Design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.028

An analogous library of 2-substituted quinoline compounds was synthesized with the aim to identify a potential drug candidate to treat visceral leishmaniasis. These molecules were tested for their in vitro and in vivo biological activity against Leishmania donovani. Metabolic stability of these compounds was also improved through the introduction of halogen substituents. Compound (26g), found to be the most active; exhibited an IC₅₀ value of 0.2 μM and >180 fold selectivity. The hydrochloride salt of (26g) showed 84.26 ± 4.44 percent inhibition at 50 mg/kg × 5 days (twice daily, oral route) dose in L. donovani/hamster model. The efficacy was well correlated with the PK data observed which indicating that the compound is well distributed.

Full text of DOI:10.1016/j.ejmech.2013.08.028

Accepted Manuscript
Design, Synthesis and Biological evaluation of 2-Substituted Quinolines as Potential
Antileishmanial Agents
Vadiraj S. Gopinath, Jakir Pinjari, Ravindra T. Dere, Aditya Verma, Preeti
Vishwakarma, Rahul Shivahare, Manjunath Moger, Palusa Sanath Kumar Goud,
Vikram Ramanathan, Prosenjit Bose, M.V.S. Rao, Suman Gupta, Sunil K. Puri,
Delphine Launay, Denis Martin
PII:
S0223-5234(13)00544-8
10.1016/j.ejmech.2013.08.028
EJMECH 6374
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 June 2013
Revised Date: 19 August 2013
Accepted Date: 25 August 2013
Please cite this article as: V.S. Gopinath, J. Pinjari, R.T. Dere, A. Verma, P. Vishwakarma, R.
Shivahare, M. Moger, P.S. Kumar Goud, V. Ramanathan, P. Bose, M.V.S. Rao, S. Gupta, S.K. Puri,
D. Launay, D. Martin, Design, Synthesis and Biological evaluation of 2-Substituted Quinolines as
Potential Antileishmanial Agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.08.028.
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Design, Synthesis and Biological evaluation of 2-Substituted Quinolines as Potential
Antileishmanial Agents
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Vadiraj S. Gopinath , Jakir Pinjari , Ravindra T. Dere , Aditya Verma , Preeti Vishwakarma ,
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Rahul Shivahare , Manjunath Moger , Palusa Sanath Kumar Goud , Vikram Ramanathan ,
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Prosenjit Bose , M.V.S. Rao , Suman Gupta* , Sunil K. Puri , Delphine Launay , Denis Martin
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Advinus Therapeutics Pvt. Ltd. Bangalore-560 058, India
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Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226 031, India
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Drugs for Neglected Diseases Initiative (DNDi), Geneva, Switzerland
*Corresponding author
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Division of Parasitology, CSIR-Central Drug Research Institute, Jankipuram extension,
Lucknow-226 031 (U.P.), India
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E-mail addresses: gupta_suman@yahoo.com, suman_gupta@cdri.res.in
Phone: +91 9415755899
Fax: +91 522 2623938
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ABSTRACT
An analogous library of 2-substituted quinoline compounds was synthesized with the aim to
identify a potential drug candidate to treat visceral leishmaniasis. These molecules were tested
for their in vitro and in vivo biological activity against Leishmania donovani. Metabolic stability
of these compounds was also improved through the introduction of halogen substituents.
Compound (26g), found to be the most active; exhibited an IC value of 0.2 ꢀM and >180 fold
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selectivity. The hydrochloride salt of (26g) showed 84.26 ± 4.44 percent inhibition at 50 mg/kg x
5days (twice daily, oral route) dose in L. donovani / hamster model. The efficacy was well
correlated with the PK data observed which indicating that the compound is well distributed.
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Keywords: 2-substituted quinolines, antileishmanial activity, luciferase assay, liver microsomes,
metabolic stability
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1. Introduction
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In 1903, Leishman and Donovan separately described a protozoan parasite found in the splenic
tissue of patients in India. Their simultaneous discovery of the protozoan, now called Leishmania
donovani, first alerted the scientific community to the life-threatening disease of Visceral
Leishmaniasis (VL) [1]. VL is a parasitic disease spread by the bite of an infected female
phlebotomine sand fly [2]. It is a geographically widespread prevalent disease in many parts of
the tropical and subtropical world causing significant morbidity or mortality [3]. This disease is a
severe public health problem in many developing countries of East Africa, the Indian
subcontinent and Latin America. According to the World Health Organization (WHO), the
pathogen is endemic in 88 countries and the magnitude of the disease is estimated to be 12
million infected people with 350 million people at risk. The rate of new cases per annum is
estimated to be 2 million worldwide [4]. Many names correspond to this group of diseases: kala-
azar, dum-dum fever, white leprosy, espundia or pian bois [5].
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In recent years, new compounds with antileishmanial activity have been developed. Pentavalent
antimonials, although first introduced 70 years ago, remain the first-line treatment in numerous
countries [6]. Amphotericin B [7], originally identified as a systemic polyene antifungal, is
currently used as an efficient second-line antileishmanial and its most recent liposomal
formulation (Ambisome ) tends to become a first line treatment despite its high cost. The
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inclusion of miltefosine (Impavido ) [8] into the therapeutic armamentarium of VL is a
landmark event for the therapy of VL, as it is the only oral treatment available to date.
Pentamidine [9] (e.g. PentamR), a diamidine, has been used in the treatment of antimonial-
resistant VL. However, treatment with these existing drugs suffers from several limitations such
as cost, toxicity, parenteral administration, emergence and spread of drug resistance, and relapses
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in HIV–Leishmania co-infected patients. A low cost injectable version of paromomycin [10] has
been registered in India for VL treatment and this drug is further evaluated both as mono and
combination therapies. Sitamaquine (1) [11] Figure 1 (WR6026), an 8-aminoquinoline
derivative, has been shown to have antileishmanial activity in Phase II studies, but confirmation
of such activity during Phase III studies is still lacking. Allopurinol [12] and rifampicin [13]
showed activity in experimental systems, but proved disappointing in clinical trials. Various
other compounds, such as atovaquone [14], licochalcone A [15], Ilmofosine [16], formycin B
[17] or Camptothecin [18] have been reported to inhibit L. donovani infection but never reached
to the clinical stage. Therefore, there is still a crying need for new efficacious and safe drugs in
the absence of an upcoming vaccine.
