New antiprotozoal agents: Their synthesis and biological evaluations

Article abstract of DOI:10.1016/j.bmcl.2013.02.054

Here we report identification of new lead compounds based on quinoline and indenoquinolines with variable side chains as antiprotozoal agents. Quinolines 32, 36 and 37 (Table 1) and indenoquinoline derivatives 14 and 23 (Table 2) inhibit the in vitro growth of the Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense subspecies and Leishmania infantum with IC ₅₀ = 0.25 μM. These five compounds have superior activity to that of the front-line drugs such as benznidazole, nifurtimox and comparable to amphotericin B. Thus these compounds constitute new 'leads' for further structure-activity studies as potential active antiprotozoal agents.

Full text of DOI:10.1016/j.bmcl.2013.02.054

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2750–2758
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New antiprotozoal agents: Their synthesis and biological
evaluations
⇑
⇑
Ram Shankar Upadhayaya , Shailesh S. Dixit, Andras Földesi, Jyoti Chattopadhyaya
Program of Chemical Biology, Institute of Cell and Molecular Biology, Biomedical Centre, Uppsala University, SE-75123 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we report identification of new lead compounds based on quinoline and indenoquinolines with var-
iable side chains as antiprotozoal agents. Quinolines 32, 36 and 37 (Table 1) and indenoquinoline deriv-
atives 14 and 23 (Table 2) inhibit the in vitro growth of the Trypanosoma cruzi, Trypanosoma brucei,
Received 12 January 2013
Revised 8 February 2013
Accepted 12 February 2013
Available online 22 February 2013
Trypanosoma brucei rhodesiense subspecies and Leishmania infantum with IC50 = 0.25 lM. These five com-
pounds have superior activity to that of the front-line drugs such as benznidazole, nifurtimox and com-
parable to amphotericin B. Thus these compounds constitute new ‘leads’ for further structure–activity
studies as potential active antiprotozoal agents.
Keywords:
Antiprotozoal agents
Trypanosoma
Ó 2013 Elsevier Ltd. All rights reserved.
Leishmania
Parasite inhibitors
Conformationally constrained quinoline
Neglected tropical diseases (NTD)1 include Chagas’ disease
(American trypanosomiasis2), human African trypanosomiasis
HAT (sleeping sickness)3 and leishmaniasis.4 These are parasitic
diseases caused by the parasitic protozoan’s Trypanosoma cruzi
(T. cruzi), Trypanosoma brucei (T. brucei) and Leishmania species,
respectively. It is a serious health problem of today mainly in trop-
ical countries and in Central and South American continent causing
two million deaths per year.5,6 The present treatment is not very
effective in the chronic phase and has toxicity, side effects7–15
and parasite resistance.10–12,16–21
Thus, there is a considerable potential in developing novel ap-
proaches for antitrypanosoma and antileishmania drugs. Molecular
modeling,22 enzymatic23 and crystallographic studies24 on tipifar-
nib (1, EC50 = 4 nM, Fig. 1) and its analogues (compounds 2 and
3)22,25–27 have shown role of quinoline and side chain in its biolog-
ical activity. In these studies, the X-ray structure of cocrystal of
compound 2 with T. brucei CYP51, has elegantly shown that the
quinolone together with its imidazole ring side chain was coordi-
nated with heme iron whereas the phenyl ring attached to quino-
lone occupying an additional CYP51 active-site cavity. Qunoline as
a pharmacophore against T. brucei and T. cruzi is also interesting
because tafenoquine (3, Fig. 1) is known to act on unique target
such as cytochrome c reductase. As a part of our research project
on antitubercular drug discovery, we have screened a library of
39 compounds based on a quinoline and indenoquinolines with
various side chains for antiprotozoal activity, which have also
shown anti-TB activity.28–32 We have thus identified five com-
pounds (14, 23, 32, 36 and 37) that have shown excellent in vitro
antitrypanosomal and antileishmanial activity as low as
IC50 = 0.25 and 0.40
drugs benznidazole33 (IC50 = 3.66
and comparable to amphotericin B35 (IC50 = 0.25
l
M, respectively, which is superior to frontline
M), nifurtimox34 (IC50 = 1.8
M)
M). The diverse
l
l
l
structures of these active compounds further suggest that both
quinoline and side chain variations are important for antiprotozoal
activity.
Following the literature procedures compound 6 was prepared
through functionalization of 4-OH of 6-bromo-2-(trifluoro-
methyl)quinolin-4-ol, 436 (Scheme 1). Compound 4 was bromi-
nated by using PBr3 in DMF to give 4,6-dibromo compound 537
which was treated with strong base LDA followed by benzaldehyde
in dry THF to obtain the desired compound 6. To achieve the target
compound 9 (Scheme 2), compound 729 was treated with m-(tri-
fluoromethyl) benzene sulfonyl chloride in presence of dry pyri-
dine to give sulfonamide 8. Carbonyl group of sulfonamide 8 was
reduced by NaBH4 to give hydroxy derivative 9. To accomplish
the synthesis of compounds 11, 12, 14 and 15 (Scheme 3),
Abbreviations: CC50, concentration of inhibitor resulting in 50% parasite growth
inhibition; DCM, dichloromethane; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; EDCꢀHCl, 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochlo-
ride; Et3N, triethylamine; EtOH, ethanol; MeOH, methanol; IC50, concentration of
inhibitor resulting in 50% inhibition; SI, Ratio of CC50 value/IC50; NMR, nuclear
magnetic resonance; SAR, structure–activity relationship; PFT, protein farnesyl-
transferase; PPA, polyphosphoric acid.
