A one-pot three-component synthesis and investigation of the in vitro mechanistic anticancer activity of highly functionalized spirooxindole-pyrrolidine heterocyclic hybrids

Article abstract of DOI:10.3390/molecules25235581

With an aim to develop more effective and affordable anticancer agents possessing a unique mechanism of action, we designed and synthesized derivatives of spirooxindole-pyrrolidine heterocyclic hybrids in good yields through a one-pot three-component (3+2) cycloaddition strategy. The synthesized compounds were characterized thoroughly for the physicochemical properties by making use of FT-IR, NMR spectroscopy, and mass spectrometry. Further, these compounds have been evaluated for the influence of anticancer activity against HepG2 cells up to 200 μg/mL concentration. The highly active molecular scaffold was tested for the in-depth mechanistic studies, and it was found that the major pathway of cell death is apoptosis which occurs through the induction of reactive oxygen species followed by the involvement of caspases.

Full text of DOI:10.3390/molecules25235581

molecules
Article
A One-Pot Three-Component Synthesis and
Investigation of the In Vitro Mechanistic Anticancer
Activity of Highly Functionalized
Spirooxindole-Pyrrolidine Heterocyclic Hybrids
Raju Suresh Kumar * , Dhaifallah M. Al-thamili, Abdulrahman I. Almansour,
Natarajan Arumugam and Faruq Mohammad
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia;
442106623@ksu.edu.sa (D.M.A.-t.); almansor@ksu.edu.sa (A.I.A.); anatarajan@ksu.edu.sa (N.A.);
fmohammad@ksu.edu.sa (F.M.)
* Correspondence: sraju@ksu.edu.sa
Academic Editor: Gian Cesare Tron
Received: 20 October 2020; Accepted: 24 November 2020; Published: 27 November 2020
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: With an aim to develop more effective and affordable anticancer agents possessing a
unique mechanism of action, we designed and synthesized derivatives of spirooxindole-pyrrolidine
heterocyclic hybrids in good yields through a one-pot three-component (3+2) cycloaddition strategy.
The synthesized compounds were characterized thoroughly for the physicochemical properties by
making use of FT-IR, NMR spectroscopy, and mass spectrometry. Further, these compounds have been
evaluated for the influence of anticancer activity against HepG2 cells up to 200 µg/mL concentration.
The highly active molecular scaffold was tested for the in-depth mechanistic studies, and it was found
that the major pathway of cell death is apoptosis which occurs through the induction of reactive
oxygen species followed by the involvement of caspases.
Keywords: multicomponent reaction; functionalized spirooxindole-pyrrolidine hybrids; anticancer
activity; controlled cell death; apoptosis; ROS generation; caspase-3 activity
1. Introduction
Carcinogenesis, also termed as tumorigenesis or oncogenesis, is a complex biological multi-step
encompassing process involving a series of physiological mechanisms, where the outcome is the
growth of cancerous tumors that has the capacity to completely arrest normal functions of an organ [1].
The inhibition of normal organ functions in cancerous cells, in general, first occurs through the cell
division, followed by an uncontrolled growth of mutated cells to form malignant tumors, which on
later stages spread to nearby cells and even migrate to different sites of the body by making use of the
lymphatic system or bloodstream. To reduce the uncontrolled growth of malignant and fast dividing
cells, many different antiproliferative drugs with enhanced pharmacological activity and bioavailability
are used; however, the prolonged use of such drugs may develop uterine and endometrial cancers [2].
To overcome these issues, various synthetic and natural anticancer drugs are being developed and
introduced into the market. As the nonselective action of such developed anticancer drugs are
associated with many side effects including the bone marrow suppression, alopecia, and nephrotoxicity,
the necessity of developing novel drug entrants with improved effectiveness to target only cancer cells
is emphasized.
The construction of novel drug entrants with improved physiochemical properties, pharmacologic
effectiveness, and no or less toxicity in cancer treatment has been an essential subject in the drug
Molecules 2020, 25, 5581; doi:10.3390/molecules25235581
www.mdpi.com/journal/molecules