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Chimanines, structurally simple 2-substituted quinolines, have been reported by Fournet et al
[19] in the early 90s and conducted ethno-pharmacological studies in South America and have
discovered that alkaloids of the chemical family of quinolines had in vitro and in vivo
antiparasitic properties. However, these molecules did not bear all the needed features of a drug-
like entity, mainly because of their weak in vitro potency and metabolic instability (Table 1 and
2). Two of these parent compounds identified by Alain Fournet [19] were selected as reference
compounds: 2-n-propyl-quinoline (2) and 3-(quinolin-2-yl) prop-2-en-1-ol (14e). A library of
substituted quinolines was thus prepared and tested in vitro with the aim of addressing these
liabilities. The most potent compound (26g) was selected for further in vivo trials.
All compounds were tested in vitro against the intracellular form of L. donovani at CSIR-CDRI,
and for druggability at Advinus Therapeutics.
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2. Chemistry:
The parent compounds 2-n-propyl-quinoline (2) and 3-(quinolin-2-yl) prop-2-en-1-ol (14e) were
prepared by the method reported by Fakhfakh et al [20]. Quinoline (3) was converted to
quinoline N-oxide (4) using m-chloroperbenzoic acid, followed by bromination to 2-
bromoquinoline (5) which on reaction with propargyl alcohol in presence of tetrakis
(triphenylphosphine) palladium(0) catalyst and DIPEA as base affords 3-(quinolin-2-yl)prop-2-
yn-1-ol (6) in good yield as shown in Scheme 1.
Different synthesis strategies were developed depending upon the position of the substitution on
the quinoline ring which was targeted.
A series of di- and tri-substituted quinolines 14(a-e) and 22 were synthesized starting from
substituted anilines 7(a-e) as depicted in Scheme 2. 4-hydroxy substituted quinolines [21] 8(a-e)
and 9(a-e) were prepared starting from substituted anilines, ethylacetoacetate and
polyphosphoric acid, which resulted in two regio isomers. The mixture of isomers, when treated
with phosphorous tribromide gave 4-bromo quinoline derivatives 10(a-e). The isomers were
separated then by column chromatography and were used for next steps. Debromination of 10(a-
e) by using n-butyl lithium and quenching with water resulted in compounds 11(a-e).
Appropriate aldehydes 12(a-e) were obtained from 11(a-e) by oxidation of methyl group with
selenium dioxide in dioxane as shown in Scheme 2. Knoevenagal condensation followed by in
situ decarboxylation of aldehydes with malonic acid gave propenoic acid derivatives 13(a-e).
The propenoic acids were reduced with ethylchloroformate and sodium borohydride gave 14(a-
e). 2-substituted quinoline acid (13e) was converted to corresponding amide (16e) and then to
nitrile (17e) as per the Scheme 2. Further 3-(quinolin-2-yl) prop-2-enoic acid (13e) was
converted to 3-fluoroprop-1-en-1-yl] quinoline (15e) by the reaction of compound (14e) with
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diethylaminosulfur trifluoride (DAST). Similarly, compound (22) was obtained starting from
compound (9c) as per Scheme 3.
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Tetra-substituted quinoline derivatives were prepared from compound 10(a,c). The substitution
at C4 position on quinoline ring of compound 10(a,c) was achieved by Suzuki coupling with
appropriate aromatic boronic acids for aromatic substitution, whereas the cycloalkyl substitution
was achieved by heating the appropriate amines with cesium carbonate using
dimethylformamide as solvent. The compounds were further converted to the final compounds
26(a-l) as per Scheme 4.
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Methoxy derivative 28(g) was synthesized by reacting 25(g) with sodium methoxide to obtain
27(g) which on reaction with ethylchloroformate and sodium borohydride gave 28(g) with a
satisfactory yield (around 60%) as per Scheme 5.
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3. Results and discussion
3.1
In vitro activity profile and pharmacology:
To identify new and potent antileishmanial agents, the simple quinoline derivatives were initially
screened against the L. donovani intracellular amastigotes using the luciferase assay. The most
active compounds, 2-n-propylquinoline (2) and 3-(quinolin-2-yl) prop-2-en-1-ol (14e) were
resynthesized and tested in a murine model of visceral leishmaniasis.
The initial modifications on the quinoline ring at C2 position, as shown in Table 1 were aimed at
finding the best side chain. In preliminary studies, prop-2-yn-1-ol (6), prop-2-enoic acid (13e),
prop-2-enfluoride (15e), prop-2-enamide (16e), and prop-2-enenitrile (17e) derivatives were
synthesized. Compound bearing an amide substituent (16e) had an IC value of 20.41 µM and
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was extensively metabolized in mouse liver microsomes (% metabolism in 30 min: Human Liver
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Microsome (HLM) = 44, Mouse Liver Microsome (MLM) = 97). The nitrile derivative (17e) had
an IC value of 28.59 µM and was even more prone to extensive metabolism (% metabolism in
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30 min: HLM = 100, MLM = 100). The Prop-2-yn-1-ol (6) compound with a propynyl alcohol
side chain had an IC value of 10.69 µM (% metabolism in 30 min: HLM = 22, MLM = 99).
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The best active compound was (15e) with an IC value of 6.68 µM but it was quickly and
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completely metabolized (% metabolism in 30 min: HLM = 100, MLM = 100). Furthermore, it
exhibited very poor solubility (>1mg/mL). The next most active compound was the prop-2-en-1-
ol (14e) which exhibited an IC value of 10.04 µM, but it showed poor metabolic stability (%
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metabolism in 30 min: HLM = 96, MLM = 100) (Table 1 and Table 2). We further modified
this latter compound to further increase potency and improve metabolic stability by introducing
halogens, amines and aromatic rings at different positions on the quinoline ring.
