Natural-Product-Inspired Microwave-Assisted Synthesis of Novel Spirooxindoles as Antileishmanial Agents: Synthesis, Stereochemical Assignment, Bioevaluation, SAR, and Molecular Docking Studies

Abstract

Leishmaniasis is a neglected tropical disease, and there is an emerging need for the development of effective drugs to treat it. To identify novel compounds with antileishmanial properties, a novel series of functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one 23a–f, 24a–f, and 25a–g were prepared from natural-product-inspired pharmaceutically privileged bioactive sub-structures, i.e., isatins 20a–h, various substituted chalcones 21a–f, and 22a–c amino acids, via 1,3-dipolar cycloaddition reactions in MeOH at 80 °C using a microwave-assisted approach. Compared to traditional methods, microwave-assisted synthesis produces higher yields and better quality, and it takes less time. We report here the in vitro antileishmanial activity against Leishmania donovani and SAR studies. The analogues 24a, 24e, 24f, and 25d were found to be the most active compounds of the series and showed IC₅₀ values of 2.43 µM, 0.96 µM, 1.62 µM, and 3.55 µM, respectively, compared to the standard reference drug Amphotericin B (IC₅₀ = 0.060 µM). All compounds were assessed for Leishmania DNA topoisomerase type IB inhibition activity using the standard drug Camptothecin, and 24a, 24e, 24f, and 25d showed potential results. In order to further validate the experimental results and gain a deeper understanding of the binding manner of such compounds, molecular docking studies were also performed. The stereochemistry of the novel functionalized spirooxindole derivatives was confirmed by single-crystal X-ray crystallography studies.

1. Introduction

According to the World Health Organization (WHO), in 2021, leishmaniasis emerged as an endemic in 99 countries/territories (out of 200 countries/territories), mainly in 4 eco-epidemiological provinces worldwide (the Americas, East Africa, North Africa, and West and South East Asia) [1,2]. It is caused by Leishmania, a protozoan parasite from the Trypanosomatidae family, which is transmitted by vectors and causes cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL); these are characterized by skin ulcers affecting the mouth, nose, and throat and “kala-azar”, respectively [3]. Kala-azar (visceral leishmaniasis) is the fatal form of the disease and is triggered by Leishmania donovani, an intramacrophage protozoan parasite transmitted by the bite of infected female phlebotomine sandflies. This lethal disease affects millions of individuals living in tropical/subtropical regions worldwide [4]. Approximately, twenty-one protozoan parasite species of Leishmania are responsible for causing leishmaniasis, and this is linked to a variety of symptomatology ranging from minor skin lesions at the bite site to the deadly visceral forms. A few standard drugs are available for the treatment of this disease, such as pentavalent antimonials, amphotericin B, its liposomal encapsulation (lamb-liposomal amphotericin B), and miltefosine. Amphotericin B emerged as an alternative second-line treatment for visceral, mucocutaneous, and cutaneous leishmaniasis, especially in the case of human HIV coinfection after resistance was reported in antimonials. In Thailand, amphotericin B is the only effective drug available for the treatment of leishmaniasis [5]. According to published research, there is no current safe and effective treatment for leishmaniasis. The antileishmanial medicines used to treat leishmaniasis at present are accompanied by various kinds of side effects, toxicity, and drug resistance [6,7]. As per the WHO report, approximately 700,000 to 1,000,000 new cases are reported every year [8]. Therefore, there is an urgent need for the advent of effective medications against this neglected tropical disease (NTD).

The spirooxindole class of bio-heterocycles are identified as privileged molecules and construct the core structural unit in several naturally occurring alkaloids such as horsfiline 1 [9,10], coerulescine 2 [11,12], marcfortine B 3 [13], spirotryprostatin A 4 and B 5 [14,15], elacomine 6 [16], formosanine 7 [17], pteropodine 8 [18], alstonisine 9 [19], rychnophyilline 10 [20], strychnofoline 11 [21], spirobrassinin 12 [22], mitraphylline 13 [23], notoamide A 14 [24], etc. (Figure 1). Spirooxindoles are blended with a wide range of biological activities such as antimicrobial [25,26], antimigraine activity [27], antitumoral [28], anti-inflammatory [29], antihelmintic activity [30], antimycobacterial [31], acetyl-cholinesterase inhibitory activities [32,33], anticancer activities [34,35,36], anesthetic [37], HIV-1 N-NRT inhibitor [38], antileishmanial [39], etc.

Figure 1.

Structures of natural-product-inspired spirooxindole alkaloids 1–14.

