Design, synthesis, ADME characterization and antileishmanial evaluation of novel substituted quinoline analogs

Article abstract of DOI:10.1016/j.bmcl.2014.03.065

In vitro ADME characterization of the lead compound 1 identified for visceral leishmaniasis was undertaken and further structural analogs were synthesized for antileishmanial screening. Compound 1 was highly permeable in intestinal PAMPA model (31 × 10^-6 cm/s) and was moderately bound to mouse and human plasma proteins (% bound 85-95%), its blood to plasma concentration ratio was less than 1, but the compound was unstable in blood. Compound 1 was found to have no CYP450 liability with CYP2C9, 2C19, 2D6 and 3A4. It showed inhibition with CYP1A2 with an IC₅₀ value of 0.50 μM. Analogs of 1 were synthesized and subsequently characterized for in vitro activity against the intracellular form of Leishmania donovani. Resulting quinolines were found to have similar efficacy as 1 against the parasite. Compounds 8b and 8f were found to be the most active with IC₅₀ values of 0.84 μM and 0.17 μM, respectively compared to 0.22 μM for compound 1. Of all the analogs tested, 8d was stable in hamster, mouse and human liver microsomes but lost the efficacy with an IC₅₀ of 6.42 μM. Based on the in vitro efficacy and DMPK profile, compounds 8b and 8f seem the best candidates to be screened in further assays.