The second modification was aimed at introducing chloro, fluoro, and methoxy substitution on
the ring (Table 3 and Table 4). Introduction of methoxy group at C6 position of the compound
(14e) resulted in compound (14d) which showed lower activity. A similar behaviour was
observed with 6-fluoro derivative compound (14b). However, substitution of chloro at C6-
position of compound (14e) resulted in compound (14a) which showed improved activity and
reduced cellular toxicity but it was completely metabolized in mouse liver microsomes (%
metabolism in 30 min: HLM = 87, MLM = 100). Di-substituted compounds (14c) and (22) were
synthesized by keeping chloro group intact at C6. Between compound (14c) having fluoro at C7
and compound (22) having fluoro C5, it was observed that the latter fluoro group at C7 position
has lower cytotoxicity and significantly improved metabolic stability (% metabolism in 30 min:
HLM = 50, MLM = 60). On these bases (chloro at C6 position and fluoro at C7 position) further
optimization was performed by adding substituents at C4 position on quinoline ring.
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Starting from compound (14c), several aryl groups were introduced at C4 position in the
quinoline ring. The most active from this series were (26a-d), (26f), (26k) and (26l). The
compound with an O-aryl group at C4 position (26h) gave better efficacy (1.96 µM) than (14c).
This indicated that substitution at C4 position was a key feature to obtain high in vitro potency.
Quinoline propenols provided improvement in metabolic stability compared to the initial
compound (14e). Insertion of morpholine at C4 resulted in improved potency as well as
selectivity as shown by compound (26a). Insertion of a fluoro substituent at C7 of compound
(26a) resulted in dramatic improvement in potency and metabolic stability (compound 26g)
(Table 5 and Table 6).
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In general, these set of modifications resulted in improved metabolic stability in human and
mouse liver microsomes but not in hamster liver microsomes (HamLM). Compound (26g) with
3-(6-Chloro-7-fluoro-4-morpholino) quinoline prop-2-en-1-ol was the most potent with an
IC value of 0.22 µM and 187 fold selectivity (% metabolism in 30 min: HLM = 35, MLM = 45)
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(Table 6).
Hence this compound was selected for detailed in vivo evaluation and
pharmacokinetics studies.
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3.2.
In vivo activity profile
Results are presented in Table 7. Based on the in vitro screening profile of >100 compounds,
three compounds (14e, 26g, 26k) were identified for determining the in vivo response in the
golden hamster model. These compounds were tested at 50 mg/kg x 5 days dose by the IP route.
One compound, (26g) has shown some anti-parasitic activity (% inhibition of parasite growth
=25.37 ± 19.14 PI). To improve solubility of this compound, the hydrochloride salt was prepared
and evaluated both by IP and PO routes. This hydrochloride salt of compound (26g) showed
significant activity at 50 mg/kg (twice daily) dose after oral administration (percentage inhibition
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(PI) recorded at day 7 post treatment was 84.26 ± 4.44). When animals were treated at the same
dose regimen via IP route, a slightly lower activity was observed (77.16 ± 5.23 PI). Three more
analogs of (26g) were tested (data not shown) at 50 mg/kg (twice daily) x 5 days, following PO
administration, but none of them showed any promising activity.
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3.3.
Pharmacokinetic study profile of compound (26g)
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The pharmacokinetics results are presented in Table 8. Compound (26g) showed an extremely
high plasma clearance (865 mL/min/kg, which is 10-fold higher than liver blood flow of 90
mL/min/kg). Its volume of distribution was high (7.5 L/kg, which was 11-fold higher than total
body water of 0.7 L/kg) indicating that the compound is well distributed. Its short intravenous
elimination half-life of about 0.5 h was driven primarily by the abnormally high clearance. The
high clearance value, in relation to liver plasma flow, suggested that the liver was not the only
site of elimination. This was corroborated by direct in vitro experiments showing that the
compound was largely stable in mouse liver microsomes (Table 6). In vitro whole blood and
plasma stability experiments showed that compound (26g) was stable in plasma but unstable in
whole blood. This could be due to metabolism with some cellular component of the whole blood.
The oral solution bioavailability was found to be very low (~1%) (Figure 2).
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4. Conclusion
Overall, a library of new compounds were synthesized and assessed for in vitro antileishmanial
activity and most of them were also evaluated for their metabolic stability. A significant number
of compounds exhibited potent in vitro activity against L. donovani, as well as a high selectivity
index (i.e., activity with low cytotoxicity). As a reminder, the parent compound (n-propyl
quinoline) had an IC value of about 40 µM whereas several new compounds were in the range
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of 0.2 to 2 µM with improved metabolic stability in liver microsomes. The compound (26g) was
found most promising one when evaluated in vitro in mouse macrophage cell line and in vivo in
golden hamster / L. donovani model. This compound (26g) was further evaluated in a range of
early DMPK and pharmacology assays. These results clearly state that the lead compound (26g)
with morpholine at C4 position, chloro at C6 position and fluoro at C7 position showed very
promising activity and metabolic stability compared to parent compound (14e), thus provide a
new structural lead to develop an orally active antileishmanial agent.
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5. Experimental section
5.1. Chemistry
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5.1.1. General
All the reagents and solvents were used as received. Starting materials were commercially
available. Reactions were performed under anhydrous conditions unless noted otherwise.
Reactions were followed by TLC analysis on Merck TLC aluminium sheets with silica gel 60
F254. The purities of compounds for biological testing were assessed by analytical HPLC.
LC/MS analysis was performed on an Agilent 1200 MSD system with an Inertsil ODS-3V (250
x 4.6 mm, 5 µm particle size). Chromatograms for electrospray ionization (ESI) positive and
negative base peak intensity and a UV total absorption chromatogram from 220-300 nm were
generated, and values for m/z are given; generally, only ions that indicate the parent mass are
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reported, and unless otherwise stated, the value quoted is (M + H) for positive-ion mode and (M
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- H) for negative-ion mode. Melting points were measured on a Buchi-546 B and were
uncorrected. Preparative HPLC (Agilent 1200 series) was performed on an Inertsil ODS-3V
column (250 x 21 mm, 5µm particle size) using a gradient of 10-90% acetonitrile/10 mm
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ammonium acetate or 0.1% TFA over 20 min at a flow rate 20 mL/min. Nuclear magnetic
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resonance ( H NMR and C NMR) spectra were recorded on a 400 MHz (VARIAN AVANCE
400 AS) spectrometer. Chemical shift data for the proton and carbon resonances were reported in
parts per million (δ) relative to internal standards (CH ) Si (δ 0.0). All coupling constants (J) are
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given in Hz. The purities of the final compounds were determined with Agilent 1200 HPLC
system.
