Moni
Sharma
a,
Kuldeep
Chauhan
a,
Shikha S.
Chauhan
a,
Ashok
Kumar
a,
Shiv Vardan
Singh
b,
Jitendra K.
Saxena
b,
Pooja
Agarwal
c,
Kumkum
Srivastava
c,
S.
Raja Kumar
c,
Sunil K.
Puri
c,
Priyanka
Shah
d,
M. I.
Siddiqi
d and
Prem M. S.
Chauhan
*a
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute Chattar Manzil, P.O. Box 173, Mahatma Gandhi Marg, Lucknow 226001, India. E-mail: prem_chauhan_2000@yahoo.com; Fax: +91 522 2623405; Tel: +91 522 2612411premsc58@hotmail.com
bDivision of Biochemistry, CSIR-Central Drug Research Institute, Lucknow 226001, India
cDivision of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226001, India
dMolecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
First published on 3rd November 2011
Analogues of a novel class of hybrid 4-anilinoquinoline triazines have been synthesized with the aim of identifying the compounds with improved antimalarial activity preserving the potency of parent drug COMPOUND LINKS
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Download mol file of compoundchloroquine (CQ). All the synthesized molecules were evaluated in vitro for their antimalarial activity against chloroquine-sensitive 3D7 and chloroquine-resistant K1 strains of P. falciparum. Molecules were also screened for their cytotoxicity towards VERO cell line. Sixteen compounds (17, 19, 26, 27, 29, 31, 32, 33, 35, 36, 37, 39, 40, 49, 50, and 52) exhibited excellent antimalarial activity with IC50 values ranging from 1.36–4.63 ng ml−1 and were also found to be nontoxic with good selectivity index. In silico activity prediction as well as enzyme inhibitory activity against P. falciparumtransketolase reveals that the molecules are also good inhibitors of the enzymeP. falciparumtransketolase. The compound 52 showed good in vivo activity by oral route and resulted in survival of 3 out of 5 mice till day 28.
Fig. 1 Important antimalarials (1–5) and transketolase inhibitors (6–8). |
In 1987 Peters et al. first introduced the bitherapy strategy for the treatment of malaria, but this bitherapy strategy in malaria failed to prevent the emergence of resistance.25 On the above basis, another strategy i.e. hybridization approach has recently been developed.26 Molecular hybridization involves the rational design of new chemical entities by the fusion (usually via a covalent linker) of two drugs, both active compounds and/or pharmacophoric units recognized and derived from known bioactive molecules.27 Furthermore, to circumvent antimalarial drug resistance, hybridization is quite an attractive strategy, particularly when the pharmacophores/active molecules being merged possess independent modes of antimalarial action. Due to its advanced mode of action and high selectivity, hybrid molecule-based chemotherapy appears as a beneficial tool in the current trend of drug discovery.
Among old and new targets of malaria chemotherapy, the host heme molecule attacked by COMPOUND LINKS
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Download mol file of compoundchloroquine is still one of the most attractive targets for drug development.28–30 The biology, mode of action and resistance of COMPOUND LINKS
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Download mol file of compoundchloroquine has been extensively studied in the literature.31–36 Moreover in retrospect, CQ is a wonder drug cheap, safe, easily affordable and most effective and therefore the use of the haloquinoline core of CQ is simply too strong to abandon. On another hand Plasmodium falciparumtransketolase, a vital enzyme of the non-oxidative branch of pentose phosphate pathway has been reported as a novel target in Plasmodium falciparum.37 The selectivity of the target arises due to adequate difference between the human and parasite's transketolase. The enzyme catalyzes the cleavage of a carbon–carbon bond adjacent to a carbonyl group in keto sugars and transfers a ketol moiety to aldosugar.38 Recently the P. falciparumtransketolase has been cloned, expressed and characterized.39 The mechanism of action of transketolase has been mediated by its cofactor COMPOUND LINKS
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Download mol file of compoundthiamine pyrophosphate (ThPP), which is coordinated to divalent metal ions.40,41COMPOUND LINKS
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Download mol file of compoundp-Hydroxyphenyl pyruvate (6), a natural analogue of transketolase substrate is proved to be a reversible and competitive inhibitor of transketolase with respect to substrate.42 Molecules such as COMPOUND LINKS
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Download mol file of compoundoxythiamine (7) and thiamine thiazolone diphosphate (8) also acting on this target have poor activity and low selectivity and therefore clinical applicability of this class of compound is inadequate.43,44
In light of all the above observations, several hybrid molecules (9–12) (Fig. 2) were recently developed by our group as well as by others in malaria chemotherapy.45–55 As a part of our continuing efforts in malaria chemotherapy and to address the urgent need for new antimalarial agents, here we further optimize the derivatives of hybrid 4-anilinoquinoline-COMPOUND LINKS
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Download mol file of compoundtriazine (16–52) (Fig. 2, Table 1) as antimalarial agents.