⇑
Corresponding authors. Tel.: +46 18 4714577; fax: +46 18 554495.
E-mail addresses: ram@boc.uu.se (R.S. Upadhayaya), jyoti@boc.uu.se (J. Chatto-
padhyaya).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.054

R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
R = NH2 binds to mammalian PFT via farnesyl diphosphate
2751
R = OCH3 binds to T.cruzi 14DM
5'
Cl
4'
N
6'
C
F3C
H3CO
O
R
2'
3'
1'
N
5
4
6
3
2
A
B
N
OCH3
7
N
O
NH
8
Cl
1
H2N
3: Tafenoquine (IC50 = 5.6 µM) against L. donovani
Target: mitochondrial dysfunction through
cytochrome c reductase
1: R = NH2, Tipifarnib (EC50 = 4 nM) T. cruzi
protein farnesyltransferase (PFT); human
(hPFT IC50= 0.7 nM)
2: R = OCH3 (EC50 = 0.6 nM) T. cruzi (PFT);
human (hPFT IC50>5000 nM)
Figure 1. Quinoline based antiparasitic lead molecules (1–3) under clinical trials.
Br
N
OH
N
Br
N
Br
Br
Br
(i)
OH
CF3
(ii)
Ref. 38
CF3
CF3
4
5
6
Scheme 1. Reagents and conditions: (i) dry DMF, PBr3 at 0 °C then at rt, 4 h, 82%; (ii) LDA, dry THF, 30 min, PhCHO, ꢁ78 °C, 2 h, 16%.
F3C
F3C
O
S
O
O
S
O
(i)
(ii)
H2N
HN
HN
Ref. 28
O
Ref. 28
CH3
N
O
F
N
O
F
O
N
O
F
OH
CH3
CH3
8
9
7
Scheme 2. Reagents and conditions: (i) 3-(trifluoromethyl)benzene-1-sulfonyl chloride, dry pyridine, rt, 12 h, 54%; (ii) NaBH4, EtOH–THF (2:1, 6 mL), rt, 2 h, 53%.
R1
OH
N
O
O
CH3
O
N
Br
Br
(i)
CH3
N
OCH3
N
OCH3
Ref. 31
10
Ref. 31
11: R1 = Benzyl
12: R1 = Benhydryl
(i)
OH
OCH3
OH
H
H
R2
O
N
N
O
Br
Br
(iii)
CH3
CH3
O
N
OCH3
N
OCH3
13: R2 = N3
14: R2 = NH2
15
(ii)
Scheme 3. Reagents and conditions: (i) iso-propanol, 1-benzyl piperazine for 11, 1-benzhydryl piperazine for 12, and NaN3 for 13, reflux, 12 h, (11, 26%; 12, 27% and 13, 61%);
(ii) dry THF, PPh3, reflux, 15 h, 58%; (iii) dry DCM, 2-methoxyphenyl isocyanate, dry Et3N, 0 °C–rt, 1 h, 9%.

2752
R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
OH
O
O
Br
Br
Br
CH3
(i)
(ii)
N
Cl
N
N
N
N
Ref. 31
Ref. 31
N
N
R1
R1
17: R1 = Benzhydryl
16
18: R1 = Benzhydryl
Ref. 31
(iii)
R2
OH
N
O
O
O
N
Br
Br
(iv)
CH3
CH3
N
N
N
N
N
N
R1
R1
20: R1: Benzhydryl; R2 = -3-Methoxy phenyl
19: R1 = Benzhydryl
Scheme 4. Reagents and conditions: (i) dry pyridine, 1-benzhydryl piperazine, reflux, 8 h, 71%; (ii) dry THF, MeMgI, rt, 5 h, 82%; (iii) dry DMF, NaH, epi-chlorohydrin, 12 h,
78%; (iv) iso-propanol, m-methoxyphenyl piperazine, reflux, 12 h, 38%.
OH
O
O
Br
Br
Br
CH3
(ii)
(i)
N
OCH2CF3
N
OCH2CF3
N
Cl
Ref. 31
Ref. 31
21
22
16
(iii)
Ref. 31
O
Br
N
N
N
H
23
Scheme 5. Reagents and conditions: (i) dry DMF, 2,2,2-trifluoroethanol, NaH, rt, 15 h, 65%; (ii) dry THF, MeMgI, rt, 14 h, 58%; (iii) dry pyridine, 4-aminopyridine, reflux, 8 h,
87%.