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development sector. Undeniably, the construction of such molecular frameworks through a facile
synthetic methodology with cost-effective considerations is still a challenge for medicinal chemists and
necessitates long-lasting efforts. One of the developed strategies in recent years is the amalgamation
of two or more diverse pharmacophores into a sole molecular framework, documented as molecular
hybridization [3–10]. The molecular hybridization becomes an effective approach to the design of new
drug entrants and is based on the identification of pharmacophoric structural subunits via the suitable
combination of these subunits, which provides a base for the design of novel heterocyclic hybrids that
preserves the preselected features of parental prototypes. The prominent advantage of employing the
molecular hybridization approach is to trigger diverse targets by a sole molecule, thus increasing the
therapeutic effectiveness and bioavailability profiles. Promisingly, noteworthy results with a diverse
group of heterocyclic hybrids have been elegantly recognized as a result of this approach [11–18].
Spiropyrrolidine oxindoles hold fascinating structural characteristics and robust bioactivity
profiles to have appeared as promising synthetic targets, as these heterocyclic hybrids serve as very
useful molecular architectures for the investigation of pharmacophore space by making use of diverse
oriented synthesis that in turn results in the development of new drug entrants [19–24]. The literature
reports discovered that the presence of α,β-unsaturated ketone moiety in a piperidone molecular unit
is an important functionality for the activity of piperidone derivatives. Besides, these compounds
are investigated to be associated with both fluorescence and antitumor capacity and thus can be
potential for cancer cell studies towards the development of novel therapeutic pathways involving
fluorescent drugs [25]. However, the literature reports provide the information that the combination
of these biologically important structural units in a single molecular framework has not been
widely studied. Therefore, based on the excellent biological profiles of spiropyrrolidine oxindoles,
piperidone, and α,β-unsaturated ketones, we wondered the molecular hybridization of these structural
subunits in a single molecule that might create novel anticancer drugs. Hence, in this research,
we aimed to discuss our efforts in the design and development of more potent analogues for the
cancer chemotherapy.
2. Results and Discussion
2.1. Chemistry
In our initial attempt, the synthesis of N-unsubstituted bisarylmethylidene-tetrahydropyridinones
3
(
a–h) was performed according to the literature report as provided by Dimmock et al. [26]
With the compounds in hand, the dipolarophiles and N-substituted
bisarylmethylidene-tetrahydropyridinones ) required for the present study were synthesized in
a 85–90% yield range through the alkylation of ) with 2-(chloromethyl)pyridine hydrochloride
in the presence of K2CO3 (Scheme 1) [27 28]. The azomethine ylide was generated in situ from
isatin and phenylglycine via decarboxylative condensation. Our approach to the synthesis
of spirooxindole molecular scaffold employing isatin and (L)-Phenylglycine was based on
a multicomponent reaction strategy involving the 1,3-dipolar cycloaddition reaction between
.
3
(a–h)
5 (a–h
3 (a–h
,
6
7
N-pyridinylmethyl-bisarylmethylidenepyridinones
from 6 and 7 as outlined in Scheme 1.
5 (a–h) and azomethine ylide generated in situ
Initially, the reaction optimization for this one-pot (3+2) cycloaddition reaction was done by
performing the reaction of an equimolar mixture of 5e , and under reflux in different solvents,
,
6
7
including ethanol, methanol, dioxane, methanol/dioxane, and acetonitrile. The reaction progress
was observed by thin layer chromatography, after the completion of the reaction, as evidenced
by the disappearance of the substrates and the formation of a sole reaction product by thin-layer
chromatography (TLC) analysis. Fifty milliliters of ice-cold water were added to the reaction mixture,
and the precipitated solid was filtered, washed repeatedly with water and then dried under vacuum to
obtain the cycloadduct 8e. The obtained spirooxindole-pyrrolidine heterocyclic hybrid was purified
either by crystallization using acetonitrile or by column chromatography employing hexane-ethyl

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acetate (v/v: 3:2) as an eluent. Among the solvents used, methanol was found to be the optimal solvent
with respect to yield (88%) and reaction time (2 h). Hence, all the subsequent reactions starting with
different aryl ring-substituted dipolarophiles 5 (b–h) were performed under this optimized reaction
condition. All these reactions furnished the corresponding spirooxindole-pyrrolidine heterocyclic
hybrids 8 (a–d and f–h) in good to excellent yields (85–90%).
Scheme 1. Synthesis of spirooxindole-pyrrolidine molecular scaffolds 8(a–h).
This cycloaddition reaction proceeded regio- and stereoselectively, as only one diastereoisomeric
cycloadduct is formed despite the presence of multiple stereocenters and afforded the compounds 8 (a–h)
in good yields. The spirooxindole-pyrrolidine molecular scaffolds were obtained through a process
which involved the formation of two C–C bonds, one C–N bond, and four contiguous stereocenters.
The spirooxindole-pyrrolidine molecular scaffolds
various instrumental techniques involving FT-IR, one-dimensional (1D) and two-dimensional (2D) NMR
spectroscopy, and mass spectrometry (Figure 1 and Electronic Supplementary Information). In the
FT-IR spectrum of 8e, the noticeable infrared absorption peaks at
max of 2360, 2341, 1698, and 1597 cm–1
8 were studied thoroughly by making use of
†
υ
were referred to N–H, C=O, and C=C groups, respectively. In the 1H NMR spectrum, the H-4 proton
furnished a doublet at 4.68 ppm (J = 11.0 Hz). This is further established by its Heteronuclear
Multiple Bond Correlation (HMBC) with the carbonyl carbon C-40 at 199.35 ppm. The H,H-Correlation
Spectroscopy (H,H-COSY) of H-4 proton allocated the doublet at 5.43 ppm (J = 11.0 Hz) to its coupling
partner H-5. The C,H-COSY correlation of H-4 and H-5 with the carbon signals at 56.81 ppm and
64.36 ppm assigned these peaks to C-4 and C-5, respectively. Further, the HMBC of H-4 allocated the
carbon signal at 57.03 ppm to C-20. The C,H-COSY correlation of C-20 assigned the doublet at 2.13 ppm
(J = 13.0 Hz) and the doublet of doublets at 3.63 ppm (J = 13.0 and 2.5 Hz) to 20-CH2. The doublet of
doublets at 2.97 ppm (J = 15.0 and 3.0 Hz) and the doublet at 3.42 ppm (J = 15.0 Hz) were assigned to
60-CH2. The remaining doublets at 3.37 ppm and 3.79 ppm (J = 13.5 Hz) were due to 70-CH2. The singlets
at 2.26 and 2.27 ppm were due to the two -H3 group substituted at the aromatic ring, while the aromatic
ring protons appeared as doublets and multiplets around 6.61 to 8.45 ppm. The carbon signals at
54.21 and 63.90 ppm were allocated to C-60 and C-70, respectively, whereas the signals at 66.87 ppm
and 72.17 ppm were due to the spiro carbons C-3 and C-2, respectively. The oxindole carbonyl carbon

