Synthesis and antimalarial activity of novel bicyclic and tricyclic aza-peroxides

Abstract

For the first time, novel bicyclic 5a–h as well as tricyclic 9a–h aza-peroxides were synthesized using a ¹O₂-mediated photo-oxygenation methodology as a key step in 52–71% yields in which one of the oxygen atoms of the 1,2,4-trioxane ring has been replaced by a nitrogen atom. The methodology is simple and is an efficient way to access 1,2-dioxa-4-aza six membered ring compounds. All these compounds were assessed for their in vitro antimalarial activity against Plasmodium falciparum. Compound 9a, 9c and 9f, the most active compound of the series, showed IC₅₀ values of 9.43, 8.83 and 5.63 ng mL⁻¹, respectively which were found to be comparable to that of the antimalarial drug chloroquine (IC₅₀ value 5.2 ng mL⁻¹). Compound 9f, the most active compound found in in vitro studies, provided 40% protection to infected mice at the dose of 96 mg kg⁻¹ × 4 days when screened for its antimalarial activity in vivo against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice by the oral route. In this assay, β-arteether and chloroquine, showed 100% suppression of parasitaemia on day 4 and provided 100% and 80% protection, respectively, to the infected mice.

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Introduction

Malaria continues to affect around 30% of the world population and causes approximately 300–500 million clinical cases per year and nearly 584 000 deaths in 2013, predominantly among children under 5 years of age and pregnant women in sub-Saharan African countries.¹ Due to the increase in the movements of human population into malarial regions, variation in climate change, and above all, proliferation of multidrug resistant Plasmodium falciparum (Pf) parasites; there has been an increase in the growth of the malarial burden experienced in recent years.²

Artemisinin and its semi-synthetic derivatives i.e. artemether, arteether and artesunic acid (Fig. 1) are currently the drugs of choice for the treatment of malaria caused by multidrug-resistant Plasmodium falciparum.³ The peroxide group present in the form of 1,2,4-trioxane moiety of artemisinin is essential for the activity of this class of compounds. A wide range of artemisinin analogues had been prepared by several groups,³ including our group,^3m–r which showed very high order of antimalarial activity in vivo against multidrug-resistant malaria.³ Unfortunately, artemisinin is associated with severe drawbacks such as limited supply, high cost, recrudescence, and recently indentified the neurotoxicity,⁴ as well as nephrotoxicity, and the reported cases of clinical drug resistance² to currently used artemisinin derivatives strongly encourage the finding, of more viable alternatives to the existing chemotherapeutic treatments.

Fig. 1 Artemisinin, its derivatives and its active pharmacophore.

In this regard, several structurally simple peroxides with promising in vivo antimalarial activity such as synthetic 1,2,4-trioxanes, 1,2,4,5-tetraoxanes, 1,2,4-trioxepanes, 1,2-dioxanes etc. have also been reported in the literature.^5–9 Many of these peroxides have shown high order of antimalarial activity more than clinically used β-arteether against multidrug-resistant malaria.^5–9 Parallelly, in recent years, several amino functionalized 1,2,4-trioxanes, reported by Singh et al., have shown high order of in vivo antimalarial activity against multi-drug resistant P. yoelii in Swiss mice via oral as well as i.m. routes.¹⁰

Several attempts have been made to modify the pharmacophoric 1,2,4-trioxane moiety of structurally simple 1,2,4-trioxanes and assess these new compounds for their antimalarial efficacy. This led to the synthesis and antimalarial testing of various classes of organic peroxides other than 1,2,4-trioxanes, such as endoperoxides, alkyl hydroperoxides, peroxyamines, peroxyketals, β-peroxy-lactones, etc.¹¹ With the peroxide bond being an indispensable requirement for the antimalarial activity, replacement of O₄ of 1,2,4-trioxane by another heteroatom, mainly by nitrogen, was of obvious interest. Particularly relevant to this was, photooxygenation of allyl amine by Adams.^12a Various other structural scaffolds e.g. I–IV, with nitrogen as a part of the ring containing the peroxide bond have been reported.^12b–i Most of these are endoperoxides formed by Diels–Alder reaction of a suitable diene with singlet oxygen.

