Quinoline hybrids and their antiplasmodial and antimalarial activities

European Journal of Medicinal Chemistry 139 (2017) 22e47

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Quinoline hybrids and their antiplasmodial and antimalarial activities Yuan-Qiang Hu a, Chuan Gao b, Shu Zhang c, Lei Xu b, Zhi Xu b, Lian-Shun Feng b, *, Xiang Wu b, **, Feng Zhao b, *** a

School of Chemistry and Materials Science, Hubei Engineering University, Hubei, PR China WuXi AppTec (Wuhan), Hubei, PR China c Pony Testing International Group (Wuhan), Hubei, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 February 2017 Received in revised form 24 July 2017 Accepted 24 July 2017 Available online 27 July 2017

Malaria, in particular infection with P. falciparum (the most lethal of the human malaria parasite species, responsible for nearly one million deaths every year), is one of the most devastating and common infectious disease throughout the world. Beginning with quinine, quinoline containing compounds have long been used in clinical treatment of malaria and remained the mainstays of chemotherapy against malaria. The emergence of P. falciparum strains resistant to almost all antimalarials prompted medicinal chemists and biologists to study their effective replacement with an alternative mechanism of action and new molecules. Combination with variety of quinolines and other active moieties may increase the antiplasmodial and antimalarial activities and reduce the side effects. Thus, hybridization is a very attractive strategy to develop novel antimalarials. This review aims to summarize the recent advances towards the discovery of antiplasmodial and antimalarial hybrids including quinoline skeleton to provide an insight for rational designs of more active and less toxic quinoline hybrids antimalarials. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Antiplasmodials Antimalarials Quinoline Quinolone N-contained heterocycles Hybrids Structure-activity relationship

Contents 1. 2.

3. 4. 5.

6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Amino acid/peptide-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.1. Amino acid/peptide-QN hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.2. Amino acid/peptide-4-amino-7-chloroquinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.3. Amino acid/peptide-8-aminoquinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Bis/tris/tetraquinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Chalcone-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Ferrocene-quinoline hybrids and other metals containing quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 5.1. Ferrocene-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 5.2. Other metals containing quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 N-contained heterocyclic compound-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 6.1. Azole-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 6.2. Azine-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 6.3. Pyrimidine-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 6.4. Isatin-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 b/g-Lactam-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 6.5. 6.6. Miscellaneous N-contained heterocyclic compound-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Peroxide-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (L.-S. Feng), [email protected] (X. Wu), [email protected] (F. Zhao). http://dx.doi.org/10.1016/j.ejmech.2017.07.061 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

8. 9. 10.

23

7.1. (Dihydro)ART-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 7.2. Endoperoxide(tetraoxane/trioxane)-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Quinolone-quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Miscellaneous quinoline hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

1. Introduction Malaria is one of the most widespread and deadliest diseases that resulted in 212 million clinical cases and 429,000 deaths in 2015 alone, especially among children (in Africa, 78% of deaths apply to children under the age of five) and pregnant women according to the World Health Organization (WHO) 2016 report [1,2]. Malaria is usually caused by protozoan parasites of the genus Plasmodium including P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi species of human malaria parasite. In particular, P. falciparum is the most fatal one, which is responsible for 95% of the case of death. The life cycle of malaria parasites is rather complex. Hereinto, the erythrocytic stage is responsible for the diverse symptoms caused by infection. In order to maintain osmotic stability and to provide space for the intracellular parasite, parasites ingest and digest more than 70% of host hemoglobin as an amino acid source inside erythrocytes. It is known that hemoglobin degradation and hemozoin formation are essential for parasite survival, making these processes important targets for antimalarials development. Heme detoxification into hemozoin was believed to be the main target of quinoline antimalarials and remained one of the most attractive drug development targets [3]. Quinoline and its derivatives, exhibited diverse of activities such as antimalarial, antibiotic, antituberculosis, anti-HIV, antiplasmodial, antitumor and anti-inflammatory properties, are emerged as an important class of bioactive heterocyclic compounds in the field of pharmaceuticals [4e8]. Quinoline-containing antimalarials, such as quinine (QN), chloroquine (CQ), amodiaquine

(AQ), mefloquine (MQ) and primaquine (PQ) (Fig. 1) are mainstays of chemotherapy against malaria which have long been used in clinical. Among them, CQ has been used in the treatment as well as prophylaxis of almost all forms of malaria owing to its highly effective, safe, well-tolerated and reasonably low cost. CQ acts against the malaria parasites by blocking haemozoin formation through p-p stacking of the 4-aminoquinoline core to the heme ring system or by docking into grooves on the haemozoin crystal and preventing further crystal growth. The toxic haematins then leave the digestive vacuole and enter into the parasite cytosol where oxidative membrane damage is induced [9]. The human malaria parasite P. falciparum is able to regulate its genes and results in strains resistant to almost all the antimalarials, especially toward CQ. Resistance to CQ is associated with mutations in the gene encoding the digestive vacuole membrane protein P. falciparum CQ resistance transporter (PfCRT), which appears to result in reduced drug concentration at the target without altering the target itself. In this case, the target remains vulnerable and the organism is susceptible to drug action if access and binding to the target can be achieved [3]. To overcome the drug resistance, great efforts have been undertaken to modify the launched antimalarials and to discover entirely novel structures. Numerous of studies revealed that modification of the basic amine side chain of quinoline-contained antimalarials can keep activities against drug-resistant P. falciparum strains, but it has generally been assumed that changes to the quinoline nucleus itself will not. In fact, modification of the ring system affects the pKa of nitrogen both in the quinoline ring and in the side-chain as well as other physical parameters such as lipophilicity, sterics, and

Fig. 1. Structures of quinoline and common quinoline-contained antimalarials.

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electronegativity, however, this is not significantly correlated with the activity in drug-resistant strains [10]. Therefore, most of studies focused on the modification of the side-chain of quinoline-contained antimalarials. One of the modern concepts of drug design principle is molecular hybridization which is a recent strategy based on the covalent fusion of two or more existing pharmacophores to create a single molecule with multiple mechanisms of action, representing an encouraging approach on the development of new drugs with potential therapeutic application on several pathologies [11,12]. The pharmacophores of the new entities may have complimentary activities and/or multiple pharmacological targets and/or one part can counterbalance the side effects caused by another part. To overcome the resistance and improve the antiplasmodial and antimalarial activities, various quinoline hybrids have been developed by combining QN, CQ, 4-aminoquinolines, and MQ with other pharmacophores via C-O, C-C, and C-N covalent bonds. Great achievements have been obtained to develop quinoline hybrids as new antimalarials, such as piperaquine, a bis-4-aminoquinoline effective against CQ resistant (CQR) strains that consists of two 4aminoquinoline moieties attached by a linker. Piperaquine has been extensively used in China and Indochina as prophylaxis and treatment, and it was at least as effective as, and better tolerated than, CQ against P. falciparum and P. vivax malaria [13]. Ferroquine (FQ) (Fig. 1) is a 4-amino-7-chloroquinoline with a ferrocenyl sidechain, which is currently in phase IIB clinical trials and may also be used for the treatment of malaria in the near future [14]. This review aims to summarize the recent advances towards the discovery of antiplasmodial and antimalarial hybrids holding quinoline skeleton to provide an insight for rational designs of more active and less toxic quinoline hybrids antimalarials. 2. Amino acid/peptide-quinoline hybrids 2.1. Amino acid/peptide-QN hybrids One of severe problems for many therapeutic agents is efficient cellular drug delivery, while “smart” synthetic drug delivery vectors have significant progress in the past several decades with strong potential for targeted delivery with low toxicity, immunogenicity and large carrying capacity. In particular, cell-penetrating peptides have been proven their applicability because of their capability as drug carriers to cross cell membranes [15]. It is reported that drug-peptide hybrids can overcome multi-drug resistance in chemotherapy, while QN can prevent cells to take up essential amino acids like tryptophan and tyrosine in which dietary amino acid supplements could improve the performance of QN [16,17]. Therefore, amino acid/peptide-quinoline hybrids, which retain activities against P. falciparum, may provide means to identify novel antimalarials with enhanced activity on resistant strains. A series of peptide-quinoline hybrids 1 (Fig. 2) were tested for their in vitro antiplasmodial activities against blood stage of strain 3D7. The preliminary results showed that the majority of the hybrids maintained potency in line with the reference QN (IC50: 18 nM) with IC50 < 100 nM, and the most active hybrid Z-L-asp(Bn)QN (IC50: 17 nM) was comparable to QN. Further analysis indicated that hybridization of small peptides to the hydroxyl group of QN did not interfere with antiplasmodial activity and provides a handle for optimization of antiplasmodial and pharmacokinetic properties. Meanwhile, it seems that the peptides did not hinder the cell permeability of the hybrids [15]. 2.2. Amino acid/peptide-4-amino-7-chloroquinoline hybrids The

structure-activity

relationship

(SAR)

studies

on

4-

Fig. 2. Chemical structures of amino acid/peptide-QN hybrids 1.

aminoquinoline derivatives revealed that the 4-amino-7chloroquinoline nucleus is obligatory for antiplasmodial and antimalarial activities, in particular, inhibition of b-hematin formation and accumulation of the drug at the target site [18]. It has been reported that the antiplasmodial and antimalarial activities were decreased mainly due to the changement of the pKa of quinoline nitrogen atom (pKa1) when the 7-chloro group was replaced by no matter an electron-donating group such as -NH2, -OCH3, or an electron-withdrawing group like -NO2. This study suggests that 7chloro is essential for potential antiplasmodial and antimalarial activities of 4-aminoquinoline class of derivatives [19e21]. Meanwhile, replacement of 4-position nitrogen atom of the 7-chloro-4aminoquinoline moiety by oxygen and sulfur significantly decreases the basicity of the quinoline nitrogen which results in detriminal antiplasmodial activity [22]. Based on the above investigations, several sets of quinoline hybrids reserving 7-chloro and 4-amino group have been synthesized and screened for their in vitro antiplasmodial and in vivo antimalarial activities. A new class 4-amino-7-chloroquinoline hybrids was obtained by modifying the trunk of the side chain of quinoline with various a-, b- and g-amino acids and varying the substituents at methylpiperazine (the pendant group), and their in vitro antiplasmodial activities against the CQ sensitive (CQS) 3D7 and CQR K1 strains of P. falciparum were assessed [22]. Compared with a-amino acid hybrids, b- and g-phenylalanine hybrids exhibited better activity. The SAR study indicated that the hybrids with heteroaromatic groups, bulky alkyl groups, polar groups and more basic groups did not improve the in vitro antiplasmodial activities. The length of the side chain positively correlated with the antiplasmodial activities, which means that increasing of the chain length improved the activity against both of the tested strains. Among the hybrids, conjugate 2 (Fig. 3, IC50: 71.16 nM) with the longest chain not only displayed the most potent antiplasmodial activity against K1 strain, which was ~4 folds more active than CQ (IC50: 255 nM), but also showed the strongest binding ability to hematin, which was comparable to CQ (LogK: 5.52 ± 0.02). Thus, it is discernible from the available data that the principle interaction may be hydrophobic as well as electrostatic between the quinoline ring and the porphyrin ring system that plays a role in hematin binding. Antimicrotubular agent paclitaxel has potential antimalarial efficacy [23]. (2R,3S)-N-benzoyl-3-phenylisoserine is the essential structural component of paclitaxel for its antimicrotubular activity, may also contributing to its antimalarial activity. Based on the above consideration, a variety of (2R,3S)-N-benzoyl-3phenylisoserine 7-chloro-4-aminoquinoline hybrids 3a-j (Fig. 3) tethered via alkyl chains, ester, amide and triazole was synthesized

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25

Fig. 3. Chemical structures of amino acid/peptide-4-amino-7-chloroquinoline hybrids 2 and 3.