Fig. 2 Hybrid antimalarials. |
Compd. No. | R1 | R2 | a IC50 (ng mL−1) | b SI | |
---|---|---|---|---|---|
3D7 | K1 | ||||
a IC50 (ng mL−1): concentration corresponding to 50% growth inhibition of the parasite. b SI: Selectivity index (IC50 values of cytotoxic activity/IC50 values of antimalarial activity). | |||||
16 | 22.80 | 1.54 | 55.26 | ||
17 | 2.47 | ND | 149.49 | ||
18 | 40.02 | 78.07 | 122.19 | ||
19 | 1.36 | 5.71 | 551.47 | ||
20 | 128.74 | 106.53 | 75.13 | ||
21 | 8.97 | 2.06 | 121.51 | ||
22 | 11.11 | 19.79 | 88.21 | ||
23 | 9.81 | 95.41 | 1325.18 | ||
24 | 15.82 | ND | 682.05 | ||
25 | 41.75 | >500 | 520.00 | ||
26 | 2.41 | 16.87 | 360.99 | ||
27 | 2.11 | 4.96 | 317.53 | ||
28 | 30.60 | 6.49 | 101.30 | ||
29 | 3.82 | 8.82 | 180.15 | ||
30 | 7.80 | 35.15 | 66.66 | ||
31 | 4.14 | 9.81 | 67.63 | ||
32 | 4.63 | 8.58 | 481.64 | ||
33 | 4.26 | ND | 117.37 | ||
34 | 8.90 | ND | 72.95 | ||
35 | 2.98 | ND | 2496.64 | ||
36 | 4.26 | 3.08 | 75.11 | ||
37 | 3.81 | 155.06 | 1307.08 | ||
38 | 5.04 | 4.86 | 168.65 | ||
39 | 2.58 | 2.30 | 624.03 | ||
40 | 4.58 | 2.60 | 412.66 | ||
41 | 12.64 | 68.23 | 74.36 | ||
42 | 20.84 | 11.64 | 35.98 | ||
43 | 17.35 | 274.95 | 588.47 | ||
44 | 14.70 | >500 | 439.54 | ||
45 | 28.12 | ND | 48.36 | ||
46 | 12.12 | 116.20 | 75.90 | ||
47 | 45.21 | ND | 1160.00 | ||
48 | 20.32 | 55.48 | 435.53 | ||
49 | 3.22 | 2.86 | 121.18 | ||
50 | 2.16 | 14.68 | 430.55 | ||
51 | 5.87 | 53.17 | 41.56 | ||
52 | 2.76 | 2.66 | 3318.84 | ||
CQ (mean ± S.D.) | 2.45 ± 1.08 | 255.86 ± 65.58 | 8983.00 |
Scheme 1 Reagents and conditions: (a) 0 °C, THF, 2 h; (b) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundp-phenylenediamine, p-TSA, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundEtOH, 3 h; (c) mono amino aryl/alkyl substituted triazines, THF, reflux, 8 h; (d) various amines, THF, reflux, 5 h. |
Among the 37 hybrid prototypes prepared (16–52) and tested, 16 compounds (17, 19, 26, 27, 29, 31, 32, 33, 35, 36, 37, 39, 40, 49, 50, and 52), exhibited excellent IC50 values ranging from 1.36–4.63 ng mL−1. Five compounds (17, 19, 26, 27, and 50), showed IC50 values in the range of 1.36–2.47 ng mL−1 which were more potent than CQ. Four compounds displayed an IC50 value ranging from 2.58–3.22 ng mL−1 which were comparable to CQ. Other molecules were found to be less active than CQ, among which nine compounds showed IC50 values ranging from 3.81–5.87 ng mL−1, ten compounds exhibited IC50 values ranging from 7.80–17.35 ng mL−1 and eight compounds displayed IC50 in the range of 20.32–45.21 ng mL−1. During the synthesis of the desired molecules, p-phenylene diamine was selected as linker between the 4-aminoquinoline moiety and COMPOUND LINKS
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Download mol file of compoundtriazine nucleus. The selection of this linker was optimized by considering the analogy between the side chain of CQ and p-phenylene diamine as demonstrated in Fig. 3.