It has been well-documented that several pharmacologically privileged molecules can be assembled into a single structurally complex molecule with more multi-faceted and enhanced biological activities that can target biological sites of interest in a specific manner to combat specific diseases [40,41]. Several biologically active alkaloidal classes of heterocycles have been reported in the literature and show promising antileishmanial activity in vitro, ex vivo, and in vivo [42,43,44,45]. Recent studies have revealed that several substituted spirooxindoles 15–19 [46,47,48,49] show promising antileishmanial activity against promastigotes and the amastigotes forms of Leishmania (L.) species either in vitro or in vivo when treated with pentamidine, amphotericin B, or miltefosine as one of the standard drugs. Therefore, in our endeavor to search for novel bio-heterocycles as antileishmanial agents, we designed Prototype A, i.e., functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one incorporating subunits of 15–19 (Figure 2), and we assessed in vitro antileishmanial activities against the promastigotes form of L. donovani, with the expectation that a new series of amino-acid-based spirooxindole derivatives would also show promising in vitro activity. So far, a literature review has revealed that there is no report available showing amino-acid-based spirooxindoles as antileishmanial agents.

Figure 2.

Design strategy for the target functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one having antileishmanial activity.

Microwave-assisted organic synthesis (MAOS) is a non-conventional, eco-friendly source of energy in chemical synthesis that can perform the reaction in a shorter time with less energy and furnish the product in a greater yield with higher purities as compared to traditional synthetic processes [50,51,52,53,54,55]. This fascinating method has a wide range of applications in drug discovery evaluation and the pharmaceutical segment for chemical synthesis. It has established an ongoing position in analytical and organic laboratory praxis [56]. Multi-component reactions (MCRs) via 1,3-dipolar cycloaddition reactions have been considered the best potential way for the synthesis of a library of spirooxindole derivatives [57,58].

Herein, we report the microwave-assisted synthesis as well as in vitro antileishmanial activity and structure–activity relationship studies of a novel series of functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one 23a–f, 24a–f, and 25a–g via 1,3-dipolar cycloaddition. This was achieved by the interaction of various isatins and amino acids with substituted chalcones in up to 98% yields in a highly regioselective and stereoselective manner. For the first time, all the compounds 23a–f, 24a–f, and 25a–g were prepared via microwave-assisted methodology. The stereochemistry of the novel functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-ones was confirmed by single-crystal X-ray crystallography studies of the bromo derivative, i.e., compound 23f. To the best of our knowledge, functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one 23a–f, 24a–f, and 25a–g were identified for the first time as promising antileishmanial agents. In this study, amphotericin B was used as the standard reference drug. We also report the validation of wet results via in silico molecular docking studies of active compounds 24a, 24e, 24f, and 25d.

2. Results

2.1. Synthesis

The 1,3-dipolar cycloaddition reaction of azomethine ylides is a versatile reaction and is well known for the assembly of numerous varieties of complex bioactive azaheterocyclic skeletons [59,60,61]. The azomethine ylide is also reported to serve as an important building block for the construction of several natural-product-inspired aza-heterocycles [62,63,64] and bioactive molecules [65].

We commenced our synthetic investigation by taking isatin 20a, chalcone 21a, and L-proline 22a as starting materials for carrying out microwave-assisted synthesis of spirooxindole-pyrrolidine 23a. Initially, the reaction was attempted under refluxing conditions. Therefore, the reaction was carried out by taking 20a (1 equiv.), 21a (1 equiv.), and 22a (1 equiv.) in MeOH under refluxing conditions for 120 min. We were delighted to get the desired spiro compound 23a in 86% yield (Table 1, entry 1). Then, we analysed the effect of the number of equivalents of the starting materials. Thus, the reaction was repeated in MeOH with 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) under refluxing conditions for 180 min, yielding 23a in 96% yield (Table 1, entry 2). It was noticed that changing the number of equivalents led to an improvement in the yield of the reaction. In order to reduce the time to complete the reaction, the reaction was subjected exactly to the same conditions as mentioned in entry no. 2 and allowed to run for 120, 60, and 30 min, which produced 23a in 89%, 67%, and 58% yields, respectively (Table 1, entries 3–5). Keeping the reaction exactly under the same conditions as mentioned in entry no. 2, the screening of different solvents (AcCN, ethylene glycol, H₂O, and ethanol) did not show an incremental effect on the yield of the reaction (Table 1, entries 6–9).

Table 1.

Optimization study: Microwave-assisted synthesis of novel functionalized spiro[indoline-3,2′–pyrrolidin]–2–one/spiro[indoline-3,3′–pyrrolizin]–2–one 23a from isatin 20a, chalcone 21a, and L-proline 22a as starting materials.