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5.1.2. General procedure for synthesis of substituted quinoline hydroxy 8(a-e) and 9(a-e):
Substituted anilines 7(a-e) (1mmol) and PPA (6mL) was heated to 80°C, ethylacetoacetate
(1.2mmol) was added drop wise for 45min. and heated to 120°C for 16h. Completion of the
reaction was monitored by TLC. The RM was poured over crushed ice and neutralized with
aqueous ammonia (25%); solid precipitated was filtered, washed with diethyl ether and dried.
Product obtained was taken directly to next step without purification.
5.1.3. General procedure for synthesis of substituted quinoline bromo 10(a-e):
Phosphorous tribromide (1.5mmol) was added drop wise to a solution of compounds 8(a-e)
(1mmol) in DMF (10mL) at 0°C. After complete addition, the reaction mass to allow to room
temperature and stirred for 3 hours. After completion of the reaction, transferred the reaction
mass to crushed ice and neutralized with aqueous ammonia. The precipitated solid obtained was
filtered, and dried. The pure compounds were isolated by column chromatography using silica
gel 230-400, eluting with 10% ethyl acetate in hexane.
5.1.4. General procedure for synthesis of substituted quinoline 11(a-e):
n-Butyl lithium (2.3M, 1.2mmol) was added to the solution of compounds 10(a-e) (1mmol) in
dry THF at -78°C and stirred for 30 min. Reaction was quenched with aqueous NH Cl solution
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(5mL) and warmed to 0°C. Ice cold water was added to the reaction mixture and extracted with
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ethyl acetate. The organic layer was washed with water, brine solution and dried over sodium
sulphate, concentrated under reduced pressure. The crude product was triturated with 20%
diethyl ether in hexane to afford the title compounds.
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5.1.5. General procedure for the synthesis of 4-(substituted) -2-methyl-substituted
quinoline derivatives 23(b,c,d,k,l): To a solution of 10(a,c) (1mmol) in THF and water (3:1),
aryl boronic acid (1.2mmol), and K CO (1.5mmol) was added under nitrogen atmosphere.
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Tetrakis triphenyl phosphine palladium (0) (0.05mmol) was added and heated to 65 C for 16 h.
After completion of the reaction, organic layer separated from the aqueous layer. The aqueous
layer was extracted with EtOAc. The combined organic layer was dried over sodium sulphate,
evaporated and purified by column chromatography, eluting with ethyl acetate in hexane.
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5.1.6. General procedure for the synthesis of 4-(substituted) -2-methyl-substituted
quinoline derivatives 23(a,e,f,g,h,i,j): To the solution of 10(a,c) (1mmol) in DMF (10mL), was
added cesium carbonate (1.5mmol) and sec-amine (1.5mmol). The reaction mass was heated to
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100 C for 4h. After completion of reaction, transferred to cold water (100 mL) and extracted
with ethyl acetate. The organic layer was dried over sodium sulphate, evaporated and purified by
column chromatography, eluting with Ethyl acetate: hexane.
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5.1.7. General procedure for synthesis of substituted quinoline aldehydes 12(a-e) and 24(a-
l): A suspension of compound 11(a-e) and 23(a-l) (1mmol) in 1, 4-dioxane (10 mL) and SeO2
(2mmol) was heated to 80ºC for 2 h. After completion of the reaction, the inorganic compounds
were filtered and poured in to ice water. The precipitated solid was then filtered and dried. The
crude product was purified by column chromatography eluting with ethyl acetate: hexane
mixture. % Yield = 55-80 %.
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5.1.8. General procedure for synthesis of substituted quinoline acids 13(a-e) and 25(a-l):
To a solution of compound 12(a-e) and 24(a-l) (1mmol) in pyridine (10mL), malonic acid
o
(1.5mmol) was added and heated to 70 C for 2 h. After completion of the reaction, the solution
was poured into ice water and acidified with hydrochloric acid to pH~4. The precipitated solid
was filtered and dried to get the compounds 13(a-e) and 25(a-l).
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5.1.9. General procedure for synthesis of quinoline propenols 14(a-e) and 26(a-l): To the
solution of compound 13(a-e) and 25(a-l) (1mmol) in THF (10mL), triethyl amine (3mmol), and
o
ethyl chloroformate (2mmol) was added at 0 C and stirred for 15 min. After completion of the
o
reaction, the solid was filtered and washed with THF (20mL). The filtrate was cooled to 5 C and
sodium borohydride solution (3mmol) was added drop wise. After completion of the reaction,
the organic layer was separated. The aqueous layer was extracted with ethyl acetate (10mLx 2).
All the organic layers were combined and dried over sodium sulphate. The crude product was
obtained after evaporation and then purified by column chromatography using ethyl acetate:
hexane (10-20 %) as eluent.
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5.1.10. General procedure for the synthesis of quinoline chalcone derivatives 29(a-g): To a
solution of compound 24(a-d) (1mmol) in acetic acid (2mL), ketone (1mmol) and sulfuric acid
o
(0.2mL) was added and heated to 60 C for 2 h. After completion of the reaction, the mixture was
cooled to RT, neutralized with NaHCO solution and extracted with ethyl acetate (25mL x 3).
3
The organic layer was washed with water (25mL) and saturated brine solution (25mL), and then
dried over sodium sulphate and evaporated. The crude product was purified by column
chromatography using basic alumina as stationary phase and eluting with EtOAc: hexane as
eluent.
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5.2.
Biology
5.2.1. Parasite
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The WHO reference strain of L. donovani (MHOM/IN/80/Dd8) obtained from Imperial College,
London (UK), was maintained as promastigotes in vitro and as amastigotes in golden hamsters.