Fig. 3 Optimization of linker and lipophilicity. |
In the previous literature, several side chain modified derivatives of CQ and their significance are also described.62 Furthermore, to enhance the lipophilicity and acquire rigidity in these molecules, the flexible linker of CQ was replaced by a rigid and more lipophilic linker i.e. p-phenylene diamine and as a consequence, the synthesized molecules were found to be more active as compared to the parent drug (CQ).
Further, the activity of these molecules depends upon the substitution pattern around the triazine nucleus and thus great strides were taken to establish the complete SAR in these molecules. During the study of the substitution pattern of aryl/alkyl amines around the triazine nucleus it was observed that the meta substituted arylamines have more impact on antimalarial activity than the para substituted arylamines. The m-fluoroanilino substituted compounds having COMPOUND LINKS
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Download mol file of compoundN-ethyl piperazine (17) and COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl)morpholine (19) unit as substituents and COMPOUND LINKS
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Download mol file of compoundp-fluoroaniline analogues with the same substituents (22, 24) at the triazine nucleus showed IC50 values of 2.47 (17), 1.36 (19) and 11.11 (22), 15.82 (24) respectively. Similarly, m-chloroanilino substituted derivatives with amino substituents such as COMPOUND LINKS
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Download mol file of compoundN-methyl piperazine, COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl)morpholine showed superior activity with IC50 values of 2.41 (26), 2.11 (27) and 3.82 ng mL−1 (29) respectively in comparison to p-substituted derivatives with similar substitution having IC50 values of 7.80 (30), 4.14 (31) and 4.63 ng mL−1 (32) respectively. The COMPOUND LINKS
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Download mol file of compoundm-anisidine substituted derivative with 4-(3-aminopropyl)morpholine substitution has an IC50 value of 2.98 ng mL−1 (35) while COMPOUND LINKS
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Download mol file of compoundp-anisidine with the same substitution showed an IC50 value of 3.18 ng mL−1 (37). These results clearly indicate the preference for m-substituted aryl amines over the p-substituted aryl amines. In the case of dimethoxy substituted derivatives (38–40) a combined effect of substituents was observed with IC50 value 5.04, 2.58, 4.58 ng mL−1.
Regardless of the cyclic/acyclic amino substitution at the triazine nucleus, arylamino substituents have more impact on antimalarial activity compared to non aromatic or reduced aromatic systems such as COMPOUND LINKS
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Download mol file of compoundtetrahydroquinoline (41–44) and COMPOUND LINKS
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Download mol file of compoundtetrahydroisoquinoline (45–48). These results indicate that aromatic system is necessary to maintain the favorable interactions that are responsible for antimalarial activity. While in place of aromatic amines, morpholino substituted derivatives (49–52) also showed very good activity with IC50 values of 3.22, 2.16, 5.87, 2.76 ng mL−1. Here the heteroatom might be playing an important role in maintaining the favorable interactions.
The effect of different acyclic and cyclic amines was also established and it was observed that COMPOUND LINKS
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Download mol file of compoundN-methyl piperazine, COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl)morpholine are significant for antimalarial activity. The activity results of the hybrid molecules with these amines in combination with various anilines were found to be consistent. Compounds 26, 33, 38 and 49, where N-methyl piperazine substituent is present, have shown promising antimalarial activity ranging from 2.41–5.04 ng mL−1. While in the case of COMPOUND LINKS
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Download mol file of compoundN-ethyl piperazine (17, 27, 31, 36, 39, and 50) the antimalarial activity ranges from 2.11–4.26 ng mL−1. Among COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl) morpholine and COMPOUND LINKS
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Download mol file of compound4-(2-aminoethyl) morpholine analogues, the latter have shown dramatically reduced activity however COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl) morpholine substituted compounds (19, 29, 32, 35, 37, 40, and 52) have an excellent activity profile ranging from 1.36–4.63 ng mL−1. In the case of COMPOUND LINKS
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Download mol file of compoundpiperidine substituted derivatives the antimalarial activity was very disappointing. The activity of COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl)morpholine is mainly attributed to the basic character of the side chain which is absent in the case of COMPOUND LINKS
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Among the 37 molecules synthesized, 30 molecules were further screened against CQ-resistant (K1) strain of P. falciparum. Several molecules showed very good in vitroantimalarial activity (Table 1). Ten molecules (17, 19, 21, 27, 36, 38, 39, 40, 49, and 52) have shown promising antimalarial activity with IC50 values ranging from 1.54–5.71 ng mL−1. In several compounds the activity against resistant parasite is even better as compared to sensitive strains. In malaria chemotherapy, the mechanism of resistance mainly depends upon the active efflux of the drug (CQ) inside the parasite food vacuole and an increase in the basic character of the molecule leads to influx of that molecule at their active site. Therefore, it could be illustrated that the activity of our synthesized molecules against resistant strain depends upon the basic character of the side chain. Finally the complete SAR study concerning the various substitution patterns around the triazine nucleus suggested that meta as well as parasubstituted anilines with halosubstitution and substitution with electron donating groups in combination with COMPOUND LINKS
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Download mol file of compound4-(3-aminopropyl)morpholine are well tolerated for antimalarial activity.
