1.1. 2,5-Bis(4-nitrophenyl)furan (2)
To
a
solution of 4-bromonitrobenzene (2.14 g, 10.6 mmol) and tetrakis-
(
triphenylphospine)palladium(0) (0.21 g) in anhydrous 1,4-dioxane (27 mL) was added 2,5-bis(tri-n-
[
29, 30]
butylstannyl)furan
(3.43 g, 5.3 mmol), and the mixture was heated overnight under nitrogen at
9
5-100 °C. The resulting orange suspension was diluted with hexane (8 mL), cooled down to room
[
11]
temperature, and filtered to give an orange solid (0.87 g, yield: 53%), mp 267-270 °C, lit. mp 269-
2
1
70 °C; H-NMR (DMSO-d ): δ 8.38 (d, J=8.8 Hz, 4H), 8.19 (d, J=8.8 Hz, 4H), 7.60 (s, 2H).
6
4
.1.2. 2,5-Bis(4-aminophenyl)furan (3)
To a suspension of 2 (0.85 g, 2.7 mmol) in EtOAc (20 mL) and absolute EtOH (4 mL) was added Pd/C
10%) (0.12 g) and the mixture was shaken under hydrogen at the atmospheric pressure for 6 h, the
resulting solution was filtered over celite and the filtrate was concentrated in vacuo to dryness to
(
[
11]
give the pure diamino as a pale yellow/tan solid (0.61 g, 90%), mp 217-219 °C, lit. mp 218-221 °C.
4
.1.3. 1,4-Bis(4-nitrophenyl)butane-1,4-dione (4)
Anhydrous ZnCl (1.09 g, 8 mmol) was placed into a one-neck, 25-mL round bottomed flask and dried
2
by melting under reduced pressure (1 torr) at 250-350 °C for 15 min. After cooling to room
temperature under vacuum, dry benzene (4 mL), dry diethylamine (0.44 g, 6 mmol) and t-BuOH
(
0.56 mL, 6 mmol) were successively added. The mixture was stirred till ZnCl was fully dissolved.
2
Then, 4'-nitroacetophenone (0.99 g, 6 mmol) and 2-bromo-4'-nitroacetophenone (0.98 g, 4 mmol)
were successively added. After stirring at room temperature for 4 days, the reaction mixture was
quenched with 5% aq. H SO whereupon a yellow solid was formed. The solid was filtered, washed
2
4
successively with H O and MeOH. The 1,4-diketone was crystallized from THF to give shiny yellow
2
.
[25]
1
crystals (1.1 g, 82%), mp 201-204 °C, lit mp 195-196 °C; H-NMR (DMSO-d ): δ 8.46 (d, J= 8.0 Hz,
6
4
H), 8.34 (d, J = 8.0 Hz, 4H), 3.61 (s, 4H).
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4
.1.4. 2,5-Bis(4-nitrophenyl)pyrrole (5)
A solution of 4 (1.08 g, 3.3 mmol) and ammonium acetate (1.27 g, 16.5 mmol) in glacial acetic acid
5.5 mL) and EtOH (8 mL) was heated under reflux for 5 h. The reaction mixture was cooled down to
(
room temperature, then poured into an ice-water mixture. The precipitated solid was collected by
filtration, washed with water, and crystallized from THF to give dark red crystals (0.95 g, 93%), mp
[
24]
1
2
90-292 °C, lit. mp 295-297 °C; H-NMR (DMSO-d ): δ 11.98 (br s, 1H), 8.34 (d, J = 8 Hz, 4H), 8.15
6
(d, J = 8 Hz, 4H), 7.11 (s, 2H).
4
.1.5. 2,5-Bis(4-aminophenyl)pyrrole (6)
To a solution of 5 (0.93 g, 3 mmol) in absolute EtOH (4.5 mL), and EtOAc (22 mL) was added 10%
palladium on carbon (0.13 g). The mixture was shaken under hydrogen at atmospheric pressure for 6
h at room temperature. The reaction mixture was filtered through a pad of celite, and the solvent
was evaporated to dryness under reduced pressure. A light brown solid was obtained, which was
[
31]
directly used in the next step without further purification (0.61 g), mp 216-218 °C decomposed, lit.
mp 200-201 °C.