OH
O
Br
Br
Br
(ii)
O
(i)
CH3
CF3
NH2
N
CF3
Ref. 31
N
Ref. 30
25
(iv)
24
26
Ref. 31
(iii)
Boc
N
O
O
O
OH
N
N
Br
Br
Br
(v)
O
CH3
CF3
N
CF3
N
CF3
N
27
28
29
Scheme 6. Reagents and conditions: (i) ethyl 4,4,4-trifluoro-3-oxobutanoate, PPA, 150 °C, 11 h, 45%; (ii) dry THF, MeMgI, 5 °C–rt, 5 h, 71%; (iii) dry DMF, NaH, rt, epi-
chlorohydrin, 10 h, 51%; (iv) EtOH:H2O (2:1), NH2OHꢀHCl, NaOH, 0 °C 15 min then 90 °C, 8 h, 94%; (v) dry DMF, DMAP, EDCꢀHCl, Boc-nipecotic acid, rt, 3 h, 28%.

R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
2753
Table 1
In vitro activity (IC50
l
M) of compounds against T. cruzi (T.c.), T. brucei brucei (T.b.), T. brucei rhodesiense (T.r.) and Leishmania infantum (L.inft.)
R5
StructureR1
IC50 (lM)
No
CC50
logP
R3
R2
N
R1
R2
R3
R5
Br
T.c.
8.07
T.b.
7.52
T.r.
L.inft.
6
Br
Br
CF3
OH
5.94
8.43
8.11
1.51
6.26
4.52
N
30
OCH3
N
N
H
0.46
32.69
25.40
30.83
N
31
Br
OCH3
H
6.96
2.16
0.44
50.80
64.00
—
NO2
R6
O
F
R7
R2
=
R1
N
R2
R1
R6
—
R7
T.c.
T.b.
T.r.
L.inft.
CC50
8.00
logP
O
S
H
N
9
OH
3.70
0.25
2.18
1.46
8.11
7.69
F3C
O
N
N
32
NO2
OH
2.18
1.81
2.52
31.44
—
N
33
34
NO2
NO2
OH
OH
2.00
2.01
2.12
1.22
0.71
2.16
8.10
—
—
N
N
OCH3
OCH3
64.00
20.32
64.00
N
35
36
NO2
OH
OH
6.01
1.00
2.40
0.51
2.08
0.25
8.00
1.70
64.00
2.20
—
N
H
H
N
N
—
6.84
O
O
NO2
H
N
H
N
37
—
OH
2.89
0.65
0.25
3.17
6.21
6.72
OCH3
H
N
H
N
38
39
40
—
—
—
OH
OH
6.23
4.33
2.04
1.79
2.00
2.07
0.53
0.53
8.11
5.28
6.82
64.00
16.00
64.00
6.72
7.86
7.86
O
OCH3
CH3O
H
N
H
N
S
H
N
H
N
OH
OH
14.67
S
OCH3
H
H
N
N
41
42
—
—
5.81
1.93
2.09
2.04
1.31
2.03
8.11
42.33
64.00
7.86
7.91
S
H3CO
N
N
N
N
N
20.32

2754
R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
Table 2
In vitro activity (IC50
lM) of conformationally locked compounds
R3
R4
Structure
R1
IC50 (lM)
No.
CC50
logP
N
R2
R1
R2
R3
R4
T.c.
T.b.
T.r.
L.inft.
O
N
11
12
Br
OCH3
OCH3
OCH3
CH3
7.25
2.24
1.99
9.51
5.63
5.85
7.56
OH
N
O
O
N
OH
N
Br
CH3
5.85
2.04
1.64
8.11
17.55
NH2
O
14
15
Br
Br
CH3
CH3
0.47
2.50
0.25
8.23
0.25
1.88
0.40
9.51
1.36
5.31
3.46
4.84
OH
OCH3
O
O
N
H
N
H
OH
OH
OCH3
N
N
N
N
OCH3
20
Br
CH3
64.0
2.03
0.86
30.05
61.44
—
22
23
Br
Br
OCH2CF3
–OH
CH3
—
6.92
0.25
8.23
0.56
3.23
0.25
8.00
5.08
19.84
0.75
5.71
4.64
N
N
H
@O
O
27
29
Br
Br
CF3
CH3
—
8.37
8.57
7.56
5.49
3.06
27.27
8.11
4.00
5.84
5.23
—
O
N
O
CF3
12.29
N
O
Boc
N
O
N
N
43
44
Br
Br
OCH3
OCH3
CH3
CH3
2.16
5.35
2.03
2.31
1.17
1.25
6.01
5.08
8.00
3.68
7.50
OH
CF3
N
24.11
O
O
OH
N
45
Br
OCH3
CH3
7.42
8.23
5.28
64.00
64.00
4.91
O
O
N
N
46
47
Br
Br
N
N
–OH
CH3
CH3
1.39
0.93
18.78
28.98
5.93
7.11
12.70
21.11
11.45
32.22
3.86
4.09
O
O
(CH2)3CH3
N
N
48
Br
CH3
1.46
8.11
5.81
5.66
21.77
5.58
O
N
N
N
49
50
Br
Br
@O
—
—
6.87
7.01
7.69
8.17
5.98
2.61
20.32
9.51
4.33
6.01
6.40
N
N
N
N
N-OH
64.00
N
N
O
N
Boc
N
51
Br
—
6.21
2.04
1.65
6.96
26.91
—
H
O
O
N
N
O
N
N
N
N
N
52
53
Br
Br
—
—
6.82
7.87
6.05
2.16
1.20
1.36
5.08
5.66
64.00
35.60
—
—
N
H
O
O

R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
2755
Table 2 (continued)
R3
R4
Structure
R1
IC50 (lM)
No.