and tested for in vitro antiplasmodial activities against erythrocytic stages of K1 and W2 strains of P. falciparum [24]. All hybrids displayed excellent activities against the tested two strains with IC50 ranging from 0.13 to 2.17 mM, but less active against W2 strain than the reference CQ (IC50: 0.05 mM). Hybrid 3d (IC50: 0.22 mM) was the most active against K1 strain, but only comparable to CQ (IC50: 0.34 mM). Thus, it can be concluded that the inherent pharmacological activity of the 4-amino-7-chloroquinoline framework was preserved, but hybridization with the (2R,3S)-N-benzoyl-3phenylisoserine moiety did not appreciably improve the in vitro antiplasmodial activities of the hybrids. Overall, these hybrids provided a promising lead entry for the development of potent antimalarials to overcome the emerging problem of drug resistance. 2.3. Amino acid/peptide-8-aminoquinoline hybrids Malaria parasites undergo an asymptomatic, obligatory developmental phase in liver, which precede the formation of red blood cell-infective forms. Therefore, the liver stage of infection has strong correlation with prevention of disease. Besides, P. vivax and P. ovale malaria can generate cryptic parasite form called hypnozoites that can remain dormant for extended periods before initiating a blood-stage infection, so agents against hepatic stages would be benefit to a malaria reeducation campaign through elimination of the long-lived hypnozoites of P. vivax and P. ovale in liver. The 8-aminoquinoline antimalarial agent PQ is the only drug used for the radical cure of relapsing P. vivax and P. ovale malaria, and PQ is active against the transit liver forms of all Plasmodium species [25]. Moreover, PQ also has a broad-range of antiplasmodial and antimalarial activities including efficacy as causal prophylactic, gametocytocide and sporontocide, and CQR parasites are not crossresistant to PQ. However, PQ is ineffective as blood-schizontocide, so it cannot be used to cure infections caused by P. falciparum. In addition, a short half-life time (t1/2) of PQ due to rapid metabolism to inactive and toxic metabolities further limited its clinical use. To overcome the above drawbacks, various peptide-PQ hybrids were examined for their in vitro antiplasmodial and in vivo antimalarial activities for the development of congeners with potent bloodschizontocidal activities that could be effective against both drugsensitive and drug-resistant malaria strains [26]. Several of 8-aminoquinoline hybrids conjugated to amino acids were tested for in vitro antiplasmodial activities against CQS D6 and CQR W2 strains of P. falciparum, in vitro cytotoxicity in VERO, and

in vivo inhibition of b-haematin formation. Meanwhile, the most promising candidates were further evaluated for in vivo bloodschizontocide antimalarial activities against P. berghei infected mice [27]. Among the hybrids, L-lys containing pseudodipeptide 4 (Fig. 4) emerged as the most potent in vitro antiplasmodial (IC50: 130 and 260 ng/mL for D6 and W2 strains, respectively) and in vivo antimalarial (100% curative activity at 25 mg/kg/day, and suppressive activity (4/6) at 10 mg/kg/day) activities. Moreover, it exhibited the highest SI (>91.5 and > 183 for D6 and W2 strains, respectively) and excellent in vivo inhibition of b-haematin formation (IC50: 5.8 mM, 16 times more potent than CQ) [27]. The SAR study revealed that the dipeptide hybrids with arg and lys residues showed potent in vitro antiplasmodial and in vivo antimalarial activities, but these hybrids decreased the activity when incorporated D-amino acid into the dipeptides, indicating that further study on Lamino acid hybrids may lead to more active conjugations. To explore the most suitable alkyl group at C-2 position in PQ skeleton, a series of PQ derivatives with different substituents at C2 position were tested for their in vitro antiplasmodial and in vivo antimalarial activities [28]. It is observed that the existence of a bulky metabolically stable t-butyl group at C-2 position in PQ core leads to significant improvement in the blood-schizontocide antimalarial activity. The SAR study further revealed that t-butyl group placed at C-2 position in PQ moiety is optimal for the superior antimalarial activity, while adamantyl, i-Pr, cyclohexyl and cyclopentyl decreased the activity [28,29]. 2-t-butylprimaquine 5 (Fig. 4) showed remarkable in vivo antimalarial activity against P. berghei (sensitive strain) and P. yoelii nigeriensis (multi-drug resistant/MDR strain), thus is effective as blood-schizontocide. Moreover, hybrid 5 is found to be the first 8-aminoquinoline completely devoid of methemoglobin toxicity related to PQ [28]. The excellent antimalarial activity may contribute to block a supposed oxidative metabolic degradation pathway known for some quinoline-containing antimalarials by the placement of a bulky metabolically stable tbutyl group at C-2 position of PQ. Activities and action mechanism of amino acid/peptide-8-aminoquinoline hybrids 4, 5 and 6b are summarized in Table 1. In addition, around 35e83% of PQ was metabolized to 4-(6methoxy-quinolin-8-ylamino)pentanoic acid in primate model, while incorporation of an amino acid residue may serve to protect the primary amino of PQ against the above metabolic pathway [26,29]. In addition, L- and D-amino acids may have different efficacy, and further modification may lead to a substantially increasement in the profile of antiplasmodial and antimalarial

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Fig. 4. Chemical structures of amino acid/peptide-8-aminoquinoline hybrids 4e7.

activities. Hence, L- and D-amino acid 2-t-butylprimaquine conjugates 6 (Fig. 4) containing a free terminal amino groups were tested for their in vivo blood-schizontocide antimalarial activities against P. berghei (sensitive strain) in mice (6 mice per group) at various concentrations (100, 50, 25, 10 mg/kg/day, oral) with CQ (10 mg/kg/ day, oral) as a positive control and blank as negative control by Jain et al. [29,30]. Surprisingly, the amino acid residues are the prominent factor affecting the in vivo antimalarial activities against P. berghei (sensitive strain) of the 2-t-butylprimaquine-amino acid hybrids: for L-amino acid hybrids, the relative activity order is Lvaline (without any antimalarial activity at the tested concentrations) < L-arginine (3/6 curative at 50 mg/kg) < L-alanine (4/6 curative at 50 mg/kg) < L-ornithine (2/6 curative at 10 mg/ kg) < L-lysine (4/6 curative at 10 mg/kg) ¼ 2-t-butylprimaquine 5; for D-amino acid hybrids, the activity order is: D-lysine (2/6 curative at 10 mg/kg) > D-ornithine (6/6 curative at 50 mg/kg, while without antimalarial activity at 25 mg/kg) ¼ D-arginine. In general, the Lamino acid 2-t-butylprimaquine hybrids exhibited higher in vivo efficacy than corresponding D-amino acid derivatives which is accordance with the above results reported by Jain et al. [27]. Further modification indicated that hybrids 7 (Fig. 4) prepared by introduction of ethyl and alkoxy groups at C-4 and C-5 positions of 6 moiety respectively showed an enhanced antimalarial activities. Among of them, L-lysine hybrid 7a exhibited the most effective

in vivo antimalarial activity with 100% cures at a dose of 5 mg/kg against P. berghei (drug-sensitive strain) infected mice, and 7b showed curative activity at 50 mg/kg against P. yoelii nigeriensis (highly virulent MDR strain, naturally resistant to CQ and MQ) in mice and emerged as the most potent hybrid against MDR strain [26]. It can be concluded that quinoline is a versatile moiety with potent antiplasmodial and antimalarial activities, and incorporation of a cationic amino acid has substantially improved antiplasmodial and antimalarial activities, which may due to their reduced penetration into the red cells [30]. Moreover, amino acid/ peptide residue may serve to protect the primary side-chain amino through blocking oxidative metabolic degradation. Therefore, amino acid/peptide-quinoline hybrids hold great promise as future antimalarials against both drug-sensitive and drug-resistant strains infection. 3. Bis/tris/tetraquinoline hybrids Many studies on bisquinoline hybrids have indicated an increase in activity against CQR strains, which is attributed to the greater number of protonation sites compared with the monoquinolines resulting in their accumulation to a higher degree in the face of a decreased pH gradient in the CQR parasites [31]. Moreover, the

Table 1 In vitro, in vivo activities and action mechanism of amino acid/peptide-8-aminoquinoline hybrids 4, 5 and 6b. Hybrids (reference)

In vitro activity (IC50: ng/mL) Strain 1

4 (PQ) [27] 5 (CQ) [28,29]

D6: 130 (2000) CQS strain: 39 (113)a

W2: 260 (2800)a

6b (PQ) [27,29]

D6: 630 (2000)a

W2: 400 (2800)a

a

c d

Action mechanism

10b,c: 4/6 cures; 25b,c: 6/6 cures 10b,c: 4/6 cures; 25b,c: 6/6 cures; 50c,d: 4/6 cures; 100c,d: 6/6 cures 10b,c: 4/6 cures; 25b,c: 6/6 cures; 50c,d: 2/6 cures; 100c,d: 4/6 cures

inhibition of BH formation block a supposed oxidative metabolic degradation pathway inhibition of BH formation

Strain 2 a

b

In vivo activity (mg/kg/day)

The activity of reference drugs in brackets. P. berghei infected model. Orally. P. yoelii nigeriensis (multi-drug resistant/MDR strain) infected model.

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

bisquinoline hybrids with the incensement in steric bulk are expected to penetrate less into the red blood cell that may reduce destabilization of red cell membrane inducing hemolysis, the main cause of toxicity. Thus, synthesis of bisquinoline hybrids appears to be an attractive strategy to develop high effective and non-toxic antimalarials [32]. Piperaquine was the first bisquinoline drug synthesized in the 1960s and was in vitro active against both CQS and CQR strains, which has been used extensively in China and Indochina as prophylaxis and treatment [13]. Several bisquinoline hybrids were synthesized by Vennerstrom et al. and the most promising conjugate was Ro 48e6910. Although Ro 48e6910 exhibited potent in vivo antimalarial activity, some cross-resistance between Ro 48e6910 and CQ in vitro was observed. Even worse, Ro 48e6910 showed phototoxicity in the pre-clinical evaluation development, so further modification should be performed to avoid the serious adverse effects and crossresistance [33]. Ro 47e7737 (Fig. 5), S,S enantiomer of Ro 48e6910, with IC50 of 4e13 nM, was not only more potent than CQ, MQ, Ro 48e6910 and its R,R enantiomer against all the tested five CQS and seven CQR strains, but also comparable to CQ against the other major human malaria parasite, P. vivax [34]. The in vivo investigations indicated that Ro 47e7737 is an orally active, fastacting bisquinoline hybrid with a long-lasting effect and has powerful prophylactic properties. Toxicity liabilities, however, particularly phototoxicity like Ro 48e6910 and the danger of attendant photocarcinogenicity, rule it out as a drug development candidate. Eleven N,N-bis(7-chloroquinolin-4-yl)heteroalkanediamine hybrids 8 (Fig. 5) incorporating oxygen and nitrogen atoms in the

27

alkane bridges were screened for their in vitro antiplasmodial, in vivo antimalarial activities and inhibition of hematin polymerization. Ten out of eleven bisquinoline hybrids exhibited potent in vitro antiplasmodial activities with IC50 in a range of 1.2e89 nM against CQS D6 and CQR W2 strains, and more active than CQ (IC50: 99 nM) against CQR W2 strain. The SAR study revealed that hybrids with alkyl ether and piperazine bridges were substantially more effective than these with alkylamine bridges against P. berghei in vivo, but none of these heteroalkane-bridged bisquinoline hybrids had sufficient antimalarial activities compared to alkanebridged bisquinolines [33,34]. The bulky bisquinoline hybrids suggested to be extruded with difficulty by a proteinaceous transporter, so the hybrids may be used to overcome CQ efflux. Based on this, various bis-, tris- and tetraquinoline hybrids with bulky heteroalkanediamines linkers were synthesized, and the activities against P. falciparum strains as well as cytotoxicity toward mammalian cells were evaluated. The increased rigidity by cyclisation reduced toxicity but did not increase activity in comparison with their linear counterparts. Conjugate 9a (Fig. 5) was the most active bisquinoline hybrid with an IC50 of 38.6 ± 17.7 nM against CQS P. falciparum FcB1R strain, but less active than tetraquinoline hybrids [35]. The most active tetraquinoline hybrid 9b (Fig. 5) with IC50 ranging from 18.3 ± 9.3 to 29.2 ± 2.6 nM against the tested five CQS and CQR P. falciparum strains was no toxic in MRC-5 cells and mouse peritoneal macrophages even at 32 mM, needs to be further investigated. The results indicated that cyclic hybrids with greater steric hindrance were absence of cytotoxicity, and displayed good activities against resistant P. falciparum strains, suggesting that the greater bulkiness resulted in a weaker efflux by PfCRT. In vitro activities and action

Fig. 5. Chemical structures of piperaquine, Ro 47e7737 and bis/tris/tetraquinoline hybrids 8e11.