Promastigotes were cultivated in medium 199 (Sigma-Aldrich) supplemented with 0.1%
gentamycin (Biovaccines Private Ltd., Chevella, India) and 10% Fetal Calf Serum [22].
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For in vivo antileishmanial study (in CSIR-CDRI, Lucknow, India), Healthy inbred hamsters
weighing 40-45g (8-10 week-old) of both sexes were used. Throughout the study, animals were
housed in controlled animal quarters (23 ± 2°C; Relative humidity: 60%) with 12h light-dark
cycles). For Pharmacokinetic Studies (in Advinus Therapeutics, Bangalore, India), Male Swiss
Albino mice, 8 to 12 weeks old, 30-40 g weight range employing a sparse sampling design were
used. Animals were fasted 4 h before dose administration and feed was provided 4 h post dose.
Animals were fed standard rodent pellet and had free access to drinking water. These studies
were performed with the approval from the Institutional Animal Ethics Committee (IAEC) in
respective places accordance with the requirement of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), India.
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5.2.3. Compound/Chemicals
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Analytical standards of compounds were supplied by the Department of Process Chemistry,
Advinus Therapeutics Limited, Bangalore, India. Cremophor® EL (BASF, Germany), N-N-
Dimethyl acetamide (Sigma-Aldrich, USA), Methyl Cellulose (Sigma-Aldrich, USA),
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acetonitrile (Rankem, India), methanol (Rankem, India), formic acid (Fluka, Germany),
ammonium formate (Fluka, Germany) and other general laboratory chemicals and solvents were
of analytical grade.
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5.2.4. Antiamastigote activity
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For assessing the activity of compounds against the amastigote stage of the parasite, mouse
macrophage cell line (J-774A.1) infected with promastigotes expressing luciferase firefly
3
reporter gene was used. Cells were seeded in a 96-well plate (4 x 10 cell/100µL/well) in RPMI-
1640 medium containing 10% foetal calf serum and the plates were incubated at 37ºC in a CO2
incubator. After 24 h, the medium was replaced with fresh medium containing stationary phase
4
promastigotes (4 x10 /100µL/well). Promastigotes invade the macrophage and are transformed
into amastigotes. Each well of the plate was washed with plain RPMI medium after 24 h of
incubation to remove the un-internalized promastigotes. The test compounds diluted serially up
to 7 points in complete medium starting from 100µM and the plates were incubated at 37ºC in a
CO incubator for 72 h. After incubation, the drug containing medium was aspirated and 50 µL
2
PBS was added in each well and mixed with an equal volume of Steady Glo reagent. After gentle
shaking for 3 minutes, the reading was taken in a luminometer [23]. The values are expressed as
relative luminescence units (RLU). Data were plotted using Excel software. IC50 values of
antileishmanial activity were calculated by nonlinear regression analysis of the concentration
response curve using the four parameters Hill equations.
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5.2.5. Cytotoxicity assay
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The cell viability was determined using the MTT (3- (4, 5-Dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide, a yellow tetrazole) assay [24]. Exponentially growing mammalian
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kidney epithelial cells (Vero Cell line) (1 x 10 cells/100µl/well) were incubated with test
compounds for 72 h. The test compounds were added at three fold dilutions up to 7 points in
complete medium starting from 400 µM concentration, and were incubated at 37ºC in a
humidified mixture of 5% CO in an incubator. Podophyllotoxin was used as a reference drug
2
and control wells containing dimethyl sulfoxide (DMSO) without compounds were also included
in the experiment. Stock solutions of compounds were initially dissolved in DMSO and further
diluted with fresh complete medium. After incubation, 25 ꢀL of MTT reagent (5mg/mL) in PBS
medium was added to each well and incubated at 37°C for 2 h. At the end of the incubation
period, the supernatant was removed by inverting the plate completely without disturbing the cell
layer and 150 µL of pure DMSO were added to each well. After 15 min of shaking the readings
were recorded a wavelength maxima of at 544 nm on a micro plate reader. The cytotoxic effect
were expressed as 50% lethal dose (CC ), i.e., as the concentration of a compound which
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reduced 50% of cell viability compared to cell in culture medium alone. CC50 values were
estimated as described by Huber and Koella [25]. The selectivity index (SI) for each compound
was calculated as ratio between, cytotoxicity (CC ) and activity (IC ) against Leishmania
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amastigotes.
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5.2.6. In vivo assay
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The method of Beveridge [26], as modified by Bhatnagar et al [27] and Gupta et al [28] was
used for in vivo screening. Golden hamsters (Inbred strain) of either sex weighing 40-45g were
7
infected intracardiacally with 1 x 10 amastigotes per animal. The infection is well adapted to
the hamster model and establishes itself in 15-20 days. Meanwhile, hamsters gain weight (85-95
g) and can be subjected to repeated spleen biopsies. Pre-treatment spleen biopsy in all the
animals was carried out to assess the degree of infection. The animals with +1 grade of infection
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(5-15 amastigotes/100 spleen cell nuclei) were included in the chemotherapeutic trials. The
infected animals were randomized into several groups on the basis of their parasitic burdens.
Five to six animals were used for each test sample. Drug treatment at 50 mg/kg dose via IP or PO
route was initiated two days after biopsy (48 h recovery) and continued for 5 consecutive days.
Miltefosine (pure) at dose of 30 mg/kg x 5 days was used as reference drug. Post-treatment
biopsies were done on day 7 of the last drug administration and amastigote counts were assessed
by Giemsa staining. Intensity of infection in both, treated and untreated animals, as also the
initial count in treated animals was compared and the efficacy was expressed in terms of
percentage inhibition (PI) using the following formula:-
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PI = 100- [ANAT x 100 / (INAT x TIUC)]
Where PI is Percent Inhibition of Amastigotes multiplication,
ANAT is Actual Number of Amastigotes in Treated animals,
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INAT is Initial Number of Amastigotes in Treated animals and
TIUC is Times Increase of parasites in Untreated Control animals.