Compound No. | IC50 (μg mL−1) | Compound No. | IC50 (μg mL−1) | Compound No. | IC50 (μg mL−1) |
---|---|---|---|---|---|
a The IC50 represents the concentration of compound that inhibits β-hematin formation by50%. | |||||
16 | 3.03 | 29 | 4.56 | 42 | 6.55 |
17 | 2.06 | 30 | 2.17 | 43 | 2.31 |
18 | 2.70 | 31 | 2.05 | 44 | 2.40 |
19 | 2.06 | 32 | 2.25 | 45 | 2.81 |
20 | 3.10 | 33 | 2.05 | 46 | 2.25 |
21 | 2.08 | 34 | 2.25 | 47 | 2.08 |
22 | 2.64 | 35 | 3.49 | 48 | 2.09 |
23 | 2.38 | 36 | 2.08 | 49 | 6.50 |
24 | 2.17 | 37 | 2.34 | 50 | 4.17 |
25 | 2.43 | 38 | 3.05 | 51 | 9.82 |
26 | 2.42 | 39 | 2.45 | 52 | 2.98 |
27 | 2.06 | 40 | 2.30 | CQ | 3.65 |
28 | 2.01 | 41 | 2.48 |
Compound No. | FlexX Score Energy(Kcal mol−1) | IC50(μM) |
---|---|---|
17 | −31.71 | 75.0 ± 1.88 |
19 | −34.29 | 76.7 ± 1.17 |
26 | −34.56 | 80.2 ± 0.94 |
27 | −31.96 | 74.5 ± 1.17 |
29 | −35.60 | 76.4 ± 2.12 |
37 | −33.31 | 72.7 ± 1.64 |
39 | −30.90 | 85.5 ± 0.42 |
p -hydroxyphenylpyruvate (control) | −16.0 | 103.0 ± 1.41 |
As obvious from the visual inspection of the bound ligands in the active site of P. falciparumtransketolase shown in Fig. 4a, it was observed that top ranking docked poses of the seven compounds fitted very well into the transketolase active site. To illustrate the probable mechanism of the protein–ligand interaction, docking of compound 19 with P. falciparumtransketolase is described in detail in Fig. 4b. Tight interaction between ligand and protein was observed as evident from several hydrogen bonds between polar atoms of compound 19 and those of active site residues Ala269, Gly30, Asp473 and Arg361.
Fig. 4 (left) All the docked poses into the binding cleft of P. falciparumtransketolase. (right) Docked pose of 19 into the active site of P. falciparumtransketolase. |
The binding site for TPP (a prosthetic group of the enzyme) is characterized by a number of hydrophobic interactions including the π–π stacking interactions with Phe441, Phe444 and the phenyl ring of TPP which are important for ligand binding. Besides, several hydrophobic interactions have also been observed with residues TPP 682, Val129, Ala132, Ile133, Ala135, His136, Ala171, Leu174, Ala175, Leu178, Leu180, Arg182, Ile194, His31, Ile421 and Phe438. Some π–π interactions have also been observed between aromatic rings of 19 with those of residues His31 and Phe438. These interactions further confirm our earlier finding for the necessity of aromatic substitution at COMPOUND LINKS
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Compound | Dose (mg kg−1 × 4 days) | % suppression on day 4 | Mice alive on day 28 |
---|---|---|---|
52 | 100 mg kg−1 × 4 days | 99.99 | 3/5 |
16 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
19 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
21 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
23 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
30 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
42 | 100 mg kg−1 × 4 days | 99.00 | 0/5 |
50 | 100 mg kg−1 × 4 days | 99.99 | 0/5 |
16 | 50 mg kg−1 × 4 days | 99.82 | 0/5 |
42 | 50 mg kg−1 × 4 days | 77.82 | 0/5 |
50 | 50 mg kg−1 × 4 days | 93.54 | 0/5 |
52 | 50 mg kg−1 × 4 days | 98.39 | 0/5 |
Footnote |
† Electronic supplementary information (ESI) available: Experimental procedures, characterization data, protocols of biological assays. See DOI: 10.1039/c1md00188d |
This journal is © The Royal Society of Chemistry 2012 |