4
.1.6. 3,4-Dihydroxybenzothioamide (7)
To a thick-walled 15 mL tube with a resealable Teflon screw-cap was added 2.34 g (17.3 mmol) of
,4-dihydroxbenzonitrile, 20 mL of triethylene glycol, 4.69 g of NaSH.xH O, and 1 mL of concentrated
3
2
H SO . The tube was sealed with the cap, and the mixture was warmed to 110 °C and stirred for 3
2
4
days at this temperature. The reaction was quenched by pouring into 400 mL saturated aqueous
NH Cl, and extracted twice with 200 mL of EtOAc. The combined organic phases were washed three
4
times with 60 mL water, dried with NaSO , and concentrated to give 1.7 g (58% yield) of 7 as a yellow
4
[
26]
[32]
solid, mp 151-153 °C, lit. mp 153-154 °C.
4
.1.7. S-(2-Naphthylmethyl)-3,4-dihydroxybenzothioimidate hydrobromide (8a)
To a stirring solution of 3,4-dihydroxybenzothioamide (10 mmol, 1.69 g) in CHCl (20 mL) was added
3
2
-(bromomethyl)naphthalene (10.5 mmol, 2.33 g). The mixture was heated up to reflux for 5 h,
cooled down to room temperature and placed in an ice bath. The resulting off-white precipitate
(
2.62 g, 67%) was filtered off, dried in vacuo and used directly in the next step, mp 232-234 °C; FT-IR
-
1
(KBr) ѵ (cm ): 3000-3400 (O-H), 3487 (N-H), 3138 (Aromatic C-H), 1655 (C=N), 1604, 1494 (Aromatic
1
C=C), 1305 (-S-CH ). H-NMR (DMSO-d ): δ 11.54 (brs, 2H), 10.79 (s, 1H), 9.90 (s, 1H), 8.12 (s, 1H),
2
6
8
.01 (m, 3H), 7.69 (d, J = 8 Hz, 1H), 7.62 (m, 2H), 7.45 (d, J = 8 Hz, 1H), 7.40 (s, 1H), 7.01 (d, 1H ), 4.93
(s, 2H).
4
.1.8. S-(2-naphthylmethyl)thiobenzimidate hydrobromide (8b)
The same procedure carried out for the preparation of 8a was employed starting with
[
11]
thiobenzamide (10 mmol, 1.37 g) to give a white solid (2.8 g, 78%), mp 211-213 °C, lit. mp 210-
12 °C.
2
4
.1.9. 2,5-Bis[4-(3,4-dihydroxybenzimidoylamino)phenyl]furan dihydrobromide(9a)
To a solution of 2,5-bis(4-aminophenyl)furan (1.2 mmol, 0.3 g) in dry MeCN (6 mL) was added dry
EtOH (17 mL), and the solution was chilled briefly in an ice-water bath. S-(2-naphthylmethyl)-3,4-
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dihydroxybenzothioimidate hydrobromide (0.98 g, 2.52 mmol) was then added, and the mixture was
stirred overnight at room temperature. The resulting solution was concentrated to near dryness,
which was triturated with ether to give a yellow/orange solid of the hydrobromide salt. The solid
-
was washed with dichloromethane and dried in vacuo (1.2 g, 68%) mp 134-136 °C; FT-IR (KBr) ѵ (cm
)
1
1
: 3000-3400 (O-H), 3320 (N-H), 3132 (Aromatic C-H), 1655 (C=N), 1510, 1607 (Aromatic C=C). H-
NMR (DMSO-d ): δ 11.21 (s, 2H), 10.31 (br s, 2H), 9.61 (s, 4H), 8.86 (s, 2H), 7.96 – 8.04 (m, 4H), 7.81
6
(
2
1
d, J = 8.1 Hz,1H), 7.54 (d, J = 8.1 Hz, 4H), 7.27 – 7.34 (m, 4H), 7.17-7.20 (m,1H), 7.03 (d, J = 8.1 Hz,
13
H). C-NMR (DMSO-d ) δ (ppm): 163.19, 152.79, 151.80, 146.03, 134.75, 129.83, 126.38, 125.26,
6
21.81, 119.20, 116.49, 116.10, 109.76. Mass m/z: 519 (M-1).