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appears at 180.42 ppm. The selected 1H and 13C chemical shifts of 8e are shown in Figure 1. A peak at
603 [M+] in the mass spectrum of 8a confirmed the structure deduced from spectroscopic studies.
Figure 1. Selected 1H and 13C NMR chemical shifts of 8e.
A probable mechanism accounting for the formation of molecular scaffolds
Scheme 2. Presumably, the reaction of isatin and (L)-Phenylglycine via decarboxylative condensation
afforded the azomethine ylide which may exist in the resonating forms and 10. Then, the regioselective
with the addition of nucleophilic
-carbon of the dipolarophile to afford
8(a–h) is shown in
9
addition of ylide
carbon of the azomethine ylide to the more electron-deficient
the cycloadducts
9
to one of the exocyclic C=C bonds of
5
(
a
β
–
h
)
,
8
(
a
–
h). The other possible regioisomer 11 formed by the addition of azomethine
ylide 10 to the dipolarophile was not observed even in traces. The nonformation of this regioisomer
may be attributed to the existence of steric hindrance experienced by the aryl and oxindole rings.
2.2. Biology
The in vitro studies of cell viability due to the action of the tested compounds
8 (a–h) at the
treatments in the concentration range of 12.5–200 g/mL over two different time periods of 24 and 48 h
µ
to HepG2 cancer cells are shown in Figures 2 and 3. It can be noted from the results analysis that all
the tested compounds maintained certain levels of activity against the cancer cells and this activity
was increased with regards to an increased concentration and an increased incubation period of the
tested compounds, as against the positive control of melphalan (15
of the cells having no treatment. Table 1 indicates the concentration required for 50% of cell death
(IC50) values following the treatment of compounds ) for the HepG2 cancer cells for 24 and 48 h
µg/mL) and the negative control
8 (a–h
periods and reveals the highest activities for the two compounds 8f and 8g and the least activity for the
compound 8e. Among the two most active compounds of 8f and 8g, whether one took the lead over the
other depended upon the incubation time. That is, under the 24 h incubation time, the compound 8f is
most active, as we observed the IC50 value of approximately 43.46
period, the compound 8g has an IC50 value of approximately 12.79
anticancer activity assay, the observed IC50 value order during the 24 h incubation period followed the
order of 8f 8g 8b 8a 8h 8c 8d 8e, while for the 48 h period it is 8g 8f 8a 8b 8h
8c > 8d > 8e.
To test the behavior of our synthesized compounds towards the noncancer cells, the two most active
compounds, 8f and 8g were tested against the L929 cells over a concentration range of 12.5–200 g/mL
µg/mL, while at the 48 h incubation
µg/mL. Thus, based on the MTT
>
>
>
>
>
>
>
>
>
>
>
>
µ
for the highest incubation period of 48 h (as shown in Figure 4 and the values tabulated in Table 1).
From the analysis of the experimental results, it can be noted that the two compounds exhibited
an approximately similar activity and the considerable activity was observed only at and above
the concentration of 100
µg/mL. The IC50 values of the compounds 8f and 8g were measured to be
72.12 and 153.89 g/mL, respectively, and these values were higher as compared to the IC50 values
µ

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observed for the same compounds but with the cancer cells (HepG2). This indicated that the controlling
of cancer cells by the treatment of compounds 8f and 8g and with negligible damage to the noncancer
cells can be possible and this is one of the important criteria in cancer chemotherapy. In addition,
since our aim was to examine the probable mechanism of cell death produced by the most active
molecular scaffold, we selected the compound 8g to perform the other molecular biology studies which
required only the least concentration for the observation of cell death.
Scheme 2. Schematic representation of the plausible mechanism for the region-selective formation of
spirooxindole-pyrrolidine molecular scaffolds 8.
Figure 2. Comparison of cell viability results in vitro from the MTT assay against the HepG2 cells
when treated with the tested compounds
8(
a–
h
) with a range of concentrations (12.5–200
µg/mL; 24 h
period). From the figure, Standard (STD) stands for melphalan (15
µg/mL); * and ** relate to the
significant (p < 0.05) and highly significant (p < 0.01) values as compared against those obtained with
the untreated measurements.

