Novel pyrazole–pyrazoline hybrids endowed with thioamide as antimalarial agents: their synthesis and 3D-QSAR studies

Abstract

One of the most viable options to tackle the growing resistance to the antimalarial drugs is hybrid molecules. It involves combination of different scaffolds in one frame that may lead to compounds with diverse biological profiles. In this context, new hybrids of three different scaffolds viz pyrazole, pyrazoline and thiosemicarbazone moiety were incorporated into one single compound and evaluated for their in vitro schizontocidal activity against the CQ-sensitive 3D7 strain of Plasmodium falciparum. Compounds with significant in vitro antimalarial activity were further evaluated for cytotoxicity against VERO cell lines. The best active compound 48 exhibited an IC₅₀ of 1.13 µM. The in vitro results were further validated by quantitative structure–activity relationship (QSAR).

• Antimalarial
• cytoxicity
• hybrid molecule
• P. falciparum (3D7 strain)
• pyrazoline

Introduction

Parasite borne diseases like malaria, sleeping sickness, amoebiasis, dysentery, etc. are rife, predominantly in the developing countries and are responsible for immense distress1. Malaria, caused by P. falciparum, continues to be a grim health concern in Africa, South America and many parts of Asia2. Curbing this enervating disease has been sternly compromised by the emergence and spread of resistance to a majority of existing antimalarial agents3. In view of this, there is exigency for the breakthrough of new, affordable and safe antimalarial agents4.

Pyrazole ring is a biologically active motif, which is of much importance to medicinal chemists owing to its pharmacological diversity. It is also an integral part of a number of well-established commercially available drugs like, zoniporide, celecoxib, pyrazomycin, lonazolac, fezolamine, difenamizole, mepirizole, etc. (Figure 1).

Figure 1. Commercially available drugs bearing pyrazole nucleus.

Taking this as cue, some researchers have also highlighted the antimalarial propensity of this ring. Cunico et al.5 synthesized and evaluated the antimalarial activity of chloroquine–pyrazole analogues (I) and highlighted the critical role of aromatic functionality of pyrazole for activity. Qirante et al.6 evaluated antimalarial activity of ligands and complexes derived from pyrazole (II). Bekhit et al.7 also developed some pyrazole derivatives (III) with antimalarial activity (Figure 2).

Figure 2. Title compounds bearing the structural motifs.

Pyrazolines, the reduced form of pyrazoles, also have several applications in the field of pharmaceutical research. They have a wide range of biological activities such as, antimicrobial8, anticancer9, CNS depressant10, etc. Lately, several researchers have highlighted the antimalarial penchant of pyrazolines. Wanare et al.11 reported pyrazoline derivatives (IV) as growth inhibitors for P. falciparum. Similarly, Acharya et al.12 and Insuasty et al.13 also developed pyrazoline analogues (V–VI) and assessed their antimalarial activity (Figure 2).

Recently, there has been a great interest in thiosemicarbazones as potential antimalarial agents14–16. The pioneering work on acetylpyridine thiosemicarbazones17 (VII) as antimalarials paved way for Pingaew et al.18 to develop benzoyl thiosemicarbazone analogues of isoquinoline (VIII) as antimalarials. Khanye et al.19 also developed gold compounds containing thiosemicarbazone ligands (IX) as antimalarials (Figure 2).

Currently World Health Organisation recommends the use of two or more agents as the best way to minimize treatment failures and the spread of drug resistance20. The presence of more than one structurally active moiety in a single molecule may show an enhanced activity21. In the design of new drugs, the development of hybrid molecules through the combination of different scaffolds in one frame may lead to compounds with interesting biological profiles. Therefore, a molecule containing more than one biologically active scaffold, each with different mode of action could be favourable for the treatment of malaria. Therefore, three different scaffolds were selected viz pyrazole, pyrazoline and thiosemicarbazone moiety and incorporated into one single compound. From our ongoing efforts on finding novel hybrid antimalarials22 we, herein report the design, synthesis and antimalarial activity of novel hybrid molecules based on pyrazole moiety linked to pyrazoline moiety having thiosemicarbazone as a part of pyrazoline moiety (Figure 2). The synthetic steps involved the formation of pyrazole carbaldehydes through Vilsmeier-Haack reaction23, followed by Claisen-Schmidt condensation of these aldehydes with acetophenones. Resulting chalcones on treatment with thiosemicarbazide gave the title compounds24^,25.

The synthesized compounds were evaluated for in vitro schizontocidal activity against the CQ-sensitive 3D7 strain of P. falciparum using modification of Trager and Jenson method26 as described by Dua et al.27. The compounds having IC₅₀ less than 5 µg/mL were further evaluated for their cytotoxicity by Mosmann method28 with certain modifications. Finally, to provide an insight into the relationship existing between the chemical structure and pharmacological activity, QSAR of the synthesised compounds was performed using the PHASE module of Schrödinger29. The generated model gave statistically significant results, with r² of 0.9992, and the structural features spread across the entire molecule. This further validated our in vitro results.

Methods

Chemistry

All chemicals and solvents were purchased from SD-fine chemical and used without further purification. Melting points were measured by open-end capillary method and are uncorrected. TLC was performed with silica gel 60 F₂₅₄ (Merck) using methanol: chloroform (1:9) as the solvent system.

Elemental analyses were performed using CHNS Elementar (Vario EL-III). The IR spectra were recorded using a Bruker alpha-T spectrophotometer. ¹H-NMR spectra were recorded on a Bruker Avance-400 MHz in DMSO-d₆ with tetramethylsilane (TMS) as the internal standard. The ¹³C-NMR of compounds was recorded on Bruker Avance-100 MHz instrument in DMSO-d₆. Chemical shifts (δ) are reported in parts per million, and coupling constants (J) in hertz. High-resolution mass spectral data were obtained on an Agilent 6540 UHD Accurate Mass (Q-ToF) Mass Spectrometer (LTQ XL/LTQ Orbitrap Discovery) coupled to Agilent 1290 series LC system (Agilent PDA detector, autosampler, and thermostat, thermostated column compartment, binary pump).