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5.2.7. Administration of test samples
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Aqueous solution of test compounds was prepared by dissolving the accurately weighed sample
in distilled water/PBS. Required quantity of the compounds were dissolved once and aliquoted in
five tubes, one for each day and kept at 4°C till used.
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5.2.8. Metabolic stability assay
Screening studies for metabolic stability were performed with hamster, mouse and human liver
microsomes, and degradation at 30 minutes in the presence and absence of cofactor was
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measured by LC/MS/MS analysis. Liver microsomes (Xenotech), NADPH (Sigma), sodium
dihydrogen orthophosphate (Merck), disodium hydrogen orthophosphate (Merck), acetonitrile
(Merck) and DMSO (Sigma) were procured for the study. Stock solution (200 µM) of test
compound was prepared in Acetonitrile:DMSO mixture (96:4). NADPH solution (cofactor) was
prepared by dissolving 100 mg of NADPH in phosphate buffer (24 mL) to produce final
NADPH concentration of 5 mM. The reaction mixture containing sodium phosphate buffer (100
µM, pH 7.4), NADPH solution (5 mM), and liver microsomes (final protein concentration 1
o
mg/mL) was incubated at 37 C for 10 min. The reaction was initiated by addition of 5 µL of
compound stock solution (1 ꢀM). Aliquots (50 µL) from the reaction mixture were withdrawn at
0 and 30 min and quenched by adding to 50 µL of stop solution comprising internal standard in a
79:20:1 mixture of acetonitrile, ethanol and acetic acid. The analyte/internal standard peak area
ratio was determined in each sample by LC/MS/MS. The results are expressed as percent drug
metabolized based on the levels at the start of the incubation (0 min sample). NADPH free
reaction were performed similarly to verify non-microsomal degradation/solubility/stability
issues. Percentage of compound metabolized after 30 min of incubation was calculated.
5.2.9. Pharmacokinetic studies
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5.2.9.1. Study design
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Tested compound was administered at 10 mg/kg (IV) and 50 mg/kg (PO). The study used a
parallel design with two groups typically comprising 9 mice each for the IV and PO dose.
Animals in IV group were administered a bolus in the tail vein. Animals in PO group were dose
via oral gavages. Blood samples were collected from the orbital plexus from 3 mice at the
following time points 0.083 (only for IV), 0.25, 0.50, 1, 2, 4, 8, 12 and 24 h into microfuge tubes
containing K EDTA (20 µL of 200 mM per mL of blood). Plasma was harvested from blood
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samples by centrifugation at 8000 rpm for 5 min immediately after collection and stored below -
70 ºC until analyzed.
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5.2.9.2. Bio-analysis
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All in vivo plasma samples were analyzed using fit-for-purpose liquid chromatography tandem
mass spectrometric (LC-MS/MS) method with suitable internal standard. The calibration curve
(CC) range was 1–1000 ng/mL in all cases. Quality control (QC) samples were included at low
(3X of lower limit of quantitation), medium (close to middle of CC) and high levels (85% of
upper limit of quantitation). The CC and QC samples were prepared by spiking 2 µL of the test
item spiking solution into 98 µL of mouse blood or plasma. After mixing, a 25 µL aliquot was
mixed with 125 µL of internal standard solution. Study samples were similarly processed. After
vortex mixing (5 min), the processed samples were centrifuged (14000 rpm, 10 min) and
supernatant transferred to auto sampler vials and 5 µL of supernatant was injected in to the LC-
MS/MS for analysis [29].
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An Applied Biosystems, Sciex API 4000 triple quadrupole mass spectrometer (LC-MS/MS)
equipped with a Turbo Ion spray source interfaced with mass spectrometer was used for analysis
of all the compounds. Shimadzu Prominence LC-20 AD HPLC was system interfaced with the
mass spectrometer. All compounds were analyzed in positive ion mode using an electro spray
ion source and MRM (Multi reaction monitoring). Chromatographic separation was achieved by
using Kromasil C8 analytical column (100 mm×4.6 mm, 5µ, Akzo Nobel, Sweden) maintained
at 40 ºC using a mobile phase comprising methanol (60%), 5 mM ammonium formate solution
(40%) and formic acid (0.05%) with a flow rate of 0.5 mL/min. Chromatograms were acquired
®
using Analyst software version 1.4.1 and the data were processed using peak area ratio method.
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Calibration curves were obtained by plotting the peak area ratio against the analyte
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concentration. A weighted (w = 1/X , where X is concentration) least squares regression analysis
was used to obtain a linear equation over the range of the calibration [30].
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Pharmacokinetic parameters were calculated using non compartmental analysis tool of validated
WinNonlin (version 5.2, Pharsight Co., Mountain View, CA) software. Estimated parameters
included apparent clearance, volume of distribution at steady state (Vss), terminal half-life (T ),
1
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area under the concentration-time curve from time zero to the last measurable concentration
(AUC0-last) and the area under the concentration-time curve extrapolated to infinity (AUC0-inf).
The maximum observed concentration of drug in plasma/blood (Cmax) and the time of maximum
observed concentration in plasma/blood (Tmax) were also reported., Bioavailability was
calculated as (AUCoral or IP/ Dose oral or IP) / (AUC / Dose )×100 [31].
IV
IV
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Acknowledgment
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The authors thank Dr. T. K. Chakraborty, Director, CSIR-CDRI, Lucknow, for his
encouragement and facilities. The transgenic L. donovani promastigotes were originally procured
from Dr. Neena Goyal, Division of Biochemistry, CSIR-Central Drug Research Institute,
Lucknow, India.
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Funding
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This work was supported by the Drug for Neglected Disease Initiative (DNDi). Specific funding
from Bill & Melinda Gates Foundation, USA, with complementary core funding from
Department for International Development (DFID), UK; Dutch Ministry of Foreign Affairs
(DGIS), Nederland; Federal Ministry of Education and Research (BMBF), Germany; Spanish
Agency for International Development Cooperation (AECID), Spain; Swiss Agency for
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Development and Cooperation (SDC), Switzerland and Médecins Sans Frontières (Doctors
without Borders), International.