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Figure 3. Cell viability comparison of MTT assay results of HepG2 cells from the treatment of
compounds ) over a range of concentration (12.5–200 g/mL; 48 h period). From the figure,
STD stands for melphalan (15 g/mL); * and ** relate to the significant (p < 0.05) and highly significant
8
(a–
h
µ
µ
(p < 0.01) values as compared to those obtained with the untreated measurements.
Figure 4. Comparison of MTT assay results of L929 noncancer cells following the incubation of two
compounds 8f and 8g with a range of concentrations (12.5–200
for melphalan of 15 g/mL; * and ** refer to the significant (p < 0.05) and highly significant (p < 0.01)
values as compared to those obtained with the untreated controls.
µg/mL) over a 48 h period. STD stands
µ

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Table 1. Comparison of the concentration required for 50% of cell death (IC50) of the tested compounds
8(a–h) following the treatments to the HepG2 and L929 cells over 24 and 48 h incubation periods.
IC50 Values (µg/mL) HepG2 Cells
IC50 Values (µg/mL) L929 Cells
Entry
Compound
24 h Treatment
48 h Treatment
48 h Treatment
1
105.78 ± 2.1
27.43 ± 4.1
NA
NA
NA
NA
NA
2
3
4
5
91.5 ± 2.1
274.61 ± 2.1
352.86 ± 2.1
897.42 ± 2.1
31.21 ± 5.3
52.14 ± 6.7
53.04 ± 5.5
62.25 ± 5.8
6
43.46 ± 2.1
25.82 ± 3.2
72.12 ± 3.3

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Table 1. Cont.
IC50 Values (µg/mL) HepG2 Cells
IC50 Values (µg/mL) L929 Cells
Entry
Compound
24 h Treatment
48 h Treatment
48 h Treatment
7
65.18 ± 2.1
12.79 ± 2.6
153.89 ± 4.2
8
120.76 ± 2.1
41.32 ± 3.2
NA
To understand the cell death mechanism followed by the treatment of compound 8g, the apoptosis,
reactive oxygen species (ROS), and caspase activity assays were performed, and the obtained results
were compared with those of the positive control (melphalan; 15 µg/mL) and the negative control
of the cells without any treatment. Figure 5 shows the flow cytometry provided apoptosis assay
results of the HepG2 cells due the treatment of the compound 8g (at IC50 concentration.) and the
positive control (melphalan) and the negative control of the cells (no treatment). The results of A-2, B-2,
and C-2 provided in Figure 5 represented the mixed fluorescence intensities from the cells treated with
propidium iodide (PI), and Annexin V-fluorescein isothiocyanate (Annexin V-FITC), the cells treated
with the compound 8g, and melphalan-treated cells, respectively. From the compound 8g-treated cells,
we observed that about 66.5% cells were live, 10% were apoptotic, and 17.5% were late apoptotic/early
necrotic (Figure 5(B-2)); these values were far better than those of the melphalan-treated cells where
we observed only 17.4% of live cells, 2.2% of apoptotic, and 46% of late apoptotic/early necrotic cells
(Figure 5(C-2)). In addition, M1 and M2 shown in Figure 5 (A-3), 5(B-3), and 5(C-3) corresponded to
the fluorescence intensities generated from the Annexin V-FITC dye by the viable and apoptotically
dying cells, respectively. We observed from the Figure 5 that about 31.7% of the compound 8g-treated
cells were apoptotic and 68.7% of these cells were live. However, 65.2% of the melphalan-treated cells
were apoptotic, and 35.6% of those cells were live. Thus, from the analysis of fluorescence intensity
results by the PI and Annexin V-FITC, it can be indicated that apoptosis is the dominating mechanism
for the observed loss of cell viability in the case of the HepG2 cancer cells.
Figure 6 shows the flow cytometric analysis towards the measurement of ROS generated following
the treatment of the compound 8g (at IC50 conc.) to the HepG2 cells when compared with those of
the positive control (melphalan) and the negative control (cells of no treatment). From the analysis
of results, it can be observed that about 97% of apoptotic cells generated the ROS in the case of the
compound 8g treated cells (Figure 6 (B-3)) and this value was far higher than that of the positive control
(melphalan)-treated cells (only 13.6% cells were apoptotic cells; Figure 6 (C-3)). The observation of
such a high value of ROS generation from the compound 8g-treated cells indicated that most of the
apoptotic cells released the ROS during their contact with the tested compound.

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Figure 5. Apoptosis assay comparison of the fluorescence assay results following the treatment of
HepG2 cells with that of the tested compound 8g ). The results were compared with those of
melphalan used as the positive control ( ) and no cell treatment used as the negative control ( ) over a
48 h period.
(B
C
A
Figure 6. Reactive oxygen species (ROS) generation as a measure of fluorescence intensity in the HepG2
cells following the negative control having no cell treatment (A), the treatment of compound 8g (B),
and the positive control melphalan (C) over a 48 h period.