Some other peroxides containing nitrogen atom in the whole molecule have also been reported, but the antimalarial activity of these aza-peroxides were not studied.^12j,k Hence, there is still room for the detailed study of aza-peroxides containing nitrogen in the peroxide vicinity and could be prominent area of investigation.

As a part of an endeavor to develop novel antimalarial agents which can serve as structurally simple substitutes for artemisinin derivatives, we were interested in replacing the oxygen atom at 4^th position of the 1,2,4-trioxane ring with nitrogen atom in order to study structure–activity relationship of newly synthesized skeleton (Fig. 2). The idea was inspired from the fact that aza-artemisinin, C-11 oxygen replaced with nitrogen atom, displayed 4 times more in vivo antimalarial activity against multidrug-resistant P. yoelii (Fig. 2).¹³ Herein, we report the synthesis and antimalarial activity of a new series of bicyclic and tricyclic aza-peroxides.

Fig. 2 Comparative study.

Result and discussion

In order to test our idea, we started our investigation with the synthesis of structurally simple bicyclic 5a–h and tricyclic 9a–h, respectively as shown below in the Scheme 1.

Scheme 1 Synthesis of compounds 5a–h and 9a–h.

The starting material allylic alcohols 1a–h,¹⁴ were subjected under Mitsunobu conditions with succinimide to furnished N-substituted succinimides 2a–h in 60–74% yields. Similarly, the corresponding N-substituted phthalimides 6a–h in 66–86% yields was also prepared with phthalimide under the same Mitsunobu conditions (Scheme 1). NaBH₄ reduction of imides 2a–h and 6a–h in a 1 : 1 mixture of THF and MeOH at 0 °C furnished hydroxy-functionalized lactams 3a–h and 7a–h, respectively in 58–74% yields (Scheme 1). Using the literature procedure, singlet oxygen-mediated photooxygenation of 3a–h and 7a–h in a 2 : 1 : 1 mixture of CH₃CN, THF and CHCl₃ at −5 to 0 °C furnished the corresponding hydroperoxides 4a–h and 8a–h which, without purification, were subjected to acid catalyzed intramolecular cyclization to yield the corresponding dioxa-aza-indenones 5a–h and dioxa-aza-fluorenones 9a–h, respectively in 52–71% yields (Scheme 1, Table 1).¹⁵

Table 1 Melting points and yields of dioxa-aza-indenones 5a–h (bicyclic) and dioxa-aza-fluorenones 9a–h (tricyclic)

Entry	Ar =	Mp (°C)	Yield^a (%)
a Yields based on hydroxy-functionalized lactams 3a–h and 7a–h as starting materials.
5a	C6H5-	Oil	67
5b	4-Fluoro-C6H4-	65–67	59
5c	4-Chloro-C6H4-	69–71	55
5d	4-Bromo-C6H4-	Oil	52
5e	4-Methyl-C6H4-	93–95	69
5f	4-Methoxy-C6H4-	78–80	63
5g	4-Cyclohexyl-C6H4-	81–83	59
5h	4-Phenyl-C6H4-	136–138	71
9a	C6H5-	85–87	60
9b	4-Fluoro-C6H4-	80–82	59
9c	4-Chloro-C6H4-	121–123	54
9d	4-Bromo-C6H4-	132–134	61
9e	4-Methyl-C6H4-	Oil	70
9f	4-Methoxy-C6H4-	115–117	63
9g	4-Cyclohexyl-C6H4-	135–137	57
9h	4-Phenyl-C6H4-	148–150	61

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The acid-catalyzed intramolecular cyclization, to synthesize the corresponding dioxa-aza-indenones 5a–h and dioxa-aza-fluorenones 9a–h, was found to be highly diastereoselective as all these compounds were obtained as single isomers instead of pairs of diastereomers.

To find out the reason for this selectivity, we calculated the energies of two diastereomers of 5a and 9a using MOE (Molecular Operating Environment). The result of this study is shown in Fig. 3.

Fig. 3 Energies of diastereomers of 5a and 9a as determined by MOE.

As can be seen from the Fig. 3; the formation of single diastereomer of 5a and 9a with aryl–vinyl group in equatorial position and 4aH in axial position is favored over the other diastereomers thereby confirms its selectivity. This was further confirmed by NOESY spectrum of 9g which shows correlation between axial proton at C1 and proton at C4a (Fig. 4).