28

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

mechanism of Ro 48e6910 and bis/tris/tetraquinoline hybrids 8e14 are summarized in Table 2. Compared with CQ, hybrid 10 (Fig. 5) was around 3 times more potent against CQR W2 and K1 strains, but less active against CQS D10 and NF4 strains [36]. In spite of the bisquinoline hybrid borofluoric acid salt 11 (Fig. 5) displayed moderate activities against CQS Nigerian with IC50 of 350 nM as well as CQR FcB1 and FcM29 strains with IC50 of 444 and 468 nM which was less active than the references artemisinin (ART, IC50: 4e8 nM) and CQ (IC50: 30e260 nM), the absence of a CQ-like resistance pathway for this hybrid made it could be acted as a starting point for searching these kinds of antimalarials [37]. A series of bisquinoline hybrids 12 (Fig. 6) were screened for antiplasmodial activities against D10 and Dd2 strains of P. falciparum. Bisquinoline hybrids featuring triethylenetetramine and N,N0 -bis(3-aminopropyl)ethylene-diamine linkers, were the most active of all hybrids which were found as potent as CQ against D10 and significantly more potent against the Dd2 strain, with good selectivity towards parasitic cells [31]. A new class of bisquinoline hybrids 13 (Fig. 6) was synthesized and evaluated for their antiplasmodial activities against CQS D10 and CQR K1 strains by Deady et al. [38]. All bisquinolines exhibited considerable activities which are exemplified by 13b with IC50 of 43 and 17 nM against CQS D10 and CQR K1 strains, superior activity compared to CQ (IC50: 40 and 540 nM, respectively) and MQ (IC50: 90 and 300 nM, respectively) [38]. The rapid metabolism of 8-aminoquinolines results in the removal of side-chain amino group to yield inactive metabolites, so it is rational to presume that the side-chain primary amino group presents as an amide or secondary amine in the bis(8aminoquinoline) hybrids and could prevent metabolic degradation, resulting in increased activities [32]. Herein, a series of bis(8aminoquinoline) hybrids 14 (Fig. 6) linked through their side-chain by a set of linkers including amino acids were synthesized and assessed for the in vitro antiplasmodial and in vivo antimalarial activities by Jain et al. [32]. Almost all the hybrids were non-toxic in VERO and exhibited excellent activities against both CQS D6 and CQR W2 strains. The most promising 14a was found 6- and 9-fold more potent than PQ against the tested two strains, and inhibited BH formation with an IC50 of 10.8 mM, far more potent than PQ (IC50: >1000 mM). The in vivo blood-schizontocidal antimalarial activity against P. berghei infected mice showed that 14a exhibited significant in vivo potency with 100% cures at 25 mg/kg and was suppressive (5/6 cures) at 10 mg/kg. WR319691 and WR319775 (Fig. 6), the derivatives of MQ, have been shown to exhibit reasonable P. falciparum potency in vitro (IC90: 22e80 ng/mL against FQ-resistant strain D6 and MDR strain C235) and reduced permeability across MDCK cell monolayers (0.14  106 and 0.3  106 cm/s, respectively) [39,40]. Single-dose

pharmacokinetics were evaluated: maximum bound and unbound brain levels of WR319691 were 97 and 0.05 ng/g vs approximately 1600 and 3.2 ng/g for MQ. The t1/2 of WR319691 in plasma was approximately 13 h vs 23 h for MQ. 4. Chalcone-quinoline hybrids Chalcones, which represent key structural motifs among biologically active molecules, have many biological properties including antitumor, antibacterial as well as antiplasmodial and antimalarial activities. The antiplasmodial and antimalarial potential of chalcones is augmented by their ability to inhibit plasmodial aspartate proteases, cysteine proteases or new permeability pathways introduced into erythrocyte cell membranes by the malaria parasite, which are potential novel chemotherapeutic targets [41]. Moreover, several chalcone derivatives were reported as alternatives in antimalarial chemotherapy. Therefore, incorporation of the chalcone moiety into quinoline skeleton is a promising strategy to search effective antimalarials. A new set of amide tethered 7-chloroquinoline chalcone hybrids and 7-chloroquinolineferrocenyl chalcone hybrids was tested for their in vitro antiplasmodial activities against CQR W2 strain [41]. The introduction of an amide as linker is based on its hydrogen bonding abilities, as this has been shown to enhance antiplasmodial efficacy against both CQS and CQR strains of P. falciparum [42]. Generally speaking, 7-chloroquinoline chalcone hybrids were much more active than 7-chloroquinolineferrocenyl chalcone hybrids, but all hybrids were less active than the references CQ and ART. In addition, the activity greatly independent upon length of the alkyl chain as well as the nature of substituents in chalcone nucleus. The most active hybrid 15 (Fig. 7, IC50: 17.8 ± 8.0 nM) was as potent as CQ (IC50: 15.9 ± 1.8 nM) against CQR W2 strain, and non-cytotoxic against mammalian cells. A targeted series of triazole-linked chalcone quinoline hybrids were synthesized and evaluated for in vitro antiplasmodial activities. Several hybrids were found to be notably active, but all hybrids were less potent than CQ against CQS D10, CQR Dd2 and W2 strains [43]. The most active hybrid 16 (Fig. 7) with IC50 ranging from 40 to 90 nM against the tested three strains worth to be further modified. Replacement of triazole linker by aminoalkyl- or piperazinyl-based linkers should be resulted in more polar hybrids with potentially enhanced (pH-dependent) solubility due to introducing the 4-amino substitution on the CQ subunit. Based on the above consideration, a new class of chalcone quinoline hybrids with aminoalkyl- or piperazinyl-based linkers was synthesized by Chibale et al., but all hybrids were less active than 16 [44]. Similar results were observed in hybrids 17 and 18 (Fig. 7) [45,46]. A novel series of keto-enamine chalcone-chloroquine hybrids 19 (Fig. 7) were evaluated for in vitro antiplasmodial and in vivo

Table 2 In vitro activities and action mechanism of Ro 48e6910 and bis/tris/tetraquinoline hybrids 8e14. Hybrids (reference)

In vitro activity (IC50: nM) Strain 1

Strain 2

Strain 3

Strain 4

Ro 48e6910 (CQ) [34] 8a (CQ) [34] 9a (CQ) [35] 9b (CQ) [35] 10 (CQ) [36] 11 (CQ) [37] 12a (CQ) [31] 13a (CQ) [38] 14a (PQ) [32]

NF54: 6 (16)a D6: 1.2 (6.5)a FcB1R: 38.6 (>100)a FcB1R: 18.3 (>100)a D10: 190 (22)a Nigerian: 350 (25)a D10: 87.6 (48.4)a D10: 43 (40)a D6: 340 (2000)a

K1: 13 (316)a W2: 9.0 (99)a W2: 50.2 (>100)a W2: 29.2 (>100)a W2: 98 (280)a FcB1: 444 (132)a Dd2: 35.4 (242.3)a K1: 17 (540)a W2: 300 (2800)a

HB3: 4

W2: 10

D6: 18.9 (>100)a D6: 18.5 (>100)a NF54: 89 (7.0)a FcM29: 468 (260)a

F32: 16.1 (>100)a F32: 17.9 (>100)a K1: 56 (154)a

a b

The activity of reference drugs in bracket. No reported.

Action mechanism

inhibition inhibition inhibition inhibition NRb NRb Inhibition inhibition inhibition

of of of of

hematin hematin hematin hematin

polymerization polymerization polymerization polymerization

of hemozoin formation of hematin polymerization of BH formation

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

29

Fig. 6. Chemical structures of bisquinoline hybrids 12e14, WR319691 and WR319775.

Fig. 7. Chemical structures of chalcone-quinoline hybrids 15e19.

antimalarial activities [47]. The SAR revealed that against CQS 3D7 strain, hybrids with aliphatic linkers were far more potent than these with aromatic linkers; for aliphatic linkers, ethylene was optimal. The in vivo antimalarial results suggested that an introduction of halogen atoms or heteroaryl groups like furan and thiophene at para position of chalcone aryl ring boost up the in vivo antimalarial activity. However, all hybrids were less active than CQ for both in vitro antiplasmodial and in vivo antimalarial activities.

5. Ferrocene-quinoline hybrids and other metals containing quinoline hybrids 5.1. Ferrocene-quinoline hybrids It has been proven that incorporation of metals into drugs can have a profound effect on the biological activities. Ferrocene is a remarkable pharmacophore, exhibiting physicochemical properties

30

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

that accommodate biological uses. Ferrocene-containing derivatives exhibited various activities such as anti-Tuberculosis, antitumor, antifungal, antileishmanial, antiplasmodial and antimalarial properties [48]. Furthermore, ferrocene was used to enhance the antimalarial activity of fluoroquinolones like ciprofloxacin, or to create novel dual-action hybrids with highly promising results [49e51]. Therefore, incorporation of the ferrocene moiety into quinoline skeleton may result in potential antiplasmodial and antimalarial candidates. Ferroquine (FQ), a ferrocene-quinoline hybrid, as the most emblematic example of the contribution of the ferrocene moiety to the development antimalarials. FQ, which has been shown to be superior to CQ in both in vitro and in vivo tests, is remarkably active against both CQS and CQR P. falciparum, P. vivax strains (by a schizont maturation assay), isolates and has displayed efficiency in pre-clinical P. berghei and P. vinckei mouse malaria models. Unlike CQ metabolites, SSR97213 (Fig. 8), the major metabolite desmethylferroquine was more active than CQ against P. falciparum indicating that the ferrocene moiety in FQ and related metabolites play an important role in the observed antiplasmodial activity. Moreover, the in vitro antiplasmodial activity of FQ did not show a relevant potential for cross-resistance between FQ and CQ or any other currently used antimalarials. FQ is 100 times more lipophilic than CQ at cytosolic pH and 50-fold more concentrated than CQ at the putative acidic pH of the parasite's food vacuole, so FQ properties may be due to a highly efficient: (i) partition into, and/or hydrophobic collapse with, parasitic membrane lipids; (ii) concentration at the lipid site of hemozoin formation; (iii) maintenance of toxic hematin in the aqueous environment, by creating a barrier at the water-lipid interface [49]. In the clinical phase I trial, FQ exhibited excellent drug profiles in the high efficacy, safety, tolerability, the maximal tolerated dose (well tolerated at doses up to 1600 mg in single dose and up to 800 mg as a repeat dose), and the pharmacokinetic properties in a single dose and as repeated doses [52]. To further develop FQ for the treatment of malaria a thorough understanding of the drug's PK and PD properties is necessary. Therefore, a single-dose, phase II trial was performed in the human malaria model. FQ was rapidly absorbed with maximal exposure after 4e8 h, and non-compartmental PK analysis resulted in estimates for t1/2 of 10.9 and 23.8 days for FQ and SSR97213, respectively. Parasite clearance as reported by parasite reduction ratio was 162.9 (95% CI: 141e188) corresponding to a parasite clearance t1/2 of 6.5 h (95% CI: 6.4e6.7 h). The PK/PD modeling resulted in a predicted minimal parasiticidal concentration of 20 ng/mL, and the single dosing tested in the study was predicted to maintain an exposure above this threshold for 37.8 days. The parameters and PK/PD model paved the way to the further rational development of FQ as an antimalarial partner drug [53]. A phase II

trial conducted in four African countries showed that 3 days combination treatment with doses of 2, 4 and 6 mg/kg/day FQ and 4 mg/kg/day artesunate resulted in 97e99% cure rates, whereas the 4 mg/kg/day FQ monotherapy produced a 79% cure rate [54]. These promising properties, however, need further evaluation in clinical phase III studies. Ferrocene moiety can be directly linked to quinoline and some of these hybrids showed better activities than CQ especially against CQR P. falciparum, but in general, none of them exhibited a global antiplasmodial either similar or better than FQ [55e59]. Two series of 4-aminoquinoline hybrids 20a,b (Fig. 8) structurally related to FQ were synthesized to study the structural basis for in vitro effects on P. falciparum (CQS strains HB3 and Dd2, CQR strain W2) and on b-hematin formation [60]. In general, hybrids 20b were more potent than 20a against the tested three strains, but less active than the reference FQ. In 20a series, a hybrid (n ¼ 0) showed potent inhibition of b-hematin formation with an IC50 of 9.3 mM, was 2e4 folds more active than FQ (IC50: 23.0 mM) and CQ (IC50: 46.3 mM). The in vitro antiplasmodial activities of trioxaferroquines (1,2,4-trioxane covalently linked with FQ) were less active than the references ART (IC50: 10 ± 5 and 18 ± 5 nM) and FQ (IC50: 6 ± 3 and 7 ± 3 nM) against CQR FcB1 and FcM29 strains [60]. The most active hybrid 21 (Fig. 8, IC50: 20 ± 3 and 17 ± 2 nM) was 7e73 times more potent than CQ (IC50: 145 ± 3 and 735 ± 4 nM) against the tested two strains, was selected for in vivo evaluation in mice infected with P. vinckei petteri, and the results showed that 21 was highly active (parasitemia was undetectable on day 4 even for mice orally treated at 10 mg/kg/day). 5.2. Other metals containing quinoline hybrids Besides ferrocene-quinoline hybrids, other metals (e.g. Ru, Rh, Au, Mn, Cu and Ni) containing quinoline hybrids also exhibited considerable antiplasmodial and antimalarial activities [61e64]. Since the chemistry of ferrocene and ruthenocene is similar, ruthenocene-quinoline hybrids may also be potential antimalarials. Based on the above consideration, four ruthenocene-quinoline hybrids were evaluated for their in vitro antiplasmodial activities against CQS D10 and CQR K1 strains by Moss et al. [61]. All hybrids exhibited excellent antiplasmodial activities against the two strains with IC50 of 6.3e176.7 nM, which were comparable to these of the corresponding ferrocene-quinoline hybrids. In particular, the FQ analog 22 (Fig. 9) was 56-fold more active than CQ diphosphate (CQDP) against CQR K1, and as potent as CQ against CQS D10, could be act as a starting point for searching these kinds of antimalarials. A new class of ruthenium, rhodium and gold-CQ complexes was synthesized and evaluated for their in vitro antiplasmodial and
nchez-Delgado et al. [62,63]. The in vivo antimalarial activities by Sa

Fig. 8. Chemical structures of SSR97213 and ferrocene-quinoline hybrids 20e21.