CC50
logP
N
R2
R1
R2
R3
R4
T.c.
T.b.
T.r.
L.inft.
O
O
N
NH
Boc
SCH3
O
O
N
N
N
N
N
N
O
54
55
Br
Br
—
—
7.39
2.40
1.44
1.52
9.51
3.17
64.00
13.59
—
N
N
N
H
O
O
O
O
O
7.52
7.33
8.30
N
H
O
O
(CH2)2CH3
N
N
N
N
56
57
Br
Br
—
—
2.38
7.38
8.17
7.31
5.14
4.33
6.01
8.23
8.00
—
—
N
O
N
N
Boc
12.70
O
compound 1030 served as a key intermediate. Oxirane 10 upon
heating at reflux with appropriate piperazine(s) in iso-propanol
gave the desired piperazine derivatives 11 and 12 as a diastereo-
meric mixture. Under similar conditions when oxirane 10 was re-
acted with NaN3 gave azido compound 13. Azide 13 was reduced
by employing PPh3 in THF (Staudinger reaction) to give amine 14
which was further converted to urea 15 by treating it with 2-
methoxyphenyl isocyanate. Benzhydryl derivative 20 (Scheme 4)
was prepared from 2-bromo-6-chloro-indeno[2,1-c]quinolin-7-
one (16).30 The C2-chloro group of chloroketone 16 was nucleo-
philically displaced by benzhydrylpiperazine to give ketone 17
which was subjected to Grignard reaction (MeMgI) to transform
to the hydroxy derivative 18 in 82% yield. Compound 18 furnished
oxirane 19 (78%) upon treatment with epi-chlorohydrin and NaH in
dry DMF. Subsequently oxirane ring of 19 was opened by m-
methoxyphenyl piperazine in iso-propanol to give compound 20.
Similarly C2-chloro of chloroketone 1630 was nucleophilically dis-
placed by 2,2,2-trifluoroethanol to give ketone 21 (65%, Scheme 5).
Compound 21 was treated with Grignard reagent (MeMgI) to ob-
tain desired hydroxy compound 22. Another target compound 23
(87%) was synthesized by reacting chloroketone 16 with the nucle-
ophile 4-aminopyridine. To engineer the bioactivity we have func-
tionalized C2 position with CF3 group (as in compounds 27 and
29): we started synthesis from 2-amino-5-bromobenzophenone
24 (Scheme 6).38 Quinoline ring was constructed by treating com-
pound 24 with ethyl 4,4,4-trifluoro-3-oxobutanoate in PPA at
150 °C to get C2–CF3 indeno[2,1-c]quinoline-7-ketone derivative
25. Compound 25 was treated with Grignard reagent MeMgI to
give hydroxyl derivative 26. This was further treated with epi-chlo-
rohydrin to get desired oxirane 27. Compound 25 was converted to
its corresponding oxime 28 (94%) by heating it with hydroxyl-
amine hydrochloride in presence of NaOH. Oxime 28 was coupled
with Boc-nipecotic acid to get the desired compound 29.
and L. infantum (MHOM/MA (BE)/67) on new compounds 6, 9, 11,
12, 14, 15, 20, 22, 23, 27, 29 and the available library of quinoline
derivatives (30,28 31,28 32–42,29 43–46,30 47,32 48,32 49,30 50,30 and
51–5732 (Tables 1 and 2). Results obtained are displayed in Tables
1 and 2 using benznidazole as the reference drug including toxicity
values against Vero cells (Table 3).
The in vitro screening of the synthesized compounds enabled us
to identify five compounds (14, 23, 32, 36 and 37) with excellent
activity in whole cell parasite culture (IC50 = 0.25 lM, details of
the screening protocol can be found in SI). Despite a limited SAR
that can be drawn from these molecules, it displays a clear poten-
tial of quinoline and indenoquinoline based compounds with var-
ious substituents for further exploration against parasites.
Compound
IC50 = 0.46
whereas compound 31 (R3 = 4-nitro-imidazole) and 6 (R3 = OH) re-
sulted in relatively inferior activity with IC50 = 6.96 and 8.07 M,
30
(R1 = OCH3;
R3 = imidazole)
has
shown
lM (Table 1) and SI 67 (Table 3) against T. cruzi,
l
respectively against T. cruzi. In compounds (9 and 32–42), position
6 of quinoline (Fig. 2) is substituted with nitro, urea, thiourea, thi-
oamide and phenyl tetrazole whereas position 2 has fluorophenyl
substitution. These chemical variations provided three active com-
pounds (32, 36 and 37) that have shown IC50 = 0.25 lM.