Fig. 4 Representation of NOESY correlations in 9g.

All of these aza-peroxides 5a–h and 9a–h were initially screened for their in vitro antimalarial activity against chloroquine-sensitive P. falciparum (3D7 strain).¹⁶ Chloroquine and β-artemether served as positive controls for in vitro screening. The results are shown in Table 2. Out of these 16 compounds, among 5a–h series; compounds 5g and 5h, and among 9a–h series; the three most active compounds i.e. 9a, 9c, 9f and the next active compound 9g were selected for in vivo testing and screened against multidrug-resistant P. yoelii nigeriensis in Swiss mice by oral routes at the dose of 96 mg kg⁻¹ × 4 days using Peter's procedure.¹⁷ β-Arteether served as a positive control for in vivo screening. The results are shown in Table 3.

Table 2 In vitro antimalarial activity of compounds 5a–h and 9a–h against Plasmodium falciparum 3D7 strain

Compd no.	log P^a	IC50 (ng mL^−1)^b
a log P values are calculated using Chemdraw ultra 11.0.b IC50: concentration corresponding to 50% inhibition of chloroquine-sensitive strain 3D7 of Plasmodium falciparum.
5a	1.92	145.97
5b	2.07	162.59
5c	2.47	38.05
5d	2.74	158.61
5e	2.40	68.11
5f	1.79	245.89
5g	3.9	32.46
5h	3.59	15.27
9a	3.54	9.43
9b	3.70	39.58
9c	4.10	8.83
9d	4.37	60.11
9e	4.03	86.59
9f	3.41	5.63
9g	5.33	17.46
9h	5.22	45.36
Artemether	3.51	0.4
Chloroquine	3.73	5.2

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Table 3 Blood schizontocidal activity of 5g, 5h, 9a, 9c, 9f and 9g against multidrug-resistant (MDR) strain P. yoelii nigeriensis in Swiss mice via oral route¹⁷

Compd	Dose^a (mg kg^−1 × 4 days)	% suppression of parasitaemia on day 4^b^,^c	Cured/treated^d
a The drug dilutions of compounds were prepared in ground oil and administered to a group of mice at each dose, from day 0–3, once daily.b Parasitaemia levels were recorded from thin blood smears on day 4 and subsequently twice a week till day 28. Percent suppression = [(C − T)/C] × 100; where C = parasitaemia in control group, and T = parasitaemia in treated group.c 100% suppression of parasitaemia means no parasites were detected in 50 oil immersion fields during microscopic observation.^18d ^“5/5” means none of the treated mice developed patent infection during the 28 days observation period and hence were recorded as cured. Similarly “0/5” means none out of 5 mice were found to be cured.
5g	96	65	0/5
5h	96	57	0/5
9a	96	84	0/5
9c	96	89	0/5
9f	96	100	2/5
9g	96	100	1/5
β-Arteether	96	100	5/5
Chloroquine	96	100	4/5

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As seen from Table 2, despite having an entirely different pharmacophoric scaffold, these new organic peroxides showed significant antimalarial activity against P. falciparum in vitro. These new peroxides showed IC₅₀ values within the range of 5.63–245.89 ng mL⁻¹. The dioxa-aza-fluorenones derived aza-peroxides 9a–h displayed noticeably superior antimalarial activities (IC₅₀ values within the range of 5.63–60.11 ng mL⁻¹) as compared to the corresponding dioxa-aza-indenones derived compounds 5a–g (IC₅₀ values within the range of 32.46–245.89 ng mL⁻¹). The only exception to this trend, the compound 5h, showed IC₅₀ value of 15.27 ng mL⁻¹, which is lower than that of the corresponding phthalimide derived analogue 9h having IC₅₀ value of 45.36 ng mL⁻¹. Further, increasing lipophilicity (higher log P values), seems to be beneficial for enhancing the antimalarial activity of compounds of this series. Thus, compounds 5a–f, having log P values in the range of 1.79–2.74, showed poor antimalarial activities (IC₅₀ = 38.05–245.89 ng mL⁻¹). With increase in the lipophilicity and hence in log P values in the range of 3.41–5.33, compounds 5g, 5h and 9a–h, showed parallel rise in antimalarial activities and displayed 50% inhibition of parasites at considerably lower concentrations (IC₅₀ = 5.63–60.11 ng mL⁻¹) except compound 9e (IC₅₀ = 86.59 ng mL⁻¹).