4
.1.10. 2,5-Bis[4-(benzimidoylamino)phenyl]furan dihydrobromide (9b)
The same procedure used for the synthesis of 9a was exploited starting with diamine 3 and 8b to
[
11]
1
give a yellow solid (79%), mp 232-234 °C, lit. mp (dihydrochloride salt) 242-248 °C. H-NMR
DMSO-d ): δ 7.30 (s, 2H), 7.57 (d, J = 8, 4H), 7.84 (t, 4H), 7.81 (t, 2H), 7.95 (d, J = 8, 4H), 8.06 (d, J = 8,
(
6
4
H), 9.16 (br s, 2H), 9.92 (br s, 2H), 11.51 (br s, 2H).
4
.1.11. 2,5-Bis[4-(3,4-dihydroxybenzimidoylamino)phenyl]pyrrole dihydrobromide (10a)
The same procedure described for 9a was adopted, using diamine 6 (1.2 mmol, 0.3 g) and S-(2-
naphthylmethyl)-3,4-dihydroxybenzothioimidate hydrobromide (0.98 g, 2.52 mmol) as starting
-
1
materials to yield a light brown/orange solid (1.3 g, 62%), mp 132-135 °C; FT-IR (KBr) ѵ (cm ): 3000-
1
3
400 (O-H), 3411, 3313 (N-H), 3163 (Aromatic C-H), 1653 (C=N), 1608, 1508 (Aromatic C=C). H-NMR
(
DMSO-d ): δ (ppm) 11.52 (s, 1H), 11.11 (s, 2H), 10.29 (br s, 2H), 9.55 (s, 4H), 8.79 (s, 2H), 7.92-8.03
6
13
(m, 4H), 7.34-7.47 (m, 8H), 7.03(d, J = 8.1 Hz, 2H), 6.78 (s, 2H). C-NMR (DMSO-d ) δ (ppm): 163.06,
6
1
51.64, 146.02, 133.20, 133.00, 132.41, 126.07, 125.63, 121.69, 119.29, 116.42, 116.09, 109.29.
Mass m/z: 518 (M-1).
4
.1.12. 2,5-Bis[4-(benzimidoylamino)phenyl]pyrrole dihydrobromide (10b)
A similar procedure to that outlined for the preparation of 9a was followed, using diamine 6 and 8b
1
to yield 10b as an orange solid ( 89%), mp 231-234 °C; H-NMR (DMSO-d ): δ 11.53 (s, 1H), 11.43(s,
6
2
7
1
H), 9.86 (s, 2H), 9.09 (s, 2H), 8.04 (d, J = 8 Hz, 4H), 7.95 (d, J= 8 Hz, 4H), 7.81 (t, 2H), 7.70 (t , 4H),
13
.52 (d, J = 8 Hz, 4H), 6.80 (s, 2H). C-NMR (DMSO-d ) δ (ppm): 162.94, 133.95, 132.83, 132.55,
6
32.32, 129.21, 128.93, 125.84, 125.46, 109.15.
4
.2. Biology
.2.1. In vitro antileishmanial activity
4
Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO). 10-Fold serial
dilutions of test compounds were prepared in culture media. The maximum concentration of DMSO
in drug susceptibility assays was 1%, which was not hazardous to the parasites and amphotericin B
was used as control.
4
.2.1.1. Strains
Leishmania major (MRHO/IR/75/ER) and L. infantum (MCAN/IR/07/Moheb-gh) promastigotes were
3
grown in 25 cm plastic cell culture flasks in Brain Heart Infusion (BHI) medium at 37 g/L which was
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sterilized by autoclave, and supplemented with 15% heat-inactivated fetal bovine serum (FBS),
penicillin (100 U/mL) and streptomycin (100 U/mL) at 26 °C. Promastigotes were maintained by
subpassaging every 5-7 days into fresh culture media.