General procedure for the synthesis of 1-Phenyl-2-(1-phenylethylidene)hydrazines 1–4

GAA (0.024 mole) and phenylhydrazine (0.05 mole) were added to a solution of substituted acetophenone (0.05 mole) in 30 mL methanol. The mixture was refluxed at 80 °C until the completion of reaction. The reaction was monitored through TLC. After completion, the reaction vessel was cooled to 0 °C. The precipitate was filtered, washed with cold methanol and dried30.

General procedure for the synthesis of 1,3-diphenyl-1H-pyrazole-4-carbaldehyde 5–8

POCl₃ (0.09 mole) was added drop wise through dropping funnel to previously cooled DMF (0.09 mole) at 0–5 °C and stirred for few minutes. A solution of compounds 1–4 (0.03 mole) in DMF was added drop wise to the reaction mixture. This was then kept at room temperature for few minutes and refluxed at 70–80 °C until the completion of reaction. The reaction was monitored through TLC30.

Following completion, the reaction mixture was cooled to room temperature, quenched with crushed ice and basified with sodium hydroxide to obtain a precipitate. The precipitate was filtered, washed thoroughly with water, dried and recrystallized from ethanol.

General procedure for the synthesis of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-one 9–28

Chalcones were synthesised by Claisen Schmidt reaction31. Aldehyde (0.05 mole) and substituted acetophenone (0.05 mole) were dissolved in 35 mL methanol. To this reaction mixture was added sodium hydroxide (0.075 mole), then the mixture was refluxed with stirring for 4–5 h. The reaction was monitored through TLC. After completion, the reaction mixture was poured in ice-cold water, acidified with 4 N HCl and the solid was filtered, dried, and recrystallized from ethanol.

General procedure for the synthesis of 5-(1,3-diphenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-1-carbothioamide 29–48

Equimolar quantities (0.01 mole) of chalcone and thiosemicarbazide were refluxed in presence of sodium hydroxide (0.025 mole) in 25 mL ethanol for 3 h. White solid separation occurs in the reaction vessel itself. After the completion of reaction, the precipitated solid was filtered, washed with water, and dried24^,25.

5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-1-carbothioamide (29)

Yield: 64%; R_f: 0.32; m.p.: 245–246 °C; FT-IR (cm⁻¹): 3413, 3269 (NH₂), 1587 (C = N), 1363 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.22 (dd, 1H, J = 3.6 & 17.6 Hz), 3.91 (dd, 1H, J = 11.2 & 17.8 Hz), 6.03 (dd, 1H, J = 3.6 & 11.4 Hz), 7.24 (t, 1H, H-4′, J = 7.2 Hz), 7.38–7.49 (m, 8H, H-3,4,5,3′,5′,3″,4″,5″), 7.78 (d, 2H, J = 7.6 Hz, H-2″,6″), 7.83–7.86 (m, 5H, H-2,6,2′,6′ & one proton of NH₂), 8.07 (bs, 1H, NH₂, D₂O exchangeable), 8.13 (s, 1H, pyrazole ring); ¹³C-NMR (δ ppm, DMSO-d₆, 100 MHz): 42.81, 56.16, 118.57, 124.76, 126.29, 126.64, 127.66, 128.37, 128.44, 129.05, 129.95, 130.93, 131.56, 133.53, 139.80, 149.68, 155.24, 176.52; Mass (m/z): 424 (M⁺+1). Anal Calcd. for C₂₅H₂₁N₅S: C, 70.90; H, 5.00; N, 16.54; Found: C, 70.92; H, 4.98; N, 16.55.

3-(4-Chlorophenyl)-5-(1,3-diphenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (30)

Yield: 60%; R_f: 0.24; m.p.: 252–253 °C; FT-IR (cm⁻¹): 3422, 3254 (NH₂), 1582 (C = N), 1364 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.21 (dd, 1H, J = 3.6 & 17.8 Hz), 3.88 (dd, 1H, J = 11.6 & 18.0 Hz), 6.02 (dd, 1H, J = 3.6 & 11.2 Hz), 7.23 (t, 1H, H-4′, J = 7.6 Hz), 7.36–7.46 (m, 5H, H-3,4,5,3′,5′), 7.48 (d, 2H, J = 8.4 Hz), H-3″,5″), 7.75 (d, 2H, J = 7.2 Hz, H-2,6), 7.82 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.86 (d, 3H, J = 8.4 Hz, H-2″,6″ & one proton of NH₂ merged), 8.09 (bs, 1H, NH₂, D₂O exchangeable), 8.14 (s, 1H, pyrazole ring); Mass (m/z): 458 (M⁺+1), 459 (M⁺+2). Anal Calcd. for C₂₅H₂₀ClN₅S: C, 65.56; H, 4.40; N, 15.29; Found: C, 70.88; H, 4.41; N, 15.30.

5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyrazole-1-carbothioamide (31)

Yield: 56%; R_f: 0.46; m.p.; 259–260 °C; FT-IR (cm⁻¹): 3422, 3252 (NH₂), 1580 (C = N), 1365 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.32 (s, 3H, CH₃), 3.19 (dd, 1H, J = 3.6 & 17.8 Hz), 3.88 (dd, 1H, J = 11.2 & 17.6 Hz), 6.02 (dd, 1H, J = 3.6 & 11.2 Hz), 7.23 (d, 2H, J = 8.0 Hz, H-3″,5″), 7.25 (t, 1H, J = 7.6 Hz, H-4′), 7.38–7.49 (m 5H, H-3,4,5,3′,5′), 7.73 (d, 2H, J = 8.4 Hz, H-2,6), 7.78 (d, 3H, J = 7.2 Hz, H-2″,6″ & one proton of NH₂ merged), 7.82 (d, 2H, J = 8.0 Hz, H-2′,6′), 8.02 (bs, 1H, NH₂, D₂O exchangeable), 8.10 (s, 1H, pyrazole ring); Mass (m/z): 438 (M⁺+1). Anal Calcd. for C₂₆H₂₃N₅S: C, 71.37; H, 5.30; N, 16.01; Found: C, 71.39; H, 5.29; N, 16.00.