Dioxa-aza-fluorenones derived aza-peroxides 9a, 9c and 9f, demonstrated impressive antimalarial activities with IC₅₀ values of 9.43 ng mL⁻¹, 8.83 ng mL⁻¹ and 5.63 ng mL⁻¹, respectively. The most active azaperoxide 9f, showed the highest in vitro antimalarial activity and provided 50% inhibition of parasites at the concentration value of 5.63 ng mL⁻¹. Clinically useful drugs, artemether and chloroquine, showed 50% inhibition of the parasites at 0.4 and 5.2 ng mL⁻¹, respectively. Thus, compound 9f is as active as chloroquine in the in vitro assay against Plasmodium falciparum 3D7 strain.

Compounds 5g, 5h, 9a, 9c, 9f and 9g were further tested in vivo against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice by oral routes at the dose of 96 mg kg⁻¹ × 4 days. As can be seen from Table 3, compounds 5g, 5h, 9a and 9c, provided 65%, 57%, 84% and 89% suppression of parasitaemia, respectively, on day 4 at the dose of 96 mg kg⁻¹ × 4 days by oral route. At the same dose, compounds 9f and 9g showed 100% suppression of parasitaemia on day 4. Further, compound 9g, provided 20% protection to the infected mice, while compounds 9f, provided 40% protection to the infected mice at 96 mg kg⁻¹ × 4 days dose at the end of 28 day observation period. Clinically useful drugs, β-arteether and chloroquine, showed 100% suppression of parasitaemia on day 4 and provided 100% and 80% protection, respectively, to the infected mice in this model. Thus, dioxa-aza-fluorenones derived compounds 9f and 9g showed moderate in vivo antimalarial activities against multidrug resistant Plasmodium yoelii nigeriensis in Swiss mice by oral route.

Finally, it is worth to compare the antimalarial activity of these aza-peroxides with 1,2,4-trioxanes prepared by Singh et al. While these aza peroxides 5a–h and 9a–h showed significant in vitro antimalarial activity; all the 1,2,4-trioxanes of prototype 10 prepared by Singh et al. were found to be in vivo active. This indicates that any change in the 1,2,4-trioxanes ring system, the basic pharmacophore responsible for antimalarial activity, would leads to decrease in the antimalarial activity of the compound which, thereby, underlines the importance of oxygen atom in 1,2,4-trioxane ring.

Conclusion

In conclusion, we report the synthesis of novel bicyclic aza-peroxides 5a–h and tricyclic aza-peroxides 9a–h, the two series of novel nitrogen analogues of 1,2,4-trioxanes in 52–71% yields. All these compounds were assessed for their in vitro antimalarial activity against Plasmodium falciparum 3D7 strain. Dioxa-aza-fluorenones derived compounds 9a, 9c and 9f showed IC₅₀ values of 9.43, 8.83 and 5.63 ng mL⁻¹, respectively which was found to be comparable to that of antimalarial drug chloroquine (IC₅₀ value 5.2 ng mL⁻¹). In vivo antimalarial activity of compound 9f, the most active compound of the series, provided 40% protection to the infected mice at 96 mg kg⁻¹ × 4 days dose at the end of 28 day observation period. To the best of our knowledge, this is a first attempt for the synthesis of dioxa-aza-indenones (bicyclic) 5a–h and dioxa-aza-fluorenones (tricyclic) 9a–h based aza-peroxides which replaces one oxygen atom of 1,2,4-trioxane moiety by incorporating nitrogen atom within active pharmacophore.

