Synthesis, Biological Evaluation, Structure-Activity Relationship, and Mechanism of Action Studies of Quinoline-Metronidazole Derivatives Against Experimental Visceral Leishmaniasis

Article abstract of DOI:10.1021/acs.jmedchem.9b00628

In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, a series of quinoline-metronidazole hybrid compounds was synthesized and tested against the murine model of visceral leishmaniasis. Among all synthesized de

Full text of DOI:10.1021/acs.jmedchem.9b00628

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Synthesis, Biological Evaluation, Structure−Activity Relationship,
and Mechanism of Action Studies of Quinoline−Metronidazole
Derivatives Against Experimental Visceral Leishmaniasis
Akanksha Upadhyay,†,∥,⊥ Pragya Chandrakar,‡,∥,⊥ Sampa Gupta,†,∥ Naveen Parmar,‡,∥
Sandeep Kumar Singh,§,∥ Mamunur Rashid,§,∥ Pragati Kushwaha,†,∥ Muhammad Wahajuddin,§,∥
Koneni V. Sashidhara,*,†,∥ and Susanta Kar*,‡,∥
†Medicinal and Process Chemistry Division, ‡Parasitology Division, and §Pharmacokinetics and Metabolism Division, CSIR-Central
Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
∥Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi 110025, India
S
* Supporting Information
ABSTRACT: In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, a series of
quinoline−metronidazole hybrid compounds was synthesized and tested against the murine model of visceral leishmaniasis.
Among all synthesized derivatives, 15b and 15i showed significant antileishmanial efficacy against both extracellular
promastigote (IC50 9.54 and 5.42 μM, respectively) and intracellular amastigote (IC50 9.81 and 3.75 μM, respectively) forms of
Leishmania donovani with negligible cytotoxicity toward the host (J774 macrophages, Vero cells). However, compound 15i
effectively inhibited the parasite burden in the liver and spleen (>80%) of infected BALB/c mice. Mechanistic studies revealed
that 15i triggers oxidative stress which induces bioenergetic collapse and apoptosis of the parasite by decreasing ATP
production and mitochondrial membrane potential. Structure−activity analyses and pharmacokinetic studies suggest 15i as a
promising antileishmanial lead and emphasize the importance of quinoline−metronidazole series as a suitable platform for the
future development of antileishmanial agents.
drugs such as sodium stibogluconate, meglumine antimoniate,
and other chemotherapeutics like amphotericin B, paromomy-
cin, and pentamidine are the mainstays of antileishmanial
chemotherapy.5−7 However, these drug treatments entail high
costs, involve prolonged administration, and even exhibit side-
effects like nephrotoxicity, ototoxicity, hepatotoxicity.6 Addi-
tionally, the gradual increase in the resistance of parasite strains
against antileishmanial drugs has limited their use for
treatment in endemic areas.8 Miltefosine is the only available
oral drug for the first-line therapy of VL9 as well as cutaneous
leishmaniasis,10 yet its usage involves several drawbacks such as
high price, potential fetotoxicity, and teratogenicity.11 In the
search for more effective drugs against VL and other “neglected
INTRODUCTION
■
World Health Organization (WHO) classifies leishmaniasis as
one of the most neglected tropical human diseases of global
health concern. Visceral leishmaniasis (VL) is a life-threatening
disease caused by the obligate intracellular parasites Leishmania
donovani, Leishmania infantum (syn. Leishmania chagasi).1 VLs
is nearly always fatal, if not treated; as this disease confers
detrimental effects on vital organs like the liver and spleen.2,3
According to the recent report of WHO, leishmaniasis is
prevalent in approximately 90 countries that include areas of
India, Nepal, Bangladesh, Sudan, South Sudan, Ethiopia, and
Brazil. It is estimated that approximately 70 000 deaths and
1.5−2 million new cases occur every year because of
leishmaniasis.4 Because of the lack of a potent vaccine,
chemotherapy emerges as the only approach to cure different
types of leishmanial infection. Pentavalent antimonial-based
Received: April 12, 2019
© XXXX American Chemical Society
A
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Figure 1. Strategy for designing of quinoline−nitroimidazole hybrids.
tropical diseases”, researchers have reassessed the therapeutic
value of nitroheterocyclic compounds. In particular, nitro-
containing imidazoles like nitroimidazo-oxazine and nitro-
imidazooxazole delamanid were shown to have effective
antileishmanial efficacy.12,13 Moreover, the 5-nitroimidazole,
fexinidazole is now in clinical trials while back-up nitro-
imidazoles, such as DNDI-VL-2098 are at an advanced stage of
preclinical development for use in the treatment of VL.14,15
Research suggests that reduction of the nitro group of
nitroimidazoles in pathogens lead to the formation of reactive
metabolic intermediates that covalently bind with DNA and
trigger cell death. In Leishmania, nitroimidazoles are activated
by a novel nitroreductase that results in the generation of
reactive nitrogen species, including nitric oxides (NO) that are
major effectors of these compounds. Because the des-nitro
forms of these compounds were found to be inactive against L.
donovani, this suggests that the nitrogroup plays a key role in
the antileishmanial activity of this series.16 Quinolines are an
important structural motif found in numerous natural products
that exhibit diverse biological activities.17−20 Quinoline
derivatives have been found to be active against promastigotes
of different Leishmania species. Compounds with antileishma-
nial activity such as sitamaquine and tafenoquine, both 8-
aminoquinolines, have been extensively studied in recent
decades.21,22 Different analogues of quinoline conjugated to
sulfonamide, hydrazide, and hydrazine are known to exert
antileishmanial activity by inducing depolarization of the
mitochondrial membrane potential (MMP) in promasti-
gotes.23 In addition to these, quinoline derivatives exerted
their effect by inducing reactive oxygen species (ROS) levels in
promastigotes.24 In view of the above considerations, herein,
we introduced the nitroimidazole moieties at the second
position of quinolines to generate a novel series of hybrids and
checked its efficacy against dual life stages of the L. donovani
parasite. Figure 1 shows the chemical structures of some
representative drug leads that contain a quinoline or
nitroimidazole in their molecular framework and forms the
basis of our designed prototype hybrid. The concept of
molecular hybridization was utilized to incorporate them in
one molecular platform, in order to produce new hybrid
architecture with better efficacy and less toxicity.
RESULTS AND DISCUSSIONS
■
Chemistry. Our multistep synthetic route is shown in
Scheme 1. The synthesis of the proposed prototype was
initiated by the tosylation of metronidazole (MET-OH) 9 with
excess use of toluene sulphonyl chloride (10) in the presence
of triethylamine in CH2Cl2(10 mL) at 0 °C, afforded the
MET-OTs 11 in good yield.25 Then, the MET-OTs 11 was
treated with 3-hydroxyacetophenone 12a and 4-hydroxyaceto-
phenone 12b in the presence of K2CO3 in DMF at room
temperature yielded the desired products 13a and 13b,
respectively.26 Further, these compounds were treated with
various substituted 2-aminoarylketones in the presence of TFA
at 120 °C for 3 h.27 Under the present reaction conditions, 13a
or 13b with various substituted 2-aminoarylketones 14
proceeded smoothly and furnished the corresponding products
15a−15m in good to moderate yield.28
In Vitro Antileishmanial Activity of Quinoline−
Metronidazole Derivatives. To assess the antileishmanial
potency of quinoline−metronidazole derivatives, inhibition of
promastigotes and intracellular amastigotes of L. donovani was
tested initially at two concentrations, that is, 25 and 50 μM
(Table 1) for 48 h. To check these, we used luciferase
expressing L. donovani parasite and cell viability of both
promastigotes and intracellular amastigotes was assessed in
terms of relative luciferase units. Based on preliminary
screening, we found compounds 15b, 15h, and 15i to be
maximally active against the promastigote stage of the parasite
B
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a
Scheme 1. Synthesis of Quinoline−Metronidazole Conjugates (15a−15m)
a
Reagents and conditions: (i) Et3N, DCM, 0 °C, 5 h. (ii) K2CO3, DMF, 35−40 °C, 24 h (iii) TFA, 120 °C, 3 h.
with inhibition rates between 93 and 95% at 50 μM (Table 1).
Most of the compounds showed less to moderate activity
against the intracellular amastigote form as well, with inhibition
ranging from 23 to 67% at 50 μM (Table 1). From the
aforementioned compounds, 15b and 15i were found to
exhibit significant activity against amastigotes, with >93%
parasite killing at 50 μM concentration (Table 1). These
quinoline−metronidazole hybrids displayed superior antileish-
manial activity in comparison to their parent subunits (i.e.,
quinoline and metronidazole) which showed 83.6 and 68.1%
antiamastigote killing, respectively, at 50 μM concentration.
The antileishmanial activity of most active compounds 15b
and 15i was measured in time as well as dose-dependent
fashion against L. donovani promastigotes and amastigotes by
MTT and luciferase assay, respectively. Dose- and time-
dependent inhibition against promastigotes were observed by
both compounds 15b and 15i, where maximum inhibition was
>93% as determined by luciferase and MTT assay (Figure
2A,B). In addition to these, half-maximal inhibition (IC50)
concentrations of 15b and 15i against promastigotes were 9.54
0.73 and 5.42 0.45 μM as determined by luciferase assay.
This was comparable with the antipromastigote IC50 of both
these compounds (9.4 1.2 and 5.3 0.65 μM for 15b and
15i, respectively) as determined by MTT assay. Similarly,
different concentrations of 15b and 15i were found to
successfully inhibit the intracellular multiplication of the
parasite in a dose-dependent fashion in J774 macrophages,
where maximum elimination of amastigotes was observed at a
dose of 50 μM by both luciferase assays (>96%, Figure 2C)
and Giemsa staining of intracellular amastigotes (>93%, Figure
2D). Half-maximal inhibition concentrations (IC50) of 15b and
15i against intracellular amastigotes, were found to be 9.81
2.05 and 3.75 0.68 μM by luciferase assay and 9.09 1.04
and 4.06 0.70 μM by Giemsa staining, respectively, at 48 h,
both of which are lesser than that of their parent subunits
metronidazole, or quinoline (IC50: 28.4 and 11.6 μM,
respectively). Moreover, they are either equivalent to, or lesser
than the IC50 of miltefosine (standard chemotherapeutic
C
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a
Table 1. In Vitro Antileishmanial Efficacy of Compounds
promastigotes
concentration (μM)
intracellular amastigotes
concentration (μM) % inhibition
66.4 7.5
31.2
s. no.