Full text of DOI:10.1016/j.bmcl.2014.03.065

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2046–2052
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, ADME characterization and antileishmanial
evaluation of novel substituted quinoline analogs
Vadiraj S. Gopinath a, Mukkavilli Rao a, Rahul Shivahare b, Preeti Vishwakarma b, Sweta Ghose a,
Ashok Pradhan a, Ramamohan Hindupur a, Koushik Das Sarma a, Suman Gupta b, , Sunil K. Puri b,
⇑
Delphine Launay c, Denis Martin c
a Advinus Therapeutics Ltd, Bangalore 560 058, India
b Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226 031, India
c Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
In vitro ADME characterization of the lead compound 1 identified for visceral leishmaniasis was under-
taken and further structural analogs were synthesized for antileishmanial screening. Compound 1 was
highly permeable in intestinal PAMPA model (31 Â 10À6 cm/s) and was moderately bound to mouse
and human plasma proteins (% bound 85–95%), its blood to plasma concentration ratio was less than
1, but the compound was unstable in blood. Compound 1 was found to have no CYP450 liability with
Received 31 January 2014
Revised 19 March 2014
Accepted 20 March 2014
Available online 28 March 2014
CYP2C9, 2C19, 2D6 and 3A4. It showed inhibition with CYP1A2 with an IC50 value of 0.50 lM. Analogs
of 1 were synthesized and subsequently characterized for in vitro activity against the intracellular form
of Leishmania donovani. Resulting quinolines were found to have similar efficacy as 1 against the parasite.
Keywords:
Substituted quinoline-chalcones
Leishmania donovani
In vitro activity
Compounds 8b and 8f were found to be the most active with IC50 values of 0.84
tively compared to 0.22 M for compound 1. Of all the analogs tested, 8d was stable in hamster, mouse
and human liver microsomes but lost the efficacy with an IC50 of 6.42 M. Based on the in vitro efficacy
lM and 0.17 lM, respec-
l
ADME assays
l
and DMPK profile, compounds 8b and 8f seem the best candidates to be screened in further assays.
Ó 2014 Elsevier Ltd. All rights reserved.
Leishmaniasis comprises a variety of parasitic illnesses caused
by several species of the protozoan kinetoplastid Leishmania
parasites. Leishmaniasis is transmitted by phlebotomine sand flies
and is spread over large geographical areas around the globe.
Leishmania organisms are endemic in more than 80 countries and
350 million people are considered to be at risk worldwide.1
Leishmaniasis is a major public health problem causing significant
morbidity and mortality in Africa, Asia and Latin America. It is
present in three clinical forms (cutaneous, muco-cutaneous and
visceral), of which visceral leishmaniasis (VL) is the most severe
form and is fatal if left untreated. In the Indian subcontinent, the
disease manifests in two forms: the cutaneous and the visceral
forms. The latter, also known as kala-azar, is caused by Leishmania
donovani transmitted by Phlebotomus argentipes in an anthroponot-
ic cycle.2 Since 2005, VL endemic countries in the Indian subconti-
nent have reinforced their commitment to eliminate VL from the
region by 2015. The target is to reduce the annual VL incidence
to less than one new case per 10,000 population.3
The available treatment options for VL are limited. Historically,
antimonials have been the primary line treatment but presently
face emerging problems of toxicity and drug resistance which
restrict their widespread use.4 Amphotericin B deoxycholate is
used more often as a second line treatment for VL cases which
respond poorly to antimonials. Amphotericin B treatment requires
prolonged hospitalization as this drug is administered by a paren-
teral route.5 Miltefosine, the only oral drug for VL, has been
recently favoured as it is highly effective with minimal side effects;
however, its teratogenicity and the appearance of drug resistance
justify the search for new chemical series in order to find an orally
safe and efficacious drug.6,7
Recently, we published an article where substituted quinoline 1
(Fig. 1) was found to have good in vivo efficacy and promising
ADME properties.8 We characterized compound 1 further in
in vitro ADME assays and synthesized its analogs in search of com-
pounds with better in vitro PK/PD properties.
In our present study, we have found that compound 1 is soluble
in buffer (100 lM) and highly permeable in parallel artificial mem-
brane permeability assay (PAMPA) model (Papp = 31 Â 10À6 cm/s),
making it a BCS class 1 or class 2 compound (Fig. 2). It showed
moderate binding to mouse (90 2%, mean SD) and human
⇑
Corresponding author. Tel.: +91 9415755899; fax: +91 522 2623938.
E-mail
addresses:
gupta_suman@yahoo.com,
suman_gupta@cdri.res.in
(S. Gupta).
http://dx.doi.org/10.1016/j.bmcl.2014.03.065
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
2047
O
N
0.35
0.30
Cl
0.25
OH
F
N
0.20
Compound 1
0.15
IC50 = 0.22 0.06 µM (against L. d onovani amastigotes), SI = 187.5
In vivo % inhibition= 84.26 4.44 (dose - 50mg/kg x 5d; Twice, PO)
sample concentration
similar to equilibrium sample
0.10
0.05
0.00
Figure 1. Structure of 1 and its in vitro and in vivo antileishmanial efficacy.
0
20
40
60
80
100
(96 1%, mean SD) plasma proteins and was stable in plasma for
6 h. It did not partition into red blood cells and blood to plasma
concentration ratio was 0.70 0.04 (mean SD) after 60 min
incubation. It was found to be unstable in mouse blood (95% deg-
radation over 60 min, Fig. 3). Compound 1 was not an inhibitor of
Concentration (μM)
Figure 2. High permeability of 1 in parallel artificial membrane permeability assay
(PAMPA).
any of the tested cytochrome P450 enzymes (CYPs) up to 10
using human liver microsomes but showed inhibition of CYP1A2
with an IC50 value of 0.51 M (n = 2, Table 1, Fig. 4). Not many
drugs that are marketed are substrates of CYP1A2 and we do not
anticipate any major drug–drug interaction within the VL patient
population.
As part of our lead optimization program, further analogs of
compound 1 were targeted, with the aim of improving its proper-
ties. This paper focuses on chalcone derivatives. Different synthetic
strategies were developed depending upon the position of the
substitution on the quinoline ring which was targeted. Tetra-
substituted quinoline derivatives were prepared from compounds
5(a–b). 4-hydroxy substituted quinolines 3(a–b) and 4(a–b) were
prepared starting from substituted anilines 2(a–b), ethyl acetoace-
tate and polyphosphoric acid, which resulted in two regioisomers.
The mixture of isomers, when treated with phosphorous
lM
l
tribromide gave 4-bromo quinoline derivatives 5(a–b) and corre-
sponding regioisomers which were separated by column
chromatography.
All the compounds were synthesized as per Scheme 1. The
anomeric substitution at C4 position on quinoline ring of
compounds 5(a–b) was achieved by Suzuki coupling with appro-
priate aromatic boronic acids, while the cycloalkyl substitution
was achieved by heating the appropriate amines with cesium car-
bonate using dimethylformamide as solvent. Quinoline aldehydes
7(a–d) were synthesized from 6(a–d) using selenium dioxide.
These aldehydes gave chalcone derivatives 8(a–g) upon condensa-
tion with different substituted ketones.
Overall, seven new compounds were synthesized and assessed
for their in vitro antileishmanial activity, cytotoxicity, solubility
R1
R1
O
O
Br
2
2
2
R
R
2
R
R
R
R
(II)
(I)
+
1
1
N
N
N
N
2(a-b)
3(a-b)
4(a-b)
5(a-b)
(III)
3
R
3
3
R
R
2
1
R
R
2
R
2
1
(IV)
R
R
(V)
4
R
N
1
R
N
N
CHO
O
7(a-d)
8(a-g)
6(a-d)
2a, 3a, 4a, 5a: R1 =F, R2 =Cl
2b, 3b, 4b, 5b: R1 =H, R2 =Cl
6a, 7a: R1 =F, R2 =Cl, R3 =morpholine
6b, 7b: R1 =F, R2 =Cl, R3 =4-fluorophenyl
6c, 7c: R1 =H, R2 =Cl, R3 =4-methoxyphenyl
6d, 7d: R1 =H, R2 =Cl, R3 =4-f luorophenyl
8a: R1 =F, R2 =Cl, R3 =morpholine, R4 =2-dimethylamino methylphenol;
8b: R1 =F, R2 =Cl, R3 =4-fluorophenyl, R4 =2-dimethylamino methylphenol;
8c: R1 =H, R2 =Cl, R3 =4-methoxyphenyl, R4 =2-dimethylamino methylphenol;
8d: R1 =H, R2 =Cl, R3 =4-fluorophenyl,R4 =m-fluoroanisole;
8e: R1 =F, R2 =Cl, R3 =4-fluorophenyl,R4 =m-fluoroanisole;
8f : R1 =H, R2 =Cl, R3 =4-fluorophenyl,R4 =2-dimethylamino methylphenol;
8g : R1 =H, R2 =Cl, R3 =4-fluorophenyl,R4 =2-Morpholine methylphenol;
Scheme 1. Synthesis of substituted quinoline chalcones 8(a–g). Reagents and conditions: (I) Polyphosphoric acid, ethyl acetoacetate, 120 °C, 16 h; (II) PBr3, DMF, 3 h; (III)
Pd(PPh3)4, aryl boronic acid, THF/water, K2CO3. 70 °C, 16 h or R3-NH/Cs2CO3/DMF, 4–16 h; (IV) SeO2, 1,4-dioxane, 70 °C; (V) substituted acetophenone derivatives, acetic acid,
sulfuric acid, 60 °C.

