Design, synthesis and in vitro antiplasmodial activity of some bisquinolines against chloroquine-resistant strain

Article abstract of DOI:10.1111/cbdd.12914

A series of novel bisquinoline compounds comprising N¹-(7-chloroquinolin-4-yl) ethane-1,2-diamine and 7-chloro-N-(2-(piperazin-1-yl)ethyl)quinolin-4-amine connected with 7-chloro-4-aminoquinoline containing various amino acids is described. We have bio-evaluated the compounds against both chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum in vitro. Among the series, compounds 4 and 7 exhibited 1.8- and 10.6-fold superior activity as compared to chloroquine (CQ; IC₅₀?=?0.255?±?0.049?μm) against the K1 strain with IC₅₀ values 0.137?±?0.014 and 0.026?±?0.007?μm, respectively. Furthermore, compound 7 also displayed promising activity against the 3D7 strain (IC₅₀?=?0.024?±?0.003?μm) of P.?falciparum when compared to CQ. All the compounds in the series displayed resistance factor between 0.57 and 4.71 as against 51 for CQ. These results suggest that bisquinolines can be explored for further development as new antimalarial agents active against chloroquine-resistant P.?falciparum.

Full text of DOI:10.1111/cbdd.12914

Received Date : 19-Jul-2016
Revised Date : 11-Nov-2016
Accepted Date : 14-Nov-2016
Article type : Research Article
Design, Synthesis and in Vitro Antiplasmodial Activity of Some Bisquinolines against
Chloroquine Resistant Strain
Srinivasarao Kondaparlaa, Pooja Agarwalb, Kumkum Srivastavab, Sunil K. Purib, S. B.
Kattia*
aMedicinal & Process Chemistry Division, CSIR-Central Drug Research Institute Sector-10,
Jankipuram Extension, Sitapur Road, Lucknow -226031.
bParasitology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram
Extension, Sitapur Road, Lucknow -226031.
Short Running Title: Antiplasmodial Activity of Some Bisquinolines.
Keywords: 4-Aminoquinoline; Antiplasmodial activity; Chloroquine; Plasmodium
falciparum; Chloroquine sensitive strain; Chloroquine resistant strain.
Abbreviations: CQ, chloroquine; DCC, N,N-dicyclohexylcarbodiimide; HOBt,
hydroxybenzotriazole; DMF, dimethylformamide.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12914
This article is protected by copyright. All rights reserved.

*Corresponding author; Tel: +91-522-2772454, Fax; +91-522-2771941
Email address; setu_katti@yahoo.com (S.B. Katti)
Abstract:
A series of novel bisquinoline compounds comprising, N1-(7-chloroquinolin-4-yl)
ethane-1,2-diamine and 7-chloro-N-(2-(piperazin-1-yl)ethyl)quinolin-4-amine connected with
7-chloro-4-aminoquinoline containing various amino acids is described. We have bio-
evaluated the compounds against both chloroquine-sensitive (3D7) and chloroquine-resistant
(K1) strains of Plasmodium falciparum in vitro. Among the series, compounds 4 and 7
exhibited 1.8 and 10.6 fold superior activity as compared to chloroquine (CQ) (IC50
=
0.255±0.049 µM) against the K1 strain with IC50 values 0.137±0.014 and 0.026±0.007 µM
respectively. Furthermore, compound 7 also displayed promising activity against the 3D7
strain (IC50 = 0.024±0.003 µM) of P. falciparum when compared to CQ. All the compounds
in the series displayed resistance factor between 0.57 and 4.71 as against 51 for CQ. These
results suggest that bisquinolines can be explored for further development as new antimalarial
agents active against chloroquine resistant P. falciparum.
1. Introduction
Even after several decades of CQ (Figure 1) discovery, it is still considered as a
mainstream drug for the control and eradication of malaria. The immense success of CQ is
due to its unique features like limited host toxicity, less side effects and affordability [1].
However, in recent years efficacy of CQ and other 4-aminoquinoline analogues has steadily
declined due to emergence of drug resistant parasites in the endemic regions [2]. Therefore, it
is a clear indication for researchers to discover new chemical entities with high therapeutic
efficacy against chloroquine resistant (CQ-R) parasite. During the literature survey it was
This article is protected by copyright. All rights reserved.