Experimental

General methods

All glass apparatus were oven dried prior to use. Melting points were taken in open capillaries on complab melting point apparatus and are presented uncorrected. Infrared spectra were recorded on a Perkin-Elmer FT-IR RXI spectrophotometer. ¹H NMR and ¹³C NMR spectra were recorded using CDCl₃ as solvent either using Bruker Supercon Magnet DPX-200 or DRX-300 spectrometers (operating at 200 and 300 MHz respectively for ¹H; 50 and 75 MHz respectively for ¹³C) or using JEOL ECS-400 spectrometer (operating at 400 MHz for ¹H and 100 MHz for ¹³C). Tetramethylsilane (δ 0.00 ppm) served as an internal standard in ¹H NMR and CDCl₃ (δ 77.0 ppm) in ¹³C NMR. Chemical shifts are reported in parts per million. Splitting patterns are described as singlet (s), doublet (d), triplet (t) and multiplet (m). Fast Atom Bombardment Mass Spectra (FAB-MS) were obtained on JEOL SX-102/DA-6000 mass spectrometer using argon/xenon (6 kV, 10 mA) as the FAB gas. Glycerol or m-nitrobenzyl alcohol was used as matrix. Electrospray mass spectrometry (ES-MS) were recorded on a MICROMASS QUATTRO II triple quadruple mass spectrometer. High Resolution Electron Impact Mass Spectra (HR-EIMS) were obtained on JEOL MS route 600H instrument as well as Xevo G2-S Q-Tof (Waters, USA). Elemental analyses were performed on Vario EL-III C H N S analyzer (Germany) and values were within ±0.4% of the calculated values, and therefore these compounds meet the criteria of ≥95% purity. Column chromatography was performed over Merck silica gel (particle size: 60–120 mesh and 100–200 mesh) procured from Qualigens™ (India), flash silica gel (particle size: 230–400 mesh). All chemicals and reagents were obtained from Sigma Aldrich (USA), Merck (India) or Spectrochem (India) and were used without further purification. All animal experiments were performed in compliance with the relevant laws and guidelines of CSIR-CDRI, Lucknow Institute, and approved by the institutional animal ethical committee(s).

General procedure for preparation of N-substituted imides 2a–h and 6a–h: preparation of 2a as a representative. To a stirred mixture of allylic alcohol 1a (2 g, 13.5 mmol), succinimide (2.67 g, 26.99 mmol) and triphenylphosphine (8.85 g, 33.74 mmol) in dry THF (60 mL) at 0 °C and under inert atmosphere, was added diethylazodicarboxylate (DEAD) (4.3 mL, 26.99 mmol) dissolved in dry THF (5 mL). The contents were stirred at 0 °C for 30 min. THF was evaporated under reduced pressure. The crude product was purified by column chromatography over silica gel (100–200 mesh) using 1.5% ethyl acetate : DCM as eluant to furnish 2.2 g (71% yield) of 2a.
Compound 2a: (1-(3-phenyl-but-2-enyl)-pyrrolidine-2,5-dione). Yield: 71%; solid, mp 129–131 °C;

FT-IR (KBr, cm⁻¹) 1724.7, 1525.3, 1369.5, 1209.5, 766.1; ¹H NMR (400 MHz, CDCl₃) δ 2.12 (s, 3H), 2.64 (s, 4H), 4.23 (d, 2H, J = 6.8 Hz), 5.63–5.67 (m, 1H), 7.17–7.30 (m, 5H, Ar);

¹³C NMR (100 MHz, CDCl₃) δ 16.23 (CH₃), 28.35 (2 × CH₂), 37.27 (CH₂), 120.43 (CH), 125.93 (2 × CH), 127.48 (CH), 128.31 (2 × CH), 139.84 (C), 142.70 (C), 176.96 (2 × C);

ESI-MS (m/z) 230 [M + H]⁺.

Anal. calcd for C₁₄H₁₅NO₂: C, 73.34%, H, 6.59%, N, 6.11%; found: C, 73.51%, H, 6.61%, N, 6.19%.

N-Substituted imides 2b–h were synthesized from allylic alcohols 1b–h, respectively by using the same procedure. Likewise, imides 6a–h were synthesized from allylic alcohols 1a–h by replacing succinimide with phthalimide.