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V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
and metabolic stability in liver microsomes from different species.
1000
750
500
250
0
A significant number of compounds exhibited potent in vitro
activity against L. donovani, as well as a good selectivity index
(i.e., potency with low cytotoxicity). The parent compound 1
Concentration (Plasma) nM
Concentration (RBC) nM
had an IC50 value of about 0.22
showed IC50 in the range of 0.17–6.42
all analogs had solubility less than 10
l
M whereas all the analogs
M (Table 2). Although,
M except 8e, their meta-
l
l
bolic stability was similar to that of compound 1 (Table 3, Fig. 5).
Compound 8d was stable in hamster, mouse and human liver
microsomes but the IC50 value (6.42
Compound 8f showed low IC50 (0.17
l
M) was relatively high.
l
M) with good selectivity
(SI = 156) but was highly metabolized in hamster liver micro-
somes followed by mice and human liver microsomes. Compound
8b, which had structural features of both compounds 8d and 8f,
0
10
20
30
40
50
60
Time (min)
Figure 3. Equal blood and plasma concentration ratio of 1 in mouse.
showed promising potency (IC50 value of 0.84 lM) and moderate
stability in mouse and human liver microsomes and was rapidly
metabolized in hamster.
In conclusion, the synthesis of compound 1 analogs could con-
tribute to overcoming the main problem of quinoline analogs i.e.
metabolic instability with potency more or less similar to com-
pound 1. Compounds 8b and 8f showed modest in vitro potency
and good ADME properties, and are being investigated as back-
up molecules for further development. Although, desired efficacy
and metabolic stability in liver microsomes could not be achieved
together in the analogs synthesized, but the work remains relevant
in the search of new antileishmanial compounds.
Table 1
Cytochrome P450 (CYP) Inhibition of 1
Test item
CYP1A2
IC50 (lM)
CYP2C9
CYP2C19
CYP2D6
CYP3A4
1
0.55, 0.47
0.005, 0.002
50
0.40
>100
0.74
21
0.10
15
0.004
Positive controla
a
Positive control:
a-naphthoflavone-CYP1A2, sulfaphenazole-CYP2C9, (N)-3-
benzylnirvanol-CYP2C19, quinidine-CYP2D6 and ketoconazole-CYP3A.
CYP1A2 - 1
CYP1A2 - α-Naphthoflavone
100
100
Set 1
Set 2
75
50
25
0
75
Set 1
Set 2
50
25
0
0.0001 0.001
0.01
0.1
1
10
0.01
0.1
1
10
100
Log [1] ( M)
Log [ -Naphthoflavone] (µM)
μ
α
CYP2C9 - 1
CYP2C9 - Sulfaphenazole
100
75
50
25
0
100
75
50
25
0
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Log [1] ( M)
Log [Sulfaphenazole] ( M)
μ
μ
Figure 4. CYP inhibition profiles of 1.