5-(1,3-Diphenyl-1H-pyrazol-4-yl)-3-(4-methoxyphenyl)-4,5-dihydropyrazole-1-carbothioamide (32)

Yield: 48%; R_f: 0.35; m.p.: 242–243 °C; FT-IR (cm⁻¹): 3422, 3251 (NH₂), 1586 (C = N), 1359 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.19 (dd, 1H, J = 3.2 & 17.8 Hz), 3.78 (s, 3H, OCH₃), 3.86 (dd, 1H, J = 11.6 & 17.6 Hz), 6.01 (dd, 1H, J = 3.2 & 11.2 Hz), 6.96 (d, 2H, J = 8.8 Hz, H-3″,5″), 7.24 (t, 1H, J = 7.2 Hz, H-4′), 7.38–7.49 (m, 5H, H-3,4,5,3′,5′), 7.78 (d, 4H, J = 8.4 Hz, H-2,6,2″,6″), 7.83 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.76 (bs, 1H, NH₂, D₂O exchangeable), 8.00 (bs, 1H, NH₂, D₂O exchangeable), 8.10 (s, 1H, pyrazole ring); Mass (m/z): 454 (M⁺+1). Anal Calcd. for C₂₆H₂₃N₅OS: C, 68.85; H, 5.11; N, 15.44; Found: C, 68.83; H, 5.09; N, 15.42.

3-(4-Aminophenyl)-5-(1,3-diphenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (33)

Yield: 54%; R_f: 0.52; m.p.: 249–250 °C; FT-IR (cm⁻¹): 3457, 3276 (NH₂), 3391, 3329 (NH₂), 1588 (C = N), 1361 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.08 (dd, 1H, J = 3.2 & 17.2 Hz), 3.78 (dd, 1H, J = 11.2 & 17.0 Hz), 5.68 (bs, 2H, NH₂, D₂O exchangeable), 5.98 (dd, 1H, J = 3.2 & 11.0 Hz), 6.52 (d, 2H, J = 7.6 Hz, H-3″,5″), 7.23 (t, 1H, J = 7.6 Hz, H-4′), 7.37–7.54 (m, 8H, H-3,4,5,3′,5′,2″,6″, one proton of NH₂), 7.78 (d, 2H, J = 8.0 Hz, H-2,6), 7.81 (d, 3H, J = 8.8 Hz, H-2′,6′& one proton of NH₂ merged), 8.02 (s, 1H, pyrazole ring); Mass (m/z): 439 (M⁺+1). Anal Calcd. for C₂₅H₂₂N₆S: C, 68.47; H, 5.06; N, 19.16; Found: C, 68.45; H, 5.04; N, 19.14.

5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-1-carbothioamide (34)

Yield: 49%; R_f: 0.28; m.p.: 271–272 °C; FT-IR (cm⁻¹): 3406, 3236 (NH₂), 1589 (C = N), 1376 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.23 (dd, 1H, J = 3.6 & 17.8 Hz), 3.90 (dd, 1H, J = 11.2 & 17.8 Hz), 6.01 (dd, 1H, J = 3.6 & 11.2 Hz),7.24 (t, 1H, J = 7.2 Hz, H-4′), 7.42–7.45 (m, 5H, H-3″,4″,5″,3′,5′), 7.51 (d, 2H, J = 8.4 Hz, H-3,5), 7.79 (d, 2H, J = 8.4 Hz, H-2′,6′), 7.82–7.85 9m, 5H, H-2,6,2″,6″ & one proton of NH₂), 8.09 (bs, 1H, NH₂, D₂O exchangeable), 8.17 (s, 1H, pyrazole ring); Mass (m/z): 458 (M⁺+1), 459 (M⁺+2). Anal Calcd. for C₂₅H₂₀ClN₅S: C, 65.56; H, 4.40; N, 15.29; Found: C, 65.55; H, 4.38; N, 15.30.

3-(4-Chlorophenyl)-5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (35)

Yield: 52%; R_f: 0.42; m.p.: 260–261 °C; FT-IR (cm⁻¹): 3426, 3264 (NH₂), 1577 (C = N), 1358 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.22 (dd, 1H, J = 3.6 & 18.0 Hz), 3.87 (dd, 1H, J = 11.6 & 17.6 Hz), 6.00 (dd, 1H, J = 3.6 & 11.4 Hz), 7.25 (t, 1H, J = 7.2 Hz, H-4′), 7.42 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.49–7.51 (d, 2H, J = 8.4 Hz, H-3″,5″), 7.50–7.52 (d, 2H, J = 8.0 Hz, H-3,5), 7.77 (d, 2H, J = 8.4 Hz, H-2′,6′), 7.83 (d, 2H, J = 8.0 Hz, H-2″,6″), 7.85 (d, 2H, J = 8.8 Hz, H-2,6), 7.91 (bs, 1H, NH₂, D₂O exchangeable), 8.12 (bs, 1H, NH₂, D₂O exchangeable), 8.19 (s, 1H, pyrazole ring); Mass (m/z): 492 (M⁺+1), 493 (M⁺+2). Anal Calcd. for C₂₅H₁₉Cl₂N₅S: C, 60.98; H, 3.89; N, 14.22; Found: C, 61.00; H, 3.87; N, 14.23.

5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyrazole-1-carbothioamide (36)

Yield: 54%; R_f: 0.56; m.p.: 272–273 °C; FT-IR (cm⁻¹): 3428, 3268 (NH₂), 1577 (C = N), 1361 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.32 (s, 3H, CH₃), 3.20 (dd, 1H, J = 3.6 & 17.8 Hz), 3.87 (dd, 1H, J = 11.6 & 17.6 Hz), 5.99 (dd, 1H, J = 3.6 & 11.4 Hz), 7.23–7.28 (m, 3H, H-4′,3″,5″), 7.41 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.51 (d, 2H, J = 8.4 Hz, H-3,5), 7.73 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.79 (d, 3H, J = 8.4 Hz, H-2″,6″ & one proton of NH₂ merged), 7.83 (d, 2H, J = 8.0 Hz, H-2,6), 8.05 (bs, 1H, NH₂, D₂O exchangeable), 8.15 (s, 1H, pyrazole ring); Mass (m/z): 472 (M⁺+1), 473 (M⁺+2). Anal Calcd. for C₂₆H₂₂ClN₅S: C, 66.16; H, 4.70; N, 14.84; Found: C, 66.15; H, 4.68; N, 14.85.