1
compound code
% inhibition
15a
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
63 7.2
43 5.9
94.7 2.1
77.8 8.2
58.2 7.1
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
5
2
15b
93.2 3.8
75.2 7.9
59.2 9.9
38.2 6.1
65.3 7.2
29.5 6.3
3
15c
41.2
6
4
15d
60.8 8.8
30.2 4.1
NSI
5
15e
10.4
NSI
NSI
NSI
2
NSI
6
15f
26 3.9
12.3 4.6
NSI
7
15g
12.1 3.3
NSI
67.7 8.2
45.3 5.2
93.8 2.5
83.2 7.3
NSI
8
15h
93.2 4.4
78.2 9.1
94.4 1.2
85.8 6.4
65.8 7.2
9
15i
10
11
12
13
14
15
16
15j
64.4
7
45.3
5
34.2 5.2
60.3 7.2
33.8 4.8
27.2 4.5
15k
63.9 7.3
41.3 5.2
61.7 7.7
38.2 5.1
59.1 6.4
34.4 5.1
62.2 5.2
41.3 3.8
80.2 5.2
42.4 3.1
99.5 0.2
97.2 0.7
15l
12.1
3
15m
63.6 7.2
23.4 4.3
68.1 5.8
31.2 3.5
83.6 4.3
64.5 3.8
99.2 0.3
98.3 0.7
metronidazole
quinoline
miltefosine
a
Percentages of inhibition are expressed as mean standard deviation for each compound of three individual experiments (at least) performed in
duplicates, NSI (no significant inhibition).
regimen for VLs, anti-amastigote IC50: 9.59 μM) (Figure
2C,D). Both these compounds were found to be noncytotoxic
and exhibited a higher safety index (SI = CC50/IC50, of 15b
and 15i, was observed as 12.6 and 17.6 in J774 cells, and 36.05
and 57.9 in Vero cells, respectively, as compared to miltefosine,
SI = 5.8 and 5.3 in J774 macrophages and Vero cells,
respectively). Both 15b and 15i showed much less toxicity
toward J774 and Vero cells as >92% cells and >86% cells were
viable up to 50 and 100 μM concentrations, respectively.
Increasing the concentration of both 15b and 15i to 100 μM
did not alter their activity against promastigotes and
amastigotes significantly and was almost equivalent to the
percentage inhibition observed at 50 μM. Thereby, the 50 μM
concentration was taken as the MIC (maximum inhibitory
concentration) for both 15b and 15i. The preliminary in vitro
screening revealed that out of the synthesized derivatives, ten
compounds exhibited better (58−95%) inhibition at 50 μM
concentrations against promastigotes. Interestingly, the hybrids
were more active against L. donovani as compared to the
parental subunits alone, that is, quinoline and metronidazole.
The unsubstituted quinoline analog 15h displayed significant
activity (93.2% inhibition) against promastigotes as shown in
Table 1. The presence of methyl substitution at R2 of the
quinoline ring enhances activity against promastigotes with
lesser cytotoxicity when evaluated and compared to miltefosine
as shown in Figure 2A (15b and 15i having IC50 = 9.54 0.73
μM and IC50 = 5.42 0.45 μM, respectively). Further, these
derivatives were tested for their activity against the intracellular
amastigotes form. In this screening, again the compounds 15b
and 15i possessing a methyl group at R2 position exhibited
robust activity against amastigotes with IC50 = 9.81 2.05 μM
and IC50 = 3.75 0.68 μM, respectively, as compared to the
standard drug miltefosine. However, the substitution at R1
position of quinoline had no influence on biological activity.
Thus, in terms of preliminary structure−activity relationship
(SAR), the methyl substitution at the fourth position, that is,
R2 of the quinoline ring enhanced activity against both the
promastigote and amastigote stages of the parasite. Also, both
para and meta substitution of the phenyl ring attached to the
quinoline ring with the nitroimidazole moiety were pivotal for
optimum activity, which could be seen in the case of
compounds 15b and 15i. A preliminary summary of SAR is
schematically represented in Figure 3. These results further led
to the evaluation of in vitro pharmacokinetics stability of 15b
and 15i before proceeding for the determination of
antileishmanial efficacy against L. donovani-infected BALB/c
mice.
D
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Figure 2. Antileishmanial activity of 15b and 15i in in vitro. (A) Percentage inhibition of parasites was assessed by luciferase assay after treatment
with 15b and 15i at different concentrations for 24 and 48 h. (B) Percent viability/survival of promastigotes was evaluated by MTT assay after
treatment with varying concentrations of 15b and 15i for 48 h. (C,D) Macrophages were infected with a 1:8 (cell: promastigote) ratio, followed by
treatment with different doses of compounds 15b and 15i for 48 h, as described in the Experimental Section. The percentage of amastigote
inhibition was determined by luciferase assay (C) and the number of intra-macrophagic amastigotes was evaluated by Giemsa staining (D) as
described in the Experimental Section. (E,F) Vero cells and J774 macrophages were incubated at varying concentrations of 15b and 15i for 48 h
and cell viability was checked by the MTT method. Data are represented as means SD of individual experiments (n = 3), which were done in
replicate. Significance levels are represented as untreated vs 15b, 15i-treated groups with 25 and 50 μM doses, **p < 0.005, ***p < 0.0001.
In Vitro Pharmacokinetic Study. In Vitro-Simulated
Gastric Fluid and Simulated Intestinal Fluid Stability. The
stability of 15b and 15i was assessed in simulated gastric fluid
(SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.5).
Both these compounds were found to be stable in gastric fluid
as 84.72 2.62% of 15b and 83.58 0.77% of 15i remained
intact after 2 h of incubation with SGF (Figure 4A).
However, the percentage remaining in the intestinal fluid
was found to be only 11.40 0.35 and 43.56 3.84% for 15b
and 15i, respectively, as observed up to 2 h (Figure 4B). These
results demonstrated that compound 15i was relatively more
stable as compared to 15b in the intestinal fluid.
In Vitro Plasma Stability. Evaluation of plasma stability of
15b and 15i in rat plasma is depicted in Figure 4C. The results
demonstrate that the compounds were found to be stable as
68.16 0.085% of 15b and 88.50 3.95% of 15i, remained
intact in rat plasma after 2 h incubation. The results also clearly
indicated that compound 15i was more stable in rat plasma as
compared to 15b.
In Vitro Microsomal Stability. In vitro microsomal stability
indicates hepatic clearance (Cl) of drug from the liver. The
result of microsomal stability of compound 15b and 15i using
rat liver microsomes (RLM) is shown in (Figure 4D). The
Figure 3. Pictorial representation of SAR of quinoline−metronidazole
hybrid.
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Figure 4. In vitro stability of compounds 15b and 15i in simulated gastric (A) and intestinal (B) fluids up to 2 h. In vitro stability of compounds
15b and 15i in rat plasma (C) and in RLMs (D).
Figure 5. Effect of 15b and 15i treatment in controlling in vivo infection: 15 day-infected BALB/c mice were treated with different doses of 15b
and 15i as indicated. Mice of different experimental groups (n = 5 mice per group), were killed for the determination of hepatic (A) and splenic (B)
parasite burden by the stamp-smear method and represented as Leishman Donovan units (LDU). The in vivo safety index of 15b and 15i was
determined by assessment of serological markers. (C,D) 15 day-infected BALB/c mice were treated with 50 mg/kg of quinoline, metronidazole,
15b, 15i and 25 mg/kg of miltefosine for 5 consecutive days. Mice of different experimental groups (n = 5 mice per group), were sacrificed at day 7
post-treatment for the determination of hepatic (C) and splenic (D) parasite burden by the stamp-smear method and represented as LDU. (E)
AST and (F) ALT at various time points in infected and treated BALB/c mice. Data represent mean SD (n = 5 mice per group), representative
of three independent experiments were performed in replicate. Significance is indicated for untreated vs 15b, 15i-treated groups with 50 mg/kg
dose; *p < 0.05, **p < 0.005, ***p < 0.0006, ns = nonsignificant.
compounds 15b and 15i were found to be intact as 72.77
2.32 and 75.97 0.63%, respectively, after 60 min incubation
in RLM. The in vitro half-life (t1/2) and related parameters of
compound 15b and 15i were calculated by plotting the natural
F
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Figure 6. 15i-treated L. donovani promastigotes underwent apoptosis: J774 macrophages infected with L. donovani promastigotes (24 h) were
further incubated with 50 μM of 15i for 48 h. (A) Th1 and (B) Th2 cytokines levels in cultured supernatant were evaluated by ELISA. (C) L.
donovani parasites were incubated with 50 μM of 15i for different time points and analyzed for membrane permeabilization using the SYTOX dye.