Entry	20a (Eq.)	21a (Eq.)	22a (Eq.)	Solvent	Condition	Time (Min.)	a Yield (%)
1.	1	1	1	MeOH	Reflux	120	86
2.	1	1.5	1.5	MeOH	Reflux	180	96
3.	1	1.5	1.5	MeOH	Reflux	120	89
4.	1	1.5	1.5	MeOH	Reflux	60	67
5.	1	1.5	1.5	MeOH	Reflux	30	58
6.	1	1.5	1.5	AcCN	Reflux	180	79
7.	1	1.5	1.5	Ethylene glycol	Reflux	180	76
8.	1	1.5	1.5	H2O	Reflux	180	9
9.	1	1.5	1.5	Ethanol	Reflux	180	77
10.	1	1.5	1.5	MeOH	MW, 80 °C	1	41
11.	1	1.5	1.5	MeOH	MW, 80 °C	3	71
12.	1	1.5	1.5	MeOH	MW, 80 °C	5	98
13.	1	1.5	1.5	AcCN	MW, 80 °C	5	73
14.	1	1.5	1.5	AcCN	MW, 100 °C	10	81
15.	1	1.5	1.5	AcCN	MW, 100 °C	15	83
16.	1	1.5	1.5	Ethylene glycol	MW, 80 °C	5	72
17.	1	1.5	1.5	Ethylene glycol	MW, 100 °C	10	77
18.	1	1.5	1.5	Ethylene glycol	MW, 100 °C	15	79
19.	1	1.5	1.5	Ethanol	MW, 80 °C	5	78
20.	1	1.5	1.5	Ethanol	MW, 100 °C	10	82
21.	1	1.5	1.5	Ethanol	MW, 100 °C	15	85

^a Isolated yield after recrystallization/column chromatography.

It is well known that microwave irradiation has been used as a fundamental tool for constructing aza-heterocycles with interesting properties, either in homogeneous or heterogeneous liquid reaction systems [66]. Utilizing the dual potential of both microwave irradiation as well as the 1,3,-dipolar cycloaddition reaction strategy; equimolar amounts of 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) dissolved in MeOH were treated under microwave irradiation conditions at 80 °C for 1 and 3 min, which produced 23a in 41% and 71% yield, respectively (Table 1, entries 10–11). Intriguingly, when the same reaction was subjected to 5 min under microwave conditions; 23a was obtained in 98% yield (Table 1, entry 12). The reactions were further screened with different solvents (AcCN, ethylene glycol, and ethanol) utilizing the same conditions as mentioned in entry no. 12 with varying times and temperatures (Table 1, entries 13–21). However, none of the reactions produced better yields than those obtained in entry no. 12. Therefore, equimolar amounts of 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) dissolved in MeOH under microwave irradiation conditions at 80 °C for 5 min was found to be the best optimized reaction condition (Table 1, entry 12).

Substituted isatins 20a–h, substituted chalcones 21a–f, and various amino acids 22a–c were subjected to microwave-assisted 1,3-dipolar cycloaddition reactions in MeOH at 80 °C for 5 min, which produced the desired chalcone-isatin-amino-acid-based spirooxindole compounds 23a–f, 24a–f, and 25a–g in excellent yields (up to 98%) in a highly diastereoselective manner (Scheme 1, Please see Supplementary Materials). In this reaction, [3 + 2] cycloaddition of substituted chalcones occurred with in situ generated azomethine ylides from microwave-assisted decarboxylative condensation of substituted isatins and various secondary amino acids.

Scheme 1.

Microwave-assisted synthesis of novel functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one 23a–f, 24a–f, and 25a–g via the 1,3-dipolar cycloaddition reaction.

The physico-chemical data, such as melting point and yield, under conventional conditions as well as in microwave-assisted conditions for all the compounds (23a–f, 24a–f, and 25a–g) are shown in Table 2.

Table 2.

Physicochemical data of spirooxindole pyrrolidines/pyrrolizines compounds 23a–f, 24a–f, and 25a–g.

Sr. No.	Isatin’s (20a–h)	Chalcones (21a–f)		Amino Acids (22a–c)	Product	Reflux (180 Min.)	Microwave Heating (5 Min.)	M.P. (°C)
	R1	R2	R3			Yields a (%)	Yields b (%)
1.	5-CH3	-F	-Cl	22a	23a	96	98	182–184
2.	5-F	-F	-OCH3	22a	23b	61	72	161–163
3.	5-F	-Br	-OCH3	22a	23c	59	74	126–128
4.	5-OCH3	-Br	-OCH3	22a	23d	82	93	107–109
5.	5-NO2	-Br	-OCH3	22a	23e	72	83	111–113
6.	5-Br	-H	-H	22a	23f	71	86	175–177
7.	5-OCH3	-NO2	-Cl	22b	24a	75	89	188–190
8.	5-NO2	-CH3	-Br	22b	24b	82	95	121–122
9.	5-CH3	-NO2	-Cl	22b	24c	79	88	135–137
10.	5-H	-F	-Cl	22b	24d	89	97	104–106
11.	5-Br	-NO2	-Cl	22b	24e	69	83	166–168
12.	7-I	-CH3	-Br	22b	24f	96	98	101–102
13.	5-Br	-F	-OCH3	22c	25a	81	91	154–156
14.	5-H	-Br	-OCH3	22c	25b	61	75	118–120
15.	5-Br	-F	-Cl	22c	25c	63	71	138–140
16.	5-F	-Cyclohexyl	-Br	22c	25d	57	73	112–114
17.	5-CH3	-F	-OCH3	22c	25e	74	87	103–105
18.	5-NO2	-F	-Cl	22c	25f	58	72	172–174
19.	5-OCH3	-F	-Cl	22c	25g	67	81	142–144

^a Isolated yields by column chromatography (conventional method). ^b Isolated yields by column chromatography (microwave-assisted synthesis).