5-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-(4-methoxyphenyl)-4,5-dihydropyrazole-1-carbothioamide (37)

Yield: 53%; R_f: 0.38; m.p.: 262–263 °C; FT-IR (cm⁻¹): 3426, 3260 (NH₂), 1585 (C = N), 1363 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.19 (dd, 1H, J = 3.6 & 17.8 Hz), 3.78 (s, 3H, OCH₃), 3.86 (dd, 1H, J = 11.6 & 18.0 Hz), 5.98 (dd, 1H, J = 3.6 & 11.4 Hz), 6.97 (d, 2H, J = 8.8 Hz, H-3″,5″), 7.25 (t, 1H, J = 7.6 Hz, H-4′), 7.42 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.51 (d, 2H, J = 8.4 Hz, H-3,5), 7.75 (bs, 1H, NH₂), 7.78–7.80 (d, 2H, J = 8.8 Hz, H-2′,6′), 7.79–7.81 (d, 2H, J = 8.4 Hz, H-2″,6″), 7.83 (d, 2H, J = 8.0 Hz, H-2,6), 8.00 (bs, 1H, NH₂, D₂O exchangeable), 8.14 (s, 1H, pyrazole ring); Mass (m/z): 488 (M⁺+1), 489 (M⁺+2). Anal Calcd. for C₂₆H₂₂ClN₅OS: C, 63.99; H, 4.54; N, 14.35; Found: C, 64.00; H, 4.55; N, 14.33.

3-(4-Aminophenyl)-5-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (38)

Yield: 56%; R_f: 0.33; m.p.: 252–253 °C; FT-IR (cm⁻¹): 3455, 3273 (NH₂), 3391, 3323 (NH₂), 1590 (C = N), 1360 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.09 (dd, 1H, J = 2.4 & 17.6 Hz), 3.78 (dd, 1H, J = 10.8 & 17.4 Hz), 5.70 (bs, 2H, NH₂, D₂O exchangeable), 5.96 (dd, 1H, J = 2.8 & 10.8 Hz), 6.52 (d, 2H, J = 8.0 Hz, H-3″,5″), 7.25 (t, 1H, J = 7.2 Hz, H-4′), 7.42 (t, 2H, J = 7.6 Hz, H-3′,5′), 7.49–7.54 (m, 5H, H-3,5,2″,6″ and proton of NH₂), 7.80–7.84 (m, 4H, H-2,6,2′,6′), 7.87 (bs, 1H, NH₂), 8.07 (s, 1H, pyrazole ring); ¹³C-NMR (δ ppm, DMSO-d₆, 100 MHz): 42.22, 55.09, 113.12, 117.68, 118.11, 124.52, 125.82, 126.27, 128.61, 128.74, 129.48, 129.52, 131.86, 132.65, 139.16, 147.92, 151.35, 155.58, 174.82. Mass (m/z): 473 (M⁺+1), 474 (M⁺+1). Anal Calcd. for C₂₅H₂₁ClN₆S: C, 63.48; H, 4.48; N, 17.77; Found: C, 63.49; H, 4.46; N, 17.79.

3-Phenyl-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (39)

Yield: 60%; R_f: 0.55; m.p.: 242–243 °C; FT-IR (cm⁻¹): 3407, 3267 (NH₂), 1591 (C = N), 1368 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.34 (s, 3H, CH₃), 3.19 (dd, 1H, J = 3.6 & 17.8 Hz), 3.89 (dd, 1H, J = 11.2 & 17.8 Hz), 6.03 (dd, 1H, J = 3.6 & 11.2 Hz), 7.23 (t, 1H, J = 7.6 Hz, H-4′), 7.26 (d, 2H, J = 7.6 Hz, H-3,5), 7.41–7.45 (m, 5H, H-3′,5′,3″,4″,5″), 7.66 (d, 2H, J = 8.0 Hz, H-2″,6″), 7.81–7.85 (m,5H, H-2,6,2′,6′ & one proton of NH₂ merged), 8.08 (bs, 1H, NH₂, D₂O exchangeable), 8.10 (s, 1H, pyrazole ring); Mass (m/z): 438 (M⁺+1). Anal Calcd. for C₂₆H₂₃N₅S: C, 71.37; H, 5.30; N, 16.01; Found: C, 71.35; H, 5.29; N, 16.00.

3-(4-Chlorophenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (40)

Yield: 60%; R_f: 0.35; m.p.: 269–270 °C; FT-IR (cm⁻¹): 3418, 3260 (NH₂), 1572 (C = N), 1361 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.32 (s, 3H, CH₃), 3.21 (dd, 1H, J = 4.0 & 11.2 Hz), 3.88 (dd, 1H, J = 10.8 & 17.0 Hz), 6.01 (dd, 1H, J = 4.0 & 11.2 Hz), 7.22 (t, 1H, J = 7.6 Hz, H-4′), 7.24 (d, 2H, J = 8.4 Hz, H-3,5), 7.41 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.46 (d, 2H, J = 8.8 Hz, H-3″,5″), 7.80 (d, 2H, J = 8.8 Hz, H-2′,6′), 7.83–7.85 (m, 5H, H-2″,6″, 2,6 and one proton of NH₂ merged), 8.01 (bs, 1H, NH₂), 8.06 (s, 1H, pyrazole ring); Mass (m/z): 472 (M⁺+1), 473 (M⁺+2). Anal Calcd. for C₂₆H₂₂ClN₅S: C, 66.16; H, 4.70; N, 14.84; Found: C, 66.15; H, 4.68; N, 14.86.