Results were represented in percentages to the maximum normalized fluorescence as obtained with 0.1% Triton X-100 used for maximal
promastigote permeabilization. (D) Exponential phase L. donovani parasite (1 × 106) was incubated at maximum parasite inhibitory concentration
of 15i (50 μM) for 12, 24, and 48 h. Parasites incubated with 0.1% DMSO for 48 h were taken as the vehicle control. After incubation,
promastigotes were stained with Annexin-V Alexa Fluor 488 and PI and analyzed by flow cytometry. (E) DNA fragmentation of parasites was
analyzed by TUNEL assay. Log phase promastigotes were incubated with either vehicle (48 h) or 15i (50 μM) for 48 h. TUNEL assay was carried
out as mentioned in the Experimental Section. Tunnel-stained parasites were then visualized by both phase contrast, as well as fluorescent
microscopy. (F) Fluorogenic substrate BOC-GRR-AMC (t-butyloxycarbonyl-Gly-Arg-Arg-AMC) was used to determine metacaspase activity. Log
phase promastigotes were treated with DMSO (0.1%), 15i (50 μM) either alone or in parasites pretreated with antipain (1 μM) or zVAD-fmk (100
μM) for 1 h. Activity is expressed as relative fluorescence unit and normalized to mg of protein obtained leishmanial cell lysates. The significance
level is determined for the untreated vs treated groups, ns = nonsignificant.
logarithm of the percentage compound remaining versus
incubation time up to 60 min. The half-life values of 15b and
However, dose-dependent antileishmanial activity was different
for both the compounds where 15i exhibited better efficacy as
compared to 15b. Parasitic inhibition by compound 15i in liver
and spleen was observed to be >62% at 25 mg/kg (lower
dose), whereas compound 15b was able to eliminate >50%
parasite from liver and spleen at 50 mg/kg dose. For 15i,
maximum inhibition of organ parasite burden was obtained at
50 mg/kg/day dose for 5 consecutive days, where we observed
>80.3 and >82.3% inhibition in both splenic and liver parasitic
load, respectively, at the 7th day of post-treatment. The
inhibition observed was much higher when compared to their
parent components (quinoline and metronidazole alone)
administered for 5 consecutive days at the dose of 50 mg/kg
(Figure 5C,D). We observed 36.6 and 40.8% inhibition of the
parasitic load in spleen and liver, respectively, for metronida-
zole, and 55.2 and 58.2% inhibition of parasite burden in
spleen and liver, respectively, for quinoline. Moreover, no
significant increase in therapeutic efficacy of 15i was observed
upon increasing the dose to 100 mg/kg (inhibition in spleen
15i were found to be 2.18
0.32 and 2.50
0.27 h,
respectively. Nevertheless, the intrinsic Cl values of com-
pounds 15b and 15i were found to be 10.7 1.56 and 9.3
0.99 μL/min/mg, respectively. The abovementioned preclin-
ical stability studies demonstrated that compound 15i has
higher stability under a different set of experimental conditions
as compared to compound 15b.
In Vivo Activity of Compound 15b and 15i Against L.
donovani-Infected Mice. L. donovani-infected BALB/c mice
(15 days) were administered with compounds 15b and 15i via
intraperitoneal route (as described in Experimental Section),
for five consecutive days with 12.5−200 mg/kg/day dose range
and then sacrificed at 7th day after completion of treatment.
The parasitic load was determined in the stamp smears
obtained from spleen and liver. Both compounds 15b and 15i
were observed to inhibit parasitic load in the organs of infected
BALB/c mice in a dose-dependent fashion (Figure 5A,B).
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and liver parasite burden was 81.7 and 82.5%, respectively) and
200 mg/kg (inhibition in spleen and liver parasite burden was
82.2 and 82.7%, respectively). However, maximum inhibition
in liver and splenic parasitic load was found to be 54.14 and
55.5%, respectively, for 15b, even at a higher dose of (200 mg/
kg). Miltefosine used as standard reference drug showed >98%
inhibition of parasite burden at a curative dose (25 mg/kg/day
for 5 days) in both the spleen and liver of infected BALB/c
mice. During the experimental time duration, all mice were
found to be alive, healthy, and no marked reduction in weight
was observed in any of the experimental groups. Additionally,
in order to assess the in vivo safety index, the major serological
markers of toxicity, alanine aminotransferase (ALT), and
aspartate aminotransferase (AST) levels were also checked in
BALB/c mice of different experimental groups. On comparing
ALT and AST levels in uninfected and L. donovani-infected
BALB/c mice at 3rd and 5th week postinfection, we found a
significant rise in the serum level of both serological markers
ALT and AST in L. donovani-infected BALB/c mice, which
were measured as 82 and 202 U/L, respectively, after 5th week
of infection. On the other hand, levels in control BALB/c mice
was measured as 31 and 97 U/L, respectively, at 5th week
(Figure 5E,F). Interestingly, treatment with 15i in infected
BALB/c mice clearly reduced the levels of both serological
markers at every time period. Nevertheless, levels of both
markers remained within the normal range (AST54 to 298
U/L and ALT17 to 77 U/L in control animal). These
results suggested that 15i did not induce any hepatotoxicity.
Effect of 15i on Host Immunomodulation As Well As
Plasma Membrane Integrity/Programmed Cell Death of
L. donovani Promastigotes. During infection, L. donovani
creates a favorable environment by shifting the T-cell
responses from healing Th1 to nonhealing Th2 mode that
favors the persistence of the parasite inside the host. Thus,
redirecting the immune response from Th2 to Th1 mode and
boosting immune cells by stimulating Th1-mediated proin-
flammatory cytokine secretion is a rational approach in
leishmanial chemotherapy. To examine whether 15i was
capable of stimulating host immunity, we chose 50 μM dose
for in vitro studies, as it induced maximum killing of both
promastigote and intracellular amastigote. To evaluate the
changes in Th1/Th2 cytokines, J774 macrophages were
infected with promastigotes, followed by treatment with 15i
for 48 h and the resulting supernatant was assessed for
cytokine estimation. Enzyme-linked immunosorbent assay
(ELISA) analysis showed no significant increase in Th1
cytokines (TNF-α and IL-12) or reduction in Th2 cytokines
(TGF-β and IL-10) in 15i-treated macrophages as compared
to infected cells (Figure 6A,B). Because 15i did not induce any
apparent host-protective cytokine response, we hypothesized
that it could exert its effect by direct killing of L. donovani via
apoptosis or necrosis.
with MIC of 15i as compared to maximum permeabilization
achieved with 0.1% Triton X 100, used as the positive control
(Figure 6C). Negligible uptake of SYTOX green excluded the
possibility of nonspecific pore formation and clearly indicated
that 15i did not kill the parasite via osmolysis-mediated
necrosis. The apparent absence of necrotic cell death by 15i
encouraged us to check whether it could trigger apoptotic cell
death machinery in the leishmanial parasite, as we previously
observed antipromastigote activity of this compound by MTT
assay. Phosphatidylserine externalization is a hallmark feature
of cells undergoing apoptosis. Therefore, we next detected
phosphatidylserine externalization by staining 15i-treated
promastigotes with both Annexin-V-coupled FITC, as well as
propidium iodide (PI) to check for apoptosis or necrosis. The
dot plots are represented in (Figure 6D), which clearly
demonstrated that treatment with 15i for 24 h could initiate
early apoptosis as 52.18% of parasites was found in both
Annexin-V/PI positive quadrants. This further increased to
62.18% at 48 h treatment with 15i. On the other hand, only
7.3% of the promastigotes underwent apoptosis when treated
with a vehicle [0.1% dimethyl sulfoxide (DMSO)] which was
almost parallel to the control parasite (where 6.7% underwent
apoptosis, Figure 6D). Programmed cell death of 15i-treated
parasites was subsequently evaluated at the nuclear level by
detecting DNA fragmentation through terminal deoxynucleo-
tidyl transferase dUTP nick end labeling (TUNEL) staining.
Promastigotes showed bright green fluorescence denoting
TUNEL positivity after treatment with 15i for 48 h (Figure
6E). In contrast, control and vehicle-treated parasites showed
red fluorescence because of counterstaining with PI (Figure
6E). Merged image of the 15i-treated parasite also appeared as
bright yellowish green because of TdT-labeled nuclei, which is
indicative of programmed cell death. In addition to DNA
degradation, changes in the structure and morphology of the
parasites were observed by confocal microscopy. Promastigotes
treated with 15i appeared to be round in shape as compared to
untreated promastigotes that were flagellated and elongated, as
observed by bright field microscopy. Caspases are the primary
executioners of apoptosis in mammalian cells, however, in
protozoans, this function is accomplished by its protozoan
counterpart, that is, metacaspases. Emerging evidence suggests
that metacaspase initiates programmed cell death in
leishmanial parasite and its activity is unaffected by caspase
inhibitors; however, it is highly sensitive to serine protease
inhibitors like antipain or TLCK.29−32 We, therefore, evaluated
the activation of metacaspases by treating L. donovani parasites
with 15i for 24 and 48 h, either in the presence or absence of
caspase and metacaspase inhibitors. Our results suggested that
15i-upregulated metacaspase activity by 3.8-fold (at 48 h), as
compared to vehicle (0.1% DMSO)-treated parasites. This 15i-
mediated increase in metacaspase activity remained unaffected
by the prior treatment of caspase inhibitors (zVAD-fmk, 100
μM) but significantly decreased in the presence of the
metacaspase inhibitor (Antipain, 1 μM) (Figure 6F).
To address this issue, we first tried to understand whether
the compound was inducing plasma membrane permeabiliza-
tion of L. donovani promastigotes by lesion or pore formation
that eventually resulted in parasitic cell death from osmolysis
or necrosis. We checked for the cytosolic entry of SYTOX
green in 15i-treated parasites, which requires large-sized
lesions in the membrane, typical of nonspecific pore formation.
L. donovani promastigotes were incubated with both IC50 as
well as MIC of 15i for different time points and then incubated
with the SYTOX green dye. Flow-cytometric analysis revealed
that only 3.63% increase in the fluorescence after 4 h treatment
15i-Induced Mitochondrial-Programmed Cell Death
of the Parasites. Mitochondrial dysfunction and loss in
MMP (ΔΨm) are early events in the induction of apoptosis in
L. donovani parasites that are commonly induced by chemo-
therapeutic agents.33 To examine whether 15i treatment in L.
donovani promastigotes could change its MMP in a time-
dependent fashion, ΔΨm was evaluated using the fluorescent
dye, JC-1. At higher potential, the JC-1 dye forms aggregates
and fluoresces red in the mitochondria of control promasti-
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Figure 7. Determination of MMP loss, Cyt c release, ATP, ROS, and NO generation in 15i-treated promastigotes. (A) Exponential phase
promastigotes were incubated with 15i (50 μM) for 6−48 h or vehicle (0.1% DMSO). Parasites were also incubated with CCCP (50 μM), as the
positive control. Changes in MMP were observed after the addition of the JC-1 dye in flow cytometry. (B) Immunoblot study to check the level of
cytochrome c in mitochondrial (upper panel) and cytoplasmic (lower panel) fractions isolated from vehicle (0.1% DMSO, 48 h) or 15i-treated
parasites (50 μM, 6−48 h). COX-IV and β-tubulin were used as the endogenous controls for mitochondrial and cytosolic fractions, respectively.