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

in vitro antiplasmodial activity of ruthenium bis-CQ hybrid 23 (IC50: 10.5e46.5 nM) was 2e5 times more active than that of CQDP (IC50: 47e104.5 nM) against P. berghei and two CQR FcB1 and FcB2 strains of P. falciparum. Moreover, CQDP reduced the parasitemia by 55% in the in vivo antimalarial test; while for hybrid 23 (Fig. 9), the reduction reached 94%, and without any sign of acute toxicity being observed up to 30 days after treatment, warrant further study. The above in vitro and in vivo tests revealed that the incorporation of the metal fragments generally produced an enhancement of the efficacy of CQ. A set of novel 5-substituted (iodo and nitro) uracils-8aminoquinoline metals (Mn, Cu, Ni) complexes was synthesized and screened for their in vitro antiplasmodial activities against P. falciparum strain by Phopin et al. [64]. All of these metal complexes showed fair antiplasmodial activities with IC50 of 100e1000 mg/mL, and the SAR was enriched. These results illustrate well the potential of the novel metalbased approach is advancing for the development of chemotherapies against malaria [62]. 6. N-contained heterocyclic compound-quinoline hybrids 6.1. Azole-quinoline hybrids The recent studies proved that length of alkyl side-chain in aminoquinolines has great influence on the antiplasmodial and antimalarial activities against the drug-resistant strains of P. falciparum, meanwhile the bulkier substituents attached to the terminal amino group increased the in vivo efficacy and simultaneously decreased the potential for cross-resistance, presumably by circumventing metabolic N-dealkylation [65,66]. Thus, incorporation of bulkier substituents such as azoles, while variation of the length of alkyl side-chain, may circumvent metabolic Ndealkylation. Azoles are one of the most important classes of nitrogen containing heterocycles that exhibited the potential antiplasmodial and antimalarial activities, so introduction of azoles into quinoline framework may lead to potential antimalarials. Two novel classes of 4-aminoquinoline-tetrazole hybrids 24 and 25 (Fig. 10) were tested for in vitro antiplasmodial activities against CQS 3D7, CQR K1 and W2 strains [66]. The results showed that hybrids 25c-g were more active than the reference CQ against the tested three strains. Among them, compound 25d (IC50: 0.4 nM) was 13 folds more potent than CQ (IC50: 52 nM) against 3D7, conjugate 25e (IC50: 1 nM) was 36 times more active than CQ (IC50: 36 nM) against K1, and hybrids 25a-g (IC50: 20e69 nM) were equal

Fig. 9. Chemical structures of ruthenium containing quinoline hybrids 22 and 23.

31

to or more active than CQ (IC50: 59 nM) against W2 strain. The SAR study revealed that substituent t-butyl on tetrazole moiety was crucial for the antiplasmodial activity, and the activity of hybrids 25 was more potent than hybrids 24; the hybrid 25a with the shortest ethylene linker was less efficacious than the hybrids 25f and 25g with longer linker. It notable that hybrids 25b and 25e have resistance index (RI: IC50(K1)/IC50(3D7)) values that are lower than 1, indicating a reduced likelihood to develop cross-resistance with CQ. The most active hybrids 25c-e (CC50: 10.5e185.1 mM) had acceptable cytotoxicity in Chinese Hamster Ovarian (CHO) celllines. The in vivo pharmacokinetic studies on 25d and 25e in mice (intravenous and oral) displayed a relatively moderate synstemic plasma clearance and high volume of distribution with elimination t1/2 in a range of 1.6e6.0 h, but relative low oral bioavailability ranging from 16.2 to 30.8%. Hybrid 25d exhibited moderate inhibition on P. berghei infected mice following oral administration (5 mg/kg), achieving 46.9% reduction in parasitemia load on day 7, but less effective than the positive control (CQ, 90.6% reduction). Several series of triazole-QN hybrids with varied length ether linkers at C-4 and different substituents at N-1 position of triazoles were screened for their in vitro antiplasmodial activities against blood-stage of P. falciparum 3D7 strain [67]. Among them, 26 (Fig. 10) was found to be the most active hybrid with an IC50 of 27 nM, 2-fold more potent than QN (IC50: 58 nM). A new class of triazole-quinoline hybrids in which the C-4 substituted triazoles was incorporated into the 7-chloroquinoline directly without a linker was evaluated for their in vitro antiplasmodial activities against CQR W2 strain and cytotoxicity to Hep G2A16 cells [68]. Although all hybrids disclosed low cytotoxicity (CC50: >100 mM), only exhibited weak to moderate antiplasmodial activities (IC50: 9.6->166.0 mM), which may due to the changement of pKa of quinoline moiety, similar results were obtained by Boechat et al. [69]. The most active conjugate 27 (Fig. 10) with IC50 of 9.6 mM, was far less active than the reference CQ (IC50: 0.19 mM) [68]. Thus, hybrids with varied length of alkyl linkers between 7chloroquinoline and triazoles may show potential antiplasmodial and antimalarial activities. A series of triazole-quinoline hybrids 28a (Fig. 10) in which alkyl chains linked with N-1 position of triazole moiety were synthesized and evaluated by Rawat et al. [70]. The SAR indicated that hybrids with aryl groups having halogen (IC50: 1.28e1.55 mM) and alkyl (IC50: 1.39e2.63 mM) side chains at different positions exhibited similar activity profile. Surprisingly, introduction of COCH3 functionality at para-position of aromatic nucleus boosts up antiplasmodial activity against both CQS and CQR strains with IC50 values of 0.91e1.65 mM. Changing the phenyl ring to biphenyl moiety also improves antiplasmodial activity. The antiplasmodial activity profile of these hybrids clearly demonstrated that hybrids with C-3 spacer showed better activities than their C-2 counterparts which may be due to the increase in lipophilicity. Further incorporation of an additional 1,3,5-triazine moiety of these hybrids yielded 28b (Fig. 10), and the SAR showed a similar trend of increasement in activity with increasing length of carbon spacer, and introduction of basic moieties like piperidine, morpholine, and dimethylamine in place of chloro group enhanced the activity, while substitution of both the position of triazine nucleus with aliphatic moieties led to decrease in activity. All the hybrids were found to be non-cytotoxic up to 48 mM in VERO cells, indicating their safety towards mammalian cells. Based on the SAR studies of these hybrids, it may be concluded that lipophilicity plays an important role in driving the activity profile of these conjugates which is in agreement with the literature [71] observation that higher Clog P values are essential for better antiplasmodial activity. A set of 4-amino-7-chloroquinoline imidazole hybrids 29 (Fig. 10) by the presence of methylene as a spacer was synthesized

32

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

Fig. 10. Chemical structures of azole-quinoline hybrids 24e32.

and tested for in vitro antiplasmodial activities against CQS D10 and CQR W2 strains [72]. All hybrids exhibited moderate to excellent activities against the tested strains. The SAR revealed that the activity was greatly influenced by the presence of the N-methyl group in imidazole moiety (R ¼ Me) and a basic head which may contributed to the entrapment of the hybrids inside the food vacuole. In the series of N-unsubstituted imidazolyl hybrids, the presence of an aryl substituent on the imidazole ring not only enhanced the antiplasmodial activity, but also reduced the value of RI in both the series lacking and bearing an aliphatic basic head. Indeed, the introduction of more basic Mannich-type heads at R1 on the imidazole ring clearly improved the activity against the CQS strain, and the RI resulted greatly increased, which could be explained by the greater basicity of the aliphatic amines. Furthermore, when imidazole moiety was replaced by oxazole, thiazole or thiophene, the antiplasmodial activity decreased. The N-methyl imidazolyl hybrids were much more potent than the corresponding N-unsubstituted imidazolyl hybrids, and the most active N-methyl imidazolyl hybrid 29a (X ¼ NMe, R1 ¼ pyrrolidinemethylene, R2 ¼ 4-ClPh, IC50: 5.5 ± 1.4 and 27.2 ± 9.3 nM) was found to be 3- and 11-fold more potent than CQ (IC50: 14.3 ± 2.1 and 317.1 ± 113.1 nM) against CQS D10 and CQR W2 strains. Several 4amino-7-chloroquinoline imidazole/pyrazole/1,3,4-thiadiazole hybrids were also evaluated for their in vitro antiplasmodial activities against CQS D10 and CQR W2 strains by Monti et al. [73]. All hybrids only displayed weak to moderate activities against the tested strains which were less active than the parent CQ. In vitro activities and action mechanism of azole-quinoline hybrids 24e32 (Fig. 10) are summarized in Table 3. A new class of 4-aminoquinoline hybrids 30 (Fig. 10) that contain a 2,4,5-trisubstituted aminoxazole unit in the lateral side chain displayed promising antiplasmodial activities against CQS 3D7 and CQR K1 strains with IC50 ranging from 3.8 to 230 nM. The most active hybrid 30a (n ¼ 3, R ¼ Me, IC50: 3.8 and 19 nM,

respectively) was found to be 5- and 13-fold more potent CQ (IC50: 20 and 250 nM, respectively) against the tested two strains. Hence, such a combination of antiplasmodial pharmacophores and other functionalities offers many attractive features for accelerating antimalarials discovery [74]. A set of novel hybrids 31 (Fig. 10) by tethering benzoheterocyclic derivatives including 1H-benzo[d]imidazole, benzo[d]oxazole and benzo[d]thiazole directly with 4-amino-7-chloroquinoline was designed to avoid toxic reactive metabolite formation and tested for antiplasmodial and antimalarial activities [75e78]. In general, the SAR revealed that the influence of the benzoheterocyclic cores on the antiplasmodial activities against MDR K1 and CQR W2 strains of P. falciparum as follows: benzo[d]thiazole > benzo[d]oxazole z 1Hbenzo[d]imidazole. Four hybrids were chosen for evaluation of in vivo antimalarial efficacy in P. berghei infected mice, all of which showed promising potency following oral administration. In particular, hybrid 31a which exhibited excellent in vitro antiplasmodial activity against the tested strains with IC50 in a range of 8e22 nM, also displayed most potent in vivo activity which could completely cured treated mice at a low multiple dose of 4  10 mg/ kg [75]. While, conjugate 31b (IC50: 12 and 14 nM) was as potent as AQ (IC50: 5 and 8 nM) and 4e24 times more potent than CQ (IC50: 49 and 344 nM) against CQR K1 and W2 strains [75]. A series of 2,8-di(trifluoromethyl)quinoline midazole hybrids were screened for antiplasmodial activities against four different strains of P. falciparum W2, D6, C235 and C2A. Among them, 32 (Fig. 10) was the most active hybrid with IC90 in a range of 43e69 ng/mL [79]. 6.2. Azine-quinoline hybrids Dihydrofolate reductase (DHFR) is one of the well validated targets, has been successfully explored as a target for malarial therapy. 1,3,5-triazine (also called s-triazine) moiety known to be

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

33

Table 3 In vitro activities and action mechanism of azole-quinoline hybrids 24e32. Hybrids (reference)

In vitro activity (IC50: nM) Strain 1 K1: 2380 (36) K1: 0.4 (36)a 3D7: 27 (58)a W2: 9600 (190)a D6: 650 (40)a D6: 580 (40)a D10: 5.5 (14.3)a 3D7: 3.8 (20)a

31a (CQ) [75] 31b (CQ) [75] 32 [79]

K1: 8 (49)a K1: 12 (49)a W2: 69c

b c

Strain 2

Strain 3

Strain 4

3D7: 8 (5.2)a

W2: 30.5 (59)a

a

24d (CQ) [66] 25d (CQ) [66] 26a (QN) [67] 27a (CQ) [68] 28aa (CQ) [70] 28ba (CQ) [70] 29aa (CQ) [72] 30a (CQ) [74]

a

Action mechanism

W2: 1120 (420)a W2: 730 (420)a W2: 27.2 (317.1)a K1: 19 (250)a W2: 22 (344)a W2: 14 (344)a D6: 43c

NF54: 15 (10)a C235: 56c

C2A: 57c

NRb NRb NRb inhibition of hematin polymerization NRb NRb interfere with the heme detoxification process of parasites Inhibition of haem dimerization causing a build up of the toxic haem, resulting in parasite death inhibition of b-haematin formation inhibition of b-haematin formation NRb

The activity of reference drugs in bracket. No reported. IC90: ng/mL.