Compound 32 (R1 = NO2 and R6 = imidazole) has been found to
be most active against both Trypanosoma spp. (IC50 = 0.25–
2.18
l
M) and L. infantum (IC50 = 2.52
lM) with excellent SI >125
(CC50 = 31.44
lM). When imidazole group in compound 32 was
substituted by piperidine (33), or by p-methoxyphenyl pyrazole
(34), or by m-methoxyphenyl pyrazole (35), led to less active com-
pounds than that of compound 32 (Table 1). Compound 32 having
imidazole makes it more selective towards T. cruzi (SI = 126) and it
was found to be 8, 256 and 24 times more potent than 33–35,
respectively against T. cruzi, thus, the imidazole moiety seems to
play an important role in determining the antiparasitic activity. It
is likely that the imidazole ring in compounds 30 and 32 enhance
the cell-membrane permeability,42,43 while the OH may form polar
interactions with the target site.
The growth inhibition assays were performed against protozo-
ans39–41 including epimastigote form of T. cruzi (Tulahuen 2 strain),
T. brucei subsp. brucei 427, T. brucei subsp. rhodesiense STIB900

2756
R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
Table 3
Interestingly, among the urea derivatives (36–42) compound 36
(R1 = m-nitro-phenyl urea) showed significant activity against Try-
Selectivity index (SI, ratio of CC50 and IC50) of compounds
panosoma
spp.
(IC50 = 0.25–1.0
lM)
and
L.
infantum
#
T.c.
T.b.
T.r.
L.inft.
(IC50 = 1.70
lM). The decrease in T. cruzi activity for compounds
6
9
0.19
4.36
0.77
3.0
2.89
2.12
0.96
2.86
3
0.48
0.47
67.02
9.19
125.76
2.20
33.16
4.05
2.15
2.16
3.7
0.20
32.0
2.51
8.60
5.44
0.64
30.26
2.41
1.34
0.47
0.77
0.943
29.62
14.42
4.31
31.37
4.03
9.55
3.66
8.94
20.25
26.66
31.37
30.18
3.94
10.43
7.77
0.61
1.11
2.68
0.56
7.34
13.19
10.58
16.48
26.66
1.85
1
0.25
120.75
2.83
10.70
5.44
2.82
71.44
6.14
3
0.73
1.91
0.19
9.38
0.59
2.16
3.4
37–41 was ascribed to the presence of the amide moiety (urea or
thiourea) with electron-donating methoxy group on the aryl ring,
while compounds 36–42 showed good activity in the range of
11
12
14
15
20
22
23
27
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
IC50 = 0.25–2.09 lM against T. brucei and T. rhodesiense (Table 1).
0.56
2.04
2.48
0.15
0.14
0.72
1.21
1.25
12.47
1.29
3.15
3.75
1.96
0.99
3.03
5.22
8.0
Thiourea derivatives 39–41 are structural isomers having OCH3
group at ortho-, meta- and para-positions, respectively. Their activ-
ities against T. cruzi show that ortho-OCH3 (as in 39) and p-OCH3
(as in 41) substitutions are preferred 2–3 times over its m-OCH3
analog (as in 40).
In addition to quinoline compounds we have also synthesized
and screened indenoquinoline derivatives. In these compounds, a
new ring D (Fig. 2) was constructed by covalently locking the C4
center of the quinoline moiety with the C20 center of the phenyl
ring (Fig. 2) with the aim to reduce the conformational flexibility
across C20–C4. This chemical construction would decrease the
entropic penalty (as shown in general structure II from more flex-
ible general structure I; Fig. 2) for the complex within the target
protein, which may in turn give improved free energy of stabiliza-
tion to the complex. Interestingly, compounds following this strat-
egy have been found to be very active against Mycobacterium
tuberculosis31,32 and protozoal diseases.44
3.65
145.45
17.37
8.8
31.52
6.64
24.84
5.48
30.18
32.31
30.77
30.91
90.14
6.83
19.29
12.12
1.93
4.53
3.74
0.72
24.52
16.30
53.33
26.17
44.44
8.94
7.28
10.64
10.27
1
7.89
3.15
1.33
4.74
1
3.70
4.50
8.62
8.23
34.65
14.91
0.63
9.13
4.33
9.38
4.52
8.66
1.80
3.45
1.08
These chemical changes produced thirteen active compounds
0.9
(IC50 <2
pound 14 showed excellent activity in range of IC50 = 0.25–
0.47 M against Trypanosoma spp. and Leishmania (SI 3–5, Table 3).
lM) (11, 12, 14, 15, 23, 43, 44, 46, 47 and 52–55). Com-
1.52
3.84
0.21
6.73
3.86
12.60
6.29
6.73
4.28
1.37
0.63
l
When the free amine in 14 was replaced by imidazole (43), benz-
hydryl piperazine (12), benzyl piperazine (11), m-trifluoromethyl-
phenyl pyrazole (44), o-methoxyphenyl urea (15) or amide (45)
antiparasitic activity decreased. This analysis suggests that hydro-
phobic nature (higher logP, Tables 1 and 2) or potential steric con-
tribution led to less active compounds.