(C) L. donovani parasites were incubated with either 0.1% DMSO or 50 μM of 15i for 6−48 h. ATP content in the cytosol was determined as
mentioned in the Experimental Section. (D) Parasites were untreated, vehicle-treated or treated with 15i compound at (50 μM) for different time
periods and probed with H2DCFDA (20 μM). ROS generation was measured by flow cytometry as described in the Experimental Section. (E) Log
phase L. donovani promastigotes were incubated with 15i (50 μM) for the respective time period followed by labeling with DAF-2DA (40 μM);
fluorescence was measured as described in the Experimental Section. Data are expressed as mean of at least 3 independent experiments done in
duplicate. The significance level is determined for the untreated vs treated groups, **p < 0.005, ***p < 0.0004, ns = nonsignificant.
gotes. MMP loss is characterized by a shift of fluorescence
signal from red to green (595−530 nm) as JC-1 at a lower
potential forms monomers and emits green fluorescence in the
cytoplasm. The maximum percentage of the control and
vehicle-treated promastigotes emitted red fluorescence, in-
dicating functionally intact mitochondria. On the other hand,
in 15i-treated parasites, the fluorescence signal shifted from red
to green in a time-dependent fashion, thereby denoting the
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absence of mitochondrial JC-1 accumulation. Flow cytometric
studies showed an increase of 33.15, 54.18, and 68.76% green
fluorescent cells at 12−48 h post treatment (Figure 7A).
CCCP (mitochondrion uncoupler) was used as a positive
control and we observed the fluorescence signal shifted from
red to green remarkably in CCCP-treated parasites, which
suggested JC-1 sensitivity toward membrane potential in
leishmanial cells. This observation strongly suggested that 15i-
mediated disruption of MMP was perpetrated by an intrinsic
pathway of apoptosis in Leishmania promastigotes. The
dissipated mitochondrial membrane results in the release of
cytochrome c from mitochondria to cytosol, which in turn
hampers the effective functioning of the electron transport
chain. Immunoblot results suggested that in vehicle-treated
promastigotes, mitochondria remains functionally intact and
cytochrome c is localized in the mitochondrial fraction (Figure
7B). On the contrary, 15i treatment rapidly induced the
cytosolic translocation of cytochrome c in the parasite which
was evident from an increased cytosolic abundance of
cytochrome c with gradual disappearance in the mitochondrial
fraction (Figure 7B). The loss in membrane potential and
leakage of cytochrome c directly affects mitochondrial electron
transport chain function and ultimately disrupts ATP
generation. Consequently, cells may undergo either necrosis
or apoptosis. To study the fate of ATP generation in 15i-
treated L. donovani promastigotes, we performed biolumines-
cence assay and found that ATP levels were higher and
comparable in control and DMSO-treated promastigotes (120
11 and 118 12 nM, respectively) but was subsequently
lower in 15i-treated parasites (Figure 7C). Maximum
inhibition was observed after 48 h treatment with >67%
reduction in the ATP level in 15i-treated parasites as compared
to vehicle-treated parasites.
Treatment with 15i at 50 μM, for 6 h, resulted in a 1.75 fold
increase in DAF-2DA fluorescence (GMFC 35.89
3.55) as compared to the vehicle control (GMFC 20.48 6.8).
A maximum 3.6-fold (GMFC 74.86 8.9) increase in
SEM
fluorescence was observed following incubation of the parasite
with 15i for 12 h when compared to the vehicle-treated
parasites (Figure 7E). These results indicated that compound
15i-mediated increase in NO and ROS generation exerts a
synergistic effect on leishmanicidal action.
In Vivo Pharmacokinetics Analysis. The mean plasma
concentration versus time profiles of 15i in mice are shown in
Figure 8 and the estimated pharmacokinetic parameters were
Figure 8. Mean plasma concentrations vs time profile of compound
15i in BALB/c mice at 50 mg/kg dose.
analyzed by WinNonlin software version 8.1 and the results are
tabulated in Table 2. After oral administration at 50 mg/kg
Table 2. Oral Pharmacokinetics Parameters of CDRI
Compound 15i at 50 mg/kg Dose in Mice
In mammalian cells, increased cellular ROS generation has
been known to be responsible for the depolarization of the
mitochondrial membrane and subsequent cell death.34,35 To
investigate whether the mitochondrial dysfunction observed in
L. donovani promastigote treated with 15i is promoted by ROS
generation, we performed flow cytometry using cell-permeable
dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA),
which is widely known as a ROS indicator. While the reduced
form of H2DCFDA does not emit fluorescence, its oxidized
fluorescent form 2′,7′-dichlorofluorescein (DCF) can be
detected by flow cytometry. Our results showed that after 12
h incubation with 15i at 50 μM concentrations, ROS
generation was increased by 31.6% in the promastigotes
which further increased to 41.4 and 58.2% at 24 and 48 h post
treatment, respectively. On the contrary, promastigotes treated
with the vehicle (0.1% DMSO) showed only 7% ROS
positivity (Figure 7D). Similar to ROS, NO is well-known
for its antimicrobial effect against a variety of protozoan,
bacterial, and viral pathogens.36−39 For identifying newer and
more effective drugs against VL, trypanosomiasis, and
tuberculosis, researchers have already reconsidered exploring
the therapeutic potential of nitroheterocyclic com-
pounds.12,13,40 Hence, we proceeded to investigate whether
15i could induce NO generation for its leishmanicidal action.
NO generation was detected by the DAF-2DA dye that, upon
binding an oxidized species of NO, results in an irreversible
DAF fluorescence, thereby implying local NO formation in
living cells at an intracellular level. For these, we treated L.
donovani promastigotes with 15i for 6−18 h followed by
incubation with 40 μM DAF-2DA and flow cytometry analysis.
parameters
Ke (h−1
t1/2 (h)
compound 15i
)
0.17 0.003
4.02 0.07
0.75 0.00
Tmax (h)
Cmax (ng/mL)
AUC0−48 (h·ng/mL)
AUC0−∞ (h·ng/mL)
Vd (L/kg)
6860.00 1955.09
18189.18 880.21
18195.95 879.93
15.98 1.02
Cl (L/h/kg)
2.75 0.13
dose, the maximum attainable concentration (Cmax) and time
taken to attain maximum concentration (Tmax) were found to
be 6860.00 1955.09 ng/mL and 0.75 0.00 h, respectively,
which showed that compound 15i has rapid absorption from
the gut. The overall systemic exposure, that is, area under the
concentration−time profile (AUC0−∞) of the compound was
found to be 18195.95
879.93 h·ng/mL. The apparent
volume of distribution (Vd) and Cl were found to be 15.98
1.02 L/Kg and 2.75 0.13 L/h/kg, respectively. However, the
time taken for the systemic levels to reduce to half (half-life,
1/2) and elimination rate constant (Ke) were found to be 4.02
0.07 h and 0.17
mentioned pharmacokinetic results confirmed that compound
15i has satisfactory oral exposure and hence may be considered
for future optimization as a lead molecule.
t
0.003 h−1, respectively. The above-
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apoptotic cascade, 15i enters the parasitic compartment
without forming pores and then sequentially disturbs the
membrane potential of mitochondria, which is an early event
of apoptosis as observed by the failure of J-aggregate formation
after JC-1 staining. Loss in MMP by 15i resulted in the leakage
of cytochrome c from mitochondria to cytosolic compartment.
Our results are in accordance with Antinarelli et al., who
previously demonstrated that different quinoline conjugates are
known to induce depolarization of the MMP in promastigotes.
Several other series of compounds are also known to exert their
antileishmanial efficacy by depolarizing MMP, for instance,
hybrid of β-carboline−quinazolinone diastereomer leads to
apoptosis in L. donovani promastigotes by depolarizing the
mitochondrial membrane.58 Antileishmanial hybrid com-
pounds are also known to exert their effect by inducing
oxidative stress. For instance, β-carboline−quinazolinone
hybrids inhibit L. donovani trypanothione reductase activity
and induce oxidative stress by generating ROS.58 Similarly,
hybrids of β-carboline and 1,3,5-triazine also induce ROS
formation in leishmania.59 In our study, we also found that 15i
has the ability to impair parasite resistance to oxidative stress
by enhancing ROS and NO generation, which is crucial for
controlling these kinetoplastid protozoan parasites as their life-
cycle phase occurring in the acidified phagolysosome of
macrophages. These dual characteristic features are attained
because of its pharmacophore as quinoline derivatives exerted
their antileishmanial effect by inducing the generation of ROS
levels and metronidazole series are known to exhibit
generation of reactive nitrogen species, including NO. A
clear correlation between in vitro activity, safety index, and in
vivo activity in conjunction with favorable pharmacokinetics
properties suggest the potential of quinoline−metronidazole
derivatives for the future development of specific clinical agents
for counteracting leishmaniasis and other neglected parasitic
diseases.
CONCLUSIONS
■
Current chemotherapeutic interventions against VL demand
more novel and safer drugs including patient compliances.