The structures of all the synthesized compounds were well characterized by FT-IR, optical rotation, ¹H-NMR, ¹³C-NMR spectroscopy, and HRMS mass spectrometric analysis (Please see Supplementary Materials). Finally, the stereochemistry of the four chiral centres of the cycloaddition reaction was unequivocally determined by single-crystal X-ray diffraction analysis of the cycloadduct 23f (Figure 3, please see Supplementary Materials). After screening over the series of other derivatives, we found that the 23f prepared in one step and obtained as an off-white solid in 86% yield, which was further subjected to crystallization; we were able to isolate the 23f in ~10–11% yield using a slow evaporation crystallization technique with DCM as a solvent at low temperature. After couple of weeks, we came up with the single-crystal X-ray structure of the 23f, the raw data of 23f were subjected to the solution using Olex2 [67], and the crystal was crystallized in a trigonal system in R-3 space group. Consequently, the three-dimensional representation of compound 23f shows that the compound has four chiral carbons, with one carbon having an R-configuration and the other three having S-configurations. The crystal structure confirmed that the trans-geometry of chalcone and the regioselectivity were also well established as a result of the concerted reaction of chalcones with the ylides.

Figure 3.

(A) ORTEP diagram of the cycloadduct 23f. (B) Structure of (1′S,2′R,3S,7a’S)-2′-benzoyl -5-bromo-1′-phenyl-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indolin-3,3′-pyrrolizin]-2-one 23f.

2.2. Single-Crystal X-ray

Furthermore, it was observed that the crystallized framework had a hexagonal architecture consisting of six molecules in a circular fashion around the disordered functionality that takes non-planar circular conformations with the presence of short contacts in the alternate configuration. The molecular arrangement of 23f, its inside functionality in a large cavity, and its size are directly proportional to the distance between the carbon atoms at the opposite sides and the Van der Waals radius of the carbon atom present in the ring. In order to understand more about the intermolecular interactions of 23f, a Hirshfeld surface analysis using Crystal Explorer 3.1 software suite was used [68]. The 3D representation of short intermolecular contact can be provided by de and di mapped on the Hirshfeld surface, which corresponds to exterior and interior distances, respectively. The d_norm, shape index, and curvedness of 23f roughly indicating the presence of strong intermolecular short contacts and stronger Van der Waals interactions. The 2D finger plot of 23f reveals significant interactions corresponding to C-C, H-C, and H-H contributes 4.5%, 8.9%, and 39.0% of the total Hirshfeld surface, respectively, which is again attributable to the presence of strong intermolecular interactions; these are more prominent that of π–π interactions (Figure 4).

Figure 4.

The 2D Finger plot of C-C, H-C, and H-H of 23f.

As can be seen from Scheme 1 and Figure 4, all synthesized spirooxindoles 23a–f, 24a–f, and 25a–g were obtained in the 71–98% yield range. It was noticed that the reactions were occurring smoothly under microwave conditions with very good to excellent yields; however, the effects of the electron-donating group (EDG) and/or the electron-withdrawing group (EWG) had a marginal influence on the yield of the reaction. Among the spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one derivatives, i.e., 23a–f, 24a–f, and 25a–g, it was noticeable that the EDG (Me, OMe, cycloalkyl, Cl, Br, and I), either on isatin or on Chalcone, produced the desired compound in excellent yield for 23a, 23d, 24d, 24f, and 25a–b. However, in the case of EWG (NO₂, F), either on isatin or on chalcone, the target compounds were obtained in a good-to-excellent yield range (Figure 5).

Figure 5.

Structures of all synthesized spirooxindoles 23a–f, 24a–f, and 25a–g.

2.3. Biological Activity

Considering the importance of amphotericin B in the control of visceral leishmaniasis, the drug was selected as a control in the present study [69]. Camptothecin, a recognized inhibitor of LTopIB, effectively inhibits topoisomerase IB [70].Therefore, both drugs were used as control drugs for performing in vitro antileishmanial activity of all synthesized spirooxindoles 23a–f, 24a–f, and 25a–g.

2.3.1. In Vitro Antileishmanial Activity

The compounds (23a–f, 24a–f, and 25a–g) were initially screened for their in vitro antileishmanial activity against promastigotes of Leishmania donovani (MHOM/IN/1983/AG83) utilizing the Trypan blue dye exclusion method [71] and the plasmid relaxation assay using amphotericin B and camphothesin as standard reference drugs, respectively [72,73].