found that a number of bisquinolines exhibited potential antiplasmodial activity particularly
against CQ-R parasite [3]. The early examples of such agents include
bis(quinolyl)piperazines such as piperaquine (Figure 1) which was first synthesized in the
year 1960. Efficacy, bioavailability and good pharmacokinetic properties led this molecule to
therapeutic application. Other drug leads such as dichloroquinazine (12,278RP) and the most
promising molecule Ro 47-7737 (Figure 1) were found to be potent, but could not be pursued
as drug candidates due to toxicity. The available literature data suggest that bisquinolines are
less likely to be affected by the efflux mechanism operating in CQ-R P. falciparum [4-5].
Figure 1 Structures of some antimalarial agents
Subsequently, Cowman et al. described the synthesis of a novel series of
bisquinolinemethanols (Figure 2 A), which exhibited better activity than CQ against CQ-R
parasite, but because of toxicity, these molecules were not pursued [6]. Later, Vennerstrom
and co-workers synthesized a series of bisquinolines (Figure 2 B) with nitrogen and oxygen
containing alkane linkers, which were found to be more potent against both CQ-sensitive
(D6) as well as CQ-resistant (W2) strains of P. falciparum in the in vitro studies [7]. Further,
Raynes et al. explored two series of bisquinolines (Figure 2 C) having bisamide linkage at 6
and 8 positions of the quinoline nucleus. These analogues were found to have moderate
activity against CQ-sensitive strain (D10) and good activity against CQ-resistant strains
(FAC8 and K1) of P. falciparum. The activity profile of these bisquinolines suggested that
the position of attachment and length of the linker chain markedly affected the activity [8].
Furthermore, Girault’s group synthesised bis, tris and tetraquinolines (Figure 2 D) by
introducing proton accepting and/or substitution sites, in order to maintain both steric
hindrance and conformational flexibility of quinoline nucleus, but they were found to be less
potent than their linear counterparts [9]. More recently, Tomar et al. have synthesized a series
This article is protected by copyright. All rights reserved.

of substituted bisquinolines (Figure 2 E, R=CH3, H; R1=H, CH3, C2H5) having excellent in
vitro activity against CQ-S parasite [10].
Figure 2 Structures of some bisquinoline antimalarials.
Previously from our group, we have reported a series of CQ analogues obtained by
modifying amine side chain derived from various amino acids (Figure 3 a). These CQ
analogues showed good activity (IC50 ranging between 11.51 and >500 nM) against CQ-
sensitive (3D7) strain and very good activity (IC50 ranging between 55.29 and >500 nM)
against CQ-resistant (K1) strain of P. falciparum [11]. Recently, Kondaparla et al. have
synthesized a series of side chain modified 4-aminoquinolines by connecting N1-(7-
chloroquinolin-4-yl)ethane-1,2-diamine to different amino acids (Gly, Ala, Val, Leu, Ile, Phe,
Trp and Met) via peptide (-CO-NH)- linkage (Figure 3 b). These molecules were found to
have moderate activity (IC50 ranging between 0.015±0.002 and 3.22±0.041 µM) against 3D7
strain and promising activity (IC50 ranging between 0.17±0.002 and >1.0±0.045 µM) against
K1 strain of P. falciparum. The results indicated that the hydrophobicity of the amino acid
side chain and pendant amine group is essential for antiplasmodial activity of 4-
aminoquinolines [12].
Based on the above information, we thought it appropriate to introduce amide bond
with heterocyclic ring system (7-chloro-4-aminoquinoline) on lateral side chain of 4-
aminoquinoline as it will provide lipophilic nature to the molecules and could give
compounds with improved antimalarial activity. The quinoline nucleus was selected based on
the observation that it is a biologically privileged scaffold and well tolerated in human
subjects [13]. In continuation of the antimalarial drug discovery programme in our group
This article is protected by copyright. All rights reserved.