Moreover, it should also overcome the barrier of drug
resistance against leishmanial strain, as it is a new challenge,
which is considered seriously by the scientific community. To
throw light on this scenario, the concept of hybridization has
been introduced where two or more pharmacophores are
brought together in a single entity with individual intrinsic
pharmacological activity. This strategy has been already
implemented with great success in the discovery of potential
leads in diseases like cancer41 and AIDS42 and efforts against
VLs are currently underway. Recently, triclosan−caffeic acid
hybrids were seen to have good antileishmanial activity against
L. (V.) panamensis.43 Hybrids of diselenide and sulfonamide
also exhibit enhanced antileishmanial activity as compared to
their parent components.44 Hence, in search of new potential
candidates, we took the initiative to bring together the
pharmacores of quinoline and metranidazole within a single
entity. This is because metronidazoles are broad spectrum
antiprotozoan drugs, which are known to exert significant
efficacy against experimental VL and have the potential to
lower parasitic burden if used in combination with other
compounds.45−49 On other hand, quinolines and its derivatives
have been reported for their activity against both in vitro and
in vivo form of experimental visceral and cutaneous
leishmaniasis (against strains like L. donovani, L. chagasi,
Leishmania major etc.) with submicromolar IC50 values.50,51
We have synthesized a series of quinoline−metronidazole
hybrids and tested their efficacy against L. donovani, the
causative agent of VL. Among all synthesized compounds 15b
and 15i were the most promising candidates as they exhibit
greater antileishmanial efficacy along with a good safety index
and in vitro pharmacokinetics stability. Nevertheless, in case of
the in vivo BALB/c model of VL, 15i performed way better in
clearing parasite burden as compared to 15b. Moreover, 15i
maintained its in vivo safety index as confirmed by AST and
ALT, which is further confirmed by the fact that all infected
animal did not show any discomfort or uneasiness during or
after therapy. Besides that, the oral pharmacokinetic profile of
15i was also good as 15i has a rapid absorption from the gut.
Antileishmanial activity can be attained either by upregulat-
ing the host immune response, which is an indirect approach
or by directly acting upon the parasitic membrane or
intracellular organelles. Several antileishmanial compounds
like 18β-glycyrrhetinic acid,52 Fucoidan,53 and oleuropein,54
exhibited leishmanicidal effects against L. donovani in vitro, and
also minimize the parasite burden in L. donovani-infected
BALB/c mice by Th1 polarization, while drugs like
miltefosine55 and amphotericin-B56 induce the apoptotic
machinery in the parasite. In order to gain a mechanistic
overview of the mode of action of 15i, we checked several
biochemical and cell-biological parameters. In our case, we
found that 15i kills the parasite by initiating an apoptotic
cascade rather than acting indirectly through host immunity.
Apoptosis was also confirmed by DNA fragmentation as
observed by the tunnel assay of 15i-treated promastigotes,
where the membrane of the parasite was intact thereby
excluding the possibility of necrosis. Our study is in line with
other antileishmanial hybrids such as cycloalka[b]thiophene
and indoles that induce DNA fragmentation and initiate
apoptotic cell death in leishmania.57 In order to initiate an
EXPERIMENTAL SECTION
■
Synthetic Methods. Reagents were obtained from commercial
sources and used without further purification. Silica gel (100−200
mesh) was used to perform column chromatography. Progress of the
chemical reactions was monitored by TLC (silica gel plates with
1
fluorescence F254). Melting points were uncorrected. The H and
13C NMR spectra were recorded at 300, 400 MHz, and 75, 100 MHz,
respectively, using CDCl3 and DMSO-d6 as a solvent. All chemical
shift values were described in ppm and their multiplicities expressed
as: m = multiplet, q = quartet, t = triplet, dd = double doublet, brd =
broad doublet, d = doublet, brs = broad singlet, and s = singlet. The
ESI-MS spectra were recorded on an ion trap LCQ Advantage Max
mass spectrometer (Thermo Electron Corporation) and HRMS
spectra were recorded by Q-TOF (Agilent 6520). All the final
compounds were >95% pure as determined by the HPLC method.
General Synthetic Method for Preparing 2-(2-Methyl-5-nitro-
imidazol-1-yl)-ethyl Ester Toluene-4-sulfonate (11). A mixture of
metronidazole 9 (3.14 g, 20 mmol) and Et3N (3.0 mL, 22 mmol) was
dissolved in CH2Cl2 (20 mL). Then, the tosyl chloride 10 (3.83 g,
20.1 mmol) in CH2Cl2 (10 mL) was added dropwise at 0 °C. The
reaction mixture was stirred at room temperature for 5 h. The
progress of the reaction was monitored by TLC. After completion of
the reaction, 30 mL of ice water was added to the reaction mixture,
layer was separated, and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with saturated
NaHCO3, dried over anhy. Na2SO4, filtered, and concentrated under
reduced pressure. Compound 11 was obtained as white crystals,
which were used for the next steps without further purification.25
General Synthetic Method for Compound Preparation (13a−
13b). A solution of 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl-4-
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methylbenzenesulfonate 11 (5 g, 0.0153 mol) and 4-hydroxyaceto-
phenone 12a/3-hydroxyacetophenone 12b (2.30 g, 0.0169 mol) in
the presence of K2CO3 in DMF (50 mL) was stirred at room
temperature for 24 h. After the completion of the reaction (monitored
by TLC), the reaction mixture was suspended in water (50 mL) and
extracted with 3-fold with EtOAc (50 mL). After drying on anhy.
Na2SO4, the combined organic layers were filtered and concentrated
to dryness under reduced pressure to afford corresponding product
13a 1-(3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-
ethanone or 13b 1-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethoxy)phenyl)ethanone as yellow solid in good yield.26
124.5, 122.1, 118.3, 114.6, 66.5, 45.2, 14.1; ESIMS (m/z): 529 [M +
H]+; HRMS (m/z): calcd for C27H21BrN4O3 [M + H]+, 529.0875;
found, 529.0868.
2-(3-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-6-
nitro-4-phenylquinoline (15e). It was obtained as a white solid
(89%); mp: 229−231 °C; 1H NMR (CDCl3, 400 MHz): δ 8.84−8.83
(m, 1H), 8.50−8.47 (m, 1H), 8.35−7.33 (m, 1H), 7.97 (s, 1H), 7.91
(s, 1H), 7.78−7.74 (m, 2H), 7.62−7.54 (m, 5H), 7.45−7.41 (m, 1H),
6.99−6.96 (m, 1H), 4.79−4.77 (m, 2H), 4.49−4.46 (m, 2H), 2.67 (s,
3H); 13C NMR (DMSO-d6, 100 MHz): δ 158.7, 158.3, 151.8, 150.7,
150.1, 145.0, 139.1, 138.5, 136.2, 132.9, 131.5, 130.1, 129.7, 129.3,
129.0, 124.2, 123.1, 122.3, 120.8, 120.5, 117.2, 112.9, 66.5, 45.2, 14.2;
ESIMS (m/z): 496 [M + H]+; HRMS (m/z): calcd for C27H21N5O5
[M + H]+, 496.1621; found, 496.1595.
6-Chloro-4-(2-chlorophenyl)-2-(3-(2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl)ethoxy)phenyl)quinoline (15f). It was obtained as a white
solid (88%); mp: 192−194 °C; 1H NMR (CDCl3, 400 MHz): δ 8.18
(d, J = 7.2 Hz, 1H), 7.97 (s, 1H), 7.76 (s, 1H), 7.72−7.66 (m, 3H),
7.60 (d, J = 6.2 Hz, 1H), 7.50−7.37 (m, 5H), 6.93−6.91 (m, 1H),
4.78−4.76 (m, 2H), 4.47−4.45 (m, 2H), 2.66 (s, 3H); 13C NMR
(CDCl3, 100 MHz): δ 158.5, 156.3, 151.9, 146.9, 146.0, 140.8, 138.5,
136.3, 133.4, 133.3, 132.7, 131.8, 131.4, 130.8, 130.3, 130.2, 127.1,
126.7, 124.5, 121.0, 120.6, 116.1, 112.9, 66.9, 46.1, 14.9; ESIMS (m/
z): 519 [M + H]+; HRMS (m/z): calcd for C27H20Cl2N4O3 [M +
H]+, 519.0991; found, 519.0976.
6-Chloro-4-(2-fluorophenyl)-2-(3-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)quinoline (15g). It was obtained as a white
solid (79%); mp: 202−204 °C; 1H NMR (CDCl3, 400 MHz): δ 8.18
(d, J = 7.1 Hz, 1H), 7.97 (s, 1H), 7.80 (s, 1H), 7.71−7.67 (m, 3H),
7.62 (s, 1H), 7.54−7.52 (m, 1H), 7.44−7.38 (m, 2H), 7.36−7.33 (m,
1H), 7.31−7.27 (m, 1H), 6.93−6.92 (m, 1H), 4.77−4.75 (m, 2H),
4.47−4.43 (m, 2H), 2.65 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ
160.9, 158.5, 156.3, 151.9, 147.0, 142.8, 140.8, 133.5, 132.7, 131.8,
131.7, 131.7, 131.1, 131.0, 130.8, 130.2, 126.8, 125.2, 125.0, 124.7,
124.6, 124.4, 121.0, 120.9, 116.5, 116.3, 116.1, 112.9, 66.94, 46.2,
14.9; ESIMS (m/z): 503 [M + H]+; HRMS (m/z): calcd for
C27H20ClFN4O3 [M + H]+ 503.1286; found, 503.1284.
1-(3-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-
ethanone (13a). It was obtained as a pale yellow solid, yield 75%;
1
mp: 130−132 °C; H NMR (400 MHz, CDCl3): δ 7.97 (s, 1H),
7.54−7.57 (m, 1H), 7.34−7.40 (m, 2H), 7.00−7.03 (m, 1H), 4.74 (t,
J = 4.7 Hz, 2H), 4.38 (t, J = 5.0 Hz, 2H),2.63 (s, 3H), 2.58 (s, 3H);
13C NMR (CDCl3, 100 MHz): δ 197.5, 158.0, 151.7, 138.6, 133.3,
129.9, 122.1, 119.7, 112.7, 66.8, 45.9, 26.7, 14.7. ESIMS (m/z): 290
[M + H]+; HRMS (m/z): calcd for C14H15N3O4 [M + H]+, 290.1135;
found, 290.1134.