1.60
1.85
1.09
4'
R
5'
6'
3'
2'
C
1'
H
5
4
O2N
F
HN
N
F
3
R1
6
A
B
2
O
7
N
O
N
O
N
R2
8
1
N
N
HO
HO
CH3
General Structure I
36: R = NO2, IC50 = 1.0 µM (T.c);
0.51 µM (T.b); 0.25 µM (T.r); 1.7 µM
(L.inft.), CC50 = 2.2 µM, CLogP 7.17
32: IC50 = 0.25 µM (T.c); 2.18 µM
(T.b);1.8 µM (T.r); 2.5 µM (L.inft.) CC50
= 31.4 µM, CLogP 4.81
37: R = OCH3, IC50 = 2.8 µM (T.c);
0.65µM (T.b); 0.25 µM (T.r); 3.1 µM
(L.inft.), CC50 = 6.2 µM, CLogP 7.19
A new covalent bond
between C4-C2' restricts
the rotation of the phenyl
group
4'
OH
5'
6'
3'
2'
C
O
NH2
Br
Br
CH3
O
1' R3
R4
D
5
4
N
3
R1
N
OCH3
N
N
6
A
B
2
H
7
N
R2
8
23: IC50 = 0.25 µM (T.c);
0.56 µM (T.b); 0.25 µM (T.r); 5.0
µM(L.inft.), CC50 = 0.75 µM, CLogP 5.90
14: IC50 = 0.47 µM (T.c.);
0.25 µM (T.b); 0.25 µM (T.r.); 0.4µM
(L.inft.), CC50 = 1.36 µM, CLogP 3.91
1
General Structure II
Figure 2. Most active five compounds (14, 23, 32, 36 and 37) are shown with substituents R1–R5. See Tables 1 and 2 for structures of all compounds along with their
antiprotozoal activity (IC50) and cytotoxicity (CC50).

R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
2757
Apart from this, replacement of the OCH3 group at R2 with 1H-
imidazole (46–48) and alkyl esters (47 and 48) at R3, produced ac-
tive compounds against T. cruzi (IC50 = 0.93–1.46 lM, SI 8–35, Ta-
ble 3). Based on this small library of compounds it can be
suggested that heterocyclic aromatic amines (imidazole) as sub-
stituents are more selective to T. cruzi (46–48) whereas, piperazine
substituents (20, 11 and 12) make molecule active against T. brucei
(Table 2).
different protozoans may improve health of those infected with
multiple parasites at a low cost, (ii) the risk of possible drug–
drug interactions would be avoided in such multiple action sin-
gle-drug therapeutic, (iii) in addition, drug potency and efficacy
might be increased and thereby potential reduction of drug-
resistance.
Acknowledgements
Compounds 30, 32 and 46–48 having an imidazole moiety
showed very similar antiprotozoal activity, suggesting a critical
role of the imidazole ring, because when imidazole in 30, 32 and
46–48 was replaced by pyrazole derivatives 34, 35 and 44 resulted
in a drastic reduction in biological activity of the latter. For further
exploration, R2 was substituted with 2-pyridyl piperazine and
other heterocyclic moieties and R3 was replaced with various ami-
no acids (51–55) and ester (56) of oxime (50). In these efforts, com-
pound (23) having 4-aminopyridine was found to have excellent
We are thankful to Drugs for Neglected Diseases Initiative
(DNDi), Geneva, Switzerland for testing our compounds against
protozoan parasites. We also thank Nageshwar Rao, Santosh La-
hore and Aftab Sayyad for initial work. Generous financial sup-
port from the European Union (Project No. 222965, Project
title: New approaches to target Tuberculosis, Call identifier:
FP7-Health-2007-B) and Uppsala University is also gratefully
acknowledged.
activity against Trypanosoma spp. (IC50 = 0.25 lM), whereas com-
pounds substituted with 2-pyridinyl-piperazine (49–55), imidaz-
ole (56 and 57) and CF3 (29) found to be less active.
Supplementary data
However, these preliminary results based on limited number of
compounds allow us to conclude that besides the common core of
conformationally-constrained system, the imidazole ring may have
a crucial role in modulating the binding to the target protein as dis-
played by their excellent inhibition concentration (IC50).
In summary, quinoline derivatives (32, 36 and 37) and con-
formationally-constrained indeno[2,1-c]quinoline derivatives (14
and 23) with various substituents presented here have shown
Supplementary data (electronic supplementary information
available: Spectral data contains (1H, 13C NMR, COSY, HMQC and
HMBC) for all new compounds including experimental and NMR
assignments showing the purity and structural integrity. Biological
assays are also given in SI) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2013.02.054.
excellent activity (IC50 = 0.25 and 0.40 lM) against T. cruzi, T.
References and notes
brucei and L. infantum in the whole cell parasite culture with
low cytotoxicity in murine macrophage host cells and in diploid
human fibroblasts MRC-5 cell line. These five compounds had
better activity than the front-line drugs such as benznidazole,
nifurtimox and comparable in activity to amphotericin B. Thus
these active analogues may act as promising ‘lead’ compounds
for further structure–activity studies. However, we know from
the available compounds that quinoline in combination of appro-
priate side chain produced active compounds. Side chains substi-
tuted with electron withdrawing groups and small cyclic or
acyclic amines were found to be more active compared to elec-
tron rich substituents. In this regard, beside the replacement of
various polar groups in the molecular structures of these com-
pounds, we foresee that further SAR and target enzyme studies
are needed with more focused library following the trail of ‘lead’
molecules, which is in progress.