Leishmania major were cultured as axenic amastigotes using a modified method based on the
[
33]
previously reported approach.
supplemented with 25 % FBS at pH 5.5 (by titration of the medium with 10 mM succinate–TRIS) after
2 h of incubation at 37 °C in the presence of 5 % CO . The cellular morphology of the parasites was
Axenic amastigote forms were obtained in BHI medium
7
2
examined using both Giemsa-stained preparations and phase-contrast light microscopy.
4
.2.1.2. Antileishmanial activity against promastigote stage
The activity of AIA compounds against the growth of L. major and L. infantum promasigotes was
[
34]
measured by the MTT colorimetric assay.
45 µL of test compounds (10-fold serial dilutions) were
added to each well, in triplicate, except three blank wells which only contained 90 µL of culture
medium without any compounds or parasites. Late logarithmically growing promastigotes of L.
6
major and L. infantum were seeded in sterile 96-well plates at a concentration of 10 parasites/mL,
3
and they were dispensed in 45 µL aliquots (45×10 parasites/well). Negative controls consisted of
promastigotes in culture media without testing compound, while the positive control contained
varying concentraꢀons of amphotericin B (reference drug purchased from ꢁigma Aldrich). ꢂhe plates
ꢃere incubated at 26 ꢄC. After 72 h of incubation, 10 µL of MTT solution (5 mg/mL) was added to
each well followed by incubation for another 6 h. When purple precipitate was clearly visible under
the microscope, 100 µL of DMSO was added to all wells, including control wells, and relative optical
density (OD) was measured with a PolarStar plate reader at 570 nm. IC s were calculated by
50
[
35]
nonlinear regression analysis processed on dose–response curves, using Quest™ IC Calculator.
50
4
.2.1.3. Antileishmanial activity against axenic amastigote stage
The susceptibility of L. major axenic amastigotes to growth inhibition by tested compounds was also
6
determined using MTT assay. Briefly, 45 µL/well of the culture which contained 10 cells/mL of L.
major axenic amastigotes were placed into 96-ꢃell flat-bottom plates in the presence or absence of
compounds. Plates were incubated at 37 °C in a humid 5% CO atmosphere for 72 h. At the end of
2
incubation, 10 µL of MTT solution was added to each well and plates were subsequently incubated
for an additional 6 h. The enzyme reaction was then stopped by addition of 100 µL of DMSO and the
absorbance of each well was measured at 570 nm using a PolarStar plate reader. The 4-parameter
curve available in the program Quest™ IC Calculator was used to determine IC values for L. major
50
50
axenic amastigotes.
4
.2.2. Cytotoxicity evaluation
The toxicity of compounds to mammalian cells was determined with the MTT assay using cultured
human fibroblast cell line. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine
5
serum, penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 °C in 5% CO . The cells (10 cells
2
/
well) were incubated into 96-well plates overnight in a humidified 5% CO atmosphere at 37 °C to
2
ensure cell adherence. After 24 h, cells were treated with increasing concentrations of each
compound. Non-treated cells were included as a negative control. After 72 h incubation with each
compound, the MTT solution (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. Then
the medium and MTT were removed, cells were washed by PBS and 200 µL of DMSO was added to
dissolve the formazan crystals. The absorbance was measured using a PolarStar plate reader at 570
This article is protected by copyright. All rights reserved.

nm. The toxic dose of compound that results in 50% inhibition of control cell growth (TD value) was
50
determined using the program Quest™ IC Calculator. The TD values were used to generate the
50
50
cytotoxic index, the ratio of human cell cytotoxicity to antileishmanial activity (TD /IC ). The
50
50
inhibitory concentration 50% (IC ) is the drug concentration that results in 50% growth reduction of
50
parasites compared to untreated controls.
Author Contribution Statement
This article was extracted from the MSc research project of Foroogh Rezaei. She performed all the
experiments by herself and wrote the article along with Lotfollah Saghaei and Razieh Sabet. Afshin
Fassihi was the supervisor of the chemistry part of the research and Gholamreza Hatam was the
supervisor of the biological part of the project.
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