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Appendix A. Supplementary information
Supplementary data related to this article can be found at………
References
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[1] L. Rogers, Note on the occurrence of leishman-donovan bodies in "cachexial fevers"
including Kala-azar, Br. Med. J. 1 (1904) 1249-1251.
4
4
50
51
[2] P. Desjeux, The increase of risk factors for leishmaniasis worldwide, Trans. R. Soc. Trop.
Med. Hyg. 95 (2001) 239-243.
4
4
52
53
[3] R.D. Pearson, A. de Queiroz Sousa, Clinical spectrum of Leishmaniasis. Clin. Infect. Dis. 22
(1996) 1-11.
4
4
54
55
[4] P. Trouiller, P. Olliaro, Drug development output from 1975 to 1996 what proportion for
tropical diseases? Int. J. Infect. Dis. 3 (1999) 61-63.
4
4
56
57
[5] S. Kumari, V.J. Ram, Visceral Leishmaniasis: clinical features, pathology, diagnosis and
chemotherapeutic developments, Drug News Prospect. 15 (2002) 417-431.
4
4
4
4
4
58
59
60
61
62
[6] M. Reddy, S.S. Gill, S.R. Kalkar, W. Wu, P.J. Anderson, P.A. Rochon, Oral drug therapy for
multiple neglected tropical diseases: a systematic review, JAMA. 298 (2007) 1911-1924.
[7] C.P. Thakur, R.K. Singh, S.M. Hassan, R. Kumar, S. Narain, A. Kumar, Amphotericin B
deoxycholate treatment of visceral leishmaniasis with newer modes of administration and
precautions: a study of 938 cases, Trans. R. Soc. Trop. Med. Hyg. 93 (1999) 319-323.
2
1

ACCEPTED MANUSCRIPT
4
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4
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4
4
4
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4
4
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4
4
4
4
4
4
4
4
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
[8] H. Sindermann, S.L. Croft, K.R. Engel, W. Bommer, H.J. Eibl, C. Unger, J. Engel,
Miltefosine (Impavido): the first oral treatment against leishmaniasis. Med. Microbiol. Immunol.
193 (2004) 173-180.
[9] T.A. Patel, D.N. Lockwood, Pentamidine as secondary prophylaxis for visceral leishmaniasis
in the immunocompromised host: report of four cases. Trop. Med. Int. Health. 14 (2009) 1064-
1070.
[10] S. Sundar, T.K. Jha, P.T. Chandreshwar, K.S. Prabhat, S.K. Bhattacharya, Injectable
Paromomycin for Visceral Leishmaniasis in India, N. Engl. J. Med. 356 (2007) 2567-2569.
[11] R. Dietze, S. F. Carvalho, L.C. Valli, J. Berman, T. Brewer, W. Milhous, J. Sanchez, B.
Schuster, M. Grogl, Phase 2 trial of WR6026, an orally administered 8-aminoquinoline, in the
treatment of visceral leishmaniasis caused by Leishmania chagasi, Am. J. Trop. Med. Hyg. 65
(2001) 685-689.
[12] B.C. Walton, J. Harper, R.A. Neal, Effectiveness of Allopurinol against Leishmania
braziliensis panamensis in Aotus trivirgatus, Am. J. Trop. Med. Hyg. 32 (1983) 46-50.
[13] R.A. Bumb, R.D. Mehta, Oral rifampicin in cases of cutaneous leishmaniasis with multiple
lesions (A pilot study), Indian J. Dermatol. Venereol. Leprol. 68 (2002) 272-272.
[14] H.W. Murray, J. Hariprashad, Activity of oral atovaquone alone and in combination with
antimony in experimental visceral leishmaniasis, Antimicrob. Agents Chemother. 40 (1996) 586-
587.
[15] H.L. Ziegler, H.S. Hansen, D. Staerk, S.B. Christensen, H. Hägerstrand, J.W. Jaroszewski,
The antiparasitic compound licochalcone A is a potent echinocytogenic agent that modifies
the erythrocyte membrane in the concentration range where antiplasmodial activity is observed,
Antimicrob. Agents Chemother. 48 (2004) 4067-4071
2
2

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4
4
4
4
4
4
4
4
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4
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5
5
5
5
5
86
87
88
89
90
91
92
93
94
95
96
97
98
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00
01
02
03
04
05
06
07
[16] S.L. Croft, R.A. Neal, E.A. Thornton, D.B. Herrmann, Antileishmanial activity of the ether
phospholipid ilmofosine, Trans. R. Soc. Trop. Med. Hyg. 87 (1993) 217-219.
[17] J.D. Berman, J.V. Gallalee, Antileishmanial activity of human red blood cells containing
Formycin A, J. Infect. Dis. 151 (1985) 698-703.
[18] C.P. Thakur, R.K. Singh, S.M. Hassan, R. Kumar, S. Narain, A. Kumar, Amphotericin B
deoxycholate treatment of visceral leishmaniasis with newer modes of administration and
precautions: a study of 938 cases, Trans. R. Soc. Trop. Med. Hyg. 93 (1999) 319-323.
[19] A. Fournet, A.A. Barrios, V. Munoz, R. Hocquemiller, A. Cave, J. Bruneton, 2- substituted
quinoline alkaloids as potential antileishmanial drugs, Antimicrob. Agents Chemother. 37 (1993)
859-863.
[20] M.A. Fakhfakh, A. Fournet, E. Prina, J.F. Mouscadet, X. Franck, R. Hocquemiller, B,
Figadère, Synthesis and biological evaluation of substituted quinolines: potential treatment of
protozoal and retroviral co-infections, Bioorg. Med. Chem. 11 (2003) 5013-5023.
[21] C.R. Hauser, G.A. Reynolds, Reactions of β-Keto Esters with Aromatic Amines. Syntheses
of 2- and 4-Hydroxyquinoline Derivatives, J. Am. Chem. Soc. 70 (1948) 2402-2404.
[22] S. Gupta, S.C. Sharma, V. M. L. Srivastava, Efficacy of picroliv in combination with
miltefosine, an orally effective antileishmanial drug against experimental visceral leishmaniasis,
Acta Trop. 94 (2005) 41-47.