General procedure for preparation of amides 3a–h and 7a–h: preparation of 3a as a representative. To the solution of N-substituted imide 2a (2 g, 8.73 mmol) in MeOH : THF (1 : 1, 40 mL) at 0 °C, was added sodiumborohydride (1.3 g, 34.93 mmol) in 5 fractions over a period of 2 h with continuous stirring. Reaction mixture was stirred at 0 °C for 1 h. MeOH : THF was evaporated under reduced pressure to half of the original volume. Water (40 mL) was added and extracted with CHCl₃ (2 × 100 mL). Organic layer was dried over anhyd sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography over silica gel (100–200 mesh) using 1% methanol : DCM as eluant to furnish 1.4 g (67% yield) of 3a.
Compound 3a: (5-hydroxy-1-(3-phenyl-but-2-enyl)-pyrrolidin-2-one). Yield: 67%; viscous oil; FT-IR (neat, cm⁻¹) 3776.4, 1687.5, 1660.4, 1209.7, 768.3;

¹H NMR (400 MHz, CDCl₃) δ 1.63–1.85 (m, 1H), 2.06 (s, 3H), 2.23–2.27 (m, 2H), 2.46–2.55 (m, 1H), 2.98 (s, 1OH), 3.91–3.95 (m, 1H), 4.24 (dd, 1H, J = 6 and 15.2 Hz), 5.16 (s, 1H), 5.64–5.67 (m, 1H), 7.17–7.30 (m, 5H, Ar).

¹³C NMR (100 MHz, CDCl₃) δ 16.10 (CH₃), 28.33 (CH₂), 28.95 (CH₂), 38.25 (CH₂), 83.06 (CH), 121.94 (CH), 125.83 (2 × CH), 127.47 (CH), 128.38 (2 × CH), 139.42 (C), 142.74 (C), 174.50 (C);

ESI-MS (m/z) 232 [M + H]⁺.

Anal. calcd for C₁₄H₁₇NO₂: C, 72.70%, H, 7.41%, N, 6.06%; found: C, 72.85%, H, 7.56%, N, 6.25%.

Amides 3b–h and 7a–h were prepared from N-substituted imides 2b–h and 6a–h, respectively by using the same procedure.

General procedure for preparation of aza-peroxides 5a–h and 9a–h: preparation of 5a as a representative. A solution of amide 3a (1 g, 4.33 mmol) and methylene blue (5 mg), in MeCN : THF : CHCl₃ (2 : 1 : 1, 60 mL) was irradiated with 500 W tungsten–halogen lamp at −10 to 0 °C, while a slow stream of O₂ was bubbled into the reaction mixture for 6 h. The obtained crude reaction mixture of hydroxy hydroperoxide 4a was concentrated under reduced pressure at r.t, dissolved in acetonitrile (100 mL) and pTSA (0.1 g) was added to it. The reaction mixture was stirred at 5 °C for 2 h. The reaction mixture was concentrated to half of its original volume under reduced pressure at rt, diluted with water (100 mL) and extracted with ethyl acetate (2 × 75 mL). Combined organic layer was dried over anhyd sodium sulphate and concentrated under reduced pressure at rt. The crude product was purified by column chromatography over silica gel (60–120 mesh) using 10% ethyl acetate : hexane as eluant furnish pure product 5a (0.69 g, 65% yield).
Compound 5a: (5-(1-phenyl-vinyl)-tetrahydro-6,7-dioxa-3a-aza-inden-3-one) viscous oil. FT-IR (neat, cm⁻¹) 1705.5, 1485.5, 1445.1, 1042.4, 773.9;

¹H NMR (300 MHz, CDCl₃) δ 1.74–1.84 (m, 1H), 2.24–2.49 (m, 3H), 3.13 (dd, 1H, J = 13.4 and 10.7 Hz), 4.23 (dd, 1H, J = 13.5 and 2.7 Hz), 5.00 (dd, 1H, J = 10.7 and 2.7 Hz), 5.34 and 5.51 (2 × s, 2H), 5.65 (dd, 1H, J = 7.3 and 2.8 Hz), 7.29–7.34 (m, 5H, Ar);

¹³C NMR (75 MHz, CDCl₃) δ 21.52 (CH₂), 28.68 (CH₂), 43.43 (CH₂), 80.24 (CH), 90.84 (CH), 117.10 (CH₂), 126.50 (2 × CH), 128.29 (CH), 128.62 (2 × CH), 138.25 (C), 143.25 (C), 173.75 (C);

ESI-MS (m/z) 246 [M + H]⁺; anal. calcd for C₁₄H₁₅NO₃: C, 68.56%, H, 6.16%, N, 5.71%; found: C, 68.32%, H, 6.53%, N, 6.01%.