targeting DHFR enzyme, so 1,3,5-triazine derivatives remain an attractive proposition, with their significant biological activities and further incorporation with commercial agents such as CQ, enabling access to a wide array of structures with interesting antiplasmodial and antimalarial activities. A series of triazine-quinoline conjugates 33 (Fig. 11) were examined for their in vitro antiplasmodial activities against CQS D6 and CQR W2 strains, while the cytotoxicity evaluation was carried against a panel of three mammalian cells (VERO, LLC-PK11 and HepG2) [80]. The SAR revealed that the spacer between 4aminoquinoline and 1,3,5-triazine moiety plays an important role in defining the activity. Generally speaking, with the increasement of the length of the linkers from C2 to C4, hybrids boost up the antiplasmodial activities. In addition, enhanced antiplasmodial activity with higher SI against W2 strain has been observed for hybrids containing aromatic substitution on 1,3,5-triazine than their aliphatic counterparts, which may be attributed to greater lipophilic character associated with aromatic hybrids. Most interestingly, the introduction of amino alcohols with terminal hydroxyl group in the 1,3,5-triazine nucleus improved the potency of some of the hybrids towards both the strains. Moreover, most of the hybrids were found to be non-cytotoxic even at 25 mM against all three cell lines, indicating their safety in mammalian. A series of 4-aminoquinoline-triazine hybrids 34 (Fig. 11) with different substitution pattern have been synthesized and evaluated

for their in vitro antiplasmodial activities against CQS D6 and CQR W2 strains [81]. Almost all of the hybrids were less active than the references CQ and ART against the tested strains, and similar results were obtained by Chauhan et al. [82]. 6.3. Pyrimidine-quinoline hybrids The pyrimidine-based compounds have wide range of biological activities such as anti-inflammatory, antitumor, antiplasmodial and antimalarial activities apart from their role in the nucleic acid synthesis [83]. Pyrimidine-based compounds have potential as antimalarials due to their ability to inhibit DHFR, so incorporated pyrimidine nucleus (DHFR inhibitor) into quinoline skeleton may show promising antiplasmodial and antimalarial activities and might be able to prevent the drug resistant to certain extent [84]. A set of hybrids 35 (Fig. 12) consisting of 4-aminoquinoline and pyrimidine was synthesized and tested for in vitro antiplasmodial activities against both CQS D6 and CQR W2 strains of P. falciparum [83]. These hybrids showed potent antiplasmodial activities with IC50 ranging from 0.005 to 0.44 mM and 0.016e1.17 mM against CQS D6 and CQR W2 strains, respectively. Amongst them, eleven hybrids showed better antiplasmodial activities against the tested two strains in comparison to CQ (IC50: 0.04 and 0.39 mM, respectively). For amino-substituted pyrimidine hybrids, the SAR demonstrated no obvious trend of activity with increasing or

Fig. 11. Chemical structures of azine-quinoline hybrids 33e34.

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decreasing carbon spacers from C2 to C6, but changing the amino groups for a particular C2, C3, C4, or C6 spacer changes the activity significantly in a decreasing order of 4-ethyl piperazine > 4-methyl piperazine > morpholine > piperidine. In addition, several hybrids with no cytotoxicity even up to 60 mM concentration, and SI were very high for most of these hybrids. Hybrid 35a with the highest activity against CQS D6 and without cytotoxicity was selected for further in vivo evaluation which has also shown excellent activity (p.o.) in a mouse model of P. berghei also without any apparent toxicity. Compared to CQ (no cure), at three doses of 30 mg/kg, hybrid 35a showed better activity, which produced almost complete suppression of parasitemia and cured 80% of the treated mice. Replacement of saturated heterocyclic ring substituents by arylamines and absence of methyl group yielded 36 (Fig. 12), which with a slight reduction against both CQS D6 and CQR W2 strains of P. falciparum [84]. The SAR of hybrids 37 (Fig. 12) indicated that the in presence of methyl group (R1 ¼ Me) in pyrimidine moiety enhanced the antiplasmodial activity compared with the corresponding hydrogen hybrids [85,86], so aliphatic substituents should be further optimized. The hybrids 38 (Fig. 12) with one or

two amino groups in pyrimidine moiety were less active or as potent as CQ and PQ against CQS D10 and CQR Dd2 strains [87,88]. Interestingly, ethylene glycol linked hybrids were much more active than those with aliphatic chains. The most active 38a was as potent as both CQ and pyrimethamine against CQS D10, possessed superior potency over both drugs against CQR Dd2. Moreover, the actions of these two drugs were additive and weakly synergistic through the hybrid 38a against the D10 and Dd2 strains, respectively, so 38a appears worthy of being further investigated [87]. In vitro activities and action mechanism of pyrimidine-quinoline hybrids 35e39 are summarized in Table 4. A class of pyrimidine-quinoline hybrids 39 (Fig. 12), which possess basic, hydrophobic and hydrogen bonding substituents (required for targeting either or both heme as well as DNA), was synthesized and screened for their in vitro antiplasmodial activities against CQS 3D7 and CQR K1 strains [89]. The SAR revealed that the activities of these hybrids as dependent upon the length of the linkers and the substituents on the pyrimidine motif. In particular, the most active hybrid 39a (IC50: 3.6 nM) was found to be 56-fold more potent than CQ against CQR K1 strain. Furthermore, 39a

Fig. 12. Chemical structures of pyrimidine-quinoline hybrids 35e39.

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showed higher affinity toward AT rich pUC18 DNA, suggesting targeting of parasite AT rich DNA by 39a in addition to b-hematin inhibition as possible mode of action of these hybrids. DNA melting experiments suggested primary groove binding and/or partial intercalative nature of interaction of 39a with CT DNA. Thus, 39a acts on multiple targets (heme, enzyme involved in biosynthesis of DNA (DHFR), parasite DNA) which accounts for its high antiplasmodial activity. 6.4. Isatin-quinoline hybrids Isatin (1H-indole-2,3-dione) is a promising new class of heterocyclic molecules with diverse biological profiles such as antituberculosis, antibacterial, anti-fungal, anti-virus, antitumor, antiHIV, anti-inflammatory, antiplasmodial and antimalarial activities, and good tolerance in humans [90], and the isatin unit is a privileged natural product scaffold onto which other pharmacores can be appended. Therefore, tethering isatin and quinoline moieties by various linkers may enhance the antiplasmodial and antimalarial activities. Especially, indolin-2-ones substituted at C-3 with an aminomethylene group bearing different amino acid moieties were described as antitumor and antibacterial agents. However, the potential of 3-methylene-indolinones as antimalarials remains to be explored. Based on that, a series of 3-methylene-substituted indolinones were synthesized and evaluated for their in vitro antiplasmodial activities against CQR W2 strain [91]. It is notable that the antiplasmodial activity could be significantly improved when a 4-aminoquinoline was coupled to the 3-methylene moiety of the indolinone-2-one scaffold, suggesting that these hybrids (IC50: 0.14e0.42 mM) are potential leads for the development of new antimalarials. Furthermore, variation in carbon chain length between indolinone and quinoline scaffolds had no major impact on activity. The most active hybrid 40 (Fig. 13, IC50: 0.14 ± 0.01 mM) was found as potent as CQ (IC50: 0.14 ± 0.01 mM) against CQR W2 strain. To further evaluation, the triazole moiety was introduced as a linker of 7-chloroquinoline-isatin hybrids 41 and 42 (Fig. 13) on behalf of its favorable properties, including moderate dipole character, hydrogen bonding capability, rigidity and stability under in vivo conditions, which are evidently responsible for its diverse biological activities [92]. Interestingly, hybrids 41 in which triazole moiety linked directly on 7-chloroquinoline were inactive at maximum concentration of 5 mM against CQR W2 strain, while 42, with varied alkyl chain length between the 7-chloroquinoline and triazole groups, showed better antiplasmodial activities with IC50 ranging from 1.21 to 3.85 mM, but less active than the references ART (IC50: 0.014 mM) and CQ (IC50: 0.099 mM). The SAR revealed that three carbons are the optimal alkyl chain length, and chloro substituent at the C-5 position of the isatin ring is also crucial for the high antiplasmodial activity. On the basis of a multi-therapeutic strategy, a novel class of 4amino-7-chloroquinoline isatin scaffolds 43 (Fig. 13) and their

35

ketone analogues was designed and synthesized for biological evaluation against CQS D10 and CQR K1 and W2 strains [93]. The thiosemicarbazone moiety provides reactive sites (the imine and thiol carbonyl) for alkylation of the enzyme cysteine thiolate, and is also likely a metal chelator in which the hydrazinic nitrogen and sulfur atoms could be involved in binding to endogenous iron within P. falciparum. This has implications for metabolism within the parasite by inhibiting metal-dependent enzymes. The hydrazinic nitrogen could also assist in the accumulation within the acidic food vacuole of P. falciparum. In addition, basic protonatable piperazine nitrogen of the Mannich bases may further increase accumulation of the molecule. The variable alkyl chain is important not only for circumversion of the CQ resistance mechanism but also for lipophilicity, which is an important parameter in the effectiveness of iron chelating antimalarials [94]. All hybrids showed moderate to excellent activities with IC50 values in the range of 0.079e1.3 and 0.050e2.0 mM against CQS D10 and CQR K1 and W2 strains, respectively. For ethylene linked hybrids, thiosemicarbazone hybrids were more active than the corresponding ketone derivatives. In general, the inhibitory potency of all hybrids against the parasites did not strongly correlate with inhibitory potency against falcipain-2, which at best was weak to moderate, suggesting other mechanisms of inhibition may also be involved or hybrids may be selectively taken up by P. falciparum. In addition, a series of piperazine-tethered isatin-quinoline hybrids 44 (Fig. 13) were screened for in vitro antiplasmodial activities against CQR W2 strain, but all hybrids were less active than CQ [95]. 6.5. b/g-Lactam-quinoline hybrids Continuous interests have been placed on b-lactam antibiotics due to their classic broad spectrum of antibacterial activities, as well as other diverse pharmacological properties such as antiinflammatory, antidiabetic, antituberculosis, anti-HIV and antiplasmodial and antimalarial activities [96,97]. Therefore, introduction of b-lactam nucleus into quinoline framework has the potential to overcome the drug-resistant strains of P. falciparum. b-lactam-4-aminoquinoline hybrids having urea/oxalamide linkers and a well modulated alkyl chain length were synthesized and screened for their in vitro antiplasmodial activities by Kumar et al. [97]. The results revealed the antiplasmodial activity dependents on the N-1 substituent of the b-lactam ring, the nature of the linkers as well as the length of the alkyl chains. Among the synthesized hybrids, 45 (Fig. 14) was found to be the most active against CQR W2 strain of P. falciparum with an IC50 of 34.97 nM, which was less active than the references ART (IC50: 10.63 nM) and QN (IC50: 18.67 nM), but more potent than the parent CQ (IC50: 59.09 nM). Thus, it can act as an ideal starting point for the synthesis of new pharmacological templates against P. falciparum. The antiplasmodial and antimalarial activities of C-3 functionalized b-lactam derivatives were influenced greatly by the

Table 4 In vitro activities and action mechanism of pyrimidine-quinoline hybrids 35e39. Hybrids (reference)

35a 36a 37a 38a 39a a b

(CQ) (CQ) (CQ) (CQ) [89]

[83] [84] [86] [87]

In vitro activity (IC50: nM)

Action mechanism

Strain 1

Strain 2

D6: 5 (40)a D6: 33 (35)a D10: 22.6 (21.8)a D10: 70 (40)a 3D7: 18 (1.1)a

W2: 30 (390)a W2: 58 (367)a Dd2: 43 (140)a Dd2: 157 (417)a K1: 3.6 (201.8)a

The activity of reference drugs in bracket. No reported.

NRb inhibition of b-haematin formation inhibition of b-haematin formation NRb acts on multiple targets (heme, enzyme involved in biosynthesis of DHFR, parasite DNA)

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Fig. 13. Chemical structures of isatin-quinoline hybrids 40e44.

substituents at N-1 of the b-lactam ring and C-3 of the substituted triazole ring [98]. For further investigation, 1,2,3-triazole tethered b-lactam and 7-chloroquinoline as mono- and bisquinoline hybrids were synthesized and evaluated as potential antimalarials [99]. The results indicated that activity against CQR P. falciparum was dependent on the N-substituents of the b-lactam ring as well as the presence of bis-triazole at the C-3 position. In general, the presence of mono- and bis-1,2,3-triazole tethered 7-chloroquinolines considerably influence the antiplasmodial profile, and b-lactambisquinoline hybrids with bis-1,2,3-triazole exhibiting better activities than the corresponding mono-scaffolds which might be attributed to either solubility enhancing properties of triazole rings or increased heme-binding of 7-chloroquinolines. Moreover, the presence of a substituent on the nitrogen of the b-lactam ring also influenced activity with the antiplasmodial potency of N-aryl substituted hybrids influenced by the presence of a C-3 triazole ring while this effect was minimal in the case of N-alkyl derivatives, and the results were in accordance with the previous observation of activity affected by an N-alkyl group on b-lactams [98]. Conjugates 46a,b (Fig. 14) were found to be the most active in mono- and bisquinoline hybrids with IC50 of 1.9 and 1.1 mM against CQR W2 strain, respectively. In order to substantiate the observed activity profile and to provide insight into the mechanisms of action of the hybrids, molecular docking studies were performed into the binding pocket of P. falciparum dihydrofolate reductase (PfDHFR) considering both the wild type (1J3I.pdb) and a quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb). The results showed a significant difference in the predicted binding energies of the complexes of monoquinoline and bisquinoline hybrids. The triazole

rings of bisquinoline hybrids were predicted to be predominantly engaged in hydrogen bonding with key amino acid residues (Ser22, Arg38, Thr36) of both PfDHFR enzymes, suggesting a role in the biological activity of these hybrids. Similarly, the absence or lower extent of intermolecular hydrogen bonding and non-bonded interactions in the monoquinoline hybrids could be responsible for the higher binding energies of their complexes with both enzymes, and these results substantiate a good relationship between the antiplasmodial activities and binding energies of the complexes. The g-lactam quinoline hybrids 47 (Fig. 14) showed no activity at the highest tested concentration 45 mM against CQS NF54 [100], while some of g-hydroxy-g-lactam quinoline hybrids 48 (Fig. 14) displayed promising activities against CQS 3D7 and DMR W2 strains [101]. 6.6. Miscellaneous N-contained heterocyclic compound-quinoline hybrids A set of pyrazolopyrimidine/triazolopyrimidine 8aminoquinoline hybrids was synthesized for biological evaluation against CQS NF54 and CQR K1 strains [102]. Systematic SAR studies indicated that both pyrazolopyrimidine and 8-aminoquinoline are essential for achieving excellent antiplasmodial activity. The most active 49 (Fig. 15, IC50: <3.9 and 6.7 nM, respectively) was found to be > 4.3- and 51-fold more potent than CQ (IC50: 17 and 347 nM, respectively), and comparable to or better than ART (IC50: 7 and 6.5 nM, respectively) against the tested two strains. The docking study revealed that hybrid 49 can retain some of the critical interactions within pfDHODH drug target.