[23] M. Sharma, K. Chauhan, R. Shivahare, P. Vishwakarma, M. Suthar, A. Sharma, S. Gupta,
J.K. Saxena, J. Lal, P. Chandra, B. Kumar, P.M.S. Chauhan. Discovery of a new class of natural
product-inspired quinazolinone hybrid as potent antileishmanial agents. J. Med. Chem. (2013)
DOI: 10.1021/jm400053v
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ACCEPTED MANUSCRIPT
5
5
5
5
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13
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15
16
17
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20
21
22
23
24
25
26
27
28
29
30
[24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55-63.
[25] W. Huber, J.C. Koella, A comparison of three methods of estimating EC50 in studies of
drug resistance of malaria parasites, Acta Trop. 55 (1993) 257-261.
[26] E. Beveridge, Chemotherapy of leishmaniasis, in: R.J. Schnitzer, F. Hawking (Eds.),
Experimental Chemotherapy, Academic press, New York, London, 1963, pp. 257-280
[27] S. Bhatnagar, P.Y. Guru, J.C. Katiyar, R. Srivastava, A. Mukherjee, M.S. Akhtar, M. Seth,
A.P. Bhaduri, Exploration of antileishmanial activity in heterocycles; results of their in vivo & in
vitro bioevaluations, Indian J. Med. Res. 89 (1989) 439-444.
[28] S. Gupta, S. Tiwari, A.P. Bhaduri, and G.K. Jain, Anilino-(2-bromophenyl) acetonitrile: a
promising orally effective antileishmanial agent, Acta Trop. 84 (2002) 165-173.
[29] M. Jemal, Z. Ouyang, Y.Q, Xia, Systematic LC-MS/MS bioanalytical method development
that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well
thought-out chromatography, Biomed. Chromatogr. 24 (2010) 2-19.
[30] T. Singtoroj, J. Tarning, A. Annerberg, M. Ashton, Y. Bergqvist, N.J. White, N.
Lindegardh, N. P. Day, A new approach to evaluate regression models during validation of
bioanalytical assays, J. Pharm. Biomed. Anal. 41 (2006) 219-227.
[31] WinNonlin® Version 4.1 User’s guide, Chapter 9, Non-compartmental Analysis. Page 215-
240, Pharsight Corporation, Mountain View, California 94040.
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Figure captions:
Figure 1: Sitamaquine (1)
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Figure 2: Mean plasma concentration-time profile of compound (26g) in male Swiss Albino
mouse after intravenous bolus (10 mg/kg) and oral gavage (50 mg/kg) administration
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Scheme 1: Synthesis of 3-(quinolin-2-yl) prop-2-yn-1-ol
Scheme 2: Synthesis of substituted quinoline derivatives
Scheme 3: Synthesis of tri-substituted (quinolin-2-yl) prop-2-en-1-ols
Scheme 4: Synthesis of tetra substituted (quinolin-2-yl) prop-2-en-1-ols
Scheme 5: Synthesis of tetra substituted methoxy (quinolin-2-yl) prop-2-en-1-ols
Table 1: Activity and cytotoxicity data for 2-substituted quinoline derivatives
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Table 2: Metabolic stability of 2-substituted quinoline derivatives
Table 3: Activity and cytotoxicity data for di and tri substituted quinoline propenols
Table 4: Metabolic stability data for di and tri substituted quinoline propenols
Table 5: Activity and cytotoxicity data for tetra substituted quinoline propenols
Table 6: Metabolic stability of tetra substituted quinoline propenols
Table 7: In vivo efficacy of 2-substituted quinoline derivatives
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Table 8: Pharmacokinetics parameters of compound (26g) in male Swiss Albino mice following
intravenous and per oral administration
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Figure 1:
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Figure 2:
Plasma Concentration (ng/mL)
000
1
IV (10 mg/kg)
PO (50 mg/kg)
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0
0
.1
0
2
4
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8
Time (h)
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Scheme 1:
(i)
(ii)
(iii)
N
N
O
N
Br
N
OH
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Reaction and conditions: (i) m-Chloroperbenzoic acid, dichloromethane (ii) POBr / DCM (iii)
3
Propargyl alcohol / Pd[P(C H ) ] / DIPEA, 70-75ºC
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5 3 4
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Scheme 2:
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o
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Reagents and conditions: (i) Polyphosphoric acid, Ethyl acetoacetate, 120 C, 16 h (ii) PBr ,
3
o
DMF, 3h; (iii) n-BuLi / THF, Water (iv) SeO , 1,4-dioxane, 70 C (v) Malonic acid, pyridine, 1 h
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(vi) Ethyl chloroformate, THF, TEA, NaBH4 (vii) DAST / dichloromethane (viii)
o
Ethylchloroformate / Aq. Ammonia (ix) POCl / 110 C / 16 h.
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Scheme 3:
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o
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Reagents and conditions: (i) Polyphosphoric acid, Ethyl acetoacetate, 120 C, 16 h (ii) PBr ,
3
o
DMF, 3h; (iii) n-BuLi / THF, Water (iv) SeO , 1,4-dioxane, 70 C (iv) Malonic acid, pyridine, 1
2
h (v) Ethyl chloroformate, THF, TEA, NaBH4
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Scheme 4:
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o
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Reagents and conditions: (i) Pd (PPh ) , Aryl boronic acid, THF/water, K CO . 70 C, 16 h or
3 4 2 3
o
R NH / Cs CO / DMF, 4-16 h. (ii) SeO , 1,4-dioxane, 70 C (iii) Malonic acid, pyridine, 1 h (iv)
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o
Ethylchloro formate, THF, TEA, NaBH , 0 C.
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Scheme 5:
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o
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Reagents and conditions: (i) Pd(PPh ) , Aryl boronic acid, THF/water, K CO . 70 C, 16 h or
3 4 2 3
o
R -NH / Cs CO / DMF, 4-16 h. (ii) SeO , 1,4-dioxane, 70 C (iii) Malonic acid, pyridine, 1 h (iv)
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2
3
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o
o
NaOMe, DMSO, 0 C (v) Ethylchloro formate, THF, TEA, NaBH , 0 C.
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