General Synthetic Method for Compound Preparation (15a−
15m). A mixture containing relevant 2-aminoarylketone (1.0 equiv)
and 1-(3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-
ethanone13a/1-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)ethanone 13b (1.0 equiv) were dissolved in TFA (25 mL) and
the solution was heated for 3 h at 120 °C. After the completion of
condensation reaction28 (monitored by TLC), the reaction mixture
was allowed to attain room temperature. Then, the reaction mixture
was neutralized with 0.4 mL of 10% NaOH, extracted with ethyl
acetate, and organic layer was washed with water. Organic layer was
then evaporated under vacuum and crude product was purified by
silica gel column chromatography to obtain 15a−15m.
2-(3-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-4-
phenylquinoline (15a). It was obtained as a white solid (85%); mp:
171−173 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.28 (d, J = 8.8 Hz,
2H), 8.11 (d, J = 8.1 Hz, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.82−7.77
(m, 2H), 7.61−7.53 (m, 6H), 7.04 (d, J = 8.8 Hz, 2H), 4.76−4.74 (m,
2H), 4.44−4.42 (m, 2H), 2.56 (s, 3H); 13C NMR (DMSO-d6, 100
MHz): δ 159.1, 155.1, 151.7, 148.4, 148.0, 138.5, 137.5, 132.8, 131.4,
129.6, 129.4, 128.7, 128.6, 128.4, 126.3, 125.1, 124.7, 118.2, 114.5,
66.4, 45.2, 14.1; ESIMS (m/z): 451 [M + H]+; HRMS (m/z): calcd
for C27H22N4O3 [M + H]+, 451.1770; found, 451.1759.
2-(4-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-4-
phenylquinoline (15h). It was obtained as a white solid (84%); mp:
1
141−143 °C; H NMR (CDCl3, 400 MHz): δ 8.19 (d, J = 8.0 Hz,
3H), 8.15−8.13 (m, 1H), 7.98−7.86 (m, 1H), 7.74−7.71 (m, 2H),
7.55−7.50 (m, 5H), 7.47−7.44 (m, 1H), 6.95−6.93 (m, 2H), 4.76−
4.73 (m, 2H), 4.42−4.39 (m, 2H), 2.62 (s, 3H); 13C NMR (CDCl3,
100 MHz): δ 158.9, 156.0, 151.8, 149.2, 148.8, 138.5, 133.4, 133.2,
129.9, 129.6, 129.1, 128.6, 128.5, 126.2, 125.7, 125.6, 118.8, 114.6,
66.8, 46.0, 14.8; ESIMS (m/z): 451 [M + H]+; HRMS (m/z): calcd
for C27H22N4O3 [M + H]+, 451.1770; found, 451.1763.
4-Methyl-2-(3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)quinoline (15b). It was obtained as a white solid (69%); mp:
1
156−158 °C; H NMR (CDCl3, 400 MHz): δ 8.17 (d, J = 7.0 Hz,
1H), 8.00−7.98 (m, 2H), 7.74−7.66 (m, 4H), 7.57−7.54 (m, 1H),
7.41−7.38 (m, 1H), 6.90 (d, J = 6.4 Hz, 1H), 4.76−4.75 (m, 2H),
4.46−4.45 (m, 2H), 2.77 (s, 3H), 2.66 (s, 3H); 13C NMR (CDCl3,
100 MHz): δ 158.3, 156.3, 151.9, 148.0, 145.0, 141.6, 138.4, 133.4,
130.3, 130.0, 129.5, 127.4, 126.3, 123.7, 120.9, 119.7, 115.6, 112.9,
66.8, 46.1, 19.0, 14.8; ESIMS (m/z): 389 [M + H]+; HRMS (m/z):
calcd for C22H20N4O3 [M + H]+, 389.1614; found, 389.1597.
6-Chloro-2-(3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-4-phenylquinoline (15c). It was obtained as a white solid
(73%); mp: 189−191 °C; 1H NMR (CDCl3, 400 MHz): δ 8.14−8.10
(m, 3H), 7.98 (s, 1H), 7.83−7.82 (m, 1H), 7.75 (s, 1H), 7.65−7.62
(m, 1H), 7.57−7.51 (m, 5H), 6.95−6.93 (m, 2H), 4.76−4.73 (m,
2H), 4.42−4.39 (m, 2H), 2.64 (s, 3H); 13C NMR (CDCl3, 100
MHz): δ 159.1, 156.2, 151.8, 148.5, 147.2, 138.4, 137.8, 133.4, 132.7,
132.0, 131.5, 130.5, 129.4, 129.1, 128.9, 128.8, 126.3, 124.5, 119.5,
114.6, 66.8, 46.0, 14.7; ESIMS (m/z): 485 [M + H]+; HRMS (m/z):
calcd for C27H21ClN4O3 [M + H]+, 485.1380; found, 485.1376.
6-Bromo-2-(3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-4-phenylquinoline (15d). It was obtained as a yellow solid
4-Methyl-2-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)quinoline (15i). It was obtained as a white solid (78%); mp:
1
124−126 °C; H NMR (CDCl3, 400 MHz): δ 8.13−8.08 (m, 3H),
7.98−7.95 (m, 2H), 7.71−7.67 (m, 1H), 7.64 (s, 1H), 7.53−7.49 (m,
1H), 6.93 (d, J = 8.8 Hz, 2H), 4.74−4.72 (m, 2H), 4.40−4.38 (m,
2H), 2.74 (s, 3H), 2.63 (s, 3H); 13C NMR (DMSO-d6, 100 MHz): δ
158.9, 155.1, 151.7, 147.3, 144.9, 138.5, 132.8, 131.6, 129.4, 129.3,
128.5, 126.6, 125.8, 123.9, 118.6, 114.5, 66.4, 45.2, 18.3, 14.1; ESIMS
(m/z): 389 [M + H]+; HRMS (m/z): calcd for C22H20N4O3 [M +
H]+, 389.1614; found, 389.1605.
6-Chloro-2-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-4-phenylquinoline (15j). It was obtained as a yellow solid
1
(83%); mp: 196−198 °C; H NMR (DMSO-d6, 400 MHz): δ 8.29
(d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.9 Hz, 1H), 8.04 (s, 2H), 7.80−7.77
(m, 1H), 7.72 (s, 1H), 7.63−7.57 (m, 5H), 7.05 (d, J = 8.5 Hz, 2H),
4.76−4.74 (m, 2H), 4.45−4.42 (m, 2H), 2.55 (s, 3H); 13C NMR
(CDCl3, 100 MHz): δ 159.2, 156.2, 151.8, 148.5, 147.3, 137.8, 133.5,
132.9, 132.0, 131.6, 130.5, 129.5, 129.1, 128.9, 128.8, 126.4, 124.6,
119.5, 114.6, 66.9, 46.1, 14.8; ESIMS (m/z): 485 [M + H]+; HRMS
(m/z): calcd for C27H21ClN4O3 [M + H]+, 485.1380; found,
485.1370.
1
(84%); mp: 103−105 °C; H NMR (DMSO-d6, 400 MHz): δ 8.28
(d, J = 8.8 Hz, 2H), 8.12−8.10 (m, 1H), 8.04 (s, 1H), 7.98 (s, 1H),
7.80−7.76 (m, 4H), 7.60−7.52 (m, 3H), 7.05 (d, J = 8.8 Hz, 2H),
4.77−4.74 (m, 2H), 4.44−4.42 (m, 2H), 2.56 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz): δ 159.2, 155.2, 151.8, 148.0, 147.2, 138.5,
136.7, 132.9, 131.7, 131.6, 131.3, 129.9, 129.5, 128.8, 126.6, 125.0,
7-Bromo-2-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-4-Phenylquinoline (15k). It was obtained as a white solid
L
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(71%); mp: 153−155 °C; 1H NMR (CDCl3, 400 MHz): δ 8.19 (d, J
= 8.4 Hz, 1H), 8.12 (d, J = 8.7 Hz, 2H), 7.97 (s, 1H), 7.80 (d, J = 8.2
Hz, 1H), 7.73−7.66 (m, 4H), 7.47−7.40 (m, 3H), 6. 49 (d, J = 8.7
Hz, 2H), 4.75−4.73 (m, 2H), 4.41−4.39 (m, 2H), 2.64 (s, 3H); 13C
NMR (CDCl3, 100 MHz): δ 158.9, 155.9, 151.7, 148.7, 147.8, 138.4,
137.2, 133.3, 132.9, 131.8, 131.1, 130.0, 129.7, 129.0, 126.3, 125.2,
122.8, 118.6, 114.5, 66.7, 45.9, 14.7; ESIMS (m/z): 529 [M + H]+;
HRMS (m/z): calcd for C27H21BrN4O3 [M + H]+, 529.0875; found,
529.0870.
6-Chloro-4-(2-chlorophenyl)-2-(4-(2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl)ethoxy)phenyl)quinoline (15l). It was obtained as a white
solid (78%); mp: 217−219 °C; 1H NMR (CDCl3, 400 MHz): δ 8.12
(d, J = 8.8 Hz, 3H), 7.98 (s, 1H), 7.78 (s, 1H), 7.66−7.63 (m, 1H),
7.60−7.55 (m, 1H), 7.54−7.49 (m, 1H), 7.44−7.40 (m, 1H), 7.35−
7.33 (m, 1H), 7.31−7.28 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 4.75−
4.73 (m, 2H), 4.41−4.39 (m, 2H), 2.64 (s, 3H); 13C NMR (CDCl3,
100 MHz): δ 160.9, 159.2, 158.4, 156.2, 151.8, 147.0, 142.6, 138.5,
133.4, 132.6, 132.2, 131.7, 131.6, 131.5, 131.0, 130.9, 130.7, 129.1,
126.5, 125.2, 125.1, 124.6, 124.4, 120.5, 116.4, 116.2, 114.6, 66.8,
46.0, 14.8; ESIMS (m/z): 519 [M + H]+; HRMS (m/z): calcd for
C27H20Cl2N4O3 [M + H]+, 519.0991; found, 519.0978.