1. WHO Neglected Diseases, http://www.who.int/neglected_diseases/en/.
2. Coura, J. R. Mem. Inst. Oswaldo Cruz 2007, 102, 113.
3. Simarro, P. P.; Diarra, A.; Ruiz Postigo, J. A.; Franco, J. R.; Jannin, J. G. PLoS Negl.
Trop. Dis. 2011, 5, e1007.
4. Handman, E.; Bullen, D. V. R. Trends Parasitol. 2002, 18, 332.
5. WHO, World health report 2004 statistical annex. World Health Organization:
2004; p 187, ISSN 1020-3311, ISBN 92 4 156265 X, http://www.who.int/whr/
2004/annex/en/index.html.
6. Pagliero, R. J.; Lusvarghi, S.; Pierini, A. B.; Brun, R.; Mazzieri, M. R. Bioorg. Med.
Chem. 2010, 18, 142.
7. WHO, Control of Chagas disease: second report of the WHO expert committee.
World Health Organization, Geneva: 2002; Vol. 905, p 109, ISBN 9241209054,
http://www.who.int/iris/handle/10665/42443.
8. Caputto, M. E.; Fabian, L. E.; Benítez, D. D.; Merlino, A.; Ríos, N.; Cerecetto, H.;
Moltrasio, G. Y.; Moglioni, A. G.; González, M.; Finkielsztein, L. M. Bioorg. Med.
Chem. 2011, 19, 6818.
9. Coura, J. R.; de Castro, S. L. Mem. Inst. Oswaldo Cruz 2002, 97, 3.
10. Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19, 495.
11. Aguirre, G.; Boiani, L.; Cerecetto, H.; Fernandez, M.; Gonzalez, M.; Denicola, A.;
Otero, L.; Gambino, D.; Rigol, C.; Olea-Azar, C.; Faundez, M. Bioorg. Med. Chem.
2004, 12, 4885.
The Implication of this work is that the compounds with
12. Aguirre, G.; Cabrera, E.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Seoane, G.;
Duffaut, A.; Denicola, A.; Gil, M. J.; Martinez-Merino, V. Eur. J. Med. Chem. 2004,
39, 421.
13. Messeder, J. C.; Tinoco, L. W.; Figueroa-Villar, J. D.; Souza, E. M.; Santa Rita, R.;
de Castro, S. L. Bioorg. Med. Chem. Lett. 1995, 5, 3079.
14. Chung, M.-C.; Güido, R. V. C.; Martinelli, T. F.; Gonçalves, M. F.; Polli, M. C.;
Botelho, K. C. A.; Varanda, E. A.; Colli, W.; Miranda, M. T. M.; Ferreira, E. I.
Bioorg. Med. Chem. 2003, 11, 4779.
15. Leite, A. C. L.; de Lima, R. S.; Moreira, D. R. d. M.; Cardoso, M. V. d. O.; Gouveia
de Brito, A. C.; Farias dos Santos, L. M.; Hernandes, M. Z.; Kiperstok, A. C.; de
Lima, R. S.; Soares, M. B. P. Bioorg. Med. Chem. 2006, 14, 3749.
16. Gómez-Ayala, S.; Castrillón, J. A.; Palma, A.; Leal, S. M.; Escobar, P.; Bahsas, A.
Bioorg. Med. Chem. 2010, 18, 4721.
17. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111.
18. Ouellette, M.; Drummelsmith, J.; Papadopoulou, B. Drug Resist. Updat. 2004, 7,
257.
19. Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.; Sagrera, G.; Seoane, G.;
Denicola, A.; Peluffo, G.; Quijano, C.; Stoppani, A. O.; Paulino, M.; Olea-Azar, C.;
Basombrio, M. A. Eur. J. Med. Chem. 2000, 35, 343.
20. Keiser, J.; Stich, A.; Burri, C. Trends Parasitol. 2001, 17, 42.
21. Molfetta, F. A.; Bruni, A. T.; Honório, K. M.; da Silva, A. B. F. Eur. J. Med. Chem.
2005, 40, 329.
22. Hucke, O.; Gelb, M. H.; Verlinde, C. L.; Buckner, F. S. J. Med. Chem. 2005, 48,
5415.
antiprotozoal activity of IC50 <5 lM, should be utilized for further
lead optimization to come up with the best potential candidate.
There are compounds which have been identified specific to Try-
panosoma spp. and some are even active against all tested species
of Trypanosoma spp. and L. infantum, which poses a challenge for
further medicinal chemistry in order to explore that how the aspe-
cific character of these compounds can be directed to one organ-
ism. The compounds of present study further suggest that a
small number of molecules with extensive diversification are not
appropriate to optimize the drug-like candidates but it provides
a fast track procedure to identify the best possible type of com-
pounds, where the more concentrated efforts can be made to de-
velop the single-point diversified library to reach the drug like
compound.
Many of the compounds reported here have shown impres-
sive activity against sensitive strain H37Rv of Mycobacterium
tuberculosis.28–32 Therefore, these compounds may enable to hit
multiple targets and that might provide new avenues for the
treatment of protozoan diseases. This may impact in many
ways: (i) such multiple action single-drug therapeutic against
23. Lepesheva, G. I.; Nes, W. D.; Zhou, W.; Hill, G. C.; Waterman, M. R. Biochemistry
2004, 43, 10789.