5-(1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyrazole-1-carbothioamide (41)

Yield: 52%; R_f: 0.30; m.p.: 259–260 °C; FT-IR (cm⁻¹): 3428, 3266 (NH₂), 1578 (C = N), 1363 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.27 (s, 3H, CH₃), 2.32 (s, 3H, CH₃), 3.16 (dd, 1H, J = 4.0 & 11.2 Hz), 3.86 (dd, 1H, J = 10.8 & 17.0 Hz), 6.01 (dd, 1H, J = 4.0 & 11.2 Hz), 7.22 (d, 2H, J = 8.4 Hz, H-3″,5″), 7.26 (d, 2H, J = 7.6 Hz, H-3,5), 7.22–7.28 (1H, H-4′ merged), 7.41 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.65 (d, 2H, J = 7.6 Hz, H-2,6), 7.73 (d, 2H, J = 7.6 Hz, H-2″,6″), 7.79 (bs, 1H, NH₂), 7.81(d, 2H, J = 8.0 Hz, H-2′,6′), 8.04 (bs, 1H, NH₂),8.08 (s, 1H, pyrazole ring); Mass (m/z): 452 (M⁺+1). Anal Calcd. for C₂₇H₂₅N₅S: C, 71.81; H, 5.58; N, 15.51; Found: C, 71.80; H, 5.60; N, 15.50.

3-(4-Methoxyphenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (42)

Yield: 55%; R_f: 0.27; m.p.: 236–237 °C; FT-IR (cm⁻¹): 3423, 3249 (NH₂), 1586 (C = N), 1365 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.29 (s, 3H, CH₃), 3.75 (s, 3H, OCH₃), 3.21 (dd, 1H, J = 4.0 & 11.2 Hz), 3.86 (dd, 1H, J = 10.8 & 17.0 Hz), 6.03 (dd, 1H, J = 4.0 & 11.2 Hz), 6.94 (d, 2H, J = 8.8 Hz, 3″,5″), 7.21 (t, 1H, J = 7.6 Hz, H-4′), 7.26 (d, 2H, J = 7.6 Hz, H-3,5), 7.41 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.72 (d, 2H, J = 7.6 Hz, H-2,6), 7.75 (bs, 1H, NH₂), 7.77 (d, 2H, J = 7.6 Hz, H-2″,6″), 7.80 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.88 (bs, 1H, NH₂), 8.03 (s, 1H, pyrazole ring); Mass (m/z): 468 (M⁺+1). Anal Calcd. for C₂₇H₂₅N₅OS: C, 69.35; H, 5.39; N, 14.98; Found: C, 69.33; H, 5.41; N, 15.00.

3-(4-Aminophenyl)-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (43)

Yield: 48%; R_f: 0.59; m.p.: 241–242 °C; FT-IR (cm⁻¹): 3449, 3271 (NH₂), 3382, 3317, (NH₂), 1591 (C = N), 1359 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.35 (s, 3H, CH₃), 3.04 (dd, 1H, J = 3.2 & 17.6 Hz), 3.76 (dd, 1H, J = 10.8 & 17.4 Hz), 5.68 (bs, 2H, NH₂, D₂O exchangeable), 5.97 (dd, 1H, J = 3.6 & 11.2 Hz), 6.51 (d, 2H, J = 8.8 Hz, H-3″,5″), 7.22 (t, 1H, J = 7.6 Hz, H-4′), 7.26 (d, 2H, J = 8.4 Hz, H-3,5), 7.40 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.48 (d, 2H, J = 8.8 Hz, H-2″,6″), 7.54 (bs, 1H, NH₂), 7.66 (d, 2H, J = 8.0 Hz, H-2,6), 7.80 (d, 2H, J = 7.6 Hz, H-2′,6′), 7.84 (bs, 1H, NH₂), 7.98 (s, 1H, pyrazole ring); Mass (m/z): 453 (M⁺+1). Anal Calcd. for C₂₆H₂₄N₆S: C, 69.00; H, 5.35; N, 18.57; Found: C, 68.98; H, 5.33; N, 18.55.

5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-dihydropyrazole-1-carbothioamide (44)

Yield: 50%; R_f: 0.49; m.p.: 255–256 °C; FT-IR (cm⁻¹): 3423, 3268 (NH₂), 1592 (C = N), 1365 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.79 (s, 3H, OCH₃), 3.19 (dd, 1H, J = 4.0 & 11.2 Hz), 3.86 (dd, 1H, J = 10.8 & 17.0 Hz), 6.04 (dd, 1H, J = 4.0 & 11.2 Hz), 7.03 (d, 2H, J = 8.4 Hz, H-3,5), 7.23 (t, 1H, J = 7.2 Hz, H-4′), 7.40–7.45 (m, 5H, H-3′,5′,3″,4″,5″), 7.73–7.77 (m, 4H, H-2,6,2″,6″), 7.80–7.82 (m, 3H, H-2′,6′ and one proton of NH₂ merged), 7.98 (bs, 1H, NH₂), 8.09 (s, 1H, pyrazole ring); Mass (m/z): 454 (M⁺+1). Anal Calcd. for C₂₆H₂₃N₅OS: C, 68.85; H, 5.11; N, 15.44; Found: C, 68.83; H, 5.09; N, 15.46.

3-(4-Chlorophenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (45)

Yield: 54%; R_f: 0.38; m.p.: 232–233 °C; FT-IR (cm⁻¹): 3428, 3265 (NH₂), 1594 (C = N), 1373 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.18 (dd, 1H, J = 3.2 & 17.6 Hz), 3.77 (s, 3H, OCH₃), 3.85 (dd, 1H, J = 11.6 & 17.8 Hz), 6.00 (dd, 1H, J = 3.6 & 11.2 Hz), 6.99 (d, 2H, J = 8.0 Hz, H-3,5), 7.22 (m, 3H, H-3″,5″,4′), 7.40 (t, 2H, J = 7.2 Hz, H-3′,5′), 7.69 (d, 2H, J = 8.0 Hz, H-2,6), 7.72 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.78 (bs, 1H, NH₂), 7.80 (d, 2H, J = 8.0 Hz, H-2″,6″), 8.02 (bs, 1H, NH₂), 8.06 (s, 1H, pyrazole ring); Mass (m/z): 488 (M⁺+1), 489(M⁺+2). Anal Calcd. for C₂₆H₂₂ClN₅OS: C, 63.99; H, 4.54; N, 14.35; Found: C, 63.98; H, 4.55; N, 14.30.