Trypan Blue Dye Exclusion Method

The promastigotes were harvested from the culture vials, counted, and 2 × 10⁶ cells/well were seeded in a 48-well culture plate. The antileishmanial screening of all the derivatives, 23a–f, 24a–f, and 25a–g, as well as the positive control, was performed at various concentrations (2 μg/mL, 4 μg/mL, 8 μg/mL, and 16 μg/mL) added in triplicate. The plate was incubated at 22 ± 1 °C in the BOD incubator for 72 h. After 72 h, each well was counted for the number of viable parasites using the Trypan blue dye exclusion method, and the percentage growth inhibition was calculated by using the formula:

$$\mathrm{Percentage} \mathrm{viability}=\frac{\mathrm{No}. \mathrm{of} \mathrm{viable} \mathrm{cells} \mathrm{in} \mathrm{treated} \mathrm{well}}{\mathrm{No}. \mathrm{of} \mathrm{viable} \mathrm{cells} \mathrm{in} \mathrm{blank} \mathrm{well}}\times 100$$

Percentage growth inhibition = 100 − percentage viability

The IC₅₀ (inhibitory concentration at which 50% of the parasites were dead) value was obtained by plotting a linear dose–response curve in SPSS software (Version 23) [71].

Plasmid Relaxation Assay

The relaxation of supercoiled plasmid DNA is the method used for the determination of LTopIB activity. Various doses of each compound were treated with one unit of pure LTopIB (the enzyme to relax 0.5 µg of supercoiled DNA for 30 min at 37 °C) for 20 min at 4 °C. The reaction mixture, including 0.5 μg of supercoiled pBluescript SK(−) plasmid, 10 mM Tris-HCl buffer pH 7.5, 5 mM MgCl₂, 0.1 mM EDTA, 15 µg/mL of bovine serum albumin, and 150 mM KCl, was then added in a final volume of 20 μL. After 30 min at 37 °C, the reaction mixtures were stopped by adding 4 μL of loading buffer, which included 5% sarkosyl, 0.12% bromophenol blue, and 25% glycerol. By electrophoresis, the topoisomers were separated on 1% agarose gels and electrophoresed at 2 V/cm for 16 h in a 0.1 M Tris-borate-EDTA buffer (pH 8.0) after being stained with ethidium bromide (0.5 µg/mL). Plotting the percentage of supercoiled DNA versus drug concentrations allowed researchers to determine the 50% inhibition concentration (IC₅₀) values of LTopIB inhibition as the 50% reduction of supercoiled DNA [72].

2.3.2. Inhibition of Leishmanial DNA Topoisomerase IB

Because of the presence of spirooxindole systems in the structure of these compounds, we aimed to assess their inhibitory potential on purified recombinant LTopIB measuring the relaxation of supercoiled plasmid DNA. In this regard, all spirooxindole derivatives were assessed for LTopIB inhibition through the prevention of DNA relaxation in a circular DNA plasmid. All compounds were tested at a single concentration of 100 µM to discard those that did not prevent DNA relaxation by LTopIB. After this initial test, potential inhibitor dose/response curves were performed to obtain their IC₅₀ values. The compounds 24a, 24e, 24f, and 25d were potent LTopIB inhibitors, and the IC₅₀ values of all nineteen compounds are shown in Table 3. The lowest IC₅₀ value corresponded to 24e (IC₅₀ = 15.7 µM).

Table 3.

In vitro antileishmanial activity of novel functionalized spiro[indoline-3,2′ -pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one based compounds 23a–f, 24a–f, and 25a–g.

Spirooxindole Derivatives, i.e., 23a–f, 24a–f, and 25a–g	IC50 (µM) a Using Trypan Blue Dye Exclusion Method	IC50 (µM) a Using Plasmid Relaxation Assay
23a	>10 µM	>100 µM
23b	>20 µM	>100 µM
23c	>20 µM	>100 µM
23d	7.78 µM	53.6 µM
23e	>20 µM	>100 µM
23f	>20 µM	>100 µM
24a	2.43 µM	17.3 µM
24b	5.36 µM	37.6 µM
24c	>20 µM	>100 µM
24d	>20 µM	>100 µM
24e	0.96 µM	15.7 µM
24f	1.62 µM	19.6 µM
25a	>10 µM	71.3 µM
25b	>10 µM	89.1 µM
25c	>10 µM	64.5 µM
25d	3.55 µM	27.2 µM
25e	>10 µM	>100 µM
25f	>20 µM	>100 µM
25g	>10 µM	78.4 µM
Amphotericin B	0.060 µM	-
Camptothecin	-	3 µM

^a IC₅₀: value indicates the effective concentration of a compound required to achieve 50% growth inhibition in µM.

All nineteen compounds exhibited moderate-to-good antileishmanial activity against Leishmania donovani. The results are shown in Table 3.