herein we report the efficient synthesis of new bis(4-aminoquinoline) analogues (Figure 3 c)
(scheme 1) and their antiplasmodial activity.
Figure 3 Some lead molecules of 4-aminoquinoline derived antimalarials
developed from this laboratory.
2. Results and Discussion
2.1 Chemistry
The desired bisquinolines 4-15 were synthesized via coupling of N1-(7-chloroquinolin-4-
yl)ethane-1,2-diamine (1) and 7-chloro-N-(2-(piperazin-1-yl)ethyl)quinolin-4-amine (3) with
respective intermediates 2a-2h using (DCC/HOBt) as a coupling reagent (scheme 1). Further,
synthesis of intermediates 1 and 3 were achieved by the nucleophilic aromatic substitution
reaction (SNAr) executed on 4,7-dichloroquinoline using 1,2-diaminoethane and 2-
aminoethylpiperazine as reactive precursors respectively, in good yields. However, when the
same reaction was performed for the preparation of intermediates 2a-2h, the desired products
could not be obtained. Subsequently, we have achieved the synthesis of required
intermediates 2a-2h by performing the reaction in phenol at 160 °C. The final compounds (4-
15) were characterized by 1H NMR, 13C NMR, ESI-MS and HRMS respectively.
Scheme 1 Synthesis of bisquinoline antimalarials.
2.2 Antiplasmodial (P. falciparum) Activity
The bisquinolines 4-15 have the IC50 values ranging between 0.024±0.003 and 1.69±0.045
µM against chloroquine sensitive (CQ-S) and 0.026±0.007 to 2.09±0.049 µM against
chloroquine resistant (CQ-R) strains of P. falciparum in vitro and the results are shown in
Table 1. From the in vitro results, it is clear that bisquinolines constructed with alkylamine
linker were more potent than the corresponding piperazine linker analogues. However,
bisquinolines 4, 5 and 8-15 displayed poor activity against the CQ-S strain with IC50 values
This article is protected by copyright. All rights reserved.

ranging between 0.155±0.017 and 1.69±0.045µM as compared to CQ (IC50 = 0.005±0.002
µM). On the other hand compound 4 showed superior antiplasmodial activity against the CQ-
R strain (IC50 = 0.137±0.014 µM) when compared to CQ. Moreover, compounds 9 and 11
exhibited activity comparable to CQ against the CQ-R strain of P. falciparum with IC50
values 0.304±0.028 µM and 0.355±0.037 µM respectively. Among all the bisquinolines,
compound 7 with an isobutyl group in the side chain displayed good inhibition of parasite
growth against the CQ-S strain (IC50 = 0.024±0.003 µM). It is important to mention here that
this compound also showed 10.6 fold superior activity against the CQ-R strain of P.
falciparum with IC50 = 0.026±0.007 µM as compared to CQ (IC50 = 0.255±0.049 µM). By
comparison, compound 13 containing a piperazine linker exhibited very poor inhibition of
parasite growth (IC50 = 1.69±0.045 µM (3D7), 2.09±0.049 µM (K1) against both strains of P.
falciparum. It may be appropriate to mention that some of our compounds have exhibited
very low resistance factor values ranging from 0.57-4.71 than the reference drug CQ which is
having resistance factor value 51. From the in vitro data it is clear that there is a correlation
between length of the bisquinoline heteroalkane linker and the antiplasmodial activity, this is
in agreement with the published report from Vennerstrom et al. [7].
2.3 Discussion
According to the obtained in vitro results, it has been observed that nature of the
linker plays a vital role on the inhibition of parasite growth against both CQ-S and CQ-R
strains of P. falciparum. In general, bisquinoline analogues 4-11 containing alkylamine
linkers were more potent than piperazine linker bisquinolines 12-15 (Table 1; eg 7 vs 13, 9
vs 14 and 10 vs 15) and this may be due to increased rigidity and decreased flexibility of the
piperazine moiety. One exception is that of 6 compared with compound 12, with the
piperazine linker compound showing activity against both strains (IC50 =0.884±0.046 μM
(3D7), 0.510±0.036 μM (K1)). Compound 4 having simple hydrogen as its side chain showed
This article is protected by copyright. All rights reserved.