5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-dihydropyrazole-1-carbothioamide (46)

Yield: 60%; R_f: 0.31; m.p.: 245–246 °C; FT-IR (cm⁻¹): 3433, 3273 (NH₂), 1581 (C = N), 1358 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 2.31 (s, 3H, CH₃), 3.15 (dd, 1H, J = 3.2 & 17.0 Hz), 3.78 (s, 3H, OCH₃), 3.84 (dd, 1H, J = 11.2 & 17.2 Hz), 6.00 (dd, 1H, J = 3.2 & 11.4 Hz), 7.00 (d, 2H, J = 8.4 Hz, H-3,5), 7.22 (3H, H-4′,3″,5″), 7.40 (t, 2H, J = 7.6 Hz, H-3′,5′), 7.69 (d, 2H, J = 8.8 Hz, H-2,6), 7.72 (d, 2H, J = 8.4 Hz, H-2″,6″), 7.80 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.79 (bs, 1H, NH₂), 8.02 (bs, 1H, NH₂),8.06 (s, 1H, pyrazole ring); Mass (m/z): 468 (M⁺+1). Anal Calcd. for C₂₇H₂₅N₅OS: C, 69.35; H, 5.39; N, 14.98; Found: C, 69.36; H, 5.40; N, 14.96.

3-(4-Methoxyphenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (47)

Yield: 61%; R_f: 0.43; m.p.: 234–235 °C; FT-IR (cm⁻¹): 3424, 3250 (NH₂), 1587 (C = N), 1365 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.15 (dd, 1H, J = 3.2 & 17.6 Hz), 3.78 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.83 (dd, 1H, J = 11.2 & 17.8 Hz), 5.99 (dd, 1H, J = 3.2 & 11.2 Hz), 6.96 (d, 2H, J = 8.4 Hz, H-3,5), 7.01 (d, 2H, J = 8.8 Hz, H-3″,5″), 7.22 (t, 1H, J = 7.2 Hz, H-4′), 7.40 (t, 2H, J = 8.0 Hz, H-3′,5′), 7.70 (d, 2H, J = 8.4 Hz, H-2′,6′), 7.75 (bs, 1H, NH₂), 7.77 (d, 2H, J = 8.8 Hz, H-2″,6″), 7.80 (d, 2H, J = 8.4 Hz, H-2,6), 7.99 (bs, 1H, NH₂), 8.06 (s, 1H, pyrazole ring); ¹³C-NMR (δ ppm, DMSO-d₆, 100 MHz): 42.78, 55.63, 55.86, 56.09, 114.49, 114.54, 118.46, 124.02, 124.40, 125.96, 126.07, 126.47, 129.38, 129.62, 129.93, 139.84, 149.51, 155.19, 159.57, 161.63, 176.14; Mass (m/z): 484 (M⁺+1). Anal Calcd. for C₂₇H₂₅N₅O₂S: C, 67.06; H, 5.21; N, 14.48; Found: C, 67.04; H, 5.20; N, 14.50.

3-(4-Aminophenyl)-5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydropyrazole-1-carbothioamide (48)

Yield: 52%; R_f: 0.40; m.p.: 255–256 °C; FT-IR (cm⁻¹): 3483, 3204 (NH₂), 3418, 3308 (NH₂), 1565 (C = N), 1347 (C = S); ¹H-NMR (δ ppm, DMSO-d₆, 400 MHz): 3.05 (dd, 1H, J = 3.2 & 17.6 Hz), 3.75 (dd, 1H, J = 11.2 & 17.4 Hz), 3.79 (s, 3H, OCH₃), 5.68 (bs, 2H, NH₂, D₂O exchangeable), 5.96 (dd, 1H, J = 3.2 & 10.8 Hz), 6.51 (d, 2H, J = 8.4 Hz, H-3″,5″), 7.01 (d, 2H, J = 8.8 Hz, H-3,5), 7.22 (t, 1H, J = 7.2 Hz, H-4′), 7.40 (t, 2H, J = H-3′,5′), 7.48 (d, 2H, J = 8.4 Hz, H-2″,6″), 7.54 (bs, 1H, NH₂), 7.70 (d, 2H, J = 8.8 Hz, H-2,6), 7.79 (d, 2H, J = 8.0 Hz, H-2′,6′), 7.81 (bs, 1H, NH₂), 7.97 (s, 1H, pyrazole ring); Mass (m/z): 469 (M⁺+1). Anal Calcd. for C₂₆H₂₄N₆OS: C, 66.64; H, 5.16; N, 17.94; Found: C, 66.66; H, 5.13; N, 17.95.

Pharmacology

In vitro antimalarial activity

In vitro drug sensitivity of the synthesized compounds was assessed using Trager and Jensen26 method as described by Dua et al.27. Chloroquine sensitive 3D7 P. falciparum strains were used for the study. Culture was maintained in A⁺ erythrocytes using RPMI 1640 medium supplemented with AB⁺ human serum (10%), sodium bicarbonate (0.2%), HEPES buffer (25 mM) and gentamycin (50 µg mL⁻¹). The culture was treated with different drug concentrations. After 72 h of incubation, blood smears were prepared and stained with JSB I and JSB II. Percentage maturation of schizonts against control was determined. Chloroquine was used as the standard reference. The inhibitory concentration value which kills 50% of the parasites (IC₅₀) was considered for anti-plasmodial activity. IC₅₀ values were determined using HN-NonLin V1.1.

Compounds with IC₅₀ less than 5 µM were further tested for cytotoxicity study to find out the selectivity index (SI) of the synthesised compounds.