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37

Fig. 14. Chemical structures of b/g-lactam-quinoline hybrids 45e48.

Pyrazoline derivatives are the electron rich nitrogen heterocycles which play a significant role in the diverse biological activities with strong efficacy. Scaffolds containing the 2-pyrazoline moiety have demonstrated a broad range of pharmacological activities including antiplasmodial and antimalarial, so incorporation of 2pyrazoline into quinoline motif may results in high potency antimalarials [103]. A new class of morpholinoquinoline pyrazoline hybrids was screened for in vitro antiplasmodial activities against CQS and QN sensitive strains of P. falciparum [103]. The most active 50 (Fig. 15, IC50: 15 nM) was found to be 4- and 55-fold more potent than CQ and QN (IC50: 62 and 826 nM, respectively), may become a new potential member of antimalarials in future. A series of novel 2-imidazololine quinoline hybrids exhibited excellent in vitro antiplasmodial activities against CQS 3D7 and CQR K1 strains of P. falciparum with ED50 in a range of 0.48e970 nM [104]. In terms of SAR, the hybrids with long linkers were more active than these with short one, as evidenced by C4 > C3 > C2. Notably, hybrids with the ferrocenyl group were the most active among their respective groups. The most active 51 (Fig. 15, ED50: 0.48 and 16 nM, respectively) was 41- and 66-fold more potent than CQ (ED50: 20 and 1030 nM, respectively) against the tested two strains, worth to further evaluation. The pyridine quinoline hybrids also showed promising antiplasmodial activities against CQS D6, CQR W2 and MDR TM91C235 strains with IC50 ranging form 7.29e375.05 nM, and without cytotoxicity in human liver carcinoma cell lines [105]. The most potent hybrid 52 (Fig. 15) was 31e33 times more potent than CQ against CQR W2 and MDR TM91C235 strains. Atorvastatin, one of the leading pyrrole drugs, not only has been

shown considerable activity against P. falciparum [106], but also promoted a synergistic effect with dihydroartemisinin (DHA) activity [107], so atorvastatin-quinoline hybrids may be potential antimalarial candidates. Based on the above consideration, a set of novel atorvastatin-quinoline hybrids has been synthesized and evaluated for their antiplasmodial activities against CQR W2 clone and cytotoxicity in BGM cells by Boechat et al. [108]. The minimal dose lethal concentration for 50% of BGM cells (MDL50) of the synthesized hybrids in a range of 136.4e1661 mM, so these hybrids displayed low cytotoxicity. All hybrids exhibited promising activities with IC50 ranging from 0.4 to 1.41 mM, comparable to CQ (IC50: 0.59 mM) and more potent than PQ and atorvastatin (IC50: 1.89 and 10.3 mM, respectively). The SAR indicated that the potency of the hybrids was influenced greatly by the length of the linkers, and increase in the chain length favored the activity, indicating further study could be focused on the optimization of the length of linkers; incensement in steric bulk of the linkers detriminal the activity, as evidenced by 53c (n ¼ 3, R ¼ H, IC50: 0.40 mM) vs 53d (n ¼ 3, R ¼ Me, IC50: 1.41 mM). The most active hybrid 53c was 1.5e26 times more potent than the references CQ, PQ and atorvastatin, and SI of 468, warrant further investigations. The oxazolidinone hybrids 54 (Fig. 15) displayed promising antiplasmodial activities against both CQS 3D7 and CQR K1 strains with IC50 ranging from 62 to 430 nM [109]. Several 1,3-thiazolidin4-one/1,3-thiazinan-4-one/rhodanine/thiazolidinedione/thioparabanic acid/thiohydantoin/thiazolidinone/mercaptopurine/aziridine/indole/azidothymidine/imidazolidine quinoline hybrids were screened for in vitro antiplasmodial activities, although almost all of them were less active than CQ, the SAR was enriched [110e121].

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Fig. 15. Chemical structures of miscellaneous N-contained heterocyclic compound-quinoline hybrids 49e54.

7. Peroxide-quinoline hybrids The antiplasmodial and antimalarial properties of ART and other peroxides like endoperoxide, tetraoxanes are currently being investigated as new frameworks to fight against CQR of malaria. The peroxides exert their antiplasmodial and antimalarial potency by interacting with heme or ferrous ions in the acid food vacuole of the parasite which results in the release of radical species. Meanwhile, the recent studies indicated that trioxane-aminoquinoline hybrids (also named as trioxaquines) and ART-QN hybrids possess enhanced antiplasmodial and antimalarial activities compared with the parent drugs. Thus, it is conceivable that hybridization of peroxides with quinoline moiety may provide valuable therapeutic intervention for the treatment of malaria. 7.1. (Dihydro)ART-quinoline hybrids ART is extracted from the leaves of Artemisia annua, or sweet wormwood, and has been used since ancient times to treat malaria by the Chinese as an herbal medicine. Semisynthetic derivatives of ART such as artemether and arteether (Fig. 16) are derived from dihydroartemisinin (DHA), the main active metabolite of ART, are highly effective against all species capable of causing human malaria, fast-acting, low host toxicity and the only alternative for the treatment of both drug-sensitive and drug-resistant P. falciparum infection [122e125]. ART-based combination therapy (ACT) has been recommended by WHO as a priority treatment in the fight against malaria. However, clinical applications of ART and its semisynthetic derivatives are impaired due to their poor solubility in oil and water, low bioavailability, relatively high recrudescence, poor pharmacokinetic properties and high cost. Chemical structures of artemisinin and its semisynthetic derivatives DHA, artemether, arteether and artesunate are shown in Fig. 16. To overcome the above deficiencies, numerous of ART

(including its semisynthetic derivatives)-quinoline hybrids were synthesized and evaluated for their antiplasmodial and antimalarial activities. Walsh et al. showed that hybrid 55 (Fig. 17), DHA covalently linked to QN via an ester linkage, had superior activity to that of ART alone, QN alone, or a 1:1 mixture of ART and QN, suggesting that the actions of both DHA and QN moieties were preserved [126]. Moreover, the activity of 55 (IC50: 8.95 nM after 48 h) was around 3 times superior to that of a 1:1 mixture of ART and QN (IC50: 31.8 nM after 48 h), indicating that the two agents joined together were more active than the same two agents administered separately. Given the lability of the ester linkage especially in vivo, it is expected that an ether/amine bond will be more stable [126]. ART semisynthetic derivatives can eliminate all species bloodstage infection, while PQ could eradicate dormant liver-stage P. vivax and P. ovale parasites and is active against the transient liver forms of all Plasmodium species, so the hybrids 56a,b (Fig. 17) with covalently linked PQ and DHA may emerged as multistage antimalarials which display the enhanced efficacy against the liver and blood stages of infection [127]. Although both hybrids were as potent as ART against cultured P. falciparum (IC50: ~10 nM) and erythrocytic stage of P. falciparum, the two hybrids 56a,b (IC50: 155 and 523 nM) displayed enhanced in vitro activities against P. berghei in liver stage, which was 18e66 times more potent that a 1:1 PQART mixture (IC50: 9714 nM), while ART has no activity. Flow cytometry-based analysis revealed that the hybrids acted by preventing intracellular parasite replication, which was in accordance with the parent PQ. The abilities of the hybrids to inhibit P. berghei liver infection (infected by intravenous injection of 10,000 luciferase-expressing P. berghei sporozoites) in mice were as active as ART (IC50: 8.2 ± 0.9 nM), and far more potent than PQ (IC50: 3300 ± 55 nM). Significant decreases in parasite loads in the livers of mice treated with a single intraperitoneal (i.p.) injection of 26.5 mmol/kg of hybrid 56a or 56b were observed, but the effect was less marked than that of PQ. The further investigation showed

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39

Fig. 16. Chemical structures of artemisinin and its semisynthetic derivatives dihydroartemisinin, artemether, arteether and artesunate.

Fig. 17. Chemical structures of (dihydro)artemisinin-quinoline hybrids 55e60.

that administration of hybrid 56a during delivered stage delayed or prevented the appearance of parasites to an extant similar to that of PQ, while hybrid 53b had no effect on infection. Similar results were observed by administered orally. The in vivo efficiency in the

treatment of C57BL/6 mice (infected by i.p. injection of 100,000 P. berghei infected RBCs) showed that the parasitemia of mice injected hybrids or ART decreased soon after initiation of treatment, nearly to zero for all mice around day 9 post-infection. The

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hybrids and ART remained blood-stage negative throughout the duration of experiment and had a 100% survival rate. However, parasitemia recurred at days 12e13 post-infection in mice treated with ART or hybrid 56a, while mice treated with hybrid 56b didn't recur. It's notable that parasitemia and survival rate were remarkable increased in oral administration of hybrid 56b in the treatment of a patent blood infection in mice (injected 10,000 GFP-expressing P. berghei sporozoites) over those for the ART-PQ mixture. Overall, both the hybrids, especially 56b, can be used to control an ongoing blood-stage infection with superior efficacy than that of ART or ART-PQ mixture. Interestingly, hybrid 56b was less active than 56a in vitro, but more potent in vivo, suggesting enhanced pharmacokinetic properties or stability. Based on the above findings, three analogues of 56a,b were synthesized and assessed for their in vitro antiplasmodial activities against blood-stage CQR W2 and liverstage P. berghei strains [25]. All hybrids were equal to or more potent than the references ART, PQ and 1:1 PQ-ART mixture against the tested two strains. The most active 56c (Fig. 17, IC50: 5.1 and 67 nM for the two strains) was 1.6- and 647-fold more potent than ART and PQ (IC50: 8.2 and 3300 nM, respectively) against blood-stage CQR W2 strain, 111 and 144 times more active than PQ (IC50: 7500 nM) and PQ-ART mixture (IC50: 9714 nM). In a word, this novel approach to design new antimalarials based on the covalent hybridization of agents acting on different stages of the parasite life cycle provided a new strategy to develop multistage antimalarials. The hybrid salt MEFAS (Fig. 17), derived from artesunate and MQ, not only showed to be more effective than the combination of artesunate and MQ, but also exhibited lower toxicity against HepG2 hepatoma cells presumably due to the salt in favor of solubility, bioavailability and pharmacokinetic properties. Most antimalarials target asexual parasites without reducing gametocyte formation or development, and obviously, antimalarials with dual roles such as target both asexual parasites and gametocytes, would improve the control of malaria. It is notable that MEFAS has been shown to be an active blood schizonticidal drug [128], also exhibited excellent capacity in blocking the infectivity of P. falciparum gametocytes: MEFAS (IC50: 4.6 mM) was significantly more active against mature gametocytes of P. falciparum than MQ (IC50: 16.5 mM) and methylene blue (IC50: 12.7 mM); MEFAS inhibited female gametocyte activation (IC50: 0.02 mM) and was 15 times more effective than artesunate (IC50: 0.30 mM), 280-fold more potent than MQ (IC50: 5.6 mM), and 35-time more active than methylene blue (IC50: 0.71 mM). Thus, MEFAS could act as a potential candidate for use in clinical trials in areas in which malaria is endemic [129]. A series of DHA-aminoquinoline hybrids were synthesized by covalently linking DHA with 4-amino-7-chloroquinoline via an ether/amine bond, and their oxalate salts were also prepared for evaluation of in vitro antiplasmodial activities [130,131]. All hybrids including their oxalate salts were less active than the parent DHA against CQS D10 and CQR Dd2 strains, but almost all of them were exhibited either comparable to or greater potency than that of CQ against CQR Dd2 strain. In general, the oxalate salts were more potent than their free base hybrids against the tested two strains, but the in vitro cytotoxicity in CHO indicated that the oxalic acid increases the toxicity of the hybrids, so there should be a balance between the activity and toxicity. Hybrid 57 (Fig. 17, IC50: 17.12 nM) and its oxalate salt (IC50: 20.76 nM) possessed the highest antiplasmodial activity, which showed 9- and 7-fold higher activity than CQ (IC50: 157.90 nM) against CQR Dd2 strain [130]. Similar hybrids were also prepared by Hoppe and O'Neill et al., and all hybrids (IC50: 5.4e35 nM) displayed excellent in vitro antiplasmodial activities against CQS D10 and CQR K1 strains, which was comparable to or better than the references CQ (IC50: 15.62e219 nM) and ART (IC50: 9.3e23 nM), but less active than