moderate activity against the 3D7 strain with IC50 value 0.155±0.017 μM, and slightly better
activity than CQ when screened against the CQ-R strain of P. falciparum. Further, in the case
of alanine analogue 5 the activity was decreased significantly against both strains (IC50
=
0.250±0.021 μM (3D7), 0.923±0.032 μM (K1) of P. falciparum. When the amino acid side
chain is changed to methionine, this afforded compound 11 with higher activity against the
3D7 strain (IC50 = 0.386±0.033 μM) and activity comparable to CQ when screened against
the K1 strain (IC50 = 0.355±0.037 μM) of P. falciparum. Among all bisquinolines of the
series, compound 7 with an isobutyl group in the side chain exhibited 10.6 fold superior
activity against the CQ-R strain (IC50 = 0.026±0.007 μM) and promising activity against the
CQ-S strain (IC50 = 0.024±0.003 μM) as compared to CQ. As already discussed, the activity
was reduced drastically against both strains (3D7 & K1) when rigid aminoethylpiperazine
moiety was incorporated instead of the flexible alkanediamine linker as in compound 13.
The structure activity relationship (SAR) studies of bis(4-aminoquinolines) suggested
that 7-chloro group and quinoline scaffolds are most favourable for antiplasmodial activity
(Figure 4). The length of the carbon chain linker in the side chain of bisquinoline plays a key
role on the antiplasmodial activity against chloroquine resistant strain of P. falciparum. It is
evident from the results that bisquinolines containing short linker such as 2 carbon chain
(ethane-1,2-diamine) were appropriate for the activity. However, bisquinolines containing
longer and conformationally rigid linker such as 1-(2-aminoethyl) piperazine have shown
poor activity. Furthermore, presence of an isobutyl group in the linker potentiates the
biological activity (Figure 4).
This article is protected by copyright. All rights reserved.

Figure 4 SAR of the synthesized potent active bisquinoline.
2.4 In Vitro cytotoxicity
The cytotoxicity of synthesized bisquinolines 4-15 was determined by the standard protocol,
MTT assay against VERO cell line (Table 1). All bisquinolines displayed the selectivity
index (SI) values ranging from 20.50 to 1011.71. The compound 7 showed good selectivity
index (SI) 1011.71 (Table 1) and equally good parasite growth inhibition against the CQ-R
strain of P. falciparum., The remaining compounds in the series showed less cytotoxic effect
with fairly considerable selectivity index (SI) and moderate antiplasmodial activity.
Table 1 Antiplasmodial activity of bisquinolines (4-15) against P. falciparum in Vitro.
3. Conclusion
In conclusion, we have synthesized a new series of bisquinolines and screened for the
antiplasmodial activity against both CQ-S and CQ-R strains of P. falciparum. From the
activity data, it may be inferred that bisquinolines with alkylamine linker were more potent
than the corresponding piperazine linker analogues. Further, compound 7 exhibited 10.6 fold
superior antiplasmodial activity against the CQ-R strain and promising activity against the
CQ-S strain of P. falciparum. Furthermore, almost all the compounds in the series showed
resistance factor values much lower than CQ. From this exploration, we have identified the
lead compound 7 [(S)-2-(7-chloroquinolin-4-ylamino)-N-(2-(7-chloroquinolin-4-ylamino)
ethyl)-4-methylpentanamide] having potent activity against both CQ-S and CQ-R strains of
P. falciparum .
This article is protected by copyright. All rights reserved.

Acknowledgment
One of the authors (K.S.R) thanks the CSIR, New Delhi, for Senior Research Fellowship.
Authors thank the Director, CDRI, for the support, and the SAIF division for the spectral
data. The CDRI Communication No. is 9379.
Conflicts of Interest
The authors have no conflict of interest to declare
Supporting Information available (Appendix S1): Biological assay & materials and
methods of synthesized compounds.
Figure Legends
Figure 1 Structures of some antimalarial agents
Figure 2 Structures of some bisquinoline antimalarials.
Figure 3 Some lead molecules of 4-aminoquinoline derived antimalarials developed from
this laboratory.
Figure 4 SAR of the synthesized potent active bisquinoline.
Scheme 1 Synthesis of bisquinoline antimalarials.
Table 1 Antiplasmodial activity of bisquinolines (4-15) against P. falciparum in Vitro.
This article is protected by copyright. All rights reserved.