2.3.3. Structure–Activity Relationship (SAR) Studies

The inhibitory concentration (IC) values for all the spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one derivatives, i.e., 23a–f, 24a–f, and 25a–g, and the positive-control drugs, were also determined against promastigotes of Leishmania donovani utilizing the Trypan blue dye exclusion method [68]. Amphotericin B was taken as a positive control. As can be seen from Table 3, compound 24e, the most active compound of the series, showed potent in vitro antileishmanial activity, with the IC₅₀ value of 0.96 µM against Leishmania donovani. Compound 24f, the next most active compound in the series (IC₅₀ = 1.62 µM), exhibited potent antileishmanial activity in comparison to the standard drug Amphotericin B (IC₅₀ = 0.060 µM). Subsequently, compounds 24a and 25d also showed promising antileishmanial activity, with IC₅₀ values of 2.43 µM and 3.55 µM, respectively. Furthermore, compounds 23d and 24b showed moderate activity (IC₅₀ ≤ 10 µM). The rest of the compounds exhibited a lesser activity profile. Thus, SAR experiments indicated that the L-phenylalanine-based spirooxindoles showed a better antileishmanial activity profile as compared to L-proline and L-tryptophan-based counterparts. In proline-based spirooxindoles 23a–f, the EDG group (OMe, Me) on the isatin moiety and the halogen (X = Br) on the chalcone functionality, i.e., 23d, provided significant activity compared to Amphotericin B. Subsequently, phenylalanine-based spirooxindoles 24a–f were found to be the best active compounds among the series. However, the presence of EDG (OMe, Me), EWG (NO₂, F), or a halogen (X = Br, I) on the isatin moiety showed promising antileishmanial activity in compounds 24a, 24b, 24e, and 24f despite having EDG, EWG, or halogen groups on chalcone, except 24c–d. Furthermore, among tryptophan-based spirooxindoles 25a–g, the presence of EDG (OMe, Me), EWG (NO₂, F), or a halogen (X = F, Br) on the isatin moiety showed moderate activity (25d) despite having EDG (OMe, Me, cyclohexyl), EWG (X = F), or a halogen (X = F, Cl, Br) on chalcone functionality. It was also observed that the presence of EDG or EWG on the spiroskeleton had no influence on the yield of the reaction.

2.4. Molecular Docking Studies

The molecular docking studies of the most active spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one derivatives, i.e., 24a, 24e, 24f, and 25d, were performed with Leishmania donovani topoisomerase I-vanadate-DNA complex protein (PDB ID: 2B9S) using Discovery Studio Visualizer Software [43].

2.4.1. Ligand Preparation

The two-dimensional structure (2d) of novel functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one-based compounds 23a–f, 24a–f, and 25a–g along with standard drugs amphotericin and camptothecin were drawn in Chem Draw Ultra 22.0 software, and then the 2D structures of the ligands were converted into MDL molfile V3000 (*mol) format. The ligand was finally optimized with a small molecule protocol, which helps to remove tautomers, isomers, and duplicate conformations.

2.4.2. Protein Preparation

The protein crystal 3D structures of the heterodimeric L. Donovani topoisomerase I- vanadate DNA complex were taken from the protein data bank (PDB), PDB ID 2B9S. The protein was minimized using the simulation protocol via the CHARMm-based smart minimizer method, and protein preparation involved five different steps: cleaning protein, inserting missing atoms, refining loops, minimizing loops, and protonating protein.

2.4.3. In Silico Studies

Analysis of the docking results was carried out by comparing the binding affinities of all the proposed docked molecules to the complex protein. The docking of the abovementioned protein was carried out by removing DNA, and the remaining protein was kept in a grid box. Then, we explored the binding orientation of active functionalized spirooxindoles in terms of their Cdocker energy and Libdock score. It is to be noted that low Cdocker energy and high Libdock score values indicate higher binding affinity toward the target protein, thereby reflecting its higher potency (Table 4).

Table 4.

Docking score, i.e., binding energy, of spirooxindole derivatives 24a, 24e, 24f, 25d, and camptothecin obtained from docking studies.

Compounds	-Cdocker Energy (kcal/mol)	CDocking Interaction Energy (kcal/mol)	Libdock Score
24a	−17.1681	26.9327	128.598
24e	−7.7614	30.2844	96.0439
24f	−7.7800	30.3540	131.125
25d	−14.6475	32.2816	83.1911
Camptothecin	−10.838	30.4772	123.320

The docking results for 24a against Leishmania donovani showed a high binding affinity docking score indicated by a total score of 128.598, and it formed three H-bonds of length 2.19 Å, 2.9 Å, and 2.16 Å to the hydrophobic nucleophilic residues, i.e., the side chains of ASP: A-353 (aspartic acid), ARG: A-190 (arginine), and ASN: B-221 (asparagine), respectively. In the docking pose of the complex, the chemical nature of binding site residues within a radius of 3 Å showed non-bonding Van der Waals interactions with HIS: A-193 (histidine), ARG: A-314 (arginine), THR: B-217 (threonine), ILE: B-220 (isoleucine), and LYS: A-352 (lysine), thus leading to more stability and activity in this compound. In addition, 24a also exhibited a π–anion interaction with ASP: A-353 and an alkyl–π–alkyl interaction with the TYR: B-222 (thyrosine) amino acid residue (Figure 6).