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The involvement of caspases by means of caspase-3 activity was tested for the HepG2 cancer cells
due to the treatment of the compound 8g (at IC50 conc.), where the results were compared against the
untreated and melphalan-treated controls as shown in Figure 7. We observed a very high number
of fluorescent cells from the tested compound 8g-treated cells (Figure 7c) as against those from the
positive control (Figure 7b) and the negative control (Figure 7a), and the same trend was quantitatively
shown in Figure 7d. This indicated that the HepG2 cells underwent the caspase-3 release before they
lost the viability, thereby confirming for the involvement of caspases for the compound 8g-treated cells.
Figure 7. Comparison of caspase-3 activity results provided by the co-incubation of HepG2 cells with
the negative control having no cell treatment (a), treated with melphalan used as the positive control
(b
), and treated with the compound 8g ) over a 48 h period. The relative fluorescence intensities of all
(c
the treatments were compared in (d).
3. Materials and Methods
The detailed materials and methods were provided in Supplementary Materials.
3.1. 4-(aryl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)-spiro[3.30]-50-(2-arylmethylidene)-10-N-
(pyridinylmethyl)piperidin-40-one 8 (a-h)
An equimolar mixture of N-substituted bisarylmethylidene-tetrahydropyridinones 5(a–h) (0.100 g,
0.27 mmol), isatin 6 (0.040 g, 0.27 mmol), and phenylglycine 7 (0.041 g, 0.27 mmol) were dissolved in
methanol (5 mL) and heated under reflux with constant stirring for 4 h. After completing the reaction
as evident from TLC, the reaction mixture was transferred into 50 mL of ice-cold water to form a
precipitate which was separated out by filtration and washed with water to obtain the product
in good yields.
8(a–h)
3.2. 4,5-Diphenylpyrrolo(spiro[2.3”]-oxindole)-spiro-[3.30]-50-(phenylmethylidene)-10-N-
(pyridinylmethyl)piperidin-40-one (8a)
Obtained as a white solid (88%); mp = 219–221 ◦C; IR (KBr): 2359, 2340, 1698, and 1599 cm−1
1H NMR (500 MHz, CDCl3): δH 2.11 (d, 1H, J = 12.5 Hz, 20-CH2), 2.51 (brs, NH), 2.96 (dd, 1H, J = 15.0
;
,