Cytotoxicity assay

Cytotoxicity of the compounds was carried out with Vero cell line using Mosmann method28 with certain modifications. The cells were incubated with different dilutions of selected compounds for 72 h using MTT as reagent for the detection of cytotoxicity. Fifty percent cytotoxic concentration (CC₅₀) values represent the concentration of compound required to kill 50% of the fibroblast cells. The SI was calculated using the formula

QSAR

In order to get better insights into the structural requirements of the title compounds to act as antimalarials QSAR of the compounds was performed using PHASE module of Schrödinger29. The structures were drawn on maestro interface. The IC₅₀ of the compounds were converted to their molar values and subsequently calculated to free energy-related terms, i.e. pIC₅₀ (Table 1). Activity threshold was set to 4.75 to discriminate between active and inactive ligands. The standardized structures were then subjected to conformational search by OPLS 2005 force field using the confogen module of the Schrödinger software. After generating the conformers, various pharmacophoric features were defined.

Table 1. IC₅₀ and SI values of the synthesised compounds.

	Substitution		IC50^a
Compound No.	R1	R2	µg/mL	µM	SI^b	Activity pIC50	Predicted activity pIC50	Fitness
29^∧	H	H	26.036	61.47	ND	4.584	4.47	2.93
30^∧	H	Cl	30.101	65.73	ND	4.521	4.63	2.94
31^∧∧	H	4-CH3	15.209	31.17	ND	4.818	4.82	2.48
32^∧∧	H	4-OCH3	5.883	12.06	ND	5.230	5.26	2.91
33^∧∧	H	4-NH2	1.508	3.22	33	5.822	5.62	2.94
34^∧	4-Cl	H	32.768	66.54	ND	4.485	4.68	2.96
35*^∧	4-Cl	Cl	35.643	77.83	ND	4.448	4.85	2.97
36^∧∧	4-Cl	4-CH3	8.905	19.63	ND	5.050	5.07	2.51
37*^∧∧	4-Cl	4-OCH3	3.559	7.86	ND	5.449	5.47	2.49
38*^∧∧	4-Cl	4-NH2	0.977	2.09	22	6.010	5.86	2.96
39^∧	4-CH3	H	24.845	52.53	ND	4.605	4.73	2.96
40^∧∧	4-CH3	Cl	12.043	27.46	ND	4.919	4.90	2.97
41^∧∧	4-CH3	4-CH3	7.721	16.36	ND	5.112	5.02	2.97
42^∧∧	4-CH3	4-OCH3	2.772	6.14	ND	5.557	5.47	2.95
43^∧∧	4-CH3	4-NH2	0.715	1.53	16	6.146	6.18	2.96
44*^∧	4-OCH3	H	20.713	43.88	ND	4.684	5.00	2.99
45*^∧∧	4-OCH3	Cl	10.091	23.06	ND	4.996	5.18	3.00
46^∧∧	4-OCH3	4-CH3	4.642	10.61	ND	5.333	5.20	3.00
47^∧∧	4-OCH3	4-OCH3	1.904	4.20	40	5.720	5.70	2.98
48^∧∧	4-OCH3	4-NH2	0.546	1.13	12	6.263	6.43	2.98

^aCalculated using HN-NonLin V1.1 Software.

^bSI: IC₅₀ values of cytotoxic activity/IC₅₀ values of antimalarial activity.

*Compounds used as test.

^∧Ligands of the inactive set.

^∧∧Ligands of the active set.

Active analogue approach was used to identify common pharmacophore hypotheses (CPHs). Common pharmacophores were selected from the conformations of the set of active ligands using a tree-based partitioning technique, which groups together similar pharmacophores according to their intersite distances. After the generation of pharmacophores their scoring, clustering and ranking was done. The purpose of scoring was to identify the suitable hypothesis, which can explain the overall aspects of the structural requirements of the receptor site. Highest scoring hypothesis was subsequently used for the development of 3D QSAR models. For the present study, ADHRR.9 hypothesis, containing one H-acceptor, one H-donor, an hydrophobic core and two aromatic ring systems, was selected (Figure 3). This hypothesis was chosen as it had structural features spread across the entire molecule.

Figure 3. Active alignment of the data set.

The 3D-QSAR models were generated by combining the generated hypothesis with their schizontocidal activity data, to identify the overall aspects of molecular requirements which govern the biological activity. For generating the 3D-QSAR model, the dataset was divided into training set (70%) and test set (30%) by using random selection criteria. The training set molecules were further taken to generate 3D-QSAR models using an atom based QSAR method with a grid spacing of 1.0 Å. QSAR models were validated by predicting the activities of the test set. A three component (PLS factor) model was found to be the best statistical parameter to obtain an optimum model.

Results and discussion

Chemistry

Synthetic pathway which leads to the synthesis of title compounds (29–48), is outlined in Scheme 1. The synthesis involves four-step reaction. In the first step, various substituted acetophenones were reacted with phenylhydrazine to yield corresponding hydrazine derivatives (1–4). The pyrazole aldehydes (5–8), an intermediate required for chalcone (9–28) formation, were obtained by using Vilsmeier Haack formylation reaction23. The key chalcone intermediates (9–28) were then synthesized through Claisen-Schmidt condensation of acetophenone derivatives in alcoholic solution containing sodium hydroxide31. For preparation of pyrazoline, a variety of methods have been applied, among which refluxing of chalcone and thiosemicarbazide in methanol using alkaline condition (sodium hydroxide; 25 mmol) was found to be the best method with greater yields and shorter reaction time24^,25. The purity of the new synthesized compounds was checked by TLC. The structures of title compounds were determined on the basis of elemental analysis and various spectroscopic methods. Elemental analysis values were within the range of ± 0.4% of the theoretical value. In IR spectra, absorptions were observed at 1347–1376 cm⁻¹ (C = S), 1572–1594 cm⁻¹ (C = N) and 3204–3276 cm⁻¹ and 3406–3483 cm⁻¹ (NH₂). In ¹H-NMR spectra, the three protons H_A, H_M and H_X of pyrazoline ring appeared as doublets of doublets (Figure 4).

Figure 4. Title compounds showing AMX pattern and the different rings.

Scheme 1. Reagents and conditions: (i) Phenyl hydrazine, GAA, methanol, reflux; (ii) DMF, POCL₃; (iii) Aromatic ketone, NaOH, methanol, reflux; (iv) Thiosemicarbazide, ethanol, reflux.