References
1. Guillon J, Grellier P, Labaied M, Sonnet P, Leger JM, Deprez-Poulain R, et al. (2004)
Synthesis, antimalarial activity, and molecular modeling of new pyrrolo[1,2-
a]quinoxalines, bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines,
and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines. Journal of Medicinal Chemistry;47: 1997-
2009 doi:10.1021/jm0310840.
2. Opsenica IM, Tot M, Gomba L, Nuss JE, Sciotti RJ, Bavari S, et al. (2013) 4-Amino-7-
chloroquinolines: probing ligand efficiency provides botulinum neurotoxin serotype A
light chain inhibitors with significant antiprotozoal activity. Journal of Medicinal
Chemistry;56: 5860-71 doi:10.1021/jm4006077.
3. Girault S, Grellier P, Berecibar A, Maes L, Mouray E, Lemiere P, et al. (2000)
Antimalarial, antitrypanosomal, and antileishmanial activities and cytotoxicity of bis(9-
amino-6-chloro-2-methoxyacridines): influence of the linker. Journal of Medicinal
Chemistry;43: 2646-54 doi:10.1021/jm990946n.
4. Ayad F, Tilley L, Deady LW (2001) Synthesis, antimalarial activity and inhibition of
haem detoxification of novel bisquinolines. Bioorganic & Medicinal Chemistry
Letters;11: 2075-7 doi:10.1016/S0960-894X(01)00383-3.
5. Raynes K, Foley M, Tilley L, Deady LW (1996) Novel bisquinoline antimalarials.
Synthesis, antimalarial activity, and inhibition of haem polymerisation. Biochemical
Pharmacology;52: 551-9 doi:10.1016/0006-2952(96)00306-1.
6. Cowman A F DL, Deharo E, Desneves J, Tilley L (1997) Synthesis and activity of some
antimalarial bisquinolinemethanols. Australian Journal of Chemistry;50: 1091-97.
This article is protected by copyright. All rights reserved.

7. Vennerstrom JL, Ager AL, Jr., Dorn A, Andersen SL, Gerena L, Ridley RG, Milhous WK
(1998)
Bisquinolines.
2.
Antimalarial
of Medicinal
N,N-bis(7-chloroquinolin-4-
Chemistry; 41:4360-64
yl)heteroalkanediamines.
doi:10.1021/jm9803828.
Journal
8. Raynes K, Galatis D, Cowman AF, Tilley L, Deady LW (1995) Synthesis and activity of
some antimalarial bisquinolines. Journal of Medicinal Chemistry;38: 204-6
doi:10.1021/jm00001a026
9. Girault S, Grellier P, Berecibar A, Maes L, Lemiere P, Mouray E, et al. (2001)
Antiplasmodial activity and cytotoxicity of bis-, tris-, and tetraquinolines with linear or
cyclic
amino
linkers.
Journal
of
Medicinal
Chemistry;44:
1658-65
doi:10.1021/jm001096a
10.Tomar N, Jain CL, Tyagi R (2013) Synthesis, Spectral analysis and Characterization of
Substituted Bisquinoline and their Antimalarial Activity. Acta Ciencia Indica. Chemistry;
39(3): 81-84
11. Sinha M, Dola VR, Agarwal P, Srivastava K, Haq W, Puri SK, Katti SB (2014)
Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant
strain. Bioorganic & Medicinal Chemistry; 22:3573-3586 doi:10.1016/j.bmc.2014.05.024
12. Kondaparla S, Agarwal P, Srivastava K, Haq W, Puri SK, Katti SB (2016) Design,
synthesis, and in vitro antiplasmodial activity of 4-aminoquinolines containing modified
amino acid conjugates. Medicinal Chemistry Research; 25:1148-1162 doi:
10.1007/s00044-016-1555-5.
This article is protected by copyright. All rights reserved.