6-Chloro-4-(2-fluorophenyl)-2-(4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)quinoline (15m). It was obtained as a yellow
solid (88%); mp: 204−206 °C; 1H NMR (CDCl3, 400 MHz): δ
8.14−8.11 (m, 3H), 7.98 (s, 1H), 7.74 (s, 1H), 7.65−7.63 (m, 1H),
7.59 (d, J = 6.3 Hz, 1H), 7.49−7.43 (m, 3H), 7.37−7.36 (m, 1H),
6.94 (d, J = 7.0 Hz, 2H), 4.76−4.74 (m, 2H), 4.42−4.40 (m, 2H),
2.64 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 159.2, 156.1, 151.8,
146.9, 145.8, 138.5, 136.3, 133.4, 133.3, 132.6, 132.1, 131.5, 131.4,
130.6, 130.2, 130.1, 129.1, 127.1, 126.4, 124.4, 120.0, 114.6, 66.8,
40.6, 14.8; ESIMS (m/z): 503 [M + H]+; HRMS (m/z): calcd for
C27H20ClFN4O3 [M + H]+, 503.1286; found, 503.1284.
Biological Assays. Maintenance of Cell Line and Parasite
Culture. Luciferase expressing L. donovani promastigotes strain
(MHOM/IN/80/Dd8) was obtained as described elsewhere.60
Briefly, the firefly luciferase gene (complete ORF) was amplified
from pGEM-Luc plasmid DNA (Promega) and cloned in the pCRII-
TOPO vector. The purified insert was obtained after digestion with
the restriction enzyme SpeI and subsequently cloned into
dephosphorylated pKS-Neo leishmania shuttle vector containing
neomycin cascade to generate the pKS-Neo-luc construct. The
orientation of the luc insert was confirmed after double digestion with
restriction enzymes, that is, EcoRI and BamHI and selected in
Escherichia coli before carrying out transfection. L. donovani
promastigotes were transfected with pKS-Neo-luc construct by
electroporation with a Gene Pulser (Bio-Rad) and the transfected
parasites were cultured in M199 medium which was supplemented
with 10% heat-inactivated fetal bovine serum (FBS) and G418
disulphate (20 μg/mL) at 24 °C. J774 macrophages cell line was
maintained in complete RPMI medium as described previously.61
Luciferase and MTT Assay. In vitro antileishmanial activity of
quinoline−metronidazole derivatives was assessed by luciferase assay
using Leishmania parasite (MHOM/IN/80/Dd8 constitutively
expressing firefly luciferase gene). Parasite multiplication was
represented as relative luminescence unit (RLU) and the IC50 value
of the synthesized derivatives and standard drugs was determined as
described earlier.61 Antipromastigote activity of 15b and 15i was also
assessed at various concentrations by MTT assay as described
elsewhere.62 The absorbance was recorded on a microplate
spectrophotometer (BioTek Instruments, Winooski, VT, USA), and
the data were represented as percentage viability of promastigotes in
comparison with control cells.
3.125 and 100 μM prior to treatment in infected macrophages. The
antiamastigote activity of 15b and 15i was also evaluated by Giemsa
staining through microscopic counting of the number of amastigotes/
100 macrophages.63
Mammalian Cell Cytotoxicity Assay. Vero cells and J774
macrophages were selected to assess the cytotoxic effects of 15b
and 15i at different concentrations (12.5−400 μM) by MTT assay as
described previously.61 The 50% cellular cytotoxic concentration
(CC50) and selectivity index (SI) of 15b and 15i were determined as
described previously.64
Animal Infection and Therapeutic Protocol. The L. donovani
strain MHOM/IN/80/Dd8 was maintained by serial passage in
hamsters for the establishment of infection. Amastigotes were then
isolated from the spleen of L. donovani-infected golden hamsters (2
months) and transformation into the promastigote form was obtained
in M199 (pH 7.4) medium, enriched with 20% FBS (heat-
inactivated), 25 mM HEPES, 2 mM glutamine, 100 μg/mL
streptomycin, and 100 IU/mL penicillin. After serial passages for 2
times, stationary-phase promastigotes were collected from the culture
medium and injected in BALB/c mice through the tail vein [2 × 107/
0.2 mL of phosphate-buffered saline (PBS)] to establish infection.
The level of infection was determined by performing autopsy in the
liver and spleen of 3−4 mice selected randomly at day 15
postinfection. Animals with similar levels of the organ parasitic load
were further selected for the in vivo experiment. Treatment was
initialized intraperitoneally (ip) for 15b and 15i and miltefosine was
administered orally in 15 day infected BALB/c mice. Five animals/
groups were used for the study and the groups were as follows:
infected untreated, infected vehicle-treated (8% DMSO in PBS),
infected miltefosine (a standard antileishmanial drug)-treated,
infected quinoline-treated, infected metronidazole-treated, infected
15b-treated, and infected 15i-treated. Miltefosine (25 mg/kg) was
suspended in triple distilled water and given orally, while 15b and 15i
were dissolved in DMSO (8%). Animals were administered with 15b
and 15i via intraperitoneal route for 5 consecutive days, and sacrificed
at day 7 post-treatment, following which the liver and spleen parasite
burden were assessed by Giemsa staining and expressed as LDU.61
Ethics Statement for in Vivo Experiments. All in vivo animal
experiments were conducted as per the strict guidelines and
recommendations provided by the specialized Committee for the
Purpose of Control and Supervision of Experiments on Animals
(CPCSEA), New Delhi, India. Animals usage was officially permitted
by the IAEC (Institutional Animal Ethical Committee) and all in vivo
experiments were performed in agreement with the IAEC of the
CSIR-CDRI (IAEC approval no: IAEC/2017/190). BALB/c mice of
weight 20 2 g were procured from the National Laboratory Animal
Center, CSIR-CDRI (Lucknow, India) and maintained in suitable
experimental conditions such as room temperature (24 2 °C) and
40−60% relative humidity while on a regular 12 h light−dark cycle.
The animals were acclimatized for a minimum period of 3 days prior
to the experiment.
Th1/Th2 Cytokine Estimation. The levels of Th1/Th2 cytokines in
the cell culture supernatants of 15i-treated infected macrophages was
assessed using ELISA kit (BD Biosciences, USA) as described
elsewhere.61
Detection of Plasma Membrane Permeability. Plasma membrane
integrity was determined using the Sytox green dye as mentioned
elsewhere65 with some modifications. Briefly, promastigotes (4 × 106/
mL) were incubated with 50 μM 15i for 0−360 min. This was
followed by washing with PBS for two times and incubation with 2
μM Sytox green at 28 °C for 15 min. The promastigotes were then
collected in fluorescence-activated cell sorting (FACS) tubes and the
fluorescence emitted upon binding of the dye to intracellular nucleic
acids was recorded by flow cytometry (BD FACSCalibur platform,
with excitation and emission wavelengths at 485 and 535 nm,
respectively). 0.1% Triton X-100 was used to obtain maximal
promastigote permeabilization and data were represented with respect
to the fluorescence obtained in this sample.
Antiamastigote Assay. Antileishmanial activity of quinoline−
metronidazole derivatives against intracellular amastigotes was
assessed by luciferase assay at different concentrations as described
previously.61 Parent molecules: metronidazole (Acros Organics,
Belgium) and quinoline (Sigma-Aldrich, USA), compounds (15b
and 15i), and standard drug miltefosine (Synphabase, Switzerland)
were subjected to serial dilution at a 2-fold concentration in RPMI
medium, maintaining the final concentrations in a range between
Apoptosis Detection by Annexin V/PI Staining. Phosphatidylser-
ine externalization in vehicle or 15i-treated L. donovani promastigotes
M
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(12−48 h) was determined by using the Apoptosis Detection Kit
(Sigma-Aldrich, USA) as described previously.61 Analysis of stained
promastigotes was performed in BD FACSCalibur platform and
CellQuest software was used for data interpretation.
TUNEL Assay. Fragmentation of DNA within the L. donovani
parasite was analyzed by terminal deoxynucleotidyltranferase (TdT)-
mediated dUTP nick end labeling (TUNEL) using APO BrdU
TUNEL Assay Kit (Molecular Probes, USA) as per the
manufacturer’s instructions. Briefly, 1 × 105 parasites were harvested
after treatment with 50 μM 15i for 48 h and samples treated with
DMSO (0.1%) were then used as the vehicle control. The experiment
was performed as described previously.66 Samples were finally
observed under a confocal microscope (LSM, Zeiss, Germany) and
analyzed on adobe Photoshop software 7.
Determination of Metacaspase-Like Protease Activity. Meta-
caspase activity was determined by using the substrate, BOC-GRR-
AMC. Briefly, 5 × 106 exponential phase parasites were either
untreated or incubated with DMSO (0.1%), 15i (50 μM) either alone
or in parasites pretreated with antipain (1 μM), zVAD-fmk (z-Val-Ala-
Asp-fmk) (100 μM) for 1 h. The cells were then pulled, subjected to
lysis in cell lysis buffer and resuspended in metacaspase buffer to
detect metacaspase activities. Furthermore, cell lysates were then
subjected to 100 μM fluorogenic substrate BOC-GRR-AMC and 1×
reaction buffer supplemented with 100 μM dithiothreitol. Samples
were then incubated at 37 °C for 2 h and the released 7-amino-4-
methylcoumarin (AMC) was detected by fluorometry (BMG
POLARstar Galaxy, Virginia, USA), with excitation and emission
wavelengths at 380 and 460 nm, respectively. The results were
represented as relative fluorescence units and normalized with cellular
protein extracts in mg.
for elution of compound 15b and 15i were set as 1.0 and 0.8 mL/min,
respectively.
In Vitro SGF and SIF Stability. SGF and SIF were prepared as per
USP specifications.68 The stability studies of compound 15b and 15i
in SGF and SIF were performed at 10 μg/mL in triplicate. Briefly,
SGF and SIF were taken in two separate glass test tubes and
preincubated in shaking water bath for 10−15 min at 37
2 °C.