The H_A proton (δ 3.26–3.30) appeared as doublet of doublet due to cisoid coupling to the H_X proton and geminally to H_M proton. The H_M proton, deshielded to some extent by aryl group (δ 3.80–3.86), was coupled transoid to H_X proton and geminally to H_A proton. The third, H_X proton (δ 5.63–5.68) was more deshielded due to effect of aryl group and electronegative nitrogen and was coupled transoid to the H_M proton and cisoid to the H_A proton. The two protons of CSNH₂ appeared at different chemical shift values (δ 7.76–7.82 and 8.04–8.06) due to anisotropic effect exerted by thiocarbomyl group.

Pharmacology

In vitro antimalarial activity of the synthesized compounds was assessed using Trager and Jensen26 method as modified by Dua et al.27 against the CQ-sensitive 3D7 strain of P. falciparum (Table 1).

Keeping all the aromatic features constant (Figure 4), the compounds bear H, Cl, CH₃ and OCH₃ on the one of the phenyl ring (B) attached to the pyrazole ring and H, Cl, CH₃, OCH₃ and NH₂ on the phenyl ring (C) attached to pyrazoline ring. The in vitro studies indicate that compounds with amino substitution on ring C gave the best inhibitory profiles, as shown by compounds 33, 38, 43 and 48 with IC₅₀ values of 3.22, 2.09, 1.53 and 1.13 µM, respectively. The most active compound, i.e. 48 bears amino at one end and methoxy group at the other. Compounds 37, 42 and 47 having methoxy substitution on ring C also showed good activity with IC₅₀ values of 7.86, 6.14 and 4.20 µM. The replacement of the methoxy by a methyl group and chloro atom decreases the antimalarial activity. The compounds with chloro substitution at both ring B and C elicit the least in vitro schizontocidal activity (Table 1).

The compounds 33, 38, 43, 47 and 48 with IC₅₀-value less than 5 µM were further tested for cytotoxicity study on VERO cells using Mosmann method28 to find out their selectivity. The compounds showed SI ranging from 12 to 40. The most active compound 48 with IC₅₀-value 1.13 µM also showed the highest SI amongst the tested compounds indicating that compounds belonging to this prototype would be less toxic (Table 1).

Following conclusions can be made on the basis of in vitro results:

• Substitution with electron releasing group showed good activity; however, electropositive group with hydrogen bonding ability were found to be most active.

• The replacement of electropositive group by electronegative group decreases the activity.

• The least activity is elicited by compound with electronegative substitution at both rings B and C.

• The compounds having in vitro antimalarial activity also showed less cytotoxicity.

QSAR

By taking into account the antimalarial activity of our synthesized compounds, a systemic pharmacophore modelling was performed and 3D-QSAR model was generated by PHASE module of Schrödinger, to support the assumption of flexible substituents responsible for better inhibition. Out of the different pharmacophore hypotheses, ADHRR.9 was selected. Active alignment of the data set is shown in Figure 3.

The statistical parameters of the 3D-QSAR analysis are summarized in Table 2.

Table 2. Statistical parameters of the best model.

Hypothesis	SD	r^2	RMSE	q^2	Pearson-r
ADHRR.9	0.1299	0.9612	0.2507	0.7982	0.9992

SD: standard deviation, r²: determination coefficient on the training set, RMSE: root mean squared error on the test set, q²: determination coefficient on the test set, r-Pearson: Pearson's correlation coefficient on the test set.

The model expresses 99% correlation (r² = 0.9992). Observed and predicted activities show a close agreement with the regression line as indicated by the fitness graph (Figure 5). This further validated the significance of the model. Validity of the model was also expressed by a determination coefficient on the test set (q²) of 0.79.The results of QSAR characteristics are given in Figure 6(a–c).

Figure 5. Fitness plot of the model.

Figure 6. Electron withdrawing, Hydrogen bond donor and hydrophobic characteristics of the hypothesis ADHRR.9.

The 3D-QSAR results covering the electron withdrawing, hydrogen bond donor (HBD) and hydrophobic characteristics were generated. The hypothesis ADHRR.9 includes one hydrogen bond acceptor, one HBD, one hydrophobic core and two aromatic ring features. The 3D-QSAR model is in agreement with the in vitro results. The blue cubes of the 3D pharmacophore regions refer to ligand regions in which the specific feature is vital for activity, while the red cubes indicate that the presence of that particular feature will reduce the activity.

Figure 6(a) represents the electron withdrawing characteristics of the data set. The QSAR shows that presence of electron withdrawing groups at position-4 of Ring C reduces the activity. Further, substantiating the in vitro results, which show that compounds 30, 35 and 40 having chloro substitution at this position have poor inhibitory profiles.

Figure 6(b) represents the HBD characteristics of the data set. HBD at position-4 of Ring C improves the activity, as is evident from compounds 33, 38, 43 and 48. In the series, these compounds have the best inhibitory profiles in the data set.

Figure 6(c) represents the hydrophobic characteristics of the data set. It demonstrates that the presence of hydrophobic substitutions at position-4 of Ring B increases the inhibitory profiles of the compounds. For instance, compounds with methoxy substitution at this position among all series are better inhibitors.

Conclusion

In conclusion, 20 compounds were synthesised, characterized and tested against 3D7 strain of P. falciparum. The active compounds had an IC₅₀ value in the range of 1.13–4.20 µM. The compounds with IC₅₀ less than 5 µM were further evaluated for their cytotoxicity assay. The most active compound, 48 shows an IC₅₀ of 1.13 µM (0.546 µg/mL) with reduced cytotoxicity. In order to support the systemic pharmacophore model, a predictive 3D-QSAR model was generated, which further substantiated the in vitro antimalarial results.

Supplementary material available online

Acknowledgements

We are thankful to Jamia Hamdard, for providing research laboratory and instrumentation facilities (NMR, mass and IR) at the institute.

Declaration of interest

The authors report no declarations of interest. Authors are thankful to University Grants Commission, Government of India, New Delhi, for financial support.