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2.5 Hz, 60-CH2), 3.35 (d, 1H, J = 14.0 Hz, 70-CH2), 3.42 (d, 1H, J = 14.5 Hz, 60-CH2), 3.65 (dd, 1H,
J = 13.0, 2.0 Hz, 20-CH2), 3.76 (d, 1H, J = 13.5 Hz, 70-CH2), 4.72 (d, 1H, J = 10.0 Hz, 4-CH), 5.47 (d, 1H,
J = 11.0 Hz, 5-CH), 6.63 (d, 1H, J = 8.0 Hz, ArH), 6.93–7.09 (m, 7H, ArH), 7.18–7.28 (m, 9H, ArH),
7.43 (d, 2H, J = 7.5 Hz, ArH), 7.47 (dd, 1H, J = 7.5, 2.0 Hz, ArH), 7.54 (d, 2H, J = 7.5 Hz, ArH), 8.26 (s, 1H,
NH), 8.42–8.43 (m, 1H, ArH). 13C NMR (125 MHz, CDCl3): δC 53.95, 56.95, 57.04, 63.68, 64.23, 67.01,
72.03, 109.11, 122.05, 122.07, 123.06, 126.85, 126.96, 127.52, 127.64, 128.21, 128.28, 128.70, 128.88, 129.01,
129.90, 130.00, 132.91, 134.82, 136.17, 137.31, 137.60, 140.80, 141.51, 148.94, 157.34, 180.53, and 199.33.
Mass: 603 [M+]. Anal. calc. for C40H34N4O2: C, 79.71; H, 5.69; N, 9.30. Found: C, 79.85; H, 5.51;
N, 9.44%.
3.3. 4-(2-Methylphenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(2-methylphenyl-methylidene)-
10-N-(pyridinylmethyl)piperidin-40-one (8b)
Obtained as a white solid (90%); mp = 190–193 ◦C; IR (KBr): 2358, 2337, 1699, and 1597 cm−1
;
1H NMR (500 MHz, CDCl3): δH 2.10 (d, 1H, J = 13.0 Hz, 20-CH2), 2.11 (s, 3H, CH3), 2.16 (s, 3H, CH3),
2.90 (dd, 1H, J = 15.0, 3.0 Hz, 60-CH2), 3.24-3.28 (m, 2H, 70-CH2, 60-CH2), 3.51 (dd, 1H, J = 13.0, 2.0
Hz, 20-CH2), 3.63 (d, 1H, J = 14.0 Hz, 70-CH2), 4.92 (d, 1H, J = 10.5 Hz, 4-CH), 5.53 (d, 1H, J = 10.0 Hz,
5-CH), 6.68-6.71 (m, 2H, ArH), 6.80 (d, 1H, J = 8.0 Hz, ArH), 6.93–7.20 (m, 6H, ArH), 7.23–7.30 (m, 8H,
ArH), 7.41–7.62 (m, 4H, ArH), 8.07 (s, 1H, ArH), 8.38–8.39 (m, 1H, ArH). 13C NMR (125 MHz, CDCl3):
δC 20.13, 21.19, 54.04, 54.64, 58.52, 64.38, 65.11, 66.11, 73.81, 109.34, 122.07, 122.50, 122.60, 125.56, 125.98,
126.70, 127.05, 127.53, 128.25, 128.45, 128.52, 128.83, 128.89, 129.56, 130.21, 130.28, 132.53, 134.01, 136.16,
136.39, 137.45, 137.94, 141.50, 141.89, 149.06, 157.73, 179.23, and 199.89. Mass: 631 [M+]. Anal. calc.
for C42H38N4O2: C, 79.97; H, 6.07; N, 8.88. Found: C, 79.85; H, 6.16; N, 8.97%.
3.4. 4-(2-Methoxyphenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(2-methoxy-
phenylmethylidene)10-N-(pyridinylmethyl)piperidin-40-one (8c)
Obtained as a reddish brown solid (86%); mp = 170–173 ◦C; IR (KBr): 2359, 2339, 1697,
1
and 1598 cm−1; H NMR (500 MHz, CDCl3): δH 2.20 (d, 1H, J = 12.5 Hz, 20-CH2), 2.95 (dd, 1H,
J = 15.0, 3.0 Hz, 60-CH2), 3.26 (d, 1H, J = 13.5 Hz, 70-CH2), 3.34 (dd, 1H, J = 15.0 Hz, 60-CH2), 3.47 (s, 3H,
OCH3), 3.64 (dd, 1H, J = 12.5, 2.5 Hz, 20-CH2), 3.72 (s, 3H, OCH3), 3.78 (d, 1H, J = 13.5 Hz, 70-CH2),
4.93 (d, 1H, J = 10.0 Hz, 4-CH), 5.67 (d, 1H, J = 10.5 Hz, 5-CH), 6.65–6.72 (m, 2H, ArH), 6.84 (d, 1H,
J = 7.5 Hz, ArH), 6.95-7.32 (m, 16H, ArH), 7.40–7.65 (m, 4H, ArH), 8.08 (s, 1H, ArH), 8.39–8.41 (m, 1H,
ArH). 13C NMR (125 MHz, CDCl3): δC 54.03, 54.72, 55.42, 55.87, 58.57, 64.43, 65.16, 66.24, 73.88, 109.31,
112.98, 122.27, 122.55, 122.64, 125.51, 126.00, 126.73, 127.11, 127.54, 128.29, 128.56, 128.85, 128.93, 129.63,
130.20, 130.38, 132.54, 13408, 136.33, 136.45, 137.41, 140.50, 141.76, 149.09, 157.70, 158.11, 160.98, 179.72,
and 199.85. Mass: 663 [M+]. Anal. calc. for C42H38N4O4: C, 76.11; H, 5.78; N, 8.45. Found: C, 76.30;
H, 5.90; N, 8.31%.
3.5. 4-(3-Nitrophenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(3-nitrophenyl methylidene)-10-N-
(pyridinylmethyl)piperidin-40-one (8d)
Obtained as a white solid (87%); mp = 193–196 ◦C; IR (KBr): 2358, 2336, 1698, 1597 cm−1; 1H NMR
(500 MHz, CDCl3): δH 1.99 (d, 1H, J = 13.0 Hz, 20-CH2), 2.99 (dd, 1H, J = 15.0, 2.5 Hz, 60-CH2),
3.34 (d, 1H, J = 15.0 Hz, 70-CH2), 3.40 (d, 1H, J = 15.0 Hz, 60-CH2), 3.70 (dd, 1H, J = 13.0, 3.0 Hz,
20-CH2), 3.77-3.92 (m, 1H, 70-CH2), 4.77 (d, 1H, J = 10.5 Hz, 4-CH), 5.49 (d, 1H, J = 11.0 Hz, 5-CH),
6.65 (d, 1H, J = 7.5 Hz, ArH), 6.96–7.06 (m, 1H, ArH), 7.10–7.27 (m, 11H, ArH), 7.40–7.48 (m, 1H, ArH),
7.52–7.82 (m, 6H, ArH), 8.05 (d, 1H, J = 8.0 Hz, ArH), 8.20 (d, 1H, J = 8.0 Hz, ArH), 8.24 (s, 1H, NH),
8.48–8.50 (m, 1H, ArH). 13C NMR (125 MHz, CDCl3): δC 54.41, 56.21, 56.48, 63.29, 64.32, 67.10, 71.51,
109.51, 122.58, 122.66, 123.29, 126.92, 127.03, 127.68, 128.20, 128.43, 128.65, 129.43, 129.77, 130.28, 132.62,
134.21, 134.65, 135.35, 135.46, 136.16, 136.34, 136.61, 138.37, 139.42, 139.98, 140.09, 141.68, 148.09, 149.54,
156.49, 181.90, and 198.37. Mass: 693 [M+]. Anal. calc. for C40H32N6O6: C, 69.35; H, 4.66; N, 12.13.
Found: C, 69.46; H, 4.74; N, 12.25%.