Compounds 5b–h and 9a–h were prepared from amides 3b–h and 7a–h, respectively using the same procedure.

In vitro antimalarial assay. The in vitro antimalarial activity of the compounds was assessed against CQ-sensitive 3D7 strain of P. falciparum and compared with that of chloroquine. The 50% inhibitory concentration (IC₅₀) were obtained following techniques of Smilkstein et al.¹⁹ In brief the parasites were maintained in vitro in RPNI medium²⁰ supplemented with gentamycin at 40 μg mL⁻¹; (Sigma), Fungizone at 0.25 μg mL⁻¹; (GIBCO) and 10% fetal bovine serum (pH 7.2), at 37 °C in a CO₂ incubator.

The compounds were dissolved in DMSO at 5 mg mL⁻¹ and required dilutions were made in a template plate with RPMI medium. 20 μL from each dilution was transferred, in duplicate, in the test plate and two wells receiving 20 μL of vehicle were kept as untreated control. For evaluation of IC₅₀ of the compounds, SYBR Green I-based fluorescence (MSF) assay was used. For the assays, fresh dilutions of all compounds in screening medium were prepared and 50 μL of highest starting concentration (10–500 ng mL⁻¹) was dispensed in duplicate wells in row ‘B’ of 96-well tissue culture plate. The highest starting concentration for chloroquine was 25 ng mL⁻¹. Subsequently two fold serial dilutions were prepared up to row ‘H’ (seven concentrations) and finally 50 μL of 2.5% parasitized cell suspension containing 0.5% parasitaemia was added to each well except 4 wells in row ‘A’ received non-infected cell suspension. These wells containing non infected erythrocytes in the absence of compound served as negative control, while parasitized erythrocytes in the presence of CQ served as positive control. After incubating the plates for 72 h, 100 μL of lysis buffer [20 mM Tris (pH 7.5), 5 mM EDTA, 0.008% (wt/vol) saponin, and 0.08% (vol/vol) Triton X-100] containing 1 × concentration of SYBR Green-I was added to each well and incubated for 1 h at room temperature. The plates were examined for the relative fluorescence units (RFUs) per well using the fluorescence plate reader (FLUOstar, BMG Labtechnologies). The IC₅₀ was determined using Logit regression analysis of dose–response curves.

In vivo antimalarial efficacy test. The blood schizontocidal activity of the test compounds was evaluated in rodent model using multidrug-resistant strain of Plasmodium yoelii nigeriensis. The colony bred Swiss mice of either sex (20 ± 2 g) were inoculated intraperitoneally with 1 × 10⁵ P. yoelii (MDR) parasites on day zero, and treatment was administered to group of five mice at each dose, from day 0 to 3, once daily. The drug dilutions of all compounds were prepared in groundnut oil so as to contain the required amount of the drug (1.2 mg kg⁻¹ for a dose of 96 mg kg⁻¹) in 0.1 mL and administered orally for each dose. Parasitaemia level were recorded from thin blood smears on day 4 and subsequently twice a week till day 28. The animals which did not develop patent infection till day 28 were recorded as cured.²¹ Mice treated with β-arteether served as positive control.

Acknowledgements

L. Y. and B. R. K. S. thanks DST, New Delhi; and UGC, New Delhi respectively for providing fellowships. M. K. T. thanks MNIT Jaipur for providing Institute fellowships. The authors thank SAIF, CSIR-CDRI, Lucknow and Materials Research Centre, MNIT Jaipur for providing analytical facilities. S. C. acknowledges MNIT Jaipur for providing Institute Seed Grant and DST-ARRS Indo-Slovenian Research Project (INT/Slovenia/P-14/2014) for funding.

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18. (a) 100% suppression of parasitaemia means no parasites were detected in 50 oil immersion microscopic fields (parasites if at all present, were below the detection limit). The parasites present below the detection limit can multiply and eventually can be detected during observation on subsequent days. In such cases though the drug is providing near 100% suppression of the parasitaemia on day 4 but will not provide full protection to the treated mice in the 28 day survival assay; (b) 100% protection means none of the treated mice developed patent infection during the 28 days observation period and hence were recorded as cured. Similarly 60% protection means only 3 out of 5 mice were cured.
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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra16781g

‡ Both authors contributed equally.

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