Afterward, aliquots of 15b and 15i were spiked separately in SGF and
SIF and 100 μL of the incubation mixture was withdrawn at 0, 5, 10,
15, 30, 45, 60, 90, and 120 min. Each sample was quenched with
acetonitrile 1:2 (% v/v), vortexed, and centrifuged for 10 000 rpm.
The supernatant was then analyzed by the UPLC system.
In Vitro Plasma Stability. The plasma stability of 15b and 15i was
performed at 10 μg/mL in triplicate. The blank rat plasma was taken
in test tubes and preincubated in shaking water bath for 10 min at 37
0.2 °C. Afterward, aliquots of compound 15b and 15i were spiked
to incubated plasma and samples were withdrawn at 0, 5, 10, 15, 30,
45, 60, 90, and 120 min. Precipitation was carried out with the
addition of acetonitrile in the ratio of 1:2 (v/v) at each time intervals,
followed by vortexing and centrifugation for 10 min at 10 000 rpm.
Supernatants were separated and subjected for analysis by UPLC.
In Vitro Microsomal Stability. Microsomal stability of 15b and 15i
was performed from RLM in triplicate. Briefly, in each glass tube,
446.25 μL of Tris buffer (50 mM, pH 7.4), magnesium chloride
(MgCl2, 40 μM), and RLM protein (0.5 mg/mL) were spiked and
incubated for at 37 0.2 °C for 5 min. Then, compounds 15b and
15i (10 μg/mL) were spiked separately in this preincubation mixture.
Positive and negative controls were also performed in RLM for
validation of assays and microsomal activity. Initiation of the reaction
was performed by adding NADPH (24 mM) followed by incubation
for 1 h. The reaction was terminated by adding ice-chilled acetonitrile
(100 μL) to the reaction mixture (50 μL) collected at predefined time
points (0, 5, 10, 15, 30, 45, and 60 min). Samples were vortex mixed
for 5 min, followed by centrifugation for 10 min at 10 000 rpm. The
20 μL of supernatant was directly analyzed by UPLC.
Measurement of MMP Change. Change in MMP (ΔΨm) in 15i-
treated promastigotes (6−48 h) was evaluated using JC-1 dye
(MitoProbe JC-1 Assay Kit, Molecular Probes, USA) as described
previously.61 The experimental data were recorded in BD
FACSCalibur platform and analyzed by CellQuest Software.
Cytochrome c Detection in the Mitochondrial and Cytosolic
Fraction of L. donovani. Mitochondria fractions were obtained from
untreated and 15i-treated L. donovani promastigotes using the
ApoAlert cell fractionation kit (Takara, Japan) as per the
manufacturer’s instructions, and stored at −20 °C at a final protein
concentration of 7−8 mg/mL. Cytosolic lysates of control and 15i-
treated promastigotes were prepared as described elsewhere.66 After
cell fractionation, mitochondrial and cytosolic levels of cytochrome c
were determined by immunoblot as described previously.66
Data Analysis. The percentage of compound remaining intact at
different time intervals after incubation was determined by the eq 1
and the in vitro metabolic half-life (t1/2) was determined by plotting
the natural logarithm of percentage compound remaining versus
time.69,70 The slope of the curve was represented as depletion rate
constant K (min−1). The half-life of metabolic stability was estimated
using the first-order eq 2. The in vitro intrinsic clearance (CLint) was
calculated through the eq 3.
concentration of drug after incubation
% Drug remaining =
Determination of ATP Generation. Promastigotes (2 × 106) were
treated with 50 μM 15i for varying time periods, and the amount of
ATP content was evaluated by using the luciferase ATP assay kit
(Invitrogen, Inc., Ltd. USA), as described previously.67
concentration of drug before incubation
(1)
× 100
ROS Measurement. Promastigotes (2 × 106 cells/mL) were
incubated with 50 μM 15i for different time points (12−48 h) and
DMSO (0.1%)-treated parasites were used as the vehicle control.
Parasites were then harvested, followed by resuspension in PBS.
Thereafter, 20 μM (DCFDA) was added to the parasites and
incubation was performed for 20 min in the dark at 37 °C.34
Thereafter, analysis was performed on BD FACSCalibur platform at
530 nm wavelength.
0.693
K
Half‐life (t1/2) =
(2)
(3)
Intrinsic clearance CLint
volume of incubation (μL)
amount of protein incubated (mg)
= K ×
NO Measurement. Promastigotes (2 × 106 cells/mL) were
incubated with 50 μM 15i for different time points (6−18 h) and
DMSO (0.1%)-treated parasites were used as the vehicle control.
Promastigotes were finally collected, followed by resuspension in PBS,
and incubated with 40 μM of DAF-2DA at 37 °C for 45 min in the
dark. The resultant fluorescence was measured and analyzed on BD
FACSCalibur platform.
UPLC Conditions for in Vitro PK Study. Compounds 15b and 15i
were analyzed on Shimadzu UPLC system equipped with the
photodiode array detector. Chromatographic separation of both
compounds was achieved with a mobile phase composition of
acetonitrile: formic acid (0.1%) on a C18 column (100 × 4.6 mm, 5.0
μm). The mobile phase ratio for compound 15b and 15i were
optimized as 50:50 (v/v) and 40:60 (v/v), respectively. The flow rates
where K represents the depletion rate constant and it was calculated
from the slope of linear regression from natural log percentage of the
substrate remaining versus incubation time.
In Vivo Pharmacokinetic Study. In vivo oral PK study of
compound 15i was performed in BALB/c male mice (n = 5). The
compound was administered at 50 mg/kg dose via oral gavage to each
mice and the oral suspension was formulated in (0.25% CMC
solution and 5% Tween 80). Blood samples were collected from the
retro-orbital plexus of mice under light ether anesthesia into heparin-
containing microfuge tubes at 0.25, 0.5, 1, 3, 5, 7, 9, 12, 24, and 48 h
of postdose. Plasma was harvested by centrifugation of blood at 10
000 rpm for 10 min and stored at −20 2 °C until bioanalysis. The
protein precipitation method was employed for sample processing by
using acetonitrile (200 μL/100 μL plasma) as protein precipitant
N
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Article
containing medicarpin as internal standard. The supernatant was
injected for LC-MS/MS.
transferase dUTP nick end labeling; FBS, fetal bovine serum;
SIF, simulated intestinal fluid; K, depletion rate constant; CLint,
intrinsic clearance; AUC, area under the curve; HPLC, high-
performance liquid chromatography; t1/2, half-time; HRMS,
high-resolution mass spectrometry; Cmax, maximum concen-
tration; IR, infrared spectroscopy; DMSO, dimethyl sulfoxide;
NMR, nuclear magnetic resonance; EtOH, ethanol;
CH3COOH, acetic acid; Cl, clearance; Vd, volume of
distribution; TLC, thin-layer chromatography; Ke, elimination
rate constant; CH3CN, acetonitrile; LDU, Leishmania
Donovan unit; FACS, fluorescence-activated cell sorting;
BOC-GRR-AMC, t-butyloxycarbonyl-Gly-Arg-Arg-AMC;
zVAD-fmk, z-Val-Ala-Asp-fmk; RLU, relative luminescence
unit
Statistical Analysis. Data are represented as the mean
SD of
three separate experiments. To calculate the statistical significance
between the groups under study, we used the software GraphPad
Prism (version 5) and the differences between the experimental sets
were analyzed by one-way ANOVA followed by Tukey’s post. P <
0.05 was considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b00628.
■
S
SMILES strings (CSV)
1H NMR and 13C NMR spectra of all compounds
(PDF)
REFERENCES
■
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: kv_sashidhara@cdri.res.in. Phone: +91-522-2772450,
ext. 4683/4684. Fax: +91-522-2771942/2771970 (K.V.S.).
*E-mail: susantakar@cdri.res.in. Phone: +91-522-2772450, ext.
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ORCID
Susanta Kar: 0000-0002-9907-539X
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(7) Jha, T. K. Drug unresponsiveness & combination therapy for
kala-azar. Indian J. Med. Res. 2006, 123, 389−398.
This study was funded by the Department of Science and
Technology (grant no. SB/FT/LS-310/2012), Council of
Scientific and Industrial Research (CSIR NWP BSC0114).
N.P., A.U., P.K., M.R. received fellowship from UGC (New
Delhi), P.C., P.V., and S.G., S.K.S. are the recipients of
fellowships from CSIR (New Delhi). The transgenic L.
donovani promastigotes was a kind gift from Dr. Neena
Goyal, Division of Biochemistry, CSIR-CDRI, Lucknow, India.
The authors are thankful to Anuradha Seth for copy-editing the
manuscript. The authors would also like to acknowledge the
Director, CSIR-CDRI, Lucknow, for facilitating the research.
Authors are also thankful to the sophisticated analytical
instrumentation facility of CSIR-CDRI for providing support
with the confocal microscopy and flow cytometry experiments.
This manuscript has CDRI Communication no. 9845.
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ABBREVIATIONS
■
VL, visceral leishmaniasis; MET-OH, metronidazole; ROI,
reactive nitrogen species; HAT, human African trypanosomia-
sis; RNI, reactive nitrogen species; IC50, half-maximal
inhibitory concentration; SAR, structure−activity relationship;
RLM, rat liver microsomes; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; ALT, alanine amino-
transferase; ip, intraperitoneal; AST, aspartate aminotransfer-
ase; MIC, maximum inhibitory concentration; CC50, 50%
reduction of viability of treated cells with respect to untreated
cells; MMP, mitochondrial membrane potential; SI, selectivity
index; ROS, reactive oxygen species; NO, nitric oxide; FITC,
fluorescein isothiocyanate; PI, propidium iodide; SGF,
simulated gastric fluid; TUNEL, terminal deoxynucleotidyl
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trypanosome drug fexinidazole shows potential for treating visceral
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N.; Ramanathan, V.; Martin, D. In vitro metabolism, disposition,
preclinical pharmacokinetics and prediction of human pharmacoki-
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