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3.6. 4-(4-Methylphenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(4-methylphenyl-methylidene)-
10-N-(pyridinylmethyl)piperidin-40-one (8e)
Obtained as a white solid (90%); mp = 195–198 ◦C; IR (KBr): 2360, 2341, 1698, and 1597 cm−1
;
1H NMR (500 MHz, CDCl3): δH 2.13 (d, 1H, J = 13.0 Hz, 20-CH2), 2.26 (s, 3H, CH3), 2.27 (s, 3H, CH3),
2.97 (dd, 1H, J = 15.0, 3.0 Hz, 60-CH2), 3.37 (d, 1H, J = 13.5 Hz, 70-CH2), 3.42 (d, 1H, J = 15.0 Hz,
60-CH2), 3.63 (dd, 1H, J = 13.0, 2.5 Hz, 20-CH2), 3.79 (d, 1H, J = 13.5 Hz, 70-CH2), 4.68 (d, 1H, J = 11.0 Hz
,
4-CH), 5.43 (d, 1H, J = 11.0 Hz, 5-CH), 6.62 (d, 1H, J = 7.5 Hz, ArH), 6.87 (d, 2H, J = 8.5 Hz, ArH),
6.91–7.10 (m, 9H, ArH), 7.15–7.19 (m, 1H, ArH), 7.20–7.26 (m, 3H, ArH), 7.31 (d, 2H, J = 8.0 Hz, ArH),
7.46–7.50 (m, 1H, ArH), 7.53–7.55 (m, 2H, ArH), 8.11 (s, 1H, NH), 8.44–8.45 (m, 1H, ArH). 13C NMR
(125 MHz, CDCl3): δC 21.07, 21.30, 54.21, 56.81, 57.03, 63.90, 64.36, 66.87, 72.17, 109.04, 122.03, 122.05,
123.05, 126.94, 127.46, 127.68, 128.27, 128.80, 128.95, 129.02, 129.08, 129.74, 130.27, 131.97, 132.06, 134.22,
136.20, 136.30, 137.73, 139.14, 140.94, 141.47, 148.97, 157.54, 180.42, 199.35. Mass: 631 [M+]. Anal. calc.
for C42H38N4O2: C, 79.97; H, 6.07; N, 8.88. Found: C, 79.81; H, 6.21; N, 8.75%.
3.7. 4-(4-Chlorophenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(4-chlorophenyl-methylidene)-10-
N-(pyridinylmethyl)piperidin-40-one (8f)
Obtained as a white solid (87%); mp = 198–202 ◦C; IR (KBr): 2359, 2340, 1698, and 1597 cm−1
1H NMR (500 MHz, CDCl3): δH 2.06 (d, 1H, J = 12.5 Hz, 20-CH2), 2.95 (dd, 1H, J = 15.0, 3.0 Hz, 60-CH2),
3.34–3.40 (m, 2H, 70-CH2, 60-CH2), 3.62 (dd, 1H, J = 13.0, 2.5 Hz, 20-CH2), 3.80 (d, 1H, J = 13.0 Hz
;
,
70-CH2), 4.65 (d, 1H, J = 10.5 Hz, 4-CH), 5.40 (d, 1H, J = 10.5 Hz, 5-CH), 6.60 (d, 1H, J = 7.5 Hz,
ArH), 6.84 (d, 2H, J = 8.5 Hz, ArH), 6.92–6.99 (m, 2H, ArH), 7.05–7.11 (m, 2H, ArH), 7.16–7.28 (m, 9H,
ArH), 7.35 (d, 1H, J = 8.0 Hz, ArH), 7.50–7.53 (m, 4H, ArH), 7.83 (s, 1H, NH), 8.47–8.48 (m, 1H, ArH).
13C NMR (125 MHz, CDCl3): δC 53.88, 56.23, 56.93, 63.70, 64.36, 67.00, 71.90, 109.23, 122.14, 122.38,
123.26, 127.09, 127.68, 127.83, 128.48, 128.56, 128.63, 128.92, 129.09, 131.27, 132.84, 133.25, 133.34, 134.92,
135.86, 136.33, 136.38, 140.54, 141.55, 149.29, 157.21, 180.40, and 198.97. Mass: 671 [M+]. Anal. calc.
for C40H32Cl2N4O2: C, 71.53; H, 4.80; N, 8.34. Found: C, 71.74; H, 4.91; N, 8.25%.
3.8. 4-(4-Fluorophenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(4-fluorophenyl-methylidene)-10-
N-(pyridinylmethyl)piperidin-40-one (8g)
Obtained as a white solid (85%); mp = 164–166 ◦C; IR (KBr): 2360, 2337, 1698, and 1597 cm−1
;
1H NMR (500 MHz, CDCl3): δH 2.07 (d, 1H, J = 12.0 Hz, 20-CH2), 2.98 (d, 1H, J = 15.0 Hz, 60-CH2),
3.32-3.34 (m, 2H, 70-CH2, 60-CH2), 3.60 (dd, 1H, J = 15.0, 5.0 Hz, 20-CH2), 3.80 (d, 1H, J = 12.5 Hz,
70-CH2), 4.66 (d, 1H, J = 10.0 Hz, 4-CH), 5.39 (d, 1H, J = 11.0 Hz, 5-CH), 6.61 (d, 1H, J = 7.0 Hz, ArH),
6.88–7.10 (m, 9H, ArH), 7.20–7.41 (m, 7H, ArH), 7.50–7.53 (m, 3H, ArH), 7.75 (s, 1H, NH), 8.44–8.48
(m, 1H, ArH). 13C NMR (125 MHz, CDCl3): δC 54.06, 56.99, 57.08, 63.94, 64.31, 66.82, 72.10, 109.11,
115.17, 115.35, 122.09, 122.25, 123.07, 126.91, 127.45, 128.31, 128.94, 129.73, 130.28, 131.94, 132.16, 134.26,
136.24, 136.39, 137.71, 140.91, 141.48, 148.93, 157.55, 158.24, 160.17, 180.39, and 199.28. Mass: 631 [M+].
Anal. calc. for C40H32F2N4O2: C, 75.22; H, 5.05; N, 8.77. Found: C, 75.38; H, 5.21; N, 8.65%.
3.9. 4-(2,4-Dichlorophenyl)-5-phenylpyrrolo(spiro[2.3”]-oxindole)spiro[3.30]-50-(2,4-dichloro-
phenylmethylidene)-10-N-(pyridinylmethyl)piperidin-40-one (8h)
Obtained as a yellow solid (89%); mp = 125–128 ◦C; IR (KBr): 2359, 2340, 1699, and 1595 cm−1
;
1H NMR (500 MHz, CDCl3): δH 2.21 (d, 1H, J = 13.0 Hz, 20-CH2), 2.98 (d, 1H, J = 14.5 Hz, 60-CH2),
3.21 (d, 1H, J = 14.0 Hz, 70-CH2), 3.29 (d, 1H, J = 14.5 Hz, 60-CH2), 3.61 (d, 1H, J = 14.0 Hz, 20-CH2),
3.71-3.76 (m, 1H, 70-CH2), 5.07 (d, 1H, J = 9.5 Hz, 4-CH), 5.52 (d, 1H, J = 9.5 Hz, 5-CH), 6.70 (t, 1H,
J = 7.5 Hz, ArH), 6.76 (d, 1H, J = 8.0 Hz, ArH), 6.93–7.34 (m, 11H, ArH), 7.41–7.48 (m, 3H, ArH),
7.60 (d, 1H, J = 7.5 Hz, ArH), 7.74 (s, 1H, NH), 7.88 (m, 1H, ArH), 8.10 (d, 1H, J = 8.5 Hz, ArH), 8.41–8.42
(m, 1H, J = 4.5 Hz, ArH). 13C NMR (125 MHz, CDCl3): δC 54.23, 54.66, 57.87, 64.30, 64.50, 65.53, 74.55,
109.39, 122.24, 122.43, 122.97, 126.83, 126.94, 127.34, 127.71, 128.04, 128.73, 128.98, 129.25, 129.83, 129.90,
130.00, 130.49, 130.98, 131.69, 133.27, 133.70, 134.10, 134.81, 135.99, 136.32, 140.45, 141.61, 149.16, 157.41,