2758
R. S. Upadhayaya et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2750–2758
24. Lepesheva, G. I.; Park, H. W.; Hargrove, T. Y.; Vanhollebeke, B.; Wawrzak, Z.;
Harp, J. M.; Sundaramoorthy, M.; Nes, W. D.; Pays, E.; Chaudhuri, M.; Villalta,
F.; Waterman, M. R. J. Biol. Chem. 2010, 285, 1773.
33. Garcia, S.; Ramos, C. O.; Senra, J. F.; Vilas-Boas, F.; Rodrigues, M. M.; Campos-
de-Carvalho, A. C.; Ribeiro-Dos-Santos, R.; Soares, M. B. Antimicrob. Agents
Chemother. 2005, 49, 1521.
25. Shibata, S.; Gillespie, J. R.; Kelley, A. M.; Napuli, A. J.; Zhang, Z.; Kovzun, K. V.;
Pefley, R. M.; Lam, J.; Zucker, F. H.; Van Voorhis, W. C.; Merritt, E. A.; Hol, W. G.;
Verlinde, C. L.; Fan, E.; Buckner, F. S. Antimicrob. Agents Chemother. 1982, 2011,
55.
34. Rivarola, H. W.; Paglini-Oliva, P. A. Curr. Drug Targets Cardiovasc. Haematol.
Disord. 2002, 2, 43.
35. Paila, Y. D.; Saha, B.; Chattopadhyay, A. Biochem. Biophys. Res. Commun. 2010,
399, 429.
26. Carvalho, L.; Luque-Ortega, J. R.; Manzano, J. I.; Castanys, S.; Rivas, L.; Gamarro,
F. Antimicrob. Agents Chemother. 2010, 54, 5344.
27. Kraus, J. M.; Verlinde, C. L. M. J.; Karimi, M.; Lepesheva, G. I.; Gelb, M. H.;
Buckner, F. S. J. Med. Chem. 2009, 52, 1639.
28. Upadhayaya, R. S.; Vandavasi, J. K.; Vasireddy, N. R.; Sharma, V.; Dixit, S. S.;
Chattopadhyaya, J. Bioorg. Med. Chem. 2009, 17, 2830.
29. Upadhayaya, R. S.; Kulkarni, G. M.; Vasireddy, N. R.; Vandavasi, J. K.; Dixit, S. S.;
Sharma, V.; Chattopadhyaya, J. Bioorg. Med. Chem. 2009, 17, 4681.
30. Upadhayaya, R. S.; Lahore, S. V.; Sayyed, A. Y.; Dixit, S. S.; Shinde, P. D.;
Chattopadhyaya, J. Org. Biomol. Chem. 2010, 8, 2180.
36. Dey, A. S.; Joullié, M. M. J. Heterocycl. Chem. 1965, 2, 113.
37. Margolis, B. J.; Long, K. A.; Laird, D. L.; Ruble, J. C.; Pulley, S. R. J. Org. Chem. 2007,
72, 2232.
38. Mamo, A.; Nicoletti, S.; Tat, N. Molecules 2002, 7, 618.
39. Gros, L.; Castillo-Acosta, V. M.; Jimenez Jimenez, C.; Sealey-Cardona, M.;
Vargas, S.; Manuel Estevez, A.; Yardley, V.; Rattray, L.; Croft, S. L.; Ruiz-Perez, L.
M.; Urbina, J. A.; Gilbert, I. H.; Gonzalez-Pacanowska, D. Antimicrob. Agents
Chemother. 2006, 50, 2595.
40. Bollini, M.; Casal, J. J.; Alvarez, D. E.; Boiani, L.; Gonzalez, M.; Cerecetto, H.;
Bruno, A. M. Bioorg. Med. Chem. 2009, 17, 1437.
31. Upadhayaya, R. S.; Shinde, P. D.; Sayyed, A. Y.; Kadam, S. A.; Bawane, A. N.;
Poddar, A.; Plashkevych, O.; Foldesi, A.; Chattopadhyaya, J. Org. Biomol. Chem.
2010, 8, 5661.
32. Upadhayaya, R. S.; Shinde, P. D.; Kadam, S. A.; Bawane, A. N.; Sayyed, A. Y.;
Kardile, R. A.; Gitay, P. N.; Lahore, S. V.; Dixit, S. S.; Foldesi, A.; Chattopadhyaya,
J. Eur. J. Med. Chem. 2011, 46, 1306.
41. Neal, R. A.; Croft, S. L. J. Antimicrob. Chemother. 1984, 14, 463.
42. Iguchi, K.; Usui, S.; Ishida, R.; Hirano, K. Apoptosis 2002, 7, 519.
43. Simonetti, G.; Baffa, S.; Simonetti, N. Int. J. Antimicrob. Agents 2001, 17,
389.
44. Dixit, S. S.; Upadhayaya, R. S.; Chattopadhyaya, J. Org. Biomol. Chem. 2012, 10,
6121.