13. Lesyk RB, Zimenkovsky BS (2004) 4-Thiazolidones: Centenarian history, current status
and perspectives for modern organic and medicinal chemistry. Current Organic
Chemistry; 8:1547–1577.
14. Kondaparla S, Soni A, Manhas A, Srivastava K, Puri SK, and Katti SB (2016) Synthesis
and antimalarial activity of new 4-aminoquinolines active against drug resistant strains.
RSC Advances;6: 105676-105689 doi: 10.1039/c6ra14016e.
15. Srivastava K and Puri SK (2009) Recent Developments in Plasmodium falciparum in
vitro model for Drug Discovery. Proceedings of the National Academy Sciences;79: 37-
47.
16. Sinha M et al. (2014) Synthesis of chiral chloroquine and its analogues as antimalarial
agents. Bioorganic & Medicinal Chemistry;22:5950-5960 doi:10.1016/j.bmc.2014.09.009.
17. Bhat HR, Singh UP, Gahtori P, Ghosh SK, Gogoi K, Prakash A, et al. (2015) Synthesis,
Docking, In Vitro and In Vivo Antimalarial Activity of Hybrid 4-aminoquinoline-1,3,5-
triazine Derivatives Against Wild and Mutant Malaria Parasites. Chemical Biology &
Drug Design;86: 265-71.
This article is protected by copyright. All rights reserved.

Table 1 Antiplasmodial activity of bisquinolines (4-15) against P. falciparum in Vitro.
Co
mpd
. no
R1
In vitro antiplasmodial activity
SIb
LogPc Resistanc
e factord
IC50(µM±SD)a
3D7
K1
H
0.155±0.017
0.137±0.014
234.83
2.95
0.88
4
5
6
7
8
9
CH3
0.250±0.021
NA
0.923±0.032
NA
428.50
NA
3.44
4.33
4.68
4.75
5.12
3.69
ND
CH(CH3)2
CH2CH(CH3)2
CHCH3CH2CH3
CH2C6H5
0.024±0.003
0.735±0.034
0.175±0.039
0.026±0.007 1011.71
1.08
1.36
1.73
1.43±0.045
104.59
166.22
0.304±0.028
10 CH2-Indolyl
11 (CH2)2SCH3
12 CH(CH3)2
0.212±0.026
0.386±0.033
0.884±0.046
1.69±0.045
0.909±0.041
0.958±0.032
2.08±0.049
0.355±0.037
0.510±0.036
2.09±0.049
1.56±0.048
0.996±0.038
95.23
92.27
20.50
34.75
33.89
55.17
4.66
3.78
4.32
4.67
5.11
4.65
4.71
0.91
0.57
1.00
1.10
1.03
13 CH2CH(CH3)2
14 CH2C6H5
15 CH2-Indolyl
This article is protected by copyright. All rights reserved.

8983
CQ
0.005±0.002
0.255±0.049
3.73
51
a
b
IC50(µM±SD) : Concentration corresponding to 50% growth inhibition of the parasite; Selectivity
c
index (SI): (CC50 for cytotoxicity to vero cells/IC50(3D7) for antiplasmodial activity); log P values
calculated using ChemBioDraw ultra software; Resistance factor (RI) = IC50 (K1)/IC50 (3D7); ND:
d
Not done, NA: Not active.
Figure 1 Structures of some antimalarial agents
This article is protected by copyright. All rights reserved.

Figure 2 Structures of some bisquinoline antimalarials.
This article is protected by copyright. All rights reserved.

Figure 3 Some lead molecules of 4-aminoquinoline derived antimalarials developed
from this laboratory.
Figure 4 SAR of the synthesized potent active bisquinoline.
This article is protected by copyright. All rights reserved.

Scheme 1 Synthesis of bisquinoline antimalarials. Reagents and conditions: a) ethylenediamine,
neat, 80-130 °C, 8h. b) amino acid, phenol, 160 °C, 1-6 h, 55-68% c) 1-(2-Aminoethyl)piperazine,
CH3OH, reflux, 4-8 h, 75% d,e) DCC/HOBt, DMF, 0 °C, 4-6 h, 69-82%.
This article is protected by copyright. All rights reserved.