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166.79, 178.05, and 198.82. Mass: 741 [M+]. Anal. calc. for C40H30Cl4N4O2: C, 64.88; H, 4.08; N, 7.57.
Found: C, 64.79; H, 4.21; N, 7.42%.
4. Conclusions
An efficient synthesis of a series of novel spirooxindole-pyrrolidine heterocyclic molecular scaffolds
in good to excellent yields has been achieved using a one-pot three-component (3+2) cycloaddition
reaction of azomethine ylide generated in situ from isatin and phenylglycine with a new class of
functionalized dipolarophiles. The structure of all the new compounds were elucidated by FT-IR,
NMR spectroscopy and mass spectrometric data. The synthesized spirooxindole heterocyclic hybrids
were tested to investigate the extent of anticancer activities against the HepG2 cancer cells in vitro
(up to 200
the positive control of melphalan (15
the highest activity was obtained for the compound 8g over a 48 h period (i.e., obtained the least IC50
value of only 12.7 g/mL). From the further investigation of the mechanism of toxicology, we found
µg/mL; 24–48 h time period). The comparative analysis of the obtained results with those of
µg/mL) and the negative control of no cell treatments showed that
µ
that the HepG2 cells experienced the apoptotic pathway with the induction of ROS generation and the
involvement of caspases. This type of cell death mechanism is a potential pathway for the treatment of
cancer cells, as noncancer cells do not have to scarify and full control over diseased cells can be possible.
Supplementary Materials: The following are available online. Detailed methodology, NMR, IR and mass spectra
of a representative compound.
Author Contributions: Conceptualization, R.S.K.; investigation, D.M.A.-t., R.S.K., A.I.A., N.A., and F.M.; writing of
the original draft preparation, R.S.K. and F.M.; supervision, R.S.K.; funding acquisition, A.I.A. All authors have
read and agreed to the published version of the manuscript.
Funding: The authors acknowledge the Deanship of Scientific Research, King Saud University, Riyadh,
Saudi Arabia for funding this work through the grant number RGP-026.
Acknowledgments: The authors thank Stellixir Biotech Pvt. Ltd., Bangalore, India for performing the
biological assays.
Conflicts of Interest: There are no conflict to declare.
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