Figure 6.

(A): Predicted 2D interactions of 24a with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 128.598, revealing H-bonding to the hydrophobic aliphatic residue, i.e., the side chain of LYS: A-352 (lysine). (B) Predicted 3D interactions of 24a with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 128.598, revealing π–anion interaction and alkyl–π–alkyl interaction with TYR: B-222 (tyrosine).

The docking results for 24e against Leishmania donovani showed a docking score of 96.0439 and showed attractive charges between nitro group substitution with ASP: A-353 (aspartic acid) and ARG: A-190 (arginine) of bond lengths of 4.58 Å and 3.80 Å, respectively, and LYS: A-352 (lysine) amino acids involved in H-bonds of lengths 2.37 Å and 2.23 Å with the carbonyl oxygen of the ligand. Furthermore, single carbon–hydrogen bond was observed with a bond length of 2.7 Å with ARG: A-190 (arginine), showing the presence of additional H-bonding (Figure 7).

Figure 7.

(A): Predicted 2D interactions of 24e with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 96.0439, revealing a conventional H-bonding carbonyl group with the amino acid residue, i.e., the side chain of LYS: A-352 (lysine). (B) Predicted 3D interactions of 24e with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 96.0439, revealing attractive charge interactions with ASP: A-353 (aspartic acid) and ARG: A-190 (arginine).

Similar to compound 24e, the docking profile for 24f against the antileishmanial target showed a docking score of 131.125 and revealed non-bonding Van der Waals interactions with GLU A:182, LYS A:251 (lysine), ASN B:221 (asparagine), THR B:217 (threonine), and TYR B:222 (tyrosine).

The π–anion interaction was observed between LYS A:352 (lysine) and isatin of bond length 3.85 Å. The conventional H-bonding of amino acids ASP: A-353 (aspartic acid) and ARG: A-190 (arginine) with bond lengths of 3.52 Å and 2.43 Å were present along with NH and carbonyl group moieties of ligand 24f, respectively. Subsequently, 24f also showed other interactions involving carbon–hydrogen bonds as well as π–cation, π–anion, and π–alkyl interactions (Figure 8).

Figure 8.

(A): Predicted 2D interactions of 24f with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 131.125, giving conventional H-bonding of the carbonyl group with the amino acid residue, i.e., the side chain of LYS: A-352 (lysine). (B) Predicted 3D interactions of 24f with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 131.125, revealing conventional H bonding interactions with ASP: A-353 (aspartic acid), and ARG: A-190 (arginine).

The docking results for 25d against Leishmania donovani (PDB ID: 2B9S) showed a high binding affinity docking score, indicated by a total score of 83.1911, and it mostly formed nonbonding Van der Waals interactions with amino acid residues GLY: A-189 (glycine), LYS: A-319 (lysine), HIS: A-193 (histidine), PHE: A-187 (phenylalanine), GLN: A-454 (glutamine), THR: B-217 (threonine), ARG: A-314 (arginine), ASN: B-221 (asparagine), ALA: A-324 (alanine), LYS: A-407 (lysine), and LYS: A-269 (lysine). It also shows a π–anion interaction between the phenyl moiety and ASP: A-353 (aspartic acid) and conventional hydrogen bonding between carbonyl oxygen and the amino acid residue, LYS: A-352 (lysine). The pair of alkyl–π–alkyl interactions HIS: A-453 (histidine) with a bond length of 5.24 Å and ARG: A-190 (arginine) with a bond length of 4.91 Å were also present, along with π–π T-shaped TYR: B-222 (thyrosine) with a bond length of 2.97 Å (Figure 9).

Figure 9.

(A): Predicted 2D interactions of 25d with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 83.1911, revealing H-bonding to the hydrophobic aliphatic residue, i.e., the side chain of LYS: A-352 (lysine). (B) Predicted 3D interactions of 25d with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 83.1911, revealing alkyl–π–alkyl interaction and π–π T-shaped interaction with TYR: B-222 (thyrosine).