V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
2049
CYP2C19 - 1
CYP2C19 - N-3-Benzylnirvanol
100
75
50
25
0
100
75
50
25
0
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Log [N-3-Benzylnirvanol] (μM)
Log [1] (μM)
CYP2D6 - 1
CYP2D6 - Quinidine
100
75
50
25
0
100
75
50
25
0
0.001
0.01
0.1
1
10
0.01
0.1
1
10
100
Log [Quinidine] (μM)
Log [1] (μM)
CYP3A4 - 1
CYP3A4 - Ketoconazole
100
75
50
25
0
100
75
50
25
0
0.001
0.01
0.1
1
10
0.01
0.1
1
10
100
Log [Ketoconazole] (μM)
Log [1] (μM)
Fig. 4 (continued)

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V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
Table 2
Antileishmanial activity and cytotoxicity data of substituted quinoline chalcones 8(a–g)
a
b
Compounds
R1
R2
R3
R4
IC50
(
lM)
CC50
(lM)
SIc
O
1
F
Cl
OH
0.22 0.06
1.99 0.82
41.25 4.2
187.5
N
O
OH
8a
8b
F
F
Cl
Cl
24.96 2.17
13
28
N
N
OH
F
0.84 0.12
23.67 2.41
N
OH
O
F
F
F
F
8c
8d
8e
8f
H
F
Cl
Cl
Cl
Cl
1.55 0.89
6.42 1.83
3.48 1.61
0.17 0.02
28.75 3.21
221.52 10.22
45.84 2.19
26.54 3.56
19
N
O
35
F
F
O
H
H
13
OH
156
N
OH
8g
H
—
Cl
—
1.16 1.01
8.10 0.60
23.54 4.11
54.56 4.81
20
7
N
O
Miltefosine
—
—
a
b
c
IC50 values against intracellular amastigote form of L. donovani.
CC50 values in vero cells (mean SD) are the average of two independent experiments.
SI, selectivity index (ratio of CC50/IC50); miltefosine was used as a reference drug.
Table 3
Solubility and metabolic stability of substituted quinoline chalcones 8(a–g) in liver microsomes
Compounds
Solubility (lM)
% remaining in
% remaining in
% remaining in
HLMa after 30 min
MLMb after 30 min
HamLMc after 30 min
1
8a
8b
8c
8d
8e
8f
>100
<10
<10
<10
<10
>100
<10
ND
65
63
63
63
77
60
58
ND
55
54
63
66
72
0
44
15
22
19
71
ND
3
43
ND
8g
ND
a
b
c
HLM, human liver microsome.
MLM, mouse liver microsome.
HamML, hamster liver microsome; ND: not determined.
Biochemistry, CSIR-Central Drug Research Institute, Lucknow, In-
dia. This work was supported by the Drugs for Neglected Diseases
initiative (DNDi), Switzeland. Specific funding from Bill & Melinda
Gates Foundation, USA, with complementary core funding from
Department for International Development (DFID), UK; Dutch
Acknowledgements
The authors thank Director, CSIR-CDRI, Lucknow, for his encour-
agement and facilities. The transgenic L. donovani promastigotes
were originally procured from Neena Goyal, Division of

V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
2051
Metabolic Stability-Compound 1
1000
100
10
% Remaining-HLM
1
% Remaining-MLM
% Remaining-HamLM
0.1
0
5
10
15
20
25
30
Time (min)
Metabolic Stability-Compound 8b
Metabolic Stability-Compound 8a
1000
100
10
1000
100
10
% Remaining-HLM
% Remaining-HLM
1
1
% Remaining-MLM
% Remaining-MLM
% Remaining-HamLM
% Remaining-HamLM
0.1
0.1
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
Metabolic Stability-Compound 8c
Metabolic Stability-Compound 8d
1000
100
10
1000
100
10
% Remaining-HLM
% Remaining-HLM
1
1
% Remaining-MLM
% Remaining-MLM
% Remaining-HamLM
% Remaining-HamLM
0.1
0.1
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
Metabolic Stability-Compound 8e
Metabolic Stability-Compound 8f
1000
100
10
1000
100
10
% Remaining-HLM
% Remaining-MLM
% Remaining-HLM
1
1
% Remaining-MLM
% Remaining-HamLM
0.1
0.1
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
Figure 5. Metabolic stability profiles of 1 and its analogs 8(a–g) in liver microsomes.

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V. S. Gopinath et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2046–2052
Ministry of Foreign Affairs (DGIS), Netherlands; Federal Ministry of
Education and Research (BMBF), Germany; Spanish Agency for
International Development Cooperation (AECID), Spain; Swiss
Agency for Development and Cooperation (SDC), Switzerland and
Médecins Sans Frontières (Doctors without Borders), International.
This is CSIR-CDRI communication number 8650.
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065.