artemether (IC50: 1.26e3.1 nM) and DHA (IC50: 3e4 nM) [132,133]. The cytotoxicity of the hybrids (IC50: 0.169e0.496 mM) in HeLa cell line was much higher than CQ (IC50: 8.54 mM) [132]. It was observed that morphological changes in parasites treated with the hybrids were similar to those of ART. The further investigation indicated that hybrids seem to share dual action mechanisms of both CQ and ART: the hybrids exhibited enhanced inhibitory activities against the polymerization of b-hematin (ART derivatives are not able to inhibit hemozoin polymerization); they caused an augment in accumulation of hemoglobin within the parasites that was in the middle of ART and CQ; and they also appeared to inhibit endocytosis as evidenced by the abatement of transport vesicles in the parasites [133]. Moreover, when the two pharmacophores were linked via an ester/amine bond, the hybrids 58 (Fig. 17) did not show an enhanced antiplasmodial activities against CQS D10 and CQR Dd2 strains compared to ART and DHA, but the cross-resistance was absent in these hybrids (RI: 0.9e1.3), which was identical with the previous studies [131,133]. Additionally, the in vitro antiplasmodial activity was generally decreased with increasing length of sidechains [134], which was proved by the previous research [135]. Considering that CQ derivatives incorporating a Schiff base hydrazone moiety showed better antiplasmodial activity and inhibition against cysteine protease falcipain-2 of P. falciparum, the scaffolds of 4-quinolylhydrazone hybridized DHA as the new hybrids 59 (Fig. 17) may also be effective against falcipain-2 [136,137]. All hybrids 59 were found to be significant inhibitor of falcipain-2 with IC50 ranging from 0.15 to 2.28 mM. It is worth to notice that chloro on the 6-position played a significant role in the inhibitory activity, as it was more suitable for the key active site of falcipain-2. The molecular docking results showed that hybrid 59a with chloro on the 6-position exhibited the highest inhibitory activity (IC50: 0.15 mM) has close hydrophobic interactions with the residues within the pocket of cysteine protease falcipain-2 [137]. The results may represent a point for finding new mechanism and effective leads blocking the cleavage activity of falcipain-2. Four hybrids with two DHA moieties linked to CQ nucleus were synthesized and screened for their antiplasmodial activities against CQS D10 and CQR Dd2 strains [138]. All hybrids exhibited excellent antiplasmodial activities against the tested CQS D10 and CQR Dd2 strains. The most active 60 (Fig. 17, IC50: 5.31 and 28.43 nM against CQS D10 and CQR Dd2 strains, respectively) was 4e5 folds more potent than CQ (IC50: 21.54 and 157.90 nM against CQS D10 and CQR Dd2 strains, respectively) against the tested two strains, could be acted as a lead for further investigation. 7.2. Endoperoxide(tetraoxane/trioxane)-quinoline hybrids Endoperoxides, which possess excellent antiplasmodial and antimalarial properties, belong to the G-factor family. The crucial structural functionality within ART, synthetic 1,2,4-trioxanes and 1,2,4,5-tetraoxanes are the endoperoxide bridges, so endoperoxidebased hybrids represent an attractive alternative to ACTs. 1,2,4,5-tetraoxane drug candidates showed outstanding antimalarial activities (via reaction with haem (or free Fe (II)) to generate cytotoxic radicals), stability, low toxicity and ADME (absorption, distribution, metabolism, and excretion) properties that overcome most of the problems encountered previously with the synthetic and semi-synthetic antimalarial endoperoxide drugs that have progressed into pre-clinical development [139]. Therefore, it has been postulated that the development of hybrids comprising of quinoline and tetraoxanes (tetraoxaquines) into single scaffold may result into new class of agents for deployment in the control and eradication of malaria as a component of combination chemotherapy [140].

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A series of hybrids 61 (Fig. 18) conjugation of 4-amino-7chloroquinoline and 1,2,4,5-tetraoxanes (tetraoxaquines) by covalent linkers were tested against CQR strain (PKL-9) of P. falciparum, and inhibitor against falcipain-2 was also performed by docking study. The results indicated that all hybrids showed significant activities against CQR strain (PKL-9), but were less active than CQ in terms of IC50. The SAR revealed that hybrids with spirocycloheptane substituent on tetraoxane moiety of tetraoxaquine showed less antiplasmodial activities as compared to other congeners having dimethyl, spiro-cyclohexane, spiro-cyclopentane. In addition, nature of linkers (1,2-diaminopropane, o-phenylenediamine and piperazine) displayed unique role on the pharmacological activity. In particular, tetraoxaquines with 1,2diaminopropane and o-phenylenediamine as linkers exhibit prominent activities than hybrids having a piperazine linker [140]. Rawat et al. also proved that the nature of linkers between tetraoxane and CQ significantly affect the antiplasmodial activities [141]. The imine containing hybrid 62a (Fig. 18, IC50: 0.47 and 0.57 mM, respectively) was more active than its reduction counterpart hybrid 62b (Fig. 18, IC50: 2.64 and 1.62 mM, respectively) against CQS D6 and CQR W2 strains, and both of them were no toxic towards the tested six cell lines. A series of 4-amino-7chloroquinoline and 1,2,4,5-tetraoxane conjugates were screened for in vitro activities against three P. falciparum strains: CQS D6, CQR  W2 and MDR TM91C235 strains by Solaja et al. [142]. Hybrid 63a (Fig. 18, IC90: 2.26e3.70 nM) showed the most potent activity and was 3.4- to 283-fold more potent than the references MQ, CQ and ART against the tested three strains, and in the in vivo metabolism studies 63a was relatively metabolically stable with t1/2>60 min in both mouse and human liver microsomes. Unfortunately, 63a (5/5 curative at 320 mg/kg/day, while only 1/5 cure at 80 mg/kg/day) only exhibited moderate in vivo activity against P. berghei (KBG 173

41

strain) infected ICR mice, which was less potent than 63b (Fig. 18, 3/ 5 cure at 80 mg/kg/day), but no toxic effects of the two hybrids upon necropsy were observed even at 960 mg/kg [143]. A series of 1,2,4,5-tetraoxane-PQ hybrids 64 (Fig. 18) were synthesized and screened for antiplasmodial and antimalarial activities against both blood-stage and liver-stage malaria parasites [25]. All hybrids exhibited excellent activities against blood-stage CQR W2 strain (IC50: 21.2e45.2 nM), and high potency against liver-stage P. berghei with most of hybrids displaying IC50 ranging from 330 to 604 nM which were more potent than PQ and 1:1 PQ-ART mixture (IC50: 7500 and 9714 nM, respectively). The in vivo study revealed that hybrid 64a irreversibly cleared the parasitemia from GFP-expressing P. berghei ANKA-infected mice, while screening of transmission-blocking activity showed that 64a was superior to PQ, and completely inhibited the appearance of oocysts in mosquito midguts when administrated at 25 mmol/kg [25]. These results indicated that 1,2,4,5-tetraoxane-PQ hybrids are excellent starting point to develop agents that convey all the desired antimalarial multistage. Trioxane OZ277 (Fig. 19) was launched in India as a combination agent with synriam for the treatment of malaria, and the nextgeneration trioxane OZ439 (Fig. 19) has completed phase IIa clinical trials, so trioxanes are emerged as excellent antiplasmodial and antimalarial candidates [142,143]. The attachment of a trioxane moiety to the 4-aminoquinoline entity gives trioxaquines, which are proved to be highly active in vitro against CQR strains of P. falciparum (IC50: 5e50 nM). As the trioxane moiety is a potential alkylating agent after reductive activation by heme and the 4aminoquinoline entity is known to easily penetrate within infected erythrocytes and then interact with heme, such hybrids are expected to combine the properties of both fragments. Thus, these trioxaquines are active on the young erythrocytic stages of

Fig. 18. Chemical structures of endoperoxide-quinoline hybrids 61e64.

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P. falciparum as ART derivatives, whereas CQ is active on the late stages. Chemical structures of OZ277, OZ439, DU-1102, DU-1302 and PA1103/SAR116242 are shown in Fig. 19. The DU-1102 (Fig. 19) was found to be highly active with a mean IC50 of 43 nM against thirty-two CQ and pyrimethamine sensitive or resistant isolates, and no significant difference (P > 0.05) between the mean IC50 of DU-1102 for CQS isolates (48 nM) and CQR isolates (40 nM) was observed. Moreover, there was no correlation between the responses to DU-1102 and CQ, but only a low correlation between the responses to DU-1102 and pyrimethamine, which are first- and second-line (pyrimethamine-sulfadoxine combination) agents in Cameroon, indicating an independent mode of action of the trioxaquine against the parasites [144,145]. Trioxaquine DU1302 (Fig. 19) was active by oral route on mice infected by the highly virulent strain P. yoelii nigeriensis, and displayed good bioavailability by oral administration, and did not induce any toxic effects in mice even at a dose of 120 mg/kg/day administered orally. In addition, parasitemia clearance was complete without recrudescence. DU1302 is active on gametocytes, the mosquito-transmissible forms of the parasites, which is not the case for CQ. Killing gametocytes is essential to limit the spread of malaria [146,147]. Generally speaking, DU1302 meets the main criteria of pharmacological activity necessary for further biological evaluation, so it can be considered as a promising candidate [148]. As expected, the trioxaquine PA1103/SAR116242 (Fig. 19) has also shown the dual mode of action: heme alkylation via the reductive activation the trioxane entity, heme stacking with the aminoquinoline moiety, and inhibition of hemozoin formation [149]. PA1103/SAR116242 is highly active and has a similar in vitro activity on CQS and CQR strains of P. falciparum (with IC50 values ranging from 7 to 24 nM), which is within the same range as ART. PA1103/ SAR116242, not only very efficient by oral route with a complete cure of mice infected with P. v. petteri (CQS strain) and P. vinckei (CQR strain) at 26e32 mg/kg, but also highly effective in humanized mice infected with P. falciparum. The excellent in vivo antimalarial activity combined with a dual mode of action (ART-like and CQ-like), good drug profiles (preliminary absorption, metabolism, and safety parameters) making PA1103/SAR116242 a promising candidate for a covalent biotherapy strategy [149]. Three cyclic endoperoxides 7-chloroquinoline hybrids 65a-c (Fig. 20) were evaluated for in vitro antiplasmodial activities against

CQS 3D7 and CQR W2 strains, and all hybrids showed moderate activities with IC50 ranging from 0.185 to 6.75 mM, but less active than the reference ART (IC50: 0.019 mM) [150]. The SAR revealed that double bond decreased the antiplasmodial activity (65a vs 65b, 6.05e6.75 mM vs 1.40e2.45 mM), while methylation of the hydroxyl group of the peroxylhemiketal moiety enhanced the antiplasmodial activity (65a vs 65c, 6.05e6.75 mM vs 0.185e0.24 mM). 8. Quinolone-quinoline hybrids Quinolone-derived antibiotics are prescribed worldwide for the treatment of various infectious diseases especially those that have failed to respond to other classes of antibiotics. Quinolones and fluoroquinolones have also been proposed for treatment of malaria since they have in vitro antiplasmodial activities against both CQS and CQR strains of P. falciparum [151]. However, the main boundedness in use is the relatively slow antiplasmodial and antimalarial action of quinolones/fluoroquinolones, so more efficient derivatives are warranted. Indeed, several quinolones as part of combination therapy could be used in combination with a rapidly acting antimalarial agent. Therefore, there is still scope to screen quinolones/ fluoroquinolones hybrids which may serve as promising antimalarials. Since amino acids have been used as carriers for drugs because of their abilities to transport into mammalian tissue, a series of novel hybrids 66 (Fig. 21) of QN linked by amino acid residues to quinolone antibiotics including oxolinic/nalidixic acids as well as levofloxacin/enrofloxacin as potential enhancers of the antiplasmodial activities were synthesized and examined for antiplasmodial activities against CQS 3D7 strain [151]. These hybrids retain in vitro antiplasmodial activities with IC50 values ranging from 12 to 207 nM against CQS 3D7 strain, and 66a (IC50: 12 nM) as the most active hybrid was more potent than the parents QN (IC50: 18 nM) and levofloxacin (IC50: >20,000 nM). Quinolon-4(1H)-imines were shown to hydrolyze to quinolones in aqueous solutions, thus raising the possibility of these compounds acting as chemically activated prodrugs. Based on that, quinolon-4(1H)-imine quinoline hybrid 67 (Fig. 21) was synthesized and screened for in vitro antiplasmodial activity against blood-stage P. falciparum W2 and liver-stage P. berghei strains [152]. The results showed that 67 was active against both erythrocytic and

Fig. 19. Chemical structures of OZ277, OZ439, DU-1102, DU-1302 and PA1103/SAR116242.