The docking results for camptothecin against Leishmania donovani showed a high binding affinity docking score indicated by a total score of 123.320 and formed two conventional H-bonds of length 2.55 Å and 1.79 Å to the hydrophobic nucleophilic residues, i.e., the side chains of ALA: A-324 (alanine) and LYS: A-319 (lysine), respectively. In the docking pose of the complex, the chemical nature of binding site residues showed non-bonding Van der Waals interactions with PHE: A-187 (phenylalanine), GLY: A-189 (glycine), THR: A-326 (threonine), SER: A-354 (serine), and GLU: A-353 (glutamic acid), which give extra stability and activity in this compound. In addition, camptothecin also exhibited the π–cation interaction with ARG: A-190 (arginine) and the alkyl–π–alkyl interaction with ALA: A-324 (alanine) and HIS: A-193 (histidine) amino acid residues (Figure 10).

Figure 10.

(A): Predicted 2D interactions of camptothecin with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 123.320, revealing H-bonding to the hydrophobic aliphatic residue, i.e., the side chain of ALA: A-324 (alanine). (B) Predicted 3D interactions of camptothecin with Leishmania donovani (PDB ID: 2B9S) with a docking total score of 128.598, showing different binding interaction and alkyl–π–alkyl interaction with ARG: A-190 (arginine).

3. Conclusions

In conclusion, we report the microwave-assisted synthesis of a novel series of functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one derivatives, i.e., 23a–f, 24a–f, and 25a–g, and these have pharmaceutically privileged chalcones and amino acids. The time required for completion of reaction in MM varied from 5 min as compared to CM, which required 3 h. We also report, for the first time, the antileishmanial activity and SAR studies of 23a–f, 24a–f, and 25a–g, which were validated by carrying out molecular docking studies of 24a, 24e, 24f, and 25d. The stereochemistry of the novel functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one derivatives were confirmed by single-crystal X-ray crystallography studies of 23f. Among all the synthesized compounds, 24a (IC₅₀ = 2.43 µM), 24e (IC₅₀ = 0.96 µM), 24f (IC₅₀ = 1.62 µM), and 25d (IC₅₀ = 3.55 µM) showed potent in vitro antileishmanial activity against Leishmania donovani in comparison to the standard drug Amphotericin B (IC₅₀ = 0.060 µM). All the compounds were tried as potential inhibitors of LTopIB, but only 24a, 24e, 24f, and 25d were able to inhibit the recombinant enzyme in vitro. Subsequently, the molecular docking studies validated the biological results. In short, our findings qualify the studied molecules as prospective antileishmanial agents with distinct pharmaceutically privileged structures that pave the way for further advanced applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28124817/s1, Figures S1–S38: ¹H NMR and ¹³C NMR Spectral Data of 23a–f, 24a–f, and 25a–g; Tables S1–S6: X-ray Crystallography: Single-crystal data of spirooxindole 23f. General experimental conditions; General Procedure for the Synthesis of Chalcones 21a–f; General Procedure (GP) for the Synthesis of Spirooxindole Derivatives, 23a–f, 24a–f, and 25a–g; Biological Methods: Parasite strain and culture conditions, In vitro antileishmanial activity, and Plasmid relaxation assay; Characterization data of spirooxindole derivatives (23a–f, 24a–f, 25a–g); X-ray Crystallography: Single-crystal data of spirooxindole 23f; optical rotation, ¹H and ¹³C NMR spectral data of all the synthesized compounds 23a–f, 24a–f and 25a–g.

Author Contributions

Conceptualization, S.C.; methodology, N.K.S., R.S. (Ritu Sharma), K.P.S., J.J., K.P. and R.S. (Richa Sharma); software, S.C.; validation, N.K.S., R.S. (Ritu Sharma), K.P.S. and R.S. (Richa Sharma); investigation, S.C., N.K.S., R.S. (Richa Sharma), K.P.S., J.J., K.P., R.S. (Richa Sharma), R.P., S.K., M.J., C.C., X.H. and P.C.; resources, S.C., P.C., C.C. and R.P.; and data curation, N.K.S., R.S. (Ritu Sharma), K.P.S., J.J., K.P., R.S. (Richa Sharma) and X.H.; writing—original draft preparation, S.C.; writing—review and editing, S.C., P.C., N.K.S., R.S. (Richa Sharma), K.P.S. and C.C.; supervision, S.C., P.C., M.J., S.K., R.P. and C.C.; project administration, S.C.; funding acquisition, S.C., P.C., R.P. and C.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds will be available from the authors.

Funding Statement

S.C. acknowledges the funding of this research work from Core Research Grant (CRG), Science and Engineering Research Board (SERB), New Delhi [CRG/2019/005102] and Indian Council of Medical Research (ICMR) Grant, New Delhi [61/8/2O2O-IMM/BMS]. R.P acknowledges the present work which has been partly supported by CSIR, New Delhi Grant No. 02(0286)/16/EMR-II; and ICMR, New Delhi Grant No. BIC/12(03)/2014 for funding. P.C. acknowledges Macau University of Science and Technology and School of Pharmacy (FRG-22-077-SP). This research was funded by the Ministry of Science and Technology in Taiwan, grant numbers MOST 110-2113-M-039-001 and MOST 111-2221-E-039-009 for C.C.