Y.-Q. Hu et al. / European Journal of Medicinal Chemistry 139 (2017) 22e47

Fig. 20. Chemical structures of cyclic endoperoxides 7-chloroquinoline hybrids 65.

exoerythrocytic forms of malaria parasites with IC50 values of 53.7 and 710 nM, so it constitutes excellent starting point for further lead optimization as dual-stage antimalarials. A set of fluoroquinolone derivatives having various alkyl and substituted triazolyl groups at N-1 position was synthesized and tested for in vitro antiplasmodial activities against CQS 3D7 strain [153]. The SAR revealed that derivatives with alkyl side chains were more active than the corresponding triazolyl analogues, and chloro in aromatic ring enhanced the activity. All the derivatives exhibited moderate activities against CQS 3D7 strain, and the fluoroquinolone quinoline hybrid 68 (Fig. 21) with IC50 of 4.06 ± 0.19 mM was 2 folds more potent than the parent ciprofloxacin (IC50: 8.82 ± 0.06 mM). 9. Miscellaneous quinoline hybrids A set of amide or amine tethered adamantine quinoline hybrids was synthesized and screened for in vitro antiplasmodial activities against CQS D6, CQR W2 and MDR TM91C235 strains [154]. The SAR revealed that the hybrids with amine linkers were much more potent than those with amide linkers, and the hybrids with C2 and C3 amine linkers were more active than those with longer linkers. Conjugates 69a,b (Fig. 22) were the most active hybrids with IC90 ranging from 6.41 to 20.20 nM, which were 2e34 times more potent than CQ (IC90: 16.38e529.86 nM) and MQ (IC90: 14.70e106.04 nM), and comparable to or better than ART (IC90: 11.5e17.40 nM) against the tested three strains. The MQ hybrid 70 (Fig. 22, IC50: 5.9 ± 1.2 nM) was around 1.5-fold more potent than MQ (IC50: 9.1 ± 2.8 nM) against CQS 3D7 strain [155]. Chemical structures of miscellaneous quinoline hybrids 69e76 are shown in

43

Fig. 22. Cinnamic acid derivatives can act as Michael acceptors and inhibit cysteine proteases through S-alkylation mainly attributed to their a,b-unsaturated carbonyl moieties. Irreversible S-alkylation of the falcipain catalytic Cys has been considered the major mechanism behind the inhibitory and in vitro antiplasmodial activity of peptidyl inhibitors. Furthermore, hybrids resulting from cinnamic acid conjugation with heterocyclic moieties from well known antimalarials present improved antiplasmodial and antimalarial activities, so conjugating cinnamic acid with quinoline may provide promising leads for the development of new antimalarials. A series of cinnamic acid quinoline conjugates 71a,b (Fig. 22) linked through a proper retro-enantio dipeptide or without a linker were synthesized as potential dual-action antimalarials. The SAR suggested that for dipeptide series 71a, replacement of the D-amino acids by their natural L-counterparts led to a decrease in both antiplasmodial and falcipaine-inhibitory activities. Hybrids with such spacer were active in vitro against blood-stage P. falciparum and hemozoin formation, implying that the dipeptide has a key role in mediating these two activities. Although hybrids with dipeptide linkers were more potent than the corresponding hybrids without a linker, all of them were less active than the references CQ and ART [156]. Based on the above results, a second-generation of cinnamic acid-CQ/PQ hybrids through a flexible and more hydrophobic amine chain were synthesized and screened for in vitro antiplasmodial activities [157e159]. CQ hybrids 72a (IC50: 11e59 nM), PQ hybrids 72b (IC50: 1.4e2.4 mM) had higher in vitro potency than their parents CQ (IC50: 138 nM) and PQ (IC50: 7.5 mM), against blood-stage W2 and liver-stage P. berghei strains. These novel hybrids represent a new entry as promising dual-stage antimalarial leads. Hydrazones have attracted continuous interests in the medicinal filed due to their wide range of pharmacological properties, such as antifungal, antiviral, antimicrobial, antiplasmodial and antimalarial activities. Several quinoline hydrazones were synthesized and evaluated for their antiplasmodial activities [160e162], and amongst them, conjugate 73 (Fig. 22, IC50: 0.9e39.6 nM) had higher in vitro antiplasmodial potency than CQ (IC50: 10e280 nM) against CQS D10, 3D7 and CQR W2, K1 strains [162]. Sulfonamide moiety is present in many bioactive substances and drugs such as sulfadoxine, so incorporation of sulfonamide pharmacophore into quinoline motif may enhance the in vitro antiplasmodial and in vivo antimalarial activities. A new class of fifteen quinoline-sulfonamide hybrids 74 (Fig. 22), with a 7chloroquinoline moiety connected by a linker group to 4substituted arylsulfonamide moieties was synthesized and

Fig. 21. Chemical structures of quinolone-quinoline hybrids 66e68.

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Fig. 22. Chemical structures of miscellaneous quinoline hybrids 69e76.

evaluated for the in vitro antiplasmodial and in vivo antimalarial activities against P. falciparum by Krettli et al. [163]. All hybrids with low cytotoxicity (MDL50: 14.9->257.0 mM) in BGM cells exhibited excellent in vitro schizonticidal blood activities against CQR W2 clone with IC50 values of 0.05e1.63 mM, and ten of them were more potent than the references CQ and sulfadoxine (IC50: 0.46 and > 15.5 mM, respectively). The SAR revealed that the hybrids with substituents at C-4 position of arylsulfonamide moiety were more active than the corresponding unsubstituted hybrids, and electron-donation group methyl enhanced the activity greatly as evidenced by the most active 74l was 9->310 folds more potent than CQ and sulfadoxine. Hybirds 74j, 74l, 74m and 74o exhibited the highest SI values of 3386.0, 2489.0, 1102.2 and 1031.3, respectively, higher than that of CQ (834.74), were selected for further evaluation of in vivo antimalarial activities in P. berghei infected mice. The results showed that 74j and 74m inhibited P. berghei parasitemia by 47% and 49% on day 5 after mice inoculation, and the most active 74m could act as a new prototype for the development of antimalarials against CQR parasites. Several pharmacophores such as thiosemicarbazones, guanyls, thiophenes, furans, azalides, 4H-chromenes, ureas and thioureas as well as some natural products exhibit excellent antiplasmodial profiles, so plenty of hybrids with these pharmacophores have been synthesized for screening of antiplasmodial activities [164e182]. For example, hybrid 75 (Fig. 22, IC50: 1.8e7.2 nM) was 8e72 times more potent than CQ (IC50: 15e525 nM) against CQS D6, CQR W2

and C235 strains [174]; conjugate 76 (Fig. 22) with IC90 ranging from 3.74 to 9.20 nM was more potent than the references CQ (IC90: 16.11e697.97 nM), MQ (IC90: 15.25e134.07 nM) and ART (IC90: 11.50e17.40 nM) against CQS D6, CQR W2 and MDR TM91C235 strains, and no cytotoxicity in HEPG2 and PMBC cells [183]. 10. Conclusion Malaria is a devasting global health threat, and nearly half of the world population is under the risk of being infected, especially among children and pregnant women. P. falciparum is the main cause of malaria, and is obligatory for 95% deaths. Therefore, new antimalarials should be effective against P. falciparum. Two main reasons behind malaria remaining such a burden to humanity are the widespread resistance of P. falciparum strains that resistant to almost all the currently in use antimalarials and its complex life cycle. One of the modern concepts of drug design principle is molecular hybridization which involves the rational design of new entities by covalent fusion of two or more different pharmacophore moieties with potential to enhance efficacy, fastacting, improve safety and/or reduce propensity to elicit resistance relative to the parent drugs. Numerous of quinoline hybrids have been developed by combining QN, CQ, 4-aminoquinolines, and MQ with other pharmacophores by C-O, C-C, and C-N covalent bonds. Great achievements have been obtained in this field, such as piperaquine, a bis-4-aminoquinoline effective against CQR strains,

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and FQ, a 4-amino-7-chloroquinoline with a ferrocenyl side-chain. Piperaquine has been used extensively in China and Indochina as prophylaxis and treatment, while FQ is in phase IIB clinical trials, and may also be used for the treatment of malaria in the near future. In addition, oxidative stress is related to the pathophysiology of malarial infection, and Plasmodium parasites digest hemoglobin leads to the production of heme which triggers the generation of reactive oxygen species resulting in anemia and death. Obviously, hybrids with antioxidant activity may alleviate the progression of malarial infection, and possibly prevent the sequelae [59]. Therefore, hybridization of quinoline with bioactive substances with antioxidant activity may give promising antimalarials. Numerous of studies revealed that modification of the basic amine side chain of quinoline-contained antimalarials can produce new derivatives active against both drug-sensitive and drugresistant P. falciparum strains, but transformation of the quinoline nucleus itself will not. In fact, the ring system affects the pKa of nitrogen both in the quinoline ring and in the side-chain as well as other physical parameters, however, this is not significantly correlated with the activity in drug-resistant strains. Therefore, upto date, most of studies focus on the modification of the sidechain of quinoline-contained antimalarials. The SAR studies on 4aminoquinolines suggest that 7-chloro and 4-amino groups in quinoline nucleus are essential for high antiplasmodial and antimalarial activities. When 7-chloro group was replaced by other groups, the decreased activities were noted which is mainly due to the change of the pKa of quinoline nitrogen atom (pKa1). The variable alkyl chain is important not only for circumversion of the CQR mechanism but also for lipophilicity, which is an important parameter in the effectiveness of iron chelating antimalarials. CQ hybrids where the alkyl chains between the nitrogen atoms are shortened to 2e4 atoms, or lengthened to 10e12 atoms, retained their activities against CQS strains, while those with intermediate chain lengths (5e8 carbon atoms) had rather lower activities. Hybrids containing a trioxane moiety linked to an aminoquinoline entity showed efficient antimalarial activity without recrudescence. Moreover, the approach to design new antimalarials based on the covalent hybridization of agents acting on different stages of the parasite life cycle provided a new strategy to develop dual-stage and multistage antimalarials. References [1] D.G. Cabrera, F. Douelle, Y. Younis, T.S. Feng, C.L. Manach, A.T. Nchinda, L.J. Street, C. Scheurer, J. Kamber, K.L. White, O.D. Montagnat, E. Ryan, K. Katneni, K.M. Zabiulla, J.T. Joseph, S. Bashyam, D. Waterson, M.J. Witty, S.A. Charman, S. Wittlin, K. Chibale, J. Med. Chem. 55 (2012) 11022e11030. [2] World Malaria Report, World Health Organization, 2016, 2016. [3] A. Paulo, M. Figueiras, M. Machado, C. Charneira, J. Lavrado, S.A. Santos, D. Lopes, J. Gut, P.J. Rosenthal, F. Nogueira, R. Moreira, J. Med. Chem. 57 (2014) 3295e3313. [4] Y.Q. Hu, S. Zhang, F. Zhao, C. Gao, L.S. Feng, Z.S. Lv, Z. Xu, X. Wu, Eur. J. Med. Chem. 133 (2017) 255e267. [5] L.S. Feng, M.L. Liu, S. Zhang, Y. Chai, B. Wang, Y.B. Zhang, K. Lv, Y. Guan, H.Y. Guo, C.L. Xiao, Eur. J. Med. Chem. 46 (2011) 341e348. [6] S. Zhang, Z. Xu, C. Gao, Q.C. Ren, L. Chang, Z.S. Lv, L.S. Feng, Eur. J. Med. Chem. 138 (2017) 501e513. [7] Z. Xu, S. Zhang, C. Gao, F. Zhao, Z.S. Lv, L.S. Feng, Chin. Chem. Lett. 28 (2017) 159e167. [8] X.D. Jia, S. Wang, M.H. Wang, M.L. Liu, G.M. Xia, X.M. Xia, X.J. Liu, Y. Chai, H.W. He, Chin. Chem. Lett. 28 (2017) 235e239. [9] N. Wang, K.J. Wicht, E. Shaban, T.A. Ngoc, M.Q. Wang, I. Hayashi, M.I. Hossain, Y. Takemasa, M. Kaiser, I.E.T.E. Sayed, T.J. Egan, T. Inokuchi, Med. Chem. Commun. 5 (2014) 927e931. [10] P.B. Madrid, J. Sherrill, A.P. Liou, J.L. Weisman, J.L. DeRisi, R.K. Guy, Bioorg. Med. Chem. Lett. 15 (2005) 1015e1018. [11] C. Viegas-Junior, A. Danuello, V. da S. Bolzani, E.J. Barreiro, C.A.M. Fraga, Curr. Med. Chem. 14 (2007) 1829e1852. [12] M. Matias, S. Silvestre, A. Falcao, G. Alves, Mini-Rev. Med. Chem. 17 